key: cord-317720-gbi11oxx authors: Lefferts, Joel A.; Gutmann, Edward J.; Martin, Isabella W.; Wells, Wendy A.; Tsongalis, Gregory J. title: Implementation of an Emergency Use Authorization Test During an Impending National Crisis date: 2020-05-14 journal: J Mol Diagn DOI: 10.1016/j.jmoldx.2020.05.001 sha: doc_id: 317720 cord_uid: gbi11oxx Abstract The laboratory response to the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic may be termed heroic.
From the identification of the novel coronavirus to implementation of routine laboratory testing around the world to the development of potential vaccines, laboratories have played a critical role in the efforts to curtail this pandemic.
In this brief report, we review our own effort at a mid-sized, rural, academic medical center to implement a molecular test for the virus; and, we share insights and lessons learned from that process which might be helpful in similar situations in the future.
The laboratory response to the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic may be termed heroic.
From the identification of the novel coronavirus to implementation of routine laboratory testing around the world to the development of potential vaccines, laboratories have played a critical role in the efforts to curtail this pandemic.
In this brief report, we review our own effort at a mid-sized, rural, academic medical center to implement a molecular test for the virus; and, we share insights and lessons learned from that process which might be helpful in similar situations in the future.
As news began to disseminate from Wuhan, China toward the end of 2019 and in early 2020 of a novel coronavirus and its rapid spread throughout that country, we began to think about the potential implications if the virus were to come to the United States.
From a laboratory perspective, we had three major initial concerns regarding clinical molecular testing for SARS-CoV-2: i) which test and which instrument would be ideal, ii) the availability of reagents and other supplies to perform the testing and uncertainty about the number of samples which we might receive, and iii) the laboratory workflow with respect to safety.
Over the next few weeks, it became clear that the spread of the novel coronavirus, now known as SARS-CoV-2, would impact more countries than just China, and on March 11 th , 2020 the World Health Organization (WHO) declared SARS-CoV-2 a pandemic.
An alarm was triggered for us when an initial attempt by the CDC to provide testing materials to state laboratories resulted in withdrawal of test kits due to contamination issues of reagents for one target region (N3) which was later removed, with resultant delays in developing early national testing capabilities.
And a clock began ticking with respect to our own laboratory's response to an evolving national crisis.
As we explored the possibilities of developing testing in-house, we were immediately met by the peremptory challenge that the Centers for Disease Control (CDC) and the State Health Laboratory Network would perform all initial testing.
While the CDC has significant expertise in dealing with such crises, a limiting factor that is often overlooked and became evident in the SARS-CoV-2 pandemic, is that State Public Health Laboratories are under-resourced to function in this expected role.
In addition, the hurdles to developing and/or validating a laboratory developed test (LDT) for SARS-CoV2 became much greater when a Public Health Emergency was declared by the secretary of Health and Human Services on January 31 st 2020 (pursuant to Section 564(b)(1)(C) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C.
§360bbb-3)) and the CDC assay was subsequently granted emergency use authorization on February 4th by the FDA.
This declaration, while fast-tracking approval of a first diagnostic test for an emerging public health threat concurrently raises the bar and regulatory hurdles for developing and/or validating laboratory-developed tests.
Concerned that the efforts of state laboratories would be further impacted by lack of resources, we began to identify sources -including the WHO, the CDC, and commercial vendors -of the required primers and probes for the reverse transcriptase polymerase chain reaction (RT-PCR) detection of the virus and placed orders for test reagents from potential suppliers.
The requested reagents included those needed for both manual and automated extraction of RNA from respiratory specimens.
Concerned that reagents might prove scarce, our plan was to evaluate and validate several extraction methods and RT-PCR assays simultaneously, with the hope that we would obtain a sufficient quantity of reagents to successfully initiate and maintain testing with one or more methods.
On February 29, 2020, the FDA issued the following directive: "Policy for Diagnostics Testing in Laboratories Certified to Perform High-Complexity Testing Under CLIA prior to Emergency Use Authorization for Coronavirus Disease-2019 during the Public Health Emergency".
The document provided guidance for high complexity testing laboratories developing SARS-CoV-2 tests for submission for Emergency Use Authorization (EUA) status with respect to required validation experiments and reporting to the FDA.
The policy stipulated that the required experimental data would include analytical sensitivity/limit of detection (LOD), clinical evaluation (accuracy), inclusivity, and cross reactivity testing.
The latter two criteria could be determined in silico but LOD and clinical evaluation would need to be determined with positive samples.
Importantly, once internally validated, clinical testing by high-complexity laboratories could begin immediately with a 15-day grace period before submission of the validation study to the FDA.
Given a lack of positive samples, the FDA allowed for the use of "contrived samples" that is, specimens in which known positive amounts of virus or viral RNA had been "spiked" into the samples.
While well-meaning, the guidelines proved problematic since our laboratory lacked the control RNA to perform these experiments and dealing with live virus required a Biosafety Level 3 (BSL3) facility, which, like most teaching hospitals, we lack.
While other control material such as synthetic controls and plasmids became available, the FDA maintained the need to use purified viral RNA and identified a vendor which could supply it.
Initially laboratories were required to spike RNA transcripts into previously extracted nucleic acid from negative samples for the CDC assay to determine the limit of detection but this had its own challenges of not representing extraction of true clinical samples and issues with degradation were identified.
We were then able, according to the FDA, spike purified viral RNA into crude patient specimens before performing the RNA extraction and RT-PCR portions of the procedure.
While this represented a positive clinical sample to the best of the laboratory's ability, it still did not fully account for the extraction efficiency from viral particles in a clinical specimen and naked RNA spiked into transport media or negative clinical specimens tended to degrade more rapidly.
During an informational conference call hosted by the FDA, laboratories requested to be able to use synthetic control materials, but the agency remained steadfast in its decision.
As laboratories began to place orders for the RNA control, another bottleneck in the development and validation of laboratory testing became evident, as the supplier could not distribute this required material at a sufficient pace.
At our institution, test development was delayed by more than two weeks because of the lack of access to this control material.
Although we had several synthetic and plasmid controls sitting in our freezer, we were not allowed to use them for the "spike-in" experiments.
Moreover, during multiple attempts to order the FDA-mandated RNA control, we were asked to complete numerous forms and applications that required signatures from institutional administrators who were already in the midst of dealing with the broader, evolving hospital virus crisis, creating an additional stress point.
After significant feedback from clinical laboratories, the FDA indicated that the use of some synthetic control materials (in vitro RNA transcripts) could be used for the "spike-in" experiments one week after we had finally obtained the recommended RNA material.
We chose to implement a verification strategy that would align with both emerging EUA guidance from the FDA and our standard requirements as a clinical molecular diagnostic laboratory operating under our usual regulatory framework of Clinical Laboratory Improvement Amendments (CLIA) certification and accreditation through the College of American Pathologists (CAP).
Our plan included the production of enough contrived clinical specimens and control material to proceed with validation or verification of the multiple (laboratory-developed and CDC EUA) tests that we were evaluating.
In an attempt to match approved RNA extraction methods with RT-PCR assays, we were able to extract enough nucleic acid samples to evaluate and/or validate multiple RT-PCR assays without the need for further extractions.
In essence we created a "validation set" of extracted samples.
The set included four known positive and four known negative patient samples that we obtained from the New Hampshire Public Health Laboratory (the authors gratefully acknowledge the support of Drs.
Christine Bean and Fengxiang Gao), several presumed negative samples that had previously been tested for other bacterial/viral infections, and several positive contrived specimens (spiked with viral RNA or known positive patient specimens).
In addition, we prepared standard operating procedures for the different extraction methods and RT-PCR assays as well as training documentation and worksheets.
While these steps were in excess of what the FDA required, we wanted to be confident about the validity of each assay.
These "verification" studies, while addressing typical performance characterisitcs of a test, were minimal compared to what a validation study would entail for a qualitative molecular test in our laboratory.
Safety risk assessments were performed for working areas of the laboratory and for specimen receiving/transport.
As a result, we modified our normal personal protective equipment (PPE) requirements (eye protection, disposable gloves and an impervious lab coat) to also include the use of face shields and masks while processing raw samples in a biosafety cabinet.
We also implemented additional precautions for waste disposal (use of a double bagging technique and trash removal by molecular staff to minimize foot traffic in the laboratory) and modifications to hand washing and hand sanitizing practices (required hand washing between glove changes and increased frequency of glove changes).
Remarkably, we began to perform patient testing less than one week after obtaining the materials to perform these validation steps.
As much as biomedical laboratories practice preparedness for situations such as this pandemic, there inevitably are unknown variables.
One "unknown" -that we now know only too well and that is thwarting larger scale implementation of testing at our medical center -is the nation-wide shortage of collection swabs and transport media.
While laboratories have the ability to use alternative transport media including saline, there is little we can do without collection devices.
As each swab type used in clinical laboratory testing was eventually approved for use in assays, national supplies became exhausted.
Viral transport media, historically a transport media used for molecular detection of viruses, was also becoming depleted across the country.
Fear and chaos resulted from not knowing all the clinical challenges this virus would present, how many people would be infected and become symptomatic, the degree of severity of those symptoms and how many people could potentially die.
As outlined above, we and other laboratory scientists quickly marshalled teams to develop and implement tests for the virus that hopefully will help shed light on these questions and prevent wide-scale panic and deaths.
We certainly hope that we do not see another pandemic such as SARS-CoV-2, but it is naïve to think another one will never occur.
To plan for potential future outbreaks, laboratories should be prepared and equipped to rapidly identify the pathogen; the evolution of third and fourth generations of massively parallel sequencing technologies should make this possible.
Molecular-based testing with a variety of real time PCR platforms should be granted rapid EUA status for use of standardized primer/probe sequences in conjunction with multiple specimen types and extraction methods.
Federal agencies should be better prepared to distribute control materials as well as test reagents to laboratories operating outside of the State Health Laboratory network.
While high complexity molecular testing should be restricted to those laboratories accredited to do such work, more flexibility to develop tests needed to curtail a pandemic should be granted to these laboratories under an EUA.
Finally, a global crisis is not the appropriate time for finger pointing among countries, agencies, vendors, laboratories, healthcare workers, and population sectors.
Politicians and medical personnel interviewed by the media should be certain that they understand laboratory testing processes before their limitations and capabilities, and the implications of a positive or negative test.
The SARS-CoV-2 crisis also highlights the importance of cooperation, communication, and collaboration among agencies of the federal government (e.g., FDA, CDC) and the laboratories at academic medical centers.
Our experience shows that the latter improvised while the former loosened strictures in an imperfect but ultimately collective effort to implement testing and keep our population safe
key: cord-016640-pvlg3nkp authors: Baron, Ellen Jo; Campbell, Sheldon title: Technical and Clinical Niches for Point of Care Molecular Devices date: 2012-04-05 journal: Advanced Techniques in Diagnostic Microbiology DOI: 10.1007/978-1-4614-3970-7_33 sha: doc_id: 16640 cord_uid: pvlg3nkp A point of care (POC) device is one that is used outside of a central laboratory environment; generally near , or at the site of the patient/client.
Point of care testing (POCT) varies from tests performed in physician’s office labs, or “satellite” or “stat” labs, to tests performed on tabletop instruments in a clinic area, to testing performed with hand-held instruments at the bedside.
In peripheral lab settings, POCT may be performed by trained laboratory staff, but clinic and bedside POCT is frequently performed by staff who lack specialized laboratory training and whose primary job is something other than doing lab tests.
testing (e.g., viewing of malaria smears via mobile phones) are also coming into use which might fi t an operational de fi nition of POCT [ 1 ] .
There are several reasons to develop a POC test for an infectious disease.
These include: The need to quickly provide highly targeted therapy.
Current algorithms for • seriously ill patients depend on empiric treatment based on the most likely pathogens for a given clinical presentation; however, this method involves broad-spectrum therapy to cover the likely contingencies.
Knowing the exact identi fi cation of the pathogen will allow more focused therapeutic decisions.
If a molecular method also detects important resistance factors in the pathogen, then a therapeutic decision can be made speci fi cally to both treat the pathogen and limit development of resistant organisms.
POCT infectious disease molecular assays may be developed to detect speci fi c • infections for which a rapid response is desirable.
Examples include common outpatient infections such as group A streptococcal pharyngitis where immediate diagnosis saves follow-up effort; or Chlamydia and gonorrhea, where rapid results may allow immediate treatment of patients who might otherwise be lost to follow-up.
There is the potential for both clinical and public health bene fi ts from this class of test.
Another potential objective of a POCT is to recognize quickly which patients • require infection control precautions as they are admitted to the healthcare institution to prevent the spread of the agent to other patients or to caregivers.
Some POC assays are meant for surveillance only and in such cases, interventions are taken to break transmission routes and prevent the development of infections.
Increasingly, healthcare institutions are being asked to become more cost-effective, and rapid applications of infection control activities have been shown to be most effective.
The potential for POCT to impact on infection-control is particularly signi fi cant for long-term care facilities and other health care settings without on-site laboratories.
Platforms employing molecular technology which are simple enough for potential POC use are just beginning to come to market.
No molecular test has yet achieved CLIA "waived" status (although some are being developed for FDA submission), so POC molecular tests are still physician's of fi ce lab or "satellite lab" tests rather than true bedside methods.
Several comparatively simple, rapid molecular methods, though, are available and increasingly used.
The most widely used molecular test at patient POC sites is the real-time PCR (RT-PCR) assay for detection of nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA) [ 2 ] .
Colonized patients can be placed into contact isolation, decolonization protocols can be initiated, and appropriate surgical prophylaxis can be used [ 3 ] .
The use of this assay within the United States Veterans' Administration hospitals is one factor credited with lowering healthcare-associated MRSA infections 59 % since universal screening and additional infection control interventions were implemented.
Selected nosocomial infections due to Clostridium dif fi cile and vancomycin-resistant enterococci (VRE) also decreased [ 4 ] .
Two further reports on the use of surveillance for MRSA illustrate the effectiveness of this intervention.
With rapid results available within hours of patient admission, the Northshore Hospital System showed 69.6 % decrease in hospital-associated MRSA disease over the study period [ 5 ] .
In contrast, another healthcare institution used a slower method for MRSA nasal surveillance with results available more than a day later and results were disappointing [ 6 ] .
Molecular POC tools are virtually the only method possible to achieve the most effective infection control.
Additional assays that detect both MRSA and methicillinsusceptible staphylococci in patients' skin and soft tissue wound sites and in nares are also available [ 7 ] .
Testing feces for the presence of toxigenic C. dif fi cile is another use of rapid molecular technology today [ 8 ] .
An RT-PCR platform and a loop-mediated isothermal ampli fi cation (LAMP) platform are FDA-cleared.
They each employ different targets.
The LAMP assay seeks a genetic locus in the TcdA gene of C. dif fi cile whereas the PCR assays either identify a portion of the toxin B gene (TcdB) [ 9 ] or a second FDA-cleared assay presumptively identi fi es the epidemic, hypervirulent 027 strain by detecting both a binary toxin sequence and a deletion in the toxin regulatory gene, in addition to the TcdB gene [ 10, 11 ] .
The same RT-PCR platform is FDA-cleared for detection of in fl uenza A, B, and in fl uenza A H1N1 novel 2009 in respiratory secretions, which is a modi fi cation of a previous test available for a limited time during the 2009 H1N1 In fl uenza A outbreak [ 12 ] .
In addition, a self-contained PCR technology using packets of reagents in plastic pouches has also been FDA-cleared for detection of respiratory viruses [ 13 ] .
Another rapid molecular test has been FDA-cleared for multiplex detection of 15 respiratory viruses, including adenovirus, 2 coronavirus strains, 5 in fl uenza strains, human metapneumovirus, parain fl uenza virus types 1-4, RSV, and rhino-enterovirus, with a time-to-result of 1-1.5 h using a novel multiplex PCR and array detection format [ 14 ] .
Although other PCR methods for virus detection and identi fi cation in respiratory secretions are available and show excellent sensitivities and speci fi cities, they are not candidates for POC tests due to their complexity, long performance time, or format that leads to inef fi ciencies when performing non-batch (such as stat) testing [ 14 ] .
An FDA-cleared RT-PCR assay can be used to detect gastrointestinal colonization with VRE using rectal swabs [ 15 ] .
The US version of the test was developed to detect the vanA gene only because vanB VREs are uncommon in the United States today, and because there are more vanB-containing non-enterococci than enterococci in feces.
A commonly used FDA-cleared PCR platform has another enterococcal assay that detects vanA and vanB, but speci fi city of the vanB marker is poor and the format is not optimized for POC [ 16 ] .
The same platform as described for VRE and staphylococci is FDA-cleared for direct detection of group B streptococci (GBS) in vaginal/rectal swabs [ 17 ] .
This assay has been used at the time of delivery to test women who never received antenatal surveillance cultures for GBS, or for women who tested negative in their surveillance cultures but whose colonization status may have changed between the time of the culture and presentation in labor [ 18 ] .
In addition, this platform has an FDA-cleared test for the presence of enterovirus in cerebrospinal fl uid [ 19 ] .
Unlike all the other tests available on this platform, the CSF enterovirus assay is designated "high complexity" due to the need for the testing person to pipette a speci fi c 200 m L volume of CSF into the cartridge.
Lightcycler ® and other RT-PCR platforms have been used to develop tests for herpes simplex and varicella zoster virus in cerebrospinal fl uid.
They could be considered borderline POC tests because if ordered stat, a highly trained laboratory scientist could theoretically perform the test and report results within the 4 h timeframe [ 20 ] .
It is unlikely, however, that in cases of severe disease, the clinicians treating the patient would be willing to wait that long before treating based on clinical presentation and CSF cell count separate from microbiology laboratory results.
A direct DNA hybridization assay for identi fi cation of Gardnerella vaginalis (as a marker for bacterial vaginosis), Candida albicans , and Trichomonas vaginalis has been in use in large physician of fi ces and reference laboratories for many years [ 21 ] .
Tests can be run in batches of 6 or fewer samples and results are available within 2 h. In countries other than the United States, the RT-PCR method is used to detect Mycobacterium tuberculosis and rifampin-resistance in M. tuberculosis using a hemi-nested PCR protocol that uses fi ve molecular beacons to bind to different regions of the ribosomal polymerase B gene in which most mutations conferring rifampin resistance are found [ 22 ] .
If all fi ve regions bind to their speci fi cally colored fl uorescent beacons, the organism is a wild-type M. tuberculosis .
If one or more of the regions fails to bind its speci fi c beacon, but at least two regions are present, the M. tuberculosis strain is reported as resistant [ 23 ] .
This test can be used with unprocessed respiratory tract secretions at the patient location and results are available within 2 h. Studies have shown that even unskilled workers can achieve high levels of accuracy with this assay [ 24 ] .
After achieving endorsement by the World Health Organization, it is being broadly disseminated throughout the resource-poor world.
It should not be overlooked that there are several POC diagnostics for detection of agents of bioterrorism.
Anthrax, Yersinia pestis , and Francisella tularensis are all easily weaponized agents, and tests have been developed and fi eld tested for their detection in both the environment (e.g., powders) and in or on patients [ 25 ] .
Several attractive targets for POC infectious disease diagnostics exist.
These include: Diagnosis of bacteremia and fungemia.
Current culture-based technology • requires incubation for at least 8 hours before the fi rst indication of a positive result, after which some organisms can be rapidly identi fi ed using molecular methods [ 26 ] .
However, appropriate therapy within the fi rst few hours often makes the difference between severe morbidity or death and recovery [ 27 ] .
Because the numbers of circulating bacteria or yeast in the bloodstream of septicemic patients can be low, the volume of blood necessary to detect small numbers of organisms has limited the application of molecular methods.
Once an effective front-end concentration system is developed, the diagnosis of these extremely severe infections can be approached as a POC test.
Meningitis and encephalitis are potentially severe infections that bene fi t from • early diagnosis so that patients can be appropriately managed.
The limited number of common pathogens associated with CNS infections makes development of such tests feasible.
Additional agents for which rapid, simple molecular tests are needed include Neisseria meningitidis , Streptococcus pneumoniae , GBS, Listeria , Haemophilus in fl uenzae , herpes simplex and varicella zoster virus.
Tests for diagnosis and management of diseases seen in outpatients, particularly • those in hard-to-manage populations.
POC tests for STDs, for respiratory viruses and group A streptococci, and for HIV and HCV viral load can potentially streamline care for these conditions, allowing same-visit management and decreasing both the effort of follow-up and the potential public health impact of patients who cannot be contacted to deliver their results.
Many clinicians and infection preventionists are concerned about the rising • incidence of multidrug-resistant gram-negative rods.
Metallo-beta-lactamases, carbapenemases, cephalosporinases, etc., pose risks to patients and problems for infection control.
A rapid molecular test to detect major determinants of resistance, regardless of the organism carrying them, would be a desirable rapid or POC test.
Several pathogens can be detected in patient samples using molecular tests within 4 h. However, there are formidable obstacles to moving molecular diagnostics into the POC setting.
There include: The impracticality of performing some methods in a random access, non-• batched mode.
The need for highly trained individuals to perform the test; and if they must be • located at the POC, the inef fi ciency of having such individuals waiting during the time between test requests.
The need for space for instruments and other supplies and physical infrastructure • that do not exist at most POC locations.
The delay incurred when an additional sample is received for testing once a testing • process has commenced that cannot be stopped in the middle.
The need to test all necessary controls with individual samples rather than groups • of patient samples.
The need for additional instruments for sample preparation or pre-ampli fi cation.
• The possibility of contamination.
• After these factors have been considered, the remaining current technologies include polymerase chain reaction, isothermal loop-mediated ampli fi cation, and direct DNA hybridization.
Other methods are in earlier stages of development but may show potential in the future.
Molecular methods, when brought to routine POC use, have the potential to provide performance equivalent to that of laboratory-based methods.
Methods must be chosen to have extreme sensitivity to detect small numbers of organisms in limited sample volumes, and further automation and miniaturization of platforms is desirable [ 28 ] .
This is the situation in a number of infectious diseases; for example, tuberculous meningitis, where the paucibacillary nature of the cerebrospinal fl uid has challenged the development of effective molecular assays [ 29 ] .
Molecular methods at POC will bring new challenges to those who administer and perform POCT.
In addition to the usual QA and QC associated with any POCT, molecular POCT will require procedures for controlling contamination with both ampli fi ed material and patient-derived materials.
QC of each stage of the analytical procedure; extraction, ampli fi cation, and detection, may make trouble-shooting more challenging.
The phenomenon of inhibited specimens may require operators to report more complex results than "positive" or "negative."
POC molecular instruments are likely to be more complex than current systems such as glucose testing systems and may, initially, lack some of the sophisticated POC management tools associated with traditional POC platforms [ 30 ] .
Molecular diagnostic technologies are transforming the diagnosis of infectious diseases.
Current and emerging clinical needs; increased acuity of inpatient care, expanded outpatient care, and an increasingly mobile population; the need to control healthcare-acquired infections, and novel antibiotic resistance mechanisms, will all drive molecular microbiology to the POC [ 31 ] .
Mobile phone based clinical microscopy for global health applications Institutional prescreening for detection and eradication of methicillin-resistant Staphylococcus aureus in patients undergoing elective orthopaedic surgery Preventing surgical-site infections in nasal carriers of Staphylococcus aureus Veterans Affairs initiative to prevent methicillinresistant Staphylococcus aureus infections Universal surveillance for methicillin-resistant Staphylococcus aureus in 3 af fi liated hospitals Intervention to reduce transmission of resistant bacteria in intensive care Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares Increased Clostridium dif fi cile virulence demands new treatment approach Rapid and sensitive loopmediated isothermal ampli fi cation (LAMP) test for Clostridium dif fi cile diagnosis challenges cytotoxin b cell test and culture as gold standard Clostridium dif fi cile infection caused by the epidemic BI/NAP1/027 strain Comparison of strain typing results for Clostridium dif fi cile isolates from North America Validation of the Cepheid Xpert Flu A real time RT-PCR detection panel for emergency use authorization Comparison of two multiplex methods for detection of respiratory viruses: FilmArray RP and xTAG RVP Strengths and weaknesses of FDA-approved/cleared diagnostic devices for the molecular detection of respiratory pathogens Evaluation of GeneOhm VanR and Xpert vanA/vanB molecular assays for the rapid detection of vancomycin-resistant enterococci Comparison of the BD GeneOhm VanR assay to culture for identi fi cation of vancomycin-resistant enterococci in rectal and stool specimens Evaluation of the Xpert(R) group B streptococcus real-time polymerase chain reaction assay compared to StrepB Carrot Broth for the rapid intrapartum detection of group B streptococcus colonization Diagnostic accuracy of a rapid real-time polymerase chain reaction assay for universal intrapartum group B streptococcus screening Multicenter beta trial of the GeneXpert enterovirus assay Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fl uid specimens Clinical evaluation of af fi rm VPIII in the detection and identi fi cation of Trichomonas vaginalis , Gardnerella vaginalis , and Candida species in vaginitis/vaginosis Rapid molecular detection of tuberculosis and rifampin resistance Rapid detection of Mycobacterium tuberculosis and rifampin resistance by use of on-demand, near-patient technology Feasibility, diagnostic accuracy, and effectiveness of decentralised use of the Xpert MTB/RIF test for diagnosis of tuberculosis and multidrug resistance: a multicentre implementation study Current and developing technologies for monitoring agents of bioterrorism and biowarfare Usefulness of real-time PCR for the diagnosis of sepsis in ICU-acquired infections Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis Point-of-care testing and molecular diagnostics: miniaturization required Rapid diagnosis of tuberculous meningitis: what is the optimal method Point-of-care testing informatics Clinical microbiology in the 21st century: keeping the pace
key: cord-256852-lrz17bdx authors: Nayyar, Gaurvika M. L.; Attaran, Amir; Clark, John P.; Culzoni, M. Julia; Fernandez, Facundo M.; Herrington, James E.; Kendall, Megan; Newton, Paul N.; Breman, Joel G. title: Responding to the Pandemic of Falsified Medicines date: 2015-06-03 journal: Am J Trop Med Hyg DOI: 10.4269/ajtmh.14-0393 sha: doc_id: 256852 cord_uid: lrz17bdx Over the past decade, the number of countries reporting falsified (fake, spurious/falsely labeled/counterfeit) medicines and the types and quantities of fraudulent drugs being distributed have increased greatly.
The obstacles in combating falsified pharmaceuticals include 1) lack of consensus on definitions, 2) paucity of reliable and scalable technology to detect fakes before they reach patients, 3) poor global and national leadership and accountability systems for combating this scourge, and 4) deficient manufacturing and regulatory challenges, especially in China and India where fake products often originate.
The major needs to improve the quality of the world's medicines fall into three main areas: 1) research to develop and compare accurate and affordable tools to identify high-quality drugs at all levels of distribution; 2) an international convention and national legislation to facilitate production and utilization of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; and 3) a highly qualified, well-supported international science and public health organization that will establish standards, drug-quality surveillance, and training programs like the U.S. Food and Drug Administration.
Such leadership would give authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines.
The organization would also advocate strongly for including targets for achieving good quality medicines in the United Nations Millennium Development Goals and Sustainable Development Goals.
Malaria is a devastating illness, particularly to young children and pregnant women in tropical countries.
A recent review reported that the active pharmaceutical ingredient (API) was absent in over one-third of close to 4,000 antimalarial drug samples tested from pharmacies in seven southeast Asian and 21 sub-Saharan African countries 1 ; over 40% of the alleged artemisinin-containing drugs were falsified, outright fakes.
A wide variety of falsified brand name and generic medicines and even falsified raw ingredients for several essential pharmaceuticals have been found in rich and poor countries.
[2] [3] [4] [5] [6] [7] Such drugs are often used for acutely ill patients, many of whom would die or suffer prolonged illness without proper treatment.
In addition to patients' loss of confidence in the health-care delivery system, microbial resistance to the drug may develop and spread if medicines contain subtherapeutic doses or no API.
The increasing global scientific and public awareness and epidemic proportions of the spreading problem are reflected in the number of articles on "fake drugs" cited in PubMed: 27 Until recently, there were a paucity of reports from pharmaceutical companies on the type and quantity of drugs that were fraudulently compounded or transferred by criminals.
Data are emerging from the Pharmaceutical Security Institute (PSI), a not-for-profit membership organization of pharmaceutical security directors, indicating that a large number of companies, products, and countries are targeted.
8 For instance, since 2008, Pfizer Pharmaceuticals (Pfizer Global Security, New York, NY) has identified a rapidly increasing number of falsified products, countries reporting falsified drugs, and breaches of the legitimate supply chain national entry points (Table 1) ; the increases have been from 40% to over 100%.
Of Pfizer products, those for erectile dysfunction are most frequently falsified 9 ; other such products target patients with Alzheimer's disease, cancer, high cholesterol, hypertension, malaria, and anxiety disorder.
Facilities where fake drugs were made or compounded were discovered with moldy walls, dirty equipment, and infested with rodents and insects ( Figure 1 ).
Falsifiers have created products that are visually indistinguishable from the genuine product, clearly demonstrating criminal intent to deceive.
This increasingly recognized problem on virtually all continents is a pandemic defined as "an epidemic occurring over a very wide area, crossing international boundaries, and usually affecting a large number of people."
10 DEFINITIONS Despite increasing awareness of the fraudulent drug epidemic, efforts to quantify and stop this peril have been stymied by multiple obstacles, not the least of which is agreement on definitions.
4, 11, 12 Poor quality drugs include substandard/ spurious/falsely labeled/falsified/counterfeit (SSFFC) medical products.
4 Falsified (also commonly called fake or counterfeit products are intentionally and fraudulently produced and contain no API, the incorrect dose of the API, or the incorrect API.
Substandard medicines are caused by unintentional or negligent errors of manufacturing or by degradation after manufacturing resulting in insufficient API, poor dissolution properties, or degradation products.
The nomenclature used by the World Health Organization (WHO), the World Trade Organization, the United Nations (U.N.) Office on Drugs and Crime, INTERPOL, and others can be confusing; hence, we are using terms agreed upon by WHO.
4 There are also properly manufactured medicines that are unlawful for reasons apart from their quality.
These can be unregistered with company branded or generic medicines that, for reasons of theft or accidental or intentional diversion, do not have the legally required marketing authorization of the country's regulators to be imported or sold there, and medicines that infringe the trademark of a legal product.
Relatively little is known about medicines that have expired and are repackaged with a new date; these topics along with diverted products are beyond the realm of this article.
This article focuses mainly on medical and public health considerations of falsified medicines that are particularly widespread in low-and middle-income countries.
[13] [14] [15] Although there are increasing reports of detection of a variety of fake drugs from around the world, paradoxically, there are virtually no reports from middle-or high-income countries quantifying the state of poor quality medicines, only anecdotal case citations.
Governments have been hampered by a confusing array of expensive detection technologies.
Few functional national regulatory authorities exist in low-income nations that lack trained staff and suitably equipped laboratories to test drug quality centrally or in peripheral pharmacies or markets.
13, 14 Furthermore, the variability or absence of national and international criminal statutes, lack of an international agreement against trafficking of poor quality medicines, and inadequate punishments for convicted offenders reflect the weak legal framework for confronting drug fraud.
One of the biggest obstacles in provision of quality-assured pharmaceuticals is the lack of effective manufacturing, regulatory, and quality processing in India and China.
In 2013, 10 global public health agencies including providers, foundations, and research institutions contributed to developing an advocacy campaign to address falsified medicines, particularly in China.
This campaign called Fight the Fakes is a step toward raising awareness about the problem, but legal action has to follow along with more public and political awareness.
15 The U.S. Institute of Medicine (IOM) has published a report "Countering the Problem of Falsified and Substandard Drugs."
16 The IOM recommendations to "stem the global trade" in such products are laudable in advising that the U.S. Food and Drug Administration (FDA), the National Institute of Standards and Technology, and other U.S. and international pharmaceutical and financing agencies be more actively involved in setting standards and financing improvements; yet this report falls far short of making a strong call for standardized, agreed-upon quality assessment technologies; an international law convention; and a more activist, internationally recognized lead organization, all three of which are essential for stopping the many health threats of fake drugs.
Global leadership to date has devolved in parts to the WHO, the U.N. Office on Drugs and Crime, and INTERPOL, each with diverse missions, responsibilities, limited authorities, and their own collaborations, funding networks, cultures, and languages.
17 No organization is leading assertively.
Of the three areas listed, an international convention and improved national regulations are likely to have the most enduring value in concert with effective leadership and other innovations.
The focus of all actions tied to drug quality must be on public and individual health, and strengthening national capacities to improve the health of their citizens.
Detection methods and technology.
A major hindrance to understanding the types, names, extent, and amount of poor quality drugs nationally and globally has been 1) the lack of agreed-upon field survey approaches 18 and 2) available lowcost tools to detect and classify bad drugs quickly at points of entry into countries, at public and private pharmacies, and in health units.
In 2014, the WHO published draft guidelines for surveys of medicine quality that are currently being revised.
19 Two or more levels of drug quality tests exist: 1) methods useable in the field that are quick, inexpensive, and easy to use and teach; these methods are targeted mainly to examine packaging and detect drug contents and 2) technologies requiring a laboratory equipped for exhaustive chemical analysis.
These approaches are summarized in Table 2 , deriving from the IOM report 16 and a recent analysis by Green and others 20 from the Centers for Disease Control and Prevention reference laboratory.
Within each method there are numerous tools and prototypes being used and new ones tested.
Current technologies for field use rely on visual packaging inspection, lot number reporting via mobile phones, thin-layer chromatography, colorimetric tests, and simplified spectroscopic methods.
Gas and liquid chromatography and mass spectrometry are some of the more advanced and complex techniques for investigating drug quality in central laboratories.
Qualitative or semiquantitative tests for an API are not substitutes for proper manufacturing control, dissolution studies, pharmacokinetics equivalence, and supply chain integrity.
A very promising recent development has been the U.S. Pharmacopeia Promoting the Quality of Medicines (PQM) program in several African, Asian, and Latin American countries using the "Minilab" (Global Pharma Health Fund e.V., Giessen, Germany).
[21] [22] [23] This training program supported by the U.S. Agency for International Development (USAID) and the President's Malaria Initiative (PMI) has trained several hundred persons in rapid chemical analysis of drugs taken from public and private pharmacy stocks.
23 A major reference training center has recently been opened in Accra, Ghana, with USAID and U.S. Pharmacopeia support as a referral testing and regional training center.
Important also is the development of the counterfeit detection (CD)-3 (US Food and Drug Administration, Forensic Chemistry Center, Cincinnati, OH), a promising handheld electronic device for peripheral use that detects fake packaging at point of sale with images and videos of the suspect samples.
24, 25 The FDA, Skoll Foundation, and other partners are supporting expansion of testing and use of this device.
We recommend that a precertification of essential diagnostics, drugs, and vaccines should be required for specific regional and global control, elimination, and eradication programs and campaigns.
More information is needed to confirm that precertification of products is occurring for the PMI and the Global Fund to Fight AIDS, Tuberculosis and Malaria, and products purchased by U.N. International Children's Emergency Fund (UNICEF) and WHO.
Essential drugs designated by WHO should also be targeted for special vigilance by quality assurance mechanisms.
No independent agency has inventoried and performed comparative quality assessments of these packaging and drug-testing devices and made recommendations to countries for their use.
Objective comparisons are needed of the diversity of field methods in terms of accuracy, reliability, costs of equipment and supplies, level of training needed, ease of use, spare part availability, and maintenance requirements.
Simplified standard survey protocols and methods for sampling drugs at country entry points (seaports, airports, and roads); at major pharmacy depots; in health units (public and private hospitals and clinics); and at more peripheral distribution sites (district and village pharmacies and individual vendors) are also needed.
19 Low-cost, portable detection tools would empower pharmaceutical inspectors in numerous countries that have oversight of the medicine supply.
Results would be available promptly rather than delayed when samples are sent to national or international laboratories as occurs now; lamentably, intervals of several years have occurred from the time specimens were collected to the time the results were available to those needing to take action.
1 Ideally, central reference laboratories vetted by WHO, FDA, or another agency would back up spot checks and random sampling of pharmaceuticals at the periphery.
Good quality medicines by law.
Falsified medicines are ultimately a problem that impacts public health.
The solution needs to reflect various incentives, either via financial gain, avoidance of punishment or both.
A multi-sectorial effort is essential for taking into account how this illegal market is interwoven with world trade agreements, business models, and associated legal ramifications.
Globalization has enlarged the international trade in medicines.
For example, India exports over US$15.5 billion in pharmaceuticals, which are among their most important exports.
26 As of 2011, 40% of drugs and 80% of APIs for drugs in the United States are imported from foreign countries.
27 An international law convention against substandard and falsified medicines would address both regulatory and criminal international governance challenges simultaneously through technical, legal, and financial mechanisms.
How would the convention work and what national benefits would it bring?
A convention would provide four legal underpinnings that do not exist, that together would advance patient safety and access to quality medicines.
12 First, a convention would define the various sorts of wrongful medicines accurately and thereby avoid misunderstandings caused by today's problematic or vague terminology (e.g., where countries seized good quality generic medicines as "counterfeit").
Second, a convention would promote the requirement that signatory countries enact national laws to designate wrongful actssuch as the intentional manufacture, trafficking, or selling of falsified medicines-as criminal offences, with attendant obligations to alert health-care workers and to prosecute or extradite the offenders to justice promptly.
Third, a convention would provide the legal and institutional framework for participating countries to agree, implement, and evolve convergent standards of medicine regulation, so as to reduce poor quality medicines in international trade.
Fourth, and for lower income countries particularly, a convention would contain mechanisms for financial and technical assistance, and, to join local and regional networks.
These actions would help build national and regional medicine regulatory authorities (MRAs) to a point where patients' access to quality medicines is protected.
Some have said that establishing recommended codes of practice that are nonbinding (soft law) are better than international norms and regulations that are binding (hard law).
28 We disagree with soft law in regard to controlling the current fake-drug pandemic.
There are precedents for using international law in this way.
A 1929 treaty that internationally criminalizes counterfeit banknotes provides an analogy for falsified medicines.
In the health field, there are treaties specifically addressing the illicit traffic of certain narcotic drugs and treaties to prevent harm-particularly, the Framework Convention on Tobacco Control and its associated protocols to stanch illicit trade.
That convention has brought over US$250 million new funding to global tobacco-control efforts, demonstrating that international law need not compete for resources, but can increase them.
29 The U.N. Office on Drugs and Crime has been developing "Draft Model Legislative Provisions on Fraudulent Medical Products" for several years but there has been no agreement on final text; the focus appears to be on criminal and judicial issues.
Challenges ahead.
Information is accruing that large quantities of falsified drugs are being manufactured in Asian countries.
China and India are two of the largest producers of good quality drugs and vaccines, many of which are purchased or funded by the USAID; UNICEF; Global Fund to Fight AIDS, Tuberculosis and Malaria; WHO; and other organizations, charities, and national agencies for global disease control and eradication programs.
However, according to the World Customs Organization, in 2006, 54% and 21% of unlawful drugs of all sorts confiscated worldwide were manufactured in India and China, respectively.
30 The circuitous travel itineraries of fake medicines have been traced across continents, such that the unsuspecting recipient countries assume a bona fide origin.
A particularly heinous example is that of multiples instances of the production, marketing, and international travel of falsified bevacizumab (Avastin ), a cancer medicine; the fake drug closely matched the appearance of the real medicine, but tests indicated salt, starch, and various cleaning solvents instead of the active ingredient with resulting endophthalmitis.
31 The Internet has opened up an unregulated opportunity for criminals to promote and sell fake drugs to unsuspecting vulnerable populations, often the aged and others seeking convenience and low cost.
A recent survey of over 10,000 online pharmacies found that 96% operated outside legal regulations and a large percentage closed operations within 3 years of operation.
32, 33 The FDA and other organizations participate with INTERPOL in annual international actions (Operation Pangea) to shutdown illegal pharmacy websites selling potentially counterfeit and illegal medical products.
More than 18,000 such illegal websites were closed during one week in 2012 with seizure of US$10.5 million of pharmaceuticals worldwide.
34 Leadership, collaboration, and national strengthening.
Arguably, the major obstacle to solving the problem of poor quality medicines has been the lack of a clearly identified lead organization with a plan of action developed in concert with countries, pharmaceutical companies (multinational corporations and innovator/biotechnology enterprises), and national and international agencies-and a sense of urgency to implement the plan with resources and partners-including pharmaceutical companies in low-income countries.
WHO has estimated that 30% of countries have inadequate medicines regulation authorities (MRAs) or none at all.
Moreover, WHO has found that 90% of African MRAs lack the capacity to undertake medicine regulatory functions and therefore cannot guarantee the quality, efficacy, and safety of medicines, 35, 36 The New Partnership for Africa's Development has found that there is either limited or declining government funding for MRAs in the East African Community Partner States.
37 Many have looked to WHO for this leadership, given its successful implementation of the public health treaty on tobacco control.
However, some argue that the U.N. system, including the WHO, is poorly suited to be in a leadership role because of sparse technical expertise in products, manufacturing, and quality systems.
U.N. agencies are beholden to member states and cannot regulate or enforce anything easily, especially, in India and China.
In this regard, WHO could serve the role of a partner rather than a leader.
The recently revitalized Rapid Alert program at WHO has begun to "track and trace" poor quality drugs as reported voluntarily by member countries.
38 Rapid Alert notices are published periodically by WHO indicating the fake drug type, lot number, quantity of product, and place detected.
Strong action by countries can stem the tide as shown in Rwanda 39 and Cambodia, 40 although unique situations and major multi-sectorial engagements exist in these countries.
One solution is creation of regional harmonization networks, addressing some elements of drug registration tied to regional economic communities; the African Medicines Registration Harmonization Initiative is one example of such a network.
The U.N. Office on Drugs and Crime (UNODC) has also made recent attempts at facilitating international cooperation against falsified medicines.
One proposal has been a trilateral coalition of the UNODC, WHO, and Interpol.
17 Still, active and transparent support from the FDA, drug companies, individual countries, and other partners will be needed; the FDA may be the most qualified as a leadership organization based on their technical expertise and global influence.
Mechanisms for training technical staff, regulating products, improving manufacturing practices, and stopping criminal production are needed to assure a good supply of medicines.
Given that the problem of substandard and falsified medicines should be approached primarily from a public health and equity perspective, it is important that the negotiations on the way forward be led by the Ministries of Health along with the Ministers of Finance and Trade, while respecting legitimate intellectual property rights.
Could and should WHO be the lead organization in curbing the spreading epidemic of falsified pharmaceuticals?
WHO's ability to take more assertive action is strengthened by the revised international health regulations.
WHO's director general can convene emergency committees in response to public health emergencies as has been done recently for the influenza A (H1N1) pandemic in 2009, the middle east respiratory syndrome (MERS-CoV) in 2013, the polio crises in 2014, and the Ebola epidemic in 2014-2015.
Illicit drug trafficking is an emergency.
The Drug Quality and Security Act, signed into law by President Obama in 2013, outlines steps to build an electronic system to identify and trace certain prescription drugs in the United States.
The results of this system are awaited.
Finally, the Millennium Development Goals (MDGs), under revision, should include measurable objectives for good quality drugs.
This will encourage national establishment of baseline status and achievable targets, particularly for essential drugs.
Establishment of MDG targets and Sustainable Development Goals (SDGs) will help greatly to solve the poor quality drug epidemic by application of available technology and good pharmaceutical vigilance and governance.
41 One incentive that would transform the current system is applying a "universal quality standard" to drug products.
For example, if India allows a substandard manufacturer to sell products in Africa, the FDA could ban import of products from India.
Although difficult to develop and implement, a combination of incentives and penalties driven at the political and economic levels is needed.
The major urgent needs to improve the quality of the world's medicines fall into three main areas: 1) research to develop and compare the most accurate and affordable tools to identify high-quality drugs at point of sale and deployment of the best methods; 2) an international convention and national legislation to facilitate production and use of high-quality drugs and protect all countries from the criminal and the negligent who make, distribute, and sell life-threatening products; 3) designation of a highly qualified, well-supported international organization, possibly the FDA or WHO, that will establish standards, training programs, drug quality surveillance, and authoritative guidance for countries in cooperation with national medical regulatory agencies, pharmaceutical companies, and international agencies, all of which have an urgent interest and investment in ensuring that patients throughout the world have access to good quality medicines.
The organization would also advocate strongly for including targets for achieving good quality medicines in the MDGs and SDGs including certification of pharmaceutical products entering countries that request such services, and participate in global disease control and elimination programs.
Poorquality antimalarial drugs in southeast Asia and sub-Saharan Africa Substandard and counterfeit medicines: a systematic review of the literature The global counterfeit drug trade: patient safety and public health risks Substandard/Spurious/ Falsely-labeled/Falsified/Counterfeit Medical Products Analysis of counterfeit Cialis tablets using Raman microscopy and multivariate curve resolution Confirmed glyburide poisoning from ingestion of "street Valium Characterization and identification of suspected counterfeit miltefosine capsules Empirical analysis of counterfeit drug penetration in global legitimate medicine supply chains: a descriptive assessment Internetordered Viagra (slidenafil citrate) is rarely genuine A Dictionary of Epidemiology New Definition for "Substandard Medicines How to achieve international action on falsified and substandard medicines Impact of poorquality medicines in the 'developing' world United States Pharmacopeia Ten Global Health Organizations United in a Worldwide Campaign to Protect Patients from Fakes Countering the Problem of Falsified and Substandard Drugs Improving global health governance to combat counterfeit drugs: a proposal for a UNODC-WHO-Interpol trilateral mechanism Guidelines for field surveys of the quality of medicines: a proposal Recommendations on the Content of a Survey Protocol for Surveys of the Quality of Medicines Integration of novel low-cost colorometric and visual fluorescent techniques for rapid identification of falsified artemether-lumefantrine and other drugs in resource poor areas Survey of the Quality of Selected Antimalarial Medicines Circulating in Madagascar, Senegal, and Uganda The global pandemic of falsified medicines: laboratory and field innovations policies and perspectives Evaluation of a new handheld instrument for the detection of counterfeit artesunate by visual florescence comparison FDA Facts: FDA's Counterfeit Detection Device CD-3 India Aims to Clock USD 15.5 Bn Pharma Exports FY13 Import of Human Drugs and Human Drug Components Global health and the law Follow the money: how the billions of dollars that flow from smokers in poor nations to companies in rich nations greatly exceed funding for global tobacco control and what might be done about it Counterfeiting, A Global Spread, a Global Threat Counterfeit bevacizumab and endophthalmitis Progress Report for State and Federal Regulators Medicines counterfeiting is a complex problem: a review of key challenges across the supply chain FDA Takes Action Against Thousands of Illegal Internet Pharmacies.
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key: cord-288567-1nmk9qhr authors: Frieden, Ilona J.; Püttgen, Katherine B.; Drolet, Beth A.; Garzon, Maria C.; Chamlin, Sarah L.; Pope, Elena; Mancini, Anthony J.; Lauren, Christine T.; Mathes, Erin F.; Siegel, Dawn H.; Gupta, Deepti; Haggstrom, Anita N.; Tollefson, Megha M.; Baselga, Eulalia; Morel, Kimberly D.; Shah, Sonal D.; Holland, Kristen E.; Adams, Denise M.; Horii, Kimberly A.; Newell, Brandon D.; Powell, Julie; McCuaig, Catherine C.; Nopper, Amy J.; Metry, Denise W.; Maguiness, Sheilagh title: Management of infantile hemangiomas during the COVID pandemic date: 2020-05-16 journal: Pediatr Dermatol DOI: 10.1111/pde.14196 sha: doc_id: 288567 cord_uid: 1nmk9qhr The COVID‐19 pandemic has caused significant shifts in patient care including a steep decline in ambulatory visits and a marked increase in the use of telemedicine.
Infantile hemangiomas (IH) can require urgent evaluation and risk stratification to determine which infants need treatment and which can be managed with continued observation.
For those requiring treatment, prompt initiation decreases morbidity and improves long‐term outcomes.
The Hemangioma Investigator Group has created consensus recommendations for management of IH via telemedicine.
FDA/EMA‐approved monitoring guidelines, clinical practice guidelines, and relevant, up‐to‐date publications regarding initiation and monitoring of beta‐blocker therapy were used to inform the recommendations.
Clinical decision‐making guidelines about when telehealth is an appropriate alternative to in‐office visits, including medication initiation, dosage changes, and ongoing evaluation, are included.
The importance of communication with caregivers in the context of telemedicine is discussed, and online resources for both hemangioma education and propranolol therapy are provided.
The novel coronavirus (COVID-19) pandemic has drastically altered health care delivery including widespread reductions in ambulatory visits to minimize exposure to and transmission of COVID-19 resulting in unprecedented adoption of virtual care via telemedicine platforms.
In light of these significant shifts in patient care, the Hemangioma Investigator Group (HIG) met with the goal of creating consensus recommendations to provide timely care for infants with infantile hemangioma (IH) via telehealth.
The use of beta-blockers in the treatment of IH has revolutionized care, and recent American Academy of Pediatrics (AAP) clinical practice guidelines (CPG) emphasize that early therapeutic intervention is critical for complicated IH to prevent medical complications or permanent disfigurement.
1 In this statement, we review FDA/EMA-approved monitoring guidelines, information derived from several clinical practice guidelines, and other publications regarding initiation and monitoring of beta-blocker therapy, including newly published information which could help inform modification of these practices.
We give recommendations to help guide decisions about when telehealth may be an alternative to in-office visits, including initiation, dosage changes, and continued evaluation for those patients requiring treatment.
We also provide tools for patient communication in context of telemedicine.
While these recommendations were prompted by the COVID-19 pandemic, we recognize that they might be relevant in analogous settings where there is a disruption of the normal delivery of medical care and potentially in settings with lack of access to practitioners with expertise in IH management.
The Hemangioma Investigator Group (HIG) met via videoconferencing on March 22, 2020 , and subdivided members into 3 groups: one to work on the introduction and discussion, one to create a table of inclusion and exclusion criteria for telemedicine use of betablockers, and one to curate available patient-education materials for practitioners and parents.
Through an iterative process of review of these 3 domains, we were able to achieve unanimous consensus regarding the content of these recommendations.
The most rapid IH growth occurs between 1 and 3 months of age, and there is a "window of opportunity" to treat problematic IHs in order to prevent morbidities.
Telemedicine has a critical role to play in facilitating early evaluation and risk stratification.
In areas where access to specialists has long been challenging, telemedicine triage has the potential to improve care for high-risk IH.
Early consultation ideally by 1 month of age or as soon as high-risk features are recognized is warranted.
Table 1 , from the AAP CPG, delineates risk categories of IH and potential associated morbidities.
Oral beta-blockers are the gold standard when systemic treatment is indicated for IH and propranolol solution is the only FDA/EMAapproved treatment.
Methods for initiation of oral propranolol have evolved over time.
[2] [3] [4] [5] [6] [7] [8] [9] Consensus recommendations prior to the FDA/EMA approval in 2014 included the following 10 : (a) screening for contraindications to propranolol, (b) performing or obtaining documentation of, a recent normal cardiovascular and pulmonary history and examination, (c) obtaining key historical data including poor feeding, dyspnea, tachypnea, diaphoresis, wheezing, heart murmur, or family history of heart block or arrhythmia, and (d) prolonged in-office monitoring.
The FDA/EMA-approved administration monitoring recommendations include in-office heart rate (HR) and blood pressure (BP) monitoring for 2 hours after the first dose of propranolol or for increasing the dose (>0.5 mg/kg/d) for infants 5 weeks of adjusted gestational age or older.
10, 11 More recent consensus statements 1,12-14 vary in specific recommendations for propranolol initiation.
Both Australian and British guidelines 13, 14 recommend full-term healthy infants without comorbidities may undergo outpatient initiation without in-office monitoring with initial doses of 1 mg/kg/d.
13, 14 Both state that a thorough medical While rare, hypoglycemia, seen primarily with intercurrent illness or decreased feeding, is a serious potentially life-threatening risk, 16 other risks include bronchospasm and wheezing, usually in the context of a respiratory illness, cold hands and feet, gastrointestinal upset, and sleep disturbances.
All of these potential adverse events require anticipatory guidance of parents, which should still be a part of clinical care, whether in person or via telemedicine.
Other non-FDA-approved beta-blocking agents, including oral atenolol and nadolol, have been used for the treatment of IH with several publications supporting their efficacy.
However, the group was unable to reach consensus recommendation regarding telemedicine for initiation of either of these medications.
Topical timolol has been widely used for treating IH with efficacy reported, particularly for small, superficial IH.
9, 15 Systemic absorption is variable but does occur, 17, 18 suggesting that similar prescreening should be performed to assure that infants are healthy and have had a normal cardiovascular and pulmonary examinations, for example, via recent history and physical examination (see discussion below).
Our recommendations regarding telemedicine initiation of betablocker therapy are summarized in Table 2 with an accompanying algorithm ( Figure 1 ).
They were developed after review of relative and absolute contraindications for propranolol, reported adverse events, FDA/EMA labeling recommendations, and published guidelines, with group consensus.
They are made with the goal of supporting practicing clinicians in delivering high-quality care in a dramatically altered Highest • Large (>5 cm) or segmental facial or scalp: a. higher risk of airway hemangiomas (if beard area), b. may be associated with PHACE syndrome, c. high risk of scarring and/or disfigurement.
• Large or segmental lumbosacral or perineal: a. may be associated with LUMBAR syndrome, b. high risk of ulceration and scarring.
• Multifocal IHs (≥5) and abdominal ultrasonography reveals hemangiomas: a. may be associated with abdominal compartment syndrome, high-output b. congestive heart failure, and hypothyroidism.
• Periocular IH causing eyelid asymmetry, lid closure or ptosis, proptosis, or other findings with potential impact on visual axis: a. risk of astigmatism, anisometropia, and amblyopia a In ordinary circumstances, infants are being seen regularly for well-child visits by primary care providers, who weigh and measure infants and perform heart and lung examinations as a standard part of their care.
If these examinations are not occurring due to disruptions in healthcare, it becomes much more difficult to ascertain whether there is a normal cardiovascular or pulmonary examination, if normal growth is occurring and other baseline characteristics.
In such cases, decisions about initiating therapy must be done on a case-by-case basis.
b During this pandemic and other unusual circumstances, in-person visits may not be possible in a timely fashion.
In these settings, triage and management decisions need to be made on a case-by-case basis, ideally in conjunction with relevant specialists as needed (eg, ENT and cardiology).
Topical timolol is efficacious for smaller, thin IH.
17, 19, 20 Although rigorous safety studies have not been performed, if used in small amounts, the rate of adverse events is very low.
19 Systemic absorption occurs to varying degrees measurable in both urine and plasma; plasma concentrations demonstrated to have measurable systemic β-blocking activity in adults have been reported.
17, 18, 21 Based on this information, we recommend that timolol application should be limited to the dose for which safety data have been most often reported, 1 drop twice daily of timolol 0.5%.
Timolol is not recommended for the treatment of thick or deep IH, both because it is less effective and systemic absorption may be greater.
18 Because of the potential for systemic exposure of topical application, pre-screening should be performed to assure that infants are healthy with normal cardiopulmonary examinations via recent history and physical examination.
As with oral beta-blockers, temporary discontinuation is recommended if patients experience respiratory or gastrointestinal symptoms.
Infants under 3 months of age, and those whose history suggests ongoing IH growth, should be monitored via frequent visits or photographs submitted by parents to assure that therapy does F I G U R E 1 Algorithm for management not need to be switched from topical to oral.
Such follow-up visits can often be done via telemedicine.
The COVID-19 pandemic has caused an abrupt shift from ambulatory visits to telemedicine platforms.
This consensus statement provides guidance on timely treatment for patients with IH requiring early intervention while prioritizing patient safety.
While we acknowledge the benefits of in-person visits when health care systems are operating normally, there was group consensus that telehealth visits could provide an alternative method of evaluation and treatment as long as safeguards are in place to minimize risks.
Our recommendations are based upon first ensuring that there are no contraindications for therapy, documentation of a recent normal physical examination, and no signs or symptoms of active illness ( Table 2 ).
We suggest that these patients are amenable to initiation of therapy through a process of limited physical examination coupled with virtual counseling and education about the natural history, treatment options, administration of medication, and potential adverse reactions to therapy.
We recognize that there are other circumstances in which these recommendations may be applicable including natural disasters (eg, earthquakes, hurricanes).
In addition, there are patients whose access to specialty care is severely limited due to geographic constraints (eg, living many hours away from a center with expertise in the evaluation and management of IH) where these recommendations may prove beneficial.
With or without telemedicine, all patients with IH require careful consideration of risks and benefits of any proposed treatment, discussion with families regarding treatment options, and recommendations and information about possible adverse events from prescribed medications (Table 3) .
For IH still in the rapid growth phase, we recommend particularly close follow-up, ideally within 1-2 weeks.
Telemedicine is particularly well-suited for these typically brief follow-up visits to assure that the IH is behaving as anticipated.
Parents should be advised to reach out to practitioners in the context of changes in the IH (eg, ulceration, ongoing growth, development/ progression of functional impairment).
If the patient develops respiratory symptoms (eg, cough, wheezing, respiratory distress), gastrointestinal symptoms (eg, vomiting, diarrhea, decreased intake), or lethargy, medication should be immediately discontinued and a physician notified.
22 Beta-blocker therapy information • https://heman gioma educa tion.org/heman gioma -treat ment/newhig-propr anolo l-educa tion-video -for-careg ivers • https://pedsd erm.net/site/asset s/files /1028/12_spd_propr anolol_color_web-final.pdf • https://heman gioma educa tion.org/syste mic-treat ment/ • https://heman gioma educa tion.org/topic al-and-local -treat ment/ a These links created or vetted by HIG members.
Sheilagh Maguiness Clinical Practice Guideline for the management of infantile hemangiomas Epidemiology of COVID-19 among children in China Syndrome: consensus-derived diagnosis and care recommendations Totté JE et al E-learning enables parents to assess an infantile hemangioma The infantile hemangioma referral score: a validated tool for physicians Effective health care program.
Diagnosis and management of infantile heamngioma A randomized, controlled trial of oral propranolol in infantile hemangioma Hemangeol (propranolol hydrochloride oral solution) Modalities of use of oral propranolol in proliferative infantile hemangiomas: an international survey among practitioners Chamlin SL et al Initiation and use of propranolol for infantile hemangioma: report of a consensus conference Evaluation of a modified outpatient model for using propranolol to treat infantile hemangiomas Baselga E et al Treatment of infantile haemangiomas: recommendations of a European expert group Oral propranolol in the treatment of proliferating infantile haemangiomas: British Society for Paediatric Dermatology consensus guidelines Consensus statement for the treatment of infantile haemangiomas with propranolol Limited utility of repeated vital sign monitoring during initiation of oral propranolol for complicated infantile hemangioma Propranolol in the treatment of infantile haemangiomas: lessons from the European Propranolol In the Treatment of Complicated Haemangiomas (PITCH) taskforce survey Safety and efficacy of topical timolol treatment of infantile haemangioma: a prospective trial Systemic timolol exposure following topical application to infantile hemangiomas Topical timolol maleate treatment of infantile hemangiomas The role of topical timolol in the treatment of infantile hemangiomas: a systematic review and meta-analysis Topical timolol for infantile hemangiomas: evidence for efficacy and degree of systemic absorption Propranolol treatment of infantile hemangiomas: anticipatory guidance for parents and caretakers
key: cord-299323-riotkgj4 authors: Seo, Yurim; Pacifici, Eunjoo title: Elements of Regulatory Dissonance: Examining FDA and EMA Product Labeling of New Vaccines (2006–2018) date: 2020-10-13 journal: Vaccine DOI: 10.1016/j.vaccine.2020.09.067 sha: doc_id: 299323 cord_uid: riotkgj4 With the ongoing globalization of the pharmaceutical industry, efforts to harmonize technical requirements of registering drugs and biologics, including vaccines, have produced a number of useful guidelines utilized around the world.
However, such efforts have not been extended to the regulatory review process or product labeling.
Prescribing information and patient information leaflet are two types of such product labeling documents.
This study examined the differences in the languages of these documents between the United States (US) and European Union (EU).
The key documents examined were the U.S. Food & Drug Administration’s (FDA) Package Inserts (PIs), U.S. Centers for Disease Control and Prevention’s (CDC) Vaccine Information Statements (VISs), and the European Medicines Agency’s (EMA) Summary of Product Characteristics (SmPCs) and Package Leaflets (PLs).
Prescribing information and patient information leaflet languages were subsequently organized into ten and seven categories, respectively.
Comparison of FDA PIs to EMA SmPCs showed little harmonization between the two regions, and CDC VISs to EMA PLs revealed even less.
Each year, regulatory agencies worldwide review new drugs and biologics, including vaccines, for marketing approval within their respective regions.
This process is essential, as it not only assesses the safety and efficacy of products but also oversees the product labeling intended for use by healthcare professionals and patients.
However, because approval processes are not globally harmonized, a pharmaceutical company seeking approval for its product in multiple regions must prepare and submit separate applications, which may be different in content and format.
Although efforts by the International Council for Harmonization (ICH) to harmonize technical requirements for registering drugs and biologics have produced a number of useful guidelines that are used around the world, such efforts have not been extended to the regulatory review process or product labeling [1] .
Currently, the United States Food & Drug Administration (FDA) and the European Medicines Agency (EMA) are considered two of the most prominent regulatory agencies around the world.
This study compared vaccine prescribing information and patient information leaflet languages between FDA/Centers for Disease Control and Prevention (CDC) and EMA.
In both the United States (US) and the European Union (EU), vaccines undergo rigorous regulatory approval procedures to ensure their safety, efficacy, and quality.
Depending on the product, this process can take anywhere from months to years, where delay in access could pose risks to public health.
This is especially important and relevant in the case of vaccines that are developed for highly contagious illnesses or in response to infectious disease outbreaks.
In the US, FDA is responsible for all drug approvals.
When a company files a Biologics License Application (BLA) for a vaccine, the application is reviewed by the Center for Biologics Evaluation and Research (CBER) [2] .
In the EU, all biologics, including vaccines, must be authorized by EMA.
There are three available pathways for pharmaceutical product approval in the EU: centralized, decentralized, and mutual recognition [3] .
The centralized route allows companies to submit a single Marketing Authorization Application (MAA) to EMA that leads to the product's approval in all countries within the European Economic Area (i.e., the 27 member states of the EU plus Iceland, Liechtenstein and Norway).
Once submitted, it undergoes review by the Committee for Medicinal Products for Human Use (CHMP) [4, 5, 6, 7] .
All products https://doi.org/10.1016/j.vaccine.2020.09.067 0264-410X/Ó 2020 Elsevier Ltd. All rights reserved.
approved by both FDA and EMA during the time frame of this study were approved through the centralized procedure.
On the other hand, the decentralized pathway allows companies to apply for simultaneous authorization in more than one, but not all, EU member states, as long as the product for which they are seeking approval has not yet been authorized for marketing in any EU nation [3] .
In this process, one country is designated as the reference member state and completes a preliminary assessment.
If the other countries are in agreement with the reference state's assessment, marketing authorization will be granted.
Finally, the mutual recognition pathway allows a company whose product has already been authorized in one EU nation (reference state) to apply for approval in other EU countries (target states) [3] .
The target states rely on the scientific assessment of the reference state to decide whether to grant a marketing authorization [7] .
The application for approval includes extensive safety and efficacy data collected during the pre-clinical and clinical phases as well as the company's proposed labeling, which is the ultimate deliverable of the approval process.
Based on this information, each agency evaluates the risk-benefit ratio of the vaccine [8, 9] .
One of the final steps of product approval is the completion of product labeling.
Regulatory agencies work with the manufacturers to finalize their proposed language, ensuring that the information is appropriate, sufficient, and accurate [10] .
There are two types of product labels -one intended for use by healthcare professionals (prescribing information), and one intended for use by patients (patient information leaflets).
Vaccine labeling for medical professionals are technical documents used by physicians, pharmacists, and other qualified medical personnel to obtain information regarding the administration, precautions, safety, potential side effects, and efficacy of a vaccine and evaluate whether it is appropriate to administer to a given patient.
On the other hand, the labels for patients present information regarding directions, indication, contraindications, side effects, and dosages in a more patient-friendly manner, allowing the patients to weigh the risks against the benefits and make an informed decision.
Product labeling is often the only vaccine information disseminated to healthcare professionals and patients; hence, it is crucial for it to be accurate and sufficient.
Discrepancies between regions can lead to unwarranted differences in the understanding and utilization of the product.
In the US, Package Inserts (PI) serve as useful communication tools to healthcare professionals, providing sufficient safety and effectiveness data necessary to administer vaccines appropriately [11, 12] .
The labels for patients are the Patient Information through FDA and Vaccine Information Statements (VIS) provided by CDC.
However, federal law requires that patients receiving any vaccine in the US be given the VIS rather than the Patient Information document [13] .
In the EU, the Summary of Product Characteristics (SmPC), required by Directive 2001/83/EC, is the official information source for medical professionals [10, 14] .
The information in the SmPC is used to draft the Package Leaflet (PL), a document intended for use by patients.
It is to be written in clear and understandable language so patients are adequately informed about the safety, effectiveness, and directions for use.
With the help of healthcare professionals, patients will use this document to discuss and make decisions about their treatment [15] .
In an effort to develop guidelines for international harmonization, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) was created in April 1990 in Brussels, and has since developed quality, safety, efficacy, and multidisciplinary guidelines [16, 17, 18 ].
An example includes the Common Technical Document (CTD), which is a harmonized electronic application for new products [18] .
The CTD is comprised of Modules 1 through 5, of which Module 1 is region specific, and Modules 2 through 5 are the same for all regions.
Included in Module 1 is the prescribing information, such as the proposed product information and labeling, which depicts the essential information on safety, efficacy, and guidelines for use [19, 20] .
The contents of such approved labeling are an important tool for both healthcare professionals and the general public, because although these products are intended to provide therapeutic benefits, they may also pose harm.
This concept is particularly applicable to vaccines, as these products are primarily given to healthy individuals, and their benefits must clearly outweigh their risks.
Although product labeling should be tailored to the needs of the local population, the core safety and efficacy information included should be the same across all regions, especially when they are derived from the same set of clinical evidence.
The objective of this study was to compare contents of the product labeling and patient leaflets between the US and EU to identify where they harmonize and where they differ.
A retrospective analysis was performed of all vaccines approved by FDA and EMA between January 1, 2006 and June 30, 2018.
The regulatory aspects examined for each vaccine were the prescribing information and patient information leaflet languages.
Prescribing information was extracted from FDA's PI and EMA's SmPC, and the patient information leaflet language from the CDC's VIS and EMA's PL.
The most recently updated versions (as of August 2018) of all regulatory documents were taken from FDA, CDC, and EMA websites (https://www.accessdata.fda.gov/scripts/cder/daf/, https:// www.cdc.gov/vaccines/hcp/vis/index.html, https://www.ema.europa.eu/en/medicines, respectively).
First, the prescribing information and patient information leaflet languages were assessed for the level of harmonization between the two regions.
Although the same topics could be found in the documents of both regions, the contents were arranged differently depending on the agency.
For example, to describe the intended use of the vaccines, FDA used the term ''Indications and Usage," while EMA used ''Therapeutic Indications."
For analysis, this labeling element was simply referred to as ''Indication" (Supplementary Table 1 ).
Ten prescribing information elements and seven patient information leaflet elements were examined (Supplementary Tables 1 and 2 ).
Detailed criteria on the type of information assessed for each element are outlined in Supplementary Tables 1 and 2.
These criteria must be the same for the corresponding element to be considered harmonized, ignoring any spelling, punctuation, grammatical, or phrasing differences.
Because the vaccines analyzed in this study were approved over a long period of time (12 years), the possibility of change in the level of harmonization over the years was explored.
The number of harmonized prescribing information and patient information leaflet elements were organized by year of approval into three groups: 2006-2009, 2010-2013, and 2014-2018 .
For vaccines whose approval dates in the US and EU did not fall into the same category, FDA's approval date was used.
The average of the number of elements harmonized was calculated for each time period and analyzed for any trends.
From January 2006 to June 2018, 39 vaccines were approved by FDA and 34 by EMA.
Of these, a total of 12 vaccines were approved by both FDA and EMA (Supplementary Table 3 ).
Of the twelve common vaccines across FDA and EMA, none had harmonized prescribing information across all ten elements ( Table 1 ).
The greatest level of harmonization across the elements was six out of ten.
The labeling elements with the greatest level of harmonization were Pregnancy Assessment and Pediatric Assessment, both of which were harmonized across seven out of twelve vaccines.
Conversely, Warnings & Precautions, Adverse Events, and Patient Counseling Information were not harmonized for any vaccine (Tables 1 and 2 ).
Similar to the prescribing information, none of the twelve vaccines demonstrated harmonized patient information across all seven elements (Table 1) .
For all vaccines, harmonization occurred across only one or two out of seven elements.
The element that was most harmonized (observed across all 12 vaccines) was Side Effects Reporting, as both CDC and EMA have established national reporting systems.
The element Use During Pregnancy was harmonized in four out of twelve vaccines.
The elements Disease Information, Contraindications, Side Effects, and What to Look Out For were not harmonized in any products across the two regions (Table 3 ).
No pattern was observed in the number of prescribing information and patient information leaflet elements harmonized over time (Fig.
1) .
This analysis of prescribing information and patient information leaflet languages demonstrated that certain differences exist between the labeling languages of the US and EU.
Despite the same clinical data submitted for market authorization application, it is evident that the resulting labeling language is different.
For example, same products assessed by each regulatory agency with the same set of clinical data may result in labels with different therapeutic goals [21, 22] .
Hence EMA's SmPC for a shingles vaccine may state that it is for the prevention of herpes zoster (shingles) and post-herpetic neuralgia, while FDA's PI states that it should be used for the prevention of herpes zoster only and explicitly indicates the vaccine is not to be used for the treatment of postherpetic neuralgia.
These observed differences indicate the lack of a harmonized process to translate the information in the marketing application to the prescribing information and patient information leaflet.
Although ICH provides guidelines on a harmonized method of application submission, there are currently no harmonized guidelines on the content of labels; hence different agencies around the world may be submitted the same set of pre-clinical and clinical trial data but use different assessment criteria that ultimately lead to divergent labeling language.
Given the findings of this study, the question arises as to what implications these differences could have for medical professionals and patients.
For instance, differences in indication, dosing, and recommended ages for use may lead to inconsistencies in not only Y. Seo and E. Pacifici Vaccine xxx (xxxx) xxx the number of doses but also whether individuals receive the vaccine at all.
An example of differences in the recommended age range for use is demonstrated by the meningococcal vaccine.
In the US, FDA sets the age range to 2 months to 55 years of age, while EMA's age range is from 2 years of age.
Moreover, the age ranges for use may be complicated by the recommended vaccine schedules set by the public health authorities of each country.
Within the age ranges set by EMA, each country in the EU can have different recommendations based on the epidemiology and culture of the respective region [23] .
In the US, children 2 to 23 months are able to receive the vaccine if they have certain conditions, including complement deficiency or human immunodeficiency virus (HIV).
However, in the EU countries, these children cannot be vaccinated due to the restrictions set by the indication statement.
What are the implications of this difference?
One of the reasons for EMA's decision not to include children ages 2 to 23 months was that antibody persistence was decreased in clinical trial participants who received the vaccine in those age ranges [24] .
In this case, the children in the US who receive the vaccine may be placed at unnecessary risk of injection-related side effects.
On the other hand, even this temporary protection provided by the vaccine may be beneficial for children who are at especially high risk of contracting the disease; in this case, the children in the EU who do not receive this vaccine may be faced with greater risk of harm.
As such, although the implications of these differences may not be clear, they could be clinically significant.
Furthermore, an important patient information leaflet element that supplements the patient's understanding of the therapeutic indication is Disease Information, which describes the disease that the vaccine is designed to prevent.
With this information, patients can understand what the disease is, the dangers of contracting the disease, and why it is important to be vaccinated against it.
However, there were notable differences in the level of detail for this element between the US and EU.
Although patients may consult other sources as well, the Disease Information section of the patient information leaflet remains the primary document to learn about the disease that the vaccine protects against.
If this section is not sufficient, patients will not be able to properly make an informed decision as to whether they should be immunized.
The dangers of having limited information on the populations at risk is that individuals within this population may not know that they are especially vulnerable to contracting the disease.
As such, they may not be fully equipped with the information necessary to make an educated decision.
The safety profile for vaccines is especially important and must be clearly established, as these products are generally given to healthy individuals.
Included in the safety profile is the element Contraindications.
An example of differences in this element is demonstrated by the shingles vaccine.
In particular, one of the contraindications listed by EMA is ''active untreated TB."
However, the US documents do not include this contraindication.
If the vaccine administration to individuals with active untreated TB causes harm, then those in the US may not be adequately informed about this risk.
On the other hand, if it is generally satisfactory for TB patients to receive the vaccine, these individuals in the EU may be placed at a disadvantage, as they would be at increased risk of contracting shingles without this vaccine.
If vaccine labels were harmonized, everyone, regardless of region, would receive the same comprehensive information necessary to make an informed decision regarding each vaccine.
Moreover, having harmonized schedules and administration would lead to simplified vaccine delivery.
This is particularly relevant for individuals traveling or re-locating from one region to another, as their vaccine needs would remain the same anywhere.
However, differences in culture, local terminology, and/or epidemiology may result in labeling language differences.
As such, the set of vaccines necessary in one country may not be important in another due to a low disease prevalence, and cultural and terminology disparities may mean that the best labeling language is not identical across all countries.
Harmonized vaccine approval and administration would be valuable for diseases that impact the global community, as it would lead to faster access to potentially beneficial vaccines.
For example, the rapid expansion of the 2019-nCoV (COVID-19) pandemic demonstrates that a swift global response is necessary, and the development of a safe and effective vaccine is crucial to control such outbreaks.
As infectious agents can readily cross national boundaries and regulatory jurisdictions, a globally harmonized approach to vaccine approval and administration would be valuable to protect the health of the populations around the world.
The lack of harmonization among global regulatory agencies means that each country implements its own system of review.
These differences lead to redundancy and decreased efficiency, as several approaches are taken to demonstrate the same concept.
This redundancy may lead to increased cost for drug manufacturers that is passed down to consumers as increased drug prices.
Having a harmonized approach to vaccine approval would lead to lower cost of vaccines and ultimately greater affordability and access.
However, different approaches to regulatory decisions are not necessarily a problem.
Although redundancy may decrease efficiency, it could increase reliability, as repeated results will confirm the same concept [25] .
Moreover, the best approach to a product approval or public health matter is not always obvious.
When presented with the same set of evidence, different agencies can have differing opinions and draw different conclusions.
Ultimately, by presenting divergent opinions, the agencies as a whole are able to explore a variety of different paths and contribute to a more thorough assessment of the issue at hand.
One limitation of the study is the small sample size, which limits the ability to detect clear trends.
The limited sample size was determined by the number of vaccines approved by the two agencies during the 12-year period.
Another limitation is the subjective interpretation of the language and approach used by the two agencies required to identify and align the labeling elements.
For example, the US PI may state that the vaccine should not be administered to anyone who experienced a ''severe allergic reaction.
.
.after a previous dose.
.
.or any component" of the vaccine, while the EU SmPC states that ''hypersensitivity to the active substance or to any of the excipients" is a contraindication to receiving the vaccine.
In this case, a severe allergic reaction and hypersensitivity were considered as equivalent, as they refer to the same immunological reaction against the vaccine.
Overall, this analysis compared clinical labeling languages for several vaccines approved by FDA and EMA.
Some of the observed differences have clinically significant implications that could affect downstream patient care.
Future research could investigate differences between the two regions in vaccination completion rates, disease rates, and vaccination-related injuries.
Moreover, future work could also evaluate what regulatory processes were implemented by the two agencies and whether they led to the observed language differences.
Moving forward, the US and EU, as well as other countries, should work together to create uniform prescribing information and patient information leaflet content so that all individuals, regardless of region, could have access to the same clinical information.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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All authors attest they meet the ICMJE criteria for authorship.
Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.09.067.
key: cord-003118-58ta20fg authors: Van Norman, Gail A. title: Expanding Patient Access to Investigational New Drugs: Overview of Intermediate and Widespread Treatment Investigational New Drugs, and Emergency Authorization in Public Health Emergencies date: 2018-06-25 journal: JACC Basic Transl Sci DOI: 10.1016/j.jacbts.2018.02.001 sha: doc_id: 3118 cord_uid: 58ta20fg Individual patients with life-threatening or severely debilitating diseases can petition the U.S. Food and Drug Administration (FDA) through their physicians to have expanded access (EA) to drugs that are in clinical trials but have not reached full FDA approval (the “single-patient” investigational new drug [IND] application).
Additionally, recent state and federal laws—so-called “right to try legislation”—allow patients to approach drug companies directly for access prior to FDA approval.
While these pathways provide potential access for individual patients to investigational drugs, different EA pathways permit entire groups of certain patients to access investigational drugs prior to FDA approval.
This review focuses on special categories of EA INDs intended for multiple patients—the intermediate-group IND and the widespread-treatment IND—as well as emergency authorization for use of investigational drugs and biological products (e.g., vaccines) in public health emergencies.
For patients with life-threatening or severely debilitating disease, the wait for approval is simply too long, and can both abolish hope for those who diseases will be quickly fatal, and lead to sustained or even permanent disability for those whose diseases linger but are without effective proven therapies.
Spurred by patient advocacy during the early days of the acquired immunodeficiency syndrome (AIDS) epidemic in the late 1980s, and facilitated by subsequent legislative efforts over the next 20 years, regulatory initiatives permit the FDA to release drugs for use in individual patients through expanded access (EA) INDs (4, 5) , in many cases allowing emergency treatment with nonapproved drugs within hours of application, and nonemergency treatment within an average of 4 days (6) .
Further, most states have enacted so-called "right to try" legislation, permitting "compassionate use" of investigational drugs by individual patients through applications directly to the manufacturer (6) .
It should be noted that although the terms "compassionate use" or "preapproval access" are often used informally to refer to the use of an investigational drug to treat a patient outside of a clinical trial, these terms are not defined or described in FDA regulations, which simply refer to expanded access to investigational drugs.
The call for EA is not limited to individual patients.
Advocacy organizations have pressed for groups of patients with rare and/or "orphan" diseases, for example, to be able to access promising new therapies prior to their approval.
Indeed, social media is increasingly becoming a consumer/patient advocacy tool for implementing FDA regulatory changes and promoting access to investigational therapeutics (7) .
In addition, once a drug has completed phase 3 testing and is awaiting approval, patients who have benefited from in-trial treatments may want continued therapy, and such use requires some form of "bridging approval" from the FDA to allow potentially large groups of patients to continue treatment while final FDA approval is pending.
A previous review discussed individual patient emergency and nonemergency access to investigational drugs (6) .
This review will focus on FDA EA for intermediate-sized groups of patients (the "intermediate-sized IND") and EA for entire classes of patients (the "widespread treatment use" IND), as well as emergency release of investigational drugs and biologics for use in public health emergencies.
Releasing investigational new drugs to individual patients who are facing certain death or disability seems to be a relatively uncomplicated decision, but allowing EA to entire groups of patients for treatment with an investigational new drug presents more complex regulatory, logistical, and ethical challenges for scientists, commercial entities, and the FDA.
The current regulatory process from IND filing to drug approval has evolved and includes not only the FDA's historical primary mission of ensuring patient safety, but also, since the latter half of the 20th century, the newer mission of ensuring that marketed drugs are actually efficacious for their advertised/approved use.
EA for a single patient may not present much of a challenge to the assertion that a drug's benefits outweigh the risks, because as presumably the patient requesting compassionate use faces an otherwise dismal clinical future, taking even significant risks with a new drug still presents potential benefits to a patient without other options.
Early in a drug's regulatory pathway, however, it is not usually possible to ensure that a drug has a reasonable risk/benefit ratio for all patients, including those in the early stages of disease.
Drug companies face bigger issues when the seeker of EA is a group of patients or an entire class of patients.
Before marketing, manufacture of the drug for clinical studies is nearly an "all cost" proposition for the commercial entity; the drug cannot be marketed to cover its costs.
Thus, companies generally only manufacture sufficient quantities (plus a small margin) to cover the requirements of clinical studies, rather than devote resources to manufacturing large quantities of a drug which has a <10% chance of ever making it to market (1, 2) .
The FDA approval process begins with the filing of an investigational new drug (IND).
Making the drug available to groups or classes of patients who might then deplete the supply of drug for clinical studies could compromise the very research that would more completely disclose a drug's risks and benefits; thus, it could possibly impede full market approval that would make the drug more widely available.
Companies have also expressed concern about how data from such "compassionate use" may be applied in the approval process.
for CMV infection (9) .
Later studies also showed that ganciclovir patients were living longer (10).
The FDA refused approval of ganciclovir for treatment of CMV retinitis, because they had no animal studies for that use, nor significant human placebocontrolled trials on which to base a marketing application.
Many questioned whether the use of ganciclovir was wise, or safe (11, 12) .
But, because of ganciclovir's known efficacy, it became paradoxically impossible to carry out human controlled trials, because such trials are only ethically justifiable if investigators are honestly uncertain about whether net positive benefits over placebo exists (13) .
Furthermore, neither patients nor doctors were willing to risk assignment to placebo and further loss of eyesight after the results were published.
Syntex then sought approval to study ganciclovir for treatment of CMV colitis, knowing that once marketing approval of the drug was obtained, FDA rules would allow "off-label" use for retinitis (14) .
At this time, EA of nonapproved drugs for treatment of more than 1 patient at a time is achievable only through the FDA, in contrast with access for individual patients, which technically can be legally obtained without the FDA by applying to the manufacturer directly (6) .
As with individual EA INDs, specific conditions for group patient access apply: 1) the patients must have a serious or immediately life-threatening disease or condition with no comparable therapy or satisfactory alternative therapy; 2) the potential benefit must justify the potential risks of the treatment; and 3) providing the treatment must not compromise or interfere with the ongoing FDA drug development program, such as by critically depleting a limited supply of investigational drug that is also needed for an ongoing study or a future study that is in the planning stages (4).
An "immediately life-threatening condition or disease" is defined by the FDA as "a stage of disease in which there is reasonable likelihood that death will occur within a matter of months or in which premature death is likely without early treatment."
A serious disease or condition is defined as being "associated with morbidity that has substantial impact on day-to-day functioning."
Furthermore, while short-lived or self-limited morbidity will usually not be a sufficient qualifying condition, the morbidity "need not be irreversible, provided it is persistent or recurrent."
The FDA states that whether a condition is serious or not "is a matter of clinical judgment, based on its impact on such factors as survival, day-to-day functioning, or the likelihood that the disease, if left untreated, will progress from a less severe condition to a more serious one" (15) .
and other animal species such as cats, armadillos, guinea pigs, swine and ferrets in which thalidomide had been tested, teratogenic effects had been induced only occasionally" (18) .
In fact, when human birth defects began to appear in the offspring of women who had ingested thalidomide during pregnancy as a sedative and to treat nausea, researchers pointed out that thalidomide had failed to demonstrate teratogenicity in rats, and at first insisted that thalidomide could not be the culprit.
In Germany, where the drug was first developed, thalidomide was held to be so safe that no prescription was required for its use, it was advertised for use in pregnant women (19) , and the drug company distributed free samples to its factory employees (18, 19) .
given the most severe rating for drugs that contribute to fetal deformities, and for drugs whose risks prioritizing those who should receive treatment, such as women and children (36) .
The fact that the treatment was made available to 2 U.S. citizens and not to Africans, who comprised most of its victims, engendered anger over the social justice of such decisions, providing, as Enserink (35) points out, a tragic validation to the satirical yet somewhat prophetic paper that had appeared in The Onion only weeks before titled "Experts: Ebola vaccine at least 50 white people away" (37) .
As of now, 12 products have been approved under the Animal Rule, 7 of which were issued quickly after the guidance was published ( Table 3 There is a reasonably well-understood pathophysiological mechanism of the toxicity of the toxic substance and its prevention or substantial reduction by the product.
The effect is demonstrated in more than 1 animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans.
The animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity.
The data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans.
Drugs, devices, and the FDA: part 1 Understanding FDA regulatory requirements for investigational new drug applications for sponsor-investigators Policy and procedures: Office of New Drugs: IND clinical holds Expanded access (compassionate use) Early access programs: benefits, challenges and key considerations for successful implementation Expanding patient access to investigational drugs: single patient INDs and the "right to try Going "social" to access experimental and potentially life-saving treatment: an assessment of the policy and on-line patient advocacy environment for expanded access Compassionate use: a story of ethics and science in the development of a new drug -propoxymethyl)guanine in patients with AIDS and other immunodeficiencies Oral ganciclovir for the prevention of cytomegalovirus disease in persons with AIDS Blinded by science: DHPG and the conflict between "clean data" and humane health care (abstract Th.G.P.11).
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The New York Times a comprehensive program for controlling and monitoring access to thalidomide The Embryo Project Encyclopedia.
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Accessed Experts: Ebola vaccine at least 50 white people away FDA product development under the animal rule: guidance for industry Working with the U.S. Food and Drug Administration to obtain approval of products under the Animal Rule Food and Drug Administration Ebola virus disease survivors: clinical and immunologic follow-up PREVAIL IV: double-blind, randomized two-phase, placebo-controlled phase II trial of GS-5734 to assess the antiviral activity, longer-term clearance of Ebola virus and safety in male Ebola survivors with evidence of Ebola virus persistence Expanded Access (Compassionate Use): IND submissions Medicine, University of Washington, 2141 8th Avenue West, Seattle, Washington 98119.
E-mail: lbsparrow@ yahoo.com.
key: cord-268283-eja8fkwv authors: Iftikhar, Hafsa; Ali, Hafiza Nayyer; Farooq, Sadia; Naveed, Hammad; Shahzad-ul-Hussan, Syed title: Identification of potential inhibitors of three key enzymes of SARS-CoV2 using computational approach date: 2020-06-09 journal: Comput Biol Med DOI: 10.1016/j.compbiomed.2020.103848 sha: doc_id: 268283 cord_uid: eja8fkwv The recent outbreak of coronavirus disease-19 (COVID-19) continues to drastically affect healthcare throughout the world.
To date, no approved treatment regimen or vaccine is available to effectively attenuate or prevent the infection.
Therefore, collective and multidisciplinary efforts are needed to identify new therapeutics or to explore effectiveness of existing drugs and drug-like small molecules against SARS-CoV-2 for lead identification and repurposing prospects.
This study addresses the identification of small molecules that specifically bind to any of the three essential proteins (RdRp, 3CL-protease and helicase) of SARS-CoV-2.
By applying computational approaches we screened a library of 4574 compounds also containing FDA-approved drugs against these viral proteins.
Shortlisted hits from initial screening were subjected to iterative docking with the respective proteins.
Ranking score on the basis of binding energy, clustering score, shape complementarity and functional significance of the binding pocket was applied to identify the binding compounds.
Finally, to minimize chances of false positives, we performed docking of the identified molecules with 100 irrelevant proteins of diverse classes thereby ruling out the non-specific binding.
Three FDA-approved drugs showed binding to 3CL-protease either at the catalytic pocket or at an allosteric site related to functionally important dimer formation.
A drug-like molecule showed binding to RdRp in its catalytic pocket blocking the key catalytic residues.
Two other drug-like molecules showed specific interactions with helicase at a key domain involved in catalysis.
This study provides lead drugs or drug-like molecules for further in vitro and clinical investigation for drug repurposing and new drug development prospects.
COVID-19 after its emergence in China, continues to spread across the globe leading to global health emergencies and accounting for devastating economic and social impacts [1, 2] .
As of April 9, 2020, over 1.7 million people have been confirmed with COVID-19 leading to over 111,000 deaths since the emergence of the infection [3] .
To date, no specific treatment regimen is available against COVID-19.
However, previously approved antiviral drug Remdesivir and an antimalarial drug Chloroquine have shown promising effects, at least in reducing the duration of symptoms of COVID-19 in limited clinical studies [4, 5] .
SARS-CoV-2 that causes this disease belongs to the family Coronaviridae and genus Coronavirus [6] .
Angiotensin-converting enzyme 2 (ACE2) that is expressed on the cellular surfaces of different organs including lungs has been identified as the primary receptor of the virus in addition to the role of another host protease enzyme, transmembrane serine protease 2 (TMPRSS2) in viral entry [7] [8] [9] .
The viral genome consists of 30,000 base pairs that encode different structural and non-structural proteins.
The structural proteins include the membrane protein, spike, envelope and nucleocapsid.
Non-structural proteins include NSP1-NSP16.
NSP3 (papain-like protease), NSP5 (3-chymotrypsin-like protease "3-CL-protease"), NSP12 (RNA-dependent RNA polymerase), NSP13 (helicase), NSP14 (N7-methyltransferase) and NSP16 (2 0 -O-methyl transferase) are important viral enzymes while NSP7-NSP10 are regulatory proteins.
The SARS-CoV-2 orf1a and orf1ab genes encode polyprotein 1a (pp1a) and polyprotein 1 ab (pp1ab), respectively.
These polyproteins are further processed by protease enzymes to produce different functional proteins.
In this regard, 3CL-protease cleaves the polyprotein at 10 different specific positions thereby producing individual functional proteins [10] .
SARS-CoV-2 is a positive sense RNA virus that requires RNAtemplated RNA synthesis.
RNA-dependent RNA polymerase (RdRp) catalyzes the synthesis of new viral RNA and plays a crucial role in the SARS-CoV-2 replication cycle [11] .
Similarly, SARS-CoV-2 helicase (NSP13) is also crucial for the viral replication and proliferation as it catalyzes the unwinding of duplex RNA and DNA into single strand nucleic acid chain during replication [12] .
Considering an urgent need of containing the pandemic, repurposing of previously approved drugs to use against SARS-CoV-2 is the first choice in the fight against this virus, as such drugs are not required to pass through most of the steps of an extensive drug testing process.
In this regard, several recent studies have been conducted using computational methods to screen libraries of approved drugs or drug-like molecules to identify potential inhibitors of different viral proteins, particularly, RdRp and 3CL-protease [13] [14] [15] [16] [17] .
Moreover, high throughput in vitro screening of FDA approved drug libraries followed by in vivo validation has also been applied on limited hits [18, 19] .
Many of the reported virtual screening based studies have targeted the FDA approved drugs and/or dug-like compound libraries.
Since a vast range of compound libraries exist therefore, multiple efforts to screen diverse compound libraries are important to explore a large chemical landscape to identify potential inhibitors or the lead structures to design inhibitors of SARS-CoV-2.
Here, we applied a computer aided drug discovery approach by targeting three important enzymes (RdRp, 3CL-protease and helicase) of SARS-CoV-2 and identified three FDA-approved drugs and three other drug-like molecules as potential therapeutics.
Structures of SARS-CoV-2 RdRp and helicase were modeled on the basis of amino acid sequence homology using SWISMODEL as crystal structure of these proteins are yet to be solved [20] .
The genome sequence of SARS-CoV2 (NCBI Reference Sequence: NC_045512.2) was used to model the structures of RdRp and helicase [21] .
The model accuracy and its stereochemical quality was assessed using the PROCHECK analysis tools, which predicts the quality of modeled structure on the basis of distribution of backbone phi/psi angles with reference to the Ramachandran plot [22] .
Recently published crystal structure of SARS-CoV-2 3CL-protease (PDB ID, 6LU7) was used for screening and docking simulations [23] .
First, a library of 4512 compounds also containing several FDA approved drugs was screened through MTiOpen screen automated virtual screening platform using the integrated Vina AutoDock program against all three proteins [24] .
Hits from virtual screening were shortlisted on the basis of binding energy based Vina empirical scoring function using À 8 kcal mol À 1 as the threshold [25] .
In this regard, 46, [50] and 35 compounds were shortlisted against 3CL-protease, RdRp and helicase, respectively and were downloaded from the ZINC database [26] .
In parallel, 62 FDA approved antiviral drugs with random targets were selected and their structure files were obtained from DrugBank in the SDF file format [27] .
Format of these compounds were converted from SMILES into PDB using an online tool (Online SMILES Translator, NCI) [28] .
The AutoDock tools 1.5.6 program was used to prepare ligand and protein structures for further docking [29] .
In ligand preparation, Gasteiger charges were computed, nonpolar hydrogen atoms were merged, torsion angles for all rotatable bonds were set as flexible and the file was saved in the pdbq format.
Similarly, protein files were prepared using automated functions of the AutoDock tools that added all hydrogen atoms, computed Gasteiger charges, merged non-polar hydrogen atoms and created pdbqt files.
Each of the shortlisted compounds was subjected to iterative docking with the respective protein using the AutoDock4.2.6 program [29] .
Docking was performed by Lamarckian genetic algorithm (LGA) method using 270,000 generations and by applying at least 100 iterations.
Pre-calculation of grid parameters for each protein was performed by using the Autogrid program.
During docking, the size of grids was kept at maximum covering the whole surface of the protein to allow the ligand to bind in an unbiased binding pocket.
The docking results were analyzed on the basis of a combination of binding energy, clustering score, shape complementarity, functional significance of the binding pocket and favorable interactions including H bonds and hydrophobic interactions.
We used integrated clustering tool of the AutoDock4.2.6 program to cluster different docked structures of a ligand on the basis of root mean square deviation (RMSD) of 1 Å.
The binding energy of every docked structure was predicted by the AutoDock program.
Thirty percent population of the docked structures in a cluster with À 7.5 kcal mol À 1 binding energy was used as a cutoff for the first ranking of iterative docking using the AutoDock tools program.
A cluster containing the highest number of docked structures was then selected.
To further illuminate H bonds and hydrophobic interactions the LigPlot þ program was used [30] .
The binding pockets with known functional significance on the basis of previously reported experimental data were considered.
Shape complementarity was superficially accessed by visual analysis of the docked structures.
To further rule out non-specificity, 100 protein families were selected randomly from pfam to check for the specificity of the drug with a particular target.
The details of the representative proteins from each family are given in Table S1 .
Eight drugs or organic molecules (Table 2) , which showed binding to any of the three target proteins of SARS-CoV-2, were subjected to docking simulation with all the 100 proteins by using the autodock4.2.6 program.
Pre-calculation of grid parameters for each protein was performed by using the autogrid program.
To rule out non-specific binding, docking was performed by Lamarckian genetic algorithm (LGA) method.
The significance measure (probability of obtaining a better binding score by chance) for a drug was calculated by comparing the binding scores obtained for specific target (three SARS-CoV2 proteins) with the binding scores for 100 structures belonging to random protein families.
Recently published X-ray structure of 3CL-protease (PDB ID: 6LU7) was used for virtual screening [23] .
A library consisting of 4574 compounds also including FDA approved drugs was screened and after Table 1 Predicted binding scores of Grazoprevir with different HCV NS3/4A X-ray structures.
Predicted binding score with Grazoprevir À 7.3 À 7.26 À 8.05 À 6.54 À 7.48 applying first ranking with predicted binding energy cutoff of À 8.0 kcal mol À 1 , 46 compounds were shortlisted.
Detailed iterative docking and subsequent analysis resulted in the identification of three SARS-CoV-2 3CL-protease binding compounds as potential inhibitors.
Rimantadine is an anti-flu FDA-approved drug with generic name as Flumadine.
The known target of Rimantadine is the M-protein of influenza-A virus [31] .
We observed that Rimantadine bound to SARS-CoV-2 3CL-protease at its substrate-binding site blocking about half of the binding pocket.
Two highly conserved amino acid residues His-41 and Cys-145 are the main catalytic residues of 3CL-protease of corona viruses, however, many residues around these catalytic residues also play key role in the binding of peptide substrate and its subsequent catalysis.
For example, His-163 and Phe-140 stabilize the position of glutamine present at the P1 position (Gln-P1) of the peptide substrate by developing two H bonds between the NΣ2 atom of His-163 and main carbonyl oxygen of Phe-140 with the OΣ1 and NΣ2 atoms of Gln-P1, respectively [32] .
In our docked structure of Rimantadine, the adamantyl ring of the drug perfectly fits in the sub-pocket of the substrate binding site occupying the space between His-163 and Ph-140 of the protease and stabilized by hydrophobic and van der Waals interactions (Fig.
1A) .
In the previously reported structure of His-41 mutant of SARS-CoV 3CL-protease in complex with its peptide substrate, carbonyl oxygen of Gln-P1 of the substrate occupies position in the oxyanion hole formed by the amide groups of Gly-143 and Cys-145 [32] .
In the docked structure the nitrogen atom of amino group of Rimantadine occupies the position close to the oxyanion hole and is stabilized by four potential H bonds with backbone nitrogen of Cys-145, carbonyl oxygen of Leu-141 and backbone nitrogen and side chain oxygen of Ser-144 (Fig.
1B) , thereby blocking the critical residues of the protease required for catalysis.
Another drug, Bagrosin was observed to bind at a potential allosteric site of the protease.
Bagrosin is an anti-epileptic drug that belongs to a class of hydantoin derivatives (anticonvulsant compounds).
It works by blocking the voltage-gated sodium channels of neurons and causes inhibition of calcium flux across neuronal membranes thereby stabilizing neurons.
Compared to other hydantoin derivatives, Bagrosin has been reported to be safer in clinical trials [33, 34] .
SARS-CoV 3CL-protease has been reported to form a homodimer resulting in better catalytic efficiency as compared to its monomeric form [35, 36] .
The recently reported X-ray structure of SARS-CoV2 3CL-protease is very similar to that of SARS-CoV forming a dimer.
The dimer formation is primarily mediated by several hydrophobic interactions between domain-3 of each monomer ( Fig.
2A ) [37] .
We observed that Bagrosin bound in a hydrophobic pocket in domain-3 of the protease and can potentially interfere with the dimer formation.
In the docked structure, the phenanthryl ring of the compound perfectly fits in a hydrophobic pocket while the polar hydantoin ring is stabilized with two H bonds with backbone nitrogen and oxygen of Lys-5 ( Fig.
2B and C) .
Another FDA approved drug, Grazoprevir was also found to bind at the allosteric site of SARS-CoV-2 protease and can potentially interfere with the dimer formation of the protease ( Fig.
2D and C) .
Grazoprevir is known as hepatitis C protease (NS3/4A) inhibitor and is used to treat hepatitis C in combination therapy [38] .
Several X-ray structures of the complexes of Grazoprevir and hepatitis C virus (HCV) NS3/4A have been reported [39, 40] .
In addition to performing docking of Grazoprevir with SARS-CoV2 3CL-protease, we also performed its docking with (HCV) NS3/4A and reproduced the binding interactions reported in different X-ray structures.
Predicted binding score obtained through docking simulations of Grazoprevir with different NS3/4A variants is given in Table 1 .
The binding score of Grazoprevir with SARS-CoV2 3CL-protease was predicted to be À 10.1, suggesting the potential stronger binding as compared to binding with HCV NS3/4A.
RdRp has been considered as an important target for the discovery of direct acting antiviral therapeutics against positive sense RNA viruses.
In this study, we used homology modeling to model the structure of SARSTable 2 Identifying the specificity of the drug-target interactions using a significance score based on binding potential to random targets.
All of its side chain parameters were within the accepted limit of structure quality (Fig.
S1 and Tables S2 and S3).
Preliminary virtual screening and subsequent analysis resulted in the identification of 30 compounds potentially binding to the protein.
These compounds were subjected to iteratively docking by selecting complete surface of the protein to map the binding.
Final analysis resulted in the identification of one compound, Casopitant, tentative generic name Rezonic.
Casopitant is a neurokinin-1 receptor antagonist and is under development to manage chemotherapy induced nausea [41] .
Two aspartate residues, Asp-760, and Asp-761 are the main catalytic residues of RdRp.
These two residues are highly conserved in all corona viruses [14] .
Casopitant bound at the catalytic site of the RdRp enzyme (Fig.
3A) .
The binding is stabilized by several favorable interactions including three H bonds between carbonyl oxygen of the acetyl piperazine ring and backbone nitrogen of Ala-762, and between the fluorine atoms of trifluoromethyl-phenyl moiety and the side chain nitrogen atoms of Arg-553 and Arg-836.
The ligand perfectly fits in the catalytic cavity completely covering the two catalytic aspartate residues (Fig.
3A and B ).
SARS-CoV-2 helicase is considered an important target for therapeutic intervention due its sequence conservation among all corona viruses [42] .
A typical CoV helicase consists of a zinc-binding domain, which is separated by stalk from the main catalytic domains 1A and 2A.
It incorporates distinct DNA and ATP binding sites.
In addition to these two important binding sites, the β19-β20 loop encompassing residues 331-357 plays a crucial role in nucleic acid unwinding [41] .
We homology modeled the structure of SARS-CoV-2 helicase using the SWISS-MODEL online tools for virtual screening.
The SARS-CoV-2 helicase sequence showed 99.8% sequence identity with helicase of SARS-CoV (PDB ID, 6JYT) that was used as a template in homology modeling.
According to the Ramachandran plot analysis, 99% of the residues were in the allowed region with over 80% in the most favorable region, validating the quality of the modeled structure ( Fig.
S1 and Tables S4 and S5).
We identified that Meclonazepam, a drug-like molecule with reported sedative, anxiolytic and anti-parasitic effects [43] , bound to the viral helicase.
According to the docking results, Meclonazepam binds to helicase at a pocket primarily formed by the β19-β20 loop and the binding is stabilized by several H bonds and hydrophobic interactions (Fig.
4A and B) .
Another drug-like molecule, Oxiphenisatin was found to bind SARS-CoV2 helicase also in a binding pocket primarily formed by the β19-β20 loop.
The binding was stabilized by several favorable interactions including five H bonds and hydrophobic interactions involving primarily the residues from the β19-β20 loop (Fig.
5 A and B) .
In order to minimize chances of false positive results in the docking simulations, we calculated a significance measure for the binding scores of prominent drug-protein pairs.
The significance measure is calculated by comparing the binding scores obtained for potential targets of a drug with the binding scores of 100 structures belonging to random protein families with the respective drug and counting the number of times out of 100 that we see better docking value when docking the respective drug with the random protein families [44] .
The significance measure enabled us to identify more promiscuous compounds where even the targets with most favorable docking score have an unfavorable significance measure (meaning that the drug is sticky and is interacting with many proteins with high probability).
We set the cutoff value of the significance measure at 0.3 also to include slightly less specific compounds taking into account the identification of leads to develop more specific potential drugs.
Drug-protein pairs with significance measure less than 0.3 were considered as specific (those with measure less than 0.1 are highly specific) as compared to the other pairs tested ( Table 2 ).
The two docked structures with binding score higher than À 0.6 showed the significance measure higher than 0.3 indicating non-specific binding of these ligands.
Over the last decade, computer aided drug discovery has emerged as a fundamental tool in drug discovery and design process [45] [46] [47] [48] .
Identification of compounds that bind and inhibit normal function of proteins involved in viral replication has been the most successful strategy in direct acting antiviral drug discovery [49] [50] [51] [52] .
In this study, we used a virtual screening based strategy to identify already approved drugs or drug-like molecules that can bind to any of the three key viral enzymes, 3CL-protease, RdRp and helicase, and potentially inhibit the function of these enzymes.
Virtual screening of over 4500 drugs and subsequent docking simulation resulted in the identification of three 3CL-protease binding drugs, one RdRp binding molecule and two helicase binding molecules.
One of the identified 3CL-protease binding drugs, Rimantadine, showed binding at the catalytic site blocking the critical catalytic residues and half of the catalytic pocket.
The two other compounds bound in a pocket present in domain III of the protease and can potentially interfere with the formation of dimerit is well established that CoV 3CL-proteases are catalytically active primarily in dimeric form [53] .
One RdRp binding compound, Casopitant was identified which perfectly fits in the catalytic cavity of the enzyme and completely blocks the two critical catalytic residues, Asp-260 and Asp-261.
Casopitant is not an approved drug but it is in the process of development towards its approval.
We identified two helicase-binding molecules, Meclonazepam and Oxiphenisatin both of these molecules bind at a pocket primarily formed by the β19-β20 loop of the enzyme.
This loop has been reported to play a crucial role in the catalytic activity, nucleic acid unwinding, of the helicase enzyme [42] .
Three of the identified compounds are already approved drugs that potentiate for their repurposing towards SARS-CoV-2 after in vitro and in vivo validation.
Moreover, all of these compounds represent leads for chemical modifications to develop new potential therapeutics.
For instance, Rimantadine binds to 3CL-protease and fits in half of the substratebinding pocket of the enzyme.
This drug also shows non-specific binding to other proteins as determined from its specificity score (0.26) after performing its docking simulation with 100 irrelevant proteins.
Its nonspecific binding could be attributed to its smaller size as several side effects of this drug have also been reported [54] .
Nevertheless, this docked structure provides necessary information for potential chemical modifications to incorporate chemical groups to fill the space of the binding pocket potentially leading to enhance the binding affinity and specificity.
In specificity score measurement, we set the cutoff value of the significance measure at 0.3 considering two aspects.
Firstly, to identify leads for chemical modification to synthesize more specific inhibitors and secondly, not to reject a ligand with good docking score even with moderate potential off target effects as in many cases a drug with several adverse effects is also acceptable when the question of life saving arises.
For example, Rimantadine is an FDA approved drug to use against flu despite its several side effects.
Recently, several studies have been conducted using computational methods to identify already approved drugs as potential inhibitors of SARS-CoV-2.
Elfiky et al.
reported the binding of four approved antiviral drugs to RdRp of SARS-CoV-2, including currently under clinical trial drug Remdesivir [14] .
RdRp is a highly significant drug target due to its conservation across positive sense RNA viruses.
However, this study was highly focused on nucleotide inhibitors and only eight already known drugs were screened against SARS-CoV-2 RdRp using a less vigorous docking program AutodockVina.
Wang et al.
recently conducted a study to screen a library consisting of diverse approved drugs against the SARS-CoV-2 3CL-protease and identified binding of several antiviral drugs, an anticancer drug, Carfilzomib and an antibiotic, Streptomycin [17] .
This study is computationally vigorous as it also includes molecular dynamics simulation to approximate binding free energy.
However, docking simulations using the autodock program provides quite a reliable approximation for further in vitro and in vivo testing as Elfiky et al.
utilized only the less vigorous program of autodock (AutoDock Vina) to identify potential inhibitors and one of their identified drugs is currently in clinical trials, suggesting the reliability of docking.
In our studies we performed computational screening by targeting three important enzymes of SARS-CoV-2 including RdRp, 3CL-protease and helicase, to identify not only the already approved drugs for repurposing but also the drug candidates or lead structures that can be chemically modified to develop potential drugs.
Moreover, we incorporated an additional aspect of specificity score obtained by performing docking simulation of every identified compound with 100 irrelevant proteins.
A similar study was recently reported by Mirza et al.
incorporating screening of drug candidates against three key enzymes of SARS-CoV-2 and reported several potential inhibitors of these enzymes [13] .
However, they performed docking by selecting a very specific area around the predicted catalytic sites of the protease and RdRp, and the ATP binding site of helicase ignoring allosteric sites.
These enzymes also incorporate allosteric sites that can be targeted for potential drug discovery.
For example, 3CL-protease is known to be catalytically active in its dimeric form and any inhibitor of dimerization could be of high significance.
Similarly, helicase incorporates several distinct sites including DNA and ATP binding sites, the zinc-binding domain and the β19-β20 loop involved in nucleic acid unwinding process.
In our studies, during docking, the whole surface of the protein was selected to allow the ligand to bind in an unbiased binding pocket and results were analyzed considering the known functional significance of the binding pocket that resulted in the identification of potential allosteric inhibitors of these enzymes as well.
Moreover, several other studies have also been reported contributing to the efforts in identifying approved drugs for repurposing or drug candidates or leads to develop drugs against SARS-CoV-2.
Overall, this computer-aided study represents a contribution to the efforts against SARS-CoV-2 and provides information about potentially repurposable drugs and drug-like molecules or lead structures that can be chemically modified to develop into potential drug candidates for further in vitro and clinical investigation.
The authors declare that they have no conflict of interest.
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key: cord-335776-e5wjsk8t authors: Costantino, Ryan C. title: The U.S. medicine chest: Understanding the U.S. pharmaceutical supply chain and the role of the pharmacist date: 2020-08-18 journal: J Am Pharm Assoc (2003) DOI: 10.1016/j.japh.2020.07.018 sha: doc_id: 335776 cord_uid: e5wjsk8t The U.S. capacity to manufacture key essential medications has diminished.
The U.S. pharmaceutical supply chain (USPSC) has diversified and now relies on international sources of active pharmaceutical ingredients and finished drug products (FDPs).
Despite years of effort raising concerns about the USPSC, pharmacists and pharmacy technicians continue to spend a substantial amount of time and energy responding to, and mitigating the impact of, medication shortages, drug recalls, and the adverse outcomes related to low-quality medications.
The extent of U.S. reliance on foreign sources of medications is largely unknown.
Pharmacists do not have a reliable way to determine the country of origin (i.e., source), capacity, or geographic location of pharmaceutical manufacturers, limiting our ability to anticipate challenges or mitigate risks to our Nation’s drug supply.
The U.S. Food and Drug Administration’s task of regulating quality and safety is challenging and will likely require additional safeguards and resources.
In addition to pharmacists’ engagement, solutions will likely need to leverage a mix of policy, economic incentives, and expanded objective surveillance testing.
The U.S. pharmaceutical supply chain is complex, global, and goes beyond FDPs.
The 2020 American Pharmacists Association House of Delegates has rightly asserted that “The quality and safety of pharmaceutical and other medical products and the global pharmaceutical and medical product supply chain are essential to the United States national security and public health.” Pharmacy professionals on the front line engage with patients, identify medication-related issues, and engage in drug-procurement decisions.
Pharmacists are essential to our nation’s overall health and must be engaged in the development and implementation of strategies to safeguard the USPSC.
The United States no longer manufactures a number of lifesaving essential medications.
1 Early warning signs of our dependency on foreign manufacturers include the 2001 anthrax attacks during which the U.S. government purchased large quantities of doxycycline from foreign sources and the closing of the last penicillin fermentation plant in 2004, largely driven out of business by global market forces.
1, 2 Over the past decades, the U.S. pharmaceutical supply chain (USPSC) has increasingly relied on international sources for pharmaceuticals and pharmacists continue to dedicate a sizable amount of energy responding to, and mitigating the impact of, medication shortages, drug recalls, and the adverse outcomes of low-quality medications.
3, 4 Concerns related to these topics have been highlighted in previous publications over the past decade yet continue to persist and may even be worsening in 2020.
5, 6 These issues may be symptoms of a larger problem related to the dissolution of our domestic drug manufacturing industry and shortcomings in our regulatory framework, impairing our ability to ensure the availability, quality, and safety of U.S. medicines.
7, 8 U.S. regulatory framework The U.S. Food and Drug Administration (FDA) provides definitions for several key terms that are helpful to understand the USPSC.
Raw materials, both organic and inorganic chemicals, are the building blocks used to manufacture active pharmaceutical ingredients (APIs) and inactive ingredients that together make up a finished drug product (FDP).
9 This FDP is what pharmacists often think of when dispensing a medication.
It is estimated that up to 90% of the raw materials and APIs needed to produce an FDP come from China and other countries such as India.
7, 8, 10, 11 In 2019, FDA reported that most API facilities (72%) and FDP facilities (53%) were located outside of the United States.
12 It is important to note that FDA does not track or inspect the raw ingredients further upstream ( Figure 1 ).
3 Furthermore, the data provided by FDA usually only capture the organization that paid the registration fee to FDA (i.e., Generic Drug User Fee Amendments fee), potentially concealing the true location of manufacturers.
Pharmacists are left with little insight regarding the quantity or volume of each drug produced at various facilities given that pharmaceutical companies often consider the information a trade secret.
3 The most conservative statement at this time would be that the extent of our reliance on foreign sources of medications is largely unknown.
This is a cause for concern because without a reliable way to determine the country of origin (i.e., source), capacity, or geographic location of pharmaceutical manufacturers, pharmacists are unable to anticipate challenges or mitigate risks to the USPSC.
In addition to the source of our medications, ensuring their quality and safety is essential to maintain public confidence, medication adherence, and avoid adverse outcomes.
13 Unfortunate events such as the 2008 heparin adulteration, 2018 angiotensin II receptor blocker (ARB) recalls, and the 2020 ranitidine removal request highlight that despite FDA's best efforts, regulating pharmaceutical products is complex.
[14] [15] [16] In the case of the heparin and ARB recalls, the root cause was contamination or poor manufacturing of APIs.
In the case of ranitidine, the issue was likely related to the unstable chemical properties of the drug molecule, which result in the generation of a known carcinogen during storage or in vivo.
17 FDA does require analytical tests and samples before new drugs are approved.
FDA also conducts postmarket surveillance testing and inspections of manufacturing facilities to assess compliance with standards that may catch some of these issues.
Domestic facilities can be inspected with no advance warnings, whereas international inspections are almost always announced in advance.
12 This raises the question as to whether domestic manufacturers are held to a different standard compared with international manufacturers.
Owing to coronavirus disease, FDA has suspended foreign inspections and scaled back domestic inspections limiting the utility of this method of oversight.
18 In response, FDA is exploring new approaches to regulatory oversight such as a quality metrics program.
19 Economic incentives may also have an impact on compliance with FDA regulations.
[20] [21] [22] In the United States, companies that sell brand name drugs receive patent protection for 20 years after their original filing, affording them an opportunity to recoup their investment as a reward for their innovation.
Because a brand name drug under patent protection has no competitor, companies have an incentive to maintain quality control to preserve the positive brand identity of their products.
The primary economic incentive for generic manufacturers involves being the first generic drug applicant to submit a complete generic drug application.
If approved, the applicant's generic drug may receive a 180-day exclusivity.
It is to be noted that this patent exclusivity is far shorter in duration than the one that brand name companies receive.
Although it still results in financial incentives for any company that receives it, it often requires generic manufacturers to operate on smaller margins to remain competitive, which may disincentivize spending on quality control.
In the case of United States vs. Ranbaxy USA, Inc, the company was found guilty of submitting false and misleading information to FDA and forced to pay a fine.
Over several years, Ranbaxy USA committed several violations, including adulteration, and failure to maintain records and meet Current Good Manufacturing Practice (cGMP) regulations.
It is a great example of how these market forces play out in the real world, and raises the question as to whether FDA's current approach to regulation, which is largely based on trust, is still viable in today's globalized pharmaceutical marketplace.
23 One solution may be to expand FDA's ongoing chemical analysis of medications.
Another might be to introduce independent objective pharmaceutical analysis (OPA) that could provide ongoing surveillance of our drug supply, and test for dissolution as well as ingredients, strength, and impurities.
This is particularly important with extended-or sustained-release products owing to limitations of bioequivalence requirements.
24, 25 To be clear, generic drugs are not inherently inferior to brand name medications, but they do receive different economic incentives, which can influence corporate behavior.
It is estimated that 90% of Americans benefit from generic medications, thanks to the groundbreaking Hatch-Waxman Act, which reduced barriers to accessing medications worldwide.
26 Most medications, brand and generic, are of high quality, and no data have demonstrated a correlation Background: The extent of our reliance on foreign sources of medications is largely unknown.
Ensuring the quality and safety of medications is essential to maintain public confidence and medication adherence and prevent adverse outcomes.
Given the increased media attention around this issue, pharmacy professionals might anticipate that the public may start seeking answers regarding the source or availability of their medication.
Pharmacists are uniquely qualified to lead our Nation's effort to safeguard the U.S. pharmaceutical supply chain through advocacy, education, and engagement.
between drug cost and quality, which highlights the need for pharmacists to be involved in pharmaceutical purchasing and logistics.
Regulatory changes that improve transparency and quality management will likely have a positive impact but may present challenges.
Increased transparency may highlight our dependency on foreign sources of medication.
If patients, organizations, or the government start to demand "American-made" medications, a shortage could arise in the short run given the lack of domestic manufacturing capacity.
It's unclear how the USPSC would respond in the long run.
Increased OPA could identify new quality or safety issues.
Regulators such as FDA may face a "Regulator's dilemma" where it has to make decisions about keeping low-quality medications on the market or risk inducing a drug shortage.
A research letter published in Circulation related to the 2018 valsartan recall highlights the challenge our health system faces addressing these supply chain issues.
27 Jackevicius et al.
27 found that although most patients taking a recalled valsartan product switched to another antihypertensive drug, approximately 10% of the patients did not refill an alternative medication.
They also reported a small but statistically significant increase in the rate of hypertension-related emergency department visits, which may be associated with the recall (from 0.11% to 0.17%; P ¼ 0.02).
As health care shifts to a value-based model it is important to remember that value is a function of quality over cost: Value ¼ Quality ðEfficacy and SafetyÞ Cost ðDirect and IndirectÞ As organizations hammer down on drug spending, there can be pressure to buy the cheapest medication available.
However, if a medication is not as effective or causes adverse reactions that result in hospitalizations is it really providing value for the cost?
28 Pharmacists, along with regulators, will need to be engaged to advise patients, prescribers, and government leaders on the best solutions to address medication shortages, recalls, and other issues that may arise.
Acetris Health, LLC, vs. United States highlights the legal challenges of regulating the USPSC.
29 The case centers around the Trade Agreements Act (TAA), a piece of legislation often used to ensure that U.S. government organizations (e.g., Department of Defense [DoD] and Department of Veterans Affairs [VA]) procure medications that are U.S.-made.
30 The court ruled that a medication manufactured in the United States was TAA-compliant even if the API came from a non-eTAA-compliant country, which in this case was India.
The court's ruling now only requires that an FDP be manufactured in the United States for the medication to be considered U.S.made.
This arguably lowers the standard or, at minimum, complicates the assessment of what is U.S.-made.
Medicare and Medicaid programs are usually exempt from TAA requirements for medications, given that these programs, run by Centers for Medicare & Medicaid Services, reimburse for medications but do not physically purchase them.
Requiring these programs or their funds to follow TAA would add leverage for other federal programs that purchase medications (e.g., DoD and VA) but may complicate or affect patients' access to medication at pharmacies that serve the Medicare/Medicaid population.
To address policy, regulatory, and legal gaps, health professionals and organizations have proposed strategies to improve the availability, quality, and safety of pharmaceuticals dispensed in the United States.
The 2020 American Pharmacists Association House of Delegates (APhA HOD) recently adopted a series of policies aimed at protecting pharmaceuticals as a strategic asset.
31 The President recently signed an executive order titled "Ensuring Essential Medicines, Medical Countermeasures, and Critical Inputs Are Made in the United States."
32 Members of the U.S. Senate and House of Representatives have also introduced legislation that would have an impact on the USPSC if passed (Table 1) .
Common themes relate to tracking the source of APIs and strengthening the requirement or incentive to purchase U.S.-made medications.
Although such actions have the potential to improve the safety and security of the USPSC, policy alone is unlikely to fix our fractured supply chain.
Mandating that any organization that purchases medication "Buy American" in the absence of Understanding the U.S. pharmaceutical supply chain SCIENCE AND PRACTICE increased domestic manufacturing capacity has the potential consequence of precipitating or exacerbating shortages.
Economic incentives coupled with domestic manufacturing would be a preferred strategy for success.
33 Advanced manufacturing technology such as pharmacy on demand also has the potential to revolutionize the manufacturing process, shifting away from traditional batch manufacturing methods to continuous-flow synthesis.
34 The technology has demonstrated the ability to reduce the time required to manufacture several different medications in a short period of time but will likely require additional research and investment.
In parallel with innovative policy, scientific, and economic support, there are several activities that pharmacists can perform to safeguard the USPSC.
Pharmacists already add value by identifying therapeutic alternatives in cases where medications are not available owing to shortage or recall.
13 Patients continue to trust their pharmacists who are often the first to hear of and report problems with medications.
35 The Pharmacists' Patient Care Process can be a helpful framework to address medication-related quality or safety issues, and pharmacists should continue to report these issues to MedWatch or other appropriate data repositories.
36, 37 Pharmacists involved with purchasing medications can screen manufacturers and ask what "backup capability" they have to produce medications.
A company with backup capability will be able to provide an uninterrupted supply of medication even if, for example, a production facility experiences a problem (e.g., natural disaster, public health crisis).
Pharmacists can review information on drug recalls on FDA's Recalls, Market Withdrawals, & Safety Alerts website, and information on drug shortages on the American Society of Health-System Pharmacists website.
38, 39 Pharmacists can also review FDA warning letters and inspection records before buying from a particular manufacturer.
A simple search in the documents for key words such as "sterility" or "data integrity" can identify red flags.
40, 41 One example of this information being put into action involves pharmacists partnering with physicians to use market intelligence to inform purchasing on the basis of corporate reputation and safety signals.
8 There may be instances where pharmacists want to play a role in purchasing medications but are unable to do so because of the corporate structure of their organization.
In these situations, the pharmacist may have to get creative and leverage informal leadership skills to enact change.
If a pharmacist has a patient safety concern or is receiving several reports from patients experiencing problems with a particular manufacturer or medication, it is still important to report these events through MedWatch or their local patient safety reporting system.
Several recent recalls for medications such as valsartan, ranitidine, and metformin were brought to FDA's attention through a citizen's petition.
42 Pharmacists should continue to engage in conversations on the appropriateness of a drug substitution, particularly in the case of narrow therapeutic drugs or high-risk situations such as post-organ transplantation.
If a substitution is made, pharmacists should follow up to assess if the patient is better, worse, or the same.
This applies both when switching from brand to generic or generic to generic.
Transitions of care can be a particularly vulnerable time for a patient.
43 One question a pharmacist might ask is, "Will the patient receive the same medication (i.e., brand or generic) in an inpatient versus outpatient setting?"
Although pharmacists often have little choice in the matter, they can certainly be aware of the issue and use their clinical judgment to determine the best course of action.
An example of this is highlighted by the role that pharmacists can play in ensuring that their patients with seizures continue to receive the same drug formulation by the same manufacturer month-to-month.
Pharmacists can also educate and advise patients and prescribers on how to deal with a recall.
In the case of ranitidine and valsartan, a pharmacist can recommend a therapeutic alternative in the same class.
In the case of metformin, pharmacists 32 could help determine if the patient received a recalled lot and assist with transitioning them to a product made by a manufacturer that was not affected by the recall.
Given the increased media attention around this issue, pharmacy professionals might anticipate that the public may start seeking answers regarding the source or availability of their medication.
Pharmacists can attempt to ask the manufacturer, distributor, or packager where the FDP and API are made, although, as discussed previously, some companies may not have, or be willing to disclose, this information.
If this is the case, the National Institutes of Health's DailyMed website may be an additional resource for this information, given that it maintains accurate drug labels that can be a source of manufacturer or distributor information.
44 The USPSC is complex, global, and goes beyond FDPs.
Protecting it will require innovative solutions, including legislation, policy, market incentives, and objective surveillance testing.
The 2020 APhA HOD has asserted that "The quality and safety of pharmaceutical and other medical products and the global pharmaceutical and medical product supply chain are essential to the United States national security and public health."
Pharmacy professionals on the front line improve the health of patients, identify medication-related issues, and engage in drug-procurement decisions.
Pharmacists are uniquely qualified to lead our nation's effort to safeguard the USPSC through advocacy, education, and engagement.
Growing reliance on China for medicines: A role for pharmacists in assuring quality Made in China: how U.S.
Dependence on Chinese medicines and components could pose a security threat.
Military officers Association of America We still don't know who makes this drug Drug shortages and labor costs: measuring the hidden costs of drug shortages on U The drug shortage crisis in the United States: causes, impact, and management strategies Medication shortages during the COVID-19 crisis: what we must do China Rx: Exposing the Risks of America's Dependence on China for Medicine Bottle of Lies: The Inside Story of the Generic Drug Boom ?fr¼207.1 #:~:text¼Finished%20drug%20product%20means%20a,hospitals%2C% 20or%20other%20sellers%20or ICRA downgrades Indian Pharma on China lockdown Dangers aside, drugmakers can't live without Chinese API Securing the U.S. drug supply chain: oversight of FDA's foreign inspection program The impact of drug shortages on patients with cardiovascular disease: causes, consequences, and a call to action Imminent risk of a global shortage of heparin caused by the African swine fever afflicting the Chinese pig herd FDA updates and press announcements on angiotensin II receptor blocker (ARB) recalls (valsartan, losartan, and irbesartan) FDA requests removal of all ranitidine products (Zantac) from the market Oral intake of ranitidine increases urinary excretion of N-nitrosodimethylamine FDA statement: coronavirus disease 2019 (COVID-19) update: foreign inspections Center for drug evaluation and research office of pharmaceutical quality: report on the state of pharmaceutical quality Trends in risks associated with new drug development: success rates for investigational drugs Returns on research and development for 1990s new drug introductions Pharmaceutical industry profits and research and development Generic drug manufacturer ranbaxy pleads guilty and agrees to pay $500 million to resolve false claims allegations, cGMP violations and false statements to the FDA Bioequivalence of generic drugs: a simple explanation for a US Food and Drug Administration requirement Your medication may not be dissolving properly IMS Institute for Healthcare Informatics.
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Danbury, CT: IMS Institute for Healthcare Informatics Population impact of generic valsartan recall Utility of lidocaine as a topical analgesic and improvements in patch delivery systems Exploring the growing U.S. reliance on China's biotech and pharmaceutical products APhA virtual house of delegates ensuring -essential-medicines-medical-countermeasures-critical-inputs-made-unitedstates A civic duty to improve access to generic pharmaceuticals Pharmacy on demand: new technologies to enable miniaturized and mobile drug manufacturing Valisure citizen petition on ranitidine Joint Commission of Pharmacy Practitioners.
Pharmacists' patient care process MedWatch: the FDA safety information and adverse event reporting program Food and Drug Administration.
Recalls, market withdrawals, & safety alerts Current drug shortages inspections-compliance-enforcement-and-criminal-investigations/ compliance-actions-and-activities/warning-letters.
Accessed May 10, 2020.
41.
U.S. Food and Drug Administration.
Inspection classification database search Pharmacy takes FDA to task in citizen petition over tainted valsartan Pharmacist impact on transitional care of highrisk patients through medication reconciliation, medication education, and postdischarge call-backs (IPITCH Study) 3537 -Protecting Our Pharmaceutical Supply Chain from China Act of 2020 3538 -Strengthening America's Supply Chain and National Security Act Stauber introduces legislation to end Chinese control over pharmaceutical supply Chain Stauber introduces legislation to put an end to America's reliance on foreign supply chains for critical minerals 4710 -Pharmaceutical Independence Long-Term Readiness Reform Act Data Innovation, Enterprise Intelligence and Data Solutions, Defense Health Management Systems Acknowledgments I would like to thank Rosemary Gibson, David Light, Robert Cunningham, and Kyleigh Hupfl for continuing to openly share their knowledge, experience, and insights with me as it relates to the USPSC.
key: cord-291626-lxa8pvt3 authors: Pelfrene, E.; Mura, M.; Cavaleiro Sanches, A.; Cavaleri, M. title: Monoclonal antibodies as anti-infective products: a promising future?
date: 2019-01-31 journal: Clinical Microbiology and Infection DOI: 10.1016/j.cmi.2018.04.024 sha: doc_id: 291626 cord_uid: lxa8pvt3 Abstract Background The paucity of licensed monoclonal antibodies (mAbs) in the infectious diseases arena strongly contrasts with the ready availability of these therapeutics for use in other conditions.
Aims This narrative review aims to assess the potential of monoclonal antibody-based interventions for infectious diseases.
Sources A review of the literature via the Medline database was performed and complemented by published official documents on licensed anti-infective mAbs.
In addition, ongoing trials were identified through a search of the clinical trial registration platform ClinicalTrials.gov.
Content We identified the few infections for which mAbs have been added to the therapeutic armamentarium and stressed their potential in representing a readily available protection tool against biothreats and newly emerging and reemerging infectious agents.
In reviewing the historical context and main features of mAbs, we assert a potentially wider applicability and cite relevant examples of ongoing therapeutic developments.
Factors hindering successful introduction of mAbs on a larger scale are outlined and thoughts are offered on how to possibly address some of these limitations.
Implications mAbs may represent important tools in treating or preventing infections occurring with reasonably sufficient prevalence to justify demand and for which existing alternatives are not deemed fully adequate.
Future initiatives need to address the prohibitive costs encountered in the development process.
The feasibility of more large-scale administration of alternative modalities merits further exploration.
In order to ensure optimal prospect of regulatory success, an early dialogue with competent authorities is encouraged.
In 2017, bezlotoxumab became the second monoclonal antibody authorized for use throughout the European Economic Area directed against an infectious agent.
It targets Clostridium difficile toxin B, and as such aims to prevent recurrences of the infection in adults at high risk of repeated bouts [1] .
This follows a much earlier EU regulatory approval of palivizumab (in 1999), which is administered to prevent serious lower respiratory tract disease requiring hospitalization caused by respiratory syncytial virus (RSV) in children at high risk for the disease [2] .
Licensing of these two monoclonal antibodies (mAbs), nearly two decades apart, illustrates the gap to date in clinical development of this therapeutic tool against infections.
This is consistent with the experience of other regulatory authorities, such as the US Food and Drug Administration (FDA), which granted similarly phrased labels in the United States for both products [3, 4] .
Additionally, the FDA recently licensed ibalizumab as a rescue therapy in heavily treatmentexperienced adults with multidrug-resistant HIV-1 infection and also previously approved raxibacumab (in 2012) and obiltoxaximab (in 2016), both intended for treatment of inhalational anthrax (in combination with appropriate antibacterial medicines) and for prophylaxis when alternative therapies are not available or are not appropriate [5e7] .
The latter two products were assessed by FDA according to its 'Animal Rule,' largely relying on efficacy data generated in a relevant animal model [8e10] .
In general, the use of mAbs is viewed as an attractive biodefense measure, countering deliberate threats [11] .
The technology platform may also offer a rapid protection against newly emerging and reemerging infectious agents when no other treatment or prophylaxis is available.
Witness to this, for example, is the ongoing research in mAbs to treat patients with Middle East respiratory syndrome [12] .
The experimental use of a tobacco plantederived mixture of mAbs (ZMapp) during an Ebola virus disease outbreak in western Africa provides an additional example in this respect [13, 14] .
Interestingly, the paucity of currently licensed mAbs in the infectious diseases arena strongly contrasts with the ready availability of these therapeutics for use in other conditions, mainly oncology and chronic inflammatory or rheumatic afflictions.
Indeed, for these disease areas, mAbs globally generate a sizeable yearly income [15] .
This narrative review revisits the potential applicability of mAbs in targeting infections and outlines their prospects for successful therapeutic development.
We undertook a search on Medline Complete regarding trials and reviews for antibody-based therapeutics in infectious diseases.
The terms used in different combinations were as follows: 'therapeutic antibodies,' 'monoclonal' and 'infectious diseases,' as well as pathogen-specific terms.
The results were limited to Englishlanguage reports and surveyed documents published from January 2008 to March 2018.
Our methodology was further complemented by published official documents on anti-infective mAbs licensed to date by EU and US regulatory authorities.
Some of the trials were identified through a search of the clinical trial registration platform ClinicalTrials.gov.
Historically, immune sera were used to treat or prevent infectious diseases before the availability of vaccines and antibiotics, with polyclonal sera (hyperimmune sera) still being used against conditions such as rabies, diphtheria, tetanus and botulism [16] .
These sera are typically administered when rapid protection is required, following known exposure in an unvaccinated individual (or vaccination is nonexistent) or when previously acquired immunity has waned.
As with hyperimmune sera, mAbs can be deployed irrespective of preexisting immune protection status and equally may benefit the immunocompromised host [17, 18] .
However, they avoid some of the disadvantages attributed to sera derived immunoglobulins, such as potential (exceedingly low) transmission of infectious hazards and batch variability [19] .
Over the years, monoclonal antibody engineering has evolved.
The first licensed monoclonal antibody, OKT3, indicated to prevent acute kidney transplant rejection, used a murine hybridoma technique [20, 21] .
Early commercial successes with mAbs were limited, however, by the availability of suitable myeloma cell lines.
Also, occurrence of adverse immune reactions, by provoking a human anti-mouse antibody response, is a recognized setback for use of the technique.
To limit such unwanted effects whilst also enhancing effector function, chimeric (mouseehuman) antibodies first replaced murine hybridomas and more recently humanized (i.e.
antibody structure containing less than 10% nonhuman sequences) and fully human mAbs have been developed [19, 21] .
New expression tools became available, such as phage display libraries, transgenic mice, Chinese hamster ovary (CHO)-based biomanufacturing and highly scalable plant technology, as well as the possibility to produce mAbs using human B cells isolated from subjects in convalescence or after vaccination [22e25].
Thus, current methods have made production more efficient, and importantly, the use of human and humanized mAbs poses fewer safety concerns as opposed to older techniques and serum therapy [19, 26] .
With exquisite specificity being the hallmark, mAbs generally harbour a low potential for off-target adverse reactions [27] , although it makes them vulnerable to escape mutants, potentially rendering this therapeutic measure ineffective [28] .
Hence, this drawback justifies the development of mAbs cocktails, with the antibody constituents binding to different epitopes [27, 29] .
Also, alternative antibody formats have been engineered, simultaneously addressing different targets involved in pathophysiologic processes.
As such, bispecific antibodies aim to ensure enhanced potency and breadth of protection [30e32].
Other developments include antigen-binding (Fab) fragments, single chain variable fragments (ScFv) and pairs linked in different ways, as to create formats combining optimal size, half-life, activity and safety [25, 33] , as well as novel delivery systems such as antibodyeantimicrobial conjugates and radioimmunoconjugates [34, 35] .
Effector functions can be modified: Fc domain changes may enhance the affinity to Fc receptors on phagocytic cells, NK cells and B cells, and hence enforce antibody-dependent killing, whilst mutations leading to increased binding affinity for neonatal Fc receptor (FcRn) contribute to an extended half-life of the antibody [25] .
In principle, mAbs could target a wide range of biologic agents, encompassing bacterial and viral pathogens, fungi and associated toxins, with their action exerted either directly (e.g.
preventing cell entry or neutralizing toxins) or via indirect mechanisms (e.g.
modulating inflammatory responses or promoting opsonic phagocytosis) [26, 34, 36] .
To date, licensed antibacterial mAbs target exotoxins, with the antigeneantibody complexes primarily removed via the reticuloendothelial system [1, 6, 7, 34] .
Instead, mAbs may bind to structural cell surface components (proteins and exopolysaccharides), with subsequent direct bactericidal clearance or immune system dependent cytotoxicity (antibody or complement dependent) [34] .
In this regard, the pharmacodynamic mode of action varies amongst the candidate products currently under development [34] .
Notwithstanding previous disappointment in translational research [37] , activities which target Staphylococcus aureus and Pseudomonas aeruginosa are the most developed so far (B. François et al., 'Safety and tolerability of a single administration of AR-301, a human monoclonal antibody, in patients with severe pneumonia caused by Staphylococcus aureus: first in man trial,' abstract 1992, paper presented at European Congress of Clinical Microbiology and Infectious Diseases 2017) [34, 38] .
Indeed, in an era of increased awareness and intensified effort to mitigate the antimicrobial resistance threat, mAbs may offer a welcome addition to the therapeutic toolkit [39] .
Although unlikely to compete directly with conventional antimicrobials in treating serious bacterial infections, mAbs might act synergistically if both measures are administered concurrently.
To date, promising results to that effect have been shown in animal models [40, 41] .
Further on, antibody administration is expected to provide less selective pressure for cross-resistance and may preserve the gut microflora [34, 42] dan important consideration in view of our expanding knowledge on the role of the human gut microbiome in both health and disease [43] .
Nevertheless, the success for a wide introduction of mAbs in treating established bacterial infections will be largely dependent on ready and specific pathogen identification.
This necessitates further investment and research into validated point-ofcare rapid diagnostics (e.g.
PCR-based and fluorescence in-situ hybridization technologies), allowing prompt targeted therapy [44] .
Also, because the effect of a single dose can last for one to a few months, the use of recombinant antibodies as prophylaxis could theoretically allow for a decrease in conventional antimicrobial usage.
Hence, in recent efforts, clinical development programmes intend to demonstrate that mAbs against bacterial pathogens will protect at risk patients (e.g.
prevention of S. aureus ventilatorassociated pneumonia in colonized, mechanically ventilated patients in the intensive care unit) (ClinicalTrials.gov NCT02940626, NCT02296320) [44] .
mAbs may provide a broad neutralizing, potent activity against highly conserved viral epitopes.
Alternatively, the antibodies can target receptors and coreceptors located on host cells [19] .
With research still in infancy, mAbs which recognize virally infected cells and induce antibody-dependent cytotoxicity may offer an additional mechanism of action [45] .
Hitherto, mAbs have been licensed as HIV rescue therapy in adults and as an RSV infection preventive in high-risk children [5, 2] .
Whilst ibalizumab targets the conformational epitope of the CD4 T-cell receptor, blocking cell entry by HIV-1, other candidates under development, either also target host antigens or alternatively directly bind HIV-1 antigens [46e48].
Research further includes the promise of bispecific and trispecific broadly neutralizing antibodies [31, 49] .
With regard to RSV, palivizumab interferes with virus attachment and fusion by binding RSV F protein [50] .
Presently RSV research is focused on extending the half-life of RSV mAbs, aiming to protect infants throughout their first RSV season with a single dose administration [51] .
Other currently studied viral targets include influenza, Ebola and Zika [36] .
Recent viral outbreaks have brought a heightened awareness to prepare against emerging infections [36] .
For such threats, mAbs could potentially be indicated as treatment of infected individuals and could as well provide targeted prophylaxis for individual protection and transmission interruption (possibly changing the dynamics of a nascent epidemic) [52] .
For example, within the context of ongoing product development targeting influenza, for which promising results were obtained in ferret models and early clinical development, broadly neutralizing mAbs might be suitable at the emergence of a pandemic outbreak, at least for specific at-risk groups such as exposed contacts and healthcare professionals [52e54].
In that scenario, they could be used to bridge the time gap before availability of strain-specific vaccines [55] .
With a potential to provide a comparably efficacious product, the replacement of currently indicated hyperimmune sera by mAbs deserves to be explored, taking into account feasibility issues related to their clinical development [16] .
For example, in the face of periodic outbreaks of diphtheria in various world regions, a global short supply of equine diphtheria antitoxin provides new impetus in developing a suitable monoclonal antibody alternative [56] .
So far, though, activities have been most advanced in respect to rabies, for which at least two different products are considered [19] .
For one of these, a single human monoclonal antibody (SII-RMAb), a recently completed noninferiority controlled trial concluded it as a potential safe and potent alternative to human rabies immunoglobulin-containing postexposure prophylaxis regimen [57] .
As illustrated by the ongoing efforts, anti-infective mAbs constitute a thriving focus of research, but in order to become a success, multiple challenges need to be overcome.
First, technical barriers remain.
For instance, proper selection of bacterial targets remains fraught with uncertainties: highly conserved outer membrane proteins may be masked and hence unavailable for binding with antibodies; conversely, epitopes located on exopolysaccharides are typically not conserved (i.e.
different serotypes exist), posing limitations to the effectiveness of a targeting single monoclonal antibody [34] .
Moreover, bacterial defence mechanisms developed in response to host immunoglobulins (e.g.
production of antibody degrading proteinases) can impede the success of antibody-based therapy.
For viral infections, a single lasting treatment is hampered in the face of multiple strains, rapid evolution and selection of escape mutants [36] .
In addition, for some pathogens, viraemia often peaks before appearance of symptoms [58, 59] .
In these circumstances, rapid point-of-care diagnostics may be fundamental to the success of the intervention, allowing prompt therapy initiation in those most at risk of developing serious illness [36, 60] .
Second, regulatory product approval pathways are not well optimized for emerging threats.
Indeed, a small number of patients and unpredictable outbreak dynamics may impede the conduct of confirmatory controlled clinical trials.
Also, human challenge studies can provide supportive evidence of protection for only a limited number of conditions [61, 62] .
In the absence of adequate risk mitigation, such an approach is not expected to be feasible for most of the emerging infections [63, 64] .
Alternatively, however, for those threats lacking sustained human-to-human transmission, assessment could be based mainly on data obtained from animal challenge studies and on pharmacokinetic/ safety evaluation of the product in healthy volunteers, on the condition that animal models can be established that are sufficiently representative of human disease [8, 65] .
The availability of protocols to test the effectiveness and safety of the therapeutic in the field at the time of an outbreak would then probably constitute a condition to the licensing.
Third, business models and potential markets do vary depending on the disease target, but on the whole, investment and product development are hindered by lack of clarity on how to position the use of mAbs amongst preventive and therapeutic options [27] .
Uncertainties remaindfor instance, in selecting appropriate trial endpoints and delineating populations best suited to the intervention [16] .
Additionally, development is constrained by high investment expenses amidst unsure future returns.
Appropriate pricing is considered a prerequisite for market success, and hence increased streamlining, optimization and innovative processes (e.g.
Quality-by-Design) may exert downward pressure on costs.
Moreover, research into new delivery methods should be expanded and intensified, ensuring convenient administration to a large number of people.
This may entail the development of novel intramuscular and inhalation formulations.
In this respect, DNA plasmid-delivered mAbs produced by muscle cells in vivo are singled out as a potential way to circumvent some of the limitations in immunoglobulin G administration [66] .
Next-generation mAbs might offer further opportunities, even allowing a single product to target multiple pathogens [67, 68] .
From a regulatory viewpoint, tools are available in the European Union to ensure a swift evaluation process and early access to the market, including specific scientific guidance or dedicated advice to companies (Table 1 ) [69] .
In this regard, obtaining and complying with European Medicines Agency's scientific advice has shown to be predictive of regulatory success [70] .
Concerning emerging infections, broad stakeholder engagement (with national and international public health agencies, private sector, funding organizations and regulatory authorities) has been advocated.
It is argued that this may entail the creation of an independent global agency, as to develop and implement a coherent strategy for global biopreparedness [71] .
Although research and clinical development of mAbs has intensified, it remains to be proven if a new dawn is on the horizon for this product class to become commonplace in the fight against an array of infections.
Inroads have been made so far, as illustrated by the few licensed mAbs against infectious agents or associated toxins.
However, for a broader anti-infective potential to be fully exploited, scientific, regulatory and commercial barriers to development need to be addressed.
Amongst other initiatives, an early dialogue with regulatory authorities is encouraged to ensure streamlined development and to enable early access for patients.
Orphan drug: medicine for diagnosis, prevention or treatment of a lifethreatening or chronically debilitating condition that is rare or where medicine is unlikely to generate sufficient profit to justify research and development costs.
Incentives apply (e.g.
fee reductions).
PRIME Priority scheme: support for development of medicines of major interest that target unmet medical need Continuous support and early interactions with reviewers; SA at key development milestones involving additional stakeholders such as HTA.
Potential for combination with one or several early access tools at time of MAA.
Early access Accelerated review Reduced evaluation time frame Rapid assessment of medicines that are of major interest for public health, especially ones that are therapeutic innovations (unmet medical need).
Conditional MAA Earlier authorization of medicines for patients with unmet medical needs, on basis of less complete clinical data Eligibility includes medicines for seriously debilitating or life-threatening diseases, emergency situations, orphan drugs.
Comprehensive data are expected to be generated after authorization within agreed time frame but risk/benefit ratio is clearly positive at time of approval.
Compassionate use Benefits seriously ill patients who cannot be treated satisfactorily or cannot enrol in ongoing clinical trials Pertains to unauthorized medicinal products for chronically, seriously debilitating or life-threatening diseases, with no satisfactory treatment authorized in EU; targeted at a group of patients rather than individual; or undergoing centralized MAA or clinical trials EMA, European Medicines Agency; FDA, US Food and Drug Administration; HTA, health technology assessment bodies; MAA, marketing authorization application; SA, scientific advice.
European Medicines Agency; Committee for Medicinal Products for Human Use.
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The views expressed in this article are the personal views of the authors and ought not be understood or quoted as being made on behalf of or reflecting the position of the European Medicines Agency or one of its committees or working parties.
key: cord-334847-lf1grybz authors: Lynch, Holly Fernandez; Dickert, Neal W; Zettler, Patricia J; Joffe, Steven; Largent, Emily A title: Regulatory Flexibility for COVID-19 Research date: 2020-07-07 journal: J Law Biosci DOI: 10.1093/jlb/lsaa057 sha: doc_id: 334847 cord_uid: lf1grybz Clinical research is critical to combatting COVID-19, but regulatory requirements for human subjects protection may sometimes pose a challenge in pandemic circumstances.
Although regulators have offered some helpful guidance for research during the pandemic, we identify further compliance challenges regarding IRB review and approval, informed consent, emergency research, and research involving incarcerated people.
Our proposals for regulatory flexibility in these areas seek to satisfy the goals of protecting participants and promoting the development of high-quality evidence to improve patient care.
These recommendations may have relevance beyond the COVID-19 pandemic to enhance the efficiency of research oversight and participant protection more broadly.
Clinical research to understand, treat, and prevent COVID-19 is both crucial and highly regulated.
Most intervention studies are subject to Food and Drug Administration (FDA) requirements and federally funded research with human subjects must follow requirements imposed by the Common Rule.
Strict regulatory compliance may be challenging amidst a public health emergency, but participant protection and high-quality science remain essential.
(1) In recognition of these considerations, FDA and the Office for Human Research Protections (OHRP) within the Department of Health and Human Services (HHS) have issued guidance on conducting research during the COVID-19 pandemic.
(2) Although this guidance offers a helpful start, gaps remain and additional regulatory flexibility is warranted in some instances.
COVID-19 research has been running at a remarkable pace,(3) challenging the capacity of both investigators and institutional review boards (IRBs).
To ensure that this research proceeds efficiently and ethically, we offer suggestions to proactively address regulatory compliance challenges regarding IRB review and approval, informed consent, and inclusion of vulnerable populations.
IRBs should also exercise the regulatory flexibility currently available to them (Table 1) .
Although the pandemic is changing rapidly, the virus remains a substantial public health threat without adequate therapeutic or prophylactic interventions, suggesting that our recommendations for flexibility will remain relevant for some time.
They will also be useful in the face of future pandemics and should be considered for research more broadly.
As part of its Coronavirus Treatment Acceleration Program (CTAP), 15 FDA is conducting "ultra-rapid protocol review," often within 24 hours, for research subject to IND and IDE requirements.
16 Many IRBs are also taking steps to speed review of COVID-19 protocols, including through disease-specific review boards, prioritization over non-COVID submissions, and more frequent meetings.
These approaches can, however, strain board capacity, especially at academic institutions where membership may not be as deep as for commercial boards.
Additionally, many members are juggling added professional and personal obligations due to the pandemic.
To address these challenges, research sites should rely on review conducted by other boards with sufficient capacity and expertise whenever possible.
This is required for multisite research subject to the Common Rule (unless a site"s board has been designated the IRB of record), but is also permitted more broadly.
17 Additionally, FDA and OHRP should consider adjusting quorum requirements through enforcement discretion or regulatory waiver procedures.
Most COVID-19 interventional research will require review at convened IRB meetings.
Quorum for such meetings requires a majority of the total board membership, including the presence of at least one member whose primary concerns are in nonscientific areas.
18 Because IRBs must comprise at least 5 members satisfying certain criteria, the minimum quorum to satisfy regulatory standards is 3 members.
19 Allowing larger boards to break into meetingspecific sub-boards, each with adequate expertise and representation, would reduce their quorum requirements.
This, in turn, would allow the assignment of fewer COVID-19 protocols per reviewer and permit members to attend fewer meetings, easing the burden on each member and allowing more time and attention for rigorous review.
This approach would entail fewer reviewers per protocol than might otherwise be the case, but if additional reviewers lack sufficient time to review all the protocols assigned to them, that tradeoff seems appropriate.
Moreover, under this flexible approach, no meeting-specific subboard would comprise fewer than 3 participating members, the minimum number of reviewers deemed acceptable by the regulations.
Although institutions already have the authority to revise their IRB charters to split larger IRBs into smaller ones, our proposal is more flexible because it would allow IRBs to make membership adjustments meeting-by-meeting as needs change in real time.
This may be especially useful as sites re-open to non-COVID research, in addition to their COVID-19 portfolios, thereby further increasing IRBs workloads.
As noted above, FDA guidance specifies acceptable procedures to obtain informed consent in the face of isolation requirements for COVID-19 patients and physical distancing requirements that may affect surrogate decisionmakers.
20 In these circumstances, FDA recommends using electronic consent, including via the COVID MyStudies App newly developed by the agency for this purpose.
It also describes an alternative process involving a combination of phone or video conferencing, provision of the consent form by someone already entering a patient"s room, signed documentation, and either a witness attestation of signature or 20 FDA, supra note 2. reasonably drawn from those data.
27 Regulators should therefore clarify the standard for disclosure, including which disclosures require reconsent and amended forms versus other mechanisms of information sharing.
At a minimum, participants should be informed of new agency warnings (e.g., recent FDA statements and actions regarding hydroxychloroquine), 28 government treatment recommendations, 29 and new product approvals or emergency use authorizations relevant to trial participation decisions.
30 In contrast, the disclosure standard should exclude new information gleaned from outside reports based on preprints without peerreview or interim findings from incomplete research, which may be more misleading than informative.
In addition, rather than expecting each IRB and investigator to engage in the difficult and potentially duplicative work of parsing new COVID-19 data for trial participants, FDA"s CTAP could maintain an up-to-date website to serve as a source of reliable guidance relevant to COVID-19 study participation.
Because COVID-19 can entail rapid development of severe, acute respiratory failure and other acute, life-threatening conditions, some protocols may need to be conducted under regulations for emergency research that allow subjects to be enrolled under an exception from informed consent (EFIC).
31 The primary challenge for COVID-19 EFIC research is that the regulations require, prior to IRB approval, consultation with individuals from communities "in which the research will be conducted and from which the subjects will be drawn."
32 This has traditionally been a lengthy process, often involving face-to-face contact with stakeholders, which will be difficult for COVID-19 research given urgency and the likely need to continue physical distancing for some time.
Regulators should therefore encourage IRBs to focus on rapid identification of high-priority community concerns from key stakeholders.
Appropriate stakeholders will be site and condition-specific; they may include community advocates and advisory boards, religious and cultural leaders, and government leaders.
Involvement of patients and family members at high risk for COVID-19 or who have experienced related conditions are also critical, as is attention to the views and concerns of minority groups and socioeconomicallydisdavantaged individuals disproportionately affected by this disease.
Regulators and IRBs should recognize that these efforts may need to be more focused than in other contexts and that researchers will predominantly need to rely on remote consultation methods, including webinars, 13 online surveys, and telephone calls.
However, it is critical that genuine efforts to solicit community views not be abandoned.
In light of open questions about how best to treat COVID-19, clinical care for patients suffering from moderate to severe disease often involves trial participation.
This makes it important to consider including people who are incarcerated, 33 given the substantial risk of infection associated with their confinement.
34 Yet, as a vulnerable population, the inclusion of incarcerated people in research is subject to additional regulatory safeguards.
Biomedical research funded by HHS may involve "prisoners" as subjects when the research examines practices that have "the intent and reasonable probability of improving the health or well-being of the subject," among other circumstances.
35 This determination must be made by OHRP and is not left up to IRBs.
36 Moreover, when this research requires assignment to control groups that may not benefit from participation, OHRP is required to consult with experts in penology medicine and ethics, as well as to publish public notice of intent to allow such research.
37 In addition to these authorizations, HHS-funded research involving "prisoners" may only be approved by an IRB that has a "prisoner representative" amongst its membership, which 15 population without any specific intention to include them.
In addition, OHRP should pursue waiver of the requirement that such research be reviewed by a specially constituted IRB, given that the inclusion of people who are incarcerated may not have been contemplated at the time of study approval and the difficulty of securing timely special review once an eligible patient presents for enrollment.
BOP should also seek a waiver to permit those in its custody to participate in these potentially beneficial studies.
To the extent that state and local rules limit the research participation of incarcerated people, adjustments to those rules may be necessary as well.
When people who are incarcerated are not specifically targeted for research, relying on traditional IRB approval standards and consent requirements should offer sufficient protection while also facilitating their access to possible research benefits.
The COVID-19 pandemic should not be viewed as an opening to opportunistically reduce participant protections.
Yet, it presents an invitation to revisit regulatory requirements and their conventional interpretations to evaluate which are truly necessary and which may constitute unjustified barriers to research.
In that regard, we must acknowledge that while human subjects research regulations and guidance are often important means of participant protection, existing approaches are not evidence-based and therefore should not be presumed to be more effective than or otherwise preferable to less burdensome alternatives.
40 OHRP should authorize the inclusion of "prisoners" in certain types of research offering the prospect of direct benefit when enrolled alongside general populations, and should seek to permit such research to proceed based on routine IRB review BOP should also seek to permit such research to proceed with individuals in its See also Bowles v finding an interpretation of an agency"s "own regulations," is "controlling unless "plainly erroneous or inconsistent with the regulation Erin Fuse Brown & Aaron Kesselheim, The Regulatory Accountability Act of 2017 -Implications for FDA Regulation and Public Health Attacking Auer and Chevron Deference: A Literature Review What Kisor Means for the Future of Auer Deference: The New Five-Step Kisor Deference Doctrine, Notice & Comment FDA, supra note 2 Developing Drugs and Biological Products for Treatment or Prevention FDA, supra note 2 EU Governments Ban Malaria Drug for COVID-19 Study on Hydroxychloroquine Use Questioned by 120 Researchers and Medical Professionals, THE GUARDIAN Cautions Against Use of Hydroxychloroquine or Chloroquine for COVID-19 Outside of the Hospital Setting or a Clinical Trial Due to Risk of Heart Rhythm Problems Biomedical Advanced Research and Development Authority (BARDA), Revocation of Emergency Use Authorization for chloroquine phosphate and hydroxychloroquine sulfate distributed from the Strategic National Stockpile Therapeutic Options we use the terms "people who are incarcerated" or "incarcerated people," except where directly quoting regulations that use the term "prisoners COVID-19 in Prisons and Jails in the United States Flattening the Curve for Incarcerated Populations -Covid-19 in Jails and Prisons, 382 NEW ENG She is the founder and co-chair of the Advancing Effective Research Ethics Oversight Consortium (AEREO), a member of a subcommittee of the U.S. Department of Health and Human Services Secretary"s Advisory Committee for Human Research Protections, a board member of Public Responsibility in Medicine and Research (PRIM&R) and the American Society of Law, Medicine, and Ethics, and a member of the NYU Working Group on Compassionate Use and Preapproval Access Dickert receives research funding from NIH, AHRQ, and the Greenwall Foundation Zettler reports serving as an expert witness retained by the Direct Purchaser Class Plaintiffs in In re Suboxone Antitrust Litigation Largent has no disclosures
key: cord-276414-kicu0tv5 authors: Bahadur Gurung, Arun; Ajmal Ali, Mohammad; Lee, Joongku; Abul Farah, Mohammad; Mashay Al-Anazi, Khalid title: In silico screening of FDA approved drugs reveals ergotamine and dihydroergotamine as potential coronavirus main protease enzyme inhibitors date: 2020-06-10 journal: Saudi J Biol Sci DOI: 10.1016/j.sjbs.2020.06.005 sha: doc_id: 276414 cord_uid: kicu0tv5 Abstract Coronaviruses with the largest viral genomes are positive-sense RNA viruses associated with a history of global epidemics such as the severe respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS) and recently the coronavirus disease 2019 (COVID-19).
There has been no vaccines or drugs available for the treatment of human coronavirus infections to date.
In the present study, we have explored the possibilities of FDA approved drugs as potential inhibitors of the coronavirus main protease, a therapeutically important drug target playing a salient role in the maturation and processing of the viral polyproteins and are vital for viral replication and transcription.
We have used molecular docking approach and have successfully identified the best lead molecules for each enzyme target.
Interestingly, the anti-migraine drugs such as ergotamine and its derivative, dihydroergotamine were found to bind to all the three target enzymes within the Cys-His catalytic dyad cleft with lower binding energies as compared to the control inhibitors (α-ketoamide 13b, SG85 and GC813) and the molecules are held within the pocket through a good number of hydrogen bonds and hydrophobic interactions.
Hence both these lead molecules can be further taken for wet-lab experimentation studies before repurposing them as anti-coronaviral drug candidates.
In silico screening of FDA approved drugs reveals ergotamine and dihydroergotamine as potential coronavirus main protease enzyme inhibitors Coronaviruses with the largest viral genomes are positive-sense RNA viruses associated with a history of global epidemics such as the severe respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS) and recently the coronavirus disease 2019 .
There has been no vaccines or drugs available for the treatment of human coronavirus infections to date.
In the present study, we have explored the possibilities of FDA approved drugs as potential inhibitors of the coronavirus main protease, a therapeutically important drug target playing a salient role in the maturation and processing of the viral polyproteins and are vital for viral replication and transcription.
We have used molecular docking approach and have successfully identified the best lead molecules for each enzyme target.
Interestingly, the antimigraine drugs such as ergotamine and its derivative, dihydroergotamine were found to bind to all the three target enzymes within the Cys-His catalytic dyad cleft with lower binding energies as compared to the control inhibitors (α-ketoamide 13b, SG85 and GC813) and the molecules are held within the pocket through a good number of hydrogen bonds and hydrophobic interactions.
Hence both these lead molecules can be further taken for wet-lab experimentation studies before repurposing them as anti-coronaviral drug candidates.
Keywords: Coronaviruses, coronaviral main protease, FDA approved drugs, ergotamine, dihydroergotamine, COVID-19, SARS-CoV-2, SARS-CoV, MERS-CoV, 2019-nCoV Coronaviruses are enveloped positive-sense RNA viruses which have crown-like appearance under the electron microscope and usually ranges from 60 to 140 nm in diameter (Richman et al., 2016) .
There are four coronaviruses (OC43, 229E, NL63 and HKU1) which cause mild respiratory distress in humans (Singhal, 2020) .
The cross-species transmission of animal beta coronaviruses to humans have been reported since last two decades-the first event which occurred in 2002-2003 when the bat originated coronaviruses crossed over to humans via palm civet cats as intermediary host and caused severe respiratory syndrome (SARS) in humans and was known as SARS coronaviruses (SARS-CoV) which infected 8422 people in China and Hong Kong and caused 916 deaths with a mortality rate of 11% (Chan-Yeung and Xu, 2003) .
In 2012, almost a decade later, another bat-originated virus emerged in Saudi Arabia and the transmission to humans was via dromedary camels.
The virus was designated as the Middle East respiratory syndrome coronavirus (MERS-CoV) which infected 2494 people and caused 858 deaths with fatality rate of 34% (Singhal, 2020) .
There is a recent global health emergency around the world with the rapid emergence and spread of 2019 novel coronavirus (2019-nCoV) or the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which has caused a pandemic known as coronavirus disease 2019 .
The outbreak was first reported in Wuhan, Hubei province, China in December 2019 (Wang et al., 2020) .
The intermediary hosts which led to the transmission of this bat-originated virus to humans is still an enigma.
According to the WHO report, there has been 25, 49,632 confirmed cases of COVID-19 and 1, 75,825 confirmed deaths to date (23/04/2020) (Coronavirus disease Pandemic, 2020).
The coronaviruses (CoVs) RNA genome has a size ranging from 27 to 31 kb and it is the largest viral RNA genomes known to date (Lai, 2001) .
The two overlapping polyproteins (pp1a and pp1ab) encoded by the CoV replicase gene are essential for viral replication and transcription (Snijder and Spaan, 1995; Yang et al., 2003) .
These polyproteins need to undergo a complex cascade of proteolytic processing for maturation which in turn regulates viral gene expression and replication (Xue et al., 2008) .
The enzyme CoV main protease (CoV M pro ; also known as 3C-like protease or 3CL pro ) catalyzes the most of the maturation cleavage events within the precursor polyproteins (Lee et al., 1991; Ziebuhr et al., 2000) .
It is a three-domain (domains I to III) cysteine protease with a chymotrypsin-like fold at the N terminus and a Cys-His catalytic located in a cleft between domains I and II (Anand et al., 2003; Yang et al., 2003) .
The CoV M pro has emerged as an attractive drug target for anti-coronaviral drug design because of its vital role in the maturation and processing of the replicase polyprotein (Xue et al., 2008; Ziebuhr et al., 2000) .
The enzyme has been targeted previously with antiviral phytochemicals (Gurung et al., 2020; Islam et al., 2020; Tahir ul Qamar et al., 2020) , marine natural products (Gentile et al., 2020) and FDA approved drugs (Kandeel and Al-Nazawi, 2020; Lobo-Galo et al., 2020) .
The lack of effective therapeutics against human coronaviral infections (Graham et al., 2013) and the high mortality rates due to the recent emergence of novel coronavirus (2019-nCoV) have necessitated the discovery of new vaccines or drugs.
In the present study, we have explored the possibility of the FDA approved drugs as potential inhibitors of M pro enzyme which will help in halting the virus replication and curtail the progression of the disease.
We have used molecular docking study to explore the binding interaction of the FDA approved drugs with three target enzymes (SARS-CoV M pro , SARS-CoV-2 M pro and MERS-CoV M pro ) and shortlisted suitable lead molecules for each target.
A set of 1390 chemical structures of FDA approved drugs were downloaded from ZINC 15 database (Sterling and Irwin, 2015) .
The 3D structures of the molecules in SDF format were retrieved and the molecules having only 2D structure available were processed into 3D structures using Open Babel version 2.4.1 software (O'Boyle et al., 2011 ) and subsequently energy-optimized using MMFF force field (Halgren, 1996) following our previously described protocol (Gurung et al., 2016) .
The molecules were prepared for docking using AutoDock Toos-1.5.6 by the addition of Gasteiger charges and hydrogen atoms and torsions for each molecule were optimally defined.
The structures of the compounds were saved in PDBQT format.
The three-dimensional structures of the enzyme targets-SARS-CoV-2 M pro (PDB ID: 6Y2F), SARS-CoV M pro (PDB ID: 3TNT) and MERS-CoV M pro (PDB ID: 5WKK) solved through high-resolution X-ray crystallographic technique at a resolution of 1.95 Å, 1.59 Å and 1.55 Å respectively were retrieved from Protein Data Bank (http://www.rcsb.org/).
Each target enzyme was prepared by removing the heteroatoms including ions, co-crystallized ligands (O6Y, G85 and AW4 corresponding to PDB IDs: 6Y2F, 3TNT and 5WKK respectively) and water molecules.
Further, an optimum number of polar hydrogen atoms and Kolmann charges were added to each protein target using AutoDock Toos-1.5.6 and the structures were saved in PDBQT format.
The binding affinity of each molecule along with the control inhibitors was evaluated against the three enzyme targets using molecular docking approach.
The binding sites for the The lowest binding energy score of each ligand was taken into account for studying their binding poses.
The molecular interactions (hydrogen bonds and hydrophobic interactions) between the target proteins and compounds were studied using LigPlot+ version 1.4.5 tool (Laskowski and Swindells, 2011) .
The binding affinities of a total of 1390 FDA approved drugs were tested against three enzyme targets-SARS-CoV-2 M pro , SARS-CoV M pro and MERS-CoV M pro .
The docking scores were benchmarked using three control inhibitors-α-ketoamide 13b (O6K), SG85 (G85) and GC813 (AW4).The top five lead molecules identified for SARS-CoV-2 M pro were Dihydroergotamine (ZINC000003978005), Avodart (ZINC000003932831), Irinotecan (ZINC000001612996), Ergotamine (ZINC000052955754) and Olysio (ZINC000164760756) with binding energies of -9.4 kcal/mol, -9.3 kcal/mol, -9.3 kcal/mol, -9.3 kcal/mol and -9.2 kcal/mol respectively ( Table 1 ).
The best lead molecule Dihydroergotamine (ZINC000003978005) binds to a pocket within the Cys145-His41 dyad and exhibits two hydrogen bonds with backbone nitrogen (N) atom of Gly143 and the binding pose further shows the participation of nine residues (Thr25, His41, Cys44, Met49, Asn142, Cys145, His164, Met165 and Glu166) in hydrophobic interactions with SARS-CoV-2 M pro ( Figure 1A) .
The control inhibitor, α-ketoamide 13b (O6K) scored binding energy of -6.9 kcal/mol with two hydrogen bonds-one with the side chain nitrogen (NE2) atom of His41 and the second one with the backbone nitrogen (N) atom of Glu166 and hydrophobic interactions via residues-Thr26, Met49, Cys145, His163, Met165, Pro168, Gln189 and Thr190 ( Figure 1B) .
For the second target-SARS-CoV M pro , the top 5 lead molecules identified were Saquinavir (ZINC000026985532), Ergotamine (ZINC000052955754), Nilotinib (ZINC000006716957), Raltegravir (ZINC000013831130) and Dihydroergotamine (ZINC000003978005) with binding energies of -9.6 kcal/mol, -9.5 kcal/mol, -9.4 kcal/mol, -9.2 kcal/mol and -9.2 kcal/mol respectively ( Table 2) .
The best lead molecule-Saquinavir (ZINC000026985532) exhibits four hydrogen bonds (Three bonds with the backbone nitrogen (N) and oxygen (O) atom of Gly143, Leu141 and Glu166 and one hydrogen bond with side-chain oxygen (OE1) atom of Gln189) and twelve residues (Thr25, Thr26, His41, Met49, Phe140, Asn142, Cys145, His164, Met165, Leu167, Thr190 and Gln192) contributing to hydrophobic interactions with the target enzyme ( Figure 2A) .
The control, inhibitor SG85 (G85) of SARS-CoV M pro exhibits binding energy of -7.9 kcal/mol with four hydrogen bonds (two hydrogen bonds with backbone N atoms of Cys145 and Glu166 and two hydrogen bonds with the side chain sulphur (SG) atom of Cys145 and side-chain oxygen (OE1) atom of Gln189) and hydrophobic interactions via sixteen residues (Thr25, Thr26, His41, Met49, Phe140, Leu141, Asn142, Gly143, Ser144, His163, His164, Met165, Asp187, Thr190, Ala191 and Gln192) ( Figure 2B) .
The top 5 leads for the third target-MERS-CoV M pro were found to be Ergotamine (ZINC000052955754), Ak-Fluor (ZINC000003860453), Avodart (ZINC000003932831), Dihydroergotamine (ZINC000003978005) and Saquinavir (ZINC000026664090) with binding energies of -9.6 kcal/mol, -9.4 kcal/mol, -9.3 kcal/mol, -9.3 kcal/mol, -9.3 kcal/mol and -8.1 kcal/mol respectively ( Table 3) .
The best lead molecule-Ergotamine (ZINC000052955754) binds to a region within the Cys148-His41 catalytic dyad pocket and establishes one hydrogen bond with side chain sulphur (SG) atom of Cys148 and hydrophobic interactions with twelve important residues (Met25, His41, Leu49, Asp190, Leu144, Cys145, Met168, Glu169, Lys191, Gln192, Val193 and His194) (Figure 3A) .
The control, inhibitor GC813 (AW4) shows binding (Bigal and Tepper, 2003; Tfelt-Hansen et al., 2000) .
Understanding the global health emergency and the immediate need for drugs and vaccines for the treatment of coronaviral infection, the present study is undertaken to identify promising inhibitors for main protease enzymes of coronaviruses through molecular docking approach.
Our study suggests that anti-migraine drugs such as Ergotamine (ZINC000052955754) and its derivative, Dihydroergotamine (ZINC000003978005) are the most potent lead molecules which can be taken for further studies in wet lab experimentations.
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key: cord-278174-znc99yos authors: Ramsey, Glenn title: Managing recalls and withdrawals of blood components date: 2004-01-31 journal: Transfusion Medicine Reviews DOI: 10.1016/j.tmrv.2003.10.005 sha: doc_id: 278174 cord_uid: znc99yos Abstract Donor centers are issuing a growing number of recalls and market withdrawals to hospital transfusion services about blood components.
More than 1 in 2,000 units were recalled in the late 1990s in the United States.
The most common reason for these notices from donor centers is postdonation donor information.
Most of these units had been transfused, and many present a “risk of a risk” (ie, a problem might have been present that might have affected the recipient).
A few regulations and standards address recalls in general terms, but transfusion services generally have wide discretion in the management of specific common recall problems.
The Food and Drug Administration (FDA) is now including posttransfusion evaluations in its guidelines for emerging infectious threats to the blood supply.
We suggest that hospital transfusion services should have standard operating procedures for managing recalls and that the hospital transfusion committee and the quality management program should provide local input or oversight.
Using the FDA’s categories of donor center biological product deviations, we provide recommendations to consider for when to notify the recipient’s physician, after postdonation information is received about a previously transfused blood component.
More study of this important everyday issue in transfusion medicine is highly desirable.
B LOOD COMPONENTS are regulated as drugs.
However, because they are derived directly from humans, they will always be subject to biological variation.
Their unit-by-unit production, testing, storage, distribution, and record keeping are also more complex than other medications, leading to further opportunities for deviations from the expected.
The US Food and Drug Administration's (FDA) current good manufacturing practices extend to after manufacturing so that, if problems are found with the finished drug, measures must be taken to correct the product or prevent consequences to patients if possible.
Over the past 10 to 15 years, the stricter application of these principles to blood components has led to a growing number of recalls and withdrawals by blood suppliers, as well as a concomitant increase in the numbers of notices sent to transfusion services about blood products they have received.
Furthermore, because platelets and red blood cells have a short shelf life and because hospitals do not keep a large reserve of blood components in storage, most of these notices about nonconforming blood products are often received after the units had been transfused.
In retrospect, many of these recalled units presented a "risk of a risk" (ie, that the problem might have affected the patient if it had actually been present but whether it was truly present is often unknown).
The lookback requirements for tracing units from donors later found to have human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections have been formalized by the FDA in rules and guidances.
1,2 (A revised rule for HIV and HCV lookbacks was proposed in 2000 but not finalized as of this writing.
3 ) HCV and HIV lookbacks have been discussed elsewhere 4,5 and will not be covered in depth here.
In contrast to HIV and HCV lookbacks, other types of notices about blood products have few specific formal rules or guidelines as to how they should be handled by the transfusion service.
The purpose of this review is to discuss the management of recalls and withdrawals of blood components.
"Recall is an effective method of removing or correcting consumer products that are in violation of laws administered by the Food and Drug Administration" (Title 21, Code of Federal Regulations (CFR), section 7.40 (21 CFR 7.40)).
The FDA classifies recalls into 3 categories, from the most serious, Class I, to the least serious, Class III ( Table 1 ).
All recalls under FDA jurisdiction are published on line in the weekly FDA Enforcement Report.
6 There is a lag time of weeks to months from the recall to publication, and the Enforcement Report does not say when the original problem occurred.
The FDA says recalls are "voluntary" by the manufacturer, although conducting recalls is required, and the FDA has the power to initiate recalls if the manufacturer does not act as required.
Recalls can include public warnings in serious situations (21 CFR 7.42).
In 1 blood component recall involving improper donor testing in a large centralized laboratory, several blood centers who had used that laboratory made community media announcements and advertisements notifying transfusion recipients to seek testing if desired.
7 Market withdrawals are also defined in Table 1 .
The FDA has stated that withdrawals because of problems beyond the control of the manufacturer may be classified as withdrawals.
For example, the US nationwide removal of Tylenol (McNeil-PPC, Ft Washington, PA) from stores because of fatal cyanide tampering in Chicago in 1982 was classified as a market withdrawal not a recall.
8 Most notices about blood components involving postdonation donor information are now categorized as market withdrawals.
Market withdrawals are not published by the FDA so their magnitude is unknown.
Ramsey and Sherman 9 analyzed recalls of blood components published by the FDA from 1990 through 1997.
During this 8-year period, recalls were announced for nearly a quarter-million blood components, comprising about 1 in 700 units available to hospitals.
Seventy-six percent were in Class III recalls, 24% were of Class II, and only 12 units were designated in Class I (because of viral or bacterial infection).
Three fourths of the recalled units involved incorrect or incomplete testing for syphilis or viral infection.
The next largest categories were for donors with reactive or previously reactive infectious disease tests, which involved 10% of recalled units.
Nearly 90% of recalled units were included in a small number (22) of large recalls of over 1,000 units each.
By 1998, the final year examined, published blood component recalls had shifted away from incorrect or incomplete infectious-disease testing (down to 20% of units) and toward donors with previously reactive infectious-disease tests (51% of units).
10 Also in contrast to 1990-1997, two thirds of the recalled units were included in a growing number of smaller recalls of under 1,000 Removal or correction of a marketed product that the FDA considers to be in violation of the law it administers and against which the agency would initiate legal action, eg, seizure.
Recall classification for use of, or exposure to, a violative product: Class I: Reasonable probability [of] serious adverse health consequences or death.
Class II: May cause temporary or medically reversible adverse health consequences or where the probability of serious adverse health consequences is remote.
Class III: Not likely to cause adverse health consequences.
Market withdrawal: Removal or correction of a distributed product which involves a minor violation that would not be subject to legal action by the FDA or which involves no violation, eg, normal stock rotation practices, routine equipment adjustments and repairs.
units each.
About 10,000 blood components were recalled in the 1998 Enforcement Reports.
When compared with the total annual blood components distributed in the United States, the risk of a unit being recalled after issue was about 1 in 2,000.
Another gauge of the types of problems found with blood components comes from the recent requirement for reporting biological product deviations (BPDs) to the FDA.
By definition, BPDs involve products that had been issued but that were later found to have safety, potency, or labeling problems.
BPDs should include all problems that lead to recalls or market withdrawals.
The FDA has summarized the first full fiscal year (FY 2002) of BPDs reported from licensed blood facilities.
11 Their statistics are presented as the number of reports, not the number of units involved, so the overall frequency of blood components involved is unknown.
One BPD for incorrect testing could involve many units, whereas BPDs for donor suitability are often reported on a unit-by-unit basis.
However, the categories and numbers of BPDs offer a useful FDA-defined framework for categorizing problems found in blood components currently.
Nearly 22,000 BPDs were reported from licensed facilities (excluding plasma centers) in FY 2002.
(Reports which actually did not need to be reported were excluded.)
Seventy-six percent were for postdonation information concerning donor high-risk behavior and history.
The most common categories here, in order, were travel to malaria and Creutzfeldt-Jacob risk areas, cancer, tattoos, disease or surgery, deferring medications, and intravenous drug use.
The next largest type of BPD, in 10% of cases, was for quality control and distribution problems, such as clotted or hemolyzed units or segments, inappropriate product release (eg, unacceptable quality control, outdated), incorrect temperature, and failure to quarantine after another problem was found.
The rest of the donor-center BPDs were in donor screening (6.0%), labeling (4.3%), routine testing (1.2%), component preparation (1.2%), collection (0.7%), miscellaneous (0.6%), donor deferral (0.2%), and viral testing (0.2%).
The most common items within each of these areas were as follows: donor screening, deferring history, but not deferred; labeling, incorrect autologous donor tag; routine testing, incorrect Rh typing; component preparation, incorrect temperature; collection, bacterial contamination; miscellaneous, HCV lookback deviation; donor deferral, previous deferral for history; and infectious disease testing, incorrect syphilis testing.
Incorrect infectious disease testing, previously the most common category of recalled units in the 1990s, was the least common category of BPDs in FY 2002.
This may reflect the current widespread use of large, dedicated centralized testing laboratories for donors.
The FDA has detailed regulations and support documents for the conduct of recalls by the manufacturer.
21 CFR 7.3 defines recalls, and 21 CFR 7.40-7.59 describe the manufacturer's obligations and the FDA's processes for monitoring and assessing recalls.
Two publications by the FDA's Office of Regulatory Affairs provide details for their inspectors about how to inspect recall operations: the Investigation Operations Manual, Chapter 8, and the Regulatory Procedures Manual, Chapter 7.
12,13 These 2 publications are on line at www.fda.gov/ora.
The FDA may conduct effectiveness checks, which are follow-up surveys of consignees such as transfusion services, to verify that recalls are carried out appropriately by the manufacturer.
In contrast, the FDA provides much less general information about the response to recalls.
One line in the CFR is addressed to consignees: "Responsibility of recipient.
Consignees that receive a recall communication should immediately carry out the instructions set forth by the recalling firm and, where necessary, extend the recall to its consignees in accordance with .
.
.
this section (21 CFR 7.49 [d])."
Therefore, if the hospital transfusion service has shipped the recalled component, or part of it, to another facility, it should conduct a recall to the second facility.
For example, some hospital transfusion services send source plasma to a manufacturer so the source plasma buyer should be notified if the original unit is recalled.
If the transfusion service is notified about an unsuitable product, but then issues it inadvertently, then a BPD report would be required.
HIV and HCV lookbacks have been referred to previously (Table 2) .
[1] [2] [3] [4] [5] Donors with reactive tests for hepatitis B virus (HBV) and human T-cell lymphotropic viruses (HTLV) I and II were ad-dressed by the FDA in a 1996 recommendation 14 (now listed under Blood Memos) and a 1997 guidance for HTLV.
15 The FDA recommended withdrawing in-dated components from donors with HBV and HTLV markers but stated that they were not recommending consignee notification for the purpose of recipient notification.
In our own practice, we perform lookback on units from donors with confirmed hepatitis B surface antigen (HBsAg) or anti-HTLV (see Management of Specific Problems), but this is not required by the FDA.
Recent FDA guidances about emerging infection issues have included provisions about posttransfusion actions.
In the January 2002 guidance 16 on Creutzfeldt-Jakob disease (CJD), the FDA stated that notifications about donors deferred for travel, bovine insulin, United Kingdom transfusion, or a single family member with CJD were intended only for product removal, and not for notification of recipients.
For other more direct donor connections to CJD, including actual donor CJD subsequent to transfusion, "consignee notification could enable the consignee to inform the physician .
.
.
so that recipient tracing and medically appropriate notification and counseling may be performed at the discretion of health care providers."
There is an ongoing study by the US Centers for Disease Control and Prevention (CDC) and the National Blood Data Resource Center to investigate transfusion recipients of blood from subsequently diagnosed CJD patients.
17 In this approved study, the recipients are not notified, but national deaths are monitored to see whether any of the recipients die of CJD.
As of August 2002, 331 transfusion recipients with 1,325 person years of follow-up had been studied.
A similar study is being conducted in Great Britain for recipients of blood from donors with later variant CJD (vCJD).
18 No secondary cases of CJD or vCJD have been reported to date.
If a notice were received in the United States about a blood component from a donor with later CJD, the CDC or the National Blood Data Resource Center should be contacted.
In the May 2003 West Nile virus (WNV) guidance, 19 if a donor has a medical diagnosis of WNV, then other units from Ϫ14 days before to ϩ28 days after the onset of illness should be traced for consideration of notifying the recipients' physicians.
If the donor is the suspected likely source of another WNV transfusion case, then other units from that donor collected from Ϫ28 days to ϩ28 days from the infectious donation should also be traced and the recipients' physicians notified.
"However, in cases when a donor is potentially associated with a case of transmission of WNV, but the epidemiological investigation has not established the specific donor as a likely source of transmission of WNV, we are not recommending notification of the transfusion service."
This is slightly misworded because units from recent donors associated with a transfused WNV patient should be sought for precautionary quarantine and retrieval from the transfusion service, but the implication is that the recipients' physicians need not be notified if the donor is not the likely source.
WNV nucleic acid testing (NAT) began in the US and Canada in the summer of 2003.
When a donor tests reactive by WNV NAT, the FDA has not specified at this writing whether to use a 14-day lookback period (as per donor WNV illness) or a 28-day period (as per donor transmission of WNV) for recent donations.
The December 2002 guidance on smallpox vaccination and blood donation 20 addressed postvaccination blood collections.
If a donor should have been ineligible, but his/her units already have been transfused, then "we recommend that medical directors consider the need for prompt record tracing and, as appropriate, notification of the treating physicians or notification of prior recipients of the affected blood and blood components previously collected from that donor."
The April 2003 FDA guidance 21 on severe acute respiratory syndrome (SARS) gave recommendations for lookback investigation.
If a blood product has been transfused from a donor who should have been ineligible at the time of donation, then "we recommend that the establishments consider notifying the treating physician of those recipients about the post donation information, including whether the donor developed suspected SARS."
Donors are deferred for 28 days after recovering from suspected SARS or for 14 days after exposure to a person with SARS or travel to SARS-risk areas.
However, the guidance states that if the donor is symptom free for more than 14 days after exposure, then product retrieval and quarantine (and thus presumably notification of the treating physician) are not necessary.
In the June 2003 draft guidance on donor syph-ilis testing, 22 lookback, quarantine, and consignee notification are not recommended for previous units from donors with later syphilis or a confirmed syphilis test.
The recent FDA draft guidance on xenotransplantation 23 and on anthrax 24 call for withdrawal of blood components inadvertently collected from donors with these unusual exposures.
The anthrax guidance has recommendations on when to notify the recipient's physician after transfusion of a suspect unit.
In the 22nd edition of the Standards of the American Association of Blood Banks (AABB), effective November 2003, chapter 7 is on deviations, nonconformances, and complications.
25 Standard 7.0 requires policies, processes, procedures and defined responsibility for detecting, investigating, and reviewing deviations.
Standard 7.1 and its subsections call for nonconforming products to be evaluated, traced, segregated if still present, and prevented from unintended use.
Blood banks and transfusion services must have processes for identification, quarantine, retrieval, and recall.
Nonconforming products that already have been released must be evaluated for quality, and when quality may have been affected, the nonconformance shall be reported to the customer.
(The "customer" is defined elsewhere as the receiver of a product or service, either another organization or another department within the same organization.
In this context of nonconforming products, the "customer" does not refer to the patient who received the product.)
Records of product nonconformances and actions about them must be maintained for 5 years (reference standards 6.2A and 6.2C).
The transfusion medicine checklist of the Laboratory Accreditation Program of the College of American Pathologists (December 2002 edition) 26 has 2 questions touching on some aspects of recalls and notifications.
TRM.42120 asks if there is a procedure to identify and quarantine all previous components from donors who now test repeatedly reactive in viral marker screening tests.
TRM.42170 asks for a "procedure consistent with [Medicare] and FDA regulations/guidelines for notification and counseling of recipients who have been transfused with a potentially infectious blood component."
The commentary for this question refers to federal requirements for notifying recipients about subsequent confirmed positive infections in their donors.
The main intent of the question is to require HIV and HCV lookback.
However, the mention of guidelines in the question may be construed to include other recent FDA guidelines as discussed earlier.
Some hospitals collect blood products, and some blood collection centers have transfusion services.
The previously mentioned regulations and standards apply to those facilities as well (ie, notices should be transmitted from the collection arm to the transfusion arm of the same establishment when necessary).
Within the extant regulations and standards summarized previously, the transfusion service has broad latitude on how to manage recalls and notifications from blood suppliers, other than HIV and HCV lookback.
The remainder of this article offers recommendations based on our experience.
However, these are only recommendations, and others may choose to use different approaches.
Given the paucity of literature about this important everyday area of activity in transfusion medicine, we hope the following will be helpful in providing a framework for others to organize their programs as suited for their hospitals.
Future study, analysis, and discourse on the management and outcomes of recalls and notifications will be very helpful.
In particular, the yield of problem investigations and the medical benefit of these notices for transfusion recipients deserve more examination.
At a minimum, we would suggest that recall management processes include the following key elements: 1.
Have a standard operating procedure.
However one chooses to manage recalls and notifications, and in however much detail desired, a procedure is a prerequisite for instructing staff in these key elements.
2.
Act immediately to quarantine and discard, or return, blood components as instructed by the supplier.
Time is of the essence when a notice is received.
Immediate action should be taken to quarantine the unit and prevent release.
Is a recalled unit of plasma being thawed in the waterbath?
In case a large number of units is involved, the transfusion service staff should have round-the-clock ac-cess to facsimile or secure electronic mail to facilitate transfer of information and prevent transcription errors from verbal messages.
For laboratories with blood bank computer systems, both computer and physical quarantine must be done, although computer quarantine may be done first to expedite prompt blockage of issue.
As noted previously, if a unit is erroneously released after a notice is received to quarantine it, then a biological product deviation report must be sent to the FDA.
3. Review and determine the medical implications of units already transfused.
This is further discussed later.
4.
Keep records of all notices and actions as required (eg, 5 years in AABB standards).
25 Telephone instructions should be logged and obeyed, but also documented pending written follow-up.
Record systems should include the capability to track investigations in progress.
Transfusion services may want to consider means to search recall records by unit number or patient or the ability to tag the unit or patient laboratory record that a recall has occurred.
Large hospitals may find a confidential database useful.
Transfusion services may wish to review their general strategies for oversight, management, and record keeping with the hospital's risk management and/or legal counsel.
Although most of these problems occur before the hospital receives the units, there could be potential medicolegal implications for the hospital and the physician.
For example, it may be advisable to consider these investigations as a subcategory of the hospital's overall incident management program for quality improvement or at least to bring serious problems to this forum.
The transfusion committee or its local equivalent may wish to provide general oversight for the recall management process.
There are several advantages to performing recall investigations under the aegis of the transfusion committee.
This educates key physicians in the range of problems found with blood components after transfusion.
It provides the medical staff's representatives a forum to review and approve the general and specific features of the procedure.
The transfusion committee also provides a logical venue to bring all or selected recalls into the hospital quality improvement program.
Some other resources of expertise in the hospital may be helpful in certain problems.
The infection control office can provide advice on transfusions, which may have posed a serious infection risk in hindsight.
Potentially, this consultation could include immediate measures such as baseline patient infectious disease testing (eg, a donor calls back to report recent previously unsuspected exposure to HIV) or antimicrobial therapy (eg, a platelet culture becomes positive after the product was already transfused).
For perplexing conundrums in posttransfusion problems, the hospital ethics committee might provide a useful forum for discussion.
The hospital public relations office should be informed when a problem could be of concern to the news media and the public, such as a large recall of blood products in the community.
Many transfusion recipients have died of their underlying illness by the time a notice arrives about one of their blood components.
In an HIV lookback, their next of kin must be notified.
However, for other problems, no further investigation is needed.
Table 3 gives suggestions for whether to inform a recipient's physician about a problem transfusion.
The list of problems is adapted from the FDA's categories of donor-center BPDs, with some additions.
Our general approach is that if a blood component might have posed a tangible infectious or other risk, then the patient's physician should evaluate whether the patient may have been affected.
On the other hand, if the problem with the product did not pose a tangible risk to the patient, then the transfusion service physician, with the oversight of the transfusion committee if desired, can exercise informed medical judgment to not notify the recipient's physician.
Blood suppliers should provide adequate information for the transfusion service to evaluate the problem and counsel the physician if needed.
Without violating the donor's confidentiality, the transfusion service may seek further information from the blood supplier if the first notice is insufficient for a decision.
Many physicians are not familiar with the details of when and why blood donors should be deferred.
When the patient's physician is informed about a (Table 4 and text).
HIV or HCV nucleic acid tests (NAT) have short seroconversion windows, but postdonation NATs have not been incorporated yet into FDA rules and guidances on HIV or HCV lookbacks.
[1] [2] [3] problem with a nonconforming blood component, some background information is often useful.
For recurring notices such as malaria-area travel, a form letter may be convenient.
For most routine notices, we have not required follow-up information from the physician.
However, for sensitive issues, the transfusion service physician may offer assistance in patient counseling if desired.
When there is concern about the possibility of infection risk from the donor, testing of the donor and/or patient may be indicated.
When a donor is deferred for a risk factor before donation, no testing is done at that time.
From the collection facility's standpoint, this may discourage test seeking by ineligible donors.
Unfortunately for the transfusion services, which have previously received blood components from that donor, the current infection status of the donor is thus left unknown.
If the donor has been tested since the donation in question, the last date of testing should be included in the notification or be sought by the transfusion service.
In serious donor risks, such as known HIV exposure, the transfusion service may ask the collection facility to seek donor testing if feasible.
Seroconversion window information is helpful for counseling and donor testing after exposure or for recipients after transfusion.
If the donor has tested negative after the seroconversion period of the test in question has elapsed, then donor infection at the time of the donation can be ruled out.
Table 4 shows seroconversion window periods for viral tests required in blood donors.
The CDC recommendation for HIV antibody testing after needlestick exposure is 6 months, 29 that is more conservative than the table figures.
In the 1996 HIV lookback rule and the 2000 proposed look-back rule for HIV and HCV, 1,3 the FDA required 12 months before a negative antibody test to rule out the need for lookback in prior donations.
NAT for HIV and HCV has much shorter window periods than antibody testing.
The CDC recommends 4 to 6 weeks of follow-up testing for HCV RNA after needlestick exposure.
29 NAT has not yet been factored into FDA lookback rules and guidances.
Blood centers have greatly reduced infectious disease testing errors and problems that predominated in recalls of the early and mid-1990s.
Today's challenge is the donor who does not reveal a deferring risk factor or condition.
An anonymous survey of blood donors for risk behaviors revealed that 1.9% had a deferrable risk at the time of their donation (1.7% after testing and confidential unit exclusion).
30 Efforts have been made to reshape screening questions to improve their comprehension by donors.
31 Computer-assisted interviews may offer donors a more comfortable way to reveal risk factors.
32 Because many current donor risk factors are based on international travel, another area for consideration is making information more widely accessible for travelers and their physicians about when not to donate blood.
For example, the CDC's key international travel health publication, the "Yellow Book," does not tell physicians and travelers about when to avoid blood donation, and for how long.
33 Likewise, when blood donor deferrals began for SARS-area travel, this was not included in CDC information pages for travelers.
34 More publicity about the consequences of travel on blood donation might reduce recall rates for geographic donor risks.
In today's regulatory climate, hospital transfusion services receive numerous recalls and market withdrawals from their blood suppliers.
Hospitals should have procedures for managing the quarantine, medical evaluation, and records of these recalls.
The transfusion committee may provide oversight for local preferences about when to inform the recipient's physician.
More study of the medical importance of recalls for transfused patients is needed.
Because the predominant reason for notices about blood products is postdonation information about the donor, this is an important area for quality improvement by blood suppliers.
New mea-sures to improve donor understanding and communication about deferring information could help reduce blood component recalls and withdrawals.
Guidance for industry: current good manufacturing practice for blood and blood components: (1) quarantine and disposition of units from prior collections from donors with repeatedly reactive screening test for antibody to hepatitis C virus (anti-HCV); (2) supplemental testing, and the notification of consignees and blood recipients of donor test results for anti-HCV Risk of human immunodeficiency virus (HIV) transmission by blood products before the implementation of HIV antibody screening Food and Drug Administration (weekly) Nordenberg T: Recalls: FDA, industry cooperate to protect consumers Blood component recalls in the United States Recommendations for the quarantine and disposition of units from prior collections from donors with repeatedly reactive screening tests for hepatitis B virus (HBV) Guidance for industry: revised preventive measures to reduce the possible risk of transmission of Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-Jakob disease (vCJD) by blood and blood products abstr) 19.
Guidance for industry: Revised recommendations for the assessment of donor suitability and blood and blood product safety in cases of known or suspected West Nile virus infection Draft guidance for industry: precautionary measures to reduce the possible risk of transmission of zoonoses by blood and blood products from xenotransplantation product recipients and their intimate contacts Updated US Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis Blooddonor perceptions of health history screening with a computerassisted self-administered interview Available at: www.cdc.gov/ncidod/sars/travel_advice.
htm
key: cord-276460-nmugz0oh authors: Katz, Louis M.; Cumming, Paul D.; Wallace, Edward L. title: Computer-Based Blood Donor Screening: A Status Report date: 2007-01-31 journal: Transfusion Medicine Reviews DOI: 10.1016/j.tmrv.2006.08.001 sha: doc_id: 276460 cord_uid: nmugz0oh There is a substantial literature suggesting that computer-assisted interviewing has advantages over face-to-face and written self-administration of interviews in venues eliciting sensitive information similar to that sought in blood donor history screening.
We review some of the recent developments in blood donor history screening, the evidence suggesting that automated interviews should be useful, and the experience to date using computer interviews for blood donation.
These data suggest that automated computer-assisted interviewing increases the elicitation of behaviors associated with the risk of transfusion-transmissible infection in donors, improves donor and staff satisfaction, and reduces errors and omissions that frequently accompany traditional interviewing methods.
Food and Drug Administration–cleared systems for computer-assisted self-interview of blood donors are briefly described.
Louis M. Katz, Paul D. Cumming, and Edward L. Wallace There is a substantial literature suggesting that computer-assisted interviewing has advantages over face-to-face and written self-administration of interviews in venues eliciting sensitive information similar to that sought in blood donor history screening.
We review some of the recent developments in blood donor history screening, the evidence suggesting that automated interviews should be useful, and the experience to date using computer interviews for blood donation.
These data suggest that automated computer-assisted interviewing increases the elicitation of behaviors associated with the risk of transfusion-transmissible infection in donors, improves donor and staff satisfaction, and reduces errors and omissions that frequently accompany traditional interviewing methods.
Food and Drug Administration-cleared systems for computer-assisted self-interview of blood donors are briefly described.
A 2007 Elsevier Inc. All rights reserved.
A SSURANCE OF A safe supply of allogeneic blood and blood products is the primary driver of activity in blood services.
Interventions with extremely high cost-benefit ratios are now common.
For example, introduction of nucleic acid testing in minipools for HIV and HCV in the late 1990s reduced the risks of transmission of these pathogens in transfused, tested allogeneic blood to 1:1.8 million and 1:1.6 million, respectively, leaving only small test-negative window period risks for these agents.
1 Small infectious risks remain, as evidenced by 2 blood recipients recently infected with HIV from a nucleic acid-tested whole blood donation.
2 There are, in addition, a host of other infections-protozoan, bacterial, viral, and prion-which are transfusion-transmissible for which no tests are currently available.
3 Protection of the allogeneic blood supply from traditional transfusion-transmissible diseases (TTD); from emerging or reemerging TTDs; and particularly from the window period residual risks for HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) depends on the effectiveness not only of laboratory screening of volunteer blood donors but also on efforts to minimize the recruitment and phlebotomy of high-risk donors.
High-risk volunteer donors are persons who (i) have a history of TTDs or have engaged in behaviors, especially sexual practices or parenteral drug use, known to be associated with TTDs and/or have close contact with others with TTDs; (ii) have traveled to or resided in locations where TTDs are prevalent: or (iii) have risky health histories, for example, prior infection, recent transfusion, or receipt of plasma derivatives associated with an appreciable incidence of infection.
Screening is the process by which blood collecting organizations identify and defer high-risk donors to avoid introducing TTDs into the allogeneic blood supply.
It is a multistep process involving recruitment of safe donors, provision of up-to-date donor education materials, checking for in-force prior deferrals, a mini-physical examination with elements focused on stigmata of parenteral drug use, and a health history interview preceding phlebotomy.
Like laboratory testing, donor history screening has improved substantially in recent years because of efforts by the blood community to recruit, identify, and retain lowrisk donors and by more active federal involvement and regulation of the whole blood and blood products collection and distribution process.
As a result, the prevalence of HIV, HBV, and HCV infections among first-time blood donors are 3%, 14% and 13%, respectively, of those in the US population.
4 There remains room for improvement, as evidenced by the finding that 2% of volunteer whole blood donors reported deferrable high-risk behaviors on an anonymous postdonation survey.
5 Forty years ago as many as 1 in 4 transfused patients developed viral hepatitis.
In the late 1960s, some 69% of all open heart surgery patients at the National Institutes of Health Clinical Center acquired hepatitis from transfused blood.
6 Introduction of the first generation hepatitis B surface antigen (HBsAg) test in 1970 reduced the rate to 7%.
6 Movement toward an all-volunteer whole blood supply in the 1970s further increased public confidence in the safety of allogeneic transfusion.
7 The transfusion-AIDS crisis in the early and mid 1980s, however, destroyed that public trust.
Although new or additional blood tests for HIV, HBV, and HCV introduced in the mid to late 1980s and early 1990s constituted major advances in protection of the allogeneic supply, all were traditional serologic tests with substantial seronegative window periods associated with residual risk of transfusionassociated infection (~1:225 000; 1:50 000, and 1:3 300 for HIV, HBV, and HCV, respectively).
8 As public confidence in the safety of the allogeneic blood supply fell in the early 1980s, in the absence of effective laboratory screening for HIV and posttransfusion non-A and non-B hepatitis, the transfusion medicine community began serious efforts to recruit and screen low-risk donors using nonlaboratory methods.
By 1983, evidence from the Centers for Disease Control that HIV was a TTD convinced the American Association of Blood Banks (AABB), American Red Cross, and the Council of Community Blood Centers (now America's Blood Centers) to initiate education and questioning of blood donors about their behaviors and experiences that were epidemiologically associated with HIV infection.
Gay organizations were asked to discourage members from donating.
Donors were given information about recognized risks and asked to self-defer if they self-identified as having risk 9 (men having sexual contact with multiple male partners, IV drug use, Haitian origin, hemophilia treatment with factor concentrates manufactured from large paid donor pools, and sexual contact with persons with these characteristics).
For reasons of assumed donor sensitivity and uncertainty about the magnitude of the AIDS threat, the 3 organizations initially did not recommend using direct questions regarding donors' sexual practices.
9 In 1985, after introduction of the first serologic test for HIV, some blood service organizations began direct questioning of male donors for male-to-male sexual activity, whereas other blood-collecting organizations introduced the test without amending their health history screening.
9 Direct questioning of donors for HIV risk was not formally required in the United States until February 1991.
Throughout the early AIDS era, the US Food and Drug Administration (FDA) worked cooperatively with the principal organizations and professional associations of the whole blood and blood products sector to regulate the transfusion medicine community through consensus.
This changed abruptly in 1992 when the FDA imposed strict regulations (current good manufacturing practices [cGMP]), similar to those required of pharmaceutical manufacturers, on the blood collecting community.
Among cGMPs was development of health history questionnaires including FDA-mandated questions on high-risk behaviors to be asked of every donor at every donation.
A primary goal of appropriate donor recruitment and screening is to select and retain donors whose characteristics predict a low incidence and prevalence of TTDs.
Retrospective studies of blood donors infected with HIV or hepatitis viruses have repeatedly shown many to have recognizable behavioral risks, which, when disclosed, justify their deferral.
10-12 Within the past several years, effort has been expended to standardize the donor history questionnaire (DHQ) and to secure approval for it from the FDA.
American Association of Blood Banks, which authored the first uniform DHQ (UDHQ) in 1993 developed, through its Interorganizational UDHQ Task Force, new long and short form UDHQs and submitted them to the FDA for approval.
In 2004, the FDA approved the new long form UDHQ as a substitute for the old UDHQ or for any of the many customized health history questionnaires previously approved by the FDA for use in individual collection facilities.
13 To date, however, the FDA has withheld approval for the new short form UDHQ, intended for frequent repeat donors, pending review of findings from ongoing studies of its performance.
Current health history questionnaires used in the blood community include facility-specific DHQs and a mix of AABB's old and new UDHQ.
All, as a condition of facility licensure, are approved by the FDA and supported by standard operating procedures (SOPs) that describe how each step in the donor interview is to be conducted and how various responses by donors to each question are to be treated, including criteria for acceptance or temporary and indefinite (ie, permanent) deferrals.
In recent years, the blood community has used donor education materials and the health history questionnaire as the initial means of protecting the blood supply from emerging and potential TTDs, for which tests are not available.
For example, in 2002, the FDA mandated inclusion of additional questions intended to identify donors with past activities, residence, or travel suggesting the possibility of exposure to variant Creutzfeldt-Jakob Disease.
In 2003, more questions were added about exposure to smallpox vaccine (vaccinia), severe acute respiratory syndrome, and West Nile Virus.
14 As a result, the number of questions on the original AABB UDHQ increased to 61.
The new long form UDHQ consists of 49 questions that were redesigned with input from cognitive scientists, using focus groups and cognitive interviews, to improve the ability to elicit the targeted information.
Historically blood-collecting organizations have administered the DHQ by having their staff ask each donor the questions in a face-to-face interview (FTFI) or using written donor self-administered questionnaires (WSAQ).
Eligibility or deferral is determined based on the responses to each question.
Staff is guided by current SOPs or, when directed by organizational SOPs, by consultation with the facility medical director or a trained designee.
More recently, some organizations have adopted various computer-assisted donor interviews ranging from staff administered questionnaires, with the staff entering donor responses into a system, to more complex computer-assisted donor self-interviewing (CASI) systems using text, often with combinations of audio and/or visual prompts.
This array of methods to administer the donor health history has prompted questions about their comparative effectiveness and efficiency.
When selecting a mode, blood-collecting organizations have the task of evaluating 3 considerations: safety, efficiency, and costs.
Each screening approach has certain advantages and disadvantages in each of the 3 dimensions.
Of the 3 modes, FTFI, WSAQ, and CASI, the latter varies the most.
In its simplest form, CASI is conducted on a stand-alone computer with text questions displayed for reading on the monitor and answers entered by the respondent using a keyboard, keypad, or mouse.
Audio presentation of the interview questions can accompany eye-readable text to improve understanding over text only.
Further enhancements of the basic CASI system include pictures illustrative of the content of the question; response inputs via touch screen; inclusion of back, help, and skip commands to improve accuracy and completeness of responses, staff review modules for assessment of completed DHQs, direct printing of the DHQ with provision for signatures, and electronic transfer of final interview data to other associated computer systems.
Survey interviewing is a common technique in behavioral research.
Consequently, behavioral researchers have a long-standing interest in evaluating the ways these interviews are conducted, especially their effects on the accuracy of subject responses to interview questions.
Since the late 1960s, survey researchers have investigated the relative effectiveness of various forms of computerassisted interviewing, primarily CASI or audio CASI (A-CASI), as alternatives to FTFI and WSAQ for eliciting accurate responses to interview questions.
In 1996, Weisband and Kiesler 15 reported a meta-analysis of 39 comparative studies from the social sciences, computer sciences, and medical literature between 1969 and 1994.
They found that computer-assisted interviewing was significantly more effective than FTFI and WSAQ, especially when the information sought was judged socially sensitive or stigmatizing.
In 1998, Turner et al, 16 studying adolescent high-risk sexual, drug use, and violent behaviors, reported them to be admitted up to 17 times more frequently when subjects were interviewed via A-CASI than by WSAQ.
The following year, Richman et al 17 reported a metaanalysis of 61 comparative studies conducted between 1967 and 1997.
A key finding of this study was that bcomputer instruments reduced social desirability distortion (ie, increased frank, accurate responses) when.
.
.used as a substitute for face-toface interviewing, particularly when.
.
.asking respondents to reveal highly sensitive behavior, such as whether they used illegal drugs or engaged in risky sexual behavior.Q Computer-assisted interviewing was significantly more effective than WSAQ when computer respondents were (i) assured anonymity, (ii) alone, and (iii) allowed to skip and backtrack on the computer.
More recent literature supports these findings.
Cooley et al 18 reports that using touch-screen A-CASI (AT-CASI) to obtain sensitive information on sexual and illicit drug behavior from patients 15 to 39 years of age at a sexually transmitted disease clinic was more effective than traditional interview techniques.
Johnson et al, 19 in a recent study comparing the effectiveness of CASI with WSAQ to elicit information on highrisk sexual behavior in a general population, reported similar findings.
A few studies have actually involved blood donors.
In 1992, Locke et al 20 reported a crossover study that compared the effectiveness of the traditional written questionnaire plus FTFI with a CASI system using the standard American Red Cross health history questionnaire.
Among 272 donors, the CASI system identified 12 reporting behaviors associated with HIV risk or symptoms compatible with AIDS, none of whom were identified when interviewed first by written questionnaire and FTFI.
In 1993, the American Institutes for Research (AIR) conducted an extensive, complex cross-over study, contracted for by the FDA, comparing the effectiveness of existing WSAQ and/or FTFI interviewing with an AT-CASI system developed by AIR for donor interviewing.
21 Of 7015 donors in the study, 27 (1.09%) of 2468 donors interviewed via computer were deferred for risky behavior, whereas only 23 of 4547 interviewed traditionally (0.51%) were rejected for similar reasons ( P = .00553).
The authors conclude that use of the computer alone would have identified more deferrals than the comparator interviews.
FDA officials deemed the study inconclusive because study procedures did not permit determination of whether the observed outcome was the result of greater sensitivity of AT-CASI to identify high-risk behavior or lower specificity.
More recently, Sellors, 22 in a randomized cross-over study of donor deferral rates conducted at 133 blood clinics in Canada, compared a handheld computerized health history questionnaire with a written questionnaire.
They found that 43.7% of deferrable donors were identified by the CASI but not by the written questionnaire.
In 2005, Katz et al 23 reported results of a before-and-after study of donor deferrals for high-risk behaviors among 2739 first-time blood donors at the Mississippi Valley Regional Blood Center.
They found that only 1 of 890 (1.12 per 1000) such donors interviewed by FTFI between 2000 and 2001 was deferred, whereas 19 of 1849 (10.28 per 1000) donors interviewed using an audiovisual touch-screen CASI (AVT-CASI) between 2001 and 2002 were deferred, a significant difference ( P = .017; odds ratio, 9.15; 95% confidence interval, 1.3-183.8).
In a similar study using essentially the same system at another regional blood center, Cumming et al 24 found that with a combination of FTFI and WSAQ, the rate of deferrals of first-time donors for high-risk behavior was 5.8 per 1000, whereas after installation of an AVT-CASI system the rate increased to 11.2 per 1000, a 93% increase.
These data demonstrate that CASI is more effective than FTFI in eliciting positive responses to sensitive questions.
It also appears CASI is more effective than WSAQ in this respect, although less so than when compared with FTFI.
Behavioral researchers and others infer that the difference between FTFI and the other 2 modes of interviewing arises in part from privacy differences and from reduction of social desirability distortion (the tendency of respondents to provide information biased toward what they perceive as the socially accepted or desirable answer).
15,17 Because CASI and WSAQ are quite similar in the privacy provided each interview subject, these researchers offered the following possible reasons why people disclose more when using a computer: computer interfaces create in respondents an inattention to audience; they provide for immersion in the immediate task along with invulnerability to criticism; they foster an impression that responses bdisappearQ into the computer; they give a sense of comfort leading to a less wary attitude; and there are other misattributions that cause respondents to be careless about their responses.
Whatever the underlying mechanism(s), the weight of available evidence indicates that CASI is more effective than WSAQ in eliciting accurate answers to sensitive questions.
To what extent does the observed increased deferral of donors admitting to high-risk behavior by CASI actually protect blood safety?
This depends mainly on the incidence (early, test-negative infections) of TTDs among donors admitting such behavior to the computer but withholding the information when using other interview formats.
From studies by Petersen and Doll, 10 Conry-Cantilena et al, 11 and Murphy et al, 12 we can conclude that the elicited behaviors are useful indices of TTD risk, so the donors should be deferred.
The studies that would be required to actually demonstrate a decrease in risk to transfusion recipients are too large to accomplish and unlikely to be undertaken.
CASI systems have other characteristics that enhance their appeal for blood donor screening: (i) increased donor and staff satisfaction, (ii) potentially improved understanding by less literate donors when audio and/or visual prompts are incorporated, (iii) reduced donor and staff errors, and (iv) reduced staff time.
Blood bankers are acutely concerned with donor reactions to the screening interview.
When interviewing of blood donors on their sexual, drug, and other socially sensitive behaviors was introduced, the intrusive nature and complexity of information sought during the interview provoked one blood banker to label the interview a bdonor interrogation.Q 25 To date, all studies of CASI systems for donor screening have included surveys assessing donor reactions to the interview format.
Locke et al, 20 in their early study, reported that although donors believed CASI interviewing was as effective as FTFI for screening, they found it more private (39% vs 7%) and more likely (61%) to elicit honest responses.
The AIR study reported that 53.5% of the donors preferred CASI for interviewing, whereas 64.4% felt it would yield more honest answers.
21 More recently, Zuck et al 26 reported that 40% of the donors participating in a pilot study of an early version of an AVT-CASI system found the computer preferable, whereas 36% preferred the FTFI mode.
Sixty-seven percent of donors believed donors would be more truthful when interviewed via AVT-CASI, although only 7% thought FTFI would yield more truthful information.
Katz et al 23 reported that 68% of donors preferred the CASI system, whereas only 10% preferred the FTFI.
In addition, 92% of donors were very satisfied with the privacy provided by the system, and 68% felt it would elicit more truthful answers.
One study, Sanchez et al, 27 has questioned increased donor satisfaction with and preference for CASI interviewing.
Thirteen percent of the blood donors they surveyed about their hypothetical reaction to CASI screening stated such a system would either discourage them from donating (5.2%) or that they were unsure (8.0%).
None of the donors in the study had actually used CASI screening, prompting Gillespie, 28 in an editorial commenting on the studies of Sanchez et al and Katz et al, to emphasize the importance of differentiating real and projected behavior and the importance of studying actual behavior in any scientific field.
From these and similar studies, 29-31 it is reasonable to conclude that most donors and interviewees are more satisfied with CASI than FTFI.
Moreover, as Katz et al 23 demonstrated, blood center staffs believe AVT-CASI is faster, personally more satisfying, and less likely to induce staff errors.
Comprehension of questions on the screening interview is essential.
When donors fail to understand a question and answer it erroneously, the interview has failed.
The ability of donors to understand medical and health-related questions is of particular importance for WSAQ and CASI because both assume donor literacy.
There is substantial evidence that this assumption is overly optimistic.
Wilson, 32 in a summary of findings from 10 studies on literacy and health, concluded that most adults with poor literacy are adept at disguising their inability and disinclined to ask for help.
The author cites the 1993 National Adult Literacy Study, which found that the average American reads at the eighth to ninth grade level, whereas most medical information found on the Internet is written at the 12th grade level.
In an analysis of medical literacy among a randomly selected sample of 18 to 45 old-year-old adults in Baltimore, Al-Tayib 33 found that 28% had a literacy level of grade 8 or less and 12% of grade 6 or less.
Eighteen percent of the study's participants with some college or a 2-year degree (sometimes cited as the prototypic blood donor) were reading at an eighth grade level when responding to a WSAQ on drug use, sexual behavior, and sexually transmitted diseases, which are topics also queried among donors.
4Information on in this table was provided by vendors with confirmation by end users of configurations in use, except for the Haemonetics system, which was not yet in use at the time of compiling the information.
Thanks to Paul Sullivan at MVRBC, Judith Woll at Community Blood CenterDayton, and Susan Rossman at Gulf Coast Regional Blood Center for their bin-useQ system descriptions.
The recent revision of AABB's UDHQ is the prime example of effort by the blood services community to adapt donor screening to reasonable levels of adult literacy.
After focus groups and cognitive interviews, many medical terms in the original UDHQ were eliminated or modified in the new questionnaire to improve donor comprehension.
The potentially iterative nature of a FTFI might seem ideal to assess and reinforce comprehension, but that depends on adequate staff sensitivity, intuition, patience, and training to perceive and remediate donor misunderstanding of the content and intent of questions.
CASI systems can include audio to mitigate literacy issues and/or bHelpQ and bSkipQ alternative responses along with the usual bYesQ and bNoQ choices for each question providing donors with assistance when needed.
Some CASI systems include visual prompts illustrating the activity or subject matter to improve donor comprehension (Table 1) .
Donor and staff errors and omissions on screening interviews result in the collection, distribution, and transfusion of unacceptable donations.
Donor errors, particularly donor failure to admit to high-risk behaviors, are thought to endanger the blood supply.
Also troublesome are errors and omissions recognized and reported to the collecting organization after donation (postdonation information [PDI]).
Errors resulting in labeling and making available for distribution nonconforming blood products are transmitted to FDA as blood product deviation reports (BPDRs), and summarized annually by the agency.
34 The donor interview process is responsible for 75% of all BPDRs, occurring at a rate 11 times that of the next largest category.
The frequency of such errors may be, in part, a function of the screening method used by the collecting organization.
Cumming et al, 24 in their recent study at a regional blood center, found that in the first year after implementing an AVT-CASI system, the elicitation of information resulting in donor deferrals increased substantially.
First-time donor PDI reports to FDA increased by 269%, compared with those during the previous FTFI-WSAQ interview, whereas repeat donor PDIs increased by 79% and then declined substantially.
The hypothesis is that AVT-CASI may have more effectively prompted donors' subsequent recall of information requested during the interview.
Goldman et al, 35 in a 2005 study of 870 Canadian donors (94% repeat) found that (i) blood donors' ability to recall questions immediately after completing their paper DHQ was poor, (ii) recall was particularly poor when items were part of a list, and (iii) recall was improved when using the AABB UDHQ and further improved by using AVT-CASI.
CASI systems have an advantage over FTFI and WSAQ for error reduction.
Properly designed CASI systems include programed quality checks and staff alerts for aberrant or incomplete donor responses, incomplete staff reviews, omission of donor and staff signatures (when done electronically), and failure to properly file donor health history records when transferred to electronic data repositories.
By automating the screening process wherever possible and embedding programed checks on human error, CASI systems substantially reduce staff errors in the screening process.
Three recent studies indicate the extent to which CASI systems are effective in this respect.
Gordon et al, 36 in assessing the effects of loss of their center's computer equipment to disaster, found the frequency of omissions on the donor record form quadrupled (.25% vs 1%) when computer-assisted interviewing fell from 98% to 30%.
Katz et al 37 reported a 61% decline in staff-related screening errors after introduction of an AVT-CASI system.
Cumming et al 24 reported staff errors and omissions decreased by 69% after AVT-CASI introduction at another regional blood center.
Thus far, there have been no studies comparing CASI systems to WSAQ in this respect.
However, data processing procedures for donor screening via WSAQ are essentially the same as those for FTFI, so it is reasonable to expect that error reduction would be similar when CASI is substituted for WSAQ.
CASI donor screening requires less staff time than FTFI.
In their study, based on an AVT-CASI version of the original UDHQ, Katz et al, 23 reported a decrease in staff time per interview of 5 minutes (68%), whereas total interview time for donors increased from 7.4 to 11.2 minutes, compared with the previous FTFI.
Interestingly, despite the increased total time, 87% of donors perceived that the time needed for CASI was favorable, compared with FTFI.
More recently, the same authors reported a further 38% reduction in average staff review time when using the CASI version of the new UDHQ (1.77-1.09 minutes).
37 There is a dearth of literature addressing the tradeoff between system costs and the value of quality improvements in donor screening realized when CASI reduces staff errors and omissions.
Cunningham 38 has estimated the costs of staff errors that occurred during donor screening at the Indiana Blood Center in 2003.
This study evaluated bhidden costs (of errors) to the blood center, including costs associated with documentation of the error upon discovery; initial evaluation and investigation of an error; lost product costs; labor expenses; and quarantine and resolution of quarantine of the involved products.Q The estimated annual cost of such errors was $87 280, and the study bprovided compelling evidence that automated donor screening processes should be considered.Q Four vendors presently offer proprietary CASI systems for blood donor screening in the United States: Talisman Medical Systems Ltd, Vienna, VA (Quality Donor System) referred to hereafter as bQDSQ; Healthcare ID Inc, Buffalo Grove, IL, (DONOR-ID), referred to hereafter as bHCIDQ; Information Data Management, Rosemont, IL (Prelude), referred to hereafter as bIDMQ; and Haemonetics' 5D Information Management Division, Edmonton, Alberta, Canada (eQue), referred to hereafter as b5D.Q Characteristics of the 4 systems, as described by vendors and, where installed and in use, verified by customers, are summarized in Table 1 .
Of the 4 systems, 2 are operational (QDS and HCID) in blood centers; the third, IDM, is being installed at present; and the fourth, 5D, is scheduled for first installation in fall 2006.
All systems can use the new AABB long form UDHQ with English and Spanish versions available.
Beyond this, they differ in several respects.
QDS is a standardized system with the vendor responsible for installation and maintenance of all screening questions, all audio and visual prompts, and all additions and changes thereto.
With HCID, IDM, and 5D, the user is responsible for installation of the UDHQ questionnaire, selecting pictures for visual display, recording audio prompts, providing the Spanish translation, and making future changes in system content.
Thus, QDS is a bturnkey softwareQ system in that it relieves center management of the tasks of installing and maintaining system content, whereas HCID, IDM, and 5D allow management to modify questions on the UDHQ, addition of local questions if desired, and the choice of developing audio and visual prompts.
All vendors use commercially available, off-shelf computer equipment specified by brand name and model number for purchase by the user.
Described generically, QDS uses finger touch-screen tablet computers with Windows (Microsoft) printers for all fixed sites and mobiles; HCID uses touch-screen laptop computers for mobiles and standard desktops for fixed sites; IDM uses standard desktop computers with personal digital assistant (PDA)/handhelds planned for early 2007, touch-screen monitors, stylus-only tablet computers, wireless scanners, and electronic signature pad; and 5D uses standard desktop computers with touch-screen monitors, fingerprint scanners, and signature pads for fixed sites.
Mobiles are planned but as yet undefined.
Typically, installation, validation, and implementation require weeks or months before center screening is fully automated.
In general, implementations have been sequential with fixed donation sites preceding mobile operations.
All systems permit the donor the alternative of answering, skipping, or requesting help on each question.
In addition the donor can backtrack and revise responses to previous questions.
When the interview is completed, each system shifts to its staff review mode, highlighting those questions that require further staff interaction with the donor.
Each highlighted question must be resolved by further donor explanation satisfactory to staff or by a deferral decision.
Staff is constrained in the review by center SOPs directing further inquiry and deferral decisions.
After, and only after, all highlighted questions have been adjudicated, the systems produce the donor record for staff and donor signature.
QDS and HCID produce printed output for signature, whereas IDM and 5D display the final interview on the monitor, to be signed electronically on a signature pad.
An imminent release of QDS provides for touch-screen input of donor and staff signatures.
No formal donor training time is required with any of the systems because operation of each system is self-evident.
QDS, for which the vendor retains responsibility for system maintenance, requires 2 to 3 hours for center staff familiarization with system setup and the staff review procedure.
HCID, IDM, and 5D, because each assigns responsibility for software maintenance and process changes to center staffs, vary the requirement with HCID using 2 to 3 days of staff training.
IDM uses 5 sessions of 4 hours each together with a short refresher course, and 5D allots 1 day for reviewers and up to 3 days for system administrators.
The means by which data are communicated among stations of the screening systems and from the screening system to the blood bank computer system or other data repository is important in minimizing staff time to update system records and avoid errors in communication between systems.
All systems can operate on a local area network (LAN), whereby a single server holds all system software and services multiple clients on the network.
At present, one center using QDS operates its fixed and mobile sites on wireless LANs.
All other centers use stand-alone stations, each of which holds the full complement of software.
The advantage of a LAN is that only the server is involved in system changes and validations, whereas with standalone stations, each station is involved, increasing staff time for implementation and maintenance.
Although the QDS 510(k) covers electronic data transfer, at present, centers using the system prefer manual entry of selected interview data into the main blood bank computer system.
IDM, HCID, and 5D, on the other hand, produce electronic donor records that can then be transferred directly to the main system.
IDM does not produce a printed donor record; 5D can generate the hard copy if required.
QDS software is web-based, whereas HCID and IDM are client-server based and 5D is web-based with client-server for staff review.
The main difference between the 2 is that with web-based software, only the computer servers need be updated and validated for system changes, whereas with client-server software, both servers and stations must be updated and validated.
A wide area network (WAN) using a single server to service multiple geographically dispersed stations has advantages over standalone or LAN software (see section below on system extensions).
All center system installations in the United States, whether consisting of standalone stations, LANs, or WANs must be submitted to the FDA for review and clearance.
All 4 proprietary systems are FDA 510(k) cleared.
Centers are permitted to install them using a changes being effected-30 days (CBE-30) license supplement that allows FDA 30 days to object before implementation.
Additions or changes in QDS interview questions are the responsibility of the vendor staff who develop required revisions or those requested by users and transmit them to center staff for installation and validation.
This change process requires 1 week, based on experience with smallpox vaccination, severe acute respiratory syndrome, West Nile symptoms, and variant Creutzfeldt-Jacob disease guidances.
Gerber et al, 39 using a QDS system, describe making such a change in 10 standalone stations within 3 days.
HCID, IDM, and 5D leave center staff responsible for changes for which there is no reported experience to date.
Available technology offers several opportunities for improvement in present AVT-CASI screening systems.
Chief among these are (i) Internet donor interviewing, (ii) WAN and wireless communications, (iii) archival databases, and (iv) staff decision aids.
Use of the Internet for e-mail communication is commonplace in the United States and already in use by blood centers for donor recruitment.
An obvious next step for blood centers, strongly supported by sponsoring organizations interested in minimizing employee donation time, is to screen donors at home or work via the Internet.
Smith and associates first suggested doing this some years ago.
40 A system for Internet interviewing has been developed for QDS and pilot-tested at the Mississippi Valley Regional Blood Center.
41 The interview is conducted via a secure Internet server requiring password-protected broadband access, audio capability, and a printer at the donor's computer.
Donors access and complete the interview without providing personal identifiers.
An encoded bar code printout with a unique identifier is produced for donors to present to the donation site.
Screening staff ask the donor 3 additional questions to verify authorship of the printout, that nothing material has changed since its completion and that it was completed in a private setting.
The staff then scans the bar code to decode the interview, insert the donor identification, complete the staff review, and print the donor card for signatures.
Most donors participating in the pilot study reported the system allowed for adequate privacy, felt the Web site was easy to access, that instructions were easy to follow, and audio and video quality was very good.
Thirty-nine percent were interviewed at home, 58% at work, and 3% elsewhere.
Sixty-one percent stated a preference for the Internet interview, 35.6% were neutral, and 3.4% preferred the on-site interview.
A prior approval supplement for implementation has been submitted to FDA.
In addition to assisting organizations sponsoring blood drives by decreasing donor time at the blood drive, the system is expected to increase donor satisfaction.
Furthermore, because Internet donor interviewing is anonymous until staff review, the interview site remote, and the interview totally private and under donor control at all times, it may increase the accuracy of elicited information.
As Fielding et al 42 discovered in a recent test of postdonation telephone interviewing of blood donors, traditional onsite WSAQ for donor screening can lead to underreporting of high-risk behavior, probably because of social desirability bias and embarrassment considerations, factors that may be minimized when donors are interviewed remotely.
Typical blood center organization includes a headquarters containing the blood bank computer system and principal collection site, multiple widely dispersed satellite collection sites, and mobile collection operations used to service donors at workplaces, schools, churches, and other convenient locations.
A CASI system for such a center typically consists of several interviewing stations at each fixed and mobile collection site, either standalone or on a LAN, each of which contains the full complement of screening software including a database capable of storing donor interview data.
Data are returned to the main site in portable computers on portable media or over phone lines.
This process is inefficient.
If activity data are required from screening terminals, staff must extract the data from each station computer or LAN servers and collate and transmit them to headquarters.
Implementation of system changes is similarly complicated.
Construction of a WAN simplifies all this as each site station becomes part of the center-wide electronic network.
WANs allow transmission of encrypted data to and from the headquarters' central server, site servers, and each station and mobile unit.
Consequently, little or no staff time is needed for daily up-and downloading of data, for collation and communication of data to and from system-wide databases, or for making system changes, which are confined to the WAN central server.
Because much US blood is collected on mobile operations in suburban and rural areas where no wired WANs are available, QDS, IDM, and 5D have WAN capability including data encryption.
At present, QDS is testing a WAN system using satellite communication between headquarters and mobile units as part of its system now installed at West Tennessee Regional Blood Center.
43 Given WAN capability with a CASI screening system, a center can install and maintain a central database containing the historical record of every donor's previous interviews, with the information accessible for staff review during subsequent interviews.
For example, centers report that FDA inspectors cite them for inconsistent donor travel histories.
A WAN configuration on a CASI system will permit staff at any collection location to access a donor's travel history during the staff review for comparison with the current information, allowing resolution of any discrepancies.
Real-time access to historical data can prevent inappropriate collections, facilitate timely discovery and action on postdonation information, and prevent unnecessary deferrals when complicated donor histories have been previously evaluated and found acceptable.
Decision aids consist of a variety of supplementary tables, flow charts, and instructions, detailing for staff how to evaluate aberrant or unclear responses to interview questions.
They range in complexity from simple checklists to complex flow charts involving multiple supplementary questions leading to a variety of decision outcomes.
Normally, such checklists or flow charts are included in the center's printed SOPs for staff guidance on handling responses to each question in the interview.
American Association of Blood Banks, as part of development of the new long form UDHQ, has produced a set of decision aids consisting of flow charts and tables to assist screening staff in their response to aberrant answers.
These lists and flow charts can be incorporated into CASI systems for use by staff when consultation or guidance is needed during review, as has been done with QDS and IDM.
Computerized health histories and donor screening increase the accuracy and efficiency of the blood donation process.
Presumably this translates into transfusion safety, although direct measurement is difficult, given low rates of donor infection.
Given that 75% of BPDRs reported to FDA involve the donor interview, decrements in errors and omissions by themselves justify broader implementation of CASI systems and are consonant with the blood community's adoption of cGMPs.
The enhancements being developed for present systems will provide further process improvements that mesh nicely with the emphasis on current good manufacturing practices in blood banking and the use of electronic health records more broadly.
Personnel communication with PD Cumming.
Current prevalence rates for HIV, HBV and HCV for US population obtained from: Morbidity and Mortality Weekly The national blood policy: A study in the politics of health.
Paper presented at the 22nd meeting of the Secretary's Advisory Committee of Blood Safety and Availability Petersen LR, Doll L: HIV Blood donor study group: Epidemiologic, laboratory and donation characteristics Risk factors for hepatitis C virus infection in the United States blood donors Draft guidance for industry-acceptable fulllength donor history questionnaire and accompanying materials for use in screening human donors of blood and blood components Weisband S, Kiesler S: Self disclosure on computer forms: meta-analysis and implications A metaanalysis study of social desirability distortion in computerassisted questionnaires, traditional questionnaires, and interviews Effects of computer-assisted self-interviews on reporting of sexual HIV risk behaviors in a general population sample: A methodological experiment Computer-based interview for screening blood donors for risk of HIV transmission American Institute for Research: Increasing the safety of the blood supply by screening donors more effectively, executive summary Comparison of deferral rates using a computerized versus written blood donor questionnaire: A randomized, cross-over study Regional scale impacts of an audiovisual touch-screen computer-assisted self interviewing (AVT) system.
Abstract of presentation submission 06-AB-862-AABB Computer-assisted audiovisual health history interviewing: Results of the pilot study of the Hoxworth Quality Donor System The crucial link between literacy and health Effects of low medical literacy on health survey measurements The blood donor screening questionnaire: Is it effective?
AABB 2005 bulletin board.
Transfusion 45s:SP202, 2005 (abstr) Cunningham KJ: Cost of quality for donor screening documentation errors Audio versus manual implementation of new questions Innovations in blood donor screening and blood collection Internet donor health history interviewing Risk-behavior reporting by blood donors with an automated telephone system The adventures of implementing a satellite base wide area network.
Abstract of presentation submission 06-AB-156-AABB The authors thank Talisman staff Chantell Murphy for her literature research; Philip S. Abrams, PhD, for his advice on architecture and other computer systems technology; and Joseph C. Johnston for general comments on the manuscript.
key: cord-253840-xudra8tp authors: Gillette, Michael; Taylor, Addison; Butulija, Djenita; Kadiyala, Himabindu; Jneid, Hani title: Reflections of the Angiotensin Receptor Blocker Recall by the FDA and Repercussions on Healthcare date: 2020-04-21 journal: Cardiovasc Drugs Ther DOI: 10.1007/s10557-020-06976-0 sha: doc_id: 253840 cord_uid: xudra8tp PURPOSE: Beginning in July of 2018, the FDA issued a voluntary recall regarding the presence of a contaminant found in the manufacturing of valsartan.
What would ensue has become a largely unprecedented sequence of alarming events since the FDA began reporting public recalls, withdrawals and safety alerts on their website in 2016.
Since then, the United States has been significantly impacted by drug recalls affecting angiotensin receptor blockers.
This report arms clinicians with additional guidance and provides a framework for responding appropriately to future similar incidents and includes an overview of the angiotensin receptor blockers, and their effects and safety profiles.
METHODS: This report includes a review of data from all pertinent clinical and scientific sources including information from the FDA’s inspection documents and recall website.
Additional information is provided on the specific bottles including all lot numbers, expiration dates, etc.
RESULTS: The recalls/withdrawals are attributable to the presence of cancer-causing contaminants identified during the manufacturing process from drug manufacturers abroad.
The root causes behind the recalls and subsequent shortage appear multifactorial, and stem to a certain extent from the outsourcing of medication manufacturing overseas and lack of quality checks and appropriate oversight.
CONCLUSIONS: This inherent issue is not likely to resolve soon and has eroded the public trust of/in the healthcare system and the pharmaceutical industry.
Patients and healthcare providers are significantly affected and should have a full understanding of the matter in order to guide appropriate response and actions.
ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10557-020-06976-0) contains supplementary material, which is available to authorized users.
Over the preceding 12-24 months, healthcare providers have encountered an alarming number of recalls related to cardiovascular medications, particularly with angiotensin receptor blockers (ARBs).
At least 17 warnings to date have been listed on the Food and Drug Administration recall website (https:// www.fda.gov/safety/recalls-market-withdrawals-safety-alerts).
ARBs, such as valsartan and losartan, represent a class of medications that in randomized controlled clinical trials (RCTs) have been shown to reduce blood pressure (BP) in hypertensive patients and impart cardiovascular benefits in diabetic nephropathy, systolic heart failure, left ventricular dysfunction, and following stroke [1] [2] [3] [4] [5] [6] [7] [8] 10] .
They are often prescribed for those individuals who develop a persistent dry cough when taking angiotensin converting enzyme inhibitors (ACE-Is), an adverse event reported to be as prevalent as 35% in some ethnic groups [5, 6] .
The first ARB, losartan, was approved by the FDA in 1995, followed by valsartan in 1996, and since then 7 additional ARBs (Table 1) have received approval for clinical use [7] .
The most recent estimates from 2013 to 2016 estimates that there are over 116 million adults in the USA with a systolic blood pressure of 130 mm Hg (mmHg) or greater [9] .
Uncontrolled hypertension can obviously result in major adverse cardiovascular (i.e.
stroke, myocardial infarction) events Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10557-020-06976-0) contains supplementary material, which is available to authorized users.
[5] [6] [7] [8] .
Diabetic nephropathy may also progress to kidney failure and end stage renal disease if not appropriately treated.
Given their established benefits, the use of ARBs to reduce cardiovascular morbidity and mortality is likely to further increase since the prevalence of heart failure alone is projected to grow by as much as 46% from 2012 to 2030, resulting in well over 8 million affected adults [9, 10] .
Given the prevalence of a myriad of conditions treated with ARBs, it is no surprise that valsartan was one of the top 10 prescribed medications in 2014 with sales grossing over 2 billion dollars per year at that time.
Losartan was then listed among the top 10 most widely prescribed drugs with nearly 60 million prescriptions a couple years later [11] .
In the current report, we discuss the Food and Drug Administration (FDA) recall of the ARBs and provide an overview of the problem, its intricacies and root causes, and its implication on healthcare in the USA.
Beginning in July of 2018, the FDA issued a voluntary recall regarding the presence of a contaminant found in the manufacture of valsartan [12, 13] .
What would ensue has become a largely unprecedented sequence of alarming events since the FDA began reporting public recalls, withdrawals, and safety alerts on their website (https://www.
fda.gov/Safety/Recalls/default.htm).
Since the first recall, the FDA has issued at least 31 additional recalls (Supplement 1) for ARBs specifically valsartan, irbesartan, and losartan due to the presence of at least 3 contaminants, N-Nitroso N-Methyl 4-amino butyric acid ( N M B A ) , N -n i t r o s o d i m e t h y l ( N D M A ) , o r Nnitrosodiethylamine (NDEA).
It is not unexpected that these 3 individual ARBs were primarily affected as they were the first to gain FDA approval and thus go off patent with valsartan and losartan previously carrying large market share and profits.
There have been no other major recalls affecting cardiovascular medications due to carcinogenic contaminants since 2016.
This voluntary recall, unfortunately, has impacted several manufacturers including major pharmaceutical companies and was traced back to factories overseas [12, 13] .
The contaminants found within the bottles were alarmingly categorized by the International Agency for Research on Cancer (IARC) and National Toxicology Program 14th Report on Carcinogens as "Group 2A: Probably carcinogenic to humans" and "Reasonably anticipated to be human carcinogens," respectively [14, 15] .
Group 2A is listed underneath the IARC's highest classification (i.e.
Group 1) for carcinogenic compounds [14] .
The FDA previously proposed that the presence of this substance was suspected to be due to changes in how the active chemical agent was manufactured.
The production of angiotensin receptor blockers can be quite complex and involves various substitutable ingredients or solvents to make the final product.
For instance, the manufacture of valsartan can be completed through at least 4 methods involving an assortment of steps and chemical compounds.
The patent report for valsartan alone included approximately 40 pages (https://patents.google.com/patent/WO2008135762A1/ en).
However, unskillful production may result in hazardous/flammable byproducts, oily intermediates, which are difficult to crystallize, and can further result in difficulties with purification and/or result in undesirable byproducts.
Other techniques may require extensive time and/or resources, which makes production less profitable.
It is thus evident that manufacturers including those abroad should undergo intense scrutiny in their design and production of pharmaceutical compounds, but this is not readily done in many countries.
Notably, not all ARBs and lots were affected, although the persistence of these two unique compounds found in manufacturing resulted in a high-profile review and delays in production, which ultimately contributed to a shortage of valsartan [15] .
The source of the cancer-causing contaminant was subsequently discovered by the FDA and linked to a chemical byproduct produced during the manufacturing process of the active ingredient from factories overseas [15] .
The FDA statement specifically states "specific chemicals and reaction conditions are present….
and may also result from the reuse of materials, such as solvents" [15].
This may not be surprising based on the variability in steps and solvents used to produce chemical compounds but reinforces the need for careful selection and oversight.
The FDA also indicated that patients could have been exposed over at least the past 4 years after manufacturers made a change in how they produced the active ingredients.
The switch in the manufacturing process may explain why the signal was not detected during preclinical drug development studies and before other companies began manufacturing after patent expiration.
Additionally, there was no way of capturing this mishap during routine inspections until recently when scientists became more knowledgeable about unintentional chemical compounds created during manufacturing.
The FDA was also unaware how many patients could have been exposed but estimated that up to 2 million people may have been exposed to medications containing carcinogenic impurities.
They also speculated that one additional case of cancer might result if 8000 people took the highest dose of an affected medication lot over 4 years (anticipated time that the affected products have been on the market) [15].
It is extremely important to note again that not all batches were affected.
In the FDA recall announcement, patients were advised to check with their pharmacies and healthcare providers first and to not stop their medication unless specifically instructed to do so.
This is particularly important given the recent outbreak of the coronavirus disease 2019 (COVID19) and speculation that ACE-Is or ARBs could increase the risk of infection through upregulation of angiotensin converting enzyme-2 receptors (ACE2) thereby leading to inappropriate discontinuation by patients or providers [16] .
Given that many of the medications affected were distributed nationwide, pharmacies and healthcare facilities were advised to check inventories and halt distribution and dispensing of affected products immediately.
Consumers were directed to contact their respective pharmacy or manufacturer's customer support hotline for queries, while some manufacturers have offered a hotline to provide instructions on how to return affected products (see Supplement 1) .
Consumers may also cross check their respective lot number with the recall lot numbers listed on the FDA website (https://www.fda.gov/ safety/recalls-market-withdrawals-safety-alerts).
Adverse reactions or quality problems experienced by patients with use of affected products should be reported online to the FDA MedWatch program through the following link (www.
fda.gov/medwatch/report.htm).
Alternatively, the reporting form could be downloaded via (www.fda.gov/MedWatch/ getforms.htm) or by calling.
Both patient and providers may request and submit the completed form.
Providers and pharmacies are ultimately tasked with addressing questions related to medication replacement or alternatives.
From a facility/provider practice perspective, the first step is to identify who was affected by the recall within their respective healthcare system and then create a personalized action plan to address appropriate alternatives.
Lists of exposed patients can be obtained if the facility has an electronic medical record/prescribing system using informaticians' help and/or by contacting pharmacies.
Although exposure could have been taken place over the past 4 years, the lists should begin by narrowing down to those who were specifically prescribed irbesartan, losartan, and valsartan beginning in July and then cross-checking with the affected lots (Supplement 1 or on the FDA website at https://www.fda.gov/safety/recallsmarket-withdrawals-safety-alerts).
Once patients are identified then the decision to replace or provide an alternative should be undertaken.
Valsartan is facing a shortage due to the recall, and it is unknown whether additional recalls/shortages may occur in the future.
Therefore, it is not unreasonable to consider an alternative ARB or other antihypertensive (i.e.
ACE-I if no contraindication or previous allergy/adverse event), although telmisartan and candesartan are also available as generic and could therefore be at similar risk theoretically.
If the indication is strictly hypertension, then it might be advisable to have the patient follow-up in clinic for repeat measurements to help determine whether regimen or dosage adjustments are needed to conform to the new BP cutoff of less than 130/80 mmHg according to the more recent American College of Cardiology (ACC)/American Heart Association (AHA) 2017 Guideline [8] .
ACE-Is provide similar BP lowering effects and may be a reasonable alternative to ARBs in the absence of prior intolerance.
Table 1 is provided to help identify other alternative ARBs based on FDA-approved indications and clinical characteristics.
It is imperative that the provider pay special attention to the indication when determining an alternative, prior allergies/adverse reactions, current medication/ antihypertensive regimen, vital signs, and other considerations (i.e.
cost/formulary alternatives, insurance coverage, dosing schedule, potential for noncompliance).
Other antihypertensives may be considered with a preference for those with direct evidence of clinical benefits based on the indication and comorbidities with careful consideration given to prior allergies/adverse reactions, lack of contraindications, laboratory findings, vitals, drug interactions, patient preference (i.e.
shared decision making), etc.
Regardless, all patients should be notified of the recall through letters and/or other forms of communication, particularly for facilities in less informed areas such as rural towns/communities.
It is noteworthy that concerns with ARBs causing cancer have dated as far back to 2003 in the effects of candesartan on mortality and morbidity in patients with chronic heart failure (CHARM) trial, which reported a surprising increase in cancer deaths with candesartan versus placebo (2.3% vs. 1.6%; p = 0.038) [17] .
Additionally, a slight numerical but nonstatistically significant increase in cancers was shown with losartan in the LIFE (8% vs. 7%; p = 0.118) and telmisartan in the TRANSCEND (8% vs. 6.9%, RR 1.17, 95% CI 0.97-1.42; p = 0.094) as well as ONTARGET trials (RR 1.04 [0.77, 1.40), respectively [1, 18, 19] .
Following these findings, a meta-analysis published by Sipahi and colleagues in 2010 evaluating different solid organ cancers in patients receiving ARBs compared with controls found a significantly higher incidence (7.2% vs 6%, risk ratio [RR] 1.08, 95% CI 1.01-1.15; p = 0.016) particularly with lung cancer occurrence [20] .
However, subsequent analyses have failed to confirm this association including a more recent comprehensive meta-analysis by Bangalore and colleagues that involved at least 70 randomized controlled trials [21] [22] [23] [24] [25] .
Interestingly, Bangalor and colleagues did report a slight increase in cancer when ARBs were combined with ACE-Is (2.3% vs. 2%; RR 1.14, 95% CI 1.02-1.28), but fortunately, combination therapy with ACE-Is is generally not recommended due to lack of efficacy and risk for other harms [25] .
Lastly, a meta-analysis was performed by the FDA capturing 31 trials with 84,461 patients on ARBs and 71,355 patients randomized to non-ARB comparators with an average follow-up of 39 months.
This ultimately ended concerns after reporting incident cancer events of 1.82 per 100 patient-years in the ARB group vs. 1.84 per 100-patient years in the non-ARB group (RR 0.99, 95% CI 0.92-1.06) [26] .
There was no association found between ARBs and cancer-related death (RR 1.04, 95% CI 0.96-1.13), breast cancer (OR 1.06, 95% CI 0.9-1.23), lung cancer (OR 1.07, 95% CI 0.89-1.29), or prostate cancer (OR 1.05, (95% CI 0.95-1.17) and eventually lead the FDA to propose guidance (https://www.fda.gov/regulatory-information/searchfda-guidance-documents/meta-analyses-randomizedcontrolled-clinical-trials-evaluate-safety-human-drugs-orbiological) for conducting meta-analysis when evaluating safety of medications [26] .
Previous meta-analyses were most likely affected by significant methodological flaws and uncertainty including heterogeneity in the trial conduct/selection.
It should be stated that the trials captured in the FDA's metaanalysis were undertaken decades before the recent findings of differences in ARB manufacturing that exposed patients to carcinogens.
In vitro data have demonstrated expression of the RAS and AT 1/2 receptors in various cancer cells/tissues including brain, lung, breast, prostate, and pancreatic cancers, and the use of ACE-Is and ARBs were shown to reduce the number of metastases, tumor growth, and vascularization in rodents [27] [28] [29] .
Although there is a link between AT receptor activation and inflammation, angiogenesis, and cell proliferation, no plausible biologic mechanism has been identified to connect antagonism of AT receptors and cancer development.
Moreover, if cancer risk is indeed associated with AT 2 activation indirectly through AT 1 antagonism, then the cancer risk should be higher with ARBs such as valsartan, olmesartan, azilsartan, and candesartan due to their much higher affinity for the AT 1 receptor [27] [28] [29] .
In summary, concerns regarding a cancer association have not been definitively established and is largely refuted in the healthcare community.
Although other ARBs were not affected by the recall such as azilsartan, candesartan, eprosartan, olmesartan, and telmisartan, it is possible that other generic ARBs could be affected.
Overall, the concerns of cancer and public outcry has been overwhelming to both patients and providers.
The angiotensin receptor neprilysin inhibitor (ARNI), valsartan/ sacubitril, remains under patent protection and is therefore unaffected by the recall.
Until the etiology of this recall is fully addressed, concerns over prescribing and utilizing ARBs will persist.
Because of previous concerns over drug shortages and the potential implications on patient outcomes and healthcare wastage, organizations such as the American Society of Health System Pharmacists (ASHP) have issued a congressional call to action.
It is imperative that providers and organizations continue to reach out to members of the congress to take immediate action and further investigate production and manufacturing processes both locally and abroad [30] .
Given the global impact of cardiovascular diseases and the use of ARBs for their medical management, it seems prudent to resort to ARBs when ACE-Is cannot be tolerated or when ARBs have proven indications.
When doing so, ARBs not affected by the recall should be utilized or other contemporary agents such as valsartan/sacubitril should be considered when clinically appropriate, particularly among heart failure patients with reduced ejection fraction [10] .
Cardiovascular morbidity and mortality in the Losartan Intervention For Endpoint reduction in hypertension study (LIFE): a randomised trial against atenolol The Study on Cognition and Prognosis in the Elderly (SCOPE): principal results of a randomized double-blind intervention trial Progression of cardiovascular damage: the role of renin-angiotensin system blockade Mechanisms by which angiotensin-converting enzyme inhibitors prevent diabetes and cardiovascular disease Angiotensin-converting enzyme inhibitorinduced cough: ACCP evidence-based clinical practice guidelines Cardiovasc Drugs Ther Initial treatment of hypertension Overview of angiotensin receptor blockers Guideline for the prevention, detection, evaluation, and management of high BP in adults: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines Heart disease and stroke statistics -2019 update: a report from the ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America Top 100 prescribed, top-selling drugs FDA warns about BP medication shortages amid recalls FDA warns of common BP medicine shortage due to recalls cancer-causing-contaminant-went-undetected-years-widelyused-blood-pressure-medicines/?noredirect=on 16.
Heart Failure Society of America/American College of Cardiology /American Heart Association statement addresses concerns re: using RAAS antagonists in COVID-19 HFSAACCAHA-statement-addresses-concerns-re-using-RAASantagonists-in-COVID-19 Effects of candesartan on mortality and morbidity in patients with chronic heart failure: the CHARM-Overall programme on behalf of the TRANSCEND Investigators, et al.
Effects of the angiotensin-receptor blocker telmisartan on cardiovascular events in high-risk patients intolerant to angiotensin-converting enzyme inhibitors: a randomized controlled trial on behalf of the ONTARGET investigators, et al.
Telmisartan, ramipril, or both in patients at high risk for vascular events Angiotensin-receptor blockade and risk of cancer: meta-analysis of randomized controlled trials On behalf of the ARB Trialists collaboration.
Effects of telmisartan, irbesartan, valsartan, candesartan, and losartan on cancers in 15 trials enrolling 138,769 individuals Use of angiotensin receptor blockers and the risk of cancer The use of telmisartan and the incidence of cancer Antihypertensive drug use and the risk of prostate cancer: a meta-analysis of 21 observational studies Antihypertensive drugs and risk of cancer: network meta-analyses and trial sequential analyses of 324,168 participants from randomized trials FDA drug safety communication: no increase in risk of cancer with certain BP drugs -angiotensin receptor blockers (ARBs) Differential regulation of in vivo angiogenesis by angiotensin II receptors Review: angiotensin II type 1 receptor blockers: class effects versus molecular effects Drug shortages harm patients Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Authors' Contributions All authors had access to this manuscript and data and played a role in writing this report.
Conflicts of Interest The authors declare that they have no conflict of interest.Ethical Approval This article does not contain any studies with human participants or animals performed by any of the authors.
key: cord-290895-tb0xald0 authors: Indu, Purushothaman; Rameshkumar, Marimuthu Ragavan; Arunagirinathan, Narasingam; Al-Dhabi, Naif Abdullah; Arasu, Mariadhas Valan; Ignacimuthu, Savarimuthu title: Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine against main protease and RNA-dependent RNA polymerase of SARS-CoV-2: A molecular docking and drug repurposing approach date: 2020-10-26 journal: J Infect Public Health DOI: 10.1016/j.jiph.2020.10.015 sha: doc_id: 290895 cord_uid: tb0xald0 BACKGROUND: Outbreak of COVID-19 has been recognized as a global health concern since it causes high rates of morbidity and mortality.
No specific antiviral drugs are available for the treatment of COVID-19 till date.
Drug repurposing strategy helps to find out the drugs for COVID-19 treatment from existing FDA approved antiviral drugs.
In this study, FDA approved small molecule antiviral drugs were repurposed against the major viral proteins of SARS-CoV-2.
METHODS: The 3D structures of FDA approved small molecule antiviral drugs were retrieved from PubChem.
Virtual screening was performed to find out the lead antiviral drug molecules against main protease (Mpro) and RNA-dependent RNA polymerase (RdRp) using COVID-19 Docking Server.
Furthermore, lead molecules were individually docked against protein targets using AutoDock 4.0.1 software and their drug-likeness and ADMET properties were evaluated.
RESULTS: Out of 65 FDA approved small molecule antiviral drugs screened, Raltegravir showed highest interaction energy value of -9 kcal/mol against Mpro of SARS-CoV-2 and Indinavir, Tipranavir, and Pibrentasvir exhibited a binding energy value of ≥ -8 kcal/mol.
Similarly Indinavir showed the highest binding energy of -11.5 kcal/mol against the target protein RdRp and Dolutegravir, Elbasvir, Tipranavir, Taltegravir, Grazoprevir, Daclatasvir, Glecaprevir, Ledipasvir, Pibrentasvir and Velpatasvir showed a binding energy value in range from -8 to -11.2 kcal/mol.
The antiviral drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine also exhibited good bioavailability and drug-likeness properties.
CONCLUSION: This study suggests that the screened small molecule antiviral drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine could serve as potential drugs for the treatment of COVID-19 with further validation studies.
In December 2019, an unknown virus caused severe pneumonia in Wuhan (Hubei province), China, and it spread rapidly throughout the globe [1] .
The unknown etiology was later identified as a new virus and tentatively named as 2019 novel coronavirus (2019-nCoV) [2] .
It has a positive-sense single stranded RNA as a genetic material with a genome size of 27-32kb [3, 4] .
Furthermore, 2019-nCoV shared 87.5% sequence similarity with two bats derived SARS like CoVs strains (bat-SL-CoVZC45 and bat-SL-CoVZXC21) and SARS-CoV-1.
Hence, it was named as SARS-CoV-2 and the disease caused by the virus was called as coronavirus disease (COVID-19) [5, 6] .
The world Health Organization has declared COVID-19 as a pandemic disease on 12 th March 2020 [7] .
As on 17 th May, 2020, a total of 4,525,497 cases were confirmed for COVID-19 infection and 307,395 deaths were occurred worldwide [8] .
Human coronaviruses commonly cause mild to severe infections in humans.
However, SARS-CoV-2 is a public concern because its disease mechanism and function aspects are still unknown [9] .
Disease symptoms of COVID-19 ranged from mild, selflimiting respiratory tract illness to severe progressive pneumonia, multi-organ failure, and sometimes leading to death.
SARS-CoV-2 infected patients were clinically managed by symptomatic and supportive therapies [10] [11] [12] .
Since there is no disease specific vaccine or drug available against COVID-19, there is an immediate need to identify anti-COVID-19 agents to control the outbreak and spread of viral infection [13] .
Numerous clinical trials and research efforts are being made by researchers worldwide for identifying potential therapies against COVID-19.
However, the current drug discovery program requires high cost and time [14] .
Computer-aided drug discovery is the most popular platform for the prediction of potential molecules before synthesis and in-vitro testing [15] .
Drug repurposing is the most effective strategy to find out the novel clinical use of already approved drugs to combat COVID-19 in a short period of time.
This strategy could reduce the time and cost for a new drug development as the major benefits are that the toxicity and pharmacokinetics of the repurposed drugs already available [16] .
Virtual drugs screening even eases the repurposing of old drugs by molecular docking analysis against viral protein targets [17] .
The crystal structure of 3-chymotrypsin-like protease (3CLpro), also known as the main protease (Mpro) of SARS-CoV-2 that has been reported as one of the best proven drug discovery target [18] .
RNA-dependent RNA polymerase (RdRp) is an important enzyme involves in the life cycle of RNA viruses, and RdRp has been targeted for various viral infections includes hepatitis C virus, Zika virus, and HCoVs [19, 20] .
In this study, FDA J o u r n a l P r e -p r o o f approved small molecule antiviral drugs were screened against protein targets of SARS-CoV-2 using a computational based approach.
In this study, 3D structures of FDA approved small molecule antiviral drugs were retrieved from PubChem and unavailable 3D structures for antiviral drugs were developed using the chemical tool box Open Babel [21] .
All the 3d structures were energy minimized using UFF minimization algorithm.
All the minimized structures were converted into PDBQT format before perform docking using Autodock 4.0.1.
The 3D structures of Mpro (6LU7) and RdRp (6M71) were downloaded from the Protein Data Bank (PDB).
Water molecules, ions, and other ligands present in the protein targets were removed using PyMOL software.
The downloaded structures of protein targets were converted into PDBQT format for molecular docking analysis.
Preliminary virtual screening was performed for screening of all antiviral agents against the two major proteins (Mpro and RdRp) using the virtual screening module of COVID-19 Docking Server.
COVID-19 Docking Server was constructed to predict the binding modes between the targets and small molecules, peptides, or antibodies by the implement of Autodock Vina and CoDockPP as docking engines [22] .
The lead drugs screened from virtual screening were individually docked against protein targets using AutoDock 4.0.1 [23] .
AutoDock Tools utilized for the preparation of input pdbqt files of Mpro and RdRp.
The grid was placed at the center.
Kollman charges and polar hydrogen atoms were essential components.
Since, two target proteins are used they form a different centre with different x,y, and z coordinates of -25 , 12 and 59 for 6LU7 and 120,123, 120 for 6M71, respectively.
The docked receptor and ligand interactions were visualized using Pymol.
J o u r n a l P r e -p r o o f Finally screened antiviral drugs were further checked for their pharmacokinetics, druglikeness, and medicinal chemistry properties using the SwissADME server.
The SMILES format of the ligand molecules were used for input in the tool [24] .
In this study, a total of 65 FDA approved small molecule antiviral drugs were virtually screened against the Mpro and RdRp protein targets.
The highest interaction energy among J o u r n a l P r e -p r o o f The antiviral drug Indinavir exhibited the highest binding energy of -11.5 kcal/mol against RdRp protein (Fig.3) .
While analyzing the interaction profile, it was found that six amino acids such as Asp-623 (2Å), Arg-553 ( Screened drugs in this study possessed molecular weight ranged from 444 to 1113kDa.
While checking the pharmacokinetic profile, it was noted that all the screened drugs showed poor gastrointestinal adsorption except Indinavir but almost all the drugs are a substrate of P-glycoprotein (Table 4 ).
While analyzing drug likeness properties, it was noted that all the drugs have few violations in these rules, but still it seems to fit into the FDA approval drugs.
The bioavailability value seems to be 0.55 for Indinavir, Raltegravir, Tipranavir, Dolutegravir and Etravirine while other drugs had shown bioavailability value of 0.17.
COVID-19 causes huge health crisis worldwide.
Since there are no approved drugs available, new treatment options are urgently required for treating COVID-19 [9] .
Computer based drug discovery methods are easing the identification of drugs and used to be aware of molecular aspects of protein targets and target-ligand interactions [25] .
Repurposing of FDA approved drugs will be the right choice at this time for discovering drugs against COVID-19 since, they have already evaluated the toxicity and safety in human against the management of different diseases [17] .
Asp-295 were involved in the interactions [28] .
In this study, most of the amino acid positions involved in the HB interactions between lead antiviral drugs screened against Mpro were similar to the amino acid interactions reported in the study of Chandel et al.
[28] .
Qamar et al.
[29] found five potential protease inhibitors, Nelfinavir, Remdesivir, Lopinavir, Ritonavir, and Ketoamide against protease on COVID-19 with a negative energy value.
Nelfinavir recorded the best binding energy of -7.54 kcal/mol and Thr75, Arg141, Gln175, and His176 were involved in the HB interactions and suggested Nelfinavir as a drug of choice for treating COVID-19.
They also suggested that other drugs screened in their study showed negative dock energy against the target proteins; equal importance can be given to them as protease inhibitor ligands.
In our study, other screened antiviral drugs such as Indinavir, Tipranavir, and Pibrentasvir showed dock energy value more than -8 kcal/mol and these drugs might also serve as an inhibitors of Mpro target of SARS-CoV-2.
Studies revealed that the repurposed drugs Remdesivir [30, 31] and a combination of Lopinavir and J o u r n a l P r e -p r o o f Ritonavir significantly improved the clinical condition of SARS-CoV patients [32] .
In a study, Lopinavir drug has been reported to have the highest interaction energy against Mpro, followed by Atazanavir, Saquinavir, Ritonavir, Nelfinavir, Darunavir, Tipranavir, Amprenavir and Fosamprenavir in antiviral drugs screening.
Finally, they recommended Indinavir and Remdesivir could be the potential therapeutic agents for COVID-19.
These both drugs have been used in the clinical practices with limited toxicity [33] .
RdRp is another important protein involved in the replication and transmission of coronavirus [34] .
In PDB, two structures for SARS-CoV-2 RdRp were released (PDB ID: 6M71 and 7BTF) [35] .
These protein models have the Root Mean Square Deviation (RMSD) value less than 2Å [36] .
In a study, the solved structure 7BTF was docked with 31 compounds and they reported that Hydroxychloroquine had showed an interaction energy value of -6.13 kcal/mol and Sofosbuvir showed -7.46 kcal/ mol.
They suggested that these drugs can bind with the active site of RdRp and induce RdRp inhibition.
Since the safety profile for these drugs has already been studied and approved by FDA, these can be successful candidates for COVID-19 [20] .
In this study, the PDB ID 6M71 has been targeted and it was observed that the antiviral drugs Elbasvir, Indinavir, Grazoprevir, Gelcaprevir, Ledispavir, Velpatasvir and Paritaprevir could serve as potential inhibitors of RdRp.
In a similar study, Chang et al.
[33] reported that the antiviral drug Remdesivir showed highest interaction energy value (-7.803 kcal/mol) followed by Galidesivir, Ribavirin and Fipiravir.
The Lipinski's rule of 5 was also checked for the screened antiviral drugs in this study by observing the five criteria such as molecular weight <500 Dalton, <5, hydrogen bonds, <10 H-bond acceptors and LogP <5 for considering as a drug-like molecule [37] .
Drug-likeness analysis showed that all the screened drugs have few violations despite they fit into the FDA approval drugs.
Early estimation of ADME in the drug discovery phase reduces the pharmacokinetics-related failure in the clinical phase [38] .
In this study, the pharmacokinetic J o u r n a l P r e -p r o o f parameters such as absorption, distribution, metabolism, and excretion (ADME) were analyzed for screened antiviral drugs.
Pharmacokinetic analysis revealed that all the screened drugs had a poor gastrointestinal adsorption except Indinavir but all served as a substrate of P-glycoprotein.
It is believed that Pgp plays an important role in the oral bioavailability, CNS distribution, and biliary and renal elimination of drugs, which are substrates of this transporter.
The drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir and Etravirine have high bioavailability value.
The repurposing of FDA approved antiviral drugs for COVID-19 could be the appropriate drug discovery option in this crisis time.
This study finding revealed that Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine had shown high binding energy against both Mpro and RdRp.
These antiviral drugs have good bioavailability and drug-likeness properties.
This study suggests that the small molecule antiviral drugs Raltegravir, Indinavir, Tipranavir, Dolutegravir, and Etravirine could be the potential drug of choice for the treatment of COVID-19 with further in vitro and in vivo evaluations.
We agree the submission of the revised manuscript entitled "Raltegravir, J o u r n a l P r e -p r o o f Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China A novel coronavirus from patients with pneumonia in China Origin and evolution of pathogenic coronaviruses Identification of Alpha and Beta Coronavirus in Wildlife Species in France: Bats, Rodents, Rabbits, and Hedgehogs Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding Coronaviridae Study Group of the International Committee on Taxonomy of Viruses.
The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 Crystal structure of SARSCoV-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors Coronavirus disease (COVID-19) Situation Report -118 In silico identification of potent FDA approved drugs against Coronavirus COVID-19 main protease: A drug repurposing approach Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical characteristics of novel coronavirus cases in tertiary hospitals in Hubei Province Informatics and computational methods in natural product drug discovery: A review and perspectives Modeling antimalarial and antihuman African trypanosomiasis compounds: A ligand-and structure-based approaches Targeting novel coronavirus 2019: A systematic drug repurposing approach to identify promising inhibitors against 3C-like proteinase and 2'-O-ribose methyltransferase Virtual screening and repurposing of FDA approved drugs against COVID-19 main protease Binding site analysis of potential protease inhibitors of COVID-19 using AutoDock Novel guanosine derivatives as anti-HCV NS5b polymerase: A QSAR and molecular docking study Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study Open Babel: An open chemical toolbox Using AutoDock for ligand-receptor docking COVID-19 Docking Server: An interactive server for docking small molecules, peptides and antibodies against potential targets of COVID-19 SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design Potential Inhibitor of COVID19 Innovation and trends in the development and approval of antiviral medicines: 1987-2017 and beyond Silico Identification of Potent COVID-19 Main Protease Inhibitors from FDA Approved Antiviral Compounds and Active Phytochemicals through Molecular Docking: A Drug Repurposing Approach Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants First case of 2019 novel coronavirus in the United States Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings Potential therapeutic agents for COVID-19 based on the analysis of protease and RNA polymerase docking Molecular Investigation of SARS-CoV-2 Proteins and Their Interactions with Antiviral Drugs Breakthrough: chloroquine phosphate has shown ap-parent efficacy in treatment of COVID-19 associated pneumonia in clinical studies SARS-CoV-2 RNA dependent RNA polymerase (RdRp) targeting: An in silico perspective Lead-and drug-like compounds: The rule-of-five revolution Clinical development success rates for investigational drugs The authors extend their appreciation to The Researchers supporting project number (RSP 2020/20) King Saud University, Riyadh, Saudi Arabia.
key: cord-033420-pjtyv0pv authors: Kalokairinou, Louiza; Zettler, Patricia J; Nagappan, Ashwini; Kyweluk, Moira A; Wexler, Anna title: The promise of direct-to-consumer COVID-19 testing: ethical and regulatory issues date: 2020-09-23 journal: J Law Biosci DOI: 10.1093/jlb/lsaa069 sha: doc_id: 33420 cord_uid: pjtyv0pv Widespread diagnostic and serological (antibody) testing is one key to mitigating the COVID-19 pandemic.
While at first, the majority of COVID-19 diagnostic testing in the USA took place in healthcare settings, quickly a direct-to-consumer (DTC) testing market also emerged.
In these DTC provision models, the test is initiated by a consumer and the sample collection occurs at home or in a commercial laboratory.
Although the provision of DTC tests has potential benefits—such as expanding access to testing and reducing the risk of exposure for consumers and medical personnel—it also raises significant ethical and regulatory concerns.
This article reviews these challenges and shows how they parallel and also diverge from prior concerns raised in the DTC health testing arena.
The first part of this paper provides an overview of the landscape of diagnostic and serological tests for COVID-19, anticipating how provision models are likely to evolve in the future.
The second part discusses five primary issues for DTC COVID-19 tests: test accuracy; potential misinterpretation of results; misleading claims and other misinformation; privacy concerns; and fair allocation of scarce resources.
We conclude with recommendations for regulators and companies that aim to ensure ethically marketed DTC COVID-19 tests.
In the wake of the COVID-19 pandemic, widespread diagnostic and serological (immunity) testing is considered key in containing the spread of the disease, as well as in easing stay-at-home measures and informing policies for restarting the economy.
1 Most tests, whether diagnostic or serological, are currently offered by healthcare providers who interface with the healthcare system.
However, a number of companies 2 have begun to offer COVID-19 testing on a direct-to-consumer (DTC) basis 3 : that is, the test is initiated by a consumer-not a healthcare professional-typically via the company's website.
While a healthcare professional may be involved at some stage of the process that involvement may be as minimal as a brief review of a questionnaire that the consumer has completed as part of the purchasing process.
As such, the consumer may have no direct contact with a healthcare professional (although some companies offer the option of post-test consultations in the case of positive results).
In addition, the results of DTC tests do not necessarily become part of patients' health records, as the companies offering such tests operate independently of the healthcare system.
The acquisition of the sample in DTC tests may occur in one of two ways: via at-home collection or in-laboratory collection.
In the former, individuals order and receive an at-home collection kit, where they acquire the sample and mail it back to the company (or a lab that processes the results on behalf of the company).
For inlaboratory collection, individuals make an appointment to visit a commercial laboratory where their sample is collected.
Both models of DTC COVID-19 tests-at-home or in-laboratory collection-are distinguishable from those offered by hospitals and 3 The term DTC testing has been used in the literature to describe different provision models of health products.
For the purposes of this article, our key criteria for considering a test to be DTC are (a) that it is promoted directly to consumers; (b) the testing process is initiated by consumers, who order the test online or by phone and/or book an appointment to a commercial lab in order to have their sample taken; (c) it requires little to no involvement of a healthcare provider, and whatever involvement does occur is usually not in person.
See health clinics, as it is the consumer who initiates the order and completes the testing process with little to no interaction with a healthcare professional.
Even though the COVID-19 pandemic has changed the way much healthcare is delivered by increasing the use of telemedicine to minimize in-person visits, the doctor-patient relationship remains central.
In this regard, the DTC testing model is distinct.
Although healthcare professionals employed by DTC companies may authorize the ordering of laboratory tests, they have little, and usually no direct contact with the consumer.
Indeed, from the perspective of the consumer, the process of ordering a DTC COVID-19 test may feel similar to purchasing other goods or services.
For example, to order the LabCorp COVID-19 diagnostic test, an individual must click through a brief questionnaire, after which they can input their credit card information, click 'order,' and thereafter receive shipping updates via email.
The DTC provision of COVID-19 tests by private companies may expand access to testing and contribute to the mitigation of the present public health crisis.
In particular, there are potential benefits to at-home testing, which eliminates the risks of exposure that individuals and medical personnel collecting samples may incur.
In addition, at-home testing reduces the demand for personal protective equipment required by sample collectors and decreases the testing burden for the healthcare system as a whole.
However, DTC tests also raise significant ethical concerns and pose regulatory challenges.
Although the regulatory environment for COVID-19 tests is not identical to that for other types of DTC health tests (such as currently marketed genetic or hormone tests), 4 many of the concerns raised by DTC COVID-19 tests parallel those wrought by other types of DTC health tests.
At the same time, there are important contextual differences that make concerns over DTC COVID-19 tests particularly urgent, such as the global population affected, the infectious nature of SARS-CoV-2, and the historic challenges to the healthcare system and economy during the pandemic.
As widening the provision of diagnostic and serological COVID-19 tests may be crucial to containing the pandemic, it is important to critically consider the key issues that might lie ahead for DTC COVID-19 testing.
In this article, we examine the main ethical and regulatory challenges of DTC diagnostic and serological tests, focusing on the US context.
The first part of this article provides an overview of the landscape of diagnostic and serological tests for COVID-19, anticipating how their provision models could evolve in the future.
The second part identifies five primary ethical and regulatory issues for DTC COVID-19 tests: uncertainty over the accuracy of test results; potential misinterpretation of test results by users; misleading product promotion and misinformation; privacy concerns; and fair allocation of scarce resources.
Each of these issues parallels prior concerns raised in the DTC health testing arena, but also diverges from them in important ways.
We conclude with recommendations for regulators, companies, and other relevant stakeholders that can help ensure high-quality, accurate, and equitably distributed COVID-19 tests, and inform the ethical provision of DTC health tests during public health crises.
COVID-19 diagnostic tests aim to determine whether a person is currently infected with SARS-CoV-2, the virus that causes COVID-19.
The most commonly used diagnostic tests during the COVID-19 pandemic are polymerase chain reaction (PCR) tests.
5 For the purposes of testing, a swab is taken from the patient and tested in a lab for the genetic material of the virus.
Although the typical sample used in these tests is obtained by a nasopharyngeal swab that reaches deep into the nose, the FDA recently issued Emergency Use Authorizations (EUAs) for diagnostic PCR tests using samples collected from shallow nasal swabs and saliva.
6 While PCR is considered to be the 'gold standard' of molecular testing due to its high reliability, 7 this high accuracy may not be applicable to certain rapid point-of-care PCR tests, which according to a recent preprint study, may miss up to 48% of infections.
8 At present, the only non-PCR diagnostic test that has received an EUA is an antigen test used on shallow nasal swabs can rapidly detect fragments of the virus.
9 However, these antigen tests have a higher probability of producing false-negative results than PCR tests, as they cannot detect all active infections.
10 Although at first, the majority of COVID-19 diagnostic testing in the USA took place in healthcare settings, a DTC testing market quickly emerged: first with companies marketing at-home tests without FDA authorization, and more recently, with such tests authorized by the FDA.
Specifically, in March 2020, companies such as Nurx, Everlywell and Carbon Health began marketing at-home collection kits directly to consumers.
11 The provision of tests by these companies involved healthcare professionals to various degrees (i.e.
to review an eligibility questionnaire), although typically only after the consumer had initiated or completed the purchase of the test.
12 The marketing of such tests largely coincided with the release of an FDA guidance explaining that the agency would permit laboratories and commercial manufacturers to develop and offer COVID-19 diagnostic tests as long as they notified FDA that their assays had been validated and submitted an EUA.
This policy, however, did not apply to at-home sample collection, which instead required that FDA issue an EUA before marketing.
After FDA clarified this policy in an update to its guidance document, 13 some companies pulled their at-home collection kits from the market.
14 At least one other did so only after facing enforcement action from local authorities.
15 DTC at-home collection kits, however, are rapidly reemerging with FDA authorization.
In April 2020, Pixel by LabCorp received an EUA for the first COVID-19 diagnostic test with at-home collection (via a shallow nasal swab kit).
16 Currently, consumers can purchase this kit for $119 after completing a brief online survey that assesses symptom level (severe, mild or none); potential exposure to coronavirus; and whether an individual is low-or high-risk.
17 One month later, a second diagnostic athome kit that utilizes saliva samples was issued an EUA.
18 At present (August 2020), there are approximately a dozen companies, including Everlywell, LetsGetChecked, Phosphorus, Hims, Vault Health, and 1Health.io, offering authorized at-home COVID-19 diagnostic tests that involve nasal swab or saliva collection.
19 Notably, some of these companies have previously faced criticisms outside the COVID-19 context.
More specifically, commentators have raised ethical concerns about companies such as Hims, whose business model permits consumers to order prescription drugs without interfacing directly with a healthcare professional.
20 concerns about Everywell's DTC tests measuring the immune response to food in order to determine food sensitivity.
21 While diagnostic tests aim to detect an active infection, serological tests aim to detect antibodies in the blood, indicating that a person had been infected and recovered from COVID-19.
The presence of such antibodies could potentially indicate that the individual has developed some level of immunity against the virus.
The blood sample for these tests is usually obtained through a finger prick.
The sample can be collected by an individual and sent to be analyzed in a lab, or be analyzed with laboratory equipment available at a point-of-care location (e.g.
in a pharmacy or physician's office) that can deliver results within a few minutes.
Although the accuracy of these tests for COVID-19 antibodies is not well established, 22 they are at present widely available through numerous laboratories with or without a referral, often in a DTC context that requires consumers to initiate the testing process outside the healthcare system.
The widespread availability of serological testing was made possible from a regulatory perspective because, until recently, FDA exercised its discretion not to enforce requirements for laboratories and commercial manufacturers operating without an EUA, as long as they provided a disclaimer that the tests had not been reviewed by FDA and that results should not be used as the sole basis for confirming that someone has the disease.
23 Following concerns about the quality of many tests on the market, in May 2020 FDA changed its approach, aligning its policy for serological tests with that for diagnostic tests.
24 This means that like with COVID-19 diagnostic tests, companies distributing at-home serological tests must obtain an EUA before marketing their products.
In addition, later the same month, FDA published a list of serological tests that could no longer be marketed or distributed.
25 This was because while the manufacturers of these tests had notified FDA that they intended to seek an EUA, eventually they either voluntarily withdrew their tests from the notification list or failed to apply for an EUA.
Currently, at least two companies offer DTC COVID-19 serological tests.
26 As opposed to at-home collection kits, in this context, consumers initiate the test by making an appointment and having their sample collected in a lab.
For example, the Quest Diagnostics serological test can be purchased online for $119 after individuals complete a brief questionnaire that assesses whether an individual is currently symptomatic for COVID-19.
27 Other companies offer tests specifically to employers interested in testing their employees for COVID-19 antibodies.
28 Although to-date there are no strictly at-home serological tests, these may be on the horizon: for example, Scanwell together with Lemonaid Health are reportedly developing an at-home antibody test that will require consumers to extract a blood sample and share a picture of the blood testing stick with a healthcare provider, who would interpret the results in a subsequent consultation.
29 In the future, it is possible that COVID-19 diagnostic and serological tests will become increasingly available both on an at-home and consumer-initiated basis, for a number of reasons.
First, there is consumer demand, which provides financial incentives for companies to offer such tests.
Second, there are public health benefits in expanding at-home testing capacity-this type of testing may allow for more individuals to get tested without exposing themselves and others to the risk of infection.
Third, FDA has recently expressed its explicit support for at-home COVID-19 testing, clarifying that such tests may be issued an EUA as long as manufacturers provide adequate data and scientific evidence supporting the safety and accuracy of such tests.
30 However, the development of such a market raises ethical concerns and regulatory challenges, which need to be addressed in order to reap the full potential of DTC testing.
Although COVID-19 testing in general-even when not provided on a DTC basismay raise many ethical and regulatory issues, the five main concerns discussed in this section are particularly pronounced in the DTC space.
More specifically, the athome COVID-19 diagnostic testing model presents heightened challenges regarding the accuracy of tests compared to testing that involves sample collection from a healthcare provider.
In addition, the absence (or minimal involvement) of a healthcare provider and, potentially of adequate information accompanying the tests, intensifies concerns about the possible misinterpretation of test results.
Furthermore, to-date, there has been a number of misleading claims made by companies advertising DTC COVID-19 tests (and misinformation from other sources), as well as numerous cases of marketing of fraudulent DTC tests.
Privacy concerns are also more pronounced in the DTC context, since some companies may not be covered by the Health Insurance Portability and Accountability Act (HIPAA).
Finally, the provision of DTC tests by commercial providers may exacerbate inequalities in access to testing at the expense of communities that are disproportionately affected by COVID-19.
The major ethical and regulatory concerns that surround the actual processes of DTC testing for both active COVID-19 infection and serological testing mirror existing concerns about DTC health monitoring and disease evaluation tests already widely available in the USA.
Many of these existing tests, such as HIV tests or the monitoring of HbAC levels for diabetes management, have proven track records in medical treatment.
However, many others-such as food sensitivity tests and genetic tests for susceptibility to multifactorial disorders-are of uncertain quality, particularly because of their inconclusive clinical validity (i.e.
the ability of a test to correctly identify that a particular variant is correlated with increased risk of disease or condition) 31 and unproven clinical utility (i.e.
the ability of the test to inform clinical management of a patient).
32 In the COVID-19 context, the public health value of accurate COVID-19 tests is clear.
However, there remain questions regarding the accuracy of DTC tests, which is dependent on various factors, including the quality of the sample collected, proper shipment, and stability of the specimen, as well as the sensitivity and specificity of the test.
Indeed, FDA has recently acknowledged that COVID-19 tests involving at-home sample collection may present unique issues regarding accuracy, and in recognition of these challenges it recently published an EUA template to help provide guidance to manufacturers of such tests.
33 Regarding the quality of the sample, although many diagnostic COVID-19 tests in the healthcare setting utilize nasopharyngeal swabs-which may be difficult for individuals to obtain themselves-such tests seem unlikely to come to the US DTC market.
The currently available DTC at-home tests utilize shallow nasal swabs or saliva samples, which are easier for individuals to obtain.
Early studies have indicated that nasal swabs and saliva samples-even when self-collected-can effectively and reliably identify infections of the SARS-CoV-2 virus, 34 suggested that saliva may actually be more sensitive than nasopharyngeal swabs.
35 Thus, while further research in this field is necessary, at-present, quality for selfcollected shallow nasal swabs and saliva samples does not appear to be a primary concern.
With regard to the shipment and stability of sample, FDA has noted that at-home tests may raise particular concerns due to the time lapse between the collection and the analysis.
In addition, samples may be subject to conditions during shipment (e.g.
high temperatures) that may compromise them.
36 However, for some nasal swabs (foam or polyester) shipped in certain ways (in a dry tube or in saline solution), FDA has recently indicated that preliminary data suggest that the samples appear to be stable.
37 Thus, at present, samples collected and shipped using these methods appear to alleviate stability concerns, although questions remain regarding stability for other forms of samples and shipment methods.
In addition, the sensitivity (i.e.
the ability of the test to detect the presence of the virus or antibodies) and specificity (i.e.
the ability of the test to detect the absence of the virus or antibodies) of the test itself are also crucial for the reliability of results.
While the PCR tests currently used in COVID-19 diagnostic testing are considered to be of high accuracy, there is always a risk of false-positive or false-negative results, and those risks may depend on factors such as testing outside the diagnostic window, and the use of inadequately validated assays.
38 In the case of serological tests for COVID-19, such concerns are more pressing, as their accuracy has not been well-established and the chance of such tests producing false-positive results may be high, especially in low prevalence populations.
39 A recent preprint study performed by a consortium of laboratories in California found that of the 12 antibody tests studied, one test produced false-positive results over 15% of the time, and three other tests more than 10% of the time.
40 Given that until recently there has been only limited oversight of these tests, the reliability of serological tests remains an important concern.
41 Although DTC COVID tests are reviewed by FDA before they enter the market this does not eliminate concerns about their accuracy.
Issues of unproven quality are potentially inherent to those tests that are marketed under an EUA, as all FDAauthorized COVID-19 tests currently are.
For FDA to issue an EUA for a test, the Federal Food, Drug, and Cosmetic Act requires, among other things, that FDA conclude it is 'reasonable to believe' that the test 'may be effective in diagnosing' the disease.
42 Consistent with this statutory language, FDA has explained that the standard for a product being issued an EUA requires 'a lower level of evidence than the 'effectiveness' standard that FDA uses for [standard] product approvals.'
43 Concerns about test quality have intensified following the US Department of Health and Human Services's (HHS) August 2020 statement that FDA will not require premarket review for laboratory developed tests (LDTs), including for COVID-19 LDTs, unless the agency first goes through notice-and-comment rulemaking to do so.
44 FDA describes LDTs as tests that are "designed, manufactured and used in a single laboratory," and historically has exercised its discretion not to enforce premarket review requirements for many LDTs.
45 But FDA also has enforced such requirements for DTC tests (even when they may meet the definition of an LDT), 46 and HHS's statement did not explain whether it was intended to apply to DTC products that may meet the definition of an LDT.
It, thus, is unclear what HHS intended its statement to cover and how FDA will treat DTC LDTs going forward.
47 It is possible that FDA will allow some DTC COVID-19 tests that are considered LDTs to be offered without an EUA.
48 As a result, more tests of uncertain quality may enter the market in the near future.
In addition, issues regarding the quality of DTC COVID-19 tests may be heightened because of the widespread, historic nature of the pandemic and the urgent need for expanding testing capacity-and accompanying political pressure.
At the same time, however, it is critical that regulators, industry, and the public recognize the need to ensure high-quality standards.
The promise of direct-to-consumer COVID-19 testing • 11 For all DTC health testing, the absence of a healthcare professional and, potentially of adequate information regarding the potential limitations of these products, has raised concerns about the risk of misinterpretation of results and of potentially inappropriate subsequent healthcare decision making.
These concerns apply even for those tests that have met relevant quality standards.
Moreover, such concerns are particularly salient for COVID-19 testing.
For diagnostic testing, false-negative results could create a false sense of security and contribute to further spread of the virus, while false-positive test results could keep people out of work, school, or childcare, exacerbating economic and educational harms.
Additionally, although the interpretation of COVID-19 diagnostic testing is relatively straightforward-indicating whether an individual has an acute manifestation of the infectionserological testing is far more difficult to interpret.
For example, while preliminary data suggest that recovery from COVID-19 might confer immunity to subsequent infection, it is still uncertain how protective such immunity is and how long it may last.
50 In addition, while many manufacturers claim that their serological tests are of high sensitivity and specificity, many have not released any data supporting their claims.
51 Considering the potential unreliability of currently available serological tests and the uncertainty over the meaning of COVID-19 immunity, it is of concern that several companies are marketing serological tests to employers interested in testing their employees, as such results could be used to make decisions about employees going back to work.
Misinterpreting or overestimating test results could expose individuals to risks of reinfection and could undermine public health mitigation efforts.
52 In this regard, DTC COVID-19 tests, whether diagnostic or serological, should be accompanied by clear guidance and information about their potential and limitations.
During the pandemic, with millions of individuals fearful of being sick, but also desperate to resume work, the scale, and immediacy of the adverse impact of misinterpreting COVID-19 test results are far greater than other commercially available DTC health-related tests.
Many concerns that have been previously raised regarding DTC promotion of healthrelated tests-such as misleading or inaccurate claims, exaggeration of benefits and minimization of risks 53 pandemic, the market has been flooded by companies making unsubstantiated and often fraudulent claims, such as falsely stating that their tests have been approved by FDA 54 or that their serological tests can diagnose the disease.
55 To-date, several state and local regulators have issued cease-and-desist orders to individuals and companies on the grounds that they have been illegally promoting and offering COVID-19 tests.
56 At a federal level, both FDA and Federal Trade Commission (FTC) have warned companies to stop making misleading claims, including about treating and preventing the coronavirus.
57 In addition to misleading information coming from companies themselves, the public has been receiving an overwhelming amount of misleading information about serological testing from other sources.
Politicians in the USA and abroad have exaggerated the potential of such tests, touting widespread serological testing as a 'game changer' 58 and a key to restarting the economy.
Some governments have reportedly considered issuing 'immunity passports' based on positive serological tests results.
59 Additionally, in a May 2020 statement, the Governor of New York stated that detecting COVID-19 antibodies means that, 'You can get to work, you can go back to school, you can do whatever you want.'
60 Yet, as explained above, it is still uncertain what positive serological test results mean for functional immunity, how protective any immunity is, and how long it may last.
Misinformation about COVID-19 tests, whether from companies or other sources, is particularly concerning.
61 Misleading product promotion capitalizes on widespread anxiety caused by the pandemic and preys on the vulnerability of consumers, many of whom are likely concerned about their health.
The massive amount of information (and misinformation) about COVID-19, and the quickly changing landscape, may make it particularly challenging for consumers to differentiate between legitimate tests and fraudulent products (which might mislead consumers about their COVID-19 health 54 status).
Moreover, misinformation regarding the potential benefits and limitations of the tests could lead individuals to misunderstand their COVID-19 health status-even if companies selling the tests are not themselves providing misleading information.
DTC testing by private companies in the realm of genetics has raised important questions regarding privacy and confidentiality of personal data.
62 In the USA, companies may not be subject to laws intended to protect the privacy of health information, such as the HIPAA.
63 Thus, the protection of personal data, including the duration of storage and the third party access to them, is largely determined by terms of service created by the companies themselves.
64 Similarly, many of the companies offering at-home COVID-19 tests, whether diagnostic or serological, may not be covered by HIPAA and the protection of personal data may, therefore, depend on companies' individual policies.
In the COVID-19 context, consumers may erroneously assume, in some cases, that privacy rules governing data in the healthcare setting (such as HIPAA) and doctorpatient confidentiality also apply to DTC companies, giving them a false sense of security.
65 Furthermore, consumers may not realize that for infectious diseases, the limits of confidentiality may be narrower as compared to other types of tests.
More specifically, companies may need to comply with relevant local and state regulations regarding the reporting of positive test results to authorities and may need to disclose protected health information without obtaining prior permission from the consumer.
66 Currently, several state and county health departments require healthcare practitioners and medical laboratories to report COVID-19 cases, providing, amongst other information, the name and address of the patient.
67 While the policies of some companies are more transparent than others, it is likely that consumers will click 'I Agree' to terms and conditions without ever reading them.
This may be especially the case with individuals ordering diagnostic tests, as it is likely that such tests will be purchased under conditions of urgency and anxiety that may not allow for careful review of the relevant contracts.
In addition, given the scarcity of tests, some people may not feel that they have the option to refuse.
Policies regarding third-party access to data are particularly relevant, especially in view of risks of discrimination in the context of employment.
More specifically, it is possible that employers may be interested in accessing serological test results, especially when they are the ones initiating the testing.
This is because confirming that employees have immunity could be relevant for recruitment decisions.
68 For these reasons, it is important for consumers to understand the limits of confidentiality of their health information and the risks of a privacy breach.
In recent years, DTC health testing has been marketed to consumers as an opportunity to access a wide range of health information directly, often completely bypassing the mainstream healthcare setting.
By offering consumers information on genetic susceptibility to multifactorial disorders, carrier status, reproductive health, and infectious diseases, many DTC companies have presented themselves as expanding access to testing and enabling consumers to take control of their health.
However, there have been longstanding concerns that such testing could eventually lead consumers back to the mainstream healthcare system for consultations regarding their test results, creating downstream costs and using resources that, in some settings, may be scarce.
69 For COVID-19 tests, competition over scarce resources may be more direct and tangible.
Currently, there are shortages of basic elements of diagnostic tests, such as swabs and reagents, both in the USA and worldwide.
Such shortages are partly responsible for the inadequate testing capacity in the USA.
70 Despite an increase in the number of tests performed daily since the first month of the pandemic, testing is still not scaled up sufficiently to meet public health needs or consumer demand.
71 In this regard, companies offering DTC COVID-19 testing could provide an alternative for individuals and expand access to testing.
However, given the scarcity of resources, they could also be in direct competition with other healthcare providers, such as hospitals.
The use of scarce resources by companies offering DTC testing during a global pandemic could also raise concerns over fair allocation of such resources.
Previous research has indicated that consumers who purchase DTC health testing tend to be of higher socioeconomic status.
For example, empirical studies of DTC genetic testing have shown that the majority of the consumer base is white, highly educated, and has a higher than average household income.
72 Consistent with such findings, in the context of COVID-19, it is possible that poor and marginalized communities will have less access to DTC tests, because of inadequate financial resources, limited information about the availability of such tests, companies' marketing strategies, or other reasons.
This is particularly concerning given that racial and ethnic minority groups have been disproportionately affected by the pandemic.
73 Currently, several state and county health departments are expanding testing capacity by launching testing sites in underserved communities.
74 In order to avoid two-tiered access to testing based on socioeconomic status or race, it is important that efforts to provide free testing to low-income communities, as well as to communities of color, are sustained and expanded across the USA.
Currently, issues of fair access to COVID-19 testing are more pressing for diagnostic tests, as they have clear clinical utility and are considered more reliable compared to serological tests.
However, considering the ongoing policy discussions in some countries regarding using serological test results as an 'immunity passport' that would allow individuals to return to work, 75 ensuring equitable access to serological testing may become crucial in the near future.
As this article highlights, many of the concerns surrounding DTC COVID-19 parallel those surrounding other DTC health tests that are widely available in the USA.
However, the scale of the present pandemic lends an increased urgency to existing issues.
Given the fast-changing landscape of the COVID-19 pandemic, this article cannot predict or address all issues associated with DTC COVID-19 testing that are likely to arise.
There are, however, several recommendations that can help inform the ethical provision of DTC health tests during this public health crisis.
First, consistent with its obligations under the Federal Food, Drug, and Cosmetic Act, 76 FDA should reassess its EUAs and remove authorization for tests that are found to be of low quality.
Enabling testing to reach the market as quickly as possible is, of course, an important goal.
But testing is not useful if consumers, healthcare professionals, and public health regulators cannot be confident in the results.
Ultimately, FDA must ensure progress in studying and developing high-quality testing continues, and assure available testing meets the conventional-rather the lower, EUAstandards.
Second, all stakeholders should be working to educate policymakers and the public with accurate, non-misleading information about the limits and potential benefits of DTC testing.
For example, companies should provide consumers adequate and clear information regarding their tests, in both communications about how to interpret test results and in promotional materials.
FTC and states should continuously monitor the market and take action when necessary to protect consumers and ensure they have accurate and non-misleading information.
In addition, for companies that are marketing DTC tests under an EUA and that make misleading claims that have a negative public health impact, FDA should make clear it will consider withdrawing the EUA.
77 Third, with regard to privacy, it is crucial that companies provide transparent and easy-to-comprehend privacy policies that are not buried amongst other terms and conditions.
Considering the vulnerability of consumers and the ongoing public health crisis, the FTC should monitor the practices of such companies closely to ensure that such terms are not disproportionate and safeguard the rights of consumers to privacy.
Fourth, equitable access to high-quality DTC tests is critical.
At a minimum, companies should make clear whether their tests are covered by health insurance and whether they provide options for individuals who cannot afford standard prices, whether or not they are insured.
But more is likely to be needed-and if mechanisms for equitable access for COVID-19 testing are successfully developed, they can inform models of access for other kinds of potentially beneficial DTC testing.
Ensuring a market of accurate and ethically marketed DTC COVID-19 tests presents significant challenges for regulators and requires the buy-in of all stakeholders, including industry.
At the same time, regulators and industry may be well-equipped to address many of these challenges, drawing on past experience regulating other DTC tests.
Regulators, industry, and the public also have an opportunity to think carefully about the risks and benefits of different models of DTC testing, and ultimately use the lessons learned from COVID-19 to improve the DTC testing market.
Saliva is More Sensitive for SARS-CoV-2 Detection in COVID-19 Patients Than Nasopharyngeal Swabs, medRxiv Food and Drug Administration, supra note 30 Potential Preanalytical and Analytical Vulnerabilities in the Laboratory Diagnosis of Coronavirus Disease Test Performance Evaluation of SARS-CoV-2 Serological Assays Antibody Tests Go to Market Largely Unregulated, Warns House Subcommittee Chair Direct-to-Consumer Genetic Testing: Perceptions, Problems, and Policy Responses All Your Data (effectively) Belong to us: Data Practices Among Direct-to-Consumer Genetic Testing Firms The Future of DTC Genomics and the Law HIPAA and Protecting Health Information in the 21st Century Privacy in the Age of Medical Big Data Genomic Privacy and Direct-to-Consumer Genetics: Big Consumer Genetic Data-What's in that Contract?
Health System Implications of Direct-to-Consumer Personal Genome Testing, 14 Public Health Genomi HIPAA for Professionals: FAQ Reporting COVID-19/SARS-CoV-2 Infections Employers Rush to Adopt Virus Screening.
The Tools May Not Help Much Testing Remains Scarce as Governors Weigh Reopening States Testing Challenge: Why It's so Hard to Overcome Testing Shortages in the United States A Dire Warning From COVID-19 Test Providers Cities Still Lack Testing Capacity Consumer Perspectives on Access to Direct-to-Consumer Genetic Testing: Role of Demographic Factors and the Testing Experience: Consumer Perspectives on Access to Direct-to-Consumer Genetic Testing Design, Methods, and Participant Characteristics of the Impact of Personal Genomics (PGen) Study, a Prospective Cohort Study of Direct-to-Consumer Personal Genomic Testing Customers The Impact of the COVID-19 Pandemic on Marginalized Populations in the United States: A Research Agenda Covid-19: Disproportionate Impact on Ethnic Minority Healthcare Workers will be Explored by Government Amid Ongoing COVID-19 Pandemic, Governor Cuomo Launches New Initiative to Expand Access to Testing in Low-Income Communities and Communities of Color Denver Launching Coronavirus Testing To Help Communities Of Color County Expands Coronavirus Testing in Hard-Hit Black, Latino communities Louiza Kalokairinou is a Postdoctoral Fellow at the Department of Medical Ethics and Health Policy, Perelman School of Medicine, University of Pennsylvania.
Louiza received a PhD degree in she worked as a Policy Officer in the Research Ethics and Integrity Sector of the European Commission.
Louiza's research focuses the ethical, legal, and social aspects of direct-to-consumer health products and emerging technologies Zettler is an Associate Professor of Law at The Ohio State University Moritz College of Drug Enforcement & Policy Center housed at the College of Law, and a Member of The Ohio State University Comprehensive Cancer Center In addition to Professor Zettler's academic work, she served as an associate chief counsel in the FDA's Office of the Chief Counsel Ashwini received a BA degree in Public Health/Sociology from NYU and Master of Bioethics from the University of Pennsylvania.
She will continue her education at UCLA this fall with a PhD in Health Policy and Management Kyweluk is a Postdoctoral Fellow at the Department of Medical Ethics and Health Policy, Perelman School of Medicine, University of Pennsylvania.
Moira holds a joint PhD/MPH from Northwestern University in Medical Anthropology.
Her research specialization is reproduction in the United States with a focus on assisted reproductive technologies, queer and transgender family building, reproductive health, and peri-conception genetic testing Anna Wexler is an Assistant Professor in the Department of Medical Ethics and Health Policy, Perelman School of Medicine, University of Pennsylvania.
She is the Principal Investigator of the Wexler Lab, which studies ethical, legal, and social issues surrounding emerging technology, with a particular focus on do-it-yourself and direct-to-consumer medicine and science This study was supported by the Office of the Director, NIH, under award number DP5OD026420.
Dr. Kyweluk's work was supported by a postdoctoral training grant from the National Human Genome Research Institute grant number T32HG009496.
key: cord-017413-ymo9h7wb authors: Nurudeen, Sahadat Kemi; Levine, Brian A.; Thornton, Melvin H. title: Selecting and Screening Donors date: 2012-10-19 journal: Principles of Oocyte and Embryo Donation DOI: 10.1007/978-1-4471-2392-7_4 sha: doc_id: 17413 cord_uid: ymo9h7wb Oocyte donation was originally established in 1983 as a treatment option for younger women with premature ovarian failure and for women with severe pelvic disease whose ovaries, as a result, were surgically inaccessible.
The indications for donor oocyte in vitro fertilization (IVF) have now expanded to include not only women with hypergonadotropic hypogonadism but also those with advanced reproductive age, diminished ovarian reserve, significant genetic disease risk, poor oocyte or embryo quality, or multiple failures in prior attempts to conceive using conventional assisted reproductive technology (ART).
Oocyte donation has also been recently used as an important source of material to promote the study of stem cell research.
In these first cases of donation, gametes were obtained primarily from women already undergoing IVF who had excess oocytes at the time of retrieval.
Today, most egg donors are not currently pursing infertility treatment themselves but are willing to donate their gametes for altruistic or commercial reasons.
Since its initiation, oocyte donation services have spread throughout the USA and to many areas of the world.
In the USA, 9,000–10,000 donor oocyte cycles occur annually.
Though donor oocyte IVF is available throughout the USA, globally the practice of oocyte donation varies due to legal restrictions in many countries (Chap.
30).
Oocyte donation was originally established in 1983 as a treatment option for younger women with premature ovarian failure and for women with severe pelvic disease whose ovaries, as a result, were surgically inaccessible [ 1, 2 ] .
The indications for donor oocyte in vitro fertilization (IVF) have now expanded to include not only women with hypergonadotropic hypogonadism but also those with advanced reproductive age, diminished ovarian reserve, signi fi cant genetic disease risk, poor oocyte or embryo quality, or multiple failures in prior attempts to conceive using conventional assisted reproductive technology (ART).
Oocyte donation has also been recently used as an important source of material to promote the study of stem cell research [ 3 ] .
In these fi rst cases of donation, gametes were obtained primarily from women already undergoing Screening of egg donors is an intricate • and multifaceted process that includes obtaining informed consent; securing a detailed medical, genetic, psychosocial, and reproductive history; performing a thorough physical examination; and testing for speci fi c infectious diseases.
Screening criteria proposed by ASRM • recommends that all donors should be in excellent health and without history of hereditary or communicable diseases.
The FDA requires a full historical • assessment of potential infectious disease risk factors as well as the performance of speci fi c tests utilizing nucleic acid testing (NAT) for HIV and hepatitis C within 30 days of egg harvest.
Anti-Müllerian hormone (AMH) repre-• sents an accurate serum marker for ovarian responsiveness to ovarian stimulation and is a useful adjunctive measure in predicting both poor response and hyperresponse in oocyte donors.
IVF who had excess oocytes at the time of retrieval [ 1 ] .
Today, most egg donors are not currently pursing infertility treatment themselves but are willing to donate their gametes for altruistic or commercial reasons.
Since its initiation, oocyte donation services have spread throughout the USA and to many areas of the world.
In the USA, 9,000-10,000 donor oocyte cycles occur annually [ 4 ] .
Though donor oocyte IVF is available throughout the USA, globally the practice of oocyte donation varies due to legal restrictions in many countries (Chap.
30 ).
The compensation and recruitment practices for oocyte donors vary worldwide and largely depend upon current legal or cultural practices in that locale.
For instance, countries, such as the UK and Canada, have strict restrictions on donor compensation, while others, such as Italy and Germany, prohibit compensation [ 5 ] .
Donor compensation guidelines do not exist in the USA, and regional differences in compensation do occur [ 6, 7 ] .
In the USA, donors are recruited mainly through the Internet, television, radio, and newspaper advertisements.
Donors are "matched" to recipient couples often based on educational credentials, extracurricular activities, phenotypic traits, and ethnic origins.
Though providing recipients with the choice of egg donors who exhibit these traits and qualities is important, the selection and screening process is much more comprehensive in order to ensure the safety and health of the donor, recipient, and the offspring.
Screening women interested in becoming oocyte donors is an intricate and multifaceted process that includes obtaining informed consent, taking a thorough medical history, performing a complete medical examination, testing for infectious diseases, providing a genetic screen, and evaluating the donor psychologically.
Ideally, programs want to secure the services of women who are in good health without any past history of risky behavior or familial diseases.
The screening process has evolved since the introduction of oocyte donation with recommendations and evidence provided by the American Society for Reproductive Medicine (ASRM), the US Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDC), and state health departments (Table 4 .1 ) [ 8, 9 ] .
The FDA fi nalized the donor eligibility and the good tissue practice rules to ensure public safety through proper screening for risk factors and testing of donors for pertinent transmissible diseases [ 9 ] .
Before putting potential oocyte donors through the screening process, it should be determined whether they meet the initial requirements for donor selection.
In general, donors should be healthy without a history of hereditary disease.
Donors are recommended to be between ages 21 and 34 years of age according to the 2008 ASRM screening guidelines [ 8 ] .
Using donors less than 21 years of age should be determined on an individual basis after a thorough psychological evaluation by a quali fi ed mental health professional [ 8 ] .
The use of donors over age 34 requires a discussion of the risk of aneuploidy and lower pregnancy rates associated with older women [10] [11] [12] .
Those individuals with employment ties to or fi nancial interests in a donor oocyte program or recruiting agency should not be used as oocyte donors due to the obvious con fl ict of interest.
Prior to participating in oocyte donation, the potential legal, medical, and psychosocial issues involved in the process should be discussed with the donor while obtaining informed consent.
Legal issues include but are not limited to understanding rights to maintain and protect anonymity, discovering outcomes of donation, and whether or not future contact with any resulting children is desired [ 13 ] .
For example, future anonymity could theoretically be reversed in court situations where the importance of the offspring knowing his/her genetic background is deemed to outweigh the donor's desire for privacy [ 13 ] .
Arguments in favor of revealing the donor's identity stem from the fact that children born through donated oocytes were not a part of the original decision-making process.
Though not yet regularly employed by centers nationally, informed consent should also contain information about the disposition of cryopreserved embryos resulting from oocyte donation, i.e., use for future pregnancies or donation for research [ 3, 13, 14 ] .
However, these cryopreserved embryos cannot be reclaimed by donors in the future [ 13 ] .
In summary, the potential donor should consider all legal matters both known and possible during the informed consent process (further discussed under Obligations and Rights).
Detailed medical risks involved with the process of oocyte donation should also be discussed while consenting.
Donors need to be aware of the acute risks and adverse effects of ovarian stimulation and oocyte retrieval, including but not limited to ovarian hyperstimulation syndrome (OHSS), intraperitoneal hemorrhage, and pelvic infection (Chap.
19 ) [ 15 ] .
There may also be risks associated with the use of anesthesia provided during the oocyte retrieval including allergic reaction to anesthetic drugs and respiratory compromise secondary to aspiration.
However, among the young healthy population of donors, this risk is very small [ 16 ] .
In fact, previous studies have suggested that donors are at lower risk for OHSS and other adverse events than patients undergoing autologous IVF [ 17 ] .
Programs may wish to provide donors with adequate supplemental insurance coverage for medical complications arising from oocyte donation, and the terms of such coverage should be disclosed to the donor (further discussed in Chap.
20 ).
The potential increased risk of ovarian cancer should also be introduced during the discussion of the possible medical risks from oocyte donation.
There have been concerns that controlled ovarian stimulation (COS) might increase the long-term risk of ovarian cancer in women using fertility drugs.
Recently published data have not con fi rmed a cause and effect relationship between the drugs used for COS and ovarian cancer [ 18 ] .
However, undoubtedly most donors have either heard about the association of fertility drugs and cancer or read that it exists in the popular media or Internet, and therefore, questions regarding the long-term safety of participation should be anticipated.
Unintended pregnancy in donors discontinuing oral contraceptives in order to participate also occurs.
Potential donors should be counseled on this possibility and offered options for prevention [ 8 ] .
Along with the discussion of pregnancy risk during informed consent, it is important to stress the importance of patient compliance with followup, use of contraception, and/or abstinence in order to lessen the risk of unplanned pregnancy.
Egg donors should be advised about the emotional and psychosocial consequences of participation.
They should understand the potential impact that providing eggs to an infertile woman may have upon their own offspring or future offspring, including whether or not to ever disclose to their own children or spouse the fact that they formerly participated.
Although highly unlikely, there are notable concerns that offspring could potentially marry and procreate with an unrecognized half-sibling [ 16 ] .
In addition, the donor's present or future spouse or partner may have an interest in the outcome of prior oocyte donation, and disclosure may have a negative effect on their relationship [ 13 ] .
Obligations, rights, and duties of donors should be thoroughly explained prior to participation.
For instance, donors may ask about the right to specify the type of recipients that receive their donation, the right to learn about the outcome of their donations, the right to contact any future offspring, and, as discussed previously, their right to anonymity.
Each of these obligations must be agreed upon prior to initiating a cycle of treatment.
Entering the oocyte donation process, some donors believe that they may direct their egg donation to include only speci fi c demographics of recipients.
Such triage might be based on the age of the recipients, or perhaps their marital status, sexual orientation, health, race, religion, or education.
The request of donors to specify to whom their gametes may be given is typically refused by donor programs, except in the obvious cases of designated friends or family members openly participating with known recipients.
The choice to not allow anonymous donors to direct the use of their gametes is currently supported by the Ethics Committee of the ASRM [ 13 ] .
Potential donors should understand that their preferences to donate only to certain types of recipients will likely not be considered in gamete donation.
Furthermore, the future use of embryos created by the donated eggs lies with the recipient and cannot be easily predicted or later controlled.
Programs should give consideration to the fact that donors may express an interest in learning the outcome of their donation.
Whether or not outcomes will be disclosed should be de fi ned prior to participation.
According to current guidelines, programs are not ethically bound to reveal whether or not a pregnancy occurred because the donation is made without regard to the outcome which is consistent with current blood and non-gamete tissue donation practices [ 13 ] .
Disclosing information about cycle success or failure may at times cause unanticipated emotional distress to donors possibly secondary to the news of offspring or, in cases of failure, cause concern about the donor's own fertility [ 13 ] .
Some argue that outcomes should be disclosed because donors deserve to know whether their gametes resulted in a successful pregnancy.
The knowledge of outcomes could be helpful in the event of planned or unplanned contact from the offspring, give donors the opportunity to tell their children about genetic half-siblings, and put psychological closure on their participation in oocyte donation [ 13 ] .
Some programs allow donors the option of learning whether a child was born, yet there exist few research studies to support disclosure or nondisclosure of pregnancy to determine which approach is preferable.
We believe that donors should be asked and documented as to whether they are willing to have contact with any offspring.
Initially, some participants may be content with simply providing their gametes.
However, in the future, some donors may have an interest in knowing their offspring.
On the other hand, donors have the right of not having potential obligations to offspring imposed on them without their consent [ 13 ] .
These are strong considerations, and asking them to anticipate their future inclination is complex given that their feelings may and are likely to change considerably during their lifetime.
There may be competing interests between donors, recipients, and subsequent offspring.
Disclosing to offspring the donor's genetic history does not necessarily require knowing the actual identity of the donor or meeting her.
However, with increasing interest in the issues surrounding future contact between donors and their offspring, there should be some acknowledgement of the potential for new situations and responsibilities to arise concerning participants as regulations and laws change in the future.
It has also been suggested that donors and recipient couples may wish to consider executing legal documents that attempt to de fi ne or limit the rights and duties of each with regard to any future offspring [ 13 ] .
The need for compliance with treatment should be stressed to oocyte donors.
This will assist in maximizing cycle outcomes and minimizing the potential medical risks.
Informed consent requires donors to be forthcoming about their personal medical history and behaviors so that any genetic and/or health issues that affect the well-being of offspring are known in advance [ 13 ] .
It is the responsibility of the donor to update the donor program with any changes to her health or risk factor status [ 8 ] .
However, it is less clear about the donor's obligations after donation to keep the program informed of any changes in her health status.
Programs may encourage donors to provide updates as they encounter medical conditions that may be pertinent to the offspring's health.
Standard operating procedures (SOP) should be in place with regard to medical updates, and programs should clearly convey this to their participating donors and the recipients of donor eggs.
Donors should be assured that their con fi dentiality will be protected as federal and local state laws permit.
The medical records containing the information about their participation will be protected and sustained according to local statutes [ 8 ] .
The FDA requires that the records of donor screening and testing be maintained for at least 10 years; ASRM actually recommends maintaining a permanent record of each donor's selection process, screening, testing, and follow-up evaluations [ 8 ] .
These records, including those with clinical outcomes, should be maintained for any potential information sharing in the future with offspring based on future statutes or permission of the donor.
When evaluating a potential oocyte donor, a comprehensive review of their past medical history is requisite.
According to the recommended screening criteria of the ASRM, the donor should be healthy and give no history to suggest hereditary or communicable disease [ 8 ] .
The goals of screening are to ensure that the donor is not at risk for suffering an untoward event during the stimulation/ retrieval process and to also ensure that the donor is not at risk of transmitting a possible blood-borne pathogen.
The ASRM Practice Committee recommendations for screening oocyte donors are not law and are strictly guidelines from the professional organization based on regulation from the FDA.
ASRM, in congruence with the FDA, has recommended that donor programs not accept donors, who in the past 5 years, have injected drugs for nonmedical reasons, have received humanderived clotting factor concentrates, or have had sex in exchange for money or drugs (Table 4.
2 ) [ 8 ] .
Donors, who in the preceding 12 months, have had sex or close contact with any person having HIV (Table 4.
2 ) [ 8 ] .
The FDA requires the further assessment of potential risk factors based upon the donor's travel history, given that many individuals may be harboring indolent communicable infections.
The criteria clearly recommend rejecting women who spent 3 months or more cumulatively in the UK from the beginning of 1980 through the end of 1996; those who are current or former US military members, civilian military employees, or dependents of a military member or civilian employee who resided at US military bases in Northern Europe (Germany, Belgium, and the Netherlands) for 6 months or more cumulatively from 1980 through 1990, or elsewhere in Europe (Greece, Turkey, Spain, Portugal, and Italy) for 6 months or more cumulatively from 1980 through 1996; those who spent 5 years or more cumulatively in Europe from 1980 until present; those who received any transfusion of blood or blood components in the UK or France between 1980 and the present; those whom sexual partners who were born or lived in certain countries in Africa (Cameroon, Central African Republic, Chad, Congo, Equatorial Guinea, Gabon, Niger, or Nigeria) after 1977; and those who have received a blood transfusion or any medical treatment that involved blood in the countries listed above after 1977 [ 9 ] .
Interestingly, the FDA makes special note about the importance of including a comprehensive review of symptoms to ensure that no donors are included who are at risk for West Nile virus.
Given that the disease can have profound neurologic sequelae, those who are at risk or have symptomatology consistent with an infection are recommended to defer donation for at least 120 days after onset of symptoms or diagnosis, whichever is later [ 9 ] .
Along the same lines of communicable neurologic disease, the FDA bans women from donating who have been diagnosed with variant Creutzfeldt-Jakob disease (vCJD) or any other form of CJD, diagnosed with dementia or any other degenerative or demyelinating disease of the central nervous system or other neurologic disease of unknown etiology, received a non-synthetic dura mater transplant, human pituitary-derived growth hormone, or have one or more blood relatives diagnosed with CJD.
Even those women who have a history of CJD in a blood relative should not be included unless the diagnosis of CJD was subsequently found to be in error, the CJD was iatrogenic, or laboratory testing (gene sequencing) demonstrates that the donor does not have a mutation associated with familial CJD [ 9 ] .
Lastly, given the potential for infection and transmission of pathogens to patients receiving organ transplants, those women who have received xenotransplants (live cells, tissues, or organs from a nonhuman animal source or human body fl uids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells, tissues, or organs), have been in close contact with a xenotransplant recipient, or have received human organ or tissue transplants or treatment with human extracts are not eligible as well [ 9 ] .
Though not listed in the published guidelines, physicians may also consider other aspects the donor's medical history that could preclude or limit participation in egg donation.
Knowledge of current medical conditions, such as polycystic ovarian syndrome, may assist clinicians in selecting appropriate treatment protocols in order to reduce the risk for ovarian hyperstimulation syndrome.
Donors with a history of obesity, endometriosis, or pelvic surgery will alert clinicians to possible diminished ovarian reserve or dif fi culty with ovarian access during retrieval.
Finally, clinicians may consider assessing the donor's family history for other inheritable traits and diseases, such as color blindness, diabetes, or premature ovarian failure.
The screening of oocyte donors must include a thorough and focused physical exam.
When performing the physical exam, the ASRM has outlined the requisite components (Table 4 .3 ) focused on assessing the donor's general health and potential for harboring an infection that may be transmitted to a recipient.
As discussed previously, the donor should also be evaluated for pelvic fi ndings that might complicate the treatment (i.e., polycystic appearing ovaries, endometriosis, and pelvic disease) with a transvaginal ultrasound.
A thorough medical history, as discussed previously, is important in determining individuals at high risk for infection.
The testing for infection among potential donors has been regulated by the FDA and American Association of Tissue Banks (Table 4 .4 ).
Testing should be performed within 30 days prior to oocyte collection, and abnormal results need to be veri fi ed prior to disclosure to the donor [ 8 ] .
During disclosure, centers should have options for counseling and medical referral if needed.
Any positive screening tests will exclude potential donors from anonymous donation except for a history of treated Neisseria gonorrhoeae and Chlamydia trachomatis infections [ 8 ] .
Oocyte donation should be deferred for 12 months after a negative test of cure for donors who have a history of these speci fi c infections [ 8 ] .
However, due to the increased risk of infertility in these women, in general, participation should be discouraged.
In the event that a concern for donor infection arises during a treatment cycle, recipients should be offered the option of embryo cryopreservation and quarantine for 180 days until the donor is tested and con fi rmed negative for infection [ 8 ] .
In such rare events, the program should also properly counsel recipients about their frozen embryo transfer (FET) pregnancy rates and the chance of seroconversion of the donor after cryopreservation of embryos in order for the recipients to make appropriate decisions regarding pursuit of FET.
Non-anonymous donors undergo the same testing as anonymous donors.
However, known donors who test positive are not automatically excluded from oocyte donation according to the current FDA guidelines as long as the physician is aware of the results [ 9 ] .
Though the FDA does not require disclosure of positive test results, ASRM recommends informing and properly counseling recipient couples prior to use of oocytes [ 8 ] .
There are no formal recommendations for testing of the male partners of recipients, but a few tests may be considered.
These include semen analysis, blood type, Rh factor, infectious disease blood work, and genetic screening depending on the male partner's ethnic background [ 8 ] .
[ 7 ] Physical evidence for risk of sexually transmitted disease such as genital ulcerative lesions, herpes simplex, chancroid, and urethral discharge Physical evidence for risk of or evidence of syphilis Physical evidence of anal intercourse including perianal condylomata Physical evidence of nonmedical percutaneous drug use such as needle tracks; the examination should include examination of tattoos, which might be covering needle tracks Physical evidence of recent tattooing, ear piercing, or body piercing Disseminated lymphadenopathy Unexplained oral thrush Blue or purple spots consistent with Kaposi sarcoma Unexplained jaundice, hepatomegaly, or icterus Large scab consistent with recent history of smallpox immunization Eczema vaccinatum, generalized vesicular rash, severely necrotic lesion (consistent with vaccinia necrosum), or corneal scarring (consistent with vaccinial keratitis) and utilize the nucleic acid testing (NAT) for viral particles.
The FDA requires the use of nucleic acid tests because of the ability to detect infection at a signi fi cantly earlier stage than traditionally used antibody or antigen testing [ 9, 19 ] .
There are tests currently available that are also sensitive for the detection of HIV group O antibodies.
Centers that do not have access to the FDA-licensed test for the group O antibodies should evaluate donors for risks associated with HIV group O infection [ 9 ] .
Participants that may be at risk include those who were born, lived in, or received blood transfusion or any medical treatment in Cameroon, Central African Republic, Chad, Congo, Equatorial Guinea, Gabon, Niger, or Nigeria after 1977 [ 8 ] .
Serologic testing for hepatitis B infection utilizes EIA for the detection of antigens and antibodies, speci fi cally the hepatitis B surface antigen and hepatitis B core antibodies (IgG and IgM) [ 8, 20 ] .
These tests will reveal any infected donors and further distinguish between acute or chronic hepatitis.
Though the majority of adults infected with the virus experience recovery, 1-5 % of immunocompetent adults are at risk for chronic hepatitis and thus cirrhosis and hepatocellular carcinoma [ 21 ] .
Viral infection acquired during childhood or the perinatal period has a risk of persistent infection ranging from 20 to 90 % [ 21 ] .
Women infected with hepatitis B infection should be excluded from egg donation and given appropriate recommendations for follow-up with a primary care physician or infectious disease specialist.
Many donors may have received hepatitis B vaccination in childhood or adolescence.
In these instances, past immunization may be con fi rmed by testing the serum for hepatitis B surface antibody [ 20 ] .
Like hepatitis B, chronic hepatitis C infection has the similar sequelae of cirrhosis and hepatocellular carcinoma, and up to three quarters of infected persons are unaware of their infection status [ 22 ] .
Testing for hepatitis C among donors requires serologic EIA testing for the hepatitis C antibody and NAT for viral particles [ 8 ] .
Serologic testing for syphilis is recommended by the FDA for screening and diagnosis because the bacterial source, Treponema pallidum , cannot be cultured [ 8, 23 ] .
Serologic testing of donors initially involves the nontreponemal assays, such as rapid plasma regain (RPR) and venereal disease research laboratory (VDRL) [ 8 ] .
These assays detect antibodies for phospholipids which are present not only on T. pallidum but also occur in autoimmune or in fl ammatory conditions [ 24 ] .
Positive nontreponemal assays should be con fi rmed with FDA-approved treponemal-based assays, such as fl uorescent treponemal antibody (FTA), T. pallidum particle agglutination (TP-PA), or EIA for speci fi c antibodies to T. pallidum [ 8 ] .
Unlike nontreponemal assays, treponemal-based assays remain positive for years after treatment and infection [ 25 ] .
Donors may be eligible for oocyte donation if the treponemal-based assays are negative.
Testing for these two sexually transmitted infections involves cervical cultures or nucleic acid ampli fi cation tests [ 8 ] .
Samples may be taken from urine or a swab from the cervix, urethral meatus, or vagina [ 8 ] .
As discussed previously, egg donors with a history of these infections should generally be discouraged from donation.
Testing potential donors for blood type and Rh factor is not considered mandatory by the FDA or ASRM.
It may be recommended to ensure compatibility with the maternal genotype.
The potential for Rh incompatibility and the obstetric implications (e.g., hemolytic disease of the fetus, hydrops fetalis, and intrauterine fetal demise) should be divulged to recipients.
The minimum genetic screening for oocyte donors involves ruling out any history of Mendelian disorders, such as autosomal dominant disorders, X-linked disorders, and autosomal recessive inheritance.
If donors are from certain ethnic backgrounds that are high risk for genetic disorders, they should be tested for their carrier status.
For example, patients of African or Mediterranean descent should have cell blood counts and hemoglobin electrophoresis to assess any risk for sickle cell anemia or beta-thalassemia among future offspring [ 26 ] .
Those individuals with Southeast Asia ancestry are at high risk for alpha-thalassemia and should be screened with DNA-based testing as they may have normal hemoglobin electrophoreses [ 27, 28 ] .
Donors of Askenazi jewish descent should be screened for Tay-Sachs disease, Gaucher disease, Fragile X syndrome, Fanconi anemia, Canavan disease, Niemann-Pick type A, Bloom syndrome, maple syrup disease, glycogen storage syndromes, familial dysautonomia, and mucolipidosis type IV [ 17, 29 ] .
We believe that all ethnicities should be screened for cystic fi brosis and spinal muscular atrophy (SMA) due to its relatively high carrier rate prevalence.
The carrier frequency of cystic fi brosis among the Caucasian, Hispanic, and African American populations is 1 in 25, 1 in 46, and 1 in 65, respectively [ 30 ] .
The carrier risk of SMA is 1 in 47 among the Caucasian population and 1 in 67 among the Ashkenazi Jewish population [ 31 ] .
Among the Asian, African American, and Hispanic populations, the carrier risk is 1 in 59, 1 in 72, and 1 in 68, respectively [ 31 ] .
Donors heterozygous for autosomal recessive disorders need not always be excluded, but screening requires testing of the carrier status of the recipient's partner.
It is also important to ascertain a history of major malformations of multifactorial or polygenic etiology that are associated with any serious functional or cosmetic handicap (e.g., cardiac and uterine malformations).
Donors are excluded if they have any known karyotypic abnormality or any signi fi cant familial disease with a major genetic component among fi rst-degree relatives (e.g., BRCA-positive breast cancer) [ 8 ] .
Routine karyotyping and testing for Tay-Sachs or Fragile X is not currently recommended by ASRM for donor genetic screening but may also be considered.
A recent retrospective study compared the ASRM screening guidelines with enhanced universal screening, which included Tay-Sachs, Fragile X, and karyotype analysis [ 32 ] .
Over a 12-year period, investigators found an additional 25 candidate exclusions with enhanced universal screening of 1,300 potential donors, making up 19 % of all genetic exclusions [ 32 ] .
Although karyotyping is not customarily recommended in the USA, it is part of donor screening in some European countries.
An IVF center in Valencia, Spain, which regularly analyzes the karyotypes potential donors, found that 1.4 % of karyotypes were abnormal over a 10-year period [ 33 ] .
At this time, enhanced genetic screening does not fall under current ASRM guidelines; however, Counsyl Medical Genomics offers a comprehensive panel of more than 100 recessive single gene disorders that would broadly expand surveillance (Table 4 .5 ) [ 34 ] .
However, until there is more research into the cost-effectiveness of testing and how best to advise patients of the many results, it cannot be routinely recommended.
Restricting the age of potential donors is important in maintaining a successful program, and retrieving a good number of oocytes is central to successful pregnancy outcomes.
Selecting an optimal donor and predicting ovarian response based solely upon age are limited by individual variation in response to ovarian stimulation.
In conjunction with age, ovarian reserve testing among prospective donors is useful in assessing the donor's ovarian reserve status, predicting ovarian response and assessing risk for OHSS.
Anti-Müllerian hormone (AMH) represents an accurate marker of ovarian response to gonadotropin stimulation in IVF cycles.
Several studies have suggested that AMH accurately predicts poor ovarian response [ 35, 36 ] .
Our center found signi fi cant correlations between AMH and the number of oocytes retrieved and estradiol levels among oocyte donors (Table 4 .6 ) [ 37 ] .
AMH may be useful in predicting IVF cycle outcomes and helpful in individualizing dosing protocols [ 37 ] ; however, further studies are needed.
Investigators have also suggested that antralfollicle count (AFC) could be used similarly to predict oocyte donor response to controlled ovarian hyperstimulation [ 38 ] .
Follicle-stimulating hormone (FSH) in conjunction with estradiol has been used for assessment of ovarian reserve; however, they have not been found to reliably predict ovarian reserve in young patients [ 39 ] .
Premature diminished ovarian reserve would be important to ascertain early in the donor screening process not only for the effect on oocyte yield but also for the knowledge of the donor.
The use of CGG triple nucleotide repeats on the Fragile X ( FMR1) gene has also been suggested as an addition to future ovarian reserve testing among oocyte donors given its association with premature ovarian failure [ 40 ] .
One pilot study suggested that FMR1 gene testing in conjunction with age-speci fi c AMH may be a useful adjunct measure [ 41 ] .
Prospective donors should undergo screening for cervical dysplasia in accordance to current recommendations by the American Society for Colposcopy and Cervical Pathology (ASSCP).
At presentation to our center, donors should have record of a normal PAP smear within the past 3 years.
Abnormal cytology or pathology should be referred for appropriate treatment according to current guidelines prior to participating.
Information regarding current and past drug and/or alcohol abuse can be obtained during the donor evaluation of past medical and psychosocial history; therefore, many programs do not routinely perform urine drug screening of donors.
Over a 4-year period of universal drug screening, one center found positive urine toxicology in 7 % of their donor population who initially denied current drug use [ 42 ] .
Though toxicology testing is not routinely performed, centers may wish to consider testing potential donors with a worrisome past medical or psychosocial history.
We routinely test all of our donors, and from 2004 to 2010, we found 3 % of women screened to have positive urine toxicology [ 43 ] .
Our center uses the Quest Diagnostics ten drug urine toxicology panel (Table 4 .7 ).
ASRM currently recommends psychological and social assessment of oocyte donors by a quali fi ed mental health professional [ 8 ] .
Typically, these [ 8 ] .
Presence of signi fi cant psychopathology, positive family history of heritable psychiatric disorders, substance abuse, two or more fi rst-degree relatives with substance abuse, current use of psychoactive medications, history of sexual or physical abuse without professional treatment, excessive stress, marital instability, impaired cognitive functioning, mental incompetence, and high-risk sexual practices may warrant exclusion of the prospective donor [ 8 ] .
Ineligible donors should be explained the reasons for their exclusion with appropriate referral as needed [ 8 ] .
In the case of a known donor, complete psychosocial evaluation and counseling is strongly recommended for both the donor and the recipients in order to fully assess the potential impact of donation, pregnancy, and even treatment failure on their future relationship.
Evaluation needs to rule out any undue fi nancial or emotional coercion or enticement [ 8 ] .
Programs should con fi rm that the donor maintains autonomy in her decision to participate and understands the potential effects on her relationships with the recipients and other family members if she withdraws or continues to participate [ 44 ] .
The details of the psychological evaluation are not speci fi cally de fi ned and vary between centers.
As a result the Mental Health Professional Group created guidelines for psychological testing of prospective oocyte donors.
The Minnesota Multiphasic Personality Inventory (MMPI) has been traditionally used to evaluate oocyte donors in order to differentiate healthy individuals from those predisposed to psychiatric disorders [ 45 ] .
The second edition of this test, MMPI-2, has been shown to distinguish between those individuals who answer truthfully and those who try to underplay any psychopathologic behavior.
Prior studies have found differences in MMPI-2 scores between donors who completed a donation cycle and those who were psychologically excluded, but were not able to reliably differentiate those accepted who would subsequently be noncompliant [ 46, 47 ] .
ASRM guidelines do not currently require psychological testing with MMPI-2, but centers often use this evaluation in addition to their current psychological screening methods to assess potential oocyte donors.
Screening and selection of egg donors is a comprehensive process in order to help protect the safety and health of donors, recipients, and future offspring.
For potential donors, it is a stepwise EMIT enzyme multiplied immunoassay technique sequence of events beginning with the review possible risks associated with treatment.
Once an egg donor consents, after understanding the risks and their rights, duties, and obligations, the process of donor screening and selection belongs to the egg donor program under the guidance and recommendations of the FDA, ASRM, and local state health departments.
Obtaining a thorough medical history, performing a complete physical exam, infectious disease testing, genetic screening, and psychosocial evaluation require a dedicated, organized, and thorough multidisciplinary team of reproductive endocrinologists, nurses, mental health professionals, and social workers.
As further research develops regarding the screening and selection process, there will likely be further changes of the guidelines discussed in this chapter, and it will be prudent to have a multidisciplinary team to help make adjustments to these changes an ongoing and ef fi cient process.
For now, following the current recommendations and guidelines will assist programs in the appropriate selection of oocyte donors and maximize the chances for a successful pregnancy in recipients.
The participation of young healthy women as egg donors has engendered more controversy and public scrutiny than perhaps any other aspect of egg donation.
It has been attacked from the beginning by critics of the method as a dangerous, exploitive, unprofessional, and sexist practice, and yet it endures.
To combat such ferocious criticism, it is imperative that practitioners of egg donation pay careful attention to every aspect of donor recruitment and participation.
Responsible practice fosters good outcomes and continued success.
There will always be a great amount of pressure to either supply donors or accept donors that may be less than ideal (e.g., PCOS, hypothalamic amenorrhea), but doctors need and must be able to say no when faced with choices that pose undue risk to all participating.
For the fi rst decade of practice, the model for screening donors was essentially lifted from the manner in which men were screened prior to donating sperm.
However, sperm and egg donation shares little in common with respect to time involvement and risk of participation, and therefore the need for more speci fi c and detailed professional guidelines and safety measures was necessary to protect the women donating eggs, the women receiving them, and most importantly the child that results from their collaborative efforts.
Dr. Thornton reviews the important basic requirements for establishing an egg donor program.
Many of these professional activities are now being provided by "agencies," which are typically not run by physicians, who then supply patients or programs with "matches."
Regardless of whether an agency supplies the donor or a program chooses to screen potential donors themselves, the outlined steps provided in this chapter are crucial to follow in order to ensure safety and health.
Ultimately, the responsibility falls to the doctors providing the hands-on care and in every case full knowledge of the donor's pedigree, health history, and reason for participating must be known.
During the developmental years of the method, egg and embryo donors were married middle-class mothers in their early 30s.
Today, we are working with the youngest women in the history of the technique, typically in their midtwenties, single, and without children.
Injury to any one of them is catastrophic and ironically may in fact leave them infertile.
We also can assume that some of these women will later experience infertility themselves and naturally will assume that their work as an egg donor contributed to their problem.
Disclosure of all potential risks, including later regret, is dif fi cult to do without frightening potential donors away.
However, I believe it is better to inform each of them of the known complications and lose a few candidates rather than to perform a procedure that later leads to lifelong regret.
It has always been my hope that every egg donor will look back on the experience with pride and satisfaction for having provided a truly unique gift of life.
New advances in the treatment of infertility in women with ovarian failure Establishment of a nonanonymous donor oocyte program: preliminary experience at the University of Southern California Human oocytes reprogram somatic cells to a pluripotent state Clinical summary report: all SART member clinics Oocyte donation: the legislative framework in Western Europe Society for Assisted Reproductive Technology Evaluation of compliance and range of fees among American Society for Reproductive Medicine-listed egg donor and surrogacy agencies Practice Committee of American Society for Reproductive Medicine, Practice Committee of Society for Assisted Reproductive Technology.
2008 guidelines for gamete and embryo donation: a practice committee report Guidance for industry: eligibility determination for donors of human cells, tissues, and cellular and tissue-based products.
US Department of Health and Human Services In fl uence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities Ethics Committee of the American Society for Reproductive M. Interests, obligations, and rights of the donor in gamete donation Informing egg donors of the potential for embryonic research: a survey of consent forms from U.S. in vitro fertilization clinics De fi ning the incidence of serious complications experienced by oocyte donors: a review of 1000 cases Practice Committee of American Society for Reproductive M. Repetitive oocyte donation Oocyte and embryo donation 2006: reviewing two decades of innovation and controversy Breast and ovarian cancer incidence after infertility and in vitro fertilisation Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) part II: immunization of adults Hepatitis B virus infection Self-reported hepatitis C virus antibody status and risk behavior in young injectors Cultivation of virulent Treponema pallidum in tissue culture Laboratory diagnosis and interpretation of tests for syphilis The architect syphilis assay for antibodies to Treponema pallidum: an automated screening assay with high sensitivity in primary syphilis Distribution of hemoglobinopathy variants by ethnicity in a multiethnic state Screening for common nondeletional alpha-thalassemias in Chinese newborns by determination of Hb Bart's using the Sebia Capillarys 2 electrophoresis system High-resolution melting analysis of the three common nondeletional alpha-thalassemia mutations in the Chinese population: Hbs Constant Spring, Quong Sze and Westmead 442: preconception and prenatal carrier screening for genetic diseases in individuals of eastern European Jewish descent Standards and guidelines for CFTR mutation testing Pan-ethnic carrier screening and prenatal diagnosis for spinal muscular atrophy: clinical laboratory analysis of >72,400 specimens Evaluating the necessity for universal screening of prospective oocyte donors using enhanced genetic and psychological testing Sperm and oocyte donor selection and management: experience of a 10 year follow-up of more than 2100 candidates A universal carrier test for the long tail of Mendelian disease Early follicular serum mullerian-inhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles Mullerian inhibiting substance is an accurate marker of ovarian response in women of advanced reproductive age undergoing IVF Anti-Mullerian hormone testing is useful for individualization of stimulation protocols in oocyte donors Antral follicle count (AFC) can be used in the prediction of ovarian response but cannot predict the oocyte/ embryo quality or the in vitro fertilization outcome in an egg donation program A moderately elevated day 3 FSH concentration has limited predictive value, especially in younger women Relevance of triple CGG repeats in the FMR1 gene to ovarian reserve Can egg donor selection be improved?
A pilot study Evaluation of urine toxicology screens in an oocyte donor population Routine toxicology screening of oocyte donors American Society for Reproductive Medicine Ethics Committee.
Family members as gamete donors and surrogates An analysis of social and psychological characteristics of women volunteering to become oocyte donors Analysis of Minnesota multiphasic personality inventory-2 pro fi les of prospective anonymous oocyte donors in relation to the outcome of the donor selection process Minnesota Multiphasic Personality Inventory (MMPI-2) pro fi les in the assessment of ovum donors
key: cord-308284-r546ypur authors: Simpson, Shmona; Chakrabarti, Ajoy; Robinson, David; Chirgwin, Keith; Lumpkin, Murray title: Navigating facilitated regulatory pathways during a disease X pandemic date: 2020-10-23 journal: NPJ Vaccines DOI: 10.1038/s41541-020-00249-5 sha: doc_id: 308284 cord_uid: r546ypur In 2018, the Bill and Melinda Gates Foundation convened over thirty subject matter experts in clinical development, manufacturing, and regulatory assessment to determine how the development and approval of medical countermeasures could be accelerated in the event of Disease X.
Disease X is the result of a presently unknown pathogen with epidemic or pandemic potential.
A key opportunity to accelerate the scientific assessment and regulatory approval of medical countermeasures exists within efficient navigation of facilitated regulatory pathways.
It was identified that not all stakeholders will be able to skillfully navigate the facilitated pathways offered by the various regulatory agencies during a public health emergency.
To democratize this knowledge, we have written an overview of the facilitated approaches which have been developed and refined by Stringent Regulatory Authorities and the World Health Organization for the primary assessment of medical products.
We discuss the conditions necessary for use of these approaches, scenarios in which certain pathways may be applicable, and the pros and cons of these approaches.
We also address opportunities available to developers in, or developers who wish to access, low-income countries that may have nascent regulatory frameworks.
The 2014-2016 Ebola epidemic significantly impacted the global health community.
Between Guinea's index case in December 2013 and the epidemic's end in June 2016, there were 28,000 cases and 11,325 deaths across eight countries 1 .
Despite years of prior research, no products were ready to deploy in time to save these lives.
The question then arose: How many lives would have been saved if effective medical countermeasures had been made available sooner?
The WHO created the Research and Development Blueprint initiative 2 , which asked multiple agencies how to shorten the time to development of medical countermeasures for the world's most deadly pathogens.
The Bill and Melinda Gates Foundation attempted to answer this question for Disease X-the result of a presently unknown pathogen with epidemic or pandemic potential.
In December 2018, the Foundation convened a Disease X working group-comprised of 30 experts in emerging infectious diseases; bioterrorism agents; non-clinical studies; clinical trials; chemistry, manufacturing, and controls; and regulatory scientific assessment 3 .
The goal was to understand how the next epidemic could be controlled using medical countermeasures that arrive sooner than has been the experience to date.
The control of Disease X requires improvements in many areas, including disease surveillance, public health infrastructure, laboratory capacity, product manufacturing, and delivery 4 -however the remit of this working group was to focus specifically on the expedited provision of medical countermeasures.
One of the key challenges identified was a perceived difficulty in navigating facilitated regulatory pathways.
In this Perspective, we summarize some facilitated regulatory pathways available to innovators tackling Disease X and suggest how these may shorten product development and regulatory assessment timelines.
We also discuss the pros and cons of these approaches and suggest in which situations they may be most applicable.
So far, we have identified 50 facilitated regulatory pathways (listed in Supplementary Note 1), in 24 countries around the world.
While many of these have critical nuances and are at various stages of refinement 5, 6 , they cannot all be adequately described in this short Perspective.
A broader set of considerations are available elsewhere 6, 7 .
Rather, some "Stringent Regulatory Authorities" (as classified by WHO) have completed primary assessment of thousands of products using these facilitated approaches.
These include products used in epidemic emergencies.
Many other National Regulatory Agencies (NRAs) have mirrored these processes, and/or rely on the scientific outcomes of these approaches to assist in their own authorization practices.
This Perspective provides a high-level overview of the facilitated approaches which have been developed and refined by Stringent Regulatory Authorities and the World Health Organization for the primary assessment of medical products.
It is possible that these represent the approaches most likely to be relied upon in the next epidemic or pandemic.
We hope this paper will enable academic innovators and small and medium enterprises to navigate the flexibility that exists within regulatory approaches for products that address life-threatening diseases of unmet need.
We encourage developers to make early contact with regulators in their focus countries to discuss which programs may be applicable.
Several potential regulatory scenarios may exist and co-exist during an epidemic: for example, (a) de-novo candidates requiring rapid development and regulatory assessment (b) de-novo products requiring assessment when the typical package of clinical efficacy data may not be available, (c) approval of de novo or repurposed products for "emergency" use only in specific populations (d) for compassionate use in specific (e.g., "named") individuals of an unauthorized medicine (e) conditional or accelerated authorization before the completion of efficacy studies or, (f) use of a licensed product outside of its approved use (e.g., for another indication, dosage regimen, or population).
Many regulatory agencies have instituted various programs to help navigate these scenarios (Table 1) .
These pathways are generally reserved for products that address a serious or life-threatening condition where there is unmet clinical need, or where the current treatment options are unsatisfactory.
Eventual authorization through one of these pathways depends on an evaluation of the known clinical benefits and risks of the product-in the context of the known risks of the disease.
For this, regulators employ a variety of complex modeling and analytic techniques to conduct an assessment of the benefits and risks of the product and any remaining uncertainties, and compare these to the risks of the disease and the available options at the time of application 8, 9 .
In an outbreak, these parameters can be highly variable; they may change depending on the pathogen, co-morbidities, evolution of the epidemic, populations and geographies affected.
However, these agile facilitated regulatory pathways recognize the higher tolerance for unknown risk the community has in these situations: they allow for flexibility on the depth of certain routine data requirements given the specifics of the disease being considered.
Not all of these pathways facilitate a shorter assessment time: some allow for assessment to be conducted at an earlier phase of the typical product development lifecycle in instances where the benefits, at that point in time, outweigh the risks (Fig 1) .
A critical element for successful use of these facilitated pathways is the engagement of regulators early and often: scientific advice and pre-submission meetings are invaluable.
Most Authorities allow rolling submissions of data and rolling reviews under these processes.
This early engagement allows for on-going alignment on development plans as further data become available.
It also allows sponsors to focus on the critical data requirements, identify opportunities for additional product development acceleration, and ultimately save substantial time.
Depending on the product and the clinical situation, a product may be eligible for any or all these facilitated programs and can be candidates in more than one program simultaneously.
Without active regulator engagement, it is often difficult to navigate these approaches and understand the challenges inherent in each.
Regulation through reliance and regionalization are critical elements for broadening the utility of facilitated pathways.
Reliance allows for NRAs to rely on the work product of a trusted Authority to inform their own regulatory decision.
Regionalization extends the utility, allowing neighboring regulatory authorities to workshare, and share work product, within their economic or cultural blocs.
This may mean that regulatory assessment and authorization in one jurisdiction, can de facto facilitate accelerated authorization in another jurisdiction thereby avoiding duplication of effort 10 .
A list of regulatory harmonization initiatives is provided in the Supplementary Note 1.
The following pathways are available under certain circumstances to expedite product development and marketing application assessment.
The United States Food and Drug Administration's (FDA) Priority Review 11 process provides feedback on a marketing application, i.e., an authorization or complete response, within, generally, 6 months, instead of the standard 10 months.
This faster application review is for products that purport to demonstrate significant efficacy or safety over a currently available therapy related to the treatment, diagnosis, or prevention of a serious condition.
This designation, which comes at the time of submission, does not affect the length of the clinical trial period.
Many other regulatory authorities have expedited review timeframes for similar situations, and one should always check to see if the medical countermeasure would qualify in the country in which the product is intended to be used.
Accelerated assessment, under similar situations, is also available at the European Medicines Agency (EMA), reducing review time from 210 days to 150 days.
Japan's Pharmaceutical and Medical Devices Agency (PMDA) also provides a Priority Review option, available to products that address (1) serious diseases, (2) conditions of unmet clinical need, or where superior safety and efficacy can be provided, and (3) orphan designated products.
This option reduces review time from 12 to 9 months 12,13 .
FDA's Fast Track is a program for products that have some initial evidence of efficacy or improved safety over an available therapy 14 .
This designation provides an opportunity for frequent meetings and communication with the FDA.
Most importantly, this program allows a "rolling review" process, in which the marketing application is submitted in pieces as each segment is completed rather than having to assemble the entire application and submit it all at once.
This allows FDA to assess each segment as it is submitted, and thus FDA only must review the last segment when it is completed rather than the entire application.
FDA's Breakthrough Designation 15 is granted where a new candidate demonstrates substantial improvement over an available therapy on a clinically significant endpoint.
Candidates demonstrating an improved safety profile over an available therapy with similar efficacy are also considered.
Efficacy can be demonstrated using a pharmacodynamic biomarker, surrogate or intermediate clinical endpoint providing they strongly suggest a clinically meaningful effect.
For example, Pfizer's 20-Valent Pneumococcal Conjugate Vaccine candidate and Janssen's prophylactic vaccine for the prevention of respiratory syncytial virus both achieved breakthrough designation following a Phase 2 and 2b studies, respectively 16, 17 .
A breakthrough therapy designation enables fast-track designation, intensive guidance on a drug development program, and organizational commitment.
Like FDA's Breakthrough program, investigational products to address unmet medical need are eligible for consideration under the European Union's PRIority MEdicines (PRIME) scheme, providing early clinical data demonstrate potential benefit.
In addition, applicants from the academic sector, small and medium enterprises can engage with the EMA quite early based on compelling non-clinical data and tolerability data from initial clinical trials.
Not exclusive to these applicants, the EMA also offers scientific advice and protocol assistance to ensure the most expeditious experience with applying for market authorization 18 .
PRIME also ties in to accelerated assessment if the data ultimately demonstrate the level of improvement needed for accelerated assessment designation.
The following pathways are reserved for candidates when the benefit risk analysis indicates that access should be granted even if the entire clinical trial process has not been completed.
Generally, these are temporary authorization statuses and are not intended to replace or circumvent ultimately finalizing the clinical trials required to support full market authorization.
As a condition of approval under these pathways, it is general a requisite that the clinical trials be continued until adequate clinical efficacy and safety either are or are not demonstrated.
FDA's Accelerated Approval allows for authorization where efficacy is demonstrated via an unvalidated surrogate endpoint or an intermediate clinical endpoint 19 .
These surrogate endpoints are likely predictors of clinical benefit and require that this be demonstrated by "adequate and well controlled" studies.
Ultimately, the requirement remains to confirm clinical benefit in post-authorization confirmatory trials that validate the approved endpoint.
When these studies are completed, the FDA will review the data and decide if the approval can be converted to a full authorization.
The accelerated approval may be revoked if clinical benefit relative to the risks cannot be confirmed.
To date, 208 compounds have been approved under this pathway.
These range from numerous antiretroviral compounds in the 1990's, to Janssen's Levaquin for aerosolized Bacillus anthracis, to the recent approval of Ismed's Arikayce for treatment of mycobacterium complex in 2018-all averaging an initial accelerated approval time of six months 20 .
Priority medicines with accelerated assessment scientific advice and protocol assistance (PRIME) •A strongly substantiated mechanism of action, preclinical data, and human tolerance data •Academic, small and medium enterprises may apply earlier for advice and protocol assistance Serious or life-threatening condition for which therapy is inadequate.
Rapid assessment, based on early clinical data Japan Pharmaceuticals and medical devices agency (PDMA) Priority review 22 •Available for orphan designated products, and those that receive conditional approval for diseases of unmet clinical need Rapid Assessment, decision received within 9 months Conditional term-limited approval 22 FDA's Expanded Access (EA) is a program designed for patients with an immediately life-threatening disease to access a product that has clinical trial data (putatively showing an acceptable benefit-risk profile)-but does not yet have marketing authorization.
Because the authorization has not yet been granted, the product is considered investigational and therefore written informed consent of the patient must be obtained when used.
Generally, EA is used in situations where alternative therapies are not available.
Which EA program is chosen reflects the perceived need for the product in terms of number of patients.
Under such programs, the product can be used for: (1) a single patient, (2) immediate-size populations that occur after the FDA has received a number of requests for single patient use, or (3) under a "treatment investigational new drug," designation.
These are generally used during the time period after the completion of pivotal trials, but before the authorization is granted.
During this period, there may be large numbers of patients that might benefit from the product during the time the marketing application is being assembled and/or the product is under review 21 .
Options for international expanded access exist in 21 CFR 312.110(b)(ii) 22 which allows for the export from the United States of investigational products for national emergencies elsewhere, with NRA approval in the receiving country.
For example, following promising animal and early-phase clinical studies (PREVAIL II Trial) the FDA supported the use of ZMapp through a standing expanded access protocol prior to completion of the submission, which allowed countries to retain access to vital therapeutic agents 23 .
Manufacturers who provide access to product under one of the "expanded access" programs may only recover the direct costs of manufacturing their investigational product and may not recoup additional costs or make a profit.
In these cases, access to product under an "expanded access" program allows for patients to receive the product when the potential benefit outweighs the known risk in the specific context of the patient(s).
This sometimes provides further valuable data to help support a full marketing application.
The EMA also offers programs to bridge the clinical trial and full authorization gap.
Unlike a typical full marketing authorization, a Conditional Marketing Authorization (CMA) 24 can be granted in instances of unmet medical need where the benefit-risk assessment is positive.
This is based on early data suggesting that the sponsor will be able to provide more complete data within an agreed timeframe to validate the tentative positive benefit-risk profile of the product.
These data generally include more comprehensive clinical efficacy and safety data 25 .
CMA's are generally granted for a 12-month period, after which they are further reviewed in light on any new data available.
If deemed of benefit to public health, the conditional authorization can be extended.
A large number of antiretroviral compounds 26-28 and treatment for multi-drug resistant tuberculosis such as Bedaquiline 29 and Delamanid 30 continue to be marketed conditionally by the EMA as trials are ongoing.
Ultimately, this pathway this may provide a route to full authorization for many of these candidates.
These provisions, both in the US and EU, require postauthorization infrastructure so that further, more comprehensive data can be captured and so that any requisite safety monitoring can be performed.
Such infrastructure often does not exist in lowincome countries.
This makes use of these pathways sometimes difficult, if not impossible, in countries without such needed infrastructure.
A key feature of these mechanisms is that they often feed into each other and are not meant to be mutually exclusive.
For example, in 2018, 60 Degrees Pharmaceutical's Tafenoquine for malaria prophylaxis was awarded both Fast Track and Priority Review designations 31 .
Similarly, a medicine in the PRIME scheme could also be granted conditional marketing authorization during clinical trials, while still benefitting from accelerated assessment and eventual full authorization 32 .
Japan's PDMA offers two conditional approval options; the first is for regenerative products which grants a term-limited approval based on exploratory Phase I and II safety and efficacy data 12 .
Confirmatory studies are required post-market, and applications must be submitted for complete market authorization within 7 years.
This is described as conditional "term limited" approval.
The second conditional approval is applied in instances of a serious disease, disease of unmet clinical need, or where it is too difficult or excessively lengthy to conduct efficacy studies.
In this instance, early phase clinical studies must show some safety and efficacy, and post-market requirements will include surveillance or clinical studies 33 .
Japan's Sakigake (or pioneer) program is available for diseases of unmet clinical need where the sponsor agrees to file the marketing application first in Japan (or simultaneously first in Japan and another country).
For a candidate to qualify for Sakigake, there must be some early phase clinical data and strong non-clinical and mechanism of action data.
This program offers pre-application consultation, expedited review of around six months, and superior support from PDMA.
The re-examination of clinical safety and efficacy data can be lengthened for an indeterminate period of time, in order to strengthen the likelihood of full market authorization 34 .
When conducting clinical trials presents insurmountable logistical, safety, or ethical challenges, the following regulatory approaches allow for efficacy to be demonstrated using an animal surrogate or clinically indicative endpoints alongside human safety data.
These are reserved for when public health need is significant and human efficacy testing is not possible.
In all instances, every attempt is made to procure follow-up analyses of human experience with the product post-authorization.
The FDA's Animal Rule 35 provides a pathway to approve novel candidates in the absence of the demonstration of efficacy in clinical trials.
Where clinical trials are impossible or unethical to conduct, determinations of efficacy are primarily based on studies in well-characterized animal models 36 .
The investigator must provide: • poof that the animal model can provide plausible inference for human efficacy, • proof that the mechanism of action is the same in the model, as it would be in humans, • rationale for the dose provided to humans (which may come from human pharmacokinetic studies and animal efficacy studies).
In these situations, safety is demonstrated using traditional toxicology studies and human experience data (usually from Phase 1 trials in healthy human volunteers).
The chemistry, manufacturing, and controls information needed to support an animal rule application follow FDA guidelines for a routine new drug or biological license application.
This program allows a promising candidate to be authorized for human use where a major public health need is justified.
This authorization is rare: to date only 6 and 8 drug and biologic approvals, respectively, have been granted under this provision 37 .
Built into the "animal rule" is an iterative process where post-authorization human safety and efficacy data can be obtained and used as the product is employed to further refine the product label.
Aside from de-novo product authorization, the animal rule may also be used to gain authorization for a new indication.
In addition to several medicines that have been approved under this provision, the anthrax vaccine BioThrax was approved in 2015 for post-exposure prophylaxis making it the first vaccine to receive approval for a new indication based on the Animal Rule 38 .
In the European Union, an analogous option exists within the provisions of the "Marketing Under Exceptional Circumstances" pathway.
In exceptional circumstances, when an applicant cannot reasonably be expected to conduct clinical trials, they may submit as much clinical safety as efficacy data as possible, alongside a proposal for post-approval studies that would support the original safety and efficacy claims.
Inability to supply a complete efficacy package may occur due to the rarity of the disease, limitations in the present state of scientific knowledge, or when it would be unethical to conduct.
Once marketed under exceptional circumstances, the candidate will be supplied with a summary of product characteristics stating that information regarding the candidate is incomplete, and the label may be updated 39 .
In 2013, the smallpox vaccine Imvanex was marketed under exceptional circumstances after exploratory data demonstrated that protective antibodies could be triggered, with only mild side effects (including in patients with HIV or atopic dermatitis).
To date, the vaccine's benefits and risks continue to be studied in vaccine recipients.
Ultimately true efficacy could only be demonstrated if there is an outbreak of the disease in the future 40 .
The following pathways are reserved for when a national or global public health emergency has been formally declared.
FDA's Emergency Use Authorization (EUA) is enabled once the United States Secretary of Health and Human Services declares a specific national public health emergency 41 .
It considers whether the "known and potential benefits of the product outweigh the known and potential risks" providing that no reasonable alternatives exist for treating the cause of the specified national public health emergency.
Data to support the application may include domestic and/or foreign clinical trial data, in vivo animal data, and in vitro data that provide plausible support for clinical efficacy.
Access to a product under an EUA is limited to the duration of the national health emergency and the specific access caveats imposed.
Thereafter the product is again considered investigational.
For example, during the 2009 H1N1 influenza outbreak, EUAs were granted for the dispensing of Tamiflu (oseltamivir phosphate) and Relenza (zanamivir), and intravenous Peramivir.
These were discontinued in 2010 once H1N1 was no longer deemed a threat to the United States 42 .
This pathway is applicable to medicines, biologics, vaccines, and medical devices, including in vitro diagnostics.
So far, the EUA has been utilized primarily for diagnostic tests for several infectious agents including influenza, anthrax, coronaviruses, ebolavirus, and zika viruses.
For those developing products to be used in countries with nascent or under-resourced regulatory agencies, the WHO has provided the Emergency Use Listing (EUL) process to provide guidance on product quality and use during a public health emergency of international concern (PHEIC) or other specified public health emergency.
Only the Director General of the WHO can authorize the use of the EUL process.
This program was developed and first used for Ebola diagnostics during the 2013-2015 West African Ebola outbreak and was previously referred to as the Emergency Use Assessment and Listing (EUAL) process.
Since then, it has been further refined and renamed, with the most recent guidance issued in early 2020 43 .
This process was used extensively in both the Ebola and Zika outbreaks for in vitro diagnostic products, although the process is clearly intended for medicines and vaccines also.
The first example of a vaccine advancing into the EUL process is the new oral polio vaccine for type 2 virus (nOPV2).
This genetically modified (designed to improve safety relative to the existing oral polio vaccine, mOPV2), oral polio vaccine is being developed by Bio Farma (Indonesia) and PATH.
On-going transmission of type 2 Vaccine-Derived Polio Virus (VDPV) in certain regions of the world has resulted in WHO maintaining its long-standing PHEIC for polio.
Successful completion of the EUL process would facilitate the use of nOPV2 in field campaigns to control VDPV events once an outbreak has been identified.
Use of the EUL process would allow this novel vaccine to be used prior to WHO Prequalification, enabling an improved vaccine to be utilized in this emergency in advance of obtaining all the data required for traditional product licensure pathway.
During this use, data will be gathered to support the traditional WHO Prequalification process and national product licensure pathways.
While the GMP requirements are generally the same as required for the WHO Prequalification program, the efficacy of a vaccine, for example, may be demonstrated by pre-clinical efficacy data in a suitable animal model, alongside clinical immunogenicity that is reasonably predictive of human clinical efficacy.
A plan to monitor safety and efficacy in the field must be included, and an EUL is granted initially for 12 months.
The manufacturer's history of successfully prequalifying products may contribute to the decision -especially if specific manufacturing site inspection is difficult to obtain in a timely manner.
Specific to vaccines, though the WHO has provided the EUL, at least one qualified NRA is responsible for providing "oversight of batch release and other post-EUL product safety and manufacturing quality assurance requirements 44 ."
This is usually the agency in the manufacturing country.
Some countries where epidemics occur are not equipped to do this for certain vaccines.
To mitigate this, national and regional regulatory agencies generally should engage with their global regulatory counterparts and WHO Prequalification in a collaborative approach to product assessment and oversight under emergency circumstances, especially when novel outbreak etiologies and novel therapeutic and prophylactic modalities are being proffered.
When clinical trials have been conducted Routinely, there is little motivation for manufacturers to produce initial quantities of product in excess of what is required for the clinical trials program.
However, in a public health emergency, manufacturers may be called upon to supply larger quantities of product much more quickly.
This may deplete clinical trial supplies.
If adequate forethought about production has not been undertaken, there may only be enough product for early clinical studies: this results in significant delay to commencement of later phase trials or early larger scale use of the product under one of these pathways, especially during an epidemic (Fig.
1) .
Mitigation may involve rapid and significant investment of resources in manufacturing, at risk, prior to clinical proof of concept in order to be able to meet demand if the early data support wider use of the product in emergency situations.
When clinical trials have not been conducted A key element to utilizing the Animal Rule and analogous facilitated programs is the need for a well-validated animal model.
A well-validated animal model may not always exist for "Disease X" and establishing a well-validated animal model can be challenging.
In addition, these pathways that utilize animal surrogates may not be applicable to most outbreak scenarios involving new pathogens.
Once an outbreak is underway it typically would be feasible and ethical to conduct clinical trials at which point authorization under the animal rule becomes less relevant.
Conversely, after an outbreak ends, the opportunity to evaluate efficacy in humans also ends, but at that point the level of urgency also decreases.
For novel pathogens, these approaches remain best reserved for instances where probability of human efficacy is higher and apparent at an earlier stage.
Without this, the chief value of this approach may be prior to an outbreak or after the outbreak has ended with the aim of supporting use rapidly in a potential future outbreak.
The advantage of many of these pathways is that demand for a product with reasonable presumption of efficacy and safety may be met earlier during a public health emergency, than in a traditional product development and marketing application assessment timeline.
In these situations, specific use under emergency authorization may include use in first responders, use in those infected, use in ring programs, and/or use in mass distribution as the situation warrants and as the caveats of the specific emergency use authorization dictate.
Manufacturers must commit to collecting further clinical efficacy and safety data, often including clinical trials where feasible and ethical, in conjunction with this emergency usebased field use.
Depending on the scientific robustness with which such data are collected and analyzed, these data may provide primary and/or supplementary data for later licensure.
A key downside of these approaches is the data may not help differentiate the potential clinical safety risks of the product from the underlying clinical complications of the disease.
Uncontrolled use may confound the long-term safety and efficacy assessments of a product due to the high morbidity and mortality rates during these emergencies.
The studies may also be confounded by other comorbidities and other factors typical of the geography, concomitant use of other interventions, or availability (or lack thereof) of other healthcare and medical supplies.
Mitigation of these challenges involves early discussions with regulators.
This ensures that there is a common understanding about the natural history of the disease (where possible) and thus a way to try to differentiate drug risk from disease risk and differentiate product efficacy from disease natural history.
In addition, where possible, these candidates should be deployed in a controlled clinical study to ensure that the efficacy and safety of the product is appropriately evaluated 45 .
Despite their challenges, these pathways can be used to potentially expedite medical countermeasure availability in a public health emergency for candidates with positive pre-clinical efficacy signals.
While the abovementioned regulatory agencies have instituted various programs to help expedite the development and assessment of products for use during public health emergencies, many low, and lower-middle income countries have nascent and underresourced regulatory agencies.
For products manufactured in, or used in, countries that cannot assure quality standards, WHO Prequalification is a system developed to help procurers of Prequalification eligible products determine if the products they are procuring meets international regulatory standards for product efficacy, safety and manufacturing quality.
Many low-income countries rely on Prequalification listing and the assessment and inspections documents WHO provides them to inform their own national regulatory decision on that specific version of the product.
This is done through the WHO-NRA Collaborative Process.
WHO cannot "authorize" a product; rather, it "lists" the versions of the products when the WHO assessment determines that the clinical and manufacturing data meet international standards.
Like routine product authorizations, routine WHO Prequalification is generally not used in public health emergencies.
Several early engagement opportunities and facilitated accelerated pathways exist when one is focusing on regulators in lowincome countries.
These national agencies are engaging more with their global regulatory counterparts and WHO Prequalification program staff in a collaborative approach to assess and authorize products (both clinical trials applications and marketing applications) under emergency circumstances, especially when novel outbreak etiologies and novel therapeutic and prophylactic modalities are being proffered.
WHO has instituted a number of pre-emergency activities, described in the latest version of the EUL process 43 .
The preemergency activities involve establishment of platforms for collaborations between WHO, subject matter experts, NRAs with special expertize, and NRAs where the products will be used (where they differ).
WHO establishes expert advisory committees to support each stage of the EUL.
In addition, these platforms are used for pre-submission meetings/activities, selection of products, and assessment of submitted data.
These activities allow for accelerated decision making upon declaration of a PHEIC or other covered public health emergency.
A key benefit of these activities is that the NRAs are involved at early phases of product development and participate in the assessment process.
Together, all NRAs and WHO can align on appropriate non-clinical model and clinical study design.
This allows for Phase 2b and 3 trials to commence quickly and with the appropriate assistance levels.
This can clarify clinical trial endpoints that would be supportive for EUL by WHO and in the target countries.
An additional opportunity for clinical trial discussions are regional health agreements in Sub-Saharan Africa 46-48 , South America 49 , and Asia 50 : these facilitate work-sharing and joint decision-making.
For example, the Africa Vaccine Regulatory Forum (AVAREF) 51 is a continental platform of regulators sponsored by WHO AFRO and WHO-Geneva which coordinates the joint regulatory and ethics board assessments of multinational clinical trials in Africa.
Innovator engagement with AVAREF, for example, allows the benefit of joint scientific advice and clinical trial assessment meetings.
In addition, AVAREF has established an emergency clinical trial assessment process for multi-country clinical trials.
Originally designed only for vaccine trials, AVAREF, despite its anagram, is also available for use with multinational clinical trials application assessment for medicines in Africa.
Outside of the WHO EUL, two other pathways exist to specifically support lower income countries with facilitated assessment, particularly of novel products.
They both bring the resources of an agency with specific expertize and WHO to conduct scientific assessments of clinical development programs and marketing authorization applications with opportunities for engagement in the discussions by NRAs from the countries where the product will most likely be used.
The use of such pathways brings low income country regulators into the development and assessment processes as a partner so that use of the outputs of the process can be utilized more readily by NRAs where the product will ultimately be used.
Article 58 of European Commission's Regulation No 726/2004 is a specific framework, in collaboration with WHO, designed to support lower income countries regarding products to be marketed outside of the European Union 52 .
The EMA will assess products of major public health interest, collaborating with the WHO and the NRAs in the countries of use.
Regulators, experts and observers from lower income countries participate in the scientific review of the product -both during the development phase and during the marketing application phase.
The EMA publishes a scientific opinion regarding the marketing application.
This allows under-resourced NRAs to make a decision that leverages the EMA's assessment (including good clinical and manufacturing practice inspections) and their engagement with EMA and the WHO during the development and marketing application assessments.
Recently, an option has been provided for total or partial fee waivers for the manufacturer 53 .
While not marketed in the EU, the malaria vaccine RTS,S/AS01 received a positive scientific opinion under Article 58 following trials in seven African countries 54 .
Likewise, in a special program to support access to innovative products in low-income countries, Swissmedic deploys the Marketing Authorization for Global Health Products (MAGHP) program.
It performs similarly to the EMA's Article 58 regarding assessment of product development packages and marketing authorization applications in conjunction with WHO and the NRAs from the countries where the product will be used.
The difference is that the product, if the assessment is positive, will receive a Swiss marketing authorization even if it will not be used in Switzerland.
In the EU, as the product is not intended for use in the EU, the result of the process is a positive opinion, but not a European marketing authorization.
Both of these programs aspire to facilitate a reduction in timelines for development and authorization of products intended solely or primarily for use in lower income countries, thus making needed medicines available faster 55 .
In the near future, regulatory agencies in lower income countries, that have not yet done so, must also become equipped to undertake post-EUL oversight and vigilance surveillance requirements.
In addition, they must develop local frameworks to allow emergency use authorization of products during public health emergencies, and that allow the use of candidates that may lack human efficacy data but have both recognized animal efficacy data and initial human safety data.
Accelerating availability of effective, safe, quality products is essential in a public health emergency.
Depending on the context, the feasibility of clinical trials, the strength of animal or clinical surrogate data, and the initial safety profile of the product, one facilitated pathway may be pursued over another or several of these pathways may be pursued simultaneously or sequentially.
In these situations, regulators are generally quite willing to discuss putative development plans and regulatory pathways with product developers.
Developers should take advantage of such opportunities: these are key to accelerated product development, marketing authorization assessment, and patient access under these facilitated pathways.
Generally, this is an iterative process, with decisions being made and modified as further data regarding the emergency and product become available.
Meeting demand via these pathways in the case of a large public health emergency will require robust pre-clinical studies and significant at-risk investment in scaling manufacturing ahead of clinical proof of concept.
Because of the rapidly changing nature of public health emergencies, and the requirement for a well-validated animal model, certain pathways may not be able to be utilized in a public health emergency.
Historically, most regulatory pathways used in public health emergencies rely on some human efficacy data.
Pathways that bring together the manufacturer, NRAs where the product is going to be used, NRAs with specific needed expertize, WHO, and regulatory and clinical experts will accelerate the availability of needed novel medical countermeasures.
The benefits of such rapid development could have major impacts, both in terms of lives saved and reduction in disease spread and intensity.
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First Malaria Vaccine Receives Positive Scientific Opinion from EMA Guidance Document Scientific Advice MAGHP AUTHOR CONTRIBUTIONS L contributed equally to the conceptualization of the manuscript.
S.S. designed and authored the manuscript, table and figure.
A.C. and M. M.L critically revised and approved the final version The authors declare no competing interests.
Supplementary information is available for this paper at https://doi.org/10.1038/ s41541-020-00249-5.Correspondence and requests for materials should be addressed to S.S.
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key: cord-018770-uy76mc2j authors: Connelly-Smith, Laura S. title: Donor Evaluation for Hematopoietic Stem and Progenitor Cell Collection date: 2019-11-28 journal: Best Practices of Apheresis in Hematopoietic Cell Transplantation DOI: 10.1007/978-3-319-55131-9_4 sha: doc_id: 18770 cord_uid: uy76mc2j With the increasing incidence of hematopoietic allogeneic cell transplantation (allo-HCT), the importance of securing a cellular product, safely from a donor, and ensuring that the product is without additional risk to the recipient, continues to be of paramount importance.
The evaluation of the donor’s medical eligibility and suitability is designed to identify and limit the risk of transmitting infectious, genetic, or neoplastic diseases to the recipient through the product.
It also aims to ensure a maximum level of safety for the donor and informs them of the risks of donation.
Several regulatory agencies, national and international registries, and accreditation bodies have facilitated the availability and safe provision of human cells, tissues, and cellularand tissue-based products not only at local institutions but also through international exchange.
Hematopoietic cell transplantation (HCT) remains a potentially curative treatment for life-threatening hematological and non-hematological diseases.
Over the last several years, the total number of HCTs performed worldwide has exceeded 60,000 a year (Niederwieser et al.
2016; Gratwohl et al.
2010 ).
Autologous hematopoietic cell transplantation (auto-HCT) accounts for the majority of all procedures performed, and in the United States, the number continues to increase at a fast rate, mainly from transplants performed for plasma cell and lymphoproliferative disorders extending to older patients (Gratwohl et al.
2010 ; Center for International Blood and Marrow Transplant Research (CIBMTR) 2016).
Allogeneic HCTs (allo-HCT) have exceeded 30,000 per year worldwide with the number of transplant recipients surpassing 8000 a year in the United States (Center for International Blood and Marrow Transplant Research (CIBMTR) 2016).
Approximately 70% of allogeneic transplants use hematopoietic progenitor cells (HPCs) from volunteered unrelated donors (URDs).
Advances in HLA typing, new immunosuppressive protocols, improved supportive care, and the administration of nonmyeloablative (NMA) or reduced-intensity conditioning (RIC) regimens contribute to the increased frequency of HCT.
The observed continuous annual increase of around 10% is mainly because of a rise in allo-HCT from URDs (Gratwohl et al.
2010 (Gratwohl et al.
, 2013 .
There has also been an increase in alternate donor sources with HLA-haploidentical donors now exceeding umbilical cord blood transplants (Center for International Blood and Marrow Transplant Research (CIBMTR) 2016).
Although CD34 + cell donation by apheresis is considered a relatively safe procedure with very low rates of serious adverse events (Schmidt et al.
2017) , the risk of both physical and psychological harm exists.
At the same time, there is also potential harm to any recipient through the infusion of the graft, especially by communicable diseases.
For allogenic donors, it is important to optimize the whole donation experience as these donors will undergo a procedure for which they will not be receiving any direct benefit.
There is however a potential sense of satisfaction derived from this altruistic act (Boo et al.
2011) .
Therefore, for URD, an excellent reputation of a safe and efficient process is needed to ensure adequate number of donors being maintained and joining the national registries (Billen et al.
2014) .
Pretransplant donor evaluation is an essential process to safeguard the quality and safety of donation.
The primary goals of allo-HCT donor evaluation are to ensure that a) there is minimal risk to the health of the donor from the collection procedure and b) to protect the recipient from transmissible diseases.
On May 25, 2005, the US Food and Drug Administration (FDA) implemented comprehensive regulations governing the collection and manufacture of human products for transplantation and immune modulation, as well as a variety of other cellularand tissue-based human products (Food and Drug Administration 2005) .
These regulations are based on the FDA's responsibility to limit the transmission of infectious diseases through the administration of these products and apply to peripheral blood stem cells, cord blood, and donor lymphocytes.
The responsibility for bone marrow regulations has been assigned to the Health Resources and Services Administration (HRSA).
The FDA regulations include the requirements for establishing donor eligibility and apply not only to products collected or manufactured within the country, but also to those imported from outside the United States (Food and Drug Administration 2005) .
Other international regulatory bodies, for example, European Directives for Donation of Tissues and Cellular Therapy Products (Human Tissue Authority (HTA) Regulations 2007) also have detailed requirements for donor evaluation to ensure the safety of the product for the recipient; however, unlike FDA regulations, they do not address donor safety issues.
Given the extensive international collaboration and exchange of HPC products, most regulatory agencies work closely with national registries, such as the National Marrow Donor Program (NMDP) and the World Marrow Donor Association (WMDA).
These national registries develop and establish appropriate guidance to ensure HPC donation is performed safely and ethically in volunteer URDs and have published their recommendations for donor evaluation (Sacchi et al.
2008; Lown et al.
2014 ; National Marrow Donor Program (NMDP) n.d.-a).
Donors are assessed as to their suitability and eligibility to donate HPCs.
Donor suitability refers to the general health or medical fitness of any autologous or allogeneic HPC donor to undergo the collection procedures.
Donors are evaluated as to their risk and overall safety to donate.
Donor eligibility refers to issues that relate to an allogeneic donor for who all screening and testing has been completed in accordance with applicable laws and regulations and who has been determined to be free of risk factors for relevant communicable diseases.
URDs are only eligible if they are unrestrictedly healthy.
Often however, physicians struggle with decision making as to the suitability of a relative as a donor that would not otherwise meet the suitability criteria for unrelated donation.
The suitability criteria for related donors (RDs) is often less strict and with considerable variability between transplant centers.
Differences between RDs and URDs may exist in mobilization and collection practices (Sacchi et al.
2008; Confer et al.
2011; O'Donnell et al.
2010; Clare et al.
2010) .
Published data suggest that the risks for serious adverse events and reactions might be higher for RDs than for URDs, but the amount of adequate prospective data in the RD setting is still limited (Halter et al.
2009; Kodera et al.
2014 ).
Many institutions have developed their own processes for the evaluation of RDs; historically, there had been no national guidance available.
In 2015, the Worldwide Network for Blood and Marrow Standing Committee on Donor Issues developed a consensus document with recommendations for donor workup and final clearance of family donors that would otherwise not be able to serve as URD because of age or preexisting diseases (Worel et al.
2015) .
The FACT-JACIE (Foundation for the Accreditation of Cellular Therapy/Joint Accreditation Committee ISCT and EBMT) international standards were founded in 1994 to address obstacles faced when transplantation involves donors and recipients in different countries.
This voluntary organization establishes international guidelines for the collection and transfer of hematopoietic stem cells.
Members include donor registries, cord blood registries, and numerous individuals working together to advance HCT.
FACT/JACIE addresses issues, including donor evaluation criteria, a donor follow-up policy, and the requirement that "Allogeneic donor suitability should be evaluated by a physician who is not the physician of the recipient."
Accreditation is the means which a center can demonstrate that it is performing a required level of practice in accordance with agreed standards of excellence.
Essentially it allows a center to certify that it operates an effective quality management system.
In many countries, however, accreditation is not mandatory for centers assessing RDs.
Improved compliance with internationally recognized donor care paradigms have been seen in centers with FACT-JACIE accreditation; however, important practice gaps in all centers irrespective of accreditation continue to be seen (Anthias et al.
2016a, b) .
Other organizations that provide additional insight into US regulations regarding donor evaluation include the AABB, the American Society for Blood and Marrow Transplant (ASBMT), the International Society for Cellular Therapy (ISCT), and the Center for International Blood and Marrow Transplant Research (CIBMTR).
Similar to blood transfusion, HPC donation has the potential to transmit a wide range of blood-borne diseases.
For example, hepatitis B (Lau et al.
1999 ), hepatitis C (Strasser and McDonald 1999; Shuhart et al.
1994) , human T-lymphotrophic virus type 1 (HTLV-1) and type 2 (HTLV-2) (Kikuchi et al.
2000; Ljungman et al.
1994) , Chagas disease (Villalba et al.
1992) , malaria (Mejia et al.
2012) , syphilis (Naohara et al.
1997) , and brucellosis (Ertem et al.
2000) have all been reported to be transmitted by HPCs.
In the United States, strict federal regulations regarding the evaluation of HPC donors are laid out in Title 21 of the Code of Federal regulations; Part 1271 (Human cells, Tissues and Cellular-and Tissue-Based Products).
Subpart C is Donor Eligibility Determination and lays out the requirements for donor screening and testing for "relevant" communicable disease agents and diseases (RCDAD) ( Table 4 .1).
Relevant communicable disease agents and diseases (RCDADs) are identified by the FDA as having the potential to cause significant pathogenicity to recipients of human cells, tissues, and cellular-and tissue-based products and are defined as infections that The FDA identifies specific RCDADs by listing them either specifically in the CFR or by publishing a guidance document to communicate any changes.
Some institutions and accreditation bodies may choose to include evaluation of other agents or diseases such as malaria.
To determine eligibility, donors need to be screened and tested for RCDADs.
Assessing the risk of disease transmission involves three components (Food and Drug Administration 2005) : 1.
Targeted screening history 2.
Examination for physical signs of disease 3.
Laboratory testing for specific pathogens or traits A screening history involves interviewing the donor about their medical history and relevant social behavior.
It includes the review of relevant medical records for clinical evidence of RCDADs.
The FDA recommends that the screening interview be a documented dialogue, administered by phone or in person, with appropriate follow-up or verification by a trained individual if the donor health history is selfadministered.
Various registries have developed HPC donor-screening questionnaires and their use recommended, to elicit medical history and to identify high-risk behaviors associated with risk of disease transmission (AABB n.d.-a; National Marrow Donor Program 2002) .
The screening history should also include communicable disease risks associated with xenotransplantation.
One such questionnaire that is freely available is the hematopoietic progenitor cell (HPC), Apheresis and HPC, Marrow Donor History Questionnaire (DHQ) (Appendix 4.1) developed by the AABB Inter-organizational DHQ-HPC Task Force to provide establishments with a standardized tool to screen allogeneic HPC donors for communicable disease risk factors in accordance with requirements of the FDA, AABB, FACT, and the NMDP (AABB n.d.-a).
These DHQ materials are periodically reviewed to ensure continued compliance with regulatory and accrediting agencies.
Companion documents provide rationale for the questioning and recommendations for evaluation of responses (AABB n.d.b) .
Institutions are notified of any changes as well as the timeline for implementation through existing publications and websites maintained by members of the task force.
When a new version of the documents is posted, the previous version is maintained for a period of time to allow facilities to transition to the new version.
The NMDP has developed similar medical history questionnaires to support its work with unrelated donors (https://network.bethematchclinical.org/workarea/downloadasset n.d.).
In the process of completing the DHQ, clinical staff must verbally interact with the donor to review and verify donor's responses to the DHQ and to ensure the DHQ was signed and dated.
All donors should have appropriate age-related donor health questionnaires with a parent or legal guardian (proxy) when required for age.
Appropriate arrangements must be made for donors with developmental delays, appropriate interpreters for nonnational-speaking patients.
Donors who are not English or native speaking in the country of assessment should have a medical interpreter who is not a family member or friend of the family.
A physical examination should be performed to identify any signs or stigmata that may indicate high-risk behavior for or infection with RCDAD(s).
The examination should include recent tattoos, piercings, or signs of intravenous drug use, as well as signs of significant illnesses to determine eligibility for the donation procedure.
Several institutions have developed a supplemental examination checklist (Appendix 4.2) to ensure a thorough examination for signs or stigmata of RCDADs.
In accordance with FDA regulation, laboratory testing using FDA-approved assays must be performed on the donor' blood for, at least, the following infectious disease agents: human immunodeficiency virus 1 and 2 (HIV 1 and 2), hepatitis B virus (HBV), hepatitis C (HCV), Treponema pallidum (syphilis), human T-cell lymphotrophic virus I and II (HTLV I and II), and cytomegalovirus (CMV).
The FDA has provided core requirements for laboratory testing (Table 4 .2).
For emerging infectious diseases including the Zika virus (ZIKV), severe acute respiratory syndrome (SARS), and West Nile virus (WNV), additional screening questions were emergently added to the donor qualification process in the United States, based upon recommendations from the FDA.
WNV is only infectious during the viremic phase and NAT testing must be performed concomitantly with product collection (or within 7 days before or after collection).
While it might not be possible to prevent the infusion of an infected product, knowing that a product was infected with WNV would provide an opportunity to develop a preemptive treatment strategy.
In the United States, WNV testing is to be performed specifically between June 1 and October 31 of each year.
For all other establishments and intending to import human cells, tissues, and cellular-and tissue-based products into the United States, testing of human cells, tissues, and cellular-and tissue-based products donors for WNV should be performed year-round.
It is also desirable to perform testing for prior infections with varicella zoster virus (VZV) and Epstein-Barr virus (EBV) and possibly others, such as (Table 4 .3) -Laboratories used for laboratory testing must be certified under the Clinical Laboratory Improvement Amendments of 1988 (42 U.S.C.
263a) and 42 CFR part 493, or equivalent requirements as determined by the Centers for Medicare and Medicaid Services (CMS) -Testing must be performed in accordance with the manufacturer's instructions for use (IFU) -For Hematopoietic Stem/Progenitor Cell (HSPC) Donors the laboratory specimen to be used for donor testing may be collected up to 30 days prior to or within 7 days after human cells, tissues, and cellular-and tissue-based products recovery.
For all other cells or tissue from the donor, laboratory testing must be performed at or up to 7 days before or after recovery toxoplasmosis.
Positive tests for exposure to these agents may not preclude donation or make the donor ineligible but may modify the transplant approach or posttransplant surveillance strategies.
All RCDAD screening results should be communicated effectively to the collection center as well as to the physician responsible for accepting the human cells, tissues, and cellular-and tissue-based products.
This notification should be part of a standard procedure and clearly documented.
Any human cells, tissues, and cellularand tissue-based products donor whose specimen tests positive (or reactive) using any of the assays is considered ineligible (exception syphilis and CMV screening).
Confirmatory tests should be considered when a positive (or reactive) screening test result is received for such purposes as donor counseling or investigating discordant test results.
If a confirmatory test is performed and is negative or nonreactive, these results would not override a positive or reactive screening test and the donor still remains ineligible.
Screening tests for syphilis are the exception.
Because of the potential for false-positive results in nontreponemal testing, if a specific treponemal confirmatory test is negative, the donor will be deemed eligible from syphilis standpoint.
A donor who tests positive or reactive for CMV is not necessarily ineligible to donate human cells, tissues, and cellular-and tissue-based products.
A positive or reactive (past or recent exposure (IgG or IgM)) CMV test result should also be communicated to the physician responsible for accepting the human cells, tissues, and cellular-and tissue-based products.
In case of a positive IgM CMV, it is best to exclude CMV seroconversion.
After completion of donor eligibility screening history, physical examination, and laboratory tests, written donor eligibility determination is required for all human cells, tissues, and cellular-and tissue-based products donors, except for autologous use.
All human cells, tissues, and cellular-and tissue-based products must not be transplanted, infused, or transferred until the donor has been determined to be eligible, unless (1) there is no other appropriate donor and the proposed donor poses less risk to the recipient than not using the donor and (2) approval is obtained from the recipient to proceed with transplantation using these cells.
This often poses concern because information about donor health is strictly confidential and can only been released with explicit permission from the donor.
If HPC collection proceeds with an ineligible donor, written justification is needed and shall be documented.
The results of these RCDAD screening tests must be reviewed prior to initiating preparative conditioning therapy in the recipient.
If the time between initial donor evaluation and collection is delayed, repeat testing may be necessary.
In the event of missing or incomplete screening test results at the time of HPC collection, the product should be labeled clearly by the collection center that the product has not been evaluated for infectious disease markers.
Donors are declared as ineligible, with processing centers having policies and procedures in place for the storage and release of "ineligible donor" products.
All donors must be medically evaluated to detect conditions that might significantly increase donor risk to unacceptable levels and to ensure their safety to donate.
Peripheral blood hematopoietic progenitor cell (HPC) donation typically involves the administration of 4 or 5 daily injections of granulocyte-colony stimulating factor (G-CSF) and/or other mobilizing agent followed by apheresis collection.
For autologous patients, mobilization commonly includes G-CSF +/− plerixafor or chemotherapy.
Side effects of HPC mobilization with G-CSF or other mobilization agent(s) and apheresis collection should be taken into consideration when assessing donor suitability.
The designated physician (or appropriately licensed supervised advanced practitioner) performs a medical history and physical examination according to standard medical practice.
Medical records should also be reviewed as part of the assessment.
The history not only provides an additional opportunity to review/ affirm questions provided on donor screening health questionnaire but looks to evaluate current health.
Typical questions to be covered during history taking are seen in Table 4 .4.
The physical examination will also include assessment of signs/stigmata of RCDADs (Appendix 4.2).
Vital sign testing, height, weight, noting Karnofsky-or Lansky-performance scores, and assessment of venous access are an essential part of the physical examination.
Laboratory testing and other investigations are also required to evaluate a donor's suitability (Table 4 .5).
The NMDP has developed several tools or lists of clinical disorders/diseases to assess an URD donor's health and RCDAD risk (National Marrow Donor Program (NMDP) n.d.-a).
Several centers often use these tools as guidance for their RDs.
Donors with atypical responses to screening questions, history, and physical examination must be evaluated on a case-by-case basis to determine the donor's eligibility and suitability.
The individual performing or evaluating the health screening, history, and PE should be knowledgeable by training or experience to accept or defer donors.
In general, donors with moderate or severe organ impairment should be deferred; this includes donors with coronary artery disease and renal or hepatic impairment.
Occasionally, a medical condition is identified that does not warrant Complete blood count (CBC) with differential and reticulocyte count Electrolytes (Na, K, CO 2 , chloride), blood urea nitrogen (BUN) and creatinine, alkaline phosphatase, lactate dehydrogenase (LDH), alanine aminotransferase (ALT, SGPT), glucose, serum total protein plus albumin, or serum protein electrophoresis ABO, Rh typing, antibody screening Infectious disease markers (IDMs) (see above) CMV antibody screening (see text) Serum beta-HCG pregnancy (if female of child-bearing potential) Malarial testing if donors travelled to malaria endemic areas Screening for hemoglobinopathy (e.g., SickleDex or equivalent) If donating for Thalassemia patient, thalassemia screening for hemoglobin A, A2, and F urinalysis Tuberculosis testing as clinically indicated Oxygen saturation Chest X-ray and EKG as clinically indicated.
Chest X-ray and EKG are not routinely required However, they may be performed at the discretion of the examining medical professional or the collection facility/donor center physicians based on medical assessment Criteria for whom to perform an EKG may include • History of diabetes mellitus (DM) • History of cardiovascular disease (CVD) • Treatment with digoxin or diuretics • Pulmonary disease (room air O 2 < 90%) • Smoking >20 pack years • Age over 40 (males) and over 50 (females) • If a delay in donor collection of more than 30 days repeat EKG may be required in certain cases such as history of DM, CVD, and treatment with digoxin or diuretics.
Otherwise for other donors this can be repeated if more than 6 months since the last EKG Criteria for who to perform a chest X-ray may include • History of pulmonary disease • Oxygen saturation <90% immediate deferral, but may require further investigation.
Any referral to a specialist or additional workup required should be expedited and the recipients team should be informed as soon as possible so that the transplant clinicians can determine whether or not the donor, if found to be suitable, would be available in a timely manner.
If a donor is deemed unsuitable but a decision is made that there is no other suitable donor available and the donor is prepared to take a reasonable risk, a justification must be documented.
In the event that the transplant procedure is delayed, collection or transplant facilities may require repeat donor assessment within a specified time.
The NMDP requires that donor assessment is always current to within 12 weeks (3 months) of the proposed collection date.
This includes a repeat administration of a screening questionnaire with additional tests to ensure continuing medical suitability based on updated information provided.
There are no mandatory tests and NMDP does not require any extended testing when less than 6 months have passed since the original physical examination date.
Laboratory markers for RCDADs however will need to be repeated within 30 days from collection of HPCs (Table 4 .2).
Additional risks for recipient safety following donation, other than infectious diseases, that need to be assessed during evaluation of the donor include autoimmune diseases (ADs), inherited diseases, and malignancy.
The development of an AI disorder from a donor with the same condition has been reported and includes thyroid disease (Olivares et al.
2002; Thomson et al.
1995) , diabetes mellitus (Lampeter et al.
1998) , psoriasis (Snowden and Heaton 1997) , and vitiligo (Campbell-Fontaine et al.
2005) .
Inherited diseases within the hematopoietic system that will be transmitted include hemoglobinopathies such as sickle cell disease, thalassemia, congenital platelet disorders, and inherited bone marrow failure syndromes.
Transmissions of malignant diseases from donors to patients have been reported in the past, most of them inadvertently from subclinical malignant disease or diseases not recognized by the current screening methods.
The risk for transmission of tumors is assumed to be of a very low incidence.
These rates do not include secondary malignancies of donor cell origin arising in the recipient after allo-HCT.
In addition, patients with a history of heparin allergy, heparin intolerance, or heparin-induced thrombocytopenia are at increased risk for complications with infusion of heparin-containing products.
This is essentially important if heparin is used as part of the anticoagulant during the apheresis collection process.
Donor evaluation provides an ideal opportunity to get full informed consent.
The donor would require a comprehensive discussion of potential risks and "theoretical donor safety" issues.
The donor should be aware that they are not obliged to donate, even if for a family member.
There should be no coercion and it is essential that allogeneic donor suitability should be evaluated by a physician who is not the physician of the recipient.
If the donor consents to donation and then chooses to pull out of their decision after the recipient has started conditioning treatment, the potential risks to the recipient should be discussed fully with the donor.
The most suitable donor for younger patients who undergo allo-HCT is often a minor sibling.
In rare cases, children may also be considered as potential donors for an adult sibling, parent, or other family member.
Worldwide data indicate that more than 30% of children undergoing HCT receive allografts from siblings under the age of 18 (Miano et al.
2007) .
The use of minors as HPC donors is considered medically safe (Pulsipher et al.
2005) and legally accepted given that no alternative approach of comparable effectiveness exists; however, donation of HPCs is not without risk (Pulsipher et al.
2013; Styczynski et al.
2012; Grupp et al.
2006 ) and appropriate medical evaluation of the donor is essential.
The source of the graft (peripheral blood vs. bone marrow) has the greatest influence on the type of adverse events that may present.
It is important to note that in children majority of grafts are of bone marrow origin.
Side effects include pain, either from G-CSF treatment, placement of central venous catheter (CVC), or the puncture wounds made when harvesting bone marrow.
Most young donors will require a CVC for apheresis, thus, exposing them to potential risks such as bleeding, infection, pneumothorax, and complications of sedation or general anesthesia (Pulsipher et al.
2005; Styczynski et al.
2012) .
Collection of peripheral blood graft requires special attention in children, with the use of growth factors being the main issue.
Long-term adverse effects from a brief treatment course with G-CSF for the harvest of HPCs via apheresis continues to be studied in ongoing investigations, but to date, no convincing evidence has shown significant health risks (Pulsipher et al.
2006 ).
The worldwide network for blood and marrow transplantation (WBMT) recommends G-CSF is used with caution and only when needed and emphasize the need for long-term follow-up for these donors (Halter et al.
2013) .
Several published findings suggest that pediatric donors may experience psychosocial issues around the time of and following donation including higher anxiety and lower self-esteem than non-donors (Packman et al.
2008) , moderate levels of post-traumatic stress, depression, behavioral problems, identity problems, guilt, and resentment (Packman et al.
1997 (Packman et al.
, 2008 Wiener et al.
2007) .
Young donors may also fear the medical aspects and pain involved in donation and experience anxiety and ambivalence about donation (Kinrade 1987; MacLeod et al.
2003) .
Although parents for the majority consent to medical interventions on behalf of their children, respecting a child's autonomy and obtaining a child's assent or appropriately regarding his or her dissent or refusal-is generally thought to be of paramount ethical importance.
Decision makers are burdened with great responsibility: their choice will have life-and-death consequences for another vulnerable child.
Recognizing that HPC donation has no physical benefit to these young donors and its associated with potential risks, the American Academy of Pediatrics Committee on Bioethics (AAPCOB) (Committee on Bioethics 2010) has published guidelines specifying when minors may ethically serve as HPC donors.
The AAPCOB has deemed that children may ethically serve as hematopoietic stem cell donors if five criteria are fulfilled (Table 4 .6).
A donor advocate with expertise in pediatric development (second physician or a child life specialist) should be appointed for all children who have not reached the age of majority (age at which a person is recognized by state law to be an adult) and who are being evaluated as hematopoietic graft donors.
The donor advocate must be independent of the team responsible for direct care of the recipient to ensure that the AAPCOB recommendations are met.
He or she should ideally be involved from the onset, starting with the decision about whether the minor should undergo HLA testing so that potential family or sibling donors with medical or psychological reasons not to donate would not be HLA typed.
Donors with medical conditions should be carefully examined by skilled professionals, and if their risks of complications with collection are increased, they should be deferred.
In the advancement of the effectiveness of different hematopoietic stem cell transplants, research is often needed to be performed on donors and/or recipients.
When the donor is a minor, the research must conform to the federal regulations governing pediatric subjects.
This may require national review when the research imposes more than minimal risk without prospect of direct benefit to donor subjects.
Several publications have addressed this area and should be considered before donors are evaluated for research (Wendler et al.
2016; Shah et al.
2015) .
With the increased availability of NMA conditioning over the last two decades (Pingali and Champlin 2015; Alyea et al.
2005) , and improvement in supportive care, the ability of many older patients to tolerate allo-HCT has now become apparent.
For older patients, an HLA-matched sibling is often a donor.
Unlike URD registries, there are no strict age limits recommended for related allogeneic donors.
There is experience available in the literature for donors up to the age of 75 years.
Many health disorders are more prevalent with increasing chronological age, including cardiovascular, cerebrovascular and peripheral vascular disease, chronic airways diseases, diabetes mellitus, malignancies, etc., and must be taken into consideration by any provider assessing the suitability of an older individual to donate.
In some reports, HPC collection by apheresis seems to be a safe procedure for donors ≥60 including those with significant comorbidities (Ghada et al.
2006 ).
However, certain complications are more frequent in the older donors and have demonstrated more procedure related complications than younger donors (Lysák et al.
2011) .
For example, one study demonstrated higher complications associated 1.
There is no medically equivalent histocompatible adult relative who is willing and able to donate 2.
There is a strong personal and emotionally positive relationship between the donor and recipient 3.
There is a reasonable likelihood that the recipient will benefit 4.
The clinical, emotional, and psychosocial risks to the donor are minimized and are reasonable in relation to the benefits expected to accrue to the donor and to the recipient 5.
Parental permission and, where appropriate, child assent have been obtained with hypocalcemia, thrombocytopenia, and problems with venous access in donors ≥55 years of age compared with younger donors (29% vs. 15%, P = 0.0096).
Venous access complications were also more frequently present in donors with circulatory system diseases (arterial hypertension, chronic venous insufficiency) compared with the donors without this medical history (11% vs. 3%, P = 0.006) (Lysák et al.
2011) .
A recent related-donor safety study, looking at health-related quality of life issues among older related HCT donors (>60 years) compared to younger adult counterparts, showed very few differences in indicators in physical and mental health donation-related experiences (Switzer et al.
2017 ).
This may suggest that older sibling donors do not experience the donation process as significantly more physically or psychologically impactful than their younger counterparts and, in some aspects, their experiences were more positive-for example, less donationrelated pain and less anxiety about donation.
There was less conclusive evidence supporting the procedure in sibling donors as old as mid-70s (Switzer et al.
2017) .
Regarding graft composition, some authors have found that in older donors may be different from that obtained in younger donors (Al-Ali et al.
2011; Richa et al.
2009; Miller 1996) with CD34 + cells in the peripheral blood and apheresis yield being lower in older donors (Richa et al.
2009; Suzuya et al.
2005) .
One study noted the failure of mobilization (collection of less than 2 × 10 6 CD34 + cells/kg of recipient body weight) rate at 7% in the older donor group (≥55 years) versus 0.8% in the younger donor group.
It was noted, however, that in donors younger than 50 years, the relationship is not statistically significant and is no longer an independent prognostic factor, also seen by other studies (Ings et al.
2006) .
Several studies have however reported contradictory results regarding donor-predicting factors for mobilization and yield and cannot confirm an independent influence of age on mobilization (Bagnara et al.
2000; Miflin et al.
1996; Rinaldi et al.
2012) .
There is some suggestion that the conflicting results are likely due to often small sample sizes and heterogeneous treatment with mobilizing regimens (Lysák et al.
2011) .
In autologous transplantation, elderly patients can have a high risk of poor mobilization (Goker et al.
2015) .
Some studies reported that CD34 + cell mobilization in patients of advanced age (70 years and older) with multiple myeloma was poor but still possible (Morris et al.
2003) .
This is contrary to that reported suggesting no differences in the mobilization kinetics between younger (<65 years) and older (≥65 years) myeloma patients (Jantunen et al.
2006 ).
Other investigations into whether age affects mobilization in autologous transplantation has also been contradictory in donors <70 years old (Bensinger et al.
1994 (Bensinger et al.
, 1995 .
Therefore, age can be a confounding factor in autologous stem cell mobilization.
Several donor factors predict outcome after allo-HCT and age is one of the important non-HLA factors affecting the survival rates after transplantation (Kollman et al.
2001) .
Clinical practice often prefers "HLA-matched siblings" as first-line donors for transplantation despite donor's age; however, the survival rates for unrelated donor transplants with young fully HLA compatible donors are similar to those using older sibling donors (Kollman et al.
2016) .
Allo-HCT from older adults have been associated with higher nonrelapse mortality (NRM) but donor age was not associated with relapse (Kollman et al.
2016 ).
Observed higher rates of grade II to IV acute GvHD after transplantation of grafts from older donors may be explained by replacement of naïve T-cells with memory T-cells as the immune system ages in the older donors (Miller 1996) .
On occasion, the only matched related donor identified may be an individual who has a known psychological/psychiatric disorder, and the decision for any physician to deem this prospective donor suitable may be very difficult indeed.
In 2013, the WBMT standing committee on donor issues held an international workshop to develop a consensus document with recommendations of suitability criteria for final donor workup in family donors and included donors with psychological-psychiatric disorders (Worel et al.
2015) .
These recommendations as well as recruitment assessment tools such as those used by NMDP registries may be helpful for physicians who have concerns about suitability in these donors (National Marrow Donor Program (NMDP) n.d. -a, n.d.-b) .
Donors with a history of substance abuse may not be automatically deferred, but require a careful history and medical assessment.
Donors should be assessed for risk factors for infectious diseases or underlying psychiatric disorders.
Compulsive dependence on a chemical can cause various physical ailments such as liver damage secondary to alcohol abuse.
In the case of infrequent substance abuse with marijuana alone, individuals are mostly suitable but may require cessation of use before donation or initiating G-CSF.
Donors with a previous history (and not currently using) of cocaine, crack, and methamphetamine (intranasal/oral) abuse might also be suitable; however, the use of these drugs has been associated with an increased risk of cardiovascular disorders, and careful assessment of the donor is required.
In intravenous drug abusers, donation is generally not recommended due to the increased risk of communicable diseases such as HIV, hepatitis B, and hepatitis C with contaminated needles.
Individuals who are on a substitution program but otherwise healthy may be suitable.
Donors with eating disorders (anorexia and/or bulimia) are suitable only if their disease is stable under appropriate treatment and their BMI is >16.0 in adults (Worel et al.
2015) .
These potential donors should be deferred if their overall physical status (including body size, demeanor, skin color, etc.)
indicates serious health concerns.
HPC donation in individuals with multiple personality disorders and psychosis is generally not recommended.
Subjects with obsessive-compulsive, attention deficit, or attention-deficit hyperactivity disorders are suitable if their disease is well controlled.
However, the donor's capacity to follow through the donation process may be affected.
In donors with underlying psychiatric disorders such as anxiety, depression, and bipolar disorders or in donors where there is concern that donors may not followthrough with donation, bone marrow harvest procedures may be questionable and apheresis collection and cryopreservation should be considered in advance before the conditioning regimen is started.
Certain medication may potentially defer a donation or render a donor ineligible (Table 4 .7) due to concern for potential RCDAD transmitted by transfusions and HCT.
Donors would be declared ineligible but may be able to donate dependent on institutional practice.
For the majority of potential donors, it is not usually the medication that they are taking that is likely to be a concern, but rather the underlying medical condition for which that treatment was prescribed, that may make a donor unsuitable to donate.
Certain medications would potentially increase donor or recipient risk, but these are often also required to treat a medical condition that would likely defer the donor as well (Table 4 .8).
For certain medication for which the donor's medical conditions are well controlled, the donor may be suitable to proceed with donation (Table 4 .8).
For donors on lithium, due to its interaction with GCSF, HPC collection using apheresis is generally not allowed and these donors may be considered and evaluated for marrow donation.
If a donor or a recipient has a past allergic reaction to heparin or a history of heparin-induced thrombocytopenia (HIT), the donor may donate by apheresis; however, the anticoagulant use for both circuit and product should be with ACD-A (i.e., citrate) alone.
• Human growth hormone.
Concern for Creutzfeldt-Jakob disease (CJD) • Donors with diabetes previously receiving bovine insulin.
Concern for new variant CJD the same agent responsible for bovine spongiform encephalopathy (BSE) or "Mad Cow Disease" • Hepatitis B immune globulin (HBIG) used to prevent infection following an exposure to HBV.
HBIG does not prevent HBV infection in every case and if a donor has taken it in the last 12 months HBV can still be transmitted • Unlicensed vaccine is usually associated with a research protocol and the effect with regard to stem cell recipients is unknown The FDA identified ZIKV as a RCDAD.
The potential risk of transmission of ZIKV by HCT/Ps was supported by evidence that ZIKV has been detected in tissues such as semen and placenta.
In March 2016, no FDA-cleared diagnostic tests for ZIKV were available and the FDA provided donor screening recommendations to reduce the risk of transmission of ZIKV by HCT/Ps (Food and Drug Administration 2016) .
All donors of HCT/Ps should be considered ineligible if they have had a medical diagnosis of ZIKV infection in the past 6 months and resided in, or travelled to, an area with active ZIKV transmission within the past 6 months.
Donors were also declared ineligible if they had sex within the past 6 months with a male who was known to have either of the risk factors.
The first few blood transfusion transmissions that have been reported were in Brazil, where four transmissions occurred from three donors.
On August 26, 2016, FDA issued revised guidance, recommending that blood centers in all states and the United States territories screen individual units of donated whole blood and blood components with a blood-screening test authorized for use by FDA under an investigational new drug (IND) application, or with a licensed test when available.
In late 2016, blood centers began implementing investigational blood tests with nucleic acid testing (Goodnough and Marques 2017) .
As of April 2017, there remained no commercially available diagnostic test cleared by FDA for the detection of ZIKV.
Current tests with IND include serologic tests (to assess whether individuals who may have recently been exposed to ZIKV were actually infected) and PCR or NAT tests (to diagnose acute/active ZIKV infection).
There is currently no mandate to perform laboratory testing for ZIKV in HCT/ Ps; however, several centers are currently using IND serological or NAT tests available to them.
In the event that laboratory testing is performed, attention should be given to the following: The donation of HPC is a well-recognized and regulated procedure that is performed on thousands of patients and donors throughout the world annually.
Donation of autologous HPC is part of a treatment plan with high-dose therapy in these patients aiming for potential cure or at least prolonged remission from their underlying malignancy.
The aim of their donated HPCs is to "rescue" the patients' marrows from the myeloablative chemotherapy received at the time of transplant for which a patient needs to be reasonably medically fit to receive.
In these patients, suitability for HPC collection is often determined at the time of deeming the patient a suitable candidate for auto-HCT.
The majority of severe complications are often associated with the pancytopenia accompanying chemomobilization.
As a result of this as well as the predictability of cytokine only mobilized collections, several centers now collect autologous donor HPCs from using G-CSF with/without plerixafor as mobilization agent(s) only.
These patients need to be assessed for suitability to donate; however, as the HPCs infused are their own, there is less concern for transmission of communicable diseases and eligibility to donate is not needed (Food and Drug Administration 2005) .
Allogeneic HPC donation is a safe procedure with very low rates of serious adverse events.
The side effects commonly faced during donation are transient for the majority of both related and unrelated donors.
However, there have been several donationrelated deaths (Halter et al.
2009 ), mostly in the related donor setting.
As the majority of fatal and serious adverse events have occurred in donors with preexisting medical issues, it is suspected that robust donor assessment procedures will reduce fatal complications.
Therefore, all donors must be carefully evaluated and fully informed prior to HPC donation by clinicians with good understanding of the potential physical and psychological complications and factors that may increase risk.
As discussed, donors must also be able to provide informed consent without coercion or pressure and for this the medical evaluation of any allogeneic donor should never be conducted by a physician in the same transplant team caring for the recipient.
In addition to suitability determination, donor eligibility determination is also essential and physicians evaluating allogeneic donors should be up to date with regulations and laws governing screening requirements for RCDADs.
These are important particularly with the emergence of new diseases such as that seen with WNV, SARS, and ZIKV.
Several regulatory agencies, registries, and accreditation bodies ensure steps taken to improve donor and patient safety alike.
National and international registries continue to provide updated recommendations for the safe selection of unrelated donors and provide tools and recent guidance that could be extrapolated and used in the related donor setting (Sacchi et al.
2008; Lown et al.
2014; Worel et al.
2015) .
Donor and collection centers should be encouraged to enroll in accreditation bodies, such as FACT/JACIE and AABB, to enable potential improvements in the standard of donor evaluation and collection as well as to ensure continuous improvements in their own quality management system.
Despite 3-5-year survival rates being nearly similar between matched URD and sibling RD HCT (Horowitz 2012) , the higher incidence of GvHD often assumes a matched sibling as the transplant physician's first choice for the majority of transplant indications.
In light of this as well as the notable increase in the use of related HLA-haploidentical transplants (Center for International Blood and Marrow Transplant Research (CIBMTR) 2016), RD will continue to need appropriate evaluations as to their medical suitability to donate.
There continues to be concern about the heterogeneity in the care of related HPC donors (O'Donnell et al.
2010) .
Changes to FACT standards (The Foundation for the Accreditation of Cellular Therapy (FACT) 2017) addressed some of these issues and there has since been some improvement in the practice of adult related-donor care (Anthias et al.
2016a, b) .
However, there still appears to be particular concerns including counseling and assessment of donors before HLA typing, with the use of unlicensed mobilization agents, and the absence of long-term donor follow-up (O'Donnell et al.
2010) .
The World Marrow Donor Association (WMDA) brings forward a compelling argument for the management of RD to be performed by donor registries by offering an established structure for donor care, and extensive experience in the medical evaluation of donors.
In particular, they suggest there should be significant consideration for registry provision of centralized donor follow-up (Anthias et al.
2015) .
Donor long-term follow-up is an important aspect of donor evaluation and further development of follow up of donors should be an integral part of a donor program to allow vigilance and surveillance of donations and improve knowledge of the risks of donation.
At the end of 2011, a US appeals court ruled that it was now legal to pay apheresis donors for their HPC (Medpage Today 2012).
Unlike bone marrow tissue, it was felt that peripheral blood HPC are no different from other body fluids like semen and plasma where national organ transplant act (NOTA) does not prohibit paid donors.
In a concession to the spirit of NOTA, it was deemed that the compensation could not be in the form of cash but rather a voucher that can be applied to things such as scholarships, education, housing, or donation to a charity.
In 2011, the WMDA put out a position statement why HPC donors should not be paid (Boo et al.
2011) .
Reasons included ethical concerns raised by remuneration, potential to damage the public will to act altruistically, the potential for coercion and exploitation of donors, increased risk to patients, and harm to local transplantation programs and international stem cell exchange, and the povssibility of benefiting some patients while disadvantaging others.
Had a transplant or other medical procedure that involved being exposed to live cells, tissues, or organs from an animal?
41.
Has your sexual partner or a member of your household ever had a transplant or other medical procedure that involved being exposed to live cells, tissues, or organs from an animal?
42.
Have any of your relatives had Creutzfeldt-Jakob disease?
Areas to be evaluated and documented during history and physical examination (H&P) of potential allogeneic/syngeneic donors of peripheral blood stem cells or marrow.
Note in Comments location, severity, and/or physical findings.
Physical evidence of non-medical percutaneous drug use such as needle tracks, including examination of tattoos, which may be covering needle tracks Comments: Physical evidence of recent tattooing, ear piercing, or body piercing Comments: Oral thrush Comments: Blue or purple spots consistent with Kaposi's sarcoma Comments: Unexplained jaundice, hepatomegaly, or icterus Comments: Physical evidence of sepsis, such as unexplained generalized rash Comments: Large scab consistent with recent smallpox immunization Comments: Generalized vesicular rash (generalized vaccina) Comments: Severely necrotic lesion consistent with vaccina necrosum Comments: Apheresis and HPC, Marrow DHQ Version 1 AABB (n.d.-b) AABB HPC, Apheresis and HPC, Marrow DHQ Version 1 AABB Medication Deferral List The impact of the age of HLA-identical siblings on mobilization and collection of PBSCs for allogeneic hematopoietic cell transplantation Comparative outcome of nonmyeloablative and myeloablative allogeneic hematopoietic cell transplantation for patients older than 50 years of age Related hematopoietic cell donor care: is there a role for unrelated donor registries?
European Group for Blood and Marrow Transplantation Centers with FACT-JACIE accreditation have significantly better compliance with related donor care standards Significant improvements in the practice patterns of adult related donor care in US transplantation centers Hemopoiesis in healthy old people and centenarians: well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and remodeling of cytokine network Peripheral blood stem cells (PBSCs) collected after recombinant granulocyte colony stimulating factor (rhG-CSF): an analysis of factors correlating with the tempo of engraftment after transplantation Factors that influence collection and engraftment of autologous peripheral-blood stem cells A review of the haematopoietic stem cell donation experience: is there room for improvement?
Remuneration of hematopoietic stem cell donors: principles and perspective of the World Marrow Donor Association Adoptive transfer of vitiligo after allogeneic peripheral blood stem cell transplant Center for International Blood and Marrow Transplant Research (CIBMTR) (2016) Summary Slides Centers for Disease Control and Prevention (n.d.) Areas with risk of Zika Virus National Notifiable Diseases Surveillance System (NNDSS) Management of related donor care: a European survey Children as hematopoietic stem cell donors WMDA guidelines for subsequent donations following initial BM or PBSCs Brucellosis transmitted by bone marrow transplantation Donor Screening Recommendations to Reduce the Risk of Transmission of Zika Virus by Human Cells, Tissues, and Cellular and Tissue-Based Products.
Guidance for Industry Zika virus response updates from the FDA Human cells, tissues, and cellular and tissue-based products; donor screening and testing, and related labeling Apheresis safety and product yield among elderly donors for allogeneic sibling hematopoietic stem cell transplantation (HST) Optimizing mobilization strategies in difficult-to-mobilize patients: the role of plerixafor Zika virus and patient blood management Hematopoietic stem cell transplantation A Global Perspective Hematopoietic stem cell transplantation activity in Europe Use of G-CSF in matched sibling donor pediatric allogeneic transplantation: a consensus statement from the Children's Oncology Group (COG) Transplant Discipline Committee and Pediatric Blood and Marrow Transplant Consortium (PBMTC) Executive Committee Severe events in donors after allogeneic hematopoietic stem cell donation Allogeneic hematopoietic stem cell donationstandardized assessment of donor outcome data: a consensus statement from the Worldwide Network for Blood and Marrow Transplantation (WBMT) Does matched unrelated donor transplantation have the same outcome as matched sibling transplantation in unselected patients?
Peripheral blood stem cell yield in 400 normal donors mobilised with granulocyte colony-stimulating factor (G-CSF): impact of age, sex, donor weight and type of G-CSF used High-dose melphalan (200 mg/m 2 ) supported autologous stem cell transplantation is safe and effective in elderly (>or =65 years) myeloma patients: comparison with younger patients treated on the same protocol Allogeneic bone marrow transplantation-related transmission of human T lymphotropic virus type I (HTLV-I) Preparation of sibling donor for bone marrow transplant harvest procedure PBSC collection from family donors in Japan: a prospective survey Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age The effect of donor characteristics on survival after unrelated donor transplantation for hematologic malignancy Transfer of diabetes type 1 by bone-marrow transplantation Hepatitis B virus infection and bone marrow transplantation Infection of donor lymphocytes with human T lymphotrophic virus type 1 (HTLV-I) following allogeneic bone marrow transplantation for HTLV-I positive adult T-cell leukaemia Unrelated adult stem cell donor medical suitability: recommendations from the World Marrow Donor Association Clinical Working Group Committee.
World Marrow Donor Association Clinical Working Group Committee Efficacy and safety of peripheral blood stem cell collection in elderly donors; does age interfere?
Pediatric sibling donors of successful and unsuccessful hematopoietic stem cell transplants (HCST): a qualitative study of their psychosocial experience Ruling on Paying Stem Cell Donors Stands Peripheral blood stem cell transplant-related Plasmodium falciparum infection in a patient with sickle cell disease Haematopoietic stem cell transplantation trends in children over the last three decades: a survey by the paediatric diseases working party of the European Group for Blood and Marrow Transplantation Stem cell mobilization in normal donors for allogeneic transplantation: analysis of safety and factors affecting efficacy The aging immune system: primer and prospectus Mobilization of CD341 cells in elderly patients (>/5 70 years) with multiple myeloma: influence of age, prior therapy, platelet count and mobilization regimen Positive seroconversion syphilis in a patient with acute lymphocytic leukemia after allogeneic bone marrow transplantation National Marrow Donor Program, Minneapolis National Marrow Donor Program (NMDP) (2016) National Marrow Donor Program (NMDP) (n.d.-b) Assessing Non-Medical Factors Affecting Donor Suitability Hematopoietic stem cell transplantation activity worldwide in 2012 and a SWOT analysis of the Worldwide Network for Blood and Marrow Transplantation Group including the global survey Practice patterns for evaluation, consent, and care of related donors and recipients at hematopoietic cell transplantation centers in the United States Autoimmune thyroiditis after bone marrow transplantation in a boy with Wiskott-Aldrich syndrome Psychosocial consequences of bone marrow transplantation in donor and nondonor siblings Sibling perceptions of the bone marrow transplantation process Pushing the envelope-nonmyeloablative and reduced intensity preparative regimens for allogeneic hematopoietic transplantation Safety and efficacy of allogeneic PBSC collection in normal pediatric donors: the pediatric blood and marrow transplant consortium experience (PBMTC) Weighing the risks of G-CSF administration, leukopheresis, and standard marrow harvest: ethical and safety considerations for normal pediatric hematopoietic cell donors Acute toxicities of unrelated bone marrow versus peripheral blood stem cell donation: results of a prospective trial from the National Marrow Donor Program Older age but not donor health impairs allogeneic granulocyte colony-stimulating factor (G-CSF) peripheral blood stem cell mobilization Efficacy and safety of peripheral blood stem cell mobilization and collection: a single-center experience in 190 allogeneic donors Haematopoietic stem cell donor registries: World Marrow Donor Association recommendations for evaluation of donor health Retrospective analysis of 37,287 observation years after peripheral blood stem cell donation Children as hematopoietic cell donors in research: when is it approvable?
Marrow transplantation from hepatitis C virus seropositive donors: transmission rate and clinical course Development of psoriasis after syngeneic bone marrow transplant from psoriatic donor: further evidence for adoptive autoimmunity Hepatitis viruses and hematopoietic cell transplantation: a guide to patient and donor management Risk of complications during hematopoietic stem cell collection in pediatric sibling donors: a prospective European Group for Blood and Marrow Transplantation Pediatric Diseases Working Party study Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors Health-related quality of life among older related hematopoietic stem cell donors (>60 Years) is equivalent to that of younger related donors (18 to 60 years): a related donor safety study The Foundation for the Accreditation of Cellular Therapy (FACT) (n.d.).
International standards for cellular therapy product collection, processing and adminstration Transmission of thyrotoxicosis of autoimmune type by sibling allogeneic bone marrow transplant Acute Chagas' disease in a recipient of a bone marrow transplant in Spain: case report Research involving pediatric stem cell donors: a way forward IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations 2016 Hematopoietic stem cell donation in children: a review of the sibling donor experience Suitability criteria for adult related donors: a consensus statement from the worldwide network for blood and marrow transplantation standing committee on donor issues
key: cord-001513-p7v5p036 authors: Ekins, Sean; Freundlich, Joel S.; Coffee, Megan title: A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus date: 2014-12-12 journal: F1000Res DOI: 10.12688/f1000research.5741.2 sha: doc_id: 1513 cord_uid: p7v5p036 We are currently faced with a global infectious disease crisis which has been anticipated for decades.
While many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the Ebola or similar filoviruses that cause haemorrhagic fever.
Two recent high throughput screens published in 2013 did however identify several hits that progressed to animal studies that are FDA approved drugs used for other indications.
The current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore.
This ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored.
A recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein 35 (VP35).
When receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on FDA approved drugs.
These previously identified FDA approved drugs with activity against Ebola were therefore docked into this protein.
The antimalarials chloroquine and amodiaquine docked favorably in VP35.
We propose that these drugs identified to date as inhibitors of the Ebola virus may be targeting VP35.
These computational models may provide preliminary insights into the molecular features that are responsible for their activity against Ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested.
The current Ebola virus (EBOV) crisis has demonstrated that globally we are not prepared to respond with therapeutics to treat existing infections or act as prophylactics as there is no Food and Drug Administration (FDA) or European Medicines Agency (EMEA) approved therapeutic.
More importantly this suggests we should have been prepared for a pathogen which has been known about for nearly forty years.
The current EBOV outbreak is already proving remarkably costly in terms of the mortality and financial ramifications 1, 2 .
The best approaches to EBOV so far have relied on public health measures for containment 3 which have been used in past outbreaks 4 .
These lessons with EBOV will undoubtedly be important for the next virus outbreak 5 but they also raise many questions 6 which point to how little we know about these viruses in general, as well as how best to share knowledge openly 7 .
There have been a relatively small number of studies that have attempted to identify compounds active against EBOV.
Two recent studies utilized high-throughput screens of a subset of FDA approved drugs against different EBOV strains (Zaire and Sudan) in vitro and in vivo.
These independent reports suggested the promise of the antimalarials amodiaquine and chloroquine in one study 8 , while the selective estrogen receptor modulators (SERMs) clomiphene and toremifene were active in another 9 .
Chloroquine to date has not progressed beyond the mouse EBOV model used in these studies.
We hypothesized that we could use these four molecules to computationally define the features that are important for activity.
The previous studies were not exhaustive screens of all FDA drugs and so we have taken this opportunity to suggest additional compounds.
Looked at from another perspective "non-antiviral" drugs may be worth following up even though their molecular mechanism is unknown.
These compounds may themselves have broad antiviral activity as reports describe modest inhibitory activity against other viruses 10-13 .
Several studies have identified non-FDA approved drugs including an in silico docking approach to identify molecules targeting the viral Nedd4-PPxY interface 14 .
These molecules were similar to the FDA benzimidazole and aminoquinoline 8, 9 compounds that were active against EBOV.
Another good example is the recent in silico docking of 5.4 million drug-like compounds docked in the viral protein VP35 protein 15 .
This identified multiple pyrrolidinones which inhibit its polymerase cofactor activity 15 .
The pyrrolidinones bind to an alpha helix which is proposed as important for viral function 16 .
With the limited knowledge of small molecules and potential targets we have studied whether the FDA-approved drugs that are active in vitro and in vivo versus EBOV could be targeting VP35.
Common features pharmacophore for EBOV actives Two papers from 2013 described compounds active as inhibitors of different EBOV strains in vitro and in vivo, namely amodiaquine and chloroquine in one study 8 , clomiphene and toremifene in another 9 .
These active molecules were used as they have both in vitro and in vivo activity to build a common features pharmacophore with Discovery Studio 4.1 (Biovia, San Diego, CA) from 3D conformations of the molecules generated with the CAESAR algorithm.
This identified key features.
The pharmacophore was then used to search various databases (for which up to 100 molecule conformations with the FAST conformer generation method with the maximum energy threshold of 20 kcal/mol, were created).
The pharmacophore was then used to search the Microsource Spectrum database (http://www.msdiscovery.com/spectrum.html) as well as the CDD FDA drugs dataset (https://www.collaborativedrug.com/ pages/public_access).
In both cases over 300 hits were retrieved initially.
The van der Waals surface of amodiaquine (which was more potent than chloroquine 8 ) was added to limit the number of hits retrieved 17-19 .
Receptor-ligand pharmacophores for the VP35 protein were generated from crystal structures (4IBB, 4IBC, 4IBD, 4IBE, 4IBF, 4IBG, 4IBI, 4IBJ, 4IBK) in the protein data bank PDB.
Pharmacophores were constructed using the receptor-ligand pharmacophore generation protocol in Discovery Studio version 4.1 (Biovia, San Diego, CA) with a maximum number of pharmacophores (10), minimum features (4), and maximum number of features (6) as are described elsewhere 20 .
in silico docking of molecules in VP35 structure PDB 4IBI was used for docking using LibDock in Discovery Studio (Biovia, San Diego CA) 21 .
The proposed binding site was centered on the ligand and a site sphere created (coordinates 2.14, 20.93, 1.71) with 9.45 Å diameter.
The protocol included 10 hotspots and docking tolerance (0.25).
The FAST conformation method was also used along with steepest descent minimization with CHARMm.
Further parameters followed the default settings.
The ligand VPL57 was removed from the binding site and re-docked.
The four FDA approved drugs with activity against Ebola were docked in the structure from an sdf file.
Molecules were visualized alongside the original ligand VPL57 and the 2D interaction plots generated.
Common features pharmacophore for EBOV actives The pharmacophore was generated using the in vivo and in vitro active amodiaquine, chloroquine, clomiphene and toremifene We have responded to the reviewers suggestions.
We have made a small labeling addition to Figure 1 and added a new Figure S2 which is an expanded view of Figure 2B .
(Supplemental Table 1 ) as these represent the most relevant FDA approved drugs to date.
This pharmacophore consists of 4 hydrophobic features and a hydrogen bond acceptor feature ( Figure 1 ).
The pharmacophore with van der Waals surface was also used to search FDA drug various libraries (Supplemental Table 2 and Supplemental Table 3 ).
The most interesting observations from this virtual screen are that various estradiol analogs score well (e.g.
estradiol valerate Fit value 4.23).
Previously estradiol was suggested to be active in the EBOV pseudotype assay in vitro 8 .
In addition, dibucaine was also retrieved (Fit value 1.58) which was also active in the EBOV pseudotype assay 8 .
Amodiaquine, chloroquine, clomiphene and toremifene can be used as positive controls for future screens.
Because the original complete sets of FDA approved compounds screened are not publically accessible it is difficult to compare hit rates versus all compounds tested to date.
The nine receptor-ligand pharmacophores created all consisted of three to four hydrophobic features and one to two hydrogen bonding features ( Table 1 ).
Eight of these pharmacophores also had a negative ionizable feature.
These suggest that the receptor-ligand based approach results in a general similarity across the nine structures, likely indicating the similar binding mode and importance of features for interfering with this generally hydrophobic pocket for protein-protein interactions.
In silico docking of molecules in VP35 structure Redocking the 4IBI ligand in the protein resulted in an RMSD of 3.02Å, which generally indicates the difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket ( Figure S1 ).
This molecule was ranked the 29 th pose and had a LibDock score of 86.62 ( Figure S1 higher scores are better).
The four FDA approved drugs were docked into the VP35 structure 4IBI.
All compounds docked similarly and overlapped with the co-crystal ligand ( Figure 2 ).
Amodiaquine and chloroquine had higher LibDock scores (> 90) than the 4IBI ligand, while clomiphene and toremifene had LibDock scores less than 70.
All four FDA approved drugs bound similarly to the pyrrolidinone ligands in the pocket formed by residues from the α-helical and β-sheet subdomains 15 .
We have highlighted proposed energetically favorable interactions of the antimalarial candidate binders with ILE295, LYS248 and GLN244, which scored favorably.
Previously published studies suggested mutation of ILE295, LYS248 resulted in near-complete loss of binding activity 15 .
Our previous experience with common feature and quantitative pharmacophore models has demonstrated their value in predicting novel actives from collections of FDA approved drugs 22-27 .
Candidate predicted actives may be assessed by their Fit Value to the pharmacophore model.
This score can be used to prioritize compounds for eventual testing.
In the current study it was hypothesized that two different classes of compounds showing activity against EBOV in vitro and in vivo may share a common pharmacophore.
Construction of this pharmacophore ( Figure 1 ) indicated four hydrophobic features and a hydrogen bond acceptor feature.
This pharmacophore (with an added van der Waals surface to limit the number of hits retrieved) was then used to screen and score other FDA drugs from score particularly well in terms of docking to VP35.
If this is the case it could provide a means to follow up with other small molecule analogs and/or additional FDA approved drugs that could target this protein-protein interaction.
As with our other tuberculosis-focused research 32,33 , and computational approaches to repositioning compounds 34 we embrace the essentiality for computational predictions to be interrogated through rigorous experimental studies.
For example at least two in silico docking studies screened commercially available compounds 14,15 .
We propose that docking FDA approved drugs could also be a viable first step to identifying potential compounds that could be used.
We are actively seeking collaborators with experience with EBOV assays to enable further translational studies.
We believe this computationally inspired approach may be applicable for other known infectious pathogens that do not have current treatments such as other viruses related to Ebola.
Ultimately we need to be able to leverage such approaches to provide antivirals for future pathogens.
F1000Research: Dataset 1.
Pharmacophores, receptor ligand models and docking data for FDA-approved drugs inhibiting the Ebola virus, 10.5256/f1000research.5741.d38449 35 .
The ligand-based pharmacophore was previously made available: http://figshare.com/articles/Ebola_active_cpds_pharmacophore/ 1190902.
The following PDB structures were used in this study (4IBB, 4IBC, 4IBD, 4IBE, 4IBF, 4IBG, 4IBI, 4IBJ, 4IBK).
For models and advice please contact Sean Ekins (ekinssean@ yahoo.com).
Author contributions S.E.
and M.C.
came up with the general idea for the study based on the published in vitro and in vivo data.
All authors contributed to the collaborative writing of this project.
S.E.
works for Collaborations in Chemistry, and consults for Collaborative Drug Discovery Inc.
The author(s) declared that no grants were involved in supporting this work.
Dr. Christopher D. Southan, Dr. Peter Madrid and Dr. Nadia Litterman are acknowledged for discussions on Ebola.
Biovia are kindly acknowledged for providing Discovery Studio.
An earlier preliminary version of this pharmacophore was described previously: http://figshare.com/articles/A_pharmacophore_of_ebola_active_ compounds/1190787.
a small database and identified 120 and 124 structures for future evaluation in vitro testing (Supplemental Table 2 and Supplemental Table 3 ).
Out of these compounds estradiol and dibucaine had been previously described as active in in vitro EBOV assays.
This suggested the pharmacophore could retrieve some structurally diverse classes of known hits 8 .
Recently identified co-crystal structures of the EBOV VP35 protein were used to derive receptor-ligand pharmacophores.
These nine receptor-ligand pharmacophores suggested the importance of hydrophobic, hydrogen bonding and negative ionizable interactions to interfere with this protein-protein interaction ( Table 1) .
Eight out of nine of the pharmacophores had one or more hydrogen bond acceptor feature.
These pharmacophores are grossly similar to our ligand based pharmacophore (derived from four FDA approved drugs that inhibit EBOV), as both types of model had multiple hydrophobic features and at least one hydrogen bond acceptor.
When we docked the antimalarials and SERMs into a representative VP35 structure these compounds were found to overlap with the X-ray ligand to differing extents.
Amodiaquine and chloroquine had LibDock scores greater than 90 and higher than that for the redocked X-ray ligand.
This indicated that VP35 may be a potential target for these two distinct classes of compounds.
However, it is important to point out that we have not compared docking to other proteins in EBOV and it could also be possible that these molecules are active elsewhere as well as via other mechanisms than by specific binding to proteins 28,29 .
Further, VP35 may be a preferred target for the antimalarials while the SERMs are not predicted to bind as well as the X-ray ligand.
The use of other docking and scoring methods may produce differences in the pose and predicted binding affinity, which could be of interest for further studies.
A combination of the promising efficacy of chloroquine (EC 50 16 μM 8 ) and amodiaquine (EC 50 8.4 μM 8 ) versus EBOV, their availability and likely low cost should prioritize their further laboratory exploration.
Mechanistic studies against VP35 and possibly other proteins should also be pursued and may be enlightened by the observation that both of these compounds also have reported activity against other viruses.
For example, chloroquine is active against human coronavirus OC43 (in vitro and in infected mice) as well as SARS (in vitro) 10,30,31 , while amodiaquine also inhibits dengue virus 2 replication and infectivity in vitro 11 .
In summary, this study has built on the previous publications that identified four FDA approved compounds active against different strains of EBOV 8,9 .
Our pharmacophore model for SERMs and aminoquinolines suggests that these compounds share multiple chemical features based on their overlap to the four hydrophobic features and a hydrogen bond acceptor ( Figure 1E ) and they may have a common mechanism or target.
We suggest that VP35 may be the likely target based on the overlap of receptor-based pharmacophores and docking into the crystal structure.
Amodiaquine and chloroquine Figure S1 .
Redocking VPL57 in 4IBI.
The 4IBI ligand was removed from the structure and redocked.
The closest pose (grey) was ranked 29 with RMSD 3.02A and LibDock score 86.62 when compared to the actual ligand in 4IBI (yellow).
Table 2 .
FDA drugs and common features pharmacophore.
The dataset of 2643 molecules was downloaded from the CDD Public Access (https://www.collaborativedrug.com/pages/public_access) as an sdf and then a 3D database was created in Discovery Studio using FAST conformer generation with up to 255 conformations.
The database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene.
The search 3D database protocol was used with the Fast search method.
In some cases the indication for the molecules is not described (ND).
Table 3 .
Microsource Spectrum and common features pharmacophore.
The dataset of 2311 molecules was provided by Microsource (http://www.msdiscovery.com/spectrum.html) as an sdf and then a 3D database was created in Discovery Studio using FAST conformer generation with up to 255 conformations.
The database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene.
The search 3D database protocol was used with the Fast search method.
have presented a crisp and lucid manuscript on a very relevant topic.
They have suggested a et al.
methodology to extract common features from four approved compounds that have recently been found to work against the Ebola virus (amodiaquine, chloroquine, clomiphene and toremifene), and define a pharmacophore, which has been used to search databases, and identify further compounds for and in vitro in vivo testing.
The methodology described here provides an excellent method to quickly screen known in silico compounds for possible therapies against Ebola in particular, and other viruses in general.
The result that SERMs show lower scores for binding to VP35 is rationalized by the finding that 'clomiphene and toremifene inhibit EBOV VLP entry with some specificity to GP' , and therefore does not probably inhibit VP35.
'Chloroquine to date has not progressed beyond the mouse EBOV model used in these studies.'
This statement is not clear, does it mean that the others have progressed beyond the mouse EBOV model?
The first few compounds in Supplemental Table 2 should be part of the main manuscript as a table.
Color coding of pharmacophore features should be in Fig 1 too (it comes earlier than Table 1 , where it is described).
The structures look better with a white background.
A 3 Å RMSD for redocking a given ligand is quite high .
The authors should consider the use of other docking methods, as a comparison.
A table of Libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score).
The major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures.
Further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future.
Response: Because this data is available easily on the website, I do not see any benefits of taking these compounds out of this supplemental table and putting them into the body of the manuscript.
It might also add more confusion cutting the table up.
Color coding of pharmacophore features should be in Fig 1 too (it comes earlier than Table 1 , where it is described).
Response: Thank you -this has now been added.
Response: I think this is a personal preference, the structures are clear in our opinion with a black background.
I have not had this suggestion previously with other publications regarding the background color.
The study was not intended as an exhaustive docking comparison, there are plenty of these in the literature as noted by the reviewer.
I agreed the redocking RMSD was high, but I also provided some justification for the result (difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket).
If others want to use different methods and perform a comparison for this target I would be supportive.
A table of Libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score).
The Libdock scores for the best poses are in the '4IBI Libdock docking data best poses" file.
A higher score is better and this has been added to the results section The major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures.
Further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future.
All of the models were generated with the proprietary software Discovery studio, and Response: all files have been provided.
The comment about such software is true, while I support using open software, I have yet to find an open source pharmacophore tool as good as that in Discovery Studio to date.
It is also more convenient to use this software generating pharmacophores, receptor-ligand pharmacophores and docking in the same place.
The types of analysis I have described could be repeated with any software, open source or proprietary.
My hope is that by making this work openly accessible others will be inspired to pursue computational approaches with EBOV.
Perhaps the community could propose the use of standards for open pharmacophore files as well.
By publishing in this journal we are making all our data open even though they are in proprietary formats, I do not think this should preclude publication.
No competing interests were disclosed.
The current Ebola crisis in West Africa has shattered all expectations by continuing to grow months following the initial case.
This has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community.
The authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model.
The key features of such a model are used to scan databases of small molecules.
They have come up with a list of putative inhibitors.
Their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model VP30-inhibitors that they have built.
In parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein 35.
Based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein.
In this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results.
As they mention in the text (page 4), the redocking of the ligand in 4IBI to the protein does not show the crystal structure binding mode accurately.
These different settings could help in a better prediction of the ligand orientations.
Concerning the docking and proposed mechanism, I wonder how different the other solved nucleocapsid proteins are from a structural and sequence point of view, in order to make the authors point that VP35 is indeed the target.
Would it be possible to explore for surface patches with similar physico-chemical features?
In the case of VP30, a potential binding pocket for small-molecule inhibitors has been suggested by .
How good or bad the overlapping with the built pharmachophore is Hartlieb (2007) et al.
for this case?
To complete the structural understanding of the action of these compounds, a figure displaying their location on the protein surface as well as the binding site for RNA would clarify their role in the inhibition of protein-protein interactions.
In summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as Ebola virus.
Although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value.
No competing interests were disclosed.
The current Ebola crisis in West Africa has shattered all expectations by continuing to grow months following the initial case.
This has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community.
The authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model.
The key features of such a model are used to scan databases of small molecules.
They have come up with a list of putative inhibitors.
Their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model VP30-inhibitors that they have built.
Response: To clarify, we have focused on VP35 not VP30.
I am not aware of an X-ray structure with ligand bound for VP30.
The same type of approach could certainly be pursued with other EBOV targets.
We produced a common features pharmacophore for the 4 compounds, and after looking at the VP35 receptor-ligand pharmacophores proposed that there may be some overlap, and then this led to docking the 4 compounds in the X-ray structures.
Our intent was not to design molecules but to use the available methods to perhaps infer a potential target/mechanism and then perhaps researchers would want to test the compounds.
We do not have access to experimentally test these predictions, but this manuscript may lead to others doing this work perhaps.
Whether one wants to use the Libdock score for (absolute) prediction of binding affinity interactions is debatable, rather this approach might help to limit or prioritize which compounds to test.
In parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein 35.
Based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein.
Response: VP35 may be a target for these compounds although we do not discount other targets or non-target related mechanisms.
In this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results.
As they mention in the text (page 4), the redocking of the ligand in 4IBI to the protein does not show the crystal structure binding mode accurately.
These different settings could help in a better prediction of the ligand orientations.
Response: As explained earlier, our study is not intended to be an exhaustive evaluation of docking tools, we have used different computational approaches to suggest that the FDA drugs may have a common pharmacophore, which seems to be similar to that of the ligands co-crystallized with VP-35.
Finally docking suggests they may fit into the pocket that the co-crystal ligands bind to.
The work proposes that the compounds could fit in the binding site, but it is unclear what additional value more docking would add unless we were going to try to predict and then generate the X-ray structure of these FDA drugs.
Certainly if experts in docking or crystallography want to pursue this target they can.
Ebola by the numbers: The size, spread and cost of an outbreak , and define a pharmacophore, which has been used to search databases, and identify further compounds for in vitro and in vivo testing.
The in silico methodology described here provides an excellent method to quickly screen known compounds for possible therapies against Ebola in particular, and other viruses in general.Thank you for your constructive comments.
Some minor comments.The result that SERMs show lower scores for binding to VP35 is rationalized by the finding that 'clomiphene and toremifene inhibit EBOV VLP entry with some specificity to GP' , and therefore does not probably inhibit VP35.Response: While the lower docking scores are noted I do not think this necessarily excludes them from inhibiting, as we know docking scores may not be that accurate and in this case, docking was used to answer the question could they fit.
It's pretty clear that a wide variety of drugs could fit based on the binding site size and accessibility.
Response: From discussions with the author on the paper that described Chloroquine as active versus EBOV and in mouse, this work has not gone beyond the mouse model in vitro of EBOV.
Response: this is indeed a very good point.
We are not experts on these proteins.
I think the proposed work could be done, the difficulty may be that there is no crystal structure (that I can see) with a ligand bound that would be a useful guide to binding in this pocket and would be essential for a receptor-ligand pharmacophore to be built.
In summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as Ebola virus.
Although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value.
Response: Thank you for your suggestions, we agree and would encourage other scientists to test whether these compounds are targeting VP35 or VP 30 as you propose, or having an alternative mechanism.
I think we would also be happy to see any of these molecules progress into other animal models of EBOV.No competing interests were disclosed.
Competing Interests:
key: cord-339122-7vvqtk84 authors: Deb, Chaarushena; Moneer, Osman; Nicholson Price, W title: Covid-19, Single-Sourced Diagnostic Tests, and Innovation Policy date: 2020-07-07 journal: J Law Biosci DOI: 10.1093/jlb/lsaa053 sha: doc_id: 339122 cord_uid: 7vvqtk84 The United States’ disastrous response to the onset of the Covid-19 pandemic has arisen in large part by an utter failure to provide adequate diagnostic tests for the presence of SARS-CoV-2.
The Centers for Disease Control were the sole testing source authorized by the Food and Drug Administration, and when the CDC failed to provide reliable tests in sufficient volume, it took weeks for other providers to be approved and to ramp up testing.
Revised policies should decrease the likelihood of sole-sourcing tests in pandemic contexts, which results in a fragile system.
The pandemic sole-sourcing failure, however, not only accelerated the pandemic, but also provides lessons for innovation policy about diagnostic testing more generally.
Sole-sourcing hurts clinical practice by limiting confirmatory testing and systemic robustness, whether in a pandemic or in regular practice.
We thus argue against relying too heavily on exclusivity-creating patents as innovation incentive for diagnostic tests—including the proposed Coons-Tillis patent reform bill which would increase patentability for many such tests.
Instead, we propose the use of reformed reimbursement to create better incentives for diagnostic test innovation.
In both pandemics and elsewhere, single-sourcing creates too great a point of failure, but targeted innovation policy can help At the heart of the United States' disastrous response to the Covid-19 pandemic is a failure of diagnostic testing.
That something so fundamental to medical care could be so botched evokes incredulity.
From primary care offices to critical care settings, every patient encounter begins with a diagnostic workup.
Diagnostic testing tools are key parts of the physician's toolkit, and confirmatory testing is essential.
But in the greatest public health challenge of the 21st century, the failures of diagnostic testing have been laid entirely bare.
The story of this failure in diagnostic testing has been told in detail and will be unpacked for years to come.
Briefly, the U.S. pandemic response lacked high quality diagnostics, failed to deliver tests in sufficient quantity, and was too slow in deploying developed tests when initially needed.
1 This lack of appropriate testing resulted in inaccurate patient identification and poor epidemiological characterization of viral spread.
2 From there, the errors further multiplied and rendered the rest of the governmental pandemic response similarly sluggish and ineffective.
This article seeks to highlight an implication of the failure that applies broadly to diagnostic testing in general: the problems inherent in relying on a sole source for a diagnostic test.
Though we focus on the question of single-sourcing in this exploration, we do not claim this factor as the only failure characterizing U.S. Covid-19 testing policy.
3 Beyond single-sourcing, the FDA stumbled through a series of missteps around its application of Emergency Use Authorization (EUA) strictures and its issuance of EUAs.
4 Nationwide testing further suffered from the CDC's initial promotion of stringent criteria (e.g., travel history, symptom severity, etc.)
to determine whether patients should even be allowed to receive tests.
The constellation of these factors resulted in fewer patients being tested and rapid spread of the virus.
5 On the other end of the spectrum, the FDA's antibody-testing policy was likely too lax for SARS-CoV-2 serology tests (designed to determine viral antibody presence in blood), leading to too many poor-quality marketed tests that could lead individuals to falsely believe they were immune to COVID-19.
6 The test generated a high proportion of false positives due to initial development with faulty reagents.
10 Beyond quality control issues, the diagnostic test further suffered from a slow reporting of results as clinicians had to send samples back to labs beyond the clinical practice setting for analysis.
11 These shortcomings exacerbated the pandemic's toll by preventing quick containment of the virus.
Crisis may have been averted had there been any alternatives to the CDC's test, but under the FDA's restrictions, there were no other approved tests available to perform any confirmatory testing and receive second opinions.
Labs were left to sit powerless until other tests were at long-last approved.
This de facto diagnostic test monopoly proved critical and pushed back pandemic control efforts by several weeks.
12 Even after the FDA permitted other labs to conduct testing, they were all required to follow the CDC's chemical formulation.
13 Non-CDC labs all required the same reagents, effectively rendering the additional approvals useless because the small reagent manufacturer could not keep up with the instantaneous jump in demand.
The approval of several competing tests earlier in the pandemic response that are not chemically identical, such as the Roche SARS-CoV-2 test, 14 could have also alleviated some of the initial supply chain pressure and underscores the need to prevent monopoly testing.
Of course, single-sourcing does not always lead to problems; the SARS-CoV-2 test adopted by the World Health Organization has worked well in the countries that used it.
15 But single-sourcing sharply raises the possibility of problems leading to catastrophic failure down the line.
The problems with single-sourcing, though, are not limited to pandemics.
Diagnostic tests are crucial to quotidian clinical practice.
And within that practice, the abilities to access high-quality diagnostics rapidly and to perform confirmatory testing are crucial.
Single-sourcing diagnostic tests jeopardizes the ability to confirm test results, but also may impact innovative efforts to develop new diagnostics based on old tools, supply robustness, and cost-effective development.
16 An appropriate model for diagnostic testing development, including innovation incentives, should go beyond simply discouraging test monopolies and promoting confirmatory testing, by also allowing for low R&D costs for test development and yielding fast delivery of high quality tests-preventing the errors highlighted in the Covid-19 testing response from occurring in both emergency and non-emergency situations.
Unfortunately, policy efforts ongoing since before the pandemic are pointing exactly the wrong direction.
After an eight-year stretch of unpatentability for certain diagnostics, efforts are afoot to change the law: Recent legislative action would allow such tests to be patented again, 17 raising concerns about limitations on initial use and confirmatory testing.
18 In this paper, we first consider how to address the problems with single-sourcing diagnostic tests in emergency medical contexts like the ongoing Covid-19 pandemic.
We then turn to applying those lessons for standard diagnostic test development, in particular considering how we can create adequate incentives for development without relying on a problematic single-source or quasimonopoly model.
The key diagnostic testing roadblock during the Covid-19 pandemic response in the U.S. appears to have been the FDA's decision to permit only the CDC to offer diagnostic testing for SARS-CoV-2.
Without approval to test, none of the many other potential testing providers could offer confirmatory testing when the CDC's test offered ambiguous results, provide testing to make up for the CDC's breathtaking shortfall in testing volume, or offer innovative advances on the basic test to improve the substantial time required to get results back from the CDC.
19 Though the FDA ultimately did relax its regulatory structure in approving diagnostic tests, 20 the policy-driven delay had profound consequences.
Accordingly, the most straightforward intervention would involve updates to the FDA's emergency use authorization process.
The 2013 Pandemic and All-Hazards Preparedness Reauthorization Act (PAHPRA) passed under the Obama administration contemplated a pandemic, but focused primarily on streamlining the administrative process necessary for the FDA to start issuing "Emergency Use Authorizations" (EUA) of unapproved medical countermeasures (MCM) and expanding the emergency uses of already FDA-approved MCMs.
21 We suggest updating the EUA model so that safety and efficacy of novel diagnostic tests can be established as quickly as possible.
Thus, in order to facilitate early deployment of multiple tests in a pandemic scenario, we suggest considering whether the EUA model be updated to: (1) streamline the EUA application process, perhaps through an alternative, temporary route administered through CMS, a regulatory body that CLIA-approved labs are already familiar with, (2) make the regulatory regime more flexible, such that a more lenient structure can be quickly put in place to combat the early stages of an epidemic, and (3) initially institute a limited liability model that would incentivize labs, like those at Stanford and the University of Washington, to begin using their test if they have good scientific data backing their safety and efficacy, especially if EUA processing delays are expected.
An absence of any regulatory oversight brings its own problems-witness antibody testing-but overly tight entry rules can also be disastrous.
FDA eventually adopted a more lenient policy incorporating some of these points, but the delay was costly.
Perhaps most significantly, therefore, we think it important for FDA to consider the potential danger of single-sourcing when shaping its early pandemic responses to avoid a situation like that faced in early 2020.
We recognize these changes are not a panaceasome labs lacked CLIA approval, creating a parallel barrier to testing-but think they deserve careful consideration.
The failure of diagnostic testing during the U.S. response to the Covid-19 pandemic provides a warning and a lesson for policy surrounding diagnostic tests more generally.
Good policy for diagnostic testing in non-emergent times requires both protecting clinician access to diagnostic testing and creating appropriate incentives to support diagnostic test development.
An incentive model for diagnostic tests should thus focus on three main aims: (i) promote the typical use case for tests, including allowing physicians to obtain second opinions, (ii) minimize the cost of research and development, and (iii) ensure rapid deployment of safe and effective tests.
The third and especially the first of these aims are hindered by single-sourcing.
And thus any sort of incentive model that relies on granting market exclusivity -such as the traditional patent system, FDA approval exclusivity, or trade secret protection -raises the same sorts of issues, if on a smaller scale, as those grimly demonstrated by the failure of U.S. SARS-CoV-2 testing at the onset of the Covid-19 pandemic, which ultimately resulted in the loss of many lives.
30 By preventing access to second opinions or incentivizing fast, but ultimately ineffective science, we risk putting patients in harm's way.
31 A better incentive system would allow for an increased number of players in the market, all vying to be the best diagnostic for each indication.
Such a system would promote diagnostic creation and implementation that mirrors actual diagnostic usage.
We first summarize (A) a recent history of diagnostic test patentability, since patents have both created incentives but hampered access and clinical practice; (B) describe current efforts to increase diagnostic test patents; and then (C) propose improved reimbursement mechanisms as a solution to the balancing act of managing appropriate economic incentives and allowing for optimal clinical practice during non-emergent conditions.
Diagnostic testing patentability has once again entered public debate, this time in Congress rather than the courts.
In the summer of 2019, Senators Thom Tillis (R-N.C.) and Chris Coons (D-Del.)
proposed draft legislation to modify existing patent law, rendering many diagnostic tests patentable.
35 The proposal no longer considers "abstract ideas," "laws of nature," or "natural phenomena" to be exceptions to patent eligibility, explicitly overturning the three Supreme Court decisions.
36 Under the proposal, courts would no longer be able to invalidate patents because they cover underlying biological relationships-precisely the stuff of diagnostic tests.
37 The desire to incentivize diagnostic test development through exclusivity rights granted by patents drives the biotech industry's support of the bill.
38 While single gene patents are unlikely to reemerge due to the critical patent requirement of novelty-the human genome has been sequenced many times over, so genes are unlikely to be patentably "new"-patenting in other areas of molecular diagnostics (e.g., polygenic risk scoring or autoantibody detection) remains a concern.
39 32 Robert Although the pandemic has sidelined unrelated legislative efforts, there remains interest in revising patent law.
If the Tillis-Coons bill passes, we may see a return to the pre-Mayo era in which certain diagnostic tests existed as patent-protected monopolies, making it hard to verify quality or to obtain confirmatory tests.
40 Outside the pandemic's recent horrifying exemplar, confirmatory tests have been stymied by patent-based single-sourcing in the past.
For instance, until 2009 PGxHealth was the sole provider of genetic testing services for Long QT Syndrome (LQTS).
41 Independent assessment by clinicians suggested discrepancies in test accuracy and quality.
42 A competing diagnostic test could have allowed clinicians to discover these shortcomings and further validate false negatives.
43 Good clinical practice also requires the availability of multiple diagnostic test options, including ideally by alternate test providers, as physicians frequently rely on second opinions to choose the best care for their patients.
Previous studies exploring the practice of second opinions in diagnostics have suggested that the ability to verify the results of an initial diagnosis may lead to a change in diagnosis or treatment.
44 Before the Supreme Court intervened, Myriad's exclusivity for BRCA1/2 genetic variant testing prevented precisely this ability.
45 The Tillis-Coons bill relies on patents to provide an incentive in the form of market exclusivity, but exclusivity is especially problematic for diagnostic tests.
(There are other problems with relying on patents to drive biomedical innovation, especially in relation to equity, but we do not focus on those challenges here).
46 Though patent holders may not always exercise their monopoly rights, the danger lies in their potential to do so.
Instead, we should create incentives for diagnostic test development by leveraging non-patent policy levers.
A more effective incentive system would allow an increased number of players in the market, all vying to be the most accurate and cost-effective test for each indication.
Such a system would promote diagnostic creation and implementation that mirrors actual physician usage.
Increasing reimbursement rates to reflect the expected value of diagnostic tests could help.
Public and private insurance providers use the Current Procedural Terminology (CPT) system to determine the level of reimbursement warranted by new diagnostics.
47 The CPT system, in which diagnostics are often treated as commodities, is based on cost and procedure instead of the diagnostic's value.
48 To incentivize diagnostic development without creating monopolies, health insurance reimbursement strategies should be updated to provide a monetary reward for the potentially substantial cost-savings and health improvements of diagnostics.
For administrative simplicity, the CPT shoehorns new tests into established reimbursement categories ("codes") that have established prices, generally evaluating new diagnostics only in comparison to existing diagnostics.
49 New tests found analogous to old tests may be "cross-walked" to the old test's price, even if the new test is far more effective-and provides much more value.
50 In December of 2019, 70% of new diagnostics were cross-walked.
51 Completely novel tests with no adequate comparison may require new pricing determinations that can take years to implement.
52 Thus, new diagnostics face relatively cheap prices that may not offset research and development costs, decreasing incentives to create new tests.
In an attempt to promote value-based pricing, some diagnostic test manufacturers have opted for a "miscellaneous" CPT code.
53 Such coding requires an assignment of value from each payer, which effectively bypasses traditional cost-based pricing but represents a logistical nightmare that dissuades most manufacturers.
Effective diagnostic tests, however, can shift healthcare spending from therapeutic to preventative care, promote precision medicines responding specifically to a patient's disease state, lower physician trial and error, and reduce hospital stays 54 -all of which reduce health-care system costs and improve patient care.
Shifting to a reimbursement model that recognizes these substantial savings could properly incentivize diagnostic tests' true value.
CMS has recently acquired a new set of tools that may facilitate a leadership role in reimbursement model changes.
The Clinical Laboratory Fee Schedule underwent a major overhaul as part of the Protecting Access to Medicare Act of 2014 (PAMA).
Though the system had not been updated in three decades, many cumbersome but necessary changes, such as a national fee schedule and private market data collection, were implemented in order to save Medicare nearly $4 billion over 10 years.
55 While there are likely to be bureaucratic challenges to implementing value-based reimbursement for diagnostics via CMS, some of the provisions of the new PAMA Fee Schedule, such as the shift to national pricing, could ease simpler regulatory updates rather than a complete overhaul of the Fee Schedule and thus make change easier.
To be sure, some diagnostics will recommend more expensive care; getting the incentives right across the board will be complicated, but reimbursement is a better and more nuanced tool than bringing back broad patent exclusivity.
56 We recognize reimbursement will not create a complete incentive regime, and may need supplementing with grants or prizes.
57 And in some instances, patents will still be present, especially for more technically complex diagnostics.
58 But as a general level, reimbursement policy reform shows substantial potential for creating incentives without relying on single-sourcing.
SARS-CoV-2 did not create new problems in biomedical innovation; instead, it dramatically exposed underlying issues, including in the development and deployment of diagnostic tests.
In particular, single-sourcing, whether through regulatory restrictions or patent quasi-monopolies, may seem like an attractive way to centralize control and to create incentives.
But for diagnostic tests which rely on confirmatory testing, innovative improvements, and robust access to supply, single-sourcing creates a host of serious problems.
Creating the right incentives demands careful attention to these problems, and ideally structuring a regime that avoids them.
Reimbursement, especially structured through public involvement, provides a promising option for such a regime.
Both to avoid future pandemic-related disasters and to improve the normal practice of medicine, it is worth rethinking how we drive and shape the development of diagnostic tests.
2019 ICD-10-PCS Conversion Table (ZIP)" hyperlink; then select "icd10pcs_conversion_table.xlsx" file) (last visited Apr Office of the Inspector General Health and Human Services, HHS OIG Data Brief Medicare Payments for Clincial Diagnostic Laboratory Test in 2015: Year 2 of Baseline Data REV.
1115REV.
(2015.
58 We acknowledge the existence of the rare cases in which early patent protection may be necessary to secure enough resources for development, most famously theorized by Edmund W. Kitch in The Nature and Function of the Patent System, 20 J.L.
& ECON 265, (1977) (discussing the "prospect theory" of patents).
The judicious use of grants and prizes may minimize the number of such cases.
key: cord-303865-vd3qr32o authors: Gianturco, Stephanie L.; Yoon, SeJeong; Yuen, Melissa V.; Mattingly, Ashlee N. title: Outsourcing facilities and their place in the U.S. drug supply chain date: 2020-08-28 journal: J Am Pharm Assoc (2003) DOI: 10.1016/j.japh.2020.07.021 sha: doc_id: 303865 cord_uid: vd3qr32o OBJECTIVE: The purpose of this commentary is to describe the ideal role of 503B outsourcing facilities in the U.S. drug supply chain.
We also address the challenges that 503B outsourcing facilities are facing that limit their utilization and offer possible solutions.
SUMMARY: Section 503B outsourcing facilities are emerging contributors in compounding owing to their ability to compound large quantities of medication without requiring patient-specific prescriptions.
As such, they play a valuable role in the U.S. drug supply chain.
The use of outsourcing facilities to compound ready-to-use drug products is gaining traction in hospitals and other health care systems.
Outsourcing facilities help hospitals that are facing time and cost constraints owing to the evolving regulatory landscape around compounding.
Although outsourcing facilities are assets to the drug supply chain, there are several challenges to their use.
The lack of a finalized 503B Bulks List has led to outsourcing facilities being overly cautious in compounding products using bulk drug substances.
In addition, the time between Food and Drug Administration (FDA) inspections is undefined, and a lack of follow-up information regarding concerns identified during an inspection may result in uncertainties about the current state of the outsourcing facility.
CONCLUSIONS: Health care providers, outsourcing facilities, and FDA need to work together to ensure that patients are provided the drugs they need in a safe and effective way.
The U.S. drug supply chain includes a variety of participants who ensure that the drug reaches the patient, while maintaining safety and efficacy.
Although it is preferable for providers to use Food and Drug Administration (FDA)eapproved, commercially available drug products, there are times when drug products need to be compounded to meet a patient-specific need.
In 2012, a meningitis outbreak linked to compounded steroid injections led to the passage of the Drug Quality and Security Act, which established 2 distinct types of pharmaceutical compounders for human patients: 503A and 503B.
1 Compounding under section 503A is performed by a physician or a pharmacist in a state-licensed pharmacy or federal facility, pursuant to a patient-specific prescription.
Limited quantities may be compounded in anticipation of a prescription if the facility has an established relationship with the patient or the prescriber.
2 Outsourcing facilities, established under section 503B, may register with FDA, and compounding is supervised by a pharmacist.
Outsourcing facilities are not required to receive a patient-specific prescription before compounding and therefore are able to compound drug products in larger quantities than 503A facilities.
3 As a requirement, all drug products must be compounded following current good manufacturing practices, must be reported to FDA every 6 months, and a portion of these must be sterile preparations.
1 Any drug product that an outsourcing facility intends to compound from a bulk drug substance (or a pure active pharmaceutical ingredient) must be on the 503B Bulks List that FDA is currently developing; a list of substances for which there is a demonstrated clinical need.
While the list is being developed, bulk drug substances were divided into 3 categories.
4 FDA does not intend to take action against outsourcing facilities that compound drug products using bulk drug substances in category 1, in which information supports their clinical use or need, and FDA was unable to identify significant safety risks.
4 However, outsourcing facilities cannot compound drug products using substances listed in category 2 (which have significant safety risks identified) and category 3 (which were not nominated with sufficient supporting information).
4 Drug products compounded under sections 503A and 503B cannot be identical to commercially available products, appear on the list of drug products that have been withdrawn or removed from the market for reasons of safety or efficacy, or appear on the list of drug products that present demonstrable difficulties for compounding.
5-7 A commercially available drug product can be compounded if it is on the drug shortage list published by FDA.
7, 8 How outsourcing facilities can take part in the U.S. drug supply chain Outsourcing facilities can provide hospitals and independent practices with ready-to-use sterile drug products because they can compound drug products on a larger scale and are not limited by the requirement of a patient-specific prescription.
As some commercial products require preparation into unitdosed forms for hospital use, this can be especially useful for hospitals that do not have their own in-house sterile compounding pharmacy.
In a January 2014 letter, FDA encouraged hospitals to purchase compounded sterile drug products from FDA-registered outsourcing facilities because these facilities are under increased federal oversight.
9 The use of outsourcing facilities has gained traction, on the basis of a recent report from the Office of Inspector General, which surveyed a stratified random sample of 601 Medicare-participating hospitals.
Of the 564 hospitals that responded, 89% exclusively obtained nonpatient specific compounded drug products from FDAregistered outsourcing facilities, 9% obtained some drug products from outsourcing facilities, and 2% exclusively obtained drug products from unregistered compounders.
10 Another role of outsourcing facilities is to ensure continued availability of drug products and to provide a bridge in the event of manufacturer discontinuation or other scenarios in which there is a shortage of a commercial drug products.
3, 5 In the event of a commercial manufacturer electing to withdraw a drug product from the market for reasons not relating to safety or efficacy, a provider may use an outsourcing facility to obtain the withdrawn drug products.
The same holds true for drug shortage events; outsourcing facilities can bridge the gap until the commercial product becomes accessible again or until the provider can find another therapy that works for the patient.
Finally, outsourcing facilities can provide stock for provider to use for in-office procedures in specialties such as dentistry, ophthalmology, and podiatry.
Having drug products readily available in their office is more efficient for some providers than the process of using a 503A compounding pharmacy, in which the patient must take the prescription to the pharmacy, have it compounded, and then bring it back to the prescriber before it can be used.
By keeping the drug product stocked in their offices, providers have control of the storage conditions, assuring the providers of drug product quality and safety, which is especially important when using sterile drug products.
The ideal role of outsourcing facilities in the U.S. drug supply chain We believe the ideal role for outsourcing facilities is to produce ready-to-use sterile drug products for hospitals.
For some hospitals, implementing best practices guidelines such as United States Pharmacopeia (USP) <797> standards can be time-consuming and costly, as addressed in the 2019 public comments for the recent revision of the USP <797>.
11 Commenters on the USP revision felt that identifying microorganisms when the sampling level exceeds a certain threshold, changing filters during batch processing, and Compounding Record requirements would be burdensome.
11 Hospitals can avoid these requirements if products are supplied by outsourcing facilities.
Another benefit of using outsourcing facilities for hospital supply is that they can provide aseptic drug products with longer beyond-use dates, which can help decrease waste, turnover, and cost for hospital pharmacies.
11 In these scenarios, outsourcing facilities may end up a permanent part of the hospital's drug supply chain.
Outsourcing facilities can also be temporary suppliers for hospitals that require a longer implementation time to adhere to regulatory standards.
In the summary of public comments received for the revised USP <797>, there was a concern of not Key Points Background: The Drug Quality and Security Act was passed in 2013, leading to the establishment of 2 distinct types of pharmaceutical compounders, 503A and 503B.
Compounding under section 503A is performed by a physician or a pharmacist in a licensed facility, pursuant to a patient-specific prescription.
Compounding under section 503B is performed by outsourcing facilities and supervised by a pharmacist; a patient-specific prescription is not required for 503B compounding.
We believe that the ideal role for outsourcing facilities is to produce ready-to-use sterile drug products for hospitals.
Challenges that limit the use of outsourcing facilities in the U.S. drug supply chain include a lack of a finalized 503B Bulks List, inconsistences in the inspection process, and lack of transparency in compliance status.
When selecting outsourcing facilities, buyers should thoroughly research the facility before selecting it for their compounded drug needs.
having enough time to implement changes, with several commenters requesting at least 18-24 months.
11 There were also commenters who expressed concerns about remediation plans that involved closing the pharmacy, which would lead to the hospital not being able to continue compounding.
11 Although this revised chapter is on hold, there may still be delays in implementing future revisions.
During states of emergency, hospitals in need of sterile drug products that are in high demand could consider using outsourcing facilities.
For the coronavirus disease pandemic, FDA has implemented a temporary policy for compounding facilities that allows them to take a more active role in compounding certain drug products for hospitalized patients.
12, 13 Challenges and potential solutions to participation in the U.S. drug supply chain Despite the opportunities that outsourcing facilities have in the U.S. drug supply chain, there are multiple challenges that currently limit their use.
Although the 503B Bulks List is in development, outsourcing facilities can compound using category 1 nominated substances.
However, there is no guarantee that these substances will be included on the final list, so resources invested in compounding them may be wasted.
Because the 503B Bulks List is not finalized, outsourcing facilities remain concerned about using nominated drug substances still under review, especially because no nominated drug substance has been added to the list thus far.
[14] [15] [16] In a recent comment to FDA, the Outsourcing Facilities Association (OFA), the trade organization that represents outsourcing facilities, detailed their concerns about how the 503B Bulks List is being developed.
They suggested additional questions FDA should consider when making decisions for the 503B Bulks List and recommended consistent updates to the interim list and more transparency of the review process.
14 Similar challenges with using outsourcing facilities to provide compounded drug products were identified in a recent report from the Pew Charitable Trusts.
17 Another challenge to the use of outsourcing facilities is inconsistencies in the inspection process.
After an initial inspection, outsourcing facilities are inspected on a risk-based schedule.
There is no specific time interval in which subsequent inspections take place, which can lead to a facility being left uninspected for a prolonged period of time.
In OFA's 2019 comment to FDA, the organization expressed concerns about the "pace at which inspections are classified and closed, [creating] an inconsistent enforcement environment."
14 They noted that at some facilities, inspections remain open for years, with some of these facilities operating under a warning letter or other actions.
14 OFA called for a clear distinction between outsourcing facilities that are compliant and those that are not.
14 At the end of an inspection, the facility may be issued an FDA Form 483, which describes the investigator's concerns for conditions that the facility may be violating.
18 The FDA Form 483 is publicly available on the FDA's website along with information on the dates of initial and most recent registration, the last FDA inspection, and whether any FDA action was taken on the basis of the inspection.
19 However, the publicly available information does not mention whether the observed conditions have been resolved.
Moreover, the form may not provide a complete picture of the facility because it "does not include observations of questionable or unknown significance at the time of the inspection."
18 These observations can include anything from lack of written procedures for production and process controls to deficiencies in aseptic processing areas, quality control units, and employee training.
Therefore, the observed objectionable conditions are important for potential buyers because they could affect product quality.
For outsourcing facilities to be part of the U.S. supply chain, they need to proactively respond to the concerns outlined in FDA Form 483 in a transparent and publicly accessible manner.
This may include resolving the problems quickly and posting a notice about their action on their website so potential buyers can verify facility status.
In addition, FDA should provide the current compliance status of facilities on their website, along with a definitive time frame in which actions will next be taken by them.
This will allow health professionals to know the status of an outsourcing facility before selecting one for use.
When selecting an outsourcing facility for compounded drugs, buyers should first check the FDA's list of registered outsourcing facilities, which is available on the FDA website.
Most hospitals look at FDA registration and the facility's history of federal enforcement actions, including product seizures or injunctions, when choosing an outsourcing facility.
10 Buyers should also review the outsourcing facility's inspection history and most recent results from FDA and the state board of pharmacy.
The FDA website also has a list of products that outsourcing facilities have reported making in the past year.
For more information about what products are available, buyers should review the outsourcing facility's website or reach out to the contact person provided on the FDA's list of registered facilities.
Buyers can also use the American Society of Health System Pharmacists contractor assessment tool to evaluate a facility's compliance with USP <797>.
20 Although it may be logistically challenging, buyers should try to visit potential outsourcing facilities themselves whenever possible to assess if they meet their needs.
20 Buyers can also speak to other buyers who have previously ordered from the facility to have a better understanding of the environment and their compounding capabilities.
Outsourcing facilities can play an important role, especially for hospital supply of ready-to-use sterile drug products.
However, health care providers should recognize that drug products from outsourcing facilities are not held to the same regulatory standards as commercially available drug products and thoroughly research a facility before deciding to use it for their compounded drug needs.
FDA and outsourcing facilities should consider being more transparent about compliance status.
FDA could also provide more frequent updates about the substance list and the outsourcing facilities' status on the FDA webpage.
Health care providers, outsourcing facilities, and FDA need to work together to ensure that patients are provided the drugs they need in a safe and effective way.
Silver Spring, MD: Food & Drug Administration, Center for Drug Evaluation and Research, Office of Compliance/OUDLC Evaluation of bulk drug substances nominated for use in compounding under section 503B of the Federal Food, Drug and Cosmetic Act: guidance for industry.
Silver Spring, MD: Food and Drug Administration, Center for Drug Evaluation and Research Interim policy on compounding using bulk drug substances under section 503B of the Federal Food, Drug and Cosmetic Act: guidance for industry.
Silver Spring, MD: Food and Drug Administration, Center for Drug Evaluation and Research, Office of Compliance/OUDLC Drug products withdrawn or removed from the market for reasons of safety or effectiveness, 21 CFR, x216 Drug products that present demonstrable difficulties for compounding under the Federal Food, Drug, and Cosmetic Act; establishment of a public docket Compounded drug products that are essentially copies of approved drug products under section 503B of the Federal Food, Drug, and Cosmetic Act Compounded drug products that are essentially copies of a commercially available drug product under section 503A of the Federal Food, Drug, and Cosmetic Act: guidance for industry Silver Spring, MD: U.S. Department of Health and Human Services, Food & Drug Administration Most hospitals obtain compounded drugs from outsourcing facilities, which must meet FDA quality standards Pharmaceutical compoundingsterile preparations Temporary policy for compounding of certain drugs for hospitalized patients by outsourcing facilities during the COVID-19 public health emergency: guidance for industry.
Silver Spring, MD: Food and Drug Administration, Center for Drug Evaluation and Research Temporary policy for compounding of certain drugs for hospitalized patients by pharmacy compounders not registered as outsourcing facilities during the COVID-19 public health emergency (Revised) FDA-2019-N-3077: Agency information collection activities; submission for Office of Management and Budget review; comment request; obtaining information to understand challenges and opportunities encountered by compounding outsourcing facilities List of bulk drug substances for which there is a clinical need under section 503B of the Federal Food, Drug, and Cosmetic Act List of bulk drug substances for which there is a clinical need under section 503B of the Federal Food, Drug and Cosmetic Act Market for compounded drugs needs greater transparency and regulatory certainty.
Philadelphia, PA: The Pew Charitable Trusts FDA form 483 frequently asked questions.
U.S. Food and Drug Administration Registered outsourcing facilities.
U.S. Food and Drug Administration ASHP guidelines on outsourcing sterile compounding services
key: cord-274061-ynqxgyw6 authors: Epstein, Jay S.; Jaffe, Harold W.; Alter, Harvey J.; Klein, Harvey G. title: Blood system changes since recognition of transfusion‐associated AIDS date: 2013-10-17 journal: Transfusion DOI: 10.1111/trf.12373 sha: doc_id: 274061 cord_uid: ynqxgyw6 nan The fact that transfusions could transmit infectious diseases, namely, bacterial infections, syphilis, and hepatitis, was recognized before TAA with progressive interventions dating back to the dawn of blood banking.
Donor testing for antibodies to syphilis began in 1938.
7 Bacterial infections, a major threat at the time of World War II, were later decreased by cold storage of whole blood and red blood cells (RBCs) in plastic containers.
5, 7 In the 1970s, transfusion-associated hepatitis (TAH) was largely prevented by near elimination of paid donation through product labeling to identify paid collections, concurrent with testing for hepatitis B virus (HBV) infections.
5 However, the medical importance of the residual hepatitis risk, mostly attributed to non-A, non-B hepatitis (NANBH), was recognized slowly.
5 With the acute threat of bacterial infections largely controlled, syphilis effectively prevented, and the full consequences of NANBH transmission unappreciated, the blood community in the late 1970s was more focused on systemic issues of economic competition and supply instabilities than on transmissible disease.
Then came AIDS!
AIDS was first reported as a "gay-related immune deficiency" in 1981, but soon was identified in other risk groups including sex workers, Haitian entrants to the United States, and injection drug users.
3, 4 Evidence for transfusion transmission emerged in 1982 when a few cases of AIDS were reported in hemophilia patients and later in transfusion recipients.
However, despite a number of high-level federal meetings, actions by the national government to contain the AIDS risk from transfusion were not undertaken until 1983.
8 Although transfusion transmission of HIV undoubtedly took place at least 5 years before the recognition of TAA due to the very long asymptomatic period of the disease, the delay in a response to TAA subsequent to these initial reports of disease in persons with hemophilia and transfusion recipients also contributed to the AIDS tragedy.
Rage within the hemophilia community, due both to the fact of transmission of a fatal infection and to the failure of authorities to provide adequate warnings and preventions, was expressed in a demand for a congressional investigation.
Members of Congress instead directed the Department of Health and Human Services (HHS) to look into the matter.
This was accomplished through a contract with the Institute of Medicine (IOM) to study the evolving HIV-related events impacting blood safety and the decision-making process in this crisis period.
In its report, entitled "HIV and the Blood Supply: An Analysis of Crisis Decision Making (1995)," 9 the IOM found no wrongdoing by organizations or officials, but identified failed opportunities to better protect public health.
These failures to act more rapidly and aggressively in the face of TAA were seen to unmask an underlying weakness in the ability of federal agencies to address a new threat in the face of substantial scientific uncertainty.
This weakness was attributed to systemic deficiencies, primarily of leadership and coordination.
In particular, the IOM criticized the federal agencies for lack of top-level leadership needed to overcome inherent bureaucratic inertia; absence of a systematic approach within advisory committees sufficient to maintain their focus; over dependency on the regulated industry as a source of data given the inherent conflict of interest; and failure to engage in forward thinking both with respect to new technologies and emerging safety threats.
As a consequence, the risk of TAA was severely underestimated; patients and care providers were not suitably warned of the risk; and resistance to a change in the status quo caused delayed intervention.
In a set of 14 recommendations directed primarily at federal agencies, the IOM called for a more responsive and integrated decision-making process including establishment of a Blood Safety Council reporting to a designated Blood Safety Director within HHS and a standing "expert panel" to assure communication of blood product risks and alternatives to their use both to care providers and to the public.
Specifically to the Food and Drug Administration (FDA), the IOM recommended that, "Where uncertainties or countervailing public health concerns preclude eliminating potential risks, the FDA should encourage, and where necessary require, the blood industry to implement partial solutions that have little risk of causing harm."
While not itself a mandate, the IOM's admonition that the FDA should institute measured precautions in the face of uncertainty has become a dominant factor in blood safety decision making.
A more vigilant and proactive FDA approach to blood safety unfortunately has had the unintended consequence of dramatically increasing the manufacturing costs and therefore the price of blood.
10 The HHS response to the IOM report established a new landscape for federal oversight of the blood system, which continues to the present day.
The present structure includes the Assistant Secretary for Health (ASH) as the blood safety director; heads of Public Health Service and related agencies as members of a Blood, Organ and Tissue Safety Executive Council (BOTSEC); and an HHS Secretary's Advisory Committee for Blood and Tissue Safety and Availability (formerly the Advisory Committee for Blood Safety and Availability).
The ASH is the acknowledged national blood safety director with final responsibility and authority for decisions regarding blood safety and availability.
An interagency Blood, Organ and Tissue Safety Working Group meets monthly by teleconference, more often when necessary, and the BOTSEC meets approximately quarterly face to face with the ASH to provide information and guidance regarding current and emerging issues involving the nation's blood supply.
11 Unlike FDA's Blood Products Advisory Committee, whose function is to provide external scientific advice relevant to regulation, the Secretary's advisory committee is empowered to discuss broad legal, ethical, social, and economic issues affecting the blood system.
To give voice to patient concerns, both advisory committees seat voting representatives of communities that have been particularly affected by TAA.
Additionally, in response to a series of congressional hearings, reports from the Government Accountability Office, and the IOM study, the FDA developed and HHS subsequently adopted a comprehensive "blood action plan" 12 designed to address the identified shortcomings, to ensure greater coordination among the department's public health agencies, and to increase the effectiveness of the FDA's scientific and regulatory activities.
Notably, the post-TAA era has witnessed an aggressive effort by the FDA to improve blood safety through enforcement of cGMP in blood product collection and processing aligned with the model of pharmaceutical manufacturing and a more formal relationship than blood establishments experienced in the past.
The FDA initiative also involved promotion of automation to reduce human errors, including use of validated blood bank software.
An intensive program of field inspections designed to assure universal regulatory compliance of blood collection establishments resulted in a number of court-enforced voluntary injunctions (consent decrees).
Known and emerging infectious threats to blood safety have continued to demand attention in the post-TAA era, repeatedly testing whether the lessons of TAA were learned.
Are we prepared to deal with potential threats from bioterrorism agents?
How much effort should be expended to prepare for an outbreak of chikungunya virus that might never happen?
What should we do about pandemic influenza and Middle East respiratory syndrome coronavirus in the absence of studies to establish the presence or absence of viremia in the course of the infections?
Does it make sense to screen all blood donations when risks of babesiosis and dengue are seasonal and geographical?
What changes to the current paradigm of donor screening and testing can be considered when pathogen reduction becomes available for all blood components?
More generally, as we become increasingly proactive in addressing infectious risks, are we misdirecting resources that could be better spent to improve blood safety in other ways?
Readers of this commentary are encouraged to ask themselves whether the lessons of TAA have been optimally incorporated during the decades of challenge and response that followed.
A sentinel event in the history of blood safety was the recognition and response to TAA.
Although the etiology remained unknown, the report of AIDS in three persons with hemophilia A in July 1982 suggested a blood-borne pathogen as the causative agent.
3 These three individuals were reported to be heterosexual, had no other known AIDS risk factors, and had all received frequent administration of Factor VIII concentrate.
The evidence for transmission of the "AIDS agent" through blood was further strengthened in December 1982 by the report of a 20-month-old infant in San Francisco who had developed unexplained immunodeficiency after transfusion of multiple blood products to treat erythroblastosis fetalis.
4 One of the blood donors was a man who was healthy at the time of donation, but subsequently died of AIDS.
To address the possibility that AIDS was associated with the receipt of blood and blood products, the Centers for Disease Control and Prevention (CDC) convened a meeting on January 4, 1983, with participation by the FDA, the National Hemophilia Foundation, blood banking officials, and patient advocacy groups.
From the CDC perspective, the purpose of the meeting was to discuss how to reduce the risk of AIDS in transfusion recipients and persons with hemophilia in the absence of a test for the etiologic agent.
Several possible strategies were presented, including deferral of blood donations by persons known to be at increased risk for AIDS and the use of surrogate tests to identify persons at increased risk of transmission, such as those with detectable antibody to hepatitis B core antigen (anti-HBc) or low CD4/CD8 T-cell ratios.
However, the meeting turned into a contentious debate about the existence of AIDS in transfusion recipients and persons with hemophilia, and no agreement was reached on a risk reduction strategy.
On March 4, 1983 , the US Public Health Service published the first recommendations for prevention of AIDS.
8 Among the recommendations was a statement that, "As a temporary measure, members of groups at increased risk for AIDS should refrain from donating plasma and/or blood."
In addition to persons with clinical evidence of AIDS and their sexual partners, those considered to be at increased risk included "sexually active homosexual or bisexual men with multiple partners; Haitian entrants to the United States; present or past abusers of IV drugs; patients with hemophilia; and sexual partners of individuals at increased risk for AIDS."
At the time, these recommendations were controversial.
In particular, restricting blood donation by homosexual men was seen as a civil rights issue, and deferring donations by Haitian entrants undoubtedly led to discrimination against Haitian Americans.
From a public health perspective, however, these measures were needed to increase blood safety.
With the identification of HIV, screening of donated blood and plasma became possible.
Bulk preparations of the virus, known at the time as HTLV-III, were provided by the National Cancer Institute to diagnostics companies for the development of antibody detection tests.
The first such screening test, developed by Abbott Laboratories, was approved by the FDA in March 1985.
Because of concerns that persons would donate blood for the purpose of learning their HIV infection status, the CDC funded the first alternative HIV test sites, where individuals could obtain free and confidential testing.
Blood banks also established the option of confidential unit exclusion to allow persons who had donated blood to confidentially indicate that the blood should not be used for transfusion.
A watershed event in blood safety was the statement by the FDA commissioner at a September 1994 workshop that nucleic acid technology should be implemented to close the window period for HIV detection by serology.
This technology had been considered too costly and cumbersome for practical application in blood banking.
The introduction of direct testing for HIV in donor blood, first by p24 antigen assays, which proved largely unproductive, 13 and then by nucleic acid tests (NATs) for viral RNA, which proved beneficial, put to rest a decade of concern about residual HIV risk from donations in the 3-to 6-week infectious "window period" before seroconversion dependent on the sensitivity of different screening tests.
The successful adaptation of NAT to donor screening, including testing of specimens in small pools of 16 to 24, established a new era in risk reduction from transfusiontransmitted viral diseases.
In addition to increasing the safety of transfused blood, HIV antibody screening of donors led to "lookback" programs in which recipients of previous unscreened donations from infected donors were identified.
These recipients were found to be at substantial risk for infection.
14, 15 Although no effective treatment was available at the time, infected recipients could be counseled to reduce the risk of HIV transmission to others.
Another retrovirus, HTLV-I, was also found to cause disease, including adult T-cell leukemia or lymphoma and HTLV-1 associated myelopathy or tropical spastic paraparesis.
The virus can be transmitted by transfusion of cellular blood products, but not plasma fraction or plasma derivatives.
16 In November 1988, the FDA issued guidance recommending antibody testing of donated whole blood and cellular components for HTLV-I.
Because of a high degree of sequence homology, the currently approved HTLV-I screening assay also detects antibodies to HTLV-II, a virus with transmission routes similar to HTLV-I but with less clear disease associations.
Although not FDA approved, Western blot and PCR tests can be used to distinguish between the two viruses.
World War II led to recognition of the frequent occurrence of hepatitis among military personnel through the confluence of contaminated water, massive immunizations, and for the first time, blood transfusion.
It was during this time that food-and water-borne "infectious hepatitis" was distinguished from parenterally transmitted "serum hepatitis" and these entities were later termed hepatitis A and B, respectively.
In 1943, Beeson 17 reported seven cases of jaundice occurring 1 to 4 months after transfusion of blood or plasma.
A dramatic outbreak of hepatitis involving 50,000 US soldiers was traced to serum-contaminated preparations of yellow fever vaccine, which conclusively documented parenteral transmission.
Decades later, this outbreak was shown by Seeff and colleagues 18 to be due to the hepatitis B virus.
The US Army extensively studied serum hepatitis during and after the war and characterized both the epidemiology and the resultant disease, but could not identify the causative agent.
The etiologic breakthrough began in the early 1960s with the discovery of the Australia antigen by Blumberg and coworkers at the National Institutes of Health (NIH).
19 This single finding changed the course of hepatitis history when in 1968, the Australia antigen was shown by the Blumberg group to be associated with viral hepatitis 20 and then by Prince and colleagues 21 to be specifically associated with hepatitis B.
In England, Dane and coworkers 22 showed by immune electron microscopy that the Australia antigen represented the envelope protein of HBV and it was renamed the hepatitis B surface antigen (HBsAg).
The serologic distinctions between hepatitis A and B were further solidified by the controversial, but definitive prospective studies by Krugman and colleagues 23 at the Willowbrook State School.
The US government played a pivotal role in these momentous events, first through the initial discovery of the Australia antigen in the intramural program at NIH and then through extensive grant support of the Blumberg laboratory at the Institute for Cancer Research in Philadelphia.
In the late 1960s and early 1970s, prospective studies at the NIH Clinical Center revealed several critical elements of TAH, including: 1.
That the primary risk factor for TAH was the use of paid donor blood 24 confirming earlier studies; 25 in 1972, this led to an FDA mandate requiring the labeling of paid donor blood, which effectively resulted in the near-universal adoption of blood collection only from unpaid volunteers, one of the most important transfusion-transmitted infectious disease interventions ever implemented.
2.
That HBsAg testing of blood donors was effective even when using insensitive techniques such as agar gel diffusion and counterelectropherseis; 26 nationwide testing for HBsAg was delayed until more practical and confirmable assays were introduced in 1972. volunteerism and first-generation HBsAg screening reduced the incidence of TAH from 30% to approximately 10% 26 and that this massive reduction was more dependent on the donor source than on blood screening because HBV was shown to account for less than 30% of total TAH.
Feinstone and coworkers at NIH, 27 it became evident that HAV was not responsible for the residual cases of TAH, giving rise to the cumbersome, but nonpresumptive designation NANBH.
28 While intensive efforts to isolate the NANBH agent in the decade from 1975 to 1985 were unsuccessful, studies at the NIH and CDC revealed that the agent was small, lipid-enveloped and most similar to the small RNA alpha and flaviviruses.
[29] [30] [31] Despite the absence of a specific test for detecting the NANBH agent, TAH incidence declined because of the more judicious use of blood fostered by the recognition that NANBH could result in cirrhosis and death 32 and by the devastating consequences of transfusion-transmitted HIV.
5 Further, in the absence of specific NANBH assays, surrogate assays were advocated.
The Transfusion Transmitted Virus Study, supported by the National Heart, Lung and Blood Institute, published a retrospective analysis of a prospective study that showed that alanine aminotransferase (ALT) testing of donors might effect a 30% reduction in TAH incidence.
33 This was confirmed by a similar analysis of the NIH prospective TAH study, 34 but implementation of ALT donor screening at the NIH failed to demonstrate the predicted benefit.
35 Similar retrospective testing of the Transfusion Transmitted Virus Study 36 and the NIH 37 prospective studies suggested that anti-HBc testing might result in a 30% to 40% reduction in TAH, and this fostered the voluntary introduction of ALT and anti-HBc donor testing in 1986 to 1987; the FDA recommended routine donor testing for anti-HBc in 1992.
Although anti-HBc screening was introduced specifically to detect HBV carriers who were HBsAg negative (now termed occult hepatitis B), it also served as a surrogate for NANB carriers and for seronegative HIV carriers because of overlapping transmission routes.
Had anti-HBc surrogate testing been introduced in the early 1980s it presumably would have prevented some cases of transfusion-transmitted AIDS and NANBH.
This delayed implementation was the basis for extensive litigation, but also served as the driver for the IOM recommendation of invoking the "precautionary principle" when weighing new donor screening interventions and this precautionary approach has significantly improved transfusion safety.
Industry has played a major role in hepatitis prevention, first by developing increasingly sensitive assays for HBsAg, by developing nucleic acid detection assays for all the major viruses, and particularly by cloning the NANB agent.
38 The latter was a monumental achievement by Chiron Corporation in collaboration with Dan Bradley at the CDC.
Using the then-novel technique of expression cloning, these investigators identified a single clone among millions tested that reacted with serum from patients with NANBH.
Houghton and associates at Chiron then "walked" the genome, characterized an antigen derived from the nonstructural region of the viral genome, and developed an antibody assay to detect this viral protein.
39 Studies at the NIH 40 confirmed that the cloned agent, designated hepatitis C virus (HCV), was detected in virtually all NANBH cases and identified an implicated donor in near 90% of these cases.
First-generation anti-HCV testing was introduced in 1990 and secondgeneration assays in 1992.
Prospective studies at the NIH Clinical Center documented the virtual eradication of TAH by 1997; 35 mathematical modeling after the introduction of NAT screening in 1999 predicts that the current risk of transfusion-related hepatitis C is approximately one case in every 2 million transfusions, approximately the same risk as being hit by lightning.
West Nile virus (WNV) was first identified in the United States in 1999 after an outbreak of encephalitis in NewYork.
Four cases of unexplained fever and encephalitis in recipients of organ transplants from a common donor proved to be caused by WNV and raised the possibility of transmission through blood transfusion.
Initial efforts to screen blood donors using signs and symptoms of WNV infection proved ineffective.
41 In 2002, a total of 4156 cases of human illness were reported, and at least 21 people contracted WNV through transfusion, six of whom died.
42 The rapid expansion of WNV across the United States and reported to BOTSEC by the CDC lent urgency to developing a screening test before the next epidemic season.
43 The FDA requested that industry develop such a test; the National Heart, Lung and Blood Institute provided $3.47 million in research support; the American Red Cross provided 35,000 archived specimens; and the FDA facilitated rapid national test implementation and ultimate approval.
Although development of blood screening tests usually takes years, the NAT assay for WNV was available for the 2003 epidemic season, building on technology platforms already developed for HIV and HCV.
West Nile virus was the first acute infection with a short asymptomatic viremia and an epidemic spread to warrant routine donor testing and demonstrated a successful collaboration of government, blood collectors, and the diagnostics industry.
44, 45 A footnote to the WNV screening success was the recognition that testing of pooled samples was insufficiently sensitive to detect low-titer viremia in blood donations, including in the infectious preseroconversion donations commonly encountered during epidemic spread.
However, universal testing of individual units in nonepidemic areas nationwide was inefficient and costly.
This problem was solved through a novel strategy of triggering individual testing based on the yield of pool testing.
This approach effectively detected and interdicted approximately 1400 potentially infectious blood donations during 2003 to 2005.
46 The emergence of variant Creutzfeldt-Jakob disease (vCJD) in the United Kingdom and France first reported in 1996 posed what has been arguably the most challenging blood safety problem for decision makers since the beginning of the AIDS epidemic.
Like AIDS, vCJD presented a new disease with unknown transmission dynamics, the potential for transmission through blood transfusion, the recognition of a novel infectious agent (prions), and near invariable fatality.
47 vCJD was linked to bovine spongiform encephalopathy, a disease recognized in the United Kingdom since 1986, so the incubation period of the disease was assumed to be lengthy.
The scope of the epidemic was and remains unknown.
48, 49 For prions, unlike for bacteria and viruses, no technology for developing diagnostic or screening assays was available.
The FDA established a Transmissible Spongiform Encephalopathies Advisory Committee to assure focused, objective, and transparent input to its decision making.
Based on the available epidemiologic data in 1999, the FDA recommended that blood components collected from donors diagnosed with vCJD be withdrawn and developed a mathematical model for indefinite donor deferral based on geographic exposure (donors who resided in the United Kingdom for a total of 6 months or more, between 1980 and 1996) that eliminated an estimated 87% of donor exposuredays to bovine spongiform encephalopathy in the United Kingdom with a projected loss of approximately 2% of donors, which was considered a difficult balance of safety and supply, necessitating close monitoring of the blood supply.
50 Based on continuing surveillance of vCJD, the geographic exclusion was expanded in 2002, providing approximately a 90% reduction in total risk-weighted person-days of donor exposure to bovine spongiform encephalopathy in western Europe including the United Kingdom with an estimated total donor loss of approximately 7%.
The question of blood transmission was answered when the United Kingdom reported four cases of vCJD infections associated with blood transfusion that occurred between 2003 and 2007.
51 All four recipients had received transfusions of nonleukoreduced RBCs between 1996 and 1999, which confirmed the long incubation.
Only time will tell whether the steps taken in the United States will prove both warranted and sufficient, but the policy reflects adoption of a "partial solution" when it appears to reduce risk and an attempt to act expeditiously and responsibly with a benefit-to-risk model to address risk in the face of scientific uncertainty.
Chagas disease, caused by the protozoa Trypanosoma cruzi, affects an estimated 8 million people globally; an estimated 300,000 people in the United States and Canada are infected.
Most infections are found in immigrants from Latin America.
Whereas most new infections are vector borne, transmission by blood transfusion is well recognized.
Six transmissions had been reported in the United States before the ability to screen blood donors.
52 As early as 1989, the FDA Blood Products Advisory Committee recognized that while only 20% to 30% of those infected with T. cruzi develop symptomatic disease, the infection is lifelong in the absence of early treatment and can be fatal.
53 In view of increasing immigration to the United States from endemic regions, the Blood Products Advisory Committee recommended testing donors when a suitable test became available.
Donor history screening proved insufficiently sensitive and specific.
Not until 2006 was a test found suitable for licensure.
Shortly thereafter, the major blood collectors undertook universal donor screening for antibodies to T. cruzi.
In retrospect, an earlier study in Los Angeles and Miami suggested that seropositivity did not equate with infectivity; none of 18 recipients of blood from a subsequently identified seropositive donor had evidence of infection.
54 Two years of screening in the United States established that whereas the seroprevalence may be as high as 1 in 13,292 donors in some regions, infections confirmed by lookback studies are rare.
55, 56 Reexamination by the FDA of its decision to recommend universal donor screening led to a novel policy of once-in-a-lifetime donor testing based on the demonstrated rarity of acute or incident T. cruzi infections in US donors.
Anthrax: bioterrorism, public concern, and the blood supply Anthrax is caused by infection with a spore-forming Gram-positive bacterium Bacillus anthracis found globally in temperate zones, but uncommon in the United States.
57 Only seven cases of cutaneous anthrax had been reported to the CDC between 1980 and 2000 when in 2001 an outbreak of bioterrorism-related anthrax resulted in 22 confirmed or suspected cases including five fatalities.
58 This episode raised public concern about the blood supply during a period of high anxiety regarding threats of bioterrorism.
Bacteremia is present during fulminant cutaneous and respiratory anthrax; however, bacteremia in asymptomatic individuals has not been described.
The period between exposure to B. anthracis and development of clinical anthrax is reported as 1 to 7 days but may be as long as 60 days.
Little information exists regarding transmission via blood transfusion from an asymptomatic individual who has been exposed to B. anthracis.
No such cases have been reported and no licensed diagnostic or blood donor screening test exists.
The FDA received several inquiries regarding the risk to the blood supply from donors in direct contact with material contaminated with B. anthracis.
After consulting with experts at the CDC, the NIH, and the US Army Medical Research Institute for Infectious Diseases, the FDA issued guidance regarding measures to reduce possible risk for transmission of anthrax from blood.
59 The guidance did not recommend any changes to standard donor screening and blood collection procedures, but emphasized that standard blood collection procedures already in place include deferral of any donor who is not in good health at the time of donation.
Nevertheless, to address public concerns as well as the dearth of scientific information regarding blood transmission, the FDA provided prudent but specific recommendations concerning donors with a confirmed medical diagnosis of anthrax or proven colonization with B. anthracis and provided criteria for product quarantine and retrieval related to reports of postdonation illness.
59 In 2009, the journal Science reported that a gamma retrovirus, xenotropic murine leukemia virus-related virus (XMRV) was isolated from blood in two-thirds of patients diagnosed with chronic fatigue syndrome (CFS) and, most alarmingly, in 3.7% of healthy subjects.
60 A second article reported a related retrovirus (pMLV) with an even higher prevalence of 6.8% among blood donors.
61 These reports generated enormous public interest and concern.
Given the possibility that XMRV could be transmitted by transfusion, immediate calls arose to screen blood donors for signs and symptoms of CFS and to test donations for XMRV.
At the same time, intensive efforts were being undertaken worldwide to resolve this potential safety concern.
A federal interagency working group met repeatedly by teleconference and electronic communication, and laboratories within the CDC, NIH, and FDA invested resources into investigating discrepant laboratory results.
62 Additionally, representatives of the CDC, the FDA, and the intra-and extramural programs at the NIH participated in a public-private interorganizational task force assembled within 60 days by the AABB (formerly American Association of Blood Banks).
63 The result was voluntary implementation of an interim AABB recommendation that blood collectors should "actively discourage potential donors who have been diagnosed by a physician with CFS, chronic fatigue and immune dysfunction syndrome, or myalgic encephalomyelitis from donating blood" and ultimately definitive laboratory evidence that XMRV or pMLV bore no association with CFS and posed no threat to the blood supply.
64, 65 Ongoing threats and challenges Several infectious threats are currently challenging federal decision makers.
Bacterial contamination of platelets is a clearly identified risk that is being addressed with "partial solutions," culture, and point-of-issue serologic testing.
66 Hepatitis E virus is known to be transfusion transmitted, but potential disease burden has not been defined.
67 The geographic and travel exclusions to limit the risk of malaria transmission continue to be refined pending development of screening assays or pathogen reduction technology.
Surveillance for the coronaviruses responsible for severe acute respiratory syndrome and Middle East respiratory syndrome is active and the possibility that these agents as well as pandemic influenza and monkey pox might be transfusion transmitted or disrupt blood donation is unresolved.
The possibility of seasonal and geographic-based donor screening with validated tests for dengue and babesiosis has been modeled even as pilot studies of screening assays are ongoing.
68, 69 Pathogen reduction technology offers an alternative approach to risk mitigation.
Such technology would change the riskbenefit paradigm both for the known infectious agents and for those likely to threaten the blood supply in the future.
70 Federal decision makers are involved in deter-mining when and how this technology should be applied to the nation's blood and blood components.
The federal response to transfusion-transmitted infections has evolved dramatically since the emergence of HIV as a transfusion-transmitted infection.
The philosophy of risk management has become more precautionary and patient focused, yet still data driven.
Regulation of notfor-profit blood collectors has become more formal and stringent.
Manufacturers of blood components are now held accountable for meeting cGMP standards similar to those that apply to the manufacture of medical devices and pharmaceutical-type drugs.
A new and arguably more responsive federal structure for addressing issues of blood safety and availability has been adopted.
The decisionmaking structure places a premium on clear lines of authority, internal and public communication, flexibility, and coordination among the federal agencies with major roles in blood safety.
Federal agencies have encouraged public discourse through workshops, joint initiatives with industry, and participation in public-private partnerships with professional societies and blood collectors.
These adjustments have allowed federal agencies to respond with appropriate urgency to the differing situations posed by emerging infectious agents in the era since recognition of TAA 30 years ago.
71 HIV's leading men 25 years after HIV discovery: prospects for cure and vaccine Epidemiologic notes and reports pneumocystis carinii pneumonia among persons with hemophilia A Possible transfusion-associated acquired immune deficiency syndrome (AIDS)-California The hazards of blood transfusion in historical perspective Transfusion-associated infections: 50 years of relentless challenges and remarkable progress Syphilis: a disease of direct transfusion HIV and the blood supply: an analysis of crisis decision making.
Washington DC: Institute of Medicine National Academy Press Staff costs associated with the implementation of a comprehensive compliance program in a community blood center Risk-based decision-making for blood safety: preliminary report of consensus conference US Department of Health and Human Services.
Improving Blood Safety and Supply in the U.S The efficiency of HIV p24 antigen screening of US blood donors: projections versus reality Risk of human immunodeficiency virus infection from blood donors who later developed the acquired immunodeficiency syndrome Risk of human immunodeficiency virus (HIV) transmission by blood transfusions before the implementation of HIV-1 antibody screening Guidelines for counseling persons with human T-lymphotropic virus Type I (HTLV-I) and Type II (HTLV-II) Jaundice occurring one to four months after transfusion of blood or plasma: report of seven cases A serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the United States Army A "new" antigen in leukemia sera Australia antigen and acute viral hepatitis Immunologic distinction between infectious and serum hepatitis Virus-like particles in serum of patients with Australia-antigen-associated hepatitis Infectious hepatitis: evidence for two distinctive clinical, epidemiological, and immunological types of infection Posttransfusion hepatitis after open-heart operations Serum hepatitis from transfusions of blood Posttransfusion hepatitis after exclusion of the commercial and hepatitis B antigen positive donor Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness Transfusion-associated hepatitis not due to viral hepatitis type A or B Posttransfusion non-A, non-B hepatitis: physiochemical properties of two distinct agents Inactivation of hepatitis B virus and non-A, non-B virus by chloroform Determining the size of non-A, non-B hepatitis virus by filtration The chronic sequelae of non-A, non-B hepatitis Serum alanine aminotransferase of donors in relation to the risk of non-A, non-B hepatitis in recipients: the transfusion-transmitted virus study The relationship of donor transaminase (ALT) to recipient hepatitis: impact on blood transfusion services Hepatitis C virus and eliminating post-transfusion hepatitis Hepatitis B virus antibody in blood donors and the occurrence of non-A, non-B hepatitis in transfusion recipients: an analysis of the Transfusion-Transmitted Virus Study Antibody to hepatitis B core antigen as a paradoxical marker for non-A, non-B hepatitis agents in donated blood Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome An assay for circulating antibodies to a major etiologic virus of non-A, non-B hepatitis Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis and update on West Nile virus infections in recipients of blood transfusions West Nile Virus Transmission Investigation Team.
Transmission of West Nile virus through blood transfusion in the United States in 2002 As West Nile virus season heats up, blood safety testing lags behind West Nile virus among blood donors in the United States Screening the blood supply for West Nile virus RNA by nucleic acid amplification testing Triggers for switching from minipool testing by nucleic acid technology to individual-donation nucleic acid testing for West Nile virus: analysis of 2003 data to inform 2004 decision making Transmissible spongiform encephalopathies Estimation of epidemic size and incubation time based on age characteristics of vCJD in the United Kingdom Uncertainty due to model choice in variant Creutzfeldt-Jakob disease projections Guidance for industry: revised preventive measures to reduce the possible risk of transmission of Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-Jakob disease (vCJD) by blood and blood products Transfusion transmission of human prion diseases Transfusion-associated Chagas disease (American trypanosomiasis) in Mexico: implications for transfusion medicine in the United States Guidance for industry: use of serological tests to reduce the risk of transmission of Trypanosoma cruzi infection in whole blood and blood components intended for transfusion Trypanosoma cruzi in Los Angeles and Miami blood donors: impact of evolving donor demographics on seroprevalence and implications for transfusion transmission Epidemiological and laboratory findings from 3 years of testing United States blood donors for Trypanosoma cruzi The United States Trypanosoma cruzi Infection Study: evidence for vector-borne transmission of the parasite that causes Chagas disease among United States blood donors Anthrax as a biological weapon: medical and public health management.
Working Group on Civilian Biodefense Summary of notifiable diseases-United States Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome Detection of MLV-related gag gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States Xenotropic murine leukemia virus-related virus (XMRV) and blood transfusion: report of the AABB interorganizational XMRV task force Failure to confirm XMRV/MLVs in the blood of patients with chronic fatigue syndrome: a multi-laboratory study A multicenter blinded analysis indicates no association between chronic fatigue syndrome/myalgic encephalomyelitis and either xenotropic murine leukemia virus-related virus or polytropic murine leukemia virus AABB Bacterial Contamination Task Force.
Survey of methods used to detect bacterial contamination of platelet products in the United States in 2011 Seroprevalence and incidence of hepatitis E virus infection in German blood donors Dengue viremia in blood donors identified by RNA and detection of dengue transfusion transmission during the 2007 dengue outbreak in Puerto Rico Babesia microti real-time polymerase chain reaction testing of Connecticut blood donors: potential implications for screening algorithms Protecting the blood supply from emerging pathogens: the role of pathogen inactivation Emerging infectious agents and the nation's blood supply: responding to potential threats in the 21st century None.
key: cord-024833-e6vcf4un authors: nan title: Forum date: 2019-12-19 journal: Pharmaceut Med DOI: 10.1007/s40290-019-00321-z sha: doc_id: 24833 cord_uid: e6vcf4un nan The US Food and Drug Administration (FDA) is introducing electronic submission of safety reports for investigational new drug (IND) applications through the FDA's Adverse Event Reporting System (FAERS).
The FDA has released new draft guidance on the process as well as supporting technical specification documents.
The changes will enable the FDA to review pre-and post-marketing safety data within in the same system, with the same data standard.
Publication of the draft guidance and technical documents should assist drug manufacturers in beginning preparations for when final draft guidance is issued and becomes effective.
IND safety reports will be submitted in a reporting format that is consistent with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) E2B guidelines.
The FDA will shortly announce when sponsors can begin voluntary submissions of IND safety reports to FAERS.
"The FDA highly encourages sponsors of all INDs, both commercial and noncommercial, to begin submitting IND safety reports to FAERS voluntarily as soon as the new submission process is available," said Dr. Janet Woodcock, Director of the FDA's Center for Drug Evaluation and Research (CDER).
IND safety reporting via FAERS will be voluntary until 2 years after the final guidance has been issued, after which it will become mandatory for commercial INDs.
The FDA has created a separate submission path to FAERS for IND safety report submissions; they will remain designated as investigational and will not be publicly available.
US Food and Drug Administration.
Digital submission of adverse event reports for investigational new drug applications reflects FDA's ongoing modernization efforts.
29 Oct 2019. https ://www.fda.gov/news-event s/press -annou nceme nts/digit al-submi ssion -adver se-event -repor ts-inves tigat ional -new-drug-appli catio ns-refle cts-fdas-ongoi ng.
Accessed 11 Dec 2019.
The US Institute for Safe Medication Practices (ISMP) and the ECRI Institute have announced that they have formed an agreement to work together to enhance patient safety with regard to medicines, medical devices and healthcare practices.
ISMP will become a subsidiary of the ECRI Institute on 2 January 2020, and together the two non-profit organisations will create one of the largest patient safety entities in the world.
Approximately 80% of US hospitals rely on data and recommendations from the ECRI Institute to protect patients from unsafe practices and ineffective products, while the ISMP's efforts to improve safety in patients have resulted in changes to clinical practice and public policy, including improvements in drug labelling, packaging, preparation and administration.
"This agreement will strengthen our critical contributions to medication safety," said ISMP President Michael Cohen, "It allows both organizations to retain their core missions while immediately extending our ability to share lifesaving information and further a vision where safe, high-quality healthcare is more readily available".
"For both organizations, this agreement furthers the mission, deepens expertise, and broadens relationships.
It's a good move for both of us and for all of the organizations we serve, and ultimately for the patients worldwide," commented ECRI Institute President and CEO Marcus Schabacker.
Institute for Safe Medication Practices.
ECRI Institute and Institute for Safe Medication Practices join forces to enhance patient safety.
13 Nov 2019. https ://www.ismp.org/news/ ecri-insti tute-and-insti tute-safe-medic ation -pract ices-joinforce s-enhan ce-patie nt-safet y. Accessed 11 Dec 2019.
The US FDA has published a draft document on best postmarketing drug safety surveillance practices, says Dr. Janet Woodcock, Director of the CDER, in an FDA statement.
The FDA evaluates over two million adverse event reports annually that are submitted to FAERS through the Med-Watch Program, and to the Vaccine Adverse Event Reporting System (VAERS) by patients or their families or healthcare providers, as well as adverse event reports submitted by pharmaceutical companies, and this information is used by the FDA's Office of Surveillance and Epidemiology, and the Center for Biologics Evaluation and Research's Office of Biostatistics and Epidemiology, to identify safety concerns and recommend actions to improve safety.
The Cures Act was introduced in 2016 to amend the Federal Food, Drug, and Cosmetic Act by eliminating the requirement for the FDA to prepare a summary analysis of reports of adverse drug reactions (ADRs) received up to 18 months after drug approval or after use of the drug by 10,000 patients.
A new requirement of the Cures Act was for the FDA to make its best practices for drug safety surveillance publicly available on the web.
The FDA has now announced the availability of a draft document entitled "Best Practices in Drug and Biological Product Postmarket Safety Surveillance for FDA Staff", which outlines the agency's approach to timely postmarketing analyses of drugs and biologics, and "includes a high-level overview of tools, methods, and signal detection and evaluation activities, using varied data sources, for drug safety surveillance to provide a broader context and a general overview of our overarching effort and commitment in this area", says Woodcock.
The FDA is constantly seeking new methods for improving its surveillance practices, and is inviting the public to comment on its draft document on postmarketing surveillance.
US Food and Drug Administration.
Statement on the agency's efforts to protect patients through postmarket drug safety surveillance practices.
6 Nov 2019. https ://www.fda.
gov/news-event s/press -annou nceme nts/state ment-agenc ys-effor ts-prote ct-patie nts-throu gh-postm arket -drug-safet y-surve illan ce-pract ices.
Accessed 11 Dec 2019.
A number of improvements are being introduced to make medicine labels clearer in an effort to reduce medication errors in Australia, says the Therapeutic Goods Administration (TGA).
The regulatory agency has developed a set of posters targeted at both healthcare professionals and consumers, which will be released over a 4-year transition period.
One of these posters is a quick reference guide for healthcare professionals entitled "Your medicine, your knowledge-Improved medicine labels", which highlights "some of the key medicine labelling changes that are important for consumers", noted the TGA.
In addition, another three posters have been developed for consumers to raise their awareness of the labelling improvements, and are intended to be displayed in waiting rooms or public areas.
The TGA noted that "medicine labels are already starting to change" and that some of the key information that consumers should be aware of will include the following: • prominence of active ingredients; • clearer medicine information, which may include a "Critical Health Information table" that will provide information in a consistent order and will be easy to recognise; • information on declarable substances (e.g.
allergens) will now be required on the medicine label; • easier dispensing of medicines from pharmacies; • updated names for Australian medicine ingredients to align with names used internationally.
The TGA highlighted that medicine sponsors will have 4 years (i.e.
until 31 August 2020) to implement these changes and fully comply with the new labelling rules.
During this transition period, the agency will release targeted communications to consumers regarding the labelling changes.
Therapeutic Goods Administration.
Labelling changes: information for health professionals.
18 Oct 2019. https :// www.tga.gov.au/label ling-chang es-infor matio n-healt h-profe ssion als.
Accessed 11 Dec 2019.
Experts speaking at the European Public Health Alliance's Universal Access and Affordable Medicines forum in November 2019 urged that drug approval in the EU should require the pharmaceutical company's agreement on a fair price, said Elisabeth Mahase in the BMJ.
At the forum, experts across the EU discussed the need for improved transparency from drug companies to enable fairer drug pricing.
"We need a change in law.
After European Medicines Agency (EMA) registration, we need to identify what is a fair price as a basis for negotiation", commented Carin Uyl-de Groot, Director of the Institute for Medical Technology Assessment at Erasmus University, Rotterdam, the Netherlands.
She said that the marketing authorisation process should include assessments of cost effectiveness and fair pricing by the EMA or another organisation after drug effectiveness and safety have been reviewed, and countries could use the fair pricing assessment as a basis for price negotiations with drug companies.
Experts also discussed the need for more research on cancer drugs before their approval, to ensure their effectiveness and to identify the specific patient population which will benefit from treatment with them.
"Only 20-30% of cancer drugs actually have an impact.
We make drugs available at a very high speed, without actually knowing how to use them", said Denis Lacombe, Director General at the European Organisation for Research and Treatment of Cancer.
He called for more research into the optimal way to use cancer drugs, and said that they should be less expensive if this information is not available.
Mahase E. EU drug approval should include price evaluation, says expert panel.
BMJ.
2019;367:I6591.
In response to increasing overseas enquiries, the National Institute for Health and Care Excellence (NICE) is relaunching its International division to advise organisations, ministries and government agencies from outside the UK on health related, evidence-based decision making.
The not-for-profit International division, operating on a fee for service basis, draws on the expertise of internal staff, academic partners and world-wide experts to provide support with cost effective, transparent resource allocation, improved quality of care and equitable access.
Clients can attend knowledge transfer seminars in the UK, overseas or via web conference, or employ the consultancy service for more intensive support with technical and institutional training including implementing new methods and programmes, and adapting NICE guidelines to local healthcare settings.
Chief Executive at NICE, Andrew Dillon said "We're delighted to be relaunching NICE International so that we can share what we've learnt over the last 20 years to help other international health and care systems optimise their use of evidence-based practice".
National Institute for Health and Care Excellence.
NICE International returns to deal with growing overseas enquiries.
5 Nov 2019. https ://www.nice.org.uk/news/artic le/ nice-inter natio nal-retur ns-to-deal-with-growi ng-overs easenqui ries.
Accessed 11 Dec 2019.
Market exclusivity extensions to promote antibiotic drug development may lead to billions of dollars in additional societal spending on prescription drugs, according to a study conducted by researchers from Harvard Medical School in Boston, US.
The researchers estimated the economic impact of the Re-Valuing Anti-Microbial Products (REVAMP) Act introduced in the US House of Representatives in June 2018 with the goal to promote research and development of novel antibiotic drugs, specifically those targeting multi-drug resistant pathogens.
The Act offers manufacturers that gain FDA approval for specific novel antibiotics a 12-month transferable market exclusivity extension voucher that could be applied to an existing brandname drug or sold to another manufacturer.
The analysis identified 10 antibiotics approved by the FDA from 2007 through 2016 that would likely have qualified for an exclusivity voucher, and each antibiotic was matched to the fast-track drug with the highest revenue losing exclusivity within 4 years of the antimicrobial's approval date.
The median annual revenue of these 10 antimicrobials prior to generic entry was estimated at $US249 million.
Accounting for a 75% spending reduction after generic entry, the median spending associated with an exclusivity voucher was $187 million.
Total spending associated with a 1-year exclusivity extension for all 10 drugs was estimated at $4.5 billion.
"Our results raise important concerns about the overall cost of transferable exclusivity vouchers", conclude the researchers.
Rome BN, Kesselheim AS.
Transferrable market exclusivity extensions to promote antibiotic development: an economic analysis.
Clin Infect Dis.
Epub 20 Oct 2019. http:// doi.org/10.1093/cid/ciz10 39.
Accessed 11 Dec 2019.
The newly developed TECHnical VERification (TECH-VER) checklist for validating health economic decision-analytical models can help identify causes of model implementation errors and be used as a training and quality control tool for the development of models, say authors of a report published in PharmacoEconomics.
EMBASE and MEDLINE databases were searched up to May 2019 for studies on the credibility, validation and verification of models for health technology assessment (HTA) or cost-effectiveness analysis.
A draft checklist was developed based on findings from studies identified in the literature review, and applied to health economic models which varied with respect to stakeholder developer, purpose and the maturity of clinical evidence.
Iterative revision of the checklist was performed and checked after implementation.
The checklist then underwent further revision after discussions with other health economists, until the final version of TECH-VER was created.
The TECH-VER requires a model reviewer, a transparent model, input sources, and detailed documentation reporting the concept, implementation, model inputs and results.
It includes five domains: input calculations, event-state calculations, result calculations, uncertainty analysis calculations, and overall checks.
The reviewer should assess the justifications of methods used in calculations.
Verification tests conducted to check the correctness of implementation of these calculations should include (in consecutive order): black-box tests to check that model calculations align with a priori expectations; white-box testing of program code line by line, or cell by cell for crucial calculations if black-box test results are unexpected; and model replication/parallel programming if issues related to unexpected results from black-box tests cannot be resolved through white-box testing.
"The TECH-VER checklist is a comprehensive checklist for the technical verification of decision analytical models, aiming to help identify model implementation errors and their root causes while improving the transparency and efficiency of the verification efforts", said the authors.
"It is the authors' aim that the TECH-VER checklist transforms itself to an open-source living document, with possible future versions, or 'bolt-on' extensions for specific applications with additional 'fit-for-purpose' tests, as well as 'tips and tricks' and some demonstrative error examples," they commented.
Buyukkaramikli NC, Rutten-van Mölken MPMH, Severens JL, et al.
TECH-VER: a verification checklist to reduce errors in models and improve their credibility.
Pharmacoeconomics.
Epub 8 Nov 2019. https ://doi.org/10.1007/s4027 3-019-00844 -y. Accessed 11 Dec 2019.
In most cases, dosage adjustments in patients with renal impairment can be effectively determined using their estimated glomerular filtration rate (eGFR).
However, the UK's Medicines and Healthcare products Regulatory Agency (MHRA) notes in a Drug Safety Update that "in some circumstances, the Cockcroft-Gault formula should be used to calculate creatinine clearance (CrCl)".
The MHRA has received queries about choosing which renal function measurement to use.
In some patient groups or clinical situations, eGFR can overestimate renal function, and can lead to prescribing higher than recommended medication doses.
The agency has received reports of ADRs when eGFR was used to determine dosage adjustments in patients with renal impairment.
One Yellow Card report involved an elderly patient treated with a direct-acting anticoagulant (DOAC) who developed a significant bleeding event; a review revealed that the dose initiated "was too high for the patient".
A cross-sectional study of 8 drugs in 80 general practices from the UK revealed that in elderly patients with reduced renal function, there was widespread prescribing outside recommendations; using eGFR overestimated renal function for up to 28% of patients.
Using CrCl is recommended for patients in the following situations: ≥ 75 years of age; at extremes of muscle mass; taking DOACs; taking nephrotoxic drugs; or taking drugs which are largely renally excreted and have a narrow therapeutic index.
Healthcare professionals are requested to report suspected ADRs via the Yellow Card scheme.
Medicines and Healthcare products Regulatory Agency.
Prescribing medicines in renal impairment: using the appropriate estimate of renal function to avoid the risk of adverse drug reactions.
Drug Safety Update.
2019;13 (3):6-7.
Gifts from pharmaceutical companies to general practitioners (GPs) in France appears to influence primary care prescribing, according to findings of a retrospective study published in the BMJ.
Data from the National Health Data System database managed by the French National Health Insurance (NHI) system, and from the French Transparency in Healthcare database, were used to investigate the association between the monetary value of drug company gifts to GPs and prescribing patterns of 41,257 GPs who worked in the private sector in France in 2016.
The amount per visit reimbursed by the NHI was significantly lower in GPs for whom no gifts were reported in the Transparency in Healthcare database than in those who received at least one drug company gift valued at ≥ €1000 in 2016 (− €5.33; p < 0.001).
GPs who received no gifts more frequently prescribed generic antibacterials, antihypertensives and HMG-CoA reductase inhibitors (statins) than GPs who received gifts valued at ≥ €1000 (all p < 0.001).
They significantly less frequently prescribed vasodilators but significantly more often prescribed ACE inhibitors versus ACE inhibitors plus angiotensin-II receptor antagonists (sartans) compared with GPs who received gifts valued at ≥ €1000.
There were no significant differences in prescribing of aspirin or generic antidepressants or proton pump inhibitors between GPs with and without gifts from drug companies.
"Our study shows that gifts to GPs are common and associated with less rational drug prescriptions for patients and more expenses for the National Health Insurance", said the authors.
The findings "suggest that French GPs who do not receive gifts from pharmaceutical companies have better drug prescription efficiency indicators (as defined by France's National Healthcare Insurance) and less costly drug prescriptions than those who receive gifts", they concluded.
In Australia, adverse events in vaccinated patients are monitored by the AusVaxSafety system, notes the Department of Health, and a report of safety during 2018 indicated that vaccines in the National Immunisation Program (NIP) are very safe.
Vaccine recipients or their carer are sent an SMS message by the 290 participating immunisation clinics, asking whether the recipient had any postvaccination adverse events.
Responses include Yes, No, or Stop (to opt out).
During 2018, > 80,000 SMS messages were sent, with > 58,000 responses.
A short survey is sent to Yes responders, requesting a description of the adverse event.
Report of a visit to the emergency department (ED) or a physician is flagged with the provider, who follows up with the recipient and notifies the TGA division if required.
Responses are closely monitored by AusVaxSafety, enabling potential problems to be detected and acted on.
Children are vaccinated against serious diseases at the schedule points of 2, 4, 6, 12 and 18 months of age and at 4 years.
NIP modifications from July 2018 include the third dose of pneumococcal vaccine at 12 months rather than 6 months of age, and Hib vaccine at 18 months of age and a quadrivalent meningococcal vaccine at 12 months of age rather than a combined Hib-meningococcal vaccine at 12 months of age.
An adverse event was reported by 9%, 12%, 7%, 12%, 13% and 19% of patients at the 2, 4, 6, 12 and 18 months and 4 years schedule points, respectively.
An ED or physician visit was reported by 0.9%, 0.9%, 0.7%, 1.2%, 1.6% and 2% of patients.
The most frequently reported adverse events were fever, irritability, and injection site pain, swelling or redness, which are similar to reports in previous years.
The proportion of serious adverse events (SAEs) was unaltered, and the overall type and proportion of adverse events was similar before and after modification of the NIP schedule.
Adolescents are vaccinated against human papillomavirus and diphtheria/tetanus/whooping cough.
Adverse events were reported by 9% of patients, with 0.6% requiring an ED or physician visit.
The most commonly reported events were tiredness and injection site pain.
Pregnant women are vaccinated against influenza and diphtheria/tetanus/whooping cough.
Adverse events were reported by 6% of patients, with 0.3% requiring an ED or physician visit.
The most commonly reported events were injection site pain, swelling or redness.
Department of Health-Australia.
Vaccine safety in Australia.
AusVaxSafety summary report 2018.
18 Nov 2019. https ://www.healt h.gov.au/sites /defau lt/files /docum ents/2019/11/vacci ne-safet y-in-austr alia-ausva xsafe tysumma ry-repor t-2018.pdf.
Accessed 11 Dec 2019.
Results of a preliminary study, reported in the Journal of Oncology Practice, show that use of the Decision Aid developed by the American Society of Clinical Oncology (ASCO) improves the accuracy of reporters in the attribution of SAEs to a drug, but has no apparent effect on determining seriousness.
Under US FDA regulations, sponsors of clinical trials relating to IND applications are required to reports SAEs that are unexpected and suspected to be drug-related.
Based on the FDA's Final Rule and related guidances, ASCO developed the one-page Decision Aid stepwise flowchart.
The crossover study included 29 physician-investigators or research staff from community or academic research sites who are involved in SAE reporting decisions at their site.
Ten clinical case studies were evaluated, with five assessed with the assistance of the Decision Aid tool.
Compared with an unassisted assessment, the Decision Aid tool did not significantly improve the accuracy of determining serious for the case studies (odds ratio [OR] 0.87; 0.31, 2.46).
However, the accuracy of attributing an SAE to the drug was significantly increased (OR 3.60; 1.15, 11.4).
Most participants assessed the tool as being helpful (93%) and allowing improved decision-making time (69%) and confidence in reporting (83%), and indicated that they would use the tool in practice (83%).
The preferred delivery method was by mobile application, followed by hard copy/ paper or via the ASCO web site.
Potential barriers to tool use included contract/protocol expectations or restrictions, and variability of the SAE review and decision-making process among sites.
"The Decision Aid shows promise as a method to improve the quality of SAE attribution", note the authors, "which may improve the detection of valid safety signals and reduce the administrative burden of uninformative investigational new drug safety reports".
They add that "study of the Decision Aid in a larger sample with analysis stratified by participant role and SAE reporting experience would further assess the tool's impact".
The accuracy of forecasting cancer drug costs can be improved by using a hybrid approach combining automated time-series forecasting and expert customisation, report researchers from Canada.
The researchers developed a forecasting framework from both a technical and a policy perspective to provide a flexible tool that can improve the accuracy of forecasts while incorporating multiple forecasts for the Canadian province of Ontario.
The optimal forecasts for the top nine drug policies that made up the first 80% of the total budget in the previous 12 months ('big budget drivers' [BBDs]) funded by the Provincial Drug Reimbursement Programs resulted in errors within ± 4%.
It was estimated that the forecast error from the automated approach for these nine BBD policies would have been approximately $Can2.2 million, compared with approximately $Can7.2 million using a manual approach.
This corresponds to savings of $Can5 million for these policies.
"The flexibility provided by the hybrid approach should ultimately lead to improved accuracy, which will help achieve our policy-related goal: to help the programme make better decisions regarding to ability to fund drugs in the oncology pipeline", note the researchers.
This approach to drug budget forecasting was implemented in Ontario for the first time in the 2017/2018 fiscal year.
The forecasts resulted in a 1% error for the overall budget, corresponding to an overestimate of expenditures by $Can3 million.
Although the high-cost CAR-T cell therapies tisagenlecleucel (Kymriah) and axicabtagene ciloleucel ( While ICER considered evidence provided certainty of the cost effectiveness of Yescarta and Kymriah, NICE and the TLV granted access to the drugs despite a high level of uncertainty in their cost effectiveness.
Gaps in clinical evidence were identified for both drugs in all five countries, primarily with regard to the lack of long-term data on overall survival and progression-free survival, and a lack of comparative data.
NICE, G-BA, TLV and the TC required further evidence to demonstrate the long-term clinical benefit and cost-effectiveness of the drugs.
Strategies have been implemented to mitigate the risk of introducing these high-cost drugs.
"The fact that both Yescarta and Kymriah have widespread access, despite significant uncertainty in clinical evidence and cost-effectiveness (where relevant), is largely reflective of the high unmet need in DLBCL, the willingness of payers to access CAR-T therapies and the high level of innovation perceived", said the authors.
"For both drugs, HTA bodies are recommending that re-assessment occurs when more clinical and economic data (from clinical trials, country-specific real world evidence and indirect comparisons) becomes available", they noted.
Many drugs used in dermatology appear to be associated with idiopathic intracranial hypertension (IIH), leading to the suggestion that the term drug-induced intracranial hypertension (DIIH) should be introduced, say authors of a systematic review published in the American Journal of Clinical Dermatology.
Investigators searched MEDLINE, EMBASE, and Cochrane Review Databases for all articles reporting potentially drug-related cases of IIH up until June 2019.
In total, 235 articles were considered to be relevant.
For all cases which met modified Dandy criteria for diagnosis of IIH, the Koh algorithm for adverse drug reactions (ADR) was used to assess the likelihood of IIH being drug-related.
Overall, 259 cases of DIIH were verifiable.
Vitamin A (retinol) and its derivatives (isotretinoin and tretinoin) were most frequently associated with DIIH (84 cases).
Other drugs or drug classes most strongly associated with DIIH (≥ 20 cases) included recombinant growth hormone and tetracyclines (including doxycycline, minocycline and tetracycline).
Lithium was also strongly associated with DIIH (15-19 cases) while corticosteroids were determined to be moderately associated with DIIH (10-14 cases).
Drugs found to be weakly associated with DIIH included amiodarone, ciclosporin, combined oral contraceptives, danazol, fluoroquinolones (including ciprofloxacin, levofloxacin, nalidixic acid and ofloxacin), gonadotropin-releasing hormones, luteinising hormone stimulants, subcutaneous implantable progestogen-only contraceptives (Nexaplanon and Norplant), stanozolol, sulfasalazine, sulfenazone, ustekinumab and valproate semisodium (divalproic acid).
"We suggest using the term 'drug-induced intracranial hypertension' (DIIH) and propose a set of diagnostic criteria for DIIH… This may ultimately assist physicians in counselling patients about the risk of DIIH when prescribing medications and recognizing this uncommon yet sightthreatening condition", said the authors.
"If DIIH is suspected by the physician, a timely referral for co-evaluation and co-management by a neurologist and an ophthalmologist is recommended", they commented.
Tan MG, Worley B, Kim WB, et al.
Drug-induced intracranial hypertension: a systematic review and critical assessment of drug-induced causes.
Am J Clin Dermatol.
Epub 18 Nov 2019. http://doi.org/10.1007/s4025 7-019-00485 -z. Accessed 11 Dec 2019.
Despite premarketing efforts to reduce the risk of medication errors (MEs) and a mandatory EU Risk Management Plan for all newly licensed medicinal products, over a quarter of centrally authorised products in the European Economic Area (EEA) are associated with ME safety concerns, according to findings of a study published in Drug Safety.
Data from the European Public Assessment Report registry were used to investigate ME safety concerns included in risk management plans of originator centrally authorised products which were authorised between 2010 and 2017 in the EEA.
For each product, safety concerns, categories for the Summary of Safety Concerns, and how MEs were addressed, were assessed.
In total, 311 centrally authorised products were approved during the study period, and 84 (27%) of the products had a total of 95 ME safety concerns.
The proportion of products with ME safety concerns ranged from 15.2% in 2011 to 36.4% in 2015.
The most frequent types of ME were drug administration error (n = 17), product dosage form confusion (n = 10) and product preparation error (n = 9).
Additional risk minimisation measures (aRMMS) were required to address 27 ME safety concerns in 23 of the products, and included educational material for healthcare professionals (85.2%) and/or patients (51.9%).
Studies evaluating the effectiveness of the additional measures were agreed upon for 78.3% of products with aRMMs for MEs.
"The high number of products with ME safety concerns and the high proportion of ME safety concerns with aRMMs suggest awareness regarding MEs at the level of the pharmaceutical industry and regulators.
There is limited knowledge regarding the effectiveness of the measures available to prevent MEs.
Therefore, studies are necessary to evaluate the suitability of the current risk minimisation framework for MEs", said the authors.
In patients hospitalised for an exacerbation of chronic obstructive pulmonary disease (COPD), there is a high prevalence of drug-related problems, according to study results reported in Drug Safety.
The study was conducted in an academic teaching hospital in China.
Drug-related problems and interventions were analysed based on the Pharmaceutical Care Network Europe (PCNE)-DRP V8.02 classification, where one problem (P) may have multiple causes (C) and lead to more than one intervention (I) but lead to only one outcome (O).
There were 640 drug-related problems in 393 patients, or an average of 1.6 per patient, which had 763 corresponding causes.
At least one problem occurred in 223 patients (56.7%).
The major type of problem identified was "treatment safety P2" (54.2%), followed by "treatment effectiveness P1" (24.1%).
The most frequently identified cause of the problem was "drug selection C1" (24.2%), followed by "dose selection C3" (21.5%) and "treatment duration C4" (17.7%).
The three major cause subcategories were drug dose too high, duration of treatment too long, and patient administers/uses the drug in a wrong way.
"This indicates that the pharmacist should provide necessary patient education on the correct use of drugs when providing medication review", note the authors.
Most patients (96.9%) were receiving polypharmacy (≥ 5 medications), with an average of 11.2 medications per patient.
The five most frequently used medication classes were antibiotics, antihypertensives, bronchodilators, corticosteroids, and expectorants.
The drug class most frequently involved in drug-related problems was antibiotics (36.7%), followed by corticosteroids (19.8%) and proton pump inhibitors (10.2%).
In multivariate logistic regression analysis, there was a significant association with drug-related problems in patients with renal dysfunction, those taking ≥ 10 drugs, and those who are hospitalised for ≥ 8 days, and an association in patients with ≥ 3 comorbidities.
Pharmacists proposed 1557 interventions to solve the drug-related problems, an average of 2.4 per problem identified.
These were mostly made at the "drug level I3" (45.5%), followed by at the "prescriber level I1" (32.6%) and at the "patient level I2" (16.2%).
Overall, 91.0% of interventions were accepted, and 80.0% were fully implemented; 91.6% of problems were solved.
"Pharmacists can have an important role in addressing the problems and optimizing the safety and effectiveness of therapies for hospitalized COPD patients", note the authors.
Ranitidine products containing n-nitrosodimethylamine (NDMA) levels above acceptable limits will be recalled in the US while investigations are ongoing into the contamination, says CDER Director Dr. Janet Woodcock.
A number of US-approved medicines containing ranitidine (commonly known as Zantac) have been tested over the past few months, and a summary of current investigation results was recently presented by Dr. Woodcock.
She noted that the CDER had "conducted tests that simulate what happens to ranitidine after it has been exposed to acid in the stomach with a normal diet and results of these tests indicate that NDMA is not formed through this process".
NDMA was also not formed when ranitidine was exposed to a simulated small intestine environment, but Dr. Woodcock noted that human trials still needed to be carried out to fully understand if ranitidine forms NDMA.
Although many NDMA levels observed in US FDA testing to date "are much lower than the levels some third-party scientists first claimed, some levels still exceed what the FDA considers acceptable for these medicines", highlighted Dr. Woodcock.
If the FDA or manufacturers find NDMA levels that are above acceptable limits (i.e.
96 ng/day, or 0.32 parts per million), then companies are being asked to voluntarily recall their ranitidine product(s).
In addition, manufacturers are being asked to voluntarily recall products containing nizatidine (commonly known as Axid, and chemically similar to ranitidine) if NDMA levels are found to be above the acceptable daily intake level.
Manufacturers are also requested to continue conducting their own laboratory testing to assess NDMA levels in products containing ranitidine or nizatidine, and samples should also be sent to the FDA so that the agency could conduct its own tests.
To date, FDA testing of a ranitidine syrup commonly used in paediatric patients has revealed some samples with NDMA levels above the acceptable limits, which are now being recalled.
Testing of a ranitidine injection is also ongoing.
Woodcock J.
Statement on new testing results, including low levels of impurities in ranitidine drugs.
1 Nov 2019. https ://www.fda.gov/news-event s/press -annou nceme nts/state ment-new-testi ng-resul ts-inclu ding-low-level s-impur ities -ranit idine -drugs .
Accessed 11 Dec 2019.
Attention-deficit hyperactivity disorder (ADHD) medication does not appear to be associated with serious cardiovascular (SCV) events in children or adolescents with ADHD or autism spectrum disorder (ASD), according to findings of a F. Hoffmann-La Roche-funded study published in CNS Drugs.
Commercial claims data (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) and Medicaid claims data (2012-2016) from the US Truven Health MarketScan database were used to conduct nested case-control studies in paediatric patients 3-18 years of age with ADHD (n = 2,240,774) or ASD (326,221).
Each case with the composite outcome of stroke, myocardial infarction (MI) or serious arrhythmias was matched with ten controls based on age, sex and insurance type.
Conditional logistic regression analysis was used to evaluate the associations between SCV events and current ADHD medication use including CNS stimulants such as methylphenidate or non-stimulants such as atomoxetine.
The risk of SCV events was not significantly increased in patients with ADHD or ASD; in the ADHD cohort 33.9% of cases and 32.2% of controls were exposed to ADHD medication (OR 1.08; 95% CI 0.78, 1.49), while in the ASD cohort 12.5% of cases and 22.1% of controls were exposed to ADHD drugs (OR 0.49; 95% CI 0.20, 1.20).
There was also no increased risk of SCV events after adjustment for covariates, or for individual SCV outcomes.
"In conclusion, in a large, contemporary insurance database, we found low rates of SCV events in children and adolescents with ADHD (3.1/100,000 person years) and ASD (5.6/100,000 person years).
Furthermore, we found no evidence of an increased SCV risk when exposed to ADHD medications," said the authors.
Magnesium sulphate is the drug most frequently associated with ADRs in women with high-risk pregnancy in Brazil, according to findings of a study published in the European Journal of Clinical Pharmacy.
ADRs were investigated in 607 women with highrisk pregnancies admitted to an obstetric intensive care unit (ICU) in Brazil between June 2016 and December 2017.
Patients were excluded if they were admitted to the ICU for non-obstetric conditions, stayed in the ICU for less than 24 h, or were readmitted to the ICU.
The overall incidence of ADRs was 27.2%.
No severe ADRs were reported but 29.7% of ADRs were of moderate severity.
The incidence of ADRs was highest in patients receiving magnesium sulphate (44.5%), and was much lower for all other drugs, including vancomycin (14.3%), meropenem (9.2%), cefalexin (5.3%), azithromycin (4.5%), methyldopa (2.6%), mineral oil (1.4%), insulin (1.1%), ferrous sulphate (0.5%) and captopril (0.3%).
ADRs reported in patients receiving magnesium sulphate included somnolence (68.6%), absent patellar reflex (21.6%) and hypotension (9.8%).
All four cases of methyldopa-related ADRs were somnolence.
Moderate hypoglycaemia was reported in one patient receiving insulin, and mild diarrhoea was reported in patients receiving azithromycin, cefalexin, meropenem, ferrous sulphate and vancomycin (one case each).
Multivariate analysis found that higher BP (adjusted odds ratio [aOR] 1.02), higher haemoglobin level (aOR 1.21) and lower body temperature (aOR 0.71) significantly increased the risk of ADRs.
Considering its therapeutic importance in preeclampsia/ eclampsia and for foetal neuroprotection, the benefits of magnesium sulphate to the mother and foetus outweigh its risks, even though its use is associated with significant ADR, said the investigators.
Xavier da Costa T, de Almeida Pimenta Cunha MD, do Vale Bezerra PK, et al.
Incidence of adverse drug reactions in high-risk pregnancy: a prospective cohort study in obstetric intensive care.
Eur J Clin Pharmacol.
Epub 25 Nov 2019. https ://doi.org/10.1007/s0022 8-019-02789 -9.
Accessed 11 Dec 2019.
The risk of interstitial lung disease will be added to the package inserts for two prostate cancer medicines-apalutamide and enzalutamide, according to Japan's Pharmaceuticals and Medical Devices Agency (PMDA).
Apalutamide (Erleada Tablets) is indicated for treatment of castration-resistant prostate cancer without remote metastasis.
Since the launch of this product in Japan in May 2019, there have been a total of four cases involving interstitial lung disease (including two cases for which a causal relationship to the drug could not be ruled out).
In addition, there has been one patient death reported to date, but again, a causal relationship between the drug and death subsequent to the patient's interstitial lung disease could not be ruled out [1] .
Enzalutamide (Xtandi Capsules, Xtandi Tablets) is indicated for the treatment of castration-resistant prostate cancer.
Over the past 3 fiscal years in Japan, a total of 19 cases involving interstitial lung disease have been reported to date (including five cases for which a causal relationship to the drug could not be ruled out).
Furthermore, three patient deaths have been reported, but a causal relationship between the drug and deaths could not be established in any of these cases [2] .
Based on its investigation of currently available evidence and consultation with expert advisors, the MHLW/PMDA concluded that the following package insert revisions are necessary for both apalutamide and enzalutamide: 13 reports) and fluticasone-associated adrenal cortical hypofunction (PRR 59.5; 95% CI 39.1, 90.4; 22 reports) .
All ten signals with the highest PRRs were already known.
There were 30 signals classified as new.
New signals unmasked by calculating the PRR by therapeutic area included herpes virus infections and bacterial infections associated with omalizumab, and herpes virus infections associated with budesonide.
Other new signals included hypertrichoses with budesonide and encephalopathy with theophylline.
Overall, 16% of the signals in paediatric patients did not appear in the total patient population.
"Although 92% of the statistical signals were already known, we observed 30 new signals, especially for omalizumab.
Calculation of the PRR by therapeutic area, age and sex revealed additional new signals, pointing to masking due to confounding by indication or effect modifi-cation… Safety signals of asthma drugs were mainly related to psychiatric disorders, especially in combination with the use of montelukast'', said the authors.
Baan EJ, de Smet VA, Hoeve CE, et al.
Exploratory study of signals for asthma drugs in children, using the EudraVigilance database of spontaneous reports.
Drug Saf.
Epub Patients with interstitial lung disease or a history of the disease" should be added to the "Careful Administration" section • A cautionary statement for interstitial lung disease should be added to the "Important Precautions" section Interstitial lung disease" should be added to the "Clinically Significant Adverse Reactions" section Pharmaceuticals and Medical Devices Agency of Japan.
Summary of investigation results -apalutamide USA) notes that development of antiviral vaccines began when Peyton Rous postulated in the early 1900s that sarcoma transmission between hens may be virus-related, followed by identification in 1964 of the first human DNA tumour virus, the Epstein-Barr virus.
Papillomavirus DNA was isolated in 1983, with > 120 types of HPV subsequently classified; the first HPV vaccine was licensed in the USA in Safety of the 9-valent human papillomavirus vaccine Near real-time surveillance to assess the safety of the 9-valent human papillomavirus vaccine From Peyton Rous to the HPV vaccine: a journey of discovery and progress according to findings of a study published in Drug Safety that was based on FAERS reports.
FAERS data from 2,042,801 reports over a 3-year period (2015-2017) were used to determine reporting odds ratios (RORs) for associations between antibacterials (intramuscular, intravenous, subcutaneous or parenteral) and AKIs.
There were 20,138 reports of AKI to FAERS during the study period cotrimoxazole (trimethoprim/sulfamethoxazole 95% CI 1.26, 2.36), and fluoroquinolones This study found 14 classes of antibiotics having significant reporting associations with AKI.
Among the antibiotics evaluated in this study, colistin had the highest AKI ROR and moxifloxacin had the lowest Comparing acute kidney injury reports among antibiotics: a pharmacovigilance study of the FDA Adverse Event Reporting System (FAERS) Angiotensin Receptor Antagonists Increase Risk of Suicide Treatment with statins does not appear to be associated with memory or cognitive disorders in elderly patients, according to findings of the Sydney Memory and Aging Study published in the Journal of the American College of Cardiology (JACC) [1] .
This prospective study investigated the effects of statins (atorvastatin, pravastatin or simvastatin) on memory and global cognition over a 6-year period in 1037 community-dwelling patients in Australia who were 70-90 years of age, and the effects of statins on brain volume over a 2-year period in a subgroup of 526 patients.
Five memory tests and 12 cognitive tests were used.
Brain volume was assessed using magnetic resonance imaging.Rates of memory decline and global cognitive decline did not differ significantly between statin users and patients who never used statins.Overall, initiation of statins was associated with a reduced rate of memory decline.
Statin use was associated with attenuated decline in memory test performance in patients with heart disorders and those with the apolipoprotein Eε4 (ApoEε4) genotype.Changes in brain volume did not differ significantly between statin users and non-users.
"Statin use in the elderly population was not associated with any acceleration in decline in memory, global cognition, or brain volumes… Protective associations were found for some aspects of memory testing in those with dementia risk factors such as heart disease and ApoEε4 gene carriage", concluded the investigators.
"Contrary to popular concern, statin use was not associated with cognitive decline in this observational study", said Drs.
Constantino Iadecola and Neal Parikh from Weill Cornell Medicine, New York, USA, in an accompanying editorial published in the JACC [2] .
"These data support the view that worries about cognitive impairment should not limit statin use and raise the possibility that statins may favorably alter cognitive trajectories in a group of elderly subjects at high risk of Alzheimer disease", they commented.
The low uptake of human papillomavirus (HPV) vaccines in the USA may be due to concerns about vaccine safety.
Two studies reported in Pediatrics investigated the safety of the 9-valent HPV vaccine [9vHPV vaccine; Gardasil 9].
The first study investigated 7244 postlicensure surveillance reports to the US FDA's Vaccine Adverse Event Reporting System (VAERS) from December 2014 until December 2017 [1] .
Most reports were submitted by the vaccine manufacturer (64.2%), with 26.8% of reports submitted by healthcare providers.
Disproportional reporting was evaluated using empirical Bayesian data mining.
Most reports (97.4%) were not classified as serious, and 9vHPV vaccine was given alone in 74.7% of reports.
Two deaths were reported, but "no information in autopsy reports or death certificates suggested a causal relationship with vaccination", note the authors.
The most commonly reported events were dizziness (7.5%), syncope (6.9%), headache (5.0%), and injection site pain (4.5%) or erythema (4.4%).As about 28 million doses of 9vHPV vaccine were distributed during the study period, the crude adverse event reporting rate was 259 per million doses overall and 7 per million doses for serious events.
The rate for syncope was 26 per million doses, which did exceed the threshold for disproportional reporting, but is a known adverse event for injectable vaccines.
Rates for all other events were < 1 per million doses.Analysis of prespecified conditions revealed 9 reports of anaphylaxis, 8 reports of Guillain-Barré syndrome, 17 reports of postural orthostatic tachycardia syndrome, 3 reports of primary ovarian insufficiency, 1 report of complex regional pain syndrome, and 2 reports of acute disseminated encephalomyelitis.
However the authors note that "most reported events did not meet diagnostic criteria or did not contain sufficient information to make a determination on the diagnosis".The authors conclude that "no new or unexpected safety concerns or reporting patterns of 9vHPV with clinically important AEs were detected".
The second study investigated reports to the Vaccine Safety Datalink (VSD) between October 2015 and October 2017 in patients 9-26 years of age, when 838,991 vaccine doses were administered [2] .
Rapid Cycle Analysis (RSA) methodology was used to compare adverse events in recently vaccinated and unvaccinated groups, with near real-time data.
Relative risks were investigated using maximised sequential probability ratio test (MaxSPRT), conditional MaxSPRT (cMaxSPRT) and exact sequential analysis (ESA) analyses.Unexpected statistical signals were detected for the following events: pancreatitis in men 18-26 years of age after any dose; appendicitis after dose 3 in boys 9-17 years of age; allergic reactions after dose 2 in women 18-26 years of age; and allergic reactions in girls 9-17 years of age after any dose.
Further investigation did not confirm signal generation, with results classified as false-positives.
Signals were also detected for syncope, injection site reactions, and nonspecific reactions, but no further investigations were conducted because they were "expected based on clinical trials of 9vHPV", note the authors, "and because the diagnoses were unlikely to indicate a serious adverse event".
They add that "with this large observational study, we contribute reassuring postlicensure data that will help bolster the safety profile of 9vHPV".Angiotensin receptor blockers (ARBs) appear to increase the risk of suicide in elderly patients compared with ACE inhibitors (ACEIs), according to findings of a Canadian case-control study published in JAMA Network Open [1] .Data from administrative claims databases in Ontario were used to investigate death by suicide during treatment with ARBs or ACEIs in patients 66 years of age and over.
Conditional logistic regression analysis was used to assess the association between suicide and exposure to ARBs versus ACEIs in 964 patients who died by suicide within 100 days of receiving an ACEI or ARB (cases) compared with 3856 ACEI-or ARB-treated patients matched by age, sex and comorbid hypertension and diabetes mellitus who did not die by suicide (controls).
Of patients exposed to ARBs (most frequently candesartan, telmisartan or valsartan), 26.0% were cases and 74.0% were controls, while of patients exposed to ACEIs (most frequently enalapril or ramipril), 18.4% were cases and 81.6% were controls.Treatment with ARBs significantly increased the risk of death by suicide compared with treatment with ACEIs (aOR 1.63; 95% CI 1.33, 2.00).
Sensitivity analysis showed similar findings after exclusion of patients with a history of self-harm (aOR 1.60; 95% CI 1.29, 1.98).
"Our findings suggest a possible increased risk of suicide associated with the use of ARBs compared with ACEIs among adults aged 66 years and older.
Given their high prevalence of use, the severity of the outcome, and the similar efficacy of these drug classes in treating the same conditions, clinicians may opt for preferential use of ACEIs over ARBs where possible", concluded the authors.
"The report by Mamdani et al.
that ARBs are associated with increases in the risk of suicide in a study using real-world data requires conceptual replication.
The strength of the methods, the importance of preventing suicide, and the number of people exposed to ARBs all support the need to encourage additional studies and to translate the combined findings into guidance about prescribing", said Dr. Ira Katz from the Office of Mental Health and Suicide Prevention, Department of Veterans Affairs, Philadelphia, Pennsylvania, USA, in an accompanying invited commentary published in JAMA Network Open [2] .
Analysis of spontaneous ADR reports to the European Medicine Agency's EudraVigilance database has identified new safety signals for asthma drugs in paediatric patients, say authors of a study published in Drug Safety.
A proportional reporting ratio (PRR) was calculated for each asthma drug-event combination (DEC) reported to EudraVigilance between 2011 and 2017.
Signals in paediatric patients up to 17 years of age were compared with those in the total patient population.Among the 372,345 reports in pediatric patients, there were 21,264 antiasthmatic-related DECs associated with single drug or fixed-dose combinations.There were 3697 unique DECs in paediatric patients, of which 385 met the criteria for a safety signal.
The signals were most frequently for psychiatric disorders (n = 65), respiratory, thoracic and mediastinal disorders (56) and nervous system disorders (29).
There were also signals related to injury, poisoning and procedural complications (49) after off-label use or accidental exposure to an antiasthmatic.The highest PRRs in paediatric patients were for montelukast-associated vasculitides (PRR 90.7; 95% CI 46.3, 177.9;
key: cord-333732-dtfmcqh6 authors: Johnson, Walter G; Marchant, Gary E title: Legislating in the Time of a Pandemic: Window of Opportunity or Invitation for Recklessness?
date: 2020-06-15 journal: J Law Biosci DOI: 10.1093/jlb/lsaa042 sha: doc_id: 333732 cord_uid: dtfmcqh6 The COVID-19 epidemic has been exacerbated by failures in diagnostic testing for the virus in the United States.
In response to these problems, two bills have been introduced in Congress to not only reform emergency use of diagnostic tests, but to fundamentally reform diagnostics regulation in non-emergencies.
There has been a long-standing recognition that current U.S. regulation of diagnostics is outdated and problematic, and the history of public health legislation is that emergencies and crises have been the primary motivating factor to break Congressional inertia and to implement new legislation.
Thus, the COVID-19 may create a useful “window of opportunity” to pass much-needed legislative reform of diagnostic regulation in both emergency and non-emergency contexts.
At the same time, rushing radical legislative changes, especially if they have not been subject to careful stakeholder engagement and Congressional deliberation in advance, is precarious and could result in reckless and disruptive changes.
We review and apply the historical lessons of legislating in response to a crisis and conclude the one but not both of the pending legislative proposals may satisfy the criteria for an appropriate opportunistic change for diagnostics regulation.
The delay in providing widespread diagnostic testing has significantly exacerbated the extent and consequences of the COVID-19 epidemic in the United States.
1 Even when tests were available, a notable proportion were reported to give inaccurate results.
2 In response to these failures, two bills were immediately introduced in Congress to revise the FDA approval process for diagnostic tests.
The bipartisan Verifying Accurate Leading-edge IVCT Development results may cause them to forgo beneficial interventions or undergo unnecessary and potentially harmful ones.
6 Ensuring these goals generally involves applying two standards, (1) analytical validity, referring to how accurately and precisely a diagnostic measures its intended analyte and (2) clinical validity, describing how well a diagnostic can characterize or predict a patient's health status.
7 Yet, applying high standards to diagnostics, especially should clinical validity call for complex clinical trials, may delay access to valuable diagnostics and undercut potentially beneficial innovation.
Balancing these interests has become more challenging with the emergence of molecular diagnostics technologies, which test for various "-omics" such as genomics or microbiomics.
Such testing enables new diagnostics possibilities such as predicting the risk and severity of a patient developing a condition or how an individual might respond to an intervention.
These innovations rely on tools such as algorithms and reference databases to measure and interpret their analyte, which pose more complicated analytical and clinical validity questions than more routine tests.
8 The current regulatory landscape struggles to comprehensively manage these concerns for molecular diagnostics, treating diagnostics differently based primarily on whether the test is sold as a kit or offered in-house by a laboratory, rather than based on risk.
Since the 1970s, the FDA has applied its three-tiered, risk-based regulatory scheme for medical devices to products called in vitro diagnostics (IVDs).
9 These IVDs are testing kits which manufacturers produce to market and sell to clinical laboratories and physician offices.
In the mid-2010s, several legislative proposals were floated to resolve these regulatory issues.
14 One particular proposal, beginning with an effort by the Diagnostic Test Working Group and evolving into the VALID Act, aimed to strike compromises by incorporating all diagnostics (including LDTs) into products called in vitro clinical tests (IVCTs) subject to relatively flexible FDA review.
This bill has gained the most support to date both within and outside of Congress and, under Commissioner Gottlieb, the FDA also supported some form of the compromise identified by the VALID Act.
15 Over a several year period, multiple successive drafts of the planned legislation were circulated to stakeholders and then revised based on the feedback.
In March 2019, one expert observer wrote that "the VALID Act enjoys bipartisan and bicameral support and, after years of discussion between Congress, FDA, labs, patients, and other interested stakeholders, seems poised to be enacted before the end of the current Congress."
16 By January 2020, the legislation was ready to be introduced in the following few several weeks passed before a revised EUA could be granted.
The largest private diagnostics developers did not receive EUAs until mid-March 2020, 23 due in part to requirements for evidence of effectiveness.
As the severity of the pandemic and political scrutiny increased, the FDA relaxed its EUA requirements for diagnostics several times.
These efforts included permitting some CLIA-certified laboratories to validate and use LDTs prior to communicating with the FDA.
24 However, the early enforcement decisions and lack of early engagement with industry had already discouraged many clinical laboratories from developing or seeking authorization for LDTs, 25 and the delayed availability of testing during the initial weeks of the pandemic significantly exacerbated the spread of the virus in the U.S.
The U.S. legislative process for any given issue is characterized by static inertia interrupted by an occasional "policy window" opened by external changes or events that suddenly catapult a specific issue to the forefront of Congressional attention.
26 The U.S. Congress is confronted by thousands of potential issues it could and should address and, even without political polarization and partisan gridlock, it is impossible for Congress to legislate on every item meriting attention.
For most issues, statutory change is not possible without some type of crisis or dramatic event to create a window of opportunity by triggering Congressional attention, and even that attention might be fleeting.
As such, advocates of change need to strike quickly.
27 The history of public health and safety legislation in the United States is characterized by this dynamic of "punctuated equilibrium" 28long periods of static intransience interrupted by brief flurries of rapid change, often instigated by some crisis or tragedy.
Indeed, virtually every major change in the legislative authority of public health regulatory agencies such as the FDA and EPA was enacted in response to public health tragedies or crises due at least in part to regulatory failures or omissions.
Peter Barton Hutt, For both the FDA and EPA then, the primary regulatory statutes were promulgated after high-profile tragedies that sparked intense and immediate public, media, and Congressional concern.
In some cases, the underlying problem was well-known to legislators, and extensive 50 Another observation is that after a crisis, the representatives introducing corrective bills tend to have more extreme positions than the authors of bills enacted during non-crisis periods, which tilts legislation adopted in response to tragedies to be more sweeping and path-breaking than legislation adopted in less urgent times.
51 The COVID-19 pandemic has created such a policy window for reforming U.S. diagnostics regulation, a debate which had been sputtering for several years but had been unable to get across "the finishing line."
The crisis here was foreseeable yet largely underrepresented in the diagnostics debate, which instead has centered on regulation for non-emergencies, while recent epidemics have seen the CDC successfully develop and deploy testing.
Now, the pandemic and the weak U.S. testing response has created a window of opportunity to discuss diagnostics regulation generally, prompting two bills to be floated.
The VITAL Act identifies FDA overregulation of diagnostics, especially LDTs, as the key issue which precipitated the poor COVID-19 testing rollout.
52 This proposal would leverage lawmaker attention to use improved laboratory quality standards, rather than FDA, to oversee LDTs and embolden clinical laboratories to develop diagnostics for any potential future need.
53 The VALID Act would instead advance a robust new regulatory regime for the FDA to manage all diagnostics as IVCTs, building on positions with bipartisan support such as risk-based premarket review of IVCTs and some form of optional management-based regulation for test developers.
54 The VALID Act contains a freshly added special standard for declared public health emergencies, allowing test developers to use their self-validated diagnostics while still seeking emergency authorization from FDA.
55 These provisions for emergencies could appeal to lawmaker interest in addressing the testing issues observed during the pandemic to catalyze more fundamental regulatory reform for diagnostics even during non-emergencies.
Though crises can throw open policy windows, legislating during a disaster also creates an invitation to recklessly establish, modify or disrupt regulatory regimes.
Emergency decisionmaking typically bypasses procedural norms around holding hearings, engaging with stakeholders, and justifying the purpose of decisions through written reports.
56 Substantively, emergency policymaking may result in regulatory norms or programs poorly calibrated to the longer-term and complex set of stakeholder interests and policy concerns at play.
Specifically in food and drug crisis decision-making, Peter Barton Hutt comments "[a]s is often true under those conditions, the legislation has been shaped as much by public emotion as rational policy design."
57 Accordingly, legislatures tend to underregulate a problem before a crisis occurs, but often overregulate after disaster strikes.
58 This overcompensation can result in mis-calibrated tradeoffs between complicated sets of interests and values, potentially limiting the effectiveness or efficiency of subsequent regulation.
The tendency towards short-term thinking and possibility of limited legislative history or analysis in emergency policymaking can make implementation, interpretation, and adjudication challenging for regulators and courts, especially in the years following the conclusion of the crisis.
59 The emergence of the 510(k) pathway for medical device clearance provides one such example, where the FDA found statutory provisions calling for "performance standards" for medium-risk devices too onerous and largely abandoned them in favor of "substantial equivalence" reviews.
60 Decreased participation in crisis decision-making can also jeopardize the long-term legitimacy of norms or programs established during emergencies.
Legislating during a crisis grants stakeholders a shorter window to provide comments or feedback on proposed norms and, depending on how the crisis impacts them, stakeholders may have less capacity to comment at all.
Lawmaking in the wake of the terrorist attacks of September 11, 2001 offers a cautionary tale for making broad oversight changes during or immediately after a crisis.
In particular, the USA PATRIOT Act was enacted barely six weeks following the attacks.
61 The statute established a multifaceted regulatory regime for counterterrorism with sweeping new surveillance powers, the added crime of "domestic terrorism" to enforce, and targeted restrictions for immigrants.
Numerous civil society and political entities retrospectively criticized these measures over civil liberties and privacy concerns, drawing on constitutional norms and policy arguments on limited effectiveness and discriminatory outcomes.
62 Ultimately, the criminal regulatory system set up appeared grounded in emotion and crisis overreaction as much as, or more than, sound policy and legal analysis, 63 and may have unnecessarily and disparately infringed on rights without effectively promoting national security.
The Patriot Act illustration offers another related lesson, that emergency decision-making can prompt an abrupt and durable shift in regulatory goals, values, and motivations.
Immediately prior to 9/11, the Federal Trade Commission (FTC) and other stakeholders had been promoting the adoption of enforceable data protection rules to promote privacy at the dawn of the internet age.
64 However, Zuboff illustrates how the crisis-driven urgency, combined with the politically damaging perception that the U.S. was unable to forecast the 9/11 attack, resulted in rapid legislative moves to bolster surveillance and data-sharing by both public and private entities.
65 Proposed privacy plans were scrapped in an instant, casting aside the growing normative support for data protection rules immediately prior to the crisis.
This shift in values and goals affected not only lawmakers, but also regulators at the FTC, who backed away from broader data protection activities and advocacy to support narrower and more acceptable data security initiatives.
66 The conversation around promoting digital privacy protections then fell dormant for over a decade.
In the case of diagnostics regulation, a similar swing in values could occur with the pandemic, modulating not only emergency rules but also more general regulatory norms for after the crisis.
The diagnostics debate has long been balanced on the fulcrums of safety versus innovation, consumer protection versus patient access, and overregulation of LDTs versus regulatory fairness between IVDs and LDTs.
However, the public health costs of a slow COVID-19 testing rollout fused with widespread frustration over the FDA's stunted approach towards suddenly created an acute crisis and called attention primarily to counterterrorism, this pandemic has scaled up into an emergency enduring for months and requires legislative attention on everything from economic policy to medical equipment production.
68 The acute demands on lawmakers from various policy domains during the months-long pandemic have no doubt contributed to Congress's decision not to legislate immediately on diagnostics regulation.
Still, there will be a need for Congress to address diagnostics, sooner or later, as a result of the perceived COVID-19 diagnostic testing problems.
The current COVID-19 crisis creates a double-edged sword between opportunity and opportunism.
Synthesizing lessons described above from prior legislative activities during emergencies suggests several criteria for evaluating whether public health reforms during a pandemic will likely lead to a measured, legitimate regulatory regime.
These criteria include satisfying procedural norms, diverse stakeholder engagement, rigorous policy analysis, and consistency with pre-crisis values and balances of complex, competing interests.
On one hand, the VITAL Act excavates a deregulatory approach to LDT oversight which some stakeholders advanced in 2015 without gaining significant traction.
69 The draft would reprioritize innovation, patient access, and easing burdens on clinical laboratories as the preeminent values for diagnostics oversight, 70 On the other hand, the VALID Act would create different standards for diagnostics in ordinary and emergency settings, though recent justifications for the bill have also focused on flexibility, innovation, and access.
Upon releasing the updated draft VALID Act in early March 2020, its sponsors promoted the proposal based on the flexible emergency norms the bill would create.
71 Here, the need and support for a "fix" to the emergency diagnostics authority problem is being leveraged to create a comprehensive regulatory scheme for non-emergency oversight of diagnostics.
The VALID Act and its earlier versions have gone through several years of deliberation and refinement, taking into account the views of the relevant stakeholders and the FDA, and building bipartisan support in both the Senate and the House.
Moreover, the VALID Act addresses the well-recognized double-track problem with the current regulatory system that has not been fixed due to legislative inertia and a crowded Congressional agenda.
The VALID Act has already been the subject of extensive policy analysis, broad stakeholder engagement, and lawmaking procedures and would keep intact a balance between various competing values and interests for diagnostics regulation struck prior to the pandemic.
Using the window of opportunity created by COVID-19, with the demonstrated urgency to reform emergency norms, to enact the comprehensive reform of diagnostics regulation provided by the VALID Act would therefore exemplify the type of crisis-based lawmaking that has been successful in the past to create much of our public health legislation.
71 Office of Rep. Larry Bucshon, Lawmakers Introduce Legislation to Expand Nation's Diagnostic Testing Capabilities (Mar.
5, 2020), https://bucshon.house.gov/news/documentsingle.aspx?DocumentID=3841 (The "legislation would .
.
.
overhaul how the FDA reviews and approves diagnostics .
.
.
[giving] laboratories greater flexibility to respond to public health emergencies while continuing to keep patients safe.").
Policy for Diagnostic Tests for Coronavirus Disease-2019 During the Public Health Emergency: Immediately in Effect Guidance for Clinical Laboratories, Commercial Manufacturers, and Food and Drug Administration Staff The Lost Month: How Failure to Test Blinded the U.S. to Covid-19 Environmental Protection Regulation An exception is the Deepwater Horizon tragedy, which did not result in any legislative response, notwithstanding substantial pressure for such change.
See Jaime Eagan, Never Waste a Good Crisis: Deepwater Horizon and a Call for Congressional Action The Catastrophic Model of Risk Regulation and the Regulatory Legacy of Three Mile Island and Love Canal, 15 PENN The Emergency Planning and Community Right to Know Act of 1986: Analysis and Update, 6 Rand Paul Introduces VITAL Act to Speed Availability of Testing in Health Emergencies See generally Cary Coglianese & David Lazer, Management-Based Regulation: Prescribing Private Management to Achieve Public Goals § 3 (proposing "Sec.
587A(5) Emergency use.").
The FDA's strategy during the pandemic ultimately began to take this type of approach Unorthodox Rulemaking, 115 COLOM.
L. REV.
1789 PUBLIC HEALTH EFFECTIVENESS OF THE FDA 510(K) CLEARANCE PROCESS: BALANCING Act: Safety, Innovation, and Resources in the Implementation of Medical Device Legislation, 12 Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct Terrorism Act of Whose Liberty?
Whose Security?
The USA PARTIOT Act in the Context of COINTELPRO and the Unlawful Repression of Political Dissent, 81 OR Seven Weeks: The Making of the USA PATRIOT Act FAIR INFORMATION PRACTICES IN THE ELECTRONIC MARKETPLACE: A REPORT TO CONGRESS Rahm Emanuel once said "[y]ou never want a serious crisis to go to waste THE FIGHT FOR A HUMAN FUTURE AT THE NEW FRONTIER OF POWER Key Things in the $2 Trillion Coronavirus Stimulus Package Proposal for Modernization of CLIA Regulations for Laboratory Developed Testing Procedures (LDPs) See Office of Sen. Rand Paul, supra note 52
key: cord-280040-xphxlaat authors: Rutala, William A.; Weber, David J. title: Disinfection and Sterilization in Health Care Facilities An Overview and Current Issues date: 2016-09-30 journal: Infectious Disease Clinics of North America DOI: 10.1016/j.idc.2016.04.002 sha: doc_id: 280040 cord_uid: xphxlaat When properly used, disinfection and sterilization can ensure the safe use of invasive and noninvasive medical devices.
The method of disinfection and sterilization depends on the intended use of the medical device: critical items (contact sterile tissue) must be sterilized before use; semicritical items (contact mucous membranes or nonintact skin) must be high-level disinfected; and noncritical items (contact intact skin) should receive low-level disinfection.
Cleaning should always precede high-level disinfection and sterilization.
Current disinfection and sterilization guidelines must be strictly followed.
In the United States in 2010 there were approximately 51.4 million inpatient surgical procedures and an even larger number of invasive medical procedures.
1 In 2009, there were more than 6.9 million gastrointestinal (GI) upper, 11.5 million GI lower, and 228,000 biliary endoscopies performed.
2 Each of these procedures involves contact by a medical device or surgical instrument with patients' sterile tissue or mucous membranes.
A major risk of all such procedures is the introduction of pathogenic microbes, which can lead to infection.
Failure to properly disinfect or sterilize equipment may lead to transmission via contaminated medical and surgical devices (eg, carbapenem-resistant Enterobacteriaceae [CRE] ).
3, 4 Achieving disinfection and sterilization through the use of disinfectants and sterilization practices is essential for ensuring that medical and surgical instruments do not transmit infectious pathogens to patients.
Because it is not necessary to sterilize all patient-care items, health care policies must identify whether cleaning, disinfection, or sterilization is indicated based primarily on each item's intended use, manufacturers recommendations, and guidelines.
Multiple studies in many countries have documented lack of compliance with established guidelines for disinfection and sterilization.
5 Failure to comply with scientifically based guidelines has led to numerous outbreaks and patient exposures.
[6] [7] [8] Because of noncompliance with recommended reprocessing procedures, the Centers for Disease Control and Prevention (CDC) and the Food and Drug Administration (FDA) issued a health advisory alerting health care providers and facilities about the public health need to properly maintain, clean, and disinfect and sterilize reusable medical devices in September 2015.
9 In this article, which is an updated and modified version of earlier articles, [10] [11] [12] [13] [14] a pragmatic approach to the judicious selection and proper use of disinfection and sterilization processes is presented, based on well-designed studies assessing the efficacy (via laboratory investigations) and effectiveness (via clinical studies) of disinfection and sterilization procedures.
Almost 50 years ago, Earle H. Spaulding 15 devised a rational approach to disinfection and sterilization of patient-care items or equipment.
This classification scheme is so clear and logical that it has been retained, refined, and successfully used by infection control professionals and others when planning methods for disinfection or sterilization.
[10] [11] [12] [13] [14] Spaulding thought that the nature of disinfection could be understood more readily if instruments and items for patient care were divided into 3 categories based on the degree of risk of infection involved in the use of the items.
The 3 categories he described were critical, semicritical, and noncritical.
This terminology is used by the CDC's "Guidelines for Environmental Infection Control in Healthcare Facilities" 16 and the CDC's "Guideline for Disinfection and Sterilization in Healthcare Facilities."
13 These categories and the methods to achieve sterilization, high-level disinfection, and low-level disinfection are summarized in Table 1 .
Although the scheme remains valid, there are some examples of disinfection studies with prions, viruses, mycobacteria, and protozoa that challenge the current definitions and expectations of high-level disinfection (HLD) and low-level disinfection.
22 In May 2015, the FDA convened a panel to discuss recent reports and epidemiologic investigations of the transmission of infections associated with the use of duodenoscopes in endoscopic retrograde cholangiopancreatography (ERCP) procedures.
23 After presentations from industry, professional societies, and invited speakers, the panel made several recommendations to include reclassifying duodenoscopes based on the Spaulding classification from semicritical to critical to support the shift from HLD to sterilization.
24 This change could be accomplished by shifting from HLD for duodenoscopes to sterilization and modifying the Spaulding definition of critical items from "objects which enter sterile tissue or the vascular system or through which blood flows should be sterile" to "objects which directly or secondarily (ie, via a mucous membrane such as duodenoscope) enter normally sterile tissue of the vascular system of through which blood flows should be sterile."
24, 25 Implementation of this Abbreviations: EPA, Environmental Protection Agency; glut, glutaraldehyde; HLD, high-level disinfection; HP, hydrogen peroxide; OPA, ortho-phthalaldehyde; PA, peracetic acid; ppm, parts per million.
a Prions (such as Creutzfeldt-Jakob disease) exhibit an unusual resistance to conventional chemical and physical decontamination methods and are not readily inactivated by conventional sterilization procedures.
17 b Consult the FDA-cleared package insert for information about the cleared contact time and temperature, and see reference 18 for discussion why greater than 2% glutaraldehyde products are used at a reduced exposure time (2% glutaraldehyde at 20 minutes, 20 C).
Increasing the temperature using an automated endoscope reprocesser (AER) will reduce the contact time (eg, ortho-phthalaldehyde 12 minutes at 20 C but 5 minutes at 25 C in AER).
Exposure temperatures for some of the aforementioned high-level disinfectants varies from 20 C to 25 C; check FDA-cleared temperature conditions.
19 Tubing must be completely filled for highlevel disinfection and liquid chemical sterilization.
Material compatibility should be investigated when appropriate (eg, hydrogen peroxide [HP] and HP with peracetic acid will cause functional damage to endoscopes).
Intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, most viruses, and most fungi but not spores and may include chlorine-based products, phenolics, and improved HP.
Intermediate-level disinfectants are not included in Table 1 as there as there is no device or surface for which intermediate-level disinfection is specifically recommended over low-level disinfection.
Adapted from Refs.
[11] [12] [13] 20, 21 recommendations requires sterilization technology that achieves a sterility assurance level of 10 À6 of complex medical instruments, such as duodenoscopes.
Ideally, this shift would eventually involve not only endoscopes that secondarily enter normally sterile tissue (eg, duodenoscopes, bronchoscopes) but also other semicritical devices (eg, GI endoscopes).
24, 25 Critical Items Critical items are so called because of the high risk of infection if such an item is contaminated with any microorganism, including bacterial spores.
Thus, it is critical that objects that enter sterile tissue or the vascular system be sterile because any microbial contamination could result in disease transmission.
This category includes surgical instruments, cardiac and urinary catheters, and implants used in sterile body cavities.
The items in this category should be purchased as sterile or be sterilized by steam sterilization if possible.
If heat sensitive, the object may be treated with ethylene oxide (ETO), hydrogen peroxide (HP) gas plasma, vaporized HP, HP vapor (HPV) plus ozone, or by liquid chemical sterilants if other methods are unsuitable.
Table 1 and Tables 2 and 3 list sterilization processes and liquid chemical sterilants and the advantages and disadvantages of each.
With the exception of 0.2% peracetic acid (12 minutes at 50 C-56 C), the indicated exposure times for liquid chemical sterilants range from 3 to 12 hours.
19 Liquid chemical sterilants can be relied on to produce sterility only if cleaning, which eliminates organic and inorganic material, precedes treatment and if proper guidelines as to concentration, contact time, temperature, and pH are met.
Another limitation to sterilization of devices with liquid chemical sterilants is that the devices cannot be wrapped during processing in a liquid chemical sterilant; thus, it is impossible to maintain sterility following processing and during storage.
Furthermore, devices may require rinsing following exposure to the liquid chemical sterilant with water that, in general, is not sterile.
Therefore, because of the inherent limitations of using liquid chemical sterilants in a nonautomated (or automated) reprocessor, their use should be restricted to reprocessing critical devices that are heat sensitive and incompatible with other sterilization methods.
In contrast to semicritical items that have been associated with greater than 100 outbreaks of infection, 6 critical items have rarely, 26 if ever, been associated with disease transmission.
For example, any deviation from proper reprocessing (such as crevices associated with the elevator channel) of an endoscope could lead to failure to eliminate contamination with a possibility of subsequent patient-to-patient transmission due to a low or nonexistent margin of safety.
This low (or nonexistent) margin of safety associated with endoscope reprocessing compares with the 17-log 10 margin of safety associated with cleaning and sterilization of surgical instruments (ie, 12-log 10 reduction via sterilization and at least a net 5-log 10 reduction based on the microbial load on surgical instruments [2-logs] 27 and microbial reduction via a washer disinfector [7-logs] ).
18 Semicritical items are those that come in contact with mucous membranes or nonintact skin.
Respiratory therapy and anesthesia equipment, gastrointestinal endoscopes, bronchoscopes, laryngoscopes, endocavitary probes, prostate biopsy probes, 28 cystoscopes, 29 hysteroscopes, infrared coagulation devices, 30 and diaphragm fitting rings are included in this category.
These medical devices should be free of all microorganisms (ie, mycobacteria, fungi, viruses, bacteria), although small numbers of bacterial spores may be present.
Intact mucous membranes, such as those of the lungs or the gastrointestinal tract, are generally resistant to infection by Abbreviations: AER, automated endoscope reprocessor; OPA, ortho-phthalaldehyde.
a All products effective in presence of organic soil, relatively easy to use, and have a broad spectrum of antimicrobial activity (bacteria, fungi, viruses, bacterial spores, and mycobacteria).
The aforementioned characteristics are documented in the literature; contact the manufacturer of the instrument and sterilant for additional information.
All products listed are cleared by the FDA as chemical sterilants except ortho-phthalaldehyde, which is an FDA-cleared HLD.
Adapted from Refs.
[10] [11] [12] [13] 20 (continued on next page) common bacterial spores but susceptible to other organisms, such as bacteria, mycobacteria, and viruses.
Semicritical items minimally require HLD using chemical disinfectants.
Glutaraldehyde, HP, ortho-phthalaldehyde (OPA), peracetic acid with HP, and chlorine (via electrochemical activation) are cleared by the FDA 19 and are dependable high-level disinfectants provided the factors influencing germicidal procedures are met (see Tables 1 and 2 ).
The exposure time for most high-level disinfectants varies from 8 to 45 minutes at 20 C to 25 C. 19 Because semicritical equipment has been associated with reprocessing errors that result in patient lookback and patient notifications, it is essential that control measures be instituted to prevent patient exposures.
7 Before new equipment (especially semicritical equipment as the margin of safety is less than that for sterilization) 25 is used for patient care on more than one patient, reprocessing procedures for that equipment should be developed.
Staff should receive training on the safe use and reprocessing of the equipment and be competency tested.
At the University of North Carolina (UNC) Hospitals, to ensure patient-safe instruments, all staff that reprocess semicritical instruments (eg, instruments which contact a mucous membrane such as vaginal probes, endoscopes, prostate probes) are required to attend a 3-hour class on HLD of semicritical instruments.
The class includes the rationale for and importance of high-level disinfection, discussion of high-level disinfectants and exposure times, reprocessing steps, monitoring minimum effective concentration, personal protective equipment, and the reprocessing environment (establish dirty-to-clean flow).
Infection control rounds or audits should be conducted annually in all clinical areas that reprocess critical and semicritical devices to ensure adherence to the reprocessing standards and policies.
Results of infection control rounds should be provided to the unit managers, and deficiencies in reprocessing should be corrected and the corrective measures documented to infection control within 2 weeks (immediately correct patient safety issues, such as exposure time to high-level disinfectant).
Noncritical items are those that come in contact with intact skin but not mucous membranes.
Intact skin acts as an effective barrier to most microorganisms; therefore, the Abbreviations: ETO, ethylene oxide; HP, hydrogen peroxide; TWA, time-weighted average.
Adapted from Refs.
[10] [11] [12] [13] 20 sterility of items coming in contact with intact skin is "not critical."
Examples of noncritical items are bedpans, blood pressure cuffs, crutches, bed rails, linens, bedside tables, patient furniture, and floors.
In contrast to critical and some semicritical items, most noncritical reusable items may be decontaminated where they are used and do not need to be transported to a central processing area.
There is virtually no documented risk of transmitting infectious agents to patients via noncritical items 31 when they are used as noncritical items and do not contact nonintact skin and/or mucous membranes.
However, these items (eg, bedside tables, bed rails) could potentially contribute to secondary transmission by contaminating hands of healthcare personnel or by contact with medical equipment that will subsequently come in contact with patients.
32 Table 1 and Table 4 list several low-level disinfectants that may be used for noncritical items.
Table 4 lists the advantages and disadvantages of the low-level disinfectants that are used on noncritical patient care items (eg, blood pressure cuffs) and noncritical environmental surfaces.
The exposure time for low-level disinfection of noncritical items is at least 1 minute.
Physicians use endoscopes to diagnose and treat numerous medical disorders.
Although endoscopes represent a valuable diagnostic and therapeutic tool in modern medicine, more health care-associated outbreaks have been linked to contaminated endoscopes than to any other reusable medical device.
6, 8 Additionally, endemic transmission of infections associated with GI endoscopes may go unrecognized for several reasons, including inadequate surveillance of outpatient procedures, long lag time between colonization and infection, low frequency of infection, and because pathogens are the usual enteric flora.
In addition, the risk of some procedures might be lower than others (eg, colonoscopy vs ERCP), whereby normally sterile areas are contaminated in the latter.
In order to prevent the spread of health care-associated infections (HAIs), all heat-sensitive endoscopes (eg, GI endoscopes, bronchoscopes, nasopharyngoscopes) must be properly cleaned and, at a minimum, subjected to HLD following each use.
HLD can be expected to destroy all microorganisms; although when high numbers of bacterial spores are present, a few spores may survive.
Recommendations for the cleaning and disinfection of endoscopic equipment have been published and should be strictly followed.
13, 35, 36 Unfortunately, audits have shown that personnel often do not adhere to guidelines on reprocessing 5 and outbreaks of infection continue to occur.
3, 6, 8, 37 Additionally, recent studies have suggested that current reprocessing guidelines are not sufficient to ensure successful decontamination.
38 In order to minimize patient risks and ensure that reprocessing personnel are properly trained, there should be initial and annual competency testing for each individual who is involved in reprocessing endoscopic instruments.
13, 35, 36 In general, endoscope disinfection or sterilization with a liquid chemical sterilant or high-level disinfectant involves 5 steps after leak testing: (1) clean: mechanically clean internal and external surfaces, including brushing internal channels and flushing each internal channel with water and a enzymatic cleaner or detergent; (2) disinfect: immerse endoscope in high-level disinfectant (or chemical sterilant) and perfuse (eliminates air pockets and ensures contact of the germicide with the internal channels) disinfectant into all accessible channels, such as the suction/biopsy channel and air/water channel, and expose for a time recommended for specific products; (3) rinse: rinse the endoscope and all channels with sterile water, filtered water (commonly used with automated endoscope reprocessors), or tap water; (4) dry: rinse the insertion tube and inner channels with alcohol and dry with forced air after disinfection and before storage; and (5) store: store the endoscope in a way that prevents recontamination and promotes drying (eg, hung vertically).
In the past 3 years, multiple reports of outbreaks have led the FDA, the CDC, and national news to raise awareness among the public and health care professionals that the complex design of duodenoscopes (used primarily for ERCP) may impede effective reprocessing.
Several recent publications have associated multidrug-resistant (MDR) bacterial infections, especially due to CRE, in patients who have undergone ERCP with reprocessed duodenoscopes.
3, 4, 25, 37, 39 Unlike other endoscope outbreaks, 6 these recent outbreaks occurred even when the manufacturer's instructions and professional guidelines were followed correctly.
3, 4 The key concern raised by these outbreaks is that current reprocessing guidelines are not adequate to ensure a patient-safe GI endoscope (one devoid of potential pathogens), as the margin of safety associated with reprocessing endoscopes is minimal or nonexistent.
There are at least 2 (and maybe 3) reasons for this reprocessing failure and why outbreaks continue to occur.
First, studies have shown that the internal channel of GI endoscopes, including duodenoscopes, may contain 10 7À10 (7-10-log 10 ) enteric microorganisms.
40, 41 Investigations have demonstrated that the cleaning step in endoscope reprocessing results in a 2-to 6-log 10 reduction of microbes and the HLD step results in another 4-to 6-log 10 reduction of mycobacteria for a total 6to 12-log 10 reduction of microbes.
[40] [41] [42] Thus, the margin of safety associated with cleaning and HLD of GI endoscopes is minimal or nonexistent (level of contamination: 4-log 10 [maximum contamination, minimal cleaning/HLD] to À5-log 10 [minimum contamination, maximum cleaning/HLD]).
Therefore, any deviation from proper reprocessing (such as crevices associated with the elevator channel) could lead to failure to eliminate contamination with a possibility of subsequent patient-to-patient transmission.
This low (or nonexistent) margin of safety associated with endoscope reprocessing compares with the 17-log 10 margin of safety associated with cleaning and sterilization of surgical instruments.
23 Second, GI endoscopes not only have heavy microbial contamination (10 7 -10 10 bacteria) but they are also complex with long, narrow channels, right-angle turns, and difficult-to-clean and disinfect components (eg, elevator channel).
The elevator channel in duodenoscopes is unique to side-viewing endoscopes.
It has a separate channel and provides orientation of catheters, guidewires, and accessories into the endoscopic visual field.
25 This channel is complex in design and has crevices that are difficult to access with a cleaning brush and may impede effective reprocessing.
43 Based on this and other recent studies, it is likely that MDR pathogens are acting as marker or indicator organisms for ineffective reprocessing of the complex design of duodenoscopes, which is an infectious risk to patients.
Third, biofilms could impact endoscope reprocessing failure and continued endoscope-related outbreaks.
44 Biofilms are multilayered bacteria plus exopolysaccharides that cement cells to surfaces.
They develop in a wet environment.
If reprocessing is performed promptly after use and the endoscope is dry, the opportunity for biofilm formation is minimal.
45, 46 However, the formation of endoscopic biofilm during clinical practice may be related to reuse of reprocessing methods, such as reuse of detergent, manual cleaning, and incomplete drying.
47 Ideally, reprocessing should be initiated within an hour of use; however, there are no evidence-based guidelines on delayed endoscope reprocessing.
48 It is unclear if biofilms contribute to failure of endoscope reprocessing.
What should we do now?
Unfortunately, there is currently no single, simple, and proven technology or prevention strategy that hospitals can use to guarantee patient safety.
Of course, we must continue to emphasize the enforcement of evidenced-based practices, including equipment maintenance, and routine audits with at least yearly competency testing of reprocessing staff.
13, 35, 36 All reprocessing personnel must be knowledgeable and thoroughly trained on the reprocessing instructions for duodenoscopes.
This training includes the new recommendations to use a small bristle cleaning brush and for additional flushing and cleaning steps of the duodenoscope elevator channel (http://medical.olympusamerica.com/sites/default/files/ pdf/150326_TJF-Q180V_Customer_letter.pdf).
Although these steps were described as validated, no public data are available on the ability of these new cleaning recommendations to yield an ERCP scope devoid of bacteria.
But we must do more or additional outbreaks will likely continue.
For example, all hospitals that reprocess duodenoscopes should select one of the enhanced methods for reprocessing duodenoscopes.
These enhanced methods have been priority ranked with the first providing the greatest margin of safety.
25 They include (1) ETO sterilization after HLD with periodic microbiologic surveillance; (2) double HLD with periodic microbiologic surveillance; (3) HLD with scope quarantine until negative culture results are returned; (4) liquid chemical sterilant processing system using peracetic acid (rinsed with extensively treated potable water) with periodic microbiologic surveillance; (5) other FDAcleared low-temperature sterilization technology (provided material compatibility and sterilization validation testing performed using the sterilizer and endoscope) after HLD, with periodic microbiologic surveillance; and (6) HLD with periodic microbiologic surveillance.
These supplemental measures to enhance duodenoscope reprocessing made in May-June 2015 25 were reinforced by the FDA in August 2015.
43 UNC Hospitals has chosen ETO sterilization after HLD with periodic microbiologic surveillance as its primary reprocessing method for duodenoscopes and if the ETO sterilizer is not available, then double HLD with periodic microbiologic surveillance.
49 There is excellent evidence in the scientific literature that environmental contamination plays an important role in the transmission of several key health care-associated pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycinresistant Enterococcus (VRE), Acinetobacter sp, norovirus, and Clostridium difficile.
[50] [51] [52] [53] All these pathogens have been demonstrated to persist in the environment for days (in some cases months), frequently contaminate the environmental surfaces in rooms of colonized or infected patients, transiently colonize the hands of health care personnel, be transmitted by health care personnel, and cause outbreaks in which environmental transmission was deemed to play a role.
Importantly, a study by Stiefel and colleagues 54 demonstrated that contact with the environment was just as likely to contaminate the hands of health care personnel as was direct contact with patients.
Further, admission to a room in which the previous patient had been colonized or infected with MRSA, VRE, Acinetobacter or C difficile has been shown to be a risk factor for newly admitted patients to develop colonization or infection.
[55] [56] [57] Improving room cleaning and disinfection and demonstrating the effectiveness of surface decontamination in reducing health care-associated infections Investigators have reported that intervention programs aimed at improving surface cleaning and disinfection reduced HAIs.
58 Such interventions have generally included multiple activities: disinfectant product substitutions and interventions to improve the effectiveness of cleaning and disinfection (eg, improved housekeeper education, monitoring the thoroughness of cleaning [eg, by use of ATP assays or fluorescent dyes] with feedback of performance to the environmental service workers, and/or use of cleaning checklists).
[58] [59] [60] [61] Health care facilities must also allow adequate time for room processing to ensure adherence to all steps recommended by institutional policies and professional organization guidelines.
The authors have found that collaboration between infection prevention and environmental services staff, nursing, and management is critical to an effective environmental cleaning program.
This collaboration includes ensuring that environmental services staff recognize the significance and relationship of adhering to proper work procedures to reduction of microbial contamination.
The assignment of cleaning responsibility (eg, medical equipment to be cleaned by nursing; environmental surfaces to be cleaned by environmental service) is also important to ensure all objects and surfaces in a patient room are decontaminated, especially the surfaces of medical equipment (eg, cardiac monitors).
Improved environmental cleaning has been demonstrated to reduce the environmental contamination with VRE, 61 MRSA, 62 and C difficile.
63 Further, all studies have only focused improvement on a limited number of high-risk objects.
Thus, a concern of published studies is that they have only demonstrated improved cleaning of a limited number of high-risk objects (or targeted objects) not an improvement in the overall thoroughness of room decontamination, which is the objective.
To the authors' knowledge only one study has objectively evaluated what constitutes high-touch objects in a patient room and no study has demonstrated epidemiologically what constitutes a high-risk object.
Examples of what the literature refers to as high-touch objects includes bed rails, intravenous (IV) poles, call buttons, door knobs, floors, and bathroom facilities 64 ; however, a study demonstrated high-touch objects in the intensive care unit were the bed rail, bed surface, and supply cart, whereas the high-touch surfaces in a patient ward were the bed rail, over-bed table, IV pump, and bed surface.
65 Importantly, the level of microbial contamination of room surfaces was not statistically different regardless of how often they were touched before and after cleaning.
Until research identifies which objects and surfaces pose the greatest risk of pathogen transmission, all noncritical surfaces that are touched must be cleaned/disinfected.
66 No-touch (or mechanical) methods for room decontamination As noted earlier, multiple studies have demonstrated that environmental surfaces and objects in rooms are frequently not properly cleaned and these surfaces may be important in transmission of health care-associated pathogens.
Further, although interventions aimed at improving cleaning thoroughness have demonstrated effectiveness, many surfaces remain inadequately cleaned and, therefore, potentially contaminated.
For this reason, several manufacturers have developed room disinfection units that can decontaminate environmental surfaces and objects.
These no-touch systems generally use one of 2 methods: either UV light or HPV/mist.
53 These technologies supplement, but do not replace, standard cleaning and disinfection because surfaces must be physically cleaned of dirt and debris.
Ultraviolet light for room decontamination UV radiation has been used for the control of pathogenic microorganisms in a variety of applications, such as control of legionellosis, as well as disinfection of air, surfaces, and instruments.
53, 67 At certain wavelengths, UV light will break the molecular bonds in DNA, thereby destroying the organism.
UV radiation has peak germicidal effectiveness in the wavelength range of 240 to 280 nm.
Mercury gas bulbs emit UV-C at 254 nm, whereas xenon gas bulbs produce a broad spectrum of radiation that encompasses the UV (100-280 nm) and the visible (380-700 nm) electromagnetic spectra.
68 The efficacy of UV radiation is a function of many different parameters such as dose, distance, direct or shaded exposure, exposure time, lamp placement, pathogen, carrier or surface tested, inoculum method, organic load and orientation of carriers (eg, parallel vs perpendicular).
Data demonstrate that several UV systems have effectiveness (eg, eliminate >3-log 10 vegetative bacteria [MRSA, VRE, Acinetobacter baumannii] and >2.4-log 10 C difficile) at relatively short exposure times (eg, 5-25 minutes for bacteria, 10-60 minutes for C difficile spores).
[68] [69] [70] The studies also demonstrated reduced effectiveness when surfaces were not in direct line-of-sight.
[68] [69] [70] [71] [72] Hydrogen peroxide systems for room decontamination Several systems that produce HP (eg, HPV, aerosolized dry mist HP) have been studied for their ability to decontaminate environmental surfaces and objects in hospital rooms.
HPV has been used for the decontamination of rooms in health care.
[73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] Studies have demonstrated that HP systems are a highly effective method for eradicating various pathogens (eg, MRSA, Mycobacterium tuberculosis, Serratia, C difficile spores, Clostridium botulinum spores) from rooms, furniture, and equipment.
Comparison of ultraviolet irradiation versus hydrogen peroxide for room decontamination UV devices and HP systems have their own advantages and disadvantages ( Table 5) , 53 and there is now ample evidence that these no-touch systems can reduce environmental contamination with health care-associated pathogens and reduce HAIs.
84 However, each specific marketed system should be studied and its efficacy demonstrated before being introduced into health care facilities.
The main advantage of both types of units is their ability to achieve substantial reductions in vegetative bacteria.
Another advantage is their ability to substantially reduce C difficile spores, as low-level disinfectants (such as quaternary ammonium compounds) have only limited or no measurable activity against spore-forming bacteria.
85 Both systems are residual free, and they decontaminate all exposed surfaces and equipment in the room.
Based on data that demonstrated a reduction of colonizations and/or infections associated with these technologies, the authors recommend they should be used for terminal room decontamination after discharge of patients on contact precautions.
Because different UV and HP systems vary substantially, infection preventionists should review the peer-reviewed literature and the advantages/disadvantages of each technology (Box 1) and choose only devices with demonstrated bactericidal capability as assessed by carrier tests and/or the ability to disinfect actual patient rooms.
Ultimately, one would select a device that also has demonstrated the ability to reduce HAIs.
84 Disinfection and sterilization are critical components of infection control.
Unfortunately, breaches of disinfection and sterilization guidelines are not uncommon.
Patient notifications due to improper reprocessing of semicritical (eg, endoscopes) and critical medical instruments have occurred regularly.
7 This referenced article also provides a method for assessing patient risk for adverse events, especially infection.
Use of a 14-step algorithm (Box 2) can guide an institution in managing potential disinfection and sterilization failures.
7,86 Human papilloma virus is an extremely common sexually acquired infection and is the most important cause of cervical cancer.
A 2014 article reported that the FDA-cleared high-level disinfectants (ie, glutaraldehyde, OPA) tested did not inactivate human papilloma virus, a nonenveloped virus.
87 These findings are inconsistent with many There is reliable biocidal activity against a wide range of health care-associated pathogens.
Room surfaces and equipment are decontaminated.
There is rapid room decontamination (w5-25 minutes) for vegetative bacteria, which reduces the downtime of the room before another patient can be admitted.
It is demonstrated to reduce HAIs (eg, C difficile, MRSA).
It is effective against C difficile, although requires longer exposure (w10-50 minutes).
HVAC system does not need to be disabled and the room does not need to be sealed.
UV is residual free and does not give rise to health or safety concerns.
There are no consumable products, so costs include only capital equipment and staff time.
There is good distribution in the room of UV energy via an automated monitoring system.
All patients and staff must be removed from the room before decontamination, thus, limiting use to terminal room decontamination.
Decontamination can only be accomplished at terminal disinfection (ie, cannot be used for daily disinfection) as room must be emptied of people.
Capital equipment costs are substantial.
It does not remove dust and stains, which are important to patients and visitors; hence, cleaning must precede UV decontamination.
It is sensitive to use parameters (eg, dose, distance, carrier or surface tested, exposure time, pathogen).
It requires that equipment and furniture be moved away from the walls.
Advantages It has reliable biocidal activity against a wide range of health care-associated pathogens.
Room surfaces and equipment are decontaminated.
It has been demonstrated to reduce HAIs (eg, C difficile, MRSA, VRE).
It is useful for disinfecting complex equipment and furniture.
It does not require that furniture and equipment be moved away from the walls.
HP is residual free and does not give rise to health or safety concerns (aeration unit converts HP into oxygen and water).
There is uniform distribution in the room via an automated dispersal system.
All patients and staff must be removed from the room before decontamination, thus, limiting use to terminal room decontamination.
HVAC system must be disabled to prevent unwanted dilution of HP during use, and the doors must be closed with gaps sealed by tape.
articles in the peer-reviewed literature, which demonstrates that high-level disinfectants, such as OPA and glutaraldehyde, inactivate nonenveloped viruses, such as hepatitis A virus, polio, adenovirus, norovirus, and so forth.
Because the high-level disinfectants are commonly used to disinfect endocavitary probes (eg, vaginal probes, rectal probes), there is an urgency to corroborating these data.
In a conversation with CDC staff regarding this issue, it was determined hospitals should continue to use the FDA-cleared high-level disinfectants consistent with the manufacturers' instructions until the data can be corroborated.
Data have demonstrated the activity of a HP mist device to inactivate human papilloma virus.
88 Although the most common way of performing HLD of contaminated endocavitary probes is by immersion in an FDA-cleared high-level disinfectant (eg, glutaraldehyde), an alternative procedure for disinfecting the endocavitary and surface probes is a HP mist system, which uses 35% HP at 56 C with the probe reaching no more than 40 C (ie, Trophon EPR, Nanosonics, Alexandria, Australia).
In one study, the results demonstrated complete inactivation (>6-log 10 reduction) of VRE and a CRE-Klebsiella pneumoniae strain both in the presence and absence of 5% fetal calf serum (FCS).
The Trophon EPR system showed good, but not complete, inactivation of Mycobacterium terrae (5.2-log 10 reduction for M terrae with FCS, a 4.6-log 10 reduction for M terrae without FCS) and C difficile spores (5.1-log 10 reduction for C difficile spores with FCS, 6.2-log 10 reduction for C difficile spores without FCS).
89 To simulate a worse-case condition, cleaning was not done before disinfection in these experiments; but proper cleaning of probes is necessary to ensure the success of high-level disinfection.
Other data have demonstrated the activity of Trophon to inactivate human papilloma virus 88 and other pathogens (eg, bacteria, mycobacteria, viruses) including a greater than 6-log 10 reduction of M terrae and C difficile spores in carrier tests and a greater than 6-log 10 reduction in M terrae on inoculated ultrasound probes.
90 These results differ slightly from those presented earlier, presumably because of the differences in testing methodology.
In the authors' study only the probe devices were inoculated (carriers of different materials were not tested); for recovery of bacteria on the probe, the probes were immersed in media (not swabbed, which would likely result in lower recovery).
89 The Trophon system processes the portion of the probe that has mucous membrane contact but also the handle of endocavitary probes, which may be contaminated; it is an alternative to high-level chemical disinfection for ultrasound probes.
The Department of Justice and the FDA have joined forces in prosecuting health care providers that reuse single-use devices.
For example, one physician was criminally prosecuted for reusing needle guides meant for single use during prostate procedures.
These prosecutions are based on conspiracy to commit adulteration and Medicare fraud.
Third-party reprocessing is allowed by the FDA as the reprocessor is considered the device manufacturer as defined under the Code of Federal Regulations Title 21 Part 820.
In 2011, The Joint Commission (TJC) recommended that laryngoscope blades be packaged in a way that prevents recontamination.
Examples of compliant storage include, but are not limited to, a peel pouch or a closed plastic bag.
Examples of noncompliant storage would include unwrapped blades in an anesthesia drawer as well as an unwrapped blade on top of or within a code cart.
The packaging not only prevents recontamination but also distinguishes a processed from a nonprocessed semicritical item, such as a specula, laryngoscope blade, or endoscope.
The use of a tagging system, in both inpatient and outpatient facilities, 91 that separates processed from nonprocessed items minimizes the risk that a nondisinfected, semicritical device would be used and potentially lead to cross-transmission of a pathogen.
7 This tagging system could involve a tag (eg, green tag, patient ready; red tag, requires reprocessing) for GI endoscopes or a plastic sheath or plastic-paper peel pouch (eg, endocavitary probes).
Ideally, hospitals and ambulatory care facilities 91 (as appropriate) should develop a strategy (eg, tagging, storage covers for patient-ready devices) that prevents patient exposures to contaminated devices.
In the United States, it is estimated that more than 4 million cystoscopies are performed each year.
29, 92 Cystoscopy is a diagnostic procedure that uses an endoscope especially designed to examine the bladder, lower urinary tract, and prostate gland or is used to collect urine samples, perform biopsies, and remove small stones.
A flexible or rigid scope can be used to carry out the procedure.
Because the procedure, and other channeled scopes (eg, hysteroscopes, some nasopharyngoscopes), involves a medical device in contact with the patients' mucous membranes, it is considered a semicritical device that must minimally be high-level disinfected.
The authors recently evaluated the disinfection of cystoscopes, and their results demonstrated that disinfection (ie, a reduction in bacterial load of greater than 7-log 10 colony-forming unit [CFU]) did not occur unless the channel was actively perfused with the glutaraldehyde.
In fact, failure to perfuse the channel led to only minimal, if any, reduction in bacterial contamination.
However, complete inactivation of 10 8 CFU of both VRE and CRE was achieved when the channel was actively perfused.
It seems that no high-level disinfectant entered the channel unless it was actively perfused, as the level of microbial contamination was not reduced by immersion.
29 This failure to perfuse the channel occurs because the air pressure in the channel is stronger than the fluid pressure at the fluid-air interface.
Recommendations are provided for cystoscope HLD and include actively perfusing the device while immersed in the high-level disinfectant.
93 Unfortunately, some cystoscope reprocessing recommendations published in the literature are incorrect.
For example, investigators have recommended complete immersion of the cystoscope into the high-level disinfectant but did not mention perfusion of the high-level disinfectant into the channel.
94 Laryngoscopes Laryngoscopes are routinely used to view the vocal cords and larynx and facilitate airway management.
It typically consists of a blade that connects to a handle, which usually contains 2 batteries that power the light source.
Limited guidelines are available for reprocessing laryngoscope blades and handles, and hospital practices vary.
[95] [96] [97] For example, some guidelines recommend and hospitals use low-level disinfection of the handle as it does not have direct contact with a mucous membrane, whereas others recommend the handle be high-level disinfected to prevent disease transmission.
Although blades have been linked to HAIs, handles have not been directly linked to HAIs.
However, reports of contamination with blood (40% of the handles positive for occult blood) and potentially pathogenic microorganisms (86% of the handles deemed ready for patient use were contaminated with pathogens, such as S aureus, Acinetobacter) suggest its potential, 97-100 and the blade and handle function together.
For this reason, it is ideal that the blades and handles be high-level disinfected or sterilized even if a protective barrier or sheath is used during the procedure.
In 2007, the state of California required that both blades and handles be HLD or sterilized.
UNC Hospitals is sterilizing the blades and handles (ie, blades via HP gas plasma, handle [without batteries] by steam).
Other methods for HLD or sterilization are acceptable, but one must ensure the blade and handle are compatible with the HLD or sterilization process chosen.
After sterilization the blades and handles are checked for function before packaging and then packaged in a Ziploc bag.
Per TJC, the laryngoscope blade and handle must be packaged in a way that prevents recontamination after processing (Frequently Asked Questions, The Joint Commission, October 24, 2011).
Examples of compliant storage include, but are not limited to, a peel pack after sterilization (long-term storage) or wrapping in a sterile towel (short-term storage).
Recent advances in video technology have led to the development of video laryngoscopes, such as the GlideScope (Verathon Medical, Bothell, WA) and McGrath (Medtronic, Minneapolis, MN) video laryngoscopes.
92 These new intubation devices assist in difficult airway management.
For the McGrath an image is displayed on a liquid-crystal display screen that is contained within a monitor mounted to the handle of the device.
A sterile, single-use disposable laryngoscope blade covers the camera and light-emitting diode assembly to prevent direct patient contact.
Even though a cover is used, HLD or sterilization via ETO or HP gas plasma (battery removed) is recommended for the McGrath MAC video laryngoscope.
93 The manufacturer states, whenever practical, a HLD or sterilization is preferred to a wipebased process.
The portable GlideScope video laryngoscope system is available in a single-use and a reusable configuration.
They should be cleaned and disinfected per the manufacturer's recommendations.
The single-use system features a reusable video baton and sterile stats that must be disposed of immediately after use.
Low-level disinfection is recommended for the video baton after each use using an Environmental Protection Agency-registered disinfectant (eg, antimicrobial disposable wipe per manufacturer's instructions) after each use.
The manufacturer recommends HLD for the video baton when it is visibly soiled.
The manufacturer recommends that the advanced video laryngoscope reusable blade be high-level disinfected and the GlideRite rigid stylets be sterilized.
101 Emerging Pathogens, Antibiotic-Resistant Bacteria, and Bioterrorism Agents Emerging pathogens are of growing concern to the general public and infection control professionals.
Relevant pathogens include Ebola, 102 MDR organisms such as CRE, Enterovirus D68, MDR pathogens, Middle East respiratory syndrome-Coronavirus, MDR M tuberculosis, human papilloma virus, norovirus, and nontuberculous mycobacteria (eg, M chelonae).
The susceptibility of each of these pathogens to chemical disinfectants/sterilants has been studied; all of these pathogens (or surrogate microbes such as feline-calicivirus for Norwalk virus, vaccinia for variola, 103 and Bacillus atrophaeus [formerly B subtilis] for B anthracis) are susceptible to currently available chemical disinfectants/sterilants.
19, 104 Standard sterilization and disinfection procedures for patient-care equipment (as recommended in this article) are adequate to sterilize or disinfect instruments or devices contaminated with blood or other body fluids from persons infected with blood-borne pathogens, emerging pathogens, and bioterrorism agents, with the exception of prions, HPV, and C difficile spores (see earlier discussion).
No changes in current procedures for cleaning, disinfecting, or sterilizing need to be made.
13 In addition, there are no data to show that antibiotic-resistant bacteria (MRSA, VRE, MDR M tuberculosis) are less sensitive to the liquid chemical germicides than antibiotic-sensitive bacteria at currently used germicide contact conditions and concentrations.
[105] [106] [107] SUMMARY When properly used, disinfection and sterilization can ensure the safe use of invasive and noninvasive medical devices.
The method of disinfection and sterilization depends on the intended use of the medical device: critical items (contact sterile tissue) must be sterilized before use; semicritical items (contact mucous membranes or nonintact skin) must be high-level disinfected; and noncritical items (contact intact skin) should receive low-level disinfection.
Cleaning should always precede HLD and sterilization.
Current disinfection and sterilization guidelines must be strictly followed.
National hospital discharge survey: 2010 table, procedures by selected patient characteristics-number by procedure category and age Burden of gastrointestinal disease in the United States: 2012 update New Delhi metallo-beta-lactamaseproducing carbapenem-resistant Escherichia coli associated with exposure to duodenoscopes Endoscopic retrograde cholangiopancreatography-associated AmpC Escherichia coli outbreak Endoscope reprocessing methods: a prospective study on the impact of human factors and automation Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy How to assess risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines Lessons learned from outbreaks and pseudo-outbreaks associated with bronchoscopy Immediate need for healthcare facilities to review procedures for cleaning, disinfecting, and sterilizing reusable medical devices Disinfection, sterilization and control of hospital waste Disinfection, Sterilization and Antisepsis: An Overview Association for Professionals in Infection Control and Epidemiology Guideline for disinfection and sterilization in healthcare facilities Disinfection and sterilization in healthcare facilities Disinfection, sterilization, and preservation.
Philadelphia: Lea & Febiger Healthcare Infection Control Practices Advisory Committee.
Guidelines for environmental infection control in health-care facilities Disinfection and sterilization of prion-contaminated medical instruments, reply to Belay Efficacy of a washer-disinfector in eliminating healthcare-associated pathogens from surgical instruments FDA-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices Disinfection and sterilization in healthcare facilities Guidelines for infection control in dental health-care settings-2003 Disinfection: is it time to reconsider Spaulding?
Brief summary of the gastroenterology and urology devices panel meeting Gastrointestinal endoscopes: a need to shift from disinfection to sterilization?
ERCP scopes: what can we do to prevent infections?
Outbreak of Pseudomonas aeruginosa surgical site infections after arthroscopic procedures: Texas Microbial contamination on used surgical instruments Disinfection of a probe used in ultrasoundguided prostate biopsy Effective high-level disinfection of cystoscopes: is perfusion of channels required?
Disinfection of an infrared coagulation device used to treat hemorrhoids Role of environmental contamination in the transmission of vancomycin-resistant enterococci Role of hospital surfaces in the transmission of emerging health care-associated pathogens: norovirus, Clostridium difficile, and Acinetobacter species Outbreaks associated with contaminated antiseptics and disinfectants Stability and bactericidal activity of chlorine solutions Multisociety guideline on reprocessing flexible GI endoscopes Society of Gastroenterology Nurses and Associates I.
Standards of infection control and reprocessing of flexible gastrointestinal endoscopes Risk of transmission of carbapenem-resistant Enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy Persistent contamination on colonoscopes and gastroscopes detected by biologic cultures and rapid indicators despite reprocessing performed in accordance with guidelines Control of a multi-hospital outbreak of KPC-producing Klebsiella pneumoniae type 2 in France Disinfection, sterilization and antisepsis: principles and practices in healthcare facilities Worst-case soiling levels for patient-used flexible endoscopes before and after cleaning FDA labeling requirements for disinfection of endoscopes: a counterpoint Brief summary of the gastroeneterology and urology devices panel meeting Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination Association for Professionals in Infection Control and Epidemiology Effectiveness of current disinfection procedures against biofilm on contaminated GI endoscopes Correlation between the growth of bacterial biofilm in flexible endoscopes and endoscope reprocessing methods Delayed reprocessing of endoscopes Outbreaks of carbapenem-resistant Enteriobacteriaceae infections associated with duodenoscopes: what can we do to prevent infections?
The role played by contaminated surfaces in the transmission of nosocomial pathogens Environmental contamination makes an important contribution to hospital infection Role of the environment in the transmission of Clostridium difficile in health care facilities Disinfectants used for environmental disinfection and new room decontamination technology Contamination of hands with methicillin-resistant Staphylococcus aureus after contact with environmental surfaces and after contact with the skin of colonized patients Methods for assessing the adequacy of practice and improving room disinfection Risk of acquiring antibiotic-resistant bacteria from prior room occupants Evaluation of hospital room assignment and acquisition of Clostridium difficile infection Does improving surface cleaning and disinfection reduce health care-associated infections?
Evaluating hygienic cleaning in health care settings: what you do not know can harm your patients Monitoring the effectiveness of hospital cleaning practices by use of an adenosine triphosphate bioluminescence assay Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product or procedure?
Impact of an environmental cleaning intervention on the presence of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci on surfaces in intensive care unit rooms Reduction of Clostridium difficile and vancomycin-resistant Enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods Environmental cleaning for the prevention of healthcare-associated infection.
Technical brief No.
22 (prepared by the ECRI Institute -Penn Agency for Healthcare Research and Quality A quantitative approach to defining "high-touch" surfaces in hospitals Microbial assessment of high-, medium-, and low-touch hospital room surfaces Applications of ultraviolet germicidal irradiation disinfection in health care facilities: effective adjunct, but not standalone technology Evaluation of a pulsed xenon ultraviolet disinfection system for reduction of healthcare-associated pathogens in hospital rooms Room decontamination with UV radiation Room decontamination using an ultraviolet-C device with short ultraviolet exposure time Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms Terminal decontamination of patient rooms using an automated mobile UV light unit Impact of hydrogen peroxide vapor room decontamination on Clostridium difficile environmental contamination and transmission in a healthcare setting Tackling contamination of the hospital environment by methicillin-resistant Staphylococcus aureus (MRSA): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination Environmental methicillinresistant Staphylococcus aureus (MRSA) disinfection using dry-mistgenerated hydrogen peroxide Use of hydrogen peroxide vapor for deactivation of Mycobacterium tuberculosis in a biological safety cabinet and a room Rapid recontamination with MRSA of the environment of an intensive care unit after decontamination with hydrogen peroxide vapour Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores Efficacy of vaporized hydrogen peroxide against exotic animal viruses Vapor-phase hydrogen peroxide as a surface decontaminant and sterilant Use of hydrogen peroxide vapour for environmental control during a Serratia outbreak in a neonatal intensive care unit Activity of a dry mist hydrogen peroxide system against environmental Clostridium difficile contamination in elderly care wards Airborne hydrogen peroxide for disinfection of the hospital environment and infection control: a systematic review Effectiveness of UV devices and hydrogen peroxide systems for terminal room decontamination: focus on clinical trials Selection of the ideal disinfectant Assessing the risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines Susceptibility of high-risk human papillomavirus type 16 to clinical disinfectants Susceptibility of HPV 16 and 18 to high-level disinfectants indicated for semi-critical ultrasound probes Effectiveness of a hydrogen peroxide mist (TrophonÒ) system in inactivating healthcare pathogens on surface and endocavitary probes Evaluation of an automated high-level disinfection technology for ultrasound transducers Disinfection and sterilization in physician practices and specialty clinics Reprocessing semicritical items: current issues and new technologies McGrath MAC video laryngoscope operator's manual Outbreak of cystoscopy related infections with Pseudomonas aeurginosa: New Mexico Recommendations to resolve inconsistent guidelines for the reprocessing of sheathed and unsheathed rigid laryngoscopes Prevention of disease transmission during flexible laryngoscopy Reassessment of the risk of healthcare-acquired infection during rigid laryngoscopy Nosocomial contamination of laryngoscope handles: challenging current guidelines Contamination of laryngoscope handles Incidence of visible and occult blood on laryngoscope blades and handles Evaluating environmental persistence and disinfection of the Ebola virus Makona variant The inactivation of viruses by germicides Sporicidal activity of a new low-temperature sterilization technology: the Sterrad 50 sterilizer Susceptibility of antibiotic-susceptible and antibiotic-resistant hospital bacteria to disinfectants Susceptibility of vancomycin-resistant enterococci to environmental disinfectants Antimicrobial activity of home disinfectants and natural products against potential human pathogens
key: cord-280571-ntgt5hy9 authors: Ginocchio, Christine C. title: Strengths and Weaknesses of FDA-Approved/Cleared Diagnostic Devices for the Molecular Detection of Respiratory Pathogens date: 2011-05-01 journal: Clin Infect Dis DOI: 10.1093/cid/cir046 sha: doc_id: 280571 cord_uid: ntgt5hy9 The rapid, sensitive, and specific identification of the microbial etiological characteristics of respiratory tract infections enhances the appropriate use of both antibiotics and antiviral agents and reduces the risk of nosocomial transmission.
This article reviews the current nucleic acid amplification tests approved by the U.S. Food and Drug Administration (FDA) for the detection of respiratory pathogens.
In addition, Emergency Use Authorization tests for the detection of 2009 influenza A H1N1 are discussed.
The advantages and limitations of the current FDA-approved/cleared tests are reviewed.
The identification of the causative agent(s) of respiratory tract infections is essential to provide an accurate diagnosis, appropriately manage patient care, and reduce the risk of nosocomial transmission within health care facilities.
With the steady rise of antibiotic resistance and limited or no options available for the treatment of multidrug or panresistant bacterial infections, pathogen identification is a key component in restricting antibiotic use to those circumstances in which antibiotic therapy is clearly indicated [1] .
Initial empiric therapeutic choices may be standardized and initiated on the basis of patient clinical status, underlying disease, and/or risk for infection with a multidrug-resistant pathogen.
However, subsequent bacterial pathogen identification and accurate susceptibility data should assist in promoting switches to targeted specific therapies and reduce the use of broad-spectrum antibiotics when not indicated, thereby promoting good antibiotic stewardship [2] .
Viral infections probably cause between 75% and 80% of respiratory tract diseases.
Nonetheless, it is estimated that 22.6 million (55%) of 41 million antibiotic prescriptions for respiratory tract infections, including both lower and upper respiratory infections, were for causes unlikely to have a bacterial etiology [3] .
Although in many cases of otitis and sinusitis and probably 20%-40% of cases of community-acquired pneumonia a bacterial superinfection may occur, respiratory infections due to a virus(es) alone are common in the adult and pediatric outpatient populations.
In addition, the vast majority of respiratory tract infections in nonimmunocompromised hospitalized children (especially those ,5 years of age) are due to 1 or more viruses, without a secondary bacterial infection.
The overuse of antibiotics for the treatment of outpatients is primarily due to the fact that in adults respiratory virus testing is either not performed or generally limited to rapid antigen direct tests (RADTs) for influenza.
Testing for the elderly, for persons with chronic obstructive pulmonary disease, and for pediatric patients is limited to RADTs for influenza and respiratory syncytial virus (RSV).
RADTs have highly variable sensitivities (10%-75%) and specificities (50%-100%) depending on the viral target, age of the patient, sample collection, and duration of symptoms prior to testing [4] [5] [6] .
In general, RADTs perform better when testing pediatric samples, because children shed higher titers of virus and for longer time periods than adults [4, [7] [8] [9] .
In addition to RADTs, there are U.S. Food and Drug Administration (FDA)-approved/cleared nonmolecular-based viral diagnostic methods with a more rapid time to result, compared with traditional viral tube culture, eg, direct fluorescent antibody (DFA) testing and rapid cell culture.
Both methods can readily detect 7 of the common respiratory viruses (adenovirus, influenza A, influenza B, parainfluenza 1, 2, and 3 [PIV-1, PIV-2, PIV-3], and RSV).
In addition, DFA testing can detect human metapneumovirus (hMPV) [6] .
The specificity of DFA testing and rapid cell culture are high, but the sensitivities of the tests can vary from a low of 50% (RSV culture) to a high of .80% (influenza A), when compared with nucleic acid amplification tests (NAATs) [5, 6, 10] .
DFA testing can be performed in as little as 30-60 min, and shell vial and R-Mix rapid cell cultures (Quidel/Diagnostic Hybrids) generally identify respiratory viruses in 24-48 h [6] .
If these tests are performed on site, the time to virus detection can be within a time frame that could affect patient management.
However, these tests are not widely available outside larger hospitals and reference laboratories.
Although these 8 viruses are responsible for a large number of respiratory tract infections, bocavirus, selected coronaviruses (229E, OC43, NL63, and HKU-1), parainfluenza 4, and rhinovirus are also important causes of respiratory disease and are generally only detected using NAATs.
Because antiviral therapies are currently limited to the treatment of influenza A, influenza B, cytomegalovirus pneumonia, and varicella zoster virus pneumonia, it is often argued that the specific identification of other viruses is not relevant, because the information would not change patient management.
From a treatment standpoint, this may currently be true; however, the respiratory viruses cause similar illnesses, and diagnosis based on clinical symptoms alone can be highly inaccurate [11] .
For example, a study by Poehling et al revealed that physicians who used only clinical symptoms recognized influenza in only 28% of hospitalized children and 17% of nonhospitalized children with laboratory-confirmed influenza [11] .
Therefore, establishing the viral etiological characteristics of the illness is often highly dependent on accurate diagnostic testing.
In addition, new therapeutic agents for respiratory viruses are in development, and clinical trials for these agents will need rapid diagnostics that detect a broad range of viral pathogens.
Once these new drugs are approved, clinical laboratories will need the tools to identify each virus so that appropriate antiviral therapy can be rapidly initiated.
In addition, during the first weeks of the 2009 influenza A H1N1 outbreak in the spring of 2009, multiple influenza viruses were cocirculating [5] .
It was necessary to subtype influenza A strains to differentiate seasonal influenza A/H1, seasonal influenza A/H3, and 2009 influenza A H1N1.
Subtyping is necessary to provide relevant information needed for appropriate selection of antiviral therapy, in particular for acutely ill patients.
Antiviral resistance testing of influenza isolates from 2009 (www.cdc.gov/flu) revealed that the circulating seasonal influenza A/H1 strains were resistant to oseltamivir (99.6%), and seasonal influenza A/H3 and 2009 influenza A H1N1 were resistant to the adamantanes (100%).
In addition, several patients with 2009 influenza A H1N1 infections developed oseltamivir resistance [12, 13] .
Other clinical factors, such as the potential for the development of more severe disease on the basis of the virus etiology, may need to be considered in the management of certain patients.
For example, studies from our laboratory have revealed that children with hMPV infections have a higher incidence of admission to an intensive care unit and more often require mechanical ventilation than children with RSV infections [14, 15] .
Especially in the treatment of inpatients, the costs of rapid viral diagnostics can be offset by the improvement in patient care and financial outcomes [16] [17] [18] [19] .
Hendrickson et al showed that rapid respiratory virus diagnosis can lead to benefits in several areas, including up to a 50% reduction in hospital days, 30% reduction in antibiotic use, and 20% reduction in unnecessary diagnostic tests and procedures [16] .
The burden of nosocomial influenza can be high, incurring additional costs for diagnostic tests, increased morbidity, and extended hospitalization [17, 18] .
Therefore, rapid diagnostic tests are needed to identify patients with influenza at admission, in order to prevent nosocomial transmission by facilitating isolation and cohorting decisions [18] .
Studies have documented substantial nosocomial transmission of hMPV in pediatric units [20] , as well as in chronic care facilities [21] , similar to what is seen with RSV.
During the height of RSV season, many institutions must cohort RSV-positive children because of a lack of private rooms.
However, dual infections with RSV and hMPV do occur.
Limiting diagnostics to RSV alone in a cohorting scenario could put other seriously ill children at risk for acquisition of a second viral infection with hMPV.
The meaning of mixed viral infections can be defined only if testing is comprehensive.
Broad test panels also allow for monitoring the epidemiologic patterns of respiratory disease and for identifying new or reemerging pathogens.
Finally, the identification of the exact respiratory virus is essential for the accurate assessment of the efficacy of vaccines.
The costs for testing using FDA-approved/cleared NAATs are highly variable and can range from approximately $30 to .$200 per test.
Factors that determine test cost include test volume, the price of the test kits, the number of tests per kit, size of the testing run, the number of controls required per testing run, and the type and amount of ancillary supplies.
Often FDA-approved/ cleared kits do not contain nucleic acid extraction reagents, so additional equipment and reagents are necessary and can add $3-$15 per test.
Batch testing of samples may reduce the cost per test; however, often batching is not practical if the time to result is delayed beyond a period that would affect either clinical management or infection control practices.
Instrumentation costs for molecular tests can be substantial, with real-time instrumentation costing on average $35,000-$85,000 per instrument, and higher volume laboratories may require multiple instruments.
Laboratories must also calculate the cost for technical time, which is again highly variable depending on the test complexity and run size.
Finally, the costs for quality control, proficiency testing, and competency assessment all affect the overall cost per test.
In deciding which tests are appropriate and at what cost for the patient population at a particular health care facility, all of the above factors need to be considered in light of the clinical impact of the test result.
The use of NAATs is an intrinsic part of infectious disease diagnostics.
The ability of NAATs to rapidly and accurately detect a novel pathogen was best exemplified during the 2009 influenza A H1N1 pandemic [5, [22] [23] [24] .
NAATs are especially suited for the identification of respiratory pathogens that are not routinely or easily cultured (eg, hMPV, bocavirus, parainfluenza 4, and Chlamydophila pneumoniae), pathogens that are dangerous to culture (eg, severe acute respiratory syndrome coronavirus), pathogens for which the time to detection by traditional means is often too delayed to affect patient care (eg, tube cell culture for influenza), and pathogens for which serologic testing is difficult to interpret (eg, C. pneumoniae).
In most cases, NAATs offer enhanced sensitivity over culture methods, RADTS, and DFA testing [5, 6, 10] .
The specificity of NAATs varies with target and assay design but is generally very high.
In addition, laboratorydeveloped NAATs validated in accordance with the Clinical Laboratory Improvement Amendments (CLIA) requirements can be used to identify new pathogens until FDA-approved/ cleared in vitro diagnostic (IVD) devices become available.
With clinical integration of real-time polymerase chain reaction (PCR) and FDA-approved/cleared simple cartridge-based NAATs, laboratories of all sizes are now able to perform molecular diagnostic tests.
The detection of Mycobacterium tuberculosis directly in a clinical sample from a patient not yet identified as M. tuberculosis positive is always clinically relevant.
Detection of such bacterial pathogens as Bordetella pertussis, Bordetella parapertussis, Legionella pneumophila, Mycoplasma pneumoniae, or C. pneumoniae usually indicates active infection.
In contrast, the detection of other bacteria, such as Streptococcus pneumoniae, may indicate infection or simply colonization.
Therefore, interpretation of the molecular detection of many bacterial pathogens must be viewed in light of the clinical specimen (sterile vs nonsterile body site) and determination of the quantity of organisms present.
In addition, molecular methods must include all potential pathogens, even though culture is still required for antimicrobial susceptibility testing.
For these reasons, there is currently a scarcity of FDA-approved/cleared assays for nonviral respiratory pathogens (Table 1 ).
For such targets as the atypical pneumonia pathogens, the interpretation of NAAT results is generally not an issue, quantitative tests are not necessarily indicated, and NAATS could replace traditional test methods.
However, the lack of FDA-approved/cleared NAATs for these pathogens most probably relates to the IVD device manufacturers' reluctance to perform costly clinical trials for targets with a relatively low prevalence rate and for which the potential volume of testing may be minimal.
In light of the rise in M. tuberculosis drug resistance worldwide, the rapid identification of patients with pulmonary tuberculosis is essential and allows for improved patient outcomes, the appropriate use of isolation facilities, the initiation of appropriate treatment, and the identification of possible infected contacts [25] [26] [27] .
In addition, studies have shown that the estimated costs associated with an inaccurate diagnosis of M. tuberculosis infection are substantial ($11,576 per patient) because of institutional isolation procedures [27] .
Direct sample testing can detect M. tuberculosis within 24-48 h of sample collection, compared with 1-4 weeks for liquid and traditional solid media culture methods.
Although acid-fast bacilli (AFB) smear microscopy is inexpensive and rapid to perform, the sensitivity of the test can be poor (45%-80% with culture-confirmed pulmonary M. tuberculosis colonization or infection) and cannot differentiate M. tuberculosis from mycobacteria other than M. tuberculosis [26] .
The 2009 Centers for Disease Control and Prevention (CDC) guidelines recommend that NAATs should be performed on at least 1 respiratory sample from patients with signs and symptoms of pulmonary M. tuberculosis colonization or infection for whom a diagnosis of M. tuberculosis colonization or infection has not been established or when the results would alter case management or infection control procedures [28] .
Mycobacterial culture is still required, because the M. tuberculosis isolate is needed for drug susceptibility testing.
Currently, there are 2 FDA-approved NAATs available for the detection of M. tuberculosis complex from respiratory samples, the Amplified Mycobacterium tuberculosis Direct Test (AMTD, Gen-Probe) [25, [29] [30] [31] and the Amplicor Mycobacterium tuberculosis Test (Amplicor, Roche Diagnostics) [32] [33] [34] [35] .
Recently, a new assay called the Xpert MTB/RIF (Cepheid) was developed to detect the presence of M. tuberculosis and rifampin resistance directly from processed clinical samples [reviewed in 36] .
The assay uses a heminested real-time PCR with molecular beacon detection and is performed on the GeneXpert instrument (Cepheid).
Benefits of this assay include low technical complexity, allowing for potential point-of-care testing; minimal hands-on time (15-20 min); test turnaround time of ,2 h; and containment of the sample, extracted nucleic acids, and amplicons in the test cartridge.
When compared with culture, the sensitivity of the assay for the detection of M. tuberculosis ranged from 98.2% (1 sample tested per patient) to 99.8% (3 samples tested per patient) for AFB smear-positive samples and from 72.5% (1 sample tested per patient) to 90.2% (3 samples tested per patient) for AFB smear-negative samples.
The specificity of the assay ranged from 98.1% to 99.2%.
The assay is currently available for diagnostic testing only in Europe.
Amplified Mycobacterium tuberculosis Direct Test (AMTD, Gen-Probe) The AMTD is a target-amplified nucleic acid probe test for the direct detection of M. tuberculosis complex (Mycobacterium bovis, M. bovis BCG, Mycobacterium africanum, Mycobacterium microti, and M. tuberculosis) ribosomal RNA (rRNA) [25, [29] [30] [31] .
AMTD is approved for testing both AFB smear-positive and smear-negative respiratory specimens collected from patients suspected of having M. tuberculosis colonization or infection.
The AMTD test is based on isothermal (42 o C) transcription-mediated amplification that uses 2 enzymes, reverse transcriptase (RT) and T7 RNA polymerase, and targetspecific primers.
A hybridization protection detection assay that uses a chemiluminescent-labeled, single-stranded DNA probe complementary to the M. tuberculosis complex-specific sequences detects the amplified rRNA using the Gen-Probe Leader luminometer.
Numerous studies have evaluated AMTD and have revealed an average sensitivity and specificity of .95% and .98%, respectively, for the detection of M. tuberculosis complex in smear-positive respiratory samples and a sensitivity and specificity of .60% and .72%, respectively, for smear-negative respiratory samples [29] [30] [31] .
Additional studies have evaluated the use of the test for several types of nonrespiratory samples, such as cerebrospinal fluid and lymph nodes [29] [30] [31] .
Amplicor Mycobacterium tuberculosis Test (Amplicor, Roche Diagnostics) The Amplicor Mycobacterium tuberculosis test is FDA approved for use with AFB smear-positive respiratory specimens from patients suspected of having M. tuberculosis infection or colonization [32] [33] [34] [35] .
The Amplicor Mycobacterium tuberculosis test uses traditional PCR to amplify a region of the gene encoding the 16S rRNA of all mycobacteria.
Members of the M. tuberculosis complex are identified after hybridization with a DNA target-specific probe, followed by substrate addition and colorimetric detection [32] .
When testing AFB smear-positive samples, the Amplicor Mycobacterium tuberculosis test demonstrated sensitivities and specificities for the detection of M. tuberculosis complex ranging from 92.9% to 100% and from 77.3% to 100%, respectively [32, 33] .
The sensitivities and specificities of Amplicor Mycobacterium tuberculosis for AFB smear-negative specimens ranged from 51.2% to 73.1% and 99% to 99.8%, respectively [32, 33] .
The Amplicor Mycobacterium tuberculosis test has also been evaluated for detection of M. tuberculosis meningitis [34] and for use with nonrespiratory sample types [35] .
Viral respiratory pathogens are particularly suited for detection using NAAT, since the number of targets is relatively limited, compared with the numerous potential bacterial pathogens that can cause respiratory disease (Table 1) .
Although there is much to learn regarding the clinical relevance of mixed viral respiratory infections, the detection of a respiratory virus is generally considered diagnostic at this time.
Today, most laboratories do not have the facilities for comprehensive tissue culture based viral diagnostics and therefore are limited to testing with less sensitive and less specific RADTs for only influenza A, influenza B, and RSV.
NAATS offer an excellent alternative to greatly expand the test menu of clinical laboratories, thereby providing rapid, accurate comprehensive diagnostics.
The first multiplex NAAT to receive clearance by the FDA was the xTAG respiratory virus panel (RVP) assay in January 2008.
The FDA-approved version of RVP detects adenovirus, influenza A (with subtyping of seasonal influenza A/H1 and seasonal influenza A/H3), influenza B, PIV-1, PIV-2, PIV-3, hMPV, rhinovirus, RSV A, and RSV B [37, 38] .
The US/Canadian research use-only version of the assay also detects 4 coronaviruses (OC43, 229E, NL63, and HKU-1), parainfluenza type 4, and enterovirus.
The test is approved for use with nasopharyngeal swab samples collected from persons symptomatic for a respiratory virus infection and placed in viral transport media.
The xTAG RVP is a multistep test that takes approximately 8-10 h to complete, depending on the number of samples to be tested (Figure 1 ).
Viral nucleic acid and an internal control (E. coli MS2 phage) are coextracted from clinical samples using the QIAamp MiniElute (Qiagen), the easyMAG (bioMérieux), or the miniMAG (bioMérieux) extraction platforms.
A multiplex reverse transcription polymerase chain reaction (RT-PCR), using primer sets specific for the test targets, amplifies the viral nucleic acid and internal control nucleic acid.
PCR products are treated with exonuclease I to degrade any remaining primers and with shrimp alkaline phosphatase to degrade any remaining nucleotides.
The next step consists of target-specific primer extension (TSPE).
When a viral target(s) is present, the target-specific primer (containing a unique tag sequence) is extended and biotin-deoxycytidine triphosphate is incorporated into the extending chain.
On completion of the TSPE, the detection of amplified products is performed using Luminex's Universal Tag sorting system.
The TSPE reaction is added directly to microwells containing spectrally distinguishable beads with antitags, which are complementary to the sequence tags on the primers.
Each tagged primer will hybridize only to its unique antitag complement associated with a specific colored bead.
A fluorescent reporter molecule (streptavidin-phycoerythrin) will bind to the biotin on the extended primers.
The beads are then analyzed with the Luminex xMAP 100/200 instruments.
Two lasers read each bead; the first identifies the virus-specific color-coded bead, and the second determines whether an amplicon is hybridized to the bead on the basis of the detection of fluorescence (mean fluorescence intensities [MFIs]) above a background threshold.
The clinical performance characteristics of the xTAG RVP assay were established by the manufacturer through prospectively collected nasopharyngeal swab samples (n 5 544) tested during the 2005-2006 influenza season at 4 North American clinical laboratories [101] .
All specimens were tested by means of viral culture and/or DFA testing for the following targets: influenza A, influenza B, RSV, PIV-1, PIV-2, PIV-3, and adenovirus.
The comparator methods for influenza A subtyping, RSV subtyping, and hMPV and rhinovirus detection were wellcharacterized RT-PCR assays followed by bidirectional sequencing.
xTAG RVP sensitivity for each target was determined as follows: The benefits of the xTAG RVP assay include the broad spectrum of viruses detected by a single test, with a cost per test comparable to real-time assays that only target up to 3 analytes.
The subtyping of influenza A viruses as seasonal influenza A/H1 or seasonal influenza A/H3 or the identification of an ''unsubtypeable'' virus has proven to be an important aid in identifying novel influenza A strains.
A limitation of the assay is the decreased sensitivity for the detection of certain adenovirus strains.
In-house validation studies by our laboratory have found that by reducing the positive cutoff MFI level from 300 to 150 for adenovirus, improved sensitivity for the detection of adenovirus was obtained without loss of specificity (unpublished data).
Additional limitations of the assay include the time to final results, number of required steps, technical handson time, and a potential for amplicon contamination.
There is a second-generation assay called RVP FAST (Luminex) available in Europe that addresses several of these issues by reducing the number of steps and the time to results by 3-4 h, making it possible to provide comprehensive results within a single shift.
Gen-Probe/Prodesse ProFlu,1ProFlu-ST, Pro hMPV1, and ProParaflu1 Assays (Gen-Probe/Prodesse) Currently, there are 3 Gen-Probe/Prodesse FDA-cleared multiplex real-time RT-PCR assays for the qualitative detection and discrimination of respiratory viruses.
The ProFlu1 assay targets the matrix gene for influenza A, nonstructural genes NS-1 and NS-2 for influenza B, and the polymerase gene for RSV A and RSV B.
The Pro hMPV1 assay targets highly conserved regions of the nucleocapsid (N) gene for hMPV and a transcript derived from Escherichia coli Bacteriophage MS2 A-protein gene (Internal Control).
The ProParaflu1 assay targets conserved regions of the hemagglutinin-neuraminidase gene for PIV-1, PIV-2, and PIV-3 and a transcript derived from E. coli Bacteriophage MS2 A-protein gene (Internal Control).
All 3 assays are approved for testing nasopharyngeal swab specimens obtained from symptomatic persons.
Viral nucleic acids from patient samples are coextracted with an internal control that monitors assay performance and the presence of amplification inhibitors that could lead to false-negative results.
Nucleic acids are extracted using a MagNA Pure LC Instrument (Roche Diagnostics Corp) and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche) or a NucliSENS easyMAG System and the Automated Magnetic Extraction Reagents (bio-Mérieux).
The purified nucleic acids are amplified by means of RT-PCR using target-specific oligonucleotide primers and Taqman probes complementary to highly conserved regions of the target gene.
The Taqman probes are labeled with a quencher dye attached to the 3'-end and a reporter dye at the 5'-end.
When amplified target is present, the probes bind and the 5'-3' exonuclease activity of Taq polymerase cleaves the probe, thus separating the reporter dye from the quencher.
Because the quencher and reporter dye are now physically separated, there is an increase in fluorescent signal upon excitation from a light source.
The fluorescent signal increases with each cycle as additional reporter dye molecules are cleaved from their respective probes.
During each PCR cycle, the fluorescent intensity is monitored by the realtime instrument, the SmartCycler II (time to results, including extraction, is approximately 3-3.5 hr for each test run).
The performance characteristics of the assays were established by the manufacturer through prospective and retrospective clinical studies used for FDA clearance of the tests.
ProFlu1 assay results obtained by testing 891 nasopharyngeal samples were compared with results of rapid shell vial culture.
ProFlu1 sensitivities and specificities for the detection of influenza A were 100% and 92.6%, respectively; for influenza B were 97.8% and 98.6%, respectively; and for RSV were 89.5% and 94.9%, respectively [102].
A study by Liao et al compared the Prodesse ProFlu-1 assay, a previous version of the ProFlu1 assay, with viral culture and RADTs for RSV (NOW RSV, Binax, Inverness Medical) and for influenza A and B (Directogen A1B, BD Diagnostics) [10] .
The specificities of all methods were found to be .99%.
The sensitivities for detection of influenza were 59% for Directogen A 1 B, 54% for viral culture, and 98% for ProFlu-1; the sensitivities for the detection of RSV were 82% for RSV NOW, 57% for viral culture, and 95% for ProFlu-1.
In another study, LeGoff et al evaluated the performance of ProFlu-1 in 353 pediatric nasopharyngeal specimens [40] .
Results were compared with DFA testing and viral culture.
The sensitivities and specificities of ProFlu-1 ranged from 97% to 100%, and ProFlu-1 detected viruses in 9% of the samples that had negative results by conventional methods.
In response to the 2009 influenza A H1N1 outbreak, an influenza A subtyping assay (ProFlu-ST) was developed by Prodesse and received Emergency Use Authorization (EUA) from the FDA in July 2009 (see EUA section).
ProFlu-ST is a qualitative multiplex real-time RT-PCR assay that targets the nucleoprotein gene of 2009 influenza A H1N1, the specific hemagglutinin genes of seasonal influenza A/H1 and seasonal influenza A/H3, and an internal control (MS2 phage).
The identification of 2009 influenza A H1N1 is aided by an algorithm that relies on seasonal influenza A/H1 virus and seasonal influenza A/H3 virus results in nasopharyngeal swab specimens from patients who receive a diagnosis of influenza A by a currently available FDA-cleared or authorized device.
This assay is intended for use in only CLIA high-complexity laboratories.
The performance of the assay was evaluated retrospectively using nasopharyngeal swab specimens that were previously tested with either the CDC rRT-PCR Flu Panel (IVD device) to detect seasonal influenza A/H1 and influenza A/H3 or the CDC rRT-PCR Swine Flu Panel (EUA).
The positive and negative agreements for the detection of seasonal influenza A/H1were 95.8% and 100%, respectively; for seasonal influenza A/H3 were 100% and 100%, respectively; and for 2009 influenza A H1N1 were 96.2% and 100%, respectively [103] .
The Pro hMPV1 assay clinical trial study for FDA clearance evaluated the assay's clinical performance using 1275 nasopharyngeal swab specimens tested by 4 clinical laboratories across the United States.
Using the Luminex RVP assay as the predicate device, the sensitivity of Pro hMPV1 was 94.1% and the specificity was 99.3% [104].
The performance of the ProParaflu1 assay was evaluated during the clinical trials for FDA clearance.
Using 857 nasopharyngeal swab specimens tested by 4 clinical laboratories across the United States [105], the sensitivities and specificities of the assay for the detection of PIV-1 were 88.9% and 99.9%, respectively; for the detection of PIV-2 were 96.3% and 99.8%, respectively, and for the detection of PIV-3 were 97.3% and 99.2%, respectively [105] .
At this time, no additional independent performance data are available.
The benefits of the Gen-Probe/Prodesse assays include ease of use, with approximately 1.5 h of hands-on time for nucleic acid extraction preparation and a 1-step RT-PCR setup.
The overall time to results is 4.5-5.5 h. Multiple SmartCycler instruments can be run simultaneously, with as many units per cycler used as needed, giving flexibility to run sizes.
Because tubes containing amplicons are never opened, the risk of amplicon contamination is minimal.
One limitation of the assays is a maximum of 3 targets plus an internal control that can be detected in a single reaction.
Therefore, a comprehensive viral diagnostic panel requires multiple PCRs.
Multiple PCRs are costly to the laboratory in both technical time and reagent cost.
However, the limited panel size could provide a mix-and-match test menu, allowing clinicians the option of selecting 1 or several panels.
The first-generation Verigene Respiratory Virus Nucleic Acid Test (VRNAT) was cleared by the FDA in May 2009.
This test has been replaced by the automated Verigene VRNAT SP , a CLIA moderately complex test that is intended for the identification of influenza A, influenza B, and RSV (types A and B inclusive) from nasopharyngeal swab specimens placed in viral transport media.
The Verigene System consists of 2 instruments (the fully automated Verigene Processor and the Verigene Reader) and single-use test cartridges (Figure 2 ).
The entire test process only requires 1 user pipetting step, less than 5 min of technical handson time, and a sample-to-result turnaround time of about 3.5 h. The basis of VRNATsp is Nanosphere's proprietary gold nanoparticle hybridization technology [41] .
The gold nanoparticles contain a high density (200) of sequence-specific oligonucleotides with a high affinity for complementary DNA, which allows for very efficient hybridization kinetics.
The clinical sample is pipetted into a single-use extraction tray, which is loaded into the Verigene processing unit.
Chaotropic agents are added to the sample to lyse cells, viral particles, and an internal control (MS2 phage) that is added prior to the extraction step.
The released nucleic acids are then captured on magnetic microparticles (MMPs).
The MMP-bound nucleic acids are washed, and the purified nucleic acids are eluted from the MMPs and transferred to the amplification tray.
A 1-step RT-PCR is performed using primers that target the influenza A matrix gene, the influenza B matrix gene and nonstructural gene, and the RSV L gene and F gene.
The RT-PCR reaction is followed by the primary hybridization step.
During primary hybridization, target DNA is simultaneously hybridized to target-specific capture DNA oligonucleotides arrayed in replicate on a solid substrate (a microarray) and to target-specific mediator DNA oligonucleotides.
After removal of uncaptured target nucleic acids and unhybridized mediator oligonucleotides, the process continues with the secondary hybridization.
Each microarray spot, where an appropriate target is hybridized to capture both an oligonucleotide and a mediator oligonucleotide, is saturated with silver-coated gold nanoparticle probes.
After hybridization, the cartridge is removed from the processor unit and the glass slide (microarray) is separated from the cartridge and inserted into the Verigene Reader.
A light-scattering technique, in which the slide is illuminated internally with light parallel to the slide surface, is used to analyze the results.
Spots where silverenhanced gold nanoparticle probes are present scatter this light, and the light scatter is detected optically and translated into a measurable signal.
The performance characteristics of the first-generation VRNAT assay were established during the clinical trials for IVD device clearance.
VRNAT was compared with viral culture/DFA testing with bidirectional sequencing to resolve discordant results.
Test sensitivities and specificities for the detection of influenza A were 100% and 99.8%, respectively; for influenza B were 100% and 99.1%, respectively; and for RSV were 95.7% and 98.2%, respectively [106] .
Comparison of VRNAT with VRNATsp revealed an overall positive agreement of 97.9% and a negative agreement value of 100% [107] .
The benefits of the VRNATsp System include the scalability of the system (addition of multiple processors and readers), individual sample processing with random access format, minimal hands-on time, and minimal technical expertise required to perform the test.
Because this test is CLIA moderate complexity, trained laboratory technicians could perform the testing.
This test would be well suited for small-to medium-sized laboratories, in particular for laboratories with little or no molecular testing experience.
Limitations of the assay include the single test format that requires a dedicated processor for each sample for 3-3.5 h. Multiple processor units would be needed for larger volume laboratories, and the additional instrumentation would increase costs for the lab, compared with using an instrument that can run multiple tests at a time.
Table 2 were rescinded as of 23 June 2010.
As of July 2010, only 2 of these tests have received FDA approval for use as an IVD device for the diagnosis of 2009 pandemic influenza A H1N1.
The first test approved was a new optimized CDC H1N1 assay that is available in CDC-qualified laboratories.
The second test is the Focus Diagnostics Simplexa Influenza A H1N1 (2009), which is performed using the 3M Integrated Cycler (3M).
The use of FDA-approved/cleared NAATs, in contrast to laboratory-developed tests, has substantial benefits for the laboratory.
Approved tests have undergone extensive analytical and clinical validations during the course of the FDA evaluations.
Therefore, performance parameters are well characterized, and the FDA monitors postapproval performance.
Most laboratories do not have the expertise and resources to perform extensive (Table 3) , thereby saving the laboratory considerable cost and technical time.
The regulatory requirements for ongoing quality assurance monitoring are also fewer for FDA-approved/cleared NAATs than for laboratory-developed tests [42] .
In addition, for regulatory reasons, some health care institutions will only allow the use of FDA-approved/cleared NAATs.
Because the clinical relevance of the assays has been established, the use of FDA-approved/cleared NAATs has a higher chance of reimbursement from both federal and private insurance payors.
However, FDA approval/clearance does not guarantee that these tests will be reimbursed, and, if reimbursed, the actual amount of the reimbursement can vary from state to state and by payor.
Finally, the simple-to-use fully automated molecular platforms, such as the GeneXpert and GeneSTAT, which incorporate all steps of the test process in a single test cartridge and have random-access sample-in result-out reporting, enable laboratories of all sizes to perform rapid, accurate, and sensitive molecular diagnostic testing.
The most important limitation of the current FDA-approved/ cleared NAATs is the lack of tests for the detection of nonviral targets.
In particular, NAATs are needed for the detection of the atypical pneumonia pathogens.
However, the costs of clinical trials for IVD device clearance can sometimes be quite prohibitive (.$10 million, not including the costs of developing and manufacturing the IVD device) and depend on the complexity and clinical relevance of the test and what is required for FDA approval (eg, number of trial sites, number of patients enrolled, clinical indications sought, need for long-term patient follow-up, associated diagnostic procedures [such as biopsy], device evaluations, predicate device comparisons, legal and regulatory components, and so forth).
Manufacturers must consider the potential number of tests that will be purchased and the difficulty of the trials for low-prevalence pathogens before committing the finances and resources to bring such tests through the approval process.
These factors will continue to limit the scope of FDA-approved/cleared NAATs until the approval process can be modified to encourage the submission of NAATs for low-prevalence targets.
The current formats of the NAATs require laboratories to choose between real-time, easier to perform, more rapid assays that have limited targets (generally up to 3) and more highly multiplex tests (.10) that require more hands-on time and technical steps and more time to result reporting.
NAATs with limited targets would require laboratories to run multiple tests if a broader range of target detection is indicated.
Multiple tests would increase both the required technical time and the cost per patient diagnosis.
Conversely, the use of multiple, lower multiplex tests allows for the selection of the most appropriate panels as related to age of the patient, clinical status, underlying disease, state of immune competence, and the suspected virus(es).
Currently in development are modified faster versions of the highly multiplex assays that have been designed to reduce technical hands-on time and the time to results.
Some of the FDA-approved/cleared real-time NAATs do not allow the user to see and evaluate the actual amplification curves.
The inability to see the curves makes it difficult to troubleshoot when problems occur.
Although the user should not be able to alter cutoff values established during the FDA trials, access to all or part of the raw data is desirable.
One caveat for most qualitative NAATs is that they cannot distinguish between live and dead organisms and therefore, depending on the time for nucleic acid clearance, can be limited in monitoring response to therapy.
In addition, with multiple viral infections, determining the relevance of each virus present in the sample is difficult, because residual virus detected may have been from a previous infection and may not be contributing to the current illness.
The development of quantitative assays and evidence of declining viral load may clarify or resolve both of these issues.
Finally, issues relating to billing and reimbursement are substantial.
The lack of specific Current Procedural Terminology (CPT) codes for each of the individual targets requires laboratories to bill for multiple targets using the same generic amplified probe CPT code (87798).
Although it is appropriate to bill for each target present in a multiplex assay, the use of the same CPT code multiple times for a single test can be problematic.
For example, many laboratory or hospital billing systems do not recognize multiple similar CPTs for a single test and will only drop 1 CPT code charge.
Some insurance payors will cover only the charge of the first CPT code, and there are also limitations on how many times per day a similar CPT code can be billed.
As a result, reimbursement for a highly multiplexed assay could be limited to 1 charge, thereby significantly increasing the costs to the laboratory and decreasing test profitability.
Under these circumstances and with pressure to reduce medical costs, many administrators will not approve the implementation of this testing if reimbursement is questionable.
Laboratories need analyte-specific CPT codes, and payors should reimburse for medically relevant NAATs.
The North Shore-Long Island Jewish Health System (NS-LIJHS) Infectious Diseases Molecular Diagnostics Laboratory opened 12 years ago.
We performed 3 tests: human immunodeficiency virus type 1 viral load, AMTD, and Chlamydia trachomatis/ Neisseria gonorrhoeae NAATs, with a total testing volume of 5000 tests per year.
Today, the laboratory performs NAATs for 40 different pathogens (including bacterial, mycobacterial, fungal, parasitic, and viral targets), with a volume of more than 225,000 tests per year, and in space that has tripled in size.
Molecular diagnostics for infectious diseases, along with molecular pathology, are 2 of the fastest growing laboratory departments in the NS-LIJHS.
In addition, as best exemplified by the 2001 anthrax bioterrorism event and the 2009 influenza A H1N1 pandemic, laboratories must be able to respond immediately by rapidly expanding testing capabilities, including sensitive and specific molecular diagnostics [43] .
At the onset of the Queens, New York, area 2009 influenza A H1N1 outbreak, the NS-LIJHS laboratories' respiratory virus testing volume increased from a baseline of 225 tests per day to more than 950 tests per day, within 3 days.
It was clear from the first week of the outbreak that the laboratory urgently needed to switch all influenza testing to molecular methods to obtain sufficient diagnostic sensitivity and to subtype the influenza strains.
Fortunately, an FDA-approved test, the Luminex xTAG RVP test, was available and met our diagnostic requirements [5, 24, 43] .
Laboratories must continue to plan for future pandemic outbreaks and biothreat events.
Therefore, laboratories are constantly under pressure to expand testing and meet the demands of their clinical staff and their diverse patient populations.
To do this, laboratories must provide clinically relevant, high-quality, cost-effective NAATS that are preferably FDA approved/cleared.
As we move forward, we request that the FDA work closely with such organizations as the Infectious Diseases Society of America and the American Society for Microbiology, with laboratory directors, and with IVD device manufacturers so that this goal can be met.
The labor-intensive, complex methods and platforms of yesterday have evolved into simpler, user friendly versions that can be applicable to all health care settings.
We encourage the FDA to allow the submission process to evolve in a similar manner.
No longer are the old ''gold standards'' of culture applicable, and new ways to evaluate these tests must be considered [44] so that accurate performance characteristics can be determined in a cost-effective manner.
The latter is especially true for low-volume but highly relevant assays, such as quantitative NAATs for transplantation monitoring.
In a reasonable and clear regulatory environment, the IVD device manufacturers will succeed in bringing their NAATS through the approval process.
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Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay Principles of the xTAG respiratory viral panel assay (RVP assay) xTAG Respiratory Virus Panel Luminex Molecular Diagnostics Enhanced viral etiological diagnosis of respiratory system infection outbreaks by use of a multitarget nucleic acid amplification assay WI: Gen-Probe/Prodesse Evaluation of the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for detection of influenza A and influenza B viruses and respiratory syncytial viruses in children WI: Gen-Probe/Prodesse WI: Gen-Probe/Prodesse WI: Gen-Probe/Prodesse Verigene Respiratory Virus Nucleic Acid Test IL: Nanosphere IL: Nanosphere Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes Quality assurance in clinical virology Laboratory surge response to pandemic (H1N1) 2009 outbreak Discrepant analysis: how can we test a test?
I sincerely thank the FDA and the Infectious Diseases Society of America for providing a forum for discussion and exchange of ideas relating to this timely and clinically relevant topic.Financial support.
This work was supported by US Department of Defense Award W81XWH-08-1-0477.
Potential conflicts of interest.
C. C. G. is a member of the Scientific Advisory Boards of Gen-Probe/Prodesse and Luminex Molecular Diagnostics.
C. C. G. has received research grants and honoraria from Gen-Probe/Prodesse, Luminex Molecular Diagnostics, and Diagnostic Hybrids and research grants from 3M Molecular Diagnostics and has been a consultant for Nanosphere.
key: cord-326922-bajpr5a2 authors: Watson, C. James; Whitledge, James D.; Siani, Alicia M.; Burns, Michele M. title: Pharmaceutical Compounding: a History, Regulatory Overview, and Systematic Review of Compounding Errors date: 2020-11-02 journal: J Med Toxicol DOI: 10.1007/s13181-020-00814-3 sha: doc_id: 326922 cord_uid: bajpr5a2 INTRODUCTION: Medications are compounded when a formulation of a medication is needed but not commercially available.
Regulatory oversight of compounding is piecemeal and compounding errors have resulted in patient harm.
We review compounding in the United States (US), including a history of compounding, a critique of current regulatory oversight, and a systematic review of compounding errors recorded in the literature.
METHODS: We gathered reports of compounding errors occurring in the US from 1990 to 2020 from PubMed, Embase, several relevant conference abstracts, and the US Food and Drug Administration “Drug Alerts and Statements” repository.
We categorized reports into errors of “contamination,” suprapotency,” and “subpotency.” Errors were also subdivided by whether they resulted in morbidity and mortality.
We reported demographic, medication, and outcome data where available.
RESULTS: We screened 2155 reports and identified 63 errors.
Twenty-one of 63 were errors of concentration, harming 36 patients.
Twenty-seven of 63 were contamination errors, harming 1119 patients.
Fifteen errors did not result in any identified harm.
DISCUSSION: Compounding errors are attributed to contamination or concentration.
Concentration errors predominantly result from compounding a prescription for a single patient, and disproportionately affect children.
Contamination errors largely occur during bulk distribution of compounded medications for parenteral use, and affect more patients.
The burden falls on the government, pharmacy industry, and medical providers to reduce the risk of patient harm caused by compounding errors.
CONCLUSION: In the US, drug compounding is important in ensuring access to vital medications, but has the potential to cause patient harm without adequate safeguards.
In the modern-day United States (US), medications are by-inlarge manufactured in commercial facilities, and this production is regulated and overseen by the US Food and Drug Administration (FDA).
Historically, however, medications were mixed-or compounded-by independent pharmacists for use by individual patients.
While traditional compounding is becoming less prevalent, it still occurs in instances where a particular patient may require a formulation of a medication that is not otherwise available.
Furthermore, a new form of large-scale compounding has become commonplace, whereby pharmacies produce bulk volumes of medications which are not available commercially, and broadly distribute them to healthcare practices and individual patients.
Compounding does not traditionally fall under the purview of FDA oversight, instead being regulated by individual states' boards of pharmacy.
This approach has resulted in a patchwork and oftentimes underfunded regulatory framework, which has subsequently harmed patients [1] [2] [3] [4] .
Morbidity and mortality frequently result either from a compounded medication that is contaminated with bacteria, fungi, or another medication during production, or from an error whereby the concentration of the drug dispensed is not as intended, which can lead to inadvertent over-or underdosing.
Patient harm caused by compounded medications has been the focus of media, medical, and legislative attention in recent years, especially following a multistate, multi-fatality outbreak of fungal meningitis caused by contaminated steroid injections compounded at a pharmacy in Framingham, MA [2, 3, 5, 6] .
This article seeks to provide a comprehensive review of the state of outpatient compounding in the US.
Compounding performed by hospital pharmacies for inpatient use is beyond the scope of this paper.
Much has been written on compounding pharmaceuticals; this paper is an effort to succinctly address the history, purpose, and regulatory framework in a unified location, as well as to perform a systematic review of all US compounding errors over the past 30 years.
To our knowledge, no systematic review of both contamination and non-contamination errors has to this point been undertaken.
We will first explore the definition and modern role of compounding.
Then, we will briefly discuss the modern US history of compounding, with a particular focus on factors influencing the current state of compounding.
Next, we will examine compounding through a legislative and regulatory lens, to better decipher how governmental oversight-or a lack thereof-may contribute to errors in compounding resulting in patient harm.
Understanding the interventions being made on a federal level can help improve the safety of compounding.
Finally, we have performed a systematic review of documented compounding errors and categorized those errors by type and patient outcome.
Whereby, we elucidate just how and with what frequency patients are harmed by compounding errors, with the ultimate aim of identifying potential strategies for reducing these adverse events.
Compounding is defined by the FDA as the combination, mixing, or alteration of drug ingredients to create medications tailored to individual patient needs [7] .
The United States Pharmacopeia (USP), which sets quality standards for drugs, describes compounding as "the preparation, mixing, assembling, altering, packaging, and labeling of a drug … in accordance with a licensed practitioner's prescription …" [8] Put simply, it is the creation of a medication that is not commercially available.
In the US, compounding is performed in both the inpatient hospital setting and in outpatient pharmacies, with a trend in recent decades towards larger scale outpatient production [9] .
As will be discussed later in this paper, compounding may now occur in newly defined "outsourcing facilities," which are designed to compound in bulk; some examples of these facilities include QuVa Pharma and Leiters [10] .
There are many indications for compounding.
Some patients may not tolerate pills and require a compounded liquid drug formulation; examples include young children taking antibiotics, feeding tube-dependent patients, or patients with dysphagia from neurologic compromise such as a stroke [11, 12] .
Patients may be allergic to binding agents, dyes, diluents, or other inactive ingredients in commercially available formulations.
Dietary restrictions, such as a ketogenic diet in pediatric epilepsy patients, may necessitate compounding of sugar-free medications [13] .
Refractory neuropathic pain may benefit from compounded analgesic topical creams containing multiple medications not commercially available in combination; examples include ketamine, baclofen, gabapentin, amitriptyline, bupivacaine, and clonidine [14] .
Painful oral lesions can be treated with "magic mouthwash" and dyspepsia can be treated with a "gastrointestinal (GI) cocktail"; these are terms that actually encompass a range of compounded preparations [15] .
Total parenteral nutrition (TPN) is needed for patients unable to take in sufficient oral nutrition, and numerous chemotherapy regimens must be compounded for cancer treatment [16, 17] .
Healthcare providers may need compounded medications to perform specialized procedures such as intraarticular or intravitreal injections.
In some instances, commercial preparations may be available but expensive, and a compounded equivalent is more affordable [18] .
Drug shortages, a longstanding healthcare problem exacerbated by crises such as the COVID-19 pandemic and the devastation of Puerto Rico by Hurricane Maria, may be addressed by compounding as well [19, 20] .
The FDA has responded to significant shortages during the COVID-19 pandemic by temporarily relaxing restrictions on compounding of commercially available drugs [21, 22] .
When a compounded medication is prescribed or administered, patient safety depends on adherence to Current Good Manufacturing Practices (CGMP), which are outlined in Chapter 795 of the USP for non-sterile preparations and Chapter 797 for sterile preparations.
Appropriateness of the prescription indication, safety, and dosing should be assessed by the pharmacist.
Ingredient quantities must be meticulously calculated, and the source quality of those ingredients assured.
Compounding facilities and equipment must be clean and monitored continuously.
Staff must routinely practice and be assessed for competency in proper hygienic measures.
Sterile preparations, by definition, require a higher level of care to prevent contamination than do non-sterile preparations, including differences in staff training and personal protective equipment (PPE), environment and air quality monitoring, and disinfection.
Compounded sterile preparations are further subdivided into low-, medium-, and high-risk depending upon the quantity of ingredients, number of manipulations required during compounding, and whether nonsterile ingredients requiring subsequent sterilization are incorporated.
Multiple medications must not be simultaneously compounded in the same workspace.
The compounding process must be reproducible such that medication quality is consistent throughout many production cycles.
Finally, prescriptions must be correctly labeled and patients instructed in appropriate use [8, [23] [24] [25] .
Failure to adhere to these standards has the potential to result in patient harm through multiple mechanisms including medication suprapotency, subpotency, contamination, and consumer misuse.
Throughout pre-industrial history, pharmacists played the critical role of admixing various materials to produce a finished therapeutic substance.
This role was, in essence, one of compounding [26, 27] .
However, the industrial revolution and the resultant mass production of pharmaceuticalscoupled with the increasing presence of synthetic proprietary medications-led to a change in pharmacists' primary role.
Instead of compounding, community pharmacists in the early 1900s turned their focus to the dispensing of previously manufactured medications as well as to general retail, including operating the soda fountains which came into vogue with the prohibition of alcoholic beverages.
In fact, by the 1930s, fewer than 1% of pharmacies in the US made a majority of their income from pharmaceutical sales [27] .
The decline in community pharmacy compounding was precipitous through the mid-1900s.
In the 1930s, 75% of prescriptions required some sort of in-pharmacy compounding.
That number fell to 25% by the 1950s, less than 5% by 1960, and to 1% by 1970 [28] .
Interestingly, there was a concurrent increase in the need for hospital pharmacy compounding during the same period; largely due to the advent of chemotherapy, TPN, and cardiac surgery which necessitated the administration of complex cardioplegic regimens.
By the 1980s, these advanced therapeutics began to spill into the outpatient setting, generating a novel home infusion industry for treatments such as TPN, antibiotics, and chemotherapeutics [29] .
As a result, the 1990s and 2000s yielded further diversification within the compounding industry as pharmacies began to compound in bulk.
This development was brought about by expanding home infusion programs, the more frequent outsourcing of hospital compounding to the outpatient setting, and the rise of hormone replacement therapy.
Large volume compounding blurs the line between traditional compounding which has state-based regulatory oversight, and the mass manufacture of pharmaceuticals which falls under wellestablished federal FDA regulations [29] [30] [31] .
The inspiration for this article is a well-documented history of medication errors attributable to pharmaceutical compounding, for which a lack of regulatory oversight persists as a common thread [3, 29, 32, 33] .
The most lethal and infamous of these cases occurred in 2012, when an outbreak of fungal meningitis occurred amongst patients who had received epidural spinal injections.
The outbreak affected 753 patients across 20 states, killing 64 [5, 6, 34, 35] .
Ultimately, the outbreak was linked to a compounding pharmacy, the New England Compounding Center (NECC, located in Framingham, MA).
Amongst other pharmaceuticals, NECC produced injectable sterile methylprednisolone acetate for epidural injections, which it then distributed nationally.
Following the outbreak (hereafter "Framingham"), the FDA determined that the pharmacy had disregarded basic sanitary standards and had not taken corrective measures despite internal knowledge of potential contamination [2, 5, 6, [34] [35] [36] .
As with many compounding pharmacies, NECC operated in a historically murky regulatory space, producing medications in bulk as would a commercial pharmaceutical manufacturer, while only being subjected to reduced state oversight given to compounding pharmacies.
In fact, in the years preceding the outbreak, the FDA had thrice investigated NECC and found sterility violations, but they were unable to enforce any interventions or penalties due to the FDA's contested regulatory jurisdiction [4] .
Both preceding and following Framingham, efforts have been made at the federal level to improve oversight of compounding; these are reviewed in depth later in this article.
Currently, there is incomplete tracking of compounded pharmaceuticals in the US, though they are estimated to comprise 1-3% of all prescriptions [30, 31, 37, 38] .
Ultimately, compounding is highly prevalent, and so clinicians must be familiar with the risks associated with compounded medications as they care for patients who may be suffering from a related adverse event.
Prior to Framingham, modern compounding pharmacies evolved within a regulatory framework that lacked distinct federal or state oversight roles.
In 1938, the Federal Food, Drug, and Cosmetic Act (FDCA) authorized FDA oversight of pharmaceutical manufacturing [39] .
However, because compounders traditionally produced drugs in response to individual prescriptions and on a much smaller scale than conventional drug manufacturers, pharmaceutical compounding developed and remained under the regulatory purview of individual state boards of pharmacy [32, 40] .
Towards the end of the twentieth century, pharmacies began bulk compounding in response to (1) the home infusion industry and (2) hospitals' financial interest in outsourcing compounding from their inpatient pharmacies to the outpatient setting [29] .
Concerned that bulk compounders were self-classifying as pharmacies to avoid the rigorous federal oversight required of drug manufacturers under the FDCA, Congress passed the 1997 Food and Drug Administration Modernization Act (FDAMA) [40] .
FDAMA addressed the changing nature of compounding pharmacies by creating a "safe harbor" exempting pharmacies from the more stringent FDCA regulations so long as compounders refrained from advertising their product and abided by requirements designed to increase drug safety [40, 41] .
Despite Congress's attempt to strengthen oversight of compounding pharmacies, litigation challenging FDAMA tempered the FDA's authority to regulate compounders.
In 2002, a narrowly divided US Supreme Court ruled in Thompson v. Western States Medical Center that the FDAMA advertising prohibition was unconstitutional on First Amendment free speech grounds [42] .
The ensuing regulatory confusion is well described in the literature, and the details are beyond the scope of this review [3, 28, 29, 31, 32, 40] .
For reference, a summary of the relevant legislation and litigation is provided in Fig.
1 .
Decades of regulatory uncertainty culminated in the 2012 Framingham incident, which revived Congressional efforts to address pharmaceutical compounding industry safety concerns.
In response to Framingham, Congress passed and President Barack Obama signed into law the bipartisan-supported Compounding Quality Act (CQA) as part of a broader legislative package (the 2013 Drug Quality and Security Act) [43] .
The CQA delineated state and federal oversight authority by defining two distinct categories of compounding pharmacies.
The first category is traditional compounding pharmacies, or "503A" pharmacies [44] .
503A pharmacies under the CQA may compound only in response to individual prescriptions.
Importantly, 503A pharmacies may not compound bulk medications either in anticipation of receiving prescriptions or with plans to distribute broadly to healthcare facilities [31, 45] .
In exchange for complying with these limitations, 503A pharmacies largely avoid the more burdensome regulations required of drug manufacturers under the FDCA, including adhering to CGMP [31, 37, [46] [47] [48] .
Accordingly, state boards of pharmacy continue to serve as the primary regulators of 503A pharmacies [45] .
The CQA created a second category of compounding pharmacy, called an "outsourcing facility."
[49] Unlike 503A pharmacies, outsourcing facilities voluntarily opt-in to this category by paying the FDA a user fee (approximately $18,298 in FY2020) [50] , and comply with stringent CGMP standards as well as reporting requirements [38, 46] .
Because they submit to more robust FDA oversight, outsourcing facilities are permitted to compound in bulk in advance of receiving a prescription, and may distribute their products across state lines [31, 51] .
Though the FDA enjoys primary regulatory authority over outsourcing facilities, states are not precluded from imposing additional requirements [51] .
Should a compounding pharmacy fail to comply with the 503A criteria or voluntarily register as an outsourcing facility, it is subject to the full breadth of regulations required of drug manufacturers under the FDCA [37] .
The distinctions between 503A pharmacies and outsourcing facilities are illustrated in Fig.
2 .
Following enactment of the CQA, states have taken numerous steps to further develop their respective oversight structures under the new framework.
A majority of states have strengthened regulations empowering state boards of pharmacy to hold 503A pharmacies accountable to higher safety practices, such as requiring conformation with recognized sterile compounding guidelines.
However, state oversight of 503A pharmacies continues to vary, with fewer than half of all states reporting annual inspections of 503A pharmacies in 2018 [52] .
The FDA has similarly adjusted its enforcement priorities [53, 54] .
For example, the CQA permits a 503A pharmacy to distribute no more than 5% of its total prescriptions out of state .
The goal of this provision is to avoid another national outbreak by reducing the likelihood that contaminated drugs cross state lines.
If a given state enters into a MOU with the FDA, 503A pharmacies in that state may distribute a higher percentage of prescriptions (now up to 50%) across state lines in exchange for that state's board of pharmacy agreeing to identify, investigate, and report associated adverse events [ 45] .
Importantly, the MOU standardizes procedures for state boards of pharmacy to report concerns to the FDA and other states; however, the agreement also grants states significant discretion in how states conduct investigations [54] .
In short, states that participate in the MOU, rather than the FDA, will undertake primary responsibility for detecting poor quality or dangerous compounded medications distributed by 503A pharmacies from their state.
The FDA also announced an effort to entice more compounding pharmacies to register as outsourcing facilities by embracing a risk-based approach [45] .
Since enactment of the CQA, far fewer pharmacies have registered as outsourcing facilities than the FDA had expected.
In fact, the FDA anticipated 50 pharmacies to register per year, but only 78 total were registered as of May 2020 (even fewer than the 91 registered in 2016) [38, 51, 56] .
To attract compounding pharmacies-some of which have cited cost of compliance with CGMP as a prohibitively expensive barrier to registering as an outsourcing facility-the FDA plans to reduce CGMP requirements for compounding pharmacies it deems as "lower risk."
[45] Though the FDA published draft guidance in 2018 describing how the agency may tailor CGMP requirements for outsourcing facilities, the FDA has yet to issue final guidance on this matter [53, 57] .
Critics warn that FDA and state efforts to implement the CQA regulatory scheme excludes compounding pharmacies from the decision-making process and may limit patients' access to compounded medications.
For example, the Preserving Patient Access to Compounded Medications Act (H.R.
1959) introduced in the US House of Representatives attempts to address complaints expressed by compounders [58] .
The proposed legislation seeks to ensure that compounders and other interested parties have an opportunity to comment on (and influence) FDA compounding regulations.
Furthermore, the proposed legislation would explicitly allow physicians who engage in in-office sterile compounding, or who otherwise maintain a supply of compounded medications for "office use," to avoid complying (where state law permits) with outsourcing facility regulations [58] .
Meanwhile, the sterility practices of some compounding pharmacies continue to raise alarm: between 2013 and 2018, the FDA issued more than 180 warning letters to compounding pharmacies, resulting in approximately 140 recalls.
As acknowledged by the agency, the FDA's transition to a risk-based approach may assist the agency in more efficiently targeting its limited resources, but it could also increase the likelihood of compounders engaging in unsafe practices that elude regulators [45] .
In sum, the CQA and subsequent state and FDA actions have somewhat clarified oversight roles after Framingham, largely by defining separate 503A pharmacies and outsourcing facilities.
Seven years after its enactment, however, uncertainty regarding the relative strength and consistency of said regulatory framework remains.
We performed a systematic review of compounding errors, including both errors that resulted in patient harm and those that did not, as reported in the academic literature.
We searched the National Center for Biotechnology Information (PubMed; U.S. National Library of Medicine: Bethesda, M a r y l a n d ) a n d E m b a s e ( E l s e v i e r : Amsterdam, The Netherlands) using the following search criteria: "'compounding AND pharmacy' AND 'error,' 'overdos*,' 'toxicol*,' 'infect*,' 'death,' 'outbreak,' 'injur*,' OR 'case report.'"
This search was limited to January 1990 through March 2020.
Additionally, we reviewed abstracts for years 1990-2019 for the following conferences using keyword searches for "compound" and "compounding": American College of Medical Toxicology (ACMT) Annual Scientific Meeting, North American Congress of Clinical Toxicology (NACCT), American College of Emergency Physicians (ACEP) Scientific Assembly, Society for Academic Emergency Medicine (SAEM) Annual Meeting, American Academy of Pediatrics (AAP) National Conference & Exhibition, and the Pediatric Academic Societies (PAS) Meeting.
We also reviewed the FDA's online "Drug Alerts and Statements" repository for alerts regarding compounding pharmacies' failures in sterility and potency standards.
Authors CJW and JDW screened reports by title and, when necessary for clarification, by abstract.
Under manual review, articles were excluded if they were obviously irrelevant, consisted of research comparing samples of compounded and commercial pharmaceuticals, were in a non-English language, regarded medications compounded outside of the US, were redundant with another included report, represented misuse of properly compounded medications, regarded veterinary patients, were compounded by an inpatient hospital pharmacy (including chemotherapeutics and parenteral nutrition), were published prior to 1990, or if the report lacked sufficient information to provide substantive value.
Redundant reports of the same error were included for analysis only once, but efforts were made to reference all identified reporting sources.
For included reports, CJW and JDW extracted information including date, type of error, cause of error, number of patients affected, age of patients affected, and clinical course of patients affected.
Incomplete data was acknowledged and by-inlarge was not grounds for exclusion from the study.
We categorized errors under the conceptual framework described by Sarah Sellers, PharmD, MPH, former board member for the FDA's Advisory Committee on Pharmacy Compounding, in testimony to the US Senate Committee on Health, Education, Labor, and Pensions, namely, that "suprapotency," "subpotency," and "contamination" are the primary risks associated with pharmaceutical compounding [59] .
We further broke down "contamination" into subgroups of "microbiologic contamination" for cases of bacterial, viral, or fungal contamination and "toxic contamination" for noninfectious contaminants.
When available, we documented patient age and outcome, route of administration, and medication-in-question, so as to better characterize the types of medications, errors, and patients most associated with adverse events.
We referenced and applied the principles for authoring review articles delineated within the Journal of Medical Toxicology when feasible and appropriate during the review process [60] .
Our search terms identified 1058 potential articles in PubMed and 721 potential articles in Embase.
The review of conference abstracts yielded additional potential cases as follows: [61, 62] .
In total, a total of 2155 articles, statements, and reports were identified and underwent our manual review (performed by CJW and JDW).
After the application of our exclusion criteria, a total of 63 errors were included.
These 63 errors are documented as harming 1155 patients.
When broken down by type, contamination accounted for 27 errors adversely affecting 1119 patients (Appendix Table 3 ) and errors in concentration accounted for 21 events adversely affecting 36 patients (Appendix Table 4 ).
There were 15 reports of identified compounding errors which potentially exposed innumerable patients but did not end up causing any known harm; these were predominantly errors of contamination (Appendix Table 5 ).
The number of patients exposed to potential harm cannot be calculated based on the available data, but reaches at least the several thousands (Framingham alone exposed 13,534 patients with 753 documented instances of patient harm).
Table 1 is a summary of the 27 included contamination errors.
With 1119 patients over 27 errors, the median number of patients affected per error is 8.
The mean number of patients affected per error is 41; however, by excluding Framingham, that number is 14.
With 81 deaths over 27 errors, the mean number of fatalities per error is 3; however, excluding Framingham drops that number to less than 1 (0.65).
The median number of deaths per error is 0.
Five out of the 27 contamination errors were from intraarticular (including epidural) steroids, and eight of 27 were from medications injected intravitreally.
A total of 25 of the 27 errors were from medications administered parenterally, in healthcare settings.
Three of 27 were from toxic contamination rather than microbiologic contamination.
Interestingly, six of 27 errors with documented adverse outcomes occurred following the CQA.
Table 2 is a summary of the 21 included sub-and supratherapeutic errors.
One report describes a subtherapeutic error affecting 9 pediatric patients who were on posttransplant immunosuppression with tacrolimus.
The remaining 20 reports involved errors of supratherapeutic drug concentrations; they affected a total of 27 patients, of which 14 (52%) were pediatric.
Of the 36 total patients affected by concentration errors, 23 (64%) were pediatric and 3 (8%) were over the age of 65 years.
Three patients died, all of whom received supratherapeutic intravenous colchicine at an alternative medicine infusion clinic for chronic back pain [91] .
Appended to this article are Appendix Tables 3, 4 , 5, which respectively catalog all contamination errors causing patient harm, all sub-and suprapotency errors causing patient harm, and all potential compounding errors identified and rectified before patient harm occurred.
Of the 15 potential errors identified before patient harm occurred, 13 came after the enactment of the CQA.
In this study, we separated compounding errors into the categories of contamination, suprapotency, and subpotency.
We found that medications with contamination errors are frequently (1) bulkproduced and distributed, (2) used parenterally, and (3) administered by physicians.
Because of their parenteral administration, medications contaminated with otherwise benign environmental flora are able to disseminate throughout the body and cause the devastating outcomes documented here.
Furthermore, because contamination errors are often associated with larger-even multistate-distribution networks, the reach of their impact is large.
Framingham was the archetypal contamination error.
It woke much of the medical and lay communities to the potential dangers of compounding.
It inspired the federal government to enact the CQA in 2013 and create an entirely new form of compounding facility-the outsourcing facility-to attempt to regulate the subsection of pharmacies who were bulk-compounding medications not available (or not available cheaply) through commercial channels, and who exported [45] Certainly, contamination has persisted despite the CQA and the FDA's efforts to oversee outsourcing facilities.
Given this ongoing concern, the medical community must bear some of the responsibility for reducing the number of medications manufactured in substandard environments.
It should be the expected standard for healthcare practices to purchase exclusively from compounding pharmacies strictly adherent to CGMP standards and formally approved as outsourcing facilities by the FDA.
Leading expert Outterson referenced the potential for this approach in 2014 [46] , and it is unclear how purchasers have responded.
While these policies may be more expensive; the physical, ethical, and even financial [92] consequences of purchasing compounded medications from organizations not sufficiently invested in safety are clearly documented here.
Subpotency and suprapotency can be considered as the single category of errors of concentration, as the sources and scope of concentration errors are largely similar.
Our findings demonstrate that subpotency is largely not a reportable issue, but that does not mean it is not a danger.
As an example, beyond the cited series of subtherapeutic tacrolimus concentrations [93] , another case series (excluded for location outside the US) identifies dozens of patients who received subtherapeutic chemotherapy treatments [94] .
These subtherapeutic errors are difficult to capture.
Identification must be done during routine serum testing, as occurred with the tacrolimus series; or on the supply side, as occurred with the chemotherapy series.
When considering subtherapeutic and supratherapeutic errors together as errors of concentration, we found a somewhat different pattern than that which we identified amongst errors of contamination.
The concentration errors we were able to identify, with a few notable exceptions [95, 96] , were caused by traditional compounding pharmacies.
These pharmacies, labeled as 503A pharmacies under the CQA, are limited in their scope to producing compounded medications only after an individual prescription is in-hand.
Per the CQA, 503A pharmacies are still solely regulated by state boards of pharmacy, meaning that oversight is patchwork across the US.
Many concentration errors are of orders of magnitude, suggesting that simple mathematical and measurement mistakes are to blame.
In addition to hoping that states will implement greater oversight of these 503A pharmacies, we call on the pharmacy industry to emphasize and standardize compounding training amongst its students and even consider a mandatory credential before allowing a pharmacist or pharmacy technician to compound a medication [26, [97] [98] [99] .
It is worth noting that 4-aminopyridine and liothyronine are fairly uncommon medications, however they accounted for a large number of compounding concentration errors.
There is nothing particularly special about these medications which make them prone to concentration errors, except for the fact that they are not readily commercially available, and so they must be compounded.
The prescribers of these medications need to carefully consider the benefits and risks of prescribing a treatment which requires compounding; especially when the risks are so great (status epilepticus with 4-aminopyridine and thyrotoxicosis with liothyronine).
Liothyronine, in particular, has had its clinical utility recently questioned.
For example, the National Health Service (UK) has recently called on general practitioners to stop prescribing liothyronine without specialist consultation, as most patients benefit equally from commercially prepared levothyroxine [100].
Given the risks of inadvertent overdose due to compounding errors, providers must consider commercially available alternatives whenever able.
In fact, it has been questioned w h e t h e r p r o v i d e r s w h o k n o w i n g l y p r e s c r i b e a compounded medication despite commercially available alternatives might be legally liable for any harm resulting from compounding errors [101] .
At the very least, it is incumbent on prescribers as well as pharmacists to educate their patients on the risks of taking a compounded medication-both from errors in concentration and contamination-and to instruct them on when to present to a healthcare provider.
Additionally, practitioners must be aware of their patients' medication lists, and consider a possible compounding error as a cause of medical illness.
Notably, we found that the majority of concentration errors were made in pediatric and geriatric patients, vulnerable populations who are already at increased risk of providers failing to diagnose toxicity from prescription medications.
In 2020 and beyond, we anticipate the demand for compounding to only increase.
The number of novel therapeutics continues to rise rapidly, as do their approved routes of administration.
The anti-angiogenesis medication bevacizumab is a classic example; it is commercially manufactured but is frequently compounded into smaller aliquots for intravitreal administration.
As we have seen, this process has unfortunately resulted in multiple outbreaks of endophthalmitis [72] [73] [74] [75] [76] .
Furthermore, regional and global disasters have recently resulted in significant pharmaceutical supply chain issues.
Examples of this phenomenon include Hurricane Maria's impact on Puerto Rican manufacturers in 2017 and the COVID-19 pandemic [120] [121] [122] [123] .
These disruptions place increased demand on alternative means of supply, including via pharmaceutical compounding.
In fact, the COVID-19 pandemic and its associated drug shortages has already resulted in the loosening of FDA restrictions, including allowing outsourcing facilities to compound copies of commercially available drugs for hospital use [21] .
Our study has its limitations.
While we made every effort to capture published cases of compounding errors, it is possible that our search criteria missed some cases that would have impacted our analyses.
While we also strove to review less-traditional sources, including conference abstracts and FDA alerts, we are not free of publication bias and are at risk for having excluded compounding errors not associated with adverse events, or with very small numbers of patients affected.
Similarly, it must be noted that compounding errors can only be identified following adverse events, laboratory screening, or industry or governmental report.
Even once identified, we were dependent on the publication of the error in order to capture it here.
As such, we are likely underreporting the frequency with which compounding errors occur.
Compounding is more relevant than ever.
Appreciating that the need for compounding is unlikely to diminish in the near future, we can only re-emphasize the critical nature of our recommendations for the federal and state governments to fully fund the oversight of outsourcing facilities, for healthcare practices to refuse medications compounded without strict adherence to CGMP and FDA regulations, for pharmacy schools to expand compounding training and certification, and for physicians to think critically about the risks of prescribing medications that are not commercially produced.
Conversely, we must remain aware that compounding pharmacies frequently provide an essential service and poorly calibrated regulations may contribute to issues of access.
Ultimately, medical providers must remain vigilant, especially when caring for members of vulnerable populations, and consider the possibility that a new-onset illness may very well be the result of a compounding error.
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108th Cong, 1st Sess (2003) (testimony of Sarah Sellers, PharmD, MPH, executive director Writing an effective review article Description of outbreaks of health-care-associated infections related to compounding pharmacies, 2000-12 compounding pharmacy-related outbreaks, 2001-2013: public health and patient safety lessons learned Outbreak of Serratia marcescens infections following injection of betamethasone compounded at a community pharmacy Exophiala infection from contaminated injectable steroids prepared by a compounding pharmacy -United States North Carolina Department of Health and Human Services Estimated deaths and illnesses averted during fungal meningitis outbreak associated with contaminated steroid injections Multistate investigation of suspected infections following steroid injections Neurologic complications including paralysis after a medication error involving implanted intrathecal catheters Sphingomonas paucimobilis bloodstream infections associated with contaminated intravenous fentanyl Outbreak of bacteremia due to Burkholderia contaminans linked to intravenous fentanyl from an institutional compounding pharmacy An outbreak of postoperative gram-negative bacterial endophthalmitis associated with contaminated trypan blue ophthalmic solution Eye opening: are compounded drugs causing harm?
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United States Food and Drug Administration Life-threatening sepsis caused by Burkholderia cepacia from contaminated intravenous flush solutions prepared by a compounding pharmacy in another state In the matter of Central Admixture Pharmacy Services, Inc. -Summary Suspension.
Maryland State Board of Pharmacy Outbreak of Systemic Inflammatory Response Syndrome Linked to a Compounding Pharmacy -Virginia Multistate outbreak of Pseudomonas fluorescens bloodstream infection after exposure to contaminated heparinized saline flush prepared by a compounding pharmacy A multistate outbreak of Serratia marcescens bloodstream infection associated with contaminated intravenous magnesium sulfate from a compounding pharmacy Outbreak of Serratia marcescens bloodstream infections in patients receiving parenteral nutrition prepared by a compounding pharmacy Compounding: inspections, recalls, and other actions against NuVision Pharmacy United States Food and Drug Administration Compounding: inspections, recalls, and other actions against Specialty Compounding, LLC; Cedar Park, TX.
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Time for compounding certification?
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The New York Times update on recovery efforts in Puerto Rico, and continued efforts to mitigate IV saline and amino acid drug shortages.
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United States Food and Drug Administration FDA alerts health care professionals not to use sterile drugs from Downing Labs (Aka NuVision Pharmacy) FDA announces Medistat RX's nationwide voluntary recall of sterile drug products.
United States Food and Drug Administration FDA alerts health care professionals not to use sterile drug products from Qualgen.
United States Food and Drug Administration United States Food and Drug Administration FDA alerts health care professionals and patients not to use sterile drug products from Vital Rx, Dba Atlantic Pharmacy and Compounding.
United States Food and Drug Administration FDA alerts health care professionals to voluntary nationwide recall of all sterile products from Coastal Meds.
United States Food and Drug Administration FDA announces Ranier's Rx Laboratory's voluntary recall of all sterile compounded drugs.
United States Food and Drug Administration FDA alerts health care professionals and patients not to use sterile drug products from Pharm D Solutions.
U.S. Food & Drug Administration FDA alerts health care professionals and patients not to use drug products intended to be sterile from Promise Pharmacy.
United States Food and Drug Administration FDA alerts patients and healthcare professionals to Infusion Options' voluntary recall due to quality issues.
United States Food and Drug Administration Dba AmEx Pharmacy, voluntary recall of all sterile compounded drugs.
United States Food and Drug Administration Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
key: cord-352579-ndcbmgfj authors: Takahashi, Takuto; Luzum, Jasmine A.; Nicol, Melanie R.; Jacobson, Pamala A. title: Pharmacogenomics of COVID-19 therapies date: 2020-08-18 journal: NPJ Genom Med DOI: 10.1038/s41525-020-00143-y sha: doc_id: 352579 cord_uid: ndcbmgfj A new global pandemic of coronavirus disease 2019 (COVID-19) has resulted in high mortality and morbidity.
Currently numerous drugs are under expedited investigations without well-established safety or efficacy data.
Pharmacogenomics may allow individualization of these drugs thereby improving efficacy and safety.
In this review, we summarized the pharmacogenomic literature available for COVID-19 drug therapies including hydroxychloroquine, chloroquine, azithromycin, remdesivir, favipiravir, ribavirin, lopinavir/ritonavir, darunavir/cobicistat, interferon beta-1b, tocilizumab, ruxolitinib, baricitinib, and corticosteroids.
We searched PubMed, reviewed the Pharmacogenomics Knowledgebase (PharmGKB(®)) website, Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines, the U.S. Food and Drug Administration (FDA) pharmacogenomics information in the product labeling, and the FDA pharmacogenomics association table.
We found several drug-gene variant pairs that may alter the pharmacokinetics of hydroxychloroquine/chloroquine (CYP2C8, CYP2D6, SLCO1A2, and SLCO1B1); azithromycin (ABCB1); ribavirin (SLC29A1, SLC28A2, and SLC28A3); and lopinavir/ritonavir (SLCO1B1, ABCC2, CYP3A).
We also identified other variants, that are associated with adverse effects, most notable in hydroxychloroquine/chloroquine (G6PD; hemolysis), ribavirin (ITPA; hemolysis), and interferon β -1b (IRF6; liver toxicity).
We also describe the complexity of the risk for QT prolongation in this setting because of additive effects of combining more than one QT-prolonging drug (i.e., hydroxychloroquine/chloroquine and azithromycin), increased concentrations of the drugs due to genetic variants, along with the risk of also combining therapy with potent inhibitors.
In conclusion, although direct evidence in COVID-19 patients is lacking, we identified potential actionable genetic markers in COVID-19 therapies.
Clinical studies in COVID-19 patients are deemed warranted to assess potential roles of these markers.
Since the report of the first cluster infections in Wuhan, a city in China in December 2019, Coronavirus disease 2019 (COVID19) has caused an unprecedented global pandemic and healthcare crisis with high mortality and morbidity.
In an urgent attempt to mitigate its devastating catastrophe, many drugs without established efficacy have been used in patients either as an offlabel/compassionate use or as a clinical trial.
Under these extenuating circumstances these agents have been used without good evidence of efficacy and/or extent of toxicities.
There is also no or limited data on the pharmacogenomics of these agents, and genomic determinants are important factors in efficacy and/or toxicity of many medications.
Pharmacogenomics may help clinicians to choose proper first-line agents and initial dosing that would be most likely achieve adequate drug exposure among critically ill patients; those who cannot afford a failure of ineffective therapy.
It is also important to minimize the risks of toxicity because COVID-19 particularly affects those with comorbidities on other drug therapies 1 .
Therefore the purpose of this review was to summarize the pharmacogenomic literature and clinical recommendations available for COVID-19 candidate drug therapies.
In selection of drugs of interest, we reviewed the following sources: guidelines by the Infectious Disease Society of America 2 and the National Institute of Health 3 .
We also reviewed interventional clinical trials for COVID-19 in ClinicalTrials.gov registered as of June 4, 2020 (searched by a term "COVID" and selected the study type "interventional [clinical trials]").
We excluded products derived of human plasma (e.g., convalescent plasma, immunoglobulin).
Our search was limited to online sources published in English.
As a result, we included potential antiviral or immune-based therapy in the following eight categories for the present review: hydroxychloroquine/chloroquine, azithromycin, RNA polymerase inhibitors, anti-retrovirus agents, interferon β-1b (IFN-β1b), IL-6/IL-1 antagonists, Janus kinase inhibitors, and corticosteroids.
We then reviewed relevant literature by searching PubMed with the terms including but not limited to "pharmacogenomics", "pharmacogenetics", "polymorphism", and "pharmacokinetics" in combination with each drug name.
We also reviewed the Pharmacogenomics Knowledgebase (PharmGKB ® ) website 4 , Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines 5 , the U.S. Food and Drug Administration (FDA) pharmacogenomics information in the product labeling 6 , and the FDA pharmacogenomics association table 7 .
None of the reviewed drugs had applicable CPIC guidelines.
Table 1 summarizes the drugs described in this review and the PharmGKB and FDA pharmacogenomic information.
Hydroxychloroquine and chloroquine The first antiviral drugs officially indicated for COVID-19 in the U.S. were hydroxychloroquine and chloroquine as an FDA Emergency Use Authorization issued on March 28, 2020 8 .
This was later revoked on June 15, 2020 based on new data suggesting that the drug's potential benefits may not outweigh its known and potential risks 9 been used for malaria and autoimmune disorders for decades.
It is thought to increase pH of phagolysosome and, thereby, interrupts virus fusion, and it also prevents binding of the virus to cell surface receptors 10 .
The immunomodulatory effects of these drugs may also play a role in managing the cytokine storm associated with advanced COVID-19 disease.
Concerns around the heart rhythm problems of these agents for treatment of COVID-19 has been announced from the FDA 11 .
This is corroborated by a large retrospective study of hospitalized patients with COVID-19 in New York metropolitan region; those treated with hydroxychloroquine and azithromycin had a higher incidence of cardiac arrest and possible higher mortality 12 .
The NIH guidelines currently do not recommend the use of hydroxychloroquine or chloroquine 3 .
Although the role of these drugs in COVID-19 may be limited, biomarkers for toxicity may potentially be valuable.
Hydroxychloroquine and chloroquine are metabolized via cytochrome P450 (CYP) enzymes including CYP2C8, CYP3A4, and CYP2D6 13 .
These drugs are substrates of organic anion transporting polypeptides (OATP), influx cellular membrane transporters encoded by SLCO 14 .
The genes involved in the pharmacokinetics of hydroxychloroquine and chloroquine are considered "very important pharmacogenes (VIPs)" by the PharmGKB, genes that are particularly important in the field of pharmacogenomics.
A study in 194 patients with systemic lupus erythematosus showed that metabolism ratio of the active metabolite of hydroxychloroquine to its parent drug was increased by~20% in those carrying variants in CYP2D6 (rs1135840: CC vs. GG was 0.90 vs. 0.69, respectively, p < 0.01) 15 .
In a cohort of 164 malariainfected patients, low-activity alleles of CYP2C8 (i.e., *2, *3, and *4) were associated with worse reduction of gametocytemia than wild-type alleles 1 day after chloroquine/primaquine treatment (−2.21 vs. −11.18 gametocytes/μL, p = 0.007, respectively) 16 .
In the same cohort, a subsequent analysis showed that variant alleles of SLCO1A2 and SLCO1B1 had lower gametocytemia clearance than the wild-type alleles after adjusting for CYP2C8 (p = 0.018 and 0.024, respectively) 14 .
Hydroxychloroquine and chloroquine are relatively safe drugs when used for malaria and autoimmune disease, although there are several toxicities of importance.
Individuals who are glucose-6phosphate dehydrogenase (G6PD) deficient are considered at higher risk of hemolytic anemia upon exposure to hydroxychloroquine or chloroquine.
However this has not been consistently shown.
No individuals developed hemolytic anemia among a group of 74 patients with G6PD deficiency who received a 3-day course of chloroquine with methylene blue, nor another group of 11 patients who received a total cumulative hydroxychloroquine exposure of 700 person-months 17, 18 .
Another well-recognized adverse effect of hydroxychloroquine and chloroquine is retinopathy, which occurs more frequently with long term use (in years) and higher doses.
Retinopathy occurred less frequently in those with the minor allele of ABCA4 c.5814A>G (OR: 0.01, 95% CI: 0.00-0.27, adjusted for clinical factors including treatment duration and dose) 19 .
Although data on the functional analysis of this variant is limited, mutations in ABCA4 are associated with various retinal diseases such as Stargardt disease, which is phenotypically similar to chloroquine-induced retinopathy 20 .
The average treatment duration in the study was approximately 11 years, thus the effects of ABCA4 c.5814A>G in short-term use of hydroxychloroquine or chloroquine for COVID-19 treatment would likely to be small.
Of note, hydroxychloroquine is also a CYP2D6 inhibitor, which may reduce CYP2D6 activity.
This is particularly of concern when other CYP2D6 substrates that also prolong QT interval, such as ondansetron and haloperidol, are used concomitantly with hydroxychloroquine and chloroquine since these combinations may significantly reduce their metabolism which may further potentiate QT prolongation.
Azithromycin is a macrolide antibacterial agent with antiinflammatory properties.
A recent observational study showed that azithromycin, when used in combination with hydroxychloroquine, may be more effective than hydroxychloroquine alone for COVID-19 21 .
Although macrolide agents are associated with numerous drug-drug interactions, azithromycin is not a significant substrate of CYP3A4, SLCO1B1, or SLCO1B3, and therefore has fewer interactions than erythromycin or clarithromycin 22 .
The pharmacokinetics of azithromycin are, however, influenced by the activity of P-glycoprotein transporter encoded by ABCB1.
Genetic variation in ABCB1 showed up to a 2-fold lower peak azithromycin concentrations in 20 healthy volunteers after a single dose (rs2032582TT/rs1045642TT vs. rs2032582GG/rs1045642CC was 468.0 vs. 911.2 ng/ml, respectively, p = 0.013) 23 .
Higher systemic exposure to azithromycin is of particular concern when it is combined with hydroxychloroquine or chloroquine because of their additive effects on QT prolongation which may result in fatal arrhythmias.
RNA polymerase inhibitors (remdesivir, ribavirin, and favipiravir) Nucleotide analogs, including remdesivir, ribavirin and favipiravir, inhibit viral RNA polymerase after metabolism to their active forms by intracellular enzymes.
Remdesivir became available for severe COVID-19 in the U.S. via FDA Emergency Use Authorization on May 1, 2020 24 , following two randomized, double-blinded, placebo-controlled trials suggesting potential benefits outweighing the known and potential risks 25, 26 .
Although no pharmacogenomic data of remdesivir are available, in vitro studies suggest that it is a substrate for drug metabolizing enzymes CYP2C8, CYP2D6, and CYP3A4, and is a substrate for OATP1B1 and Pglycoprotein transporters 27 .
Thus, known variants of these genes could theoretically affect the pharmacokinetics of remdesivir [28] [29] [30] .
All of these genes are considered VIPs by PharmGKB.
Ribavirin is indicated for hepatitis C virus infection and is also under investigation for COVID-19.
Genetic polymorphisms in influx cellular transporters of ribavirin result in up to 30% variability in the trough concentration; troughs were significantly higher in those with SLC29A1 variants (homozygous for the variants 2070 ng/ml vs. wild-type 1837 ng/ml; p = 0.02), while significantly lower in those with SLC28A2 variants (homozygous for the variants 1595 ng/ml vs. heterozygous 1933 ng/ml vs. homozygous wildtype 2229 ng/ml; p = 0.04) and SLC28A3 variants (homozygous 2294 ng/ml vs. heterozygous 1813 ng/ml; p = 0.01) 31 .
It is wellrecognized that various ITPA (inosine triphosphatase) variants have protective effects against hemolytic anemia, which is the most common and dose-limiting adverse effect of ribavirin 32 .
Decreased ITPA activity in red blood cells leads to accumulation of inosine triphosphate and protects against ribavirin-induced hemolysis.
In a meta-analysis of 20 studies, hemoglobin decline was associated with wild-type alleles of ITPA; rs1127354 CC (OR: 12.8, 95% CI: 7.4-22.1), rs7270101 AA (OR: 3.4, 95% CI: 2.1-5.6), and rs6051702 AA (OR: 4.4, 95% CI: 2.8-7.0) 33 .
A model incorporating ITPA genotypes and clinical factors was predictive of the degree of ribavirin-related hemoglobin decrease 34 .
Of note, hemolytic anemia was also reported from a short-term use of ribavirin for respiratory virus infection 35 .
In contrast, the ITPA variants in rs6139030 were identified to be a risk of thrombocytopenia in a genome-wide association study among 303 patients with hepatitis C who received ribavirin and peg-interferon (OR: 3.9, 95% CI: 2.8-5.5, p = 1.33 ×10 -15 ) 36 .
Favipiravir was developed and approved in Japan in 2014 exclusively for a resistant, novel influenza pandemic and is now under investigation for COVID-19.
Although no published studies specifically have addressed its pharmacogenomics, it is metabolized by aldehyde oxidase and partly via xanthine oxidase 37 .
Notably, variants of aldehyde oxidase are associated with pharmacodynamic outcomes in other drugs which are substrates of aldehyde oxidase such as azathioprine or allopurinol 38 .
Anti-retrovirus agents (lopinavir/ritonavir, darunavir/cobicistat) Lopinavir is a viral protease inhibitor that is primarily used in the treatment of human immunodeficiency virus (HIV), but it has also been used in COVID-19.
HIV-protease inhibitors including lopinavir/ritonavir are currently not recommended for COVID-19 because of lack of efficacy in a small randomized controlled trial and a concern of inadequate drug exposure relative to that required for SARS-CoV-2 inhibition 3 .
Ritonavir inhibits the inactivation of lopinavir via the CYP3A4 pathway and boosts the concentrations of lopinavir and is marketed as a combination product.
Besides CYP3A, several pathways are involved in the pharmacokinetics of lopinavir, including other CYP enzymes and membrane drug transporters.
A pharmacogenomic analysis of lopinavir/ritonavir, including 1380 variants in 638 HIV-infected Caucasians, identified four significant variants.
Clearance of lopinavir in a population PK model was higher in individuals with SLCO1B1*4/*4 and lower in individuals with two or more variant alleles of SLCO1B1*5, ABCC2 or a CYP3A tag compared to the reference group (12.6 vs. 3.9 vs. 5.4 l/h, respectively, p < 0.01) 39 .
Another genetic association study explored 290 variants for their effects on lopinavir/ritonavir related toxicity among 104 Caucasian patients with HIV; variants in the CETP, MCP-1, ABCC2, LEP, and SLCO1B3 genes were associated with dyslipidemia and hyperbilirubinemia, and a variant in IL-6 was associated with diarrhea (all p < 0.01) 40 .
Darunavir, also a protease inhibitor used in HIV treatment, is a substrate of CYP3A4 that is used simultaneously with a CYP3A4 inhibitor, cobicistat, in a clinical trial for COVID-19 2 .
Several haplotypes in CYP3A4 have been discovered that affect the activity and/or expression of CYP3A4 30 .
Those haplotypes could hypothetically affect concentrations of these drugs, but they have yet to be studied specifically with darunavir.
Although there is no direct evidence that darunavir is a substrate for SLCO3A1, a 12% significantly lower darunavir clearance was observed in carriers of an SLCO3A1 variant (p < 0.05) 41 .
A family of IFNs, particularly IFN-β1b, has showed efficacy against SARS-and/or MERS-coronaviruses and are currently being investigated for COVID-19 either alone or in combination with other therapy (e.g., lopinavir/ritonavir) 2 .
As it is commonly seen in other biologic drugs, pharmacogenomics determinants are not well-delineated for IFN-β1b.
In contrast, reduced efficacy and increased adverse effects secondary to immunogenicity is a specific concern among biologics.
In a cohort of Swedish patients with multiple sclerosis who received IFN-β1b, the risk of biologically relevant neutralizing antibody development was higher in patients with the HLA-DRB1*04 allele (OR: 3.53, 95% CI: 1.64-7.61) and lower with HLA-DRB1*15 (OR: 0.33, 95% CI: 0.16-0.71) 42 .
In a two-stage genome-wide association study among 56 cases and 126 controls of IFN-β1b-treated patients with multiple sclerosis, a higher risk of drug-induced liver injury was identified (p = 2.3 × 10 −8 , OR: 8.3, 95% CI: 3.6-19.2) in patients with variants of IRF6, which encodes for an interferon regulatory factor and is involved in promotion of liver damage 43 .
The results were confirmed in an independent cohort of multiple sclerosis patients for an association with increased peak levels of aspartate aminotransferase (p = 7.6 × 10 −5 ) and alkaline phosphatase (p = 4.9 × 10 −4 ) 43 .
IL-6 and IL-1 antagonists (tocilizumab, sarilumab, siltuximab, anakinra) Severe COVID-19 is associated with a cytokine-release syndrome with elevated interleukin-6 (IL-6) 44 .
Tocilizumab, an inhibitor of the IL-6 receptor, is commonly used for rheumatoid arthritis (RA) and cytokine-release syndrome induced by chimeric antigen receptor-T cell therapy, and now it is under investigation for COVID-19.
Although several genetic biomarkers have been reported in the efficacy of tocilizumab in RA, including FCGR3A, IL6R, CD69, GALNT18 [45] [46] [47] , potential translation of these data to COVID-19 is highly speculative.
No studies have addressed pharmacogenomics of tocilizumab in patients with cytokine-release syndrome, which is similar to the physiology in COVID-19.
The only genetic variants potentially involving tocilizumab's pharmacokinetics are in the FCGR3A gene, where differences in efficacy is postulated to be due to changes in systemic exposure.
In 87 patients with RA treated with tocilizumab, FCGR3A rs396991TT genotype showed higher response at 12 months (vs. GT; OR: 5.1; 95% CI: 1.2-21.3; p = 0.03).
This variant may affect the affinity of the Fc fragment of IgG receptor to tocilizumab and alter its systemic clearance 45 .
Polymorphisms of IL6R are considered to affect intracellular signaling pathway of IL-6 receptor bound to tocilizumab, which may also be applicable to other conditions with upregulated IL-6 pathway 46 .
In contrast, variants in CD69 and GALNT18 are thought to have limited direct effects on tocilizumab.
Variants in those genes are more likely to affect the downstream signaling pathways of the immune system in RA patients, which may limit generalization to non-RA patients 47 .
At this point there is limited evidence that pharmacogenomic biomarkers would be helpful in determining response to tocilizumab therapy in COVID-19.
No relevant pharmacogenomic data is reported in other IL-6 or IL-1 antagonists (i.e., sarilumab, siltuximab, anakinra).
A group of Janus kinase inhibitors is another potentially effective immunomodulator for COVID-19 that is currently in clinical trials.
Ruxolitinib is approved by the FDA for myeloproliferative diseases and graft-versus-host-disease, and baricitinib for RA.
No published studies have addressed the effects of genetic variants on either of the two drugs in any patient population.
However, their pharmacokinetics pathways involve a few potentially important pharmacogenes.
Ruxolitinib is a major and baricitinib is a minor substrate of CYP3A4 48, 49 .
Ruxolitinib is also partly metabolized by CYP2C9.
Both of these genes are considered VIPs in the PharmGKB and subject to notable genetic polymorphisms 30, 50 .
Although the main pharmacogene of baricitinib, SLC22A8 encoding OAT3 transporter 49 , is not a VIP, influence of variants on its activity is previously reported in another substrate drug 51 .
The potential role of corticosteroids in the treatment of COVID-19infected patients is mainly limited to those with acute respiratory distress syndrome (ARDS).
In patients with SARS-coronavirus infection, corticosteroid use was associated with delayed viral clearance but also showed possible benefits in those with ARDS 52 .
Many variants have been associated with corticosteroids response and toxicities across multiple disease conditions, including genes involved in the receptor binding (e.g., CRHR1, NR3C1), chaperone/ cochaperone protein (e.g., ST13, STIP1, FKBP5), metabolizing enzymes (e.g., CYP3A4, CYP3A5, CYP3A7, GSTT1), and transporters (e.g., MDR1, ABCB1) 53 .
The mechanistic and metabolic pathways of steroids are complex, and genomic determinants with sufficient evidence for clinical application to COVID-19 were not identified.
Variants associated with corticosteroids in PharmGKB with level of evidence higher than "low" (level 3) were only assessed in combination therapy (e.g., with chemotherapy) or inhaled corticosteroids.
No pharmacogenetic information specifically on the effectiveness of corticosteroids for ARDS was found.
Summary of clinical implications of pharmacogenomics for COVID19 We found evidence that several genetic variants may alter the pharmacokinetics of hydroxychloroquine, azithromycin, ribavirin, lopinavir/ritonavir and possibly tocilizumab, which hypothetically may affect clinical response and toxicity in the treatment of COVID-19.
Although the level of evidence for most is weak, and has not been directly studied in patients with COVID-19, some of these potential pharmacogenetic associations are worth further exploration.
QT prolongation could hypothetically be exacerbated by a combination of drug-drug, drug-gene, and drug-disease interactions in the treatment of COVID-19.
As previously described in this review, hydroxychloroquine, chloroquine and azithromycin can individually increase risk for QT prolongation, and those drugs have been used in combination in COVID-19 patients.
This combination therapy showed higher odds of cardiac arrest in hospitalized patients with COVID-19 in comparison to those on neither therapy (odds ratio 2.13 [95% CI: 1.12-4.05]) 12 .
Hydroxychloroquine is a CYP2D6 inhibitor, which calls for a vigilance in drug-drug interactions in patients with COVID-19, who are at risk of polypharmacy.
In particular, some QT-prolonging drugs, that are commonly used in hospitalized patients, are substrates of CYP2D6 which may be inhibited by hydroxychloroquine.
This may dramatically increase the risk of QT prolongation.
The CYP2D6*4 nonfunctional allele is present in nearly 20% of patients with European ancestry 54 , and thus patients with the CYP2D6*4 allele may have even higher risk.
Patients with the ABCB1 2677GG/ 3435CC genotype have higher peak concentrations of azithromycin, a QT-prolonging drug 23 .
Higher concentrations of azithromycin in combination with hydroxychloroquine may be particularly dangerous.
Baseline QT interval evaluation prior to therapy is important since there is a greater risk of prolongation in individuals with high baseline values.
The prevalence of high baseline QT intervals was reported to be 14.5% among those aged ≥40 years in a US population study 55 .
And finally, the potential combination of these arrhythmogenic risks may be further exacerbated in the setting of COVID-19, in which the disease is associated with myocardial injury and arrhythmias 56 .
The association between IFN-β1b and a variant in IRF6 for the adverse outcome of liver damage was one of the strongest pharmacogenetic associations identified in this review 43 .
The previous studies of IFN-β1b/IRF6 were performed in patients with multiple sclerosis, not COVID-19.
Therefore the IFN-β1b/IRF6 pharmacogenetic association should be investigated in patients with COVID-19, especially since a large portion of patients with COVID-19 have abnormal liver function tests and liver injury 57 .
Individuals homozygous for SLCO1B1*4 showed a greater than 2fold increase in lopinavir clearance.
Notably, this variant has a differing allele frequency across racial groups; it is 14% in European, 6% in African, and 0.3% in East Asian descents.
Although G6PD deficiency is associated with hydroxychloroquine and chloroquine related hemolytic anemia, the evidence suggests that the strength of the association is unclear and possibly because there are varying degrees of G6PD deficiency conferred by the genetic variations.
The ITPA variants, associated with protective effects against ribavirinrelated hemolytic anemia, has PharmGKB level 2 evidence (moderate strength), may have a role in improving ribarivin safety.
Older age, race, male gender, obesity and comorbid conditions have been established as risk factors for COVID-19 susceptibility and death 58, 59 .
Whether they are also precise risk factors for drug treatment failure have yet to be established.
It has yet to be determined if host genomics or confounding factors (e.g., access to healthcare, environment) also play a role.
Several studies though suggest that higher susceptibility to COVID-19 is associated with genetic variants, including those in the ACE1, ACE2, TMPRSS2, GSTT1 genes [60] [61] [62] .
Interestingly, several important ACE2 variants on the X-linked locus were identified in male as hemizygous, which may explain the higher mortality in male 63 .
Although no direct evidence of pharmacogenomics data in patients with COVID-19 was available at this time, there are plausible mechanisms by which genetic determinants may play a role.
Studies must be conducted in COVID-19 before pharmacogenomic testing can be recommended.
These data support the collection of DNA samples for pharmacogenomic studies of the hundreds of currently ongoing clinical trials of COVID-19 therapies.
One of the biggest success stories in the field of pharmacogenomics was for a drug used to treat another, highly lethal, infectious disease: abacavir for HIV.
The application of a pharmacogenetic test (HLA-B*57:01) nearly eliminates a potentially fatal hypersensitivity reaction to abacavir 64 .
Thus, the pharmacogenetic test for abacavir is now standard of care in the treatment of HIV.
It took years after the discovery of HIV to develop such a pharmacogenetic success for abacavir.
While it is understandable, at these very early stages since the discovery of COVID-19, the pharmacogenetic data are very limited.
The use of modern genomic tools coupled with a proactive assessment of the most likely gene-drug candidates could lead to a quicker understanding of the role of pharmacogenetics for COVID-19.
Important in silico efforts are underway to repurpose old drugs for COVID-19 and some of these drugs may have established pharmacogenomic markers in other disease 65, 66 .
It is worth noting potential limitations of the use of pharmacogenetics in the treatment of COVID-19.
In an acute illness such as COVID-19, pharmacogenetics would only be useful if the genetic test results were already available (i.e., pre-emptive pharmacogenetic testing) or rapidly available (i.e., point-of-care genetic testing).
Several institutions have already implemented pre-emptive pharmacogenetic testing, and some patients may have results readily available 67 .
Point-of-care pharmacogenetic tests are available but generally the potential variants of relevance in COVID-19 therapies are not available on this type of testing platform 68 .
In the face of unprecedented challenges posed by the COVID-19 pandemic, collaborative efforts among the medical communities are more important than ever to improve the efficacy of these treatments and ensure safety.
Some large national COVID-19 trials are evaluating pharmacogenomics, which will inform the role of pharmacogenomics markers for future clinical use.
No datasets were generated or analyzed during the current study.
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and P.A.J.
conceived the project.
T.T.
conducted initial literature search and drafted the manuscript.
T.T.
and P.A.J.
further refined the manuscript with input from J.A.L.
and M.R.N.
All authors contributed further literature search, provided critical feedback, and collectively composed the final manuscript.
P.A.J was in charge of overall direction and planning.
The authors declare no competing interests.
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key: cord-022053-idft1p6d authors: Pecora, Nicole; Milner, Danny A. title: New Technologies for the Diagnosis of Infection date: 2017-07-21 journal: Diagnostic Pathology of Infectious Disease DOI: 10.1016/b978-0-323-44585-6.00006-0 sha: doc_id: 22053 cord_uid: idft1p6d nan FilmArray (BioFire/BioMérieux) BD Max (BD Diagnostics) Luminex Next-Generation Sequencing Conclusion T he clinical microbiology laboratory is constantly evolving and progressing.
New technologies have revolutionized the characterization and diagnosis of pathogens.
The purpose of this chapter is to review the features, benefits, and limitations of some of the key new methodologies in infectious disease diagnostics.
The chapter will be organized by technology.
New technologies have the most impact in the fields of bacteriology and virology, but there are also promising new developments in mycology and parasitology.
The mainstay of bacterial and mycologic diagnosis continues to be culture by traditional techniques, although increasingly these are automated ( Fig.
6.1 ).
After an organism is isolated, however, a host of newer diagnostics have enhanced, and in some cases replaced, the biochemical test algorithm that has historically defined clinical bacteriology.
The advent of matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry is one of the main forces behind this shift, allowing a cultured organism to be identified in minutes instead of hours to days or even weeks in the case of very slow-growing organisms, such as mycobacteria.
Although still not in routine clinical use, the application of MALDI-TOF to direct patient samples, such as blood and urine, and applications such as antibiotic resistance are exciting developments.
Beyond bacteria, MALDI-TOF is becoming established for the identification of yeasts and, with the development of more extensive and higher quality libraries, is expected to also do so for other fungi.
Nucleic acid-based technologies have also had a big impact.
In particular, there are a growing number of test platforms that provide rapid diagnostics directly from primary specimens.
Many of these are multiplexed to look at several pathogens at one time.
The sequencing of the regions of ribosomal RNA (rRNA), such as the 16S rRNA gene in bacteria, already considered the gold standard for bacterial identification, is entering more and more into clinical diagnostics ( Fig.
6.2) .
Similarly, the routine sequencing of the intergenic transcribed spacer (ITS) and 28S rRNA regions in fungi is a welcome addition to current laborious and morphology-heavy techniques for diagnosing fungal illnesses.
The entry of next-generation sequencing (NGS) into clinical microbiology has also had a dramatic effect on both the profiling of individual specimens (i.e., members of a potential outbreak or highly drug resistant strains) as well as communities (i.e., microbiome analysis in health and disease).
Finally, the emerging technology of polymerase chain reaction (PCR)-electrospray ionization (ESI) mass spectrometry holds great promise for all major phyla of pathogens.
ITS ITS1 ITS2 ITS5 ITS1 ITS2 ITS3 ITS4 NL1 NL4 ITS1 ITS2 D1/ testing.
Immunohistochemistry (IHC) in anatomic pathology is based on the same principle as these rapid tests and has a wide range of uses for the detection and sometimes speciation of organisms in formalin-fixed, paraffin-embedded tissue (FFPE) sections.
Organisms commonly identified this way include spirochetes, mycobacteria, DNA viruses, Aspergillus, Candida, and Toxoplasma, although special reference laboratories (e.g., The Infectious Disease Pathology Branch at the Centers for Disease Control and Prevention) have a wide range of antibodies for common to exotic pathogens for tissue confirmation.
MALDI-TOF, a mass spectrometry-based method, allows for a rapid (minutes) assessment of the characteristic complement of ribosomal proteins in an isolate, providing a species-level identification in many cases.
After a microbial colony forms, it can be spotted onto a MALDI plate in matrix compound and loaded onto the mass spectrophotometer.
This topic has been reviewed in great detail.
[3] [4] [5] Briefly, a laser is focused onto the sample/matrix spot, causing sublimation, ionization, migration through a time of flight tube, and detection by a mass spectrometer (MS).The readout is a spectrum of peaks representing mass to charge ratios (M/Z) of different analytes.
The mass range (approximately 2 to 20 kDa) is highly enriched for ribosomal proteins, which make up a large share of the typical MALDI spectra on clinical instruments.
Spectra are compared with curated databases and the result is returned along with a metric describing the strength of the identification.
This technology has been widely adopted in Europe and is gradually entering clinical microbiology labs in the United States.
There are currently two US Food and Drug Administration (FDA)-approved systems: the Vitek MS (BioMérieux, Durham, North Carolina) and the MALDI Biotyper (Bruker Daltonics Inc., Billerica, Massachusetts).
Although the technology behind these two systems is essentially equivalent, they differ in their curated libraries, software, identification algorithms, and scoring systems.
5 For example, the Vitek MS reports isolates with a confidence value of 1% to 100%, whereas the Biotyper returns a score that can range from 0 to 3.000.
For most applications a score of 2.000 or greater is necessary for a species level identification.
Because of this, it can be difficult to directly compare results between the two systems.
There have been several studies using both platforms back to back.
One study on a large collection of 642 strains of bacteria, yeasts, and molds demonstrated comparable performance, although noting that the library coverage of the test cohort was greater with the Biotyper (572 strains) than the Vitek MS (406) and that the Vitek was more likely to output a misidentification for strains not represented in the library.
6 For FDA-approved applications, both the Vitek MS and the Bruker may be used for a large library of aerobic gram-negative and gram-positive organisms, as well as several anaerobes and yeast.
In general, performance has been reported to equal or exceed traditional biochemical systems, although it seems to be stronger for gram-negative than gram-positive organisms.
5, 7 Lackluster performance, in particular, has been noted for several grampositive rods.
In a study with the Biotyper, the system was unable to identify several isolates of Nocardia, Tsukamurella, Kocuria, or Gordonia, although 89.7% of Listeria and 80% of Rhodococcus were correctly classified.
8 Members of Corynebacteria, Lactobacillus, and some Listeria also seem to present some difficulty, both in terms of difficulty discriminating between species and in requiring a lowered score cutoff (1.7) for species-level identification.
9 In similar studies using the Vitek platform, there were also reported problems in speciation of Listeria.
10 The misidentification of a Kocuria as a Corynebacterium has also been reported.
11 Among both gram-positive and gram-negative organisms, MALDI-TOF has several well-characterized difficulties discriminating certain species and complexes, including Streptococcus pneumoniae/ Streptococcus mitis, Escherichia coli/Shigella, and members of the Enterobacter cloacae complex.
3 Finally, it is important to note that the Biotyper requires a Security-Relevant Library for the identification of such organisms as Francisella tularensis, Burkholderia pseudomallei, and Brucella spp.
12 MALDI is rapidly becoming the method of choice for identifying anaerobic bacteria.
13 Several studies using large, diverse collections of anaerobes have demonstrated strong performances by both the BioTyper and the Vitek MS. 5 In one recent multicenter trial bringing together 651 anaerobic isolates for analysis by the Vitek MS 2.0, 91.2% of the isolates were correctly identified to species level.
14 The most notable difficulty was in identifying Fusobacterium nucleatum (42.9% identified correctly).
The system also showed lower performance for certain species of Bacteroides, Actinomyces, and Prevotella.
Similarly, in a study using the Biotyper platform to identify 197 anaerobes isolated from blood cultures, 86.8% of the strains were correctly identified to the species level (using a score of 2) and 94.9% to the genus level.
15 Fusobacterium spp.
was again problematic, with only 23% being correctly identified to the species level.
With formic acid/ethanol pretreatment, this number increased to 76.9%.
In addition, 20.8% of the non-fragilis Bacteroides spp.
was misidentified as other Bacteroides, with high confidence scores, and only 52.9% of the gram-positive anaerobic cocci were correctly identified to the species level.
The latter was mostly a function of poor performance on several species of Peptoniphilus.
15 Interestingly, in a dedicated study looking at the performance of the Biotyper (2.0) on a collection of 277 isolates of Bacteroides spp., 97.5% were correctly identified to the species level.
16 The identification of yeasts is an FDA-approved application for both the Biotyper and Vitek MS.
Both systems have demonstrated strong performance in multiple studies, reviewed by Cassagne et al.
17 One recent study with the Biotyper, for example, used a library of 303 clinical isolates representing a diverse array of yeasts and found an agreement with standard methods in 84.8%.
18 In an additional 26 isolates the Biotyper returned a result that was confirmed as true by sequencing in 21.
The Biotyper failed to make identification in an additional 20 isolates.
Bader et al.
used an even larger cohort of clinical yeast isolates (1192) to compare the performance of traditional methods with the Bruker Biotyper and the Vitek SARAMIS (research use only database).
They found an overall agreement between the methods of 95.1% and noted not only that both MALDI methods were able to discriminate species that traditional methods could not, but that the savings in time and money were substantial.
19 More recently, Chao et al.
analyzed a collection of 200 clinical yeast isolates with both the Biotyper and the Vitek MS. 20 They found that the Biotyper slightly outperformed the Vitek MS (92.5% vs 79.5% correctly identified to the species or complex level).
This compared with rates of 89% and 74% for two common phenotypic methods (Phoenix 100 YBC and the Vitek 2 Yeast ID, respectively).
This trend was also observed by Mancini et al., who used a collection of 197 clinical yeast isolates, which included 157 Candida or related species and 40 non-Candida species.
They found that the Biotyper identified 89.8% of isolates, whereas the Vitek MS identified 84.3% using the standard commercially available database.
Importantly, the Vitek MS had a much higher rate of misidentification (12.1% vs 1%).
21 In general, it has been found that a formic acid/ethanol on the organism in question).
5, 7 Other benefits to MALDI in routine clinical microbiology include low costs of reagents and less production of waste materials (reduced consumables).
There are a few shortcomings of this technology.
For the most part, MALDI is limited to cultured isolates, so although it offers tremendous TAT advantages it is inherently limited by the time to growth of the organism.
Sparse database coverage over certain phyla (e.g., filamentous fungi, some anaerobes) presents an issue in some cases, although commercially available libraries continue to be expanded.
Another major caveat of MALDI is that it is not yet a practical method for assessing antibiotic resistance.
Direct detection of resistance-conferring enzymes is not practical within the assay's limits of detection; however, some promising studies have been published looking at the ability of MALDI to detect breakdown products after incubation in the presence of antibiotic.
3 However, resistance testing is unlikely to be incorporated into a standard MALDI microbial identification platform in the near future.
Mass spectroscopy has been applied to human tissues, including FFPE, for a variety of purposes, but, to date, there is not a commercially available or routine method for using MS in the detection of organisms in FFPE.
44, 45 With the advent of rapid and inexpensive nucleic acid methods for FFPE, this field largely lies dormant, although incredible potential for detection and other data extraction from FFPE through MS may be possible in the future.
For more than 30 years, a wide spectrum of assays based on the detection of nucleic acids has been developed for the diagnostics of infectious disease.
They range from simple probes to qualitative and quantitative target amplification to sequencing.
For example, the first nucleic acid probe-based assay was launched in 1985 to test for legionnaires' disease (Gen-Probe).
Real-time platforms, such as the Smartcycler and the Lightcycler, have an established place in both detection and quantification of pathogens, although the major impact has mostly been in virology, where the nucleic acid-based techniques were quickly adopted to overcome the difficulties inherent in viral culture.
For example, hepatitis C virus (HCV) and HIV viral loads are routine applications of these technologies.
There have been a number of newer nucleic acid-based technologies that have had a tremendous impact on microbial diagnostics, many of which are also geared towards bacterial pathogens.
These include highly sensitive probes for use in direct specimens, to alternative amplification methods, rapid assays of single targets, and multiplexed systems that allow for the detection of many organisms in one assay.
Sequencing assays are becoming more commonplace, with targets ranging from 16S rRNA to whole genomes and even metagenomic studies looking at the make-up of complex microbial ecosystems within the human host.
In this section the technologies behind several of the most important new assays will be briefly introduced, followed by a discussion of their application to clinical syndromes, such as respiratory and gastrointestinal (GI) disease, bloodstream infections, meningitis, and reproductive health/sexually transmitted disease.
Probe hybridization assays were one of the earliest diagnostic techniques using nucleic acids as a target.
The most commonly used probes are marketed by Hologic Gen-Probe Inc. (Marlborough, extraction method and lowered identity cutoffs (i.e., 1.7 on the Biotyper) are optimal for the analysis of yeasts.
22 Although not an FDA-approved application, MALDI has tremendous promise for the identification of mycobacteria and is already the preferred identification method of choice for these organisms in some clinical laboratories.
5 Caveats include the need for enhanced sample preparation methods for efficient cell breakage for biosafety reasons and to ensure high-quality spectra.
3, 23, 24 Several studies have shown promising results.
Using the Biotyper equipped with the mycobacterial library version 1.0 and the Vitek MS, 84.7% to 93.8% of isolates were identified correctly to the species level.
24, 25 Bruker has subsequently released a new version of the mycobacterial library, version 3.0, containing 149 different species.
It reportedly demonstrated high performance (>95% correct identification to species level) in a group of 1045 clinical samples.
26 Version 3.0 of the Biotyper mycobacterial library was also described by Rodriguez-Sanchez et al., who found 91.7% identification to species level on a group of 109 non-tuberculosis mycobacteria.
27 There is a great deal of interest in the identification of fungi other than yeast by MALDI, also not an FDA-approved application.
Concerns with this group include not only efficient cell lysis but also the need for higher-quality reference databases.
17 Few studies have assessed the Biotyper or Vitek MS platforms on fungal isolates using only the commercially available databases.
Iriart et al.
analyzed a group of 236 clinical isolates with the Vitek MS (192 yeast and 44 Aspergillus) and found that 93.2% of the isolates were correctly identified, including 81.8% of the Aspergillus.
28 This compared with 94.1% and 88.6%, respectively, by routine laboratory methods.
When the authors limited the study to only those species that were present in the database, 100% of the Aspergillus isolates were correctly identified.
28 In another study the Vitek MS was able to correctly discriminate closely related Aspergillus species, Aspergillus fumigatus and Aspergillus lentulus.
29 Schulthess et al.
did a prospective study on 200 isolates using the Biotyper/Filamentous Fungi Library 1.0.
They achieved a 79% species and 83.5% genus level identification.
Particularly poor performance was noted for Mucor and Scopulariposis.
30 Chen et al.
evaluated 50 clinical mold isolates with the Biotyper and found that several were not identified or identified with a low score (<1.7).
31 Twenty-eight isolates of Penicillium marneffei were not identified due to the lack of reference spectra in the database; however, the rate of identification went up to 85.7% after a single P. marneffei spectrum was added to the database.
This result is consistent with a number of studies indicating high performance of MALDI on fungal isolates using custom databases.
[32] [33] [34] [35] [36] [37] Although the use of MALDI-TOF in the microbiology lab has been confined, for the most part, to cultured organisms, there have been several studies exploring the feasibility of its use in primary samples.
For example, the use of MALDI directly on positive blood cultures is attractive for both its rapidity and the breadth of detection possible with an open system.
Sample processing directly from blood is not trivial and not yet FDA approved, but each major platform has a dedicated process and the results are promising with high clinical accuracy (80% to 90%).
3, 38, 39 Other promising applications include direct analysis of urine and cerebrospinal fluid (CSF), although, as with blood, additional screening and processing steps are required.
3, [40] [41] [42] [43] In summary, MALDI-TOF in the clinical microbiology lab has been developed to complement and in many cases replace the traditional biochemical diagnosis of bacteria and yeasts and can shorten turnaround time (TAT) by hours to days (depending There are a variety of focused assays in the clinical microbiology lab and anatomic pathology samples that query one or a handful of genes by amplification of the target.
The majority of these relies on the PCR and includes both qualitative and quantitative assays, such as those run on real-time PCR platforms (i.e., the Lightcycler [Roche Diagnostics, Indianapolis, Indiana] and the Smartcycler [Cepheid, Sunnyvale, California]).
Target detection is typically by fluorescent probes, such as TaqMan.
60 Single-target PCR has been applied to fresh, frozen, and FFPE in anatomic pathology for a large range of microorganisms but largely through home-brew or in-house assays and/or performance by large specialty reference labs, due to the challenges of control tissue, validation, and complexity of interpretation.
Recent developments in this area are based on the same technology but are innovative in their convenience, detection method, and, in some instances, sensitivity.
For example, using standard real-time PCR technology, FDA-approved assays, such as the Prodesse kits (Hologic Gen-Probe), provide convenient detection of small sets of respiratory viruses.
The Xpert system (Cepheid) has had a large impact on the clinical lab due to its ease of use (sample-to-result platform), excellent performance, and rapid TAT (approximately 1 hour).
Detection is by melt curve analysis rather than fluorescence.
This platform offers a variety of cartridges (e.g., C. difficile, MRSA, MTB/RIF) focused on quick answers to major clinical decision points.
[61] [62] [63] [64] Newer assays from Cepheid are in development for FFPE, which will include microbiologic applications.
For novel detection methods and increased sensitivity, the T2MR platform (T2 Biosystems, Lexington, Massachusetts) is one of the most innovative technologies.
It is a complete sampleto-result platform that relies on detection of pathogen-specific amplicons by hybridization with tagged superparamagnetic particles.
65 The T2Candida Panel (T2 Biosystems) is the only FDA-approved system that directly analyzes blood (without culturing) and has demonstrated impressive results in clinical trials (91% sensitivity with a 4.2-hour TAT).
66 T2 Biosystems is currently developing a bacterial panel as well, which is reported to target Acinetobacter baumannii, E. faecium, K. pneumoniae, P. aeruginosa, and SA.
67 Although PCR remains the most common means of nucleic acid amplification, other technologies have been developed that address the presence of single targets.
Transcription-mediated amplification (TMA) has been applied to mycobacteria, HCV, E. coli, and Listeria, where RNA is bound by a target oligo and then acted upon, in sequence, by reverse transcriptase (RT) (to produce cDNA), RNase H (to degrade RNA template), RT again to produce a double-stranded DNA promoter sequence, and then RNA polymerase to produce many copies of single-stranded RNA-then the process repeats itself.
Another isothermal reaction occurs in loop-mediated isothermal amplification, which uses a group of four to six primers and Bst DNA polymerase, with the creation of complimentary sites to generate exponentially amplified loop structures.
These assays have been developed for a wide range of targets and produce larger quantities of DNA than traditional PCR in a matter of minutes to hours.
This technique has been applied to viruses, bacteria, protists, fungi, and mycobacteria in a point of care manner.
[68] [69] [70] [71] [72] LAMP techniques are limited by the challenges of complex primer design, limited ability to multiplex, and common inhibitors of the enzymes in certain human samples, including blood.
A last isothermal amplification is Massachusetts) and consist of single-stranded DNA probes tagged with acridinium esters that hybridize to rRNA.
Tests are read in a luminometer.
Because there is no amplification, probe-based assays generally suffer low sensitivity and are reserved for culture confirmation, where organisms are present in high numbers.
AdvanDX (Woburn, Massachusetts) has produced a series of probe-based assays using peptide nucleic acid (PNA) probes tagged with fluorescent markers.
46 PNA probes have several advantages over traditional DNA probes, such as increased stability and ability to penetrate the bacterial cell wall.
Fluorescent tags have the added advantage of allowing for multicolor detection and the creation of limited panels.
These are rapid assays (less than 1 hour) and are mostly targeted towards rapid diagnostics of positive blood culture bottles.
[51] [52] [53] [54] In situ hybridization (ISH) of Epstein Barr encoding region for confirmation of Epstein-Barr virus infection on FFPE is routinely used in cancer diagnostics, but few, if any, other common viral infections are confirmed this way, with IHC being preferred.
Application of RNA-ISH to Aspergillus and Candida in FFPE showed less sensitivity than real-time PCR with sequencing (gold standard), although some FISH-positive, PCRnegative cases with obvious fungal elements were seen, suggesting refinements of this technique may be valuable for rapid identification of these common organisms, especially if mucormycosis is in the differential.
55 A unique probe-based approached to nucleic acid detection is the RNA hybridization and digital counting technology offered by NanoString (Seattle, Washington).
This method uses a capture probe (usually a specific 50-mer) and a reporter probe (a second, adjacent, specific 50-mer attached to a unique molecular color barcode) to bind up individual molecules of RNA in a sample, capture them on a solid state matrix, and use digital imaging to count the barcodes in the sample.
The current version can measure up to 500 independent capture:reporter pairs.
The technology can be applied to any RNA target (human or microbial), and the great advantage in infectious disease is the extremely low input quantities that can be detected without interference from other RNA.
Because of the counting algorithm, with proper probe selection in a given mixture, accurate quantification of RNA is possible without amplification.
One pitfall to this technology is that the presence of any given target in a disproportionate ratio to other targets will flood the digital analysis (i.e., consider hemoglobin relative to cytokines in peripheral blood).
Relative to sequencing, the technology remains expensive, but the benefit of quantification of extremely limited sample with minimal processing makes this an attractive target for future test development.
For microorganisms, broad panels using signature genes and/or focused panels using genes in antibiotic metabolism pathways can be created.
Studies in a range of diseases have shown that RNA from any source, including FFPE, is sufficient for NanoString analysis, [56] [57] [58] In one study, quantitative expression of Plasmodium falciparum schizonts from human tissue (both frozen and FFPE) were measured and, using imputation, the entire expression profile for the organisms determined.
59 pathogens include E. coli and Shigella, Salmonella, Campylobacter, and Clostridium perfringens.
Typical viral pathogens included in these panels are adenovirus, norovirus, and rotavirus, representing the three most common symptomatic viral diarrheas.
Diagnosis is moving more and more towards multiplex panels that cover major bacterial pathogens, as well as some parasitic and viral agents.
[84] [85] [86] In addition to standard agents of infectious diarrhea, there are special considerations when testing for disease caused by C. difficile.
These panels are, again, extremely valuable because the tools to diagnose these disease in anatomic pathology are often very limited and too invasive for most patient situations.
Parasitic enteric infections represent a major challenge in developing countries but remain rare in countries in which most new technologies are being developed.
Despite this disparate situation, efforts have been made to approach parasitic infections, and thus the inherent challenges have become apparent.
Extraction from stool for suspected parasite infections requires that protists (ciliates, ameoba, flagellates, coccidia), helminth larvae (nematodes), and helminth eggs (nematode, trematode, and cestode) be lysed sufficiently to release DNA efficiently.
This challenge has led to eight or more different extraction protocols with a range of preparation requirements, including spin columns, automated magnetic bead assays, and "bead beating" methods.
Molecular targets for subsequent detection assays as specific single targets across this range of organisms has included 18S rDNA, ITS, COX 1, pOV-46, 5.8S rDNA, Segment A, COWP, actin, HDP2, DL1, and 28S rDNA.
87 In commercially available, multiplex PCR assays, the majority of the platforms target only Cryptosporidium, Giardia, Entamoeba histolytica, and, rarely, Cyclospora.
Because these first three represent 80% to 99% of parasites isolated by all methods from stool in developed nations, additions of other much less common pathogens is not common.
Inclusion of nonpathogenic ameba is common in these assays but of questionable value.
Genitourinary samples (fluid from cervical samples, urethral swabs) are another sample that benefits from multiplex testing as public health screening of sexually transmitted diseases are extremely valuable for individual patient diagnosis, as well as epidemiologic survey, especially with the inclusion of HIV.
Neisseria gonorrhea, Chlamydia trachomatis, and HPV in the Hologic Panther system can be performed on a single sample, and this system also can test for Trichomonas.
The FilmArray from BioFire (Salt Lake City, Utah) detects Chlamydia, Neisseria, syphilis, Trichomonas, Mycoplasma/Ureaplasma, herpes simplex, and chancroid in a single sample.
88 Although relatively uncommon, meningitis and encephalitis (both nosocomial and community-acquired) carry high morbidity and mortality.
[89] [90] [91] Time is of the essence when diagnosing a causative agent, particularly when trying to determine whether it is a bacterial pathogen that needs immediate antibiotic coverage along with herpes simplex, which needs immediate antivirals.
Common bacterial pathogens include S. pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, and Listeria monocytogenes, although there are also several other, less frequently isolated, pathogens.
Viral pathogens in these panels include the primary actionable viruses (Cytomegalovirus, herpes simplex, varicella zoster virus), as well as other common viruses (Enterovirus, Parechovirus, and human herpesvirus (HHV)-6).
Platforms also may include Cryptococcus species of fungi, which is very valuable in immunosuppressed populations.
92 Current diagnostics are heavily focused on CSF biochemical profile (glucose, protein, lactate), cell count, and Gram stain.
The latter is the most specific, but the limit of detection is approximately 10 (4) accomplished by the introduction of helicase, which unwinds DNA, and single-stranded DNA binding proteins, which keep the templates separated.
This is followed by primer hybridization and amplification to produce more single-stranded DNA and thus a continuous reaction.
Assays to measure herpes simplex virus (HSV)-1 and HSV-2 using helicase have been developed with similar performance to traditional molecular methods and assays are available for Bordetella, group A Streptococcus, Trichomonas, and malaria.
73 Commonly used platforms that use these technologies include the Tigris and Panther systems by Gen-Probe, which are based on TMA, SmMIT-LAMP based on LAMP, and Ampli-Vue based on helicase.
Use of these assays in FFPE tissue has shown some promise with improved performance over PCR for some organisms (such as HCV in TMA), equivalent performance to PCR (such as human papillomavirus [HPV] in head and neck cancers with LAMP), and detection of limited signatures (such as microcarcinomas in small biopsies with helicase assays).
[74] [75] [76] The ability to detect multiple organisms in primary samples using PCR-based panels has both provided clinicians with tremendous diagnostic opportunities and presented new challenges.
77 Major platforms include FilmArray (BioMérieux), Verigene (Nanosphere), xTAG/xMAP (Luminex, Austin, Texas), with technologies that range from real-time PCR to solid and liquid phase arrays.
For a comprehensive listing of platforms see www.captodayonline.com/ productguides.
For an in-depth discussion of the major technologies see the articles included in the reference list.
78, 79 These systems are generally geared towards syndromic testing, with panels directed towards respiratory, GI, and sexually transmitted diseases, as well as meningitis and bloodstream infections.
After discussing the specific challenges of each of the major syndromes, this section will focus on commonly used FDA-approved panels that are currently available from a number of manufacturers and that scan for a wide array of microbes, encompassing viral, bacterial, and fungal organisms.
Panels aimed at respiratory pathogens are focused most heavily on viruses but also incorporate several bacterial agents.
80, 81 Commonly covered organisms include Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella species.
M. pneumoniae and C. pneumoniae, both very common causes of communityacquired pneumonia, are particularly well-suited to nucleic acid amplification tests because they are difficult to culture.
82 Although less common, Bordetella pertussis is considered to be the most common vaccine-preventable disease in the United States, is increasing in incidence, and is also difficult to culture.
83 The "bread and butter" of respiratory panels (RPs) focus on viruses with influenza A (and B), adenovirus, and respiratory syncytial virus (RSV) being always including, along with a mixture of other coronavirus, enterovirus, metapneumovirus, and the parainfluenza group.
These panels are extremely powerful from the clinician's point of view but also provide a larger range of diagnostics for respiratory viruses than can be found in most anatomic pathology labs, which are largely limited to the DNA viruses.
Although not all of these viral diagnoses are chemotherapeutically actionable, confirmation of a viral cause is valuable for prognosis and resource allocation.
Diarrheal diseases are a global public health concern.
Although these diseases are a common cause of illness and hospitalization in the United States, they impose an enormous toll in the developing world, particularly on children.
The most common bacterial 99.5% to 100%), but, as has been common with many panels and individual tests, laboratories have difficulty obtaining natural clinical cases of E. histolytica.
103 Currently, the only FDA-approved multiplex assay for agents of meningitis and encephalitis is the FilmArray meningitis panel.
It covers 14 pathogens, including the following bacteria: E. coli K1, H. influenza, L. monocytogenes, N. meningitides, S. agalactiae, and S. pneumoniae.
Although the panel was only recently approved by the FDA (October 2015) , there are a few reports of its performance.
The pre-FDA evaluation was conducted both on archived samples and prospectively on a multicenter collection of 1560 samples of CSF.
Among 235 archived samples (32 with bacteria), the percent positive and negative agreement was 100% for bacterial targets.
Among the 1560 prospective samples, there were only eight with bacterial pathogens (none with L. monocytogenes or N. meningitides).
Of those that were present, the FilmArray ME panel did not identify the only S. agalactiae.
The archived arm of the evaluation included two S. agalactiae samples, both of which were correctly identified.
106 Since FDA approval, one US study has been published on the performance of the panel in several Texas medical centers.
In that study of 48 patients with community-acquired meningitis and a negative Gram stain, the FilmArray detected two samples with bacterial pathogens, both S. pneumoniae.
Culture detected only one of these, although the other one was positive for streptococcal urinary antigen.
107 Finally, the FilmArray blood culture identification (BCID) panel tests for an array of 19 bacterial targets, including: Enterococcus, L. monocytogenes, SA, Streptococcus (multiple), A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae.
It also tests for five species of Candida and three bacterial resistance genes: mecA, vanA/B, and kpc.
It has performed well on both monomicrobial and polymicrobial specimens in several clinical studies.
108, 109 The BD Max is a sample-to-result real-time PCR-based platform.
Amplicons are detected via fluorescent probes.
A distinguishing feature is the capability to run laboratory-developed tests alongside FDA-approved panels.
Current FDA-approved assays include GBS (for group B Streptococci), CT/GC/TV (for vaginitis), MRSA XT (for extended MRSA coverage), Cdiff (for C. difficile), and StaffSR (for Staphylococcus aureas and MRSA).
The bacterial panel includes Salmonella spp., Campylobacter spp.
(jejuni/coli), Shigellosis disease-causing agents (Shigella spp.
and EIEC), as well as Shiga-toxin producing E. coli.
It has demonstrated high sensitivity for the organisms in its library in several studies.
[110] [111] [112] The parasite panel includes the three most common protist parasites in humans in developed countries, including Giardia lamblia, Cryptosporidium, and E. histolytica, but has the difficult challenge of very low population prevalence relative to the bacterial assays, making cost a challenge over other methods for uptake.
113 Verigene is another widely used multiplex PCR platform incorporating PCR of multiple viral and bacterial targets followed by hybridization to a solid-phase microarray.
Detection occurs via sandwich hybridization with another analyte-specific probe bound to a gold nanoparticle.
The signal is amplified via deposition of colloidal silver that is then detected by light diffraction.
Verigene organisms per milliliter.
90 There is a great need for rapid molecular diagnostics in this area.
In anatomic pathology, sample with culture and/or morphology with special stains and/or IHC can resolve many of these infections; however, optimal patient care and best outcomes occur in those patients who can be diagnosed early with these panels and not require an invasive biopsy.
Sepsis continues to be a tremendous cause of mortality, ranking as one of the top 10 causes of death in the United States.
93 Organisms commonly associated with bloodstream infections include SA, E. coli, Enterococcus spp., K. pneumoniae, CNS, P. aeruginosa, Candida albicans, E. cloacae, and Serratia marcescens.
94 The current gold standard for diagnosis is culture, with several automated systems (such as the BacT/Alert [BioMérieux Inc.]) available to clinical laboratories.
95 Although these systems are quite sensitive, approximately only one-third of patients with sepsis have positive cultures.
96 Some of this lack of sensitivity may be due to pretreatment with antibiotics, and some of it may be due to the low numbers of organisms present in many clinically significant bacteremias.
97 In addition to sensitivity, speed is also a major issue.
The rapid diagnosis of bloodstream infections is critical for patient outcomes, with estimates of a 7.6% decrease in survival for every hour that appropriate antimicrobials are delayed after the onset of hypotension.
98 The need for sensitive, fast detection, ideally accompanied by some assessment of antibiotic resistance determinants, has inspired a number of new technologies geared towards improved speed and sensitivity of blood culture diagnostics.
Several of these have been reviewed.
[99] [100] [101] Platforms and Technologies FilmArray (BioFire/BioMérieux) The FilmArray system consists of nested PCR followed by highresolution melt curve analysis.
102 All steps of the assay, from cell lysis to the final analysis, take place within a pouch containing freeze-dried reagents that can be stored at room temperature.
FilmArray has a short TAT of approximately 1 hour.
Disadvantages of the system include the relatively high price of the pouches and restriction of the platform to one test at a time.
Laboratories must purchase multiple FilmArray platforms if they desire to run tests in parallel.
The FilmArray panel was the first FDA-approved RP to include bacterial pathogens, covering B. pertussis, C. pneumoniae, and M. pneumoniae, along with 18 common respiratory viruses.
102 For GI testing, the FilmArray is the most comprehensive of the current FDA-approved panels, covering an array of 22 bacteria, viral, and parasitic targets, including the common agents listed above, as well as Plesiomonas shigelloides, Yersinia enterocolitica, and several species of Vibrio.
It can also differentiate between enteroaggregative, enteropathogenic, enterotoxigenic, Shiga toxin-producing, and enteroinvasive E. coli (EIEC).
Studies have shown a sensitivity of 95.9% to 100% and a specificity of 96.6% to 100% for bacterial pathogens.
103, 104 In many cases the FilmArray detected pathogens in samples that were negative and was far more likely to diagnose mixed infections than standard techniques.
104, 105 For viral pathogens the FilmArray GI panel has shown value in the younger age groups (patients younger than 12 years) for most tested pathogens (sensitivity: 95.5% to 100%; specificity: 99.1% to 99.9%), whereas Norovirus appears to be valuable across all age groups (sensitivity: 94.5%; specificity: 98.8%).
103 Performance for parasitic pathogens in this panel is equally high for Cryptosporidium, Cyclospora, and Giardia (sensitivity: 100%; specificity: Vibrio cholerae, and Y. enterocolitica.
In one study comparing the xTAG with the FilmArray and conventional techniques, the two platforms behaved comparably, with the xTAG slightly trailing the FilmArray in the diagnosis of mixed infections (27% for Fil-mArray vs 14.1% for xTAG vs 8.3% for routine testing).
104 The performance of xTAG for viral pathogens when compared with realtime RT-PCR and multiplex realtime RT-PCR, showed a wide variation in sensitivity across viruses for xTAG (20% to 100%), with performance for RSV-A, RSV-B, H1N1, and influenza B being 13.3%, 47.3%, 54.2%, and 20%, respectively.
121 In headto-head comparisons of FilmArray, eSensor RVP, and xTAG, the three methods were essentially equivalent, with xTAG having the best performance for influenza A.
122 Some of the caveats of the xTAG system are addressed with newer Luminex platforms, such as the Aries, which is a sampleto-result platform for testing primary clinical specimens.
The assay consists of real-time PCR using the Luminex MultiCode technology.
This system uses primers that incorporate isobases, isoC and isoG, which, when incorporated, can pair only with each other.
One isobase is tagged with a fluorescent reporter, and the other is tagged with a quencher.
Upon the successful generation of product, both isobases are incorporated and fluorescence decreases.
A subsequent melt curve analysis is performed, and fluorescence is restored.
Unlike the xTAG technology, Multi-Code can be run on either commercially available real-time PCR systems or within the sample-to-result Aries platform from BD.
There is only one FDA-approved test for this panel, an HSV-1 and HSV-2 panel for use in testing cutaneous and mucocutaneous lesions.
Although all of the nucleic acid-based systems described above demonstrate high levels of sensitivity and specificity, they are all inherently limited to a select group of organisms based on the probe/primer sets they contain.
Open assays using broad-range primers that target nearly universal sequences are nearly all based on the 1977 discovery by Carl Woese that the 16S rRNA subunit could be used to determine the phylogenetic structure of the domains of life.
123 Although this idea represented a fundamental shift in bacteriology, it took many years for it to be widely accepted, and even longer for the use of 16S rRNA sequencing (16Sseq) to be used as a standard means for identifying bacteria in the clinical laboratory.
124 However, since 2000, 16Sseq has become the gold standard for determining bacterial species, and its analysis has led to a great deal of restructuring and renaming among phyla.
125 Briefly, the 16S gene encodes a portion of the small ribosomal (30S) subunit and is composed of nine variable regions alternating with nine conserved regions (Fig.
6.2) .
The gene is sequenced using primers that bind to the highly conserved regions and then compared with a reference database to identify an isolate.
The choice of database is typically the most challenging part of the assay.
There are a number of databases that can be used to identify the sequence, ranging from large, public, uncurated or partially curated databases, such as Genbank and the Ribosome Database Project (RDP, University of Michigan) to commercial, highly curated collections with a much more limited scope.
Identification algorithms include Ripseq (Pathogenomix, Santa Cruz, California), the Integrated Database Network System has a TAT of approximately 2 hours and has the advantage of offering flexible pricing by which the laboratory is charged only for the tests they request, instead of the entire panel.
Disadvantages include the need to select the appropriate cartridge based on Gram stain results for bloodstream diagnostics.
Respiratory, GI, and bloodstream panels are available.
The Verigene platform can detect 13 respiratory viruses with high sensitivity (including multiple types of influenza, parainfluenza) and three Bordetella species (B. pertussis, Bordetella parapertussis/ Bordetella bronchiseptica, and Bordetella holmesii), 114, 115 although one advantage of this system is the flexibility to test limited panels based on a given population.
The GI panel is somewhat more limited than the FilmArray, covering 22 targets, including 13 bacteria, 5 viruses, and 4 parasites.
Bacteria include the Campylobacter group, Salmonella spp., Shigella spp., Vibrio group, and Y. enterocolitica, as well as Shiga toxin.
In one study of 725 stool samples the Verigene demonstrated a 98.5% agreement rate with culture.
116 For the diagnosis of bloodstream infections, the Verigene system has separate cartridges for gram-positive and gram-negative organisms, necessitating an accurate Gram stain to pick the appropriate test and making polymicrobial testing a bit more complicated.
Each panel assesses a few extra organisms than the FilmArray single panel.
In addition, the gram-negative panel queries several more resistance targets than the FilmArray, including CTX-M, IMP, NDM, OXA, and VIM.
117 Verigene in RPs has excellent performance for the influenza viruses (A and B), including the ability to subtype viruses with perfect agreement with gold standard methods (molecular); however, mixed infections with non-influenza viruses dampen performance.
118 Headto-head comparisons of the limited Verigene panel (influenza A and B, RSV) with FilmArray RP showed equivalent detection of the three major pathogens at a slightly reduced cost for the Verigene; thus analysis of the clinical population and the desires of the local clinical team for reported virus and actionable diagnoses may help in choosing between these technologies.
114 xTAG The xTAG assay system from Luminex is a liquid array based on multiplex PCR followed by amplicon extension using a targetspecific primer.
The extension step incorporates a hybridization tag onto the amplicon, along with a biotin label.
Elongated, tagged amplicons are then incubated with polystyrene microbeads of various fluorescent profiles, each coated with a specific anti-tag sequence.
Bead/amplicon pairs are then stained with another streptavidin-linked fluorophore.
The beads are analyzed by a system of two lasers on the xMAP instrument.
The first determines which beads are bound to amplicons by way of the streptavidinfluorophore.
The second laser indicates which specific target was amplified by way of the unique fluorescence of that bound bead.
119 Although the Luminex platforms generally have the potential to detect the greatest number of targets, drawbacks include the need for a separate nucleic acid extraction step and longer TAT (4 hours or longer) then the other multiplex platforms.
There are two xTAG platforms focused on respiratory testing: the original xTAG platform, restricted to viral pathogens, and the next-generation system (NxTAG), which included several bacterial pathogens as well, including Legionella pneumophila.
120 The xTAG Gastrointestinal Pathogens Panel (GPP) tests for nine bacterial, three viral, and three parasitic targets.
Among bacterial loci, the Luminex xTAG covers Campylobacter, C. difficile toxin A/B, E. coli O157, enterotoxigenic E. coli (ETEC) LT/ST, Shiga toxin-producing E. coli (STEC) stx1/stx2, Salmonella, Shigella, Sanger sequencing, which is generally unable to assess polymicrobial infections, although some analysis software packages have algorithms to deconvolute traces of 16Sseq from two or three different organisms.
148 NGS is a high-throughput method that generally refers to a massively parallel process that generates tremendous quantities of raw sequence reads.
There are a number of commercially available platforms that vary in the quantity, quality, and length of the reads that they produce.
The majority of these include a step for clonal amplification of the template and produce short (50 to 400 bp) reads.
For excellent reviews see articles provided in the reference list.
149, 150 The advent of NGS was a groundbreaking event in clinical medicine and is a key component of initiatives for "precision medicine" for cancer.
Beyond human genomic analysis, NGS has also had a tremendous influence on clinical microbiology that extends from infectious disease testing and epidemiology to analysis of the human microbiome in health, disease, and intervention.
[151] [152] [153] Within the diagnostic clinical microbiology laboratory, NGS of microbial whole genomes has been applied as an alternative to traditional methods, such as pulsed-field gel electrophoresis, for clonality analysis in outbreak tracking.
154 The refinement made possible with total chromosomal single-nucleotide polymorphism analysis, for example, allowed one group to assess the spread of variants of a colonizing population of carbapenem-resistant K. pneumoniae from one patient to several others in multiple transmission events.
These variants were differentiated from each other by fewer than a dozen nucleotides.
155 Other applications that are becoming more and more routine include the characterization of unusual pathogens, the tracking of antibiotic resistance genes, and even the characterization of the plasmids that carry them through horizontal gene transfer.
[156] [157] [158] [159] Although not yet routine, NGS has been used to diagnose infections directly from primary samples in cases in which all conventional techniques have failed.
160 In one report, after 4 months of progressive neurologic symptoms and a thorough but unrevealing infectious disease workup, NGS was finally able to diagnose a case of neuroleptospirosis directly from the CSF of a 14-year-old boy.
161 In addition to the detection and characterization of pathogens, NGS has also proven to be an invaluable tool for the characterization of the human microbiome.
The influence of the microbiome on human health and disease has become progressively more established.
Examples are many and include the development of obesity, the metabolism of therapeutic drugs, and the development of inflammatory bowel disease.
[162] [163] [164] The two most common ways of assessing the microbiome are by 16S rRNA or shotgun metagenomics.
NGS 16S analysis entails sequencing one portion of 16S rRNA from a mixed community (e.g., stool sample) with subsequent clustering of sequences either against a reference dataset or into operational taxonomic units (OTUs).
It gives a picture of taxonomic diversity in a sample and allows for the analysis of changing taxonomic composition in the face of various perturbations and interventions, such as antibiotic administration.
Shotgun metagenomics refers to the sequencing of total DNA from a mixed community.
This technique allows for more refined organism identification and an analysis of biochemical pathways, resistance genes, etc.
but requires a greater amount of raw sequence.
165, 166 (IDNS) (SmartGene, Raleigh, North Carolina), and MicroSEQ (Applied Biosystems, Thermo-Fisher, Waltham, Massachusetts).
The first two products are solely bioinformatic reference solutions.
The last includes both reagents and access to a curated database.
None of these products are FDA approved.
The Clinical and Laboratory Standards Institute (CLSI) has published guidelines for the identification of organisms by nucleic acid sequencing in which the recommended percentage identity to make a match and guidance for organisms requiring additional targets (e.g., rpoB, hsp65) for accurate speciation are laid forth.
126 The use of 16Sseq in the clinical microbiology laboratory has been shown to be particularly useful in such applications as the identification of unusual and/or slow-growing organisms and the identification of organisms from specimens in which culture is not possible or has failed, such as in specimens from antibiotictreated patients and fixed specimens.
[127] [128] [129] [130] For example, the identification of mycobacterial species identified by acid-fast staining of FFPE is a commonly used application of 16S sequencing, although additional targets, such as IS6110 and hsp65, are often needed to differentiate species.
131, 132 Specimen types most successfully analyzed by direct 16Sseq include heart valves, CSF, and synovial fluid.
[133] [134] [135] [136] [137] [138] Fungal pathogens are also identified using rRNA sequencing, although it is a less established practice than it is for bacterial targets.
The fungal rRNA 18S corresponds to bacterial 16S, although it has fewer hypervariable regions and is generally not as useful for isolate discrimination.
139 The most common regions for fungal identification are the ITS regions 1 and 2, as well as the D1 and D2 hypervariable regions of the 28S rRNA large subunit component (Fig.
6.2) .
[139] [140] [141] When using these targets to identify an isolate, criteria required for percentage identity and percentage difference between competing matches are not as clear for fungi as they are for bacteria.
126 The lack of high-quality databases has been a major difficulty in fungal sequencing, with estimates of up to 20% of the entries in GenBank being incorrectly identified down to species level.
142 To address this, several of the private databases that are available for bacterial sequencing also include curated or partially curated fungal databases (IDNS and RipSeq).
143 The MicroSEQ system also includes a fungal component, but it is limited to the D2 region of 28S.
Recently, several public databases have become available with high-quality, curated content.
Databases that are most focused on species of medical importance include the International Society for Human and Animal Mycology (ISHAM) ITS database contains 3200 sequences representing 524 human/ animal pathogenic fungal species and is a major resource for clinical laboratories.
144 It can be searched via a web-based platform (its.mycologylab.org).
The National Center for Biotechnology Information RefSeq ITS database contains 3060 sequences representing 270 families from 39 classes.
The smaller 28S RefSeq database contains approximately 500, representing greater than 100 families from 21 classes.
145 This technique is particularly promising for identifying fungi from fixed tissue.
55, 146, 147 Important caveats of rRNA sequencing include high cost and complexity, issues with uncurated databases (described above), and the potential for false positives.
This is particularly the case when broad-range sequencing is applied to primary specimens to "fish" for an infectious agent in the absence of any corroborating culture or gram stain.
Furthermore, rRNA should not be used to determine whether an infection has "cleared," due to the potential for lingering nucleic acid even in a successfully treated infection.
Analysis should be restricted to normally sterile sites for tests using In FFPE in which an organism has been seen but not identified (such as bacteria, fungal elements, viral cytopathic or necrotic effect, or unidentified parasitic elements), NGS has enormous potential to rectify theses diagnoses but is currently limited by cost per sample and the enormous amounts of raw human data in comparison with the relatively small amount of the organism's sequence.
As costs fall for sequencing and rapid bioinformatic methods for elucidating causative sequence emerge, NGS will become a primary tool for confirmation of microbial infections.
One last technology to consider is the hybrid system, PLEX-ID by Abbott, which combines a PCR step to amplify through a set of primers (PLEX-ID Viral IC, PLEX-ID BAC Spectrum BC, or PLEX-ID Flu) a broad range of organisms and then detects the PCR products through mass spectroscopy.
This approach combines amplification of many sample types, including body fluids, tissue, FFPE, culture isolates, and blood, with a highly sensitive detection system to make subtle species determinations across organism types, including bacterial, fungal, viral, and some parasitic.
[167] [168] [169] [170] As the database of spectra improves and the multiplex primers improve, this system will gain popularity, although it is still limited by initial organism burden and procedure time (up to 8 hours).
The microbiology laboratory remains the most important companion for anatomic pathologists who have a potentially infectious diagnosis under the microscope.
Anecdotal and small studies have demonstrated that the yield from molecular tests that are performed blindly (without visualization) versus with pathologist's review is miniscule and extremely expensive.
Therefore anatomic pathologists and their microbiologic colleagues must have a constant line of communication from the moment the specimen is collected until final reporting to ensure interpretation of all modalities is maximally beneficial to patient care.
This dialogue should also include discussions of molecular modalities that are applied to clinical and anatomic pathology specimens and changes, advantages, pitfalls, and benefits of those modalities in an ever-evolving manner.
As with any approach to medicine, evidence-based use of the most efficient and inexpensive method that produces the highest-impact result for the patient is the constant goal.
MS technology for microorganism identification in a public health reference laboratory Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Nocardia, Rhodococcus, Kocuria, Gordonia, Tsukamurella, and Listeria species Evaluation of the Bruker MALDI Biotyper for identification of Gram-positive rods: development of a diagnostic algorithm for the clinical laboratory Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria Kocuria rhizophila misidentified as Corynebacterium jeikeium and other errors caused by the Vitek MS system call for maintained microbiological competence in the era of matrix-assisted laser desorption ionization-time of flight mass spectrometry Importance of using Bruker's securityrelevant library for Biotyper identification of Burkholderia pseudomallei, Brucella species, and Francisella tularensis Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a new possibility for the identification and typing of anaerobic bacteria Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK(R) MS system Anaerobic blood culture isolates in a Norwegian university hospital: identification by MALDI-TOF MS vs 16S rRNA sequencing and antimicrobial susceptibility profiles ESCMID Study Group on Antimicrobial Resistance in Anaerobic Bacteria.
Species identification of clinical isolates of Bacteroides by matrixassisted laser-desorption/ionization time-of-flight mass spectrometry Performance of MALDI-TOF MS platforms for fungal identification MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption ionization time-offlight mass spectrometry Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts Comparative evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry systems for identification of yeasts of medical importance Interlaboratory comparison of sample preparation methods, database expansions, and cutoff values for identification of yeasts by matrix-assisted laser desorption ionization-time of flight mass spectrometry using a yeast test panel Clinical performance evaluation of the Sofia RSV FIA rapid antigen test for diagnosis of respiratory syncytial virus infection Serological markers in early stages of human immunodeficiency virus infection in haemophiliacs Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology MS for the diagnosis of infectious diseases A side by side comparison of Bruker Biotyper and VITEK MS: utility of screening methods and matrix-assisted laser desorption ionizationtime of flight mass spectrometry Direct identification of urinary tract pathogens from urine samples using the Vitek MS system based on matrix-assisted laser desorption ionization-time of flight mass spectrometry Mass spectrometry: pneumococcal meningitis verified and Brucella species identified in less than half an hour Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis Sample preparation for mass spectrometry analysis of formalin-fixed paraffin-embedded tissue: proteomic analysis of formalin-fixed tissue Urinary pellet sample preparation for shotgun proteomic analysis of microbial infection and host-pathogen interactions PNA for rapid microbiology Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures Direct identification of major blood culture pathogens, including Pseudomonas aeruginosa and Escherichia coli, by a panel of fluorescence in situ hybridization assays using peptide nucleic acid probes Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes Rapid detection of Enterococcus spp.
direct from blood culture bottles using Enterococcus QuickFISH method: a multicenter investigation Development of a peptide nucleic acid probe to Trichosporon species and identification of trichosporonosis by use of in situ hybridization in formalin-fixed and paraffin-embedded (FFPE) sections Design and evaluation of peptide nucleic acid probes for specific identification of Candida albicans Identification of Mycobacterium species from BACTEC MGIT positive cultures with Oligo-FISH and PNA-FISH methods Development and evaluation of nucleic acid-based techniques for an auxiliary diagnosis of invasive fungal infections in formalin-fixed and paraffin-embedded (FFPE) tissues Comparison of quantitative real time PCR with sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens Development and verification of the PAM50-based Prosigna breast cancer gene signature assay Evaluating robustness and sensitivity of the NanoString Technologies nCounter Platform to enable multiplexed gene expression analysis of clinical samples Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice Comparison of sample preparation methods, instrumentation platforms, and contemporary commercial databases for identification of clinically relevant mycobacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry Identification of mycobacteria from solid and liquid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry in the clinical laboratory Poster 1190: Mycobacteria Identification by MALDI Biotyper System: Evolution of Database Content and Evaluation Criteria Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for identification of nontuberculous mycobacteria Routine identification of medical fungi by the new Vitek MS matrix-assisted laser desorption ionization-time of flight system with a new time-effective strategy Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory Evaluation of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Bruker Biotyper for identification of Penicillium marneffei, Paecilomyces species, Fusarium solani, Rhizopus species, and Pseudallescheria boydii Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry MALDI-TOF mass spectrometry identification of filamentous fungi in the clinical laboratory Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi Detection of microorganisms in blood specimens using matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a review Sepsis: a roadmap for future research Direct identification of urinary tract pathogens from urine samples, combining urine of human papillomaviruses (HPV) in oropharyngeal squamous cell carcinoma (OPSCC) Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies Point-counterpoint: large multiplex PCR panels should be first-line tests for detection of respiratory and intestinal pathogens Emerging technologies for the clinical microbiology laboratory Basic concepts of microarrays and potential applications in clinical microbiology Molecular diagnosis of respiratory viruses Molecular Pathology in Clinical Practice Community-acquired pneumonia requiring hospitalization among U.S. adults Pertussis: microbiology, disease, treatment, and prevention Laboratory diagnosis of bacterial gastroenteritis Molecular testing for viral and bacterial enteric pathogens: gold standard for viruses, but don't let culture go just yet?
Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis Advances in molecular diagnosis of parasitic enteropathogens Multiplex PCR testing for nine different sexually transmitted infections Nosocomial bacterial meningitis State-of-the-art microbiologic testing for community-acquired meningitis and encephalitis Epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis Diagnostic performance of a multiplex PCR assay for meningitis in an HIV-infected population in Uganda Two decades of mortality trends among patients with severe sepsis: a comparative meta-analysis The clinical and prognostic importance of positive blood cultures in adults Laboratory detection of bacteremia and fungemia Predicting bacteremia in patients with sepsis syndrome.
Academic Medical Center Consortium Sepsis Project Working Group Quantitative aspects of septicemia A cross comparison of technologies for the detection of microRNAs in clinical FFPE samples of hepatoblastoma patients Plasmodium falciparum gene expression measured directly from tissue during human infection Molecular microbiology Comparison of the next-generation Xpert MRSA/SA BC assay and the GeneOhm StaphSR assay to routine culture for identification of Staphylococcus aureus and methicillin-resistant S. aureus in positive-blood-culture broths Multicenter evaluation of the Xpert Norovirus assay for detection of Norovirus genogroups I and II in fecal specimens Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares Charnot-Katsikas A.
Comparison of Cepheid Xpert(R) Flu/ RSV XC and BioFire FilmArray(R) for detection of influenza A, influenza B, and respiratory syncytial virus T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial T2Bacteria: rapid and sensitive detection and identification of sepsis pathogens in whole blood specimens by T2MR (poster) Evaluation of two commercial loop-mediated isothermal amplification assays for detection of avian influenza H5 and H7 hemagglutinin genes A comparison of in-house real-time LAMP assays with a commercial assay for the detection of pathogenic bacteria Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar Up-date on diagnostic strategies of invasive aspergillosis The Use of a Commercial Loop-Mediated Isothermal Amplification Assay (TB-LAMP) for the Detection of Tuberculosis Clinical evaluation of a helicase-dependant amplification (HDA)-based commercial assay for the simultaneous detection of HSV-1 and HSV-2 Comparison of transcription mediated amplification (TMA) and reverse transcription polymerase chain reaction (RT-PCR) for detection of hepatitis C virus RNA in liver tissue Comparative evaluation of the Nanosphere Verigene RV+ assay and the Simplexa Flu A/B & RSV kit for detection of influenza and respiratory syncytial viruses Evaluation of the Verigene EP IUO test for the rapid detection of bacterial and toxin causes of gastrointestinal infection Evaluation of FilmArray and Verigene systems for rapid identification of positive blood cultures Evaluation and verification of the nanosphere Verigene RV+ assay for detection of influenza A/B and H1/H3 subtyping Principles of the xTAG respiratory viral panel assay (RVP Assay) Clinical evaluation of the Luminex NxTAG respiratory pathogen panel Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG(R) RVP fast assay for the detection of respiratory viruses Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP fast multiplex assays for detection of respiratory viruses Phylogenetic structure of the prokaryotic domain: the primary kingdoms Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline Broad-range 16S rRNA gene polymerase chain reaction for diagnosis of culturenegative bacterial infections Utility of 16S rDNA sequencing for identification of rare pathogenic bacteria Identification of bacteria in formalin-fixed, paraffin-embedded heart valve tissue via 16S rRNA gene nucleotide sequencing 16S rRNA sequencing in routine bacterial identification: a 30-month experiment Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological 'sarcoid' may contain such DNA Broad-range PCR and sequencing in routine diagnosis of infective endocarditis Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock Emerging technologies for rapid identification of bloodstream pathogens ID learning unit-diagnostics update: current laboratory methods for rapid pathogen identification in patients with bloodstream infections Nonculture techniques for the detection of bacteremia and fungemia an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens Spectrum of enteropathogens detected by the FilmArray GI panel in a multicentre study of community-acquired gastroenteritis Multi-center clinical evaluation of a multiplex meningitis/encephalitis PCR panel for simultaneous detection of bacteria, yeasts and viruses in cerebrospinal fluid specimens General Meeting Enhancing pathogen identification in patients with meningitis and a negative Gram stain using the BioFire FilmArray((R)) meningitis/ encephalitis panel Performance evaluation of the Verigene(R) (Nanosphere) and FilmArray(R) (BioFire(R)) molecular assays for identification of causative organisms in bacterial bloodstream infections Clinical evaluation of the FilmArray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp.
(C. jejuni and C. coli), and Shiga toxin 1 and 2 genes Comparison of the BD MAX enteric bacterial panel to routine culture methods for detection of Campylobacter, enterohemorrhagic Escherichia coli (O157), Salmonella, and Shigella isolates in preserved stool specimens Comparison of the BD MAX(R) enteric bacterial panel assay with conventional diagnostic procedures in diarrheal stool samples Evaluation of the BD Max enteric parasite panel for clinical diagnostics Comparison of three commercial RT-PCR systems for the detection of respiratory viruses Next-generation sequencing for infectious disease diagnosis and management: a report of the Association for Molecular Pathology Clinical detection and characterization of bacterial pathogens in the genomics era Routine use of microbial whole genome sequencing in diagnostic and public health microbiology Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemaseproducing Enterobacteriaceae Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing Genomically informed surveillance for carbapenem-resistant enterobacteriaceae in a health care system Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences A cloudcompatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples Actionable diagnosis of neuroleptospirosis by next-generation sequencing Mirror, mirror on the wall: which microbiomes will help heal them all The microbiome revolution The human microbiome: at the interface of health and disease Sequencing the human microbiome in health and disease Characterization of the gut microbiome using 16S or shotgun metagenomics Broad-range direct detection and identification of fungi by use of the PLEX-ID PCR-electrospray ionization mass spectrometry (ESI-MS) system Polymerase-chain reaction/electrospray ionization-mass spectrometry for the detection of bacteria and fungi in bronchoalveolar lavage fluids: a prospective observational study Broad-range PCRelectrospray ionization mass spectrometry for detection and typing of adenovirus and other opportunistic viruses in stem cell transplant patients Detection and identification of yeasts from formalin-fixed, paraffinembedded tissue by use of PCR-electrospray ionization mass spectrometry Advantages and limitations of direct PCR amplification of bacterial 16S-rDNA from resected heart tissue or swabs followed by direct sequencing for diagnosing infective endocarditis: a retrospective analysis in the routine clinical setting Using PCR-based detection and genotyping to trace Streptococcus salivarius meningitis outbreak strain to oral flora of radiology physician assistant Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections Whipple disease diagnosed with PCR using formalin-fixed paraffin-embedded specimens of the intestinal mucosa Use of broad range16S rDNA PCR in clinical microbiology Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi DNA barcoding of fungi causing infections in humans and animals Intraspecific ITS variability in the kingdom fungi as expressed in the international sequence databases and its implications for molecular species identification Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS [corrected] software for identification of filamentous fungi in a clinical laboratory International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database-the quality controlled standard tool for routine identification of human and animal pathogenic fungi Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation Utility of DNA sequencing for direct identification of invasive fungi from fresh and formalin-fixed specimens Molecular methods to improve diagnosis and identification of mucormycosis Direct sequencing and RipSeq interpretation as a tool for identification of polymicrobial infections High-throughput sequencing technologies A glimpse into past, present, and future DNA sequencing Making the leap from research laboratory to clinic: challenges and opportunities for next-generation sequencing in infectious disease diagnostics
key: cord-269975-1ebmq7t8 authors: Duplantier, Allen J.; Shurtleff, Amy C.; Miller, Cheryl; Chiang, Chih-Yuan; Panchal, Rekha G.; Sunay, Melek title: Combating biothreat pathogens: ongoing efforts for countermeasure development and unique challenges date: 2020-05-27 journal: Drug Discovery Targeting Drug-Resistant Bacteria DOI: 10.1016/b978-0-12-818480-6.00007-2 sha: doc_id: 269975 cord_uid: 1ebmq7t8 Research to discover and develop antibacterial and antiviral drugs with potent activity against pathogens of biothreat concern presents unique methodological and process-driven challenges.
Herein, we review laboratory approaches for finding new antibodies, antibiotics, and antiviral molecules for pathogens of biothreat concern.
Using high-throughput screening techniques, molecules that directly inhibit a pathogen’s entry, replication, or growth can be identified.
Alternatively, molecules that target host proteins can be interesting targets for development when countering biothreat pathogens, due to the modulation of the host immune response or targeting proteins that interfere with the pathways required by the pathogen for replication.
Monoclonal and cocktail antibody therapies approved by the Food and Drug Administration for countering anthrax and under development for treatment of Ebola virus infection are discussed.
A comprehensive tabular review of current in vitro, in vivo, pharmacokinetic and efficacy datasets has been presented for biothreat pathogens of greatest concern.
Finally, clinical trials and animal rule or traditional drug approval pathways are also reviewed.
Opinions; interpretations; conclusions; and recommendations are those of the authors and are not necessarily endorsed by the US Army.
The concept of bioterrorism and the intentional release of biothreat agents for purposes of harm to human and agricultural interests stimulates discussion of some unanswerable questions.
Questions ranging from protection of a nation's security to military defense tactics, all point to the gravity of the problem for which scientists are working together in many areas of study such as the development of novel medical countermeasures to combat lethal infections, the prevention of the spread of disease in the general populace, and design of field-worthy diagnostic tools.
A biothreat organism is generally thought to be one causing severe or lethal disease or has potential to induce panic over the prospect of infection therewith; one with high pathogenicity and/or contagious infectivity; one with strong environmental stability or probable transmission as an aerosol; one with ease of large-scale production for far-reaching dissemination; and one that can be controlled for directing the release to only the intended target rather than accidental harm to the perpetrator [1] .
Improved preparedness for intentional release of bacteria, viruses, and toxins will not only protect military positions and strategies but will also increase ability to combat disease in naturally occurring epidemics of diseases caused by some of these organisms.
the priorities for the development of medical countermeasures against these organisms have been defined through international discussions [2] [3] [4] .
Currently classified as Tier 1 select agents are those pathogens of grave concern, whereas other useful classification categories are in use by US government entities such as National Institute of Allergy and Infectious Diseases and the US Centers for Disease Control and Prevention (CDC) list, denoting the pathogens as Category A, B, and C agents [5] .
Various biothreat pathogens addressed in this chapter are grouped by general category and disease associated therewith (Table 7 .1).
There are many organisms on the CDC list; consequently, not all of them are addressed in this chapter [4] .
The authors of this chapter have endeavored to provide a comprehensive survey of the literature and described the development Hemorrhagic fever A Crimean-Congo hemorrhagic fever virus Hemorrhagic fever A, B, C Denote additional categorization into Category A, B, and C pathogens, per NIAID [5] .
a Tier 1 agents of human pathogenicity are presented [4] .
Two more Tier 1 agents are rinderpest virus and foot-and-mouth disease virus, which are of agricultural concern (not covered in this chapter).
b Important non-Tier 1 agents for which countermeasures are described in this chapter [4, 5] .
See www.selectagents.gov for a comprehensive list of non-Tier 1 Select Agents and Toxins.SARS, Severe acute respiratory syndrome; MERS, Middle East respiratory syndrome-related coronavirus.
of medical countermeasures against high-priority bacterial and viral biothreat agents where the most progress has been made, and/or the most novel ground has been broken.
Bacteria cause disease in humans by invading tissue, altering the host immune response, and/or producing toxins or virulence factors.
Many of the bacteria described here are difficult to treat clinically.
The potential bacterial threat agents that pose the greatest risk to national security are ones that can be easily disseminated and result in high morbidity and mortality rates.
The former Soviet Union is known to have weaponized at least 30 viral and bacterial agents, including several vaccine or drug-resistant strains [6] .
Each agent has unique properties that present both a distinct threat and challenge for detection, prevention, and control.
Bacillus anthracis and Clostridium botulinum are Gram-positive bacterial agents of grave biothreat concern.
B. anthracis is a spore-forming bacterium that causes cutaneous, respiratory, or intestinal forms of anthrax disease, which is an acute, rapidly progressing infection in any form.
The B. anthracis spores are highly stable both in the environment and in the exposed individuals and can be easily disseminated via the aerosol route, thus making it a dangerous bacterium [7] .
The anthrax attacks in 2001 caused widespread panic, damage, disease, and death, which increased national awareness to the threat of bioterrorism.
The bacterium produces a lethal toxin that disrupts the host innate responses during the early stages of infection and ultimately leads to septicemia and death of the host (Fig.
7.1A) .
Antibiotic treatment requires a lengthy dosing regimen and is effective only if it is initiated during the early stage of the infection.
Two monoclonal antibody (mAB)-based anthrax antitoxin therapeutics [Abthrax (raxibacumab) and Anthim (obiltoxaximab)] have been approved by the US Food and Drug Administration (FDA) and included in the Strategic National Stockpile for treating inhalational anthrax [8] .
BioThrax, the only licensed anthrax vaccine, is indicated for preexposure prophylaxis of disease in persons at high risk of exposure and postexposure prophylaxis of disease following suspected or confirmed B. anthracis exposure [8] .
Botulinum neurotoxin (BoNT), produced by C. botulinum, is extremely potent, lethal, and easy to produce, transport, and misuse.
The toxin itself is the select agent, but the Clostridium organism, as an isolate capable of producing the toxin, is also classified as a Tier 1 select agent.
There are seven serotypically distinct BoNTs (serotypes A-G) and they act by blocking neurotransmitter release and thereby preventing transmission of nerve impulses, which can lead to botulism, hallmarks of which are paralysis and respiratory arrest [9] (Fig.
7.1B) .
Current treatment is limited to Botulism Immune Globulin Intravenous, human-derived antibotulism toxin antibodies for the treatment of infant botulism types A and B, and Botulism Antitoxin Heptavalent (A-G), a mixture of immune globulin fragments developed from equine plasma for the symptomatic treatment of adult and pediatric botulism.
The US Army has developed a similar antitoxin based on equine neutralizing antibodies that is effective against a number of serotypes, but there is a limited supply and risk of horse serum sensitivity.
An investigational vaccine also exists, but it offers limited protection and painful side effects [10] .
Many of the bacterial agents of biothreat concern are intracellular Gram-negative organisms.
Intracellular bacteria are particularly difficult to treat because the intracellular niche protects bacteria from the innate or adaptive immune surveillance.
These bacteria can enter host cells through phagocytosis, and to prevent their destruction in the endocytic pathways, intracellular bacteria have adapted to survive in a host lysosome and replicate within the acidic endolysosomal compartment (e.g., Coxiella burnetii).
Another intracellular bacteria Brucella spp.
can traffic from a mature lysosome to endoplasmic reticulum-derived compartments, while bacteria such as Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis can prevent acidification and maturation of the phagosome and escape to the cytosol, where they can replicate and then disseminate to neighboring cells [12] [13] [14] .
One characteristic feature of the B. mallei and B. pseudomallei intracellular life cycle is the fusion of infected mononuclear cells, forming multinucleated giant cells (MNGCs).
Although the role of B. pseudomallei-induced MNGCs is unclear, it is believed that cell fusion facilitates localized dissemination of the bacteria [15, 16] (Fig.
7.1C) .
Brucella spp.
are nonmotile bacteria that cause brucellosis, a world-wide chronic debilitating disease in both humans and animals.
Although not typically fatal, Brucella spp.
are stable and infectious as aerosols and can lead to sterility and abortions [17] .
The nonmotile bacillus B. mallei is the causative agent of glanders that usually infects equids but is highly infectious to humans at low doses, producing septicemia, severe pulmonary infection, and chronic inflammation of the skin and eyes.
B. mallei can be easily aerosolized, and even with antibiotic treatment there are high mortality rates [18] .
The motile bacterium B. pseudomallei, the causative agent of melioidosis, is a close relative to B. mallei and can lead to severe illness in humans, such as pulmonary infection and septic shock.
B. pseudomallei is an environmental saprophyte that is naturally resistant to many antibiotics [19] .
Q-fever is caused by direct contact with the nonmotile bacterium C. burnetii that was previously weaponized because of its ease of aerosolization, its environmental stability, and its ability to infect animals or humans with a single bacterium [20] .
Q-fever is not typically lethal but can be incapacitating, causing fever and difficulty breathing, and antibiotic therapy is not always effective, thus leading to persistent infections.
F. tularensis is the causative agent of tularemia and is highly infectious, resulting in an acute, rapidly progressing local or systemic infection [21] .
Y. pestis, the causative agent of plague, is a nonmotile bacterium that can be disseminated by aerosol, transmitted from person-to-person, and is characterized by a severe clinical disease course with potentially high case-fatality rates.
There is a limited window for effective treatment against plague, since the resulting respiratory and circulatory collapse from septic shock is usually fatal [22] .
There are a great number of viruses on the list of select agents and toxins, and some of these can only be handled in the laboratory at the highest biocontainment level (biosafety level 4).
The causative agents of some of the most lethal hemorrhagic fever infections are filoviruses, paramyxoviruses, and arenaviruses.
Filoviruses such as Ebola virus (EBOV) and Marburg virus infect humans and nonhuman primates (NHPs) and have caused large outbreaks in recent years.
These viruses are likely transmitted in nature by fruit bats and are spread from person to person via contact with body fluids or fomites [23] .
Marburg virus was reportedly weaponized through activities carried out by the former Soviet Union [24] .
Because of the large scale of recent EBOV outbreaks, this virus may have become available to nefarious people through access to corpses and contaminated clinical waste.
Two more viruses transmitted in nature at least in part by fruit bats are the Nipah and Hendra paramyxoviruses that belong to the Henipaviridae family and cause severe neurological and/or respiratory diseases in humans [25] .
These viruses can infect many domestic and agricultural animal species and are frequently transmitted between humans via droplets or fomites, leading to concerns of new human outbreaks in areas of Malaysia, Bangladesh, India, and Australia where case-fatality rates range from 40% to 100% [26] .
Arenaviruses, specifically Lassa virus (LASV) and Junin virus (JUNV), Machupo, and other South American viruses are transmitted not by bats but by peridomestic rodent species [27] .
None of the filoviruses or henipaviruses has any FDA-approved therapeutics or vaccines available for prevention or treatment of human disease, and while ribavirin is sometimes used to treat Lassa fever, it is not a terribly effective drug against this viral infection [28] .
Variola virus (VARV) is the causative agent of smallpox, a human viral disease for which tecovirimat was recently approved as a therapeutic by the FDA, a successful therapeutic development story [29] .
This pathogen has been eradicated since 1979 through successful vaccine campaigns [30] , but because the vaccine is no longer administered in most countries, populations may be susceptible in the event that VARV or an intentionally modified or related poxvirus with similar virulence factors and similar human lethality is resurrected [31] .
Arthropod-transmitted alphaviruses and bunyaviruses are also biothreat concerns.
For the alphaviruses specifically, the Venezuelan encephalitis viruses (VEEV), Eastern encephalitis viruses, and Western equine encephalitis viruses belonging to the family Togaviridae are found in the Americas and cause equine disease [32, 33] .
These new world alphaviruses cause encephalitis-like symptoms, are stable in the environment, grow to high titers easily in cell culture, are highly infectious by aerosol, and affect humans with incapacitating neurological disease, sometimes with high morbidity rates [33] .
Bunyaviruses such as Rift Valley fever virus and the related tick-transmitted nairovirus causing Crimean-Congo hemorrhagic fever also cause human diseases with high morbidity and mortality rates.
Some investigational new drug vaccines exist for these agents.
These viruses replicate quickly in humans and cause rapid disease; therefore the timing for therapeutic intervention is short, making treatment postinfection very challenging.
The lack of approved therapeutics available to combat biothreats may be in part attributed to the unique challenges for the discovery and development process of evaluating drugs that target select agents.
Foremost is the implementation of high-throughput screening (HTS) efforts for the discovery of new compounds against authentic or wildtype biothreat bacterial and viral pathogens [34] .
Specifically, the requirement of work to be performed in high-level biocontainment laboratories (BSL3 or BSL4) is a major limiting factor since laboratories with these capabilities are not widely available.
In addition, highly trained personnel that can handle infectious agents use robotic instruments and adhere to operational, engineering, and government regulations are a critical requirement for working with biothreat agents [34] .
In the United States, strict guidelines have been instated for generating government-approved methods and processes for inactivation of pathogens before plates/samples can be brought out of biocontainment suites for further experimentation, and to track the inactivated material [35] .
Other challenges that need to be considered include the prevention of pathogen aerosolization while handling screening plates in biocontainment laboratories and ensuring that inactivation chemicals and methods are compatible with downstream procedures.
New therapeutics effective against both natural and engineered resistant forms of bacterium are vital to the biodefense armory.
Screening for novel antimicrobials is traditionally done by scoring for growth inhibition in vitro, using the standard Clinical & Laboratory Standards Institute guidelines.
This generally involves performing a dose-response assay in a multiwell plate format and monitoring growth in the absence or presence of the test compounds.
The compound concentration that shows no visible growth is considered the minimum inhibitory concentration (MIC).
Over the years, this approach has led to the discovery of only a limited number of novel antimicrobial compounds and resistance has already been generated against most of the antibiotics used in the clinic.
One disadvantage to this approach is the inability to identify potent immunomodulatory compounds against intracellular pathogens that require the host for replication.
New approaches to understanding bacterial pathogenesis have enabled researchers to elucidate mechanisms that could be targeted to control and clear infection in lieu of simply targeting in vitro bacterial viability.
Targeting the host under in vivo-like conditions (e.g., in cell culture or animal models) will be a key feature of study design to combatting intracellular pathogens that require the host for invasion and replication and will likely identify new host-directed therapeutics.
The development of host-directed therapeutic (HDT) strategy relies on an understanding of the interactions between pathogens and their hosts and appropriate tools and HTS assays to screen and identify therapeutics.
Technological progress in assay miniaturization has emerged from a combination of advanced robotic systems, high-throughput microscopy, automated image analysis, and data analysis using powerful bioinformatics tools, and this has led to the development of high-content imaging (HCI), allowing for large-scale quantification of multiple cellular phenotypes at the system level.
Such phenotypic screening platforms rely on physiologically relevant host cell types that are permissive to pathogen infection and have the potential to identify compounds that modulate relevant biological processes in an unbiased, target, and mechanism-agnostic fashion.
This cell-based approach has the added advantage that compounds that have greater mammalian cell membrane permeability, reduced cellular toxicity, and target the host proteins will be readily identified in the context of their desirable function in cells.
Pharmacologically active compounds can be selected that inhibit the uptake or intracellular replication of the bacterium or disrupt the host-pathogen interactions.
The general workflow for high-throughput, imagebased phenotypic screening approach to identify HDTs is outlined in Fig.
7 .2 [36, 37] .
Using bacterial antigen-specific antibody to detect bacteria, this method can quantitate the number of intracellular or cell-associated bacteria and the effect of the compounds in reducing the bacterial number (% inhibition of bacterial infection), and cellular toxicity (based on loss in cell number).
Alternatively, one can use HCI to quantitate the morphological changes of MNGCs based on nuclei number and MNGC size/area and use this phenotype to screen and identify compounds that prevent bacterial spread [38] .
To overcome the problem of multidrug resistant bacteria, there is a growing focus on identifying small molecules that target drug resistant mechanisms or virulence factors, or agents that prevent/disrupt biofilm formation.
Virulence factors, such as secretion systems in gram negative bacterial pathogens, are promising therapeutic targets.
Specifically, the Type 3 Secretion System (T3SS) present in Y. pestis is responsible for injecting effectors that target the cytoskeleton and proinflammatory signaling pathways.
A number of techniques have been used to screen and identify potential T3SS inhibitors that can be adapted for biothreat pathogens.
These include an enzyme-linked immunosorbent assay (ELISA)-based detection of proteins secreted from enteropathogenic Escherichia coli (EPEC), inhibition of sheep erythrocyte lysis by EPEC, inhibition of induction of a yopE luciferase fusion in Yersinia pseudotuberculosis, and a Pseudomonas aeruginosa cell-based bioluminescent reporter screen [40] [41] [42] [43] .
Using a high-throughput luminescence screening assay, three compounds were identified that inhibit Y. pestis T3SS-mediated cytotoxicity that relieves the growth inhibition associated with in vitro activation of T3SS [44] .
Another promising approach to disarm the bacteria is to prevent/disrupt biofilms, a barrier produced by bacteria to protect itself from the aggressive host environment.
Small molecule therapeutics that specifically disrupt or prevent the biofilm formation could be used in combination with antibiotics.
The common method to quantitate biofilms is a colorimetric-based assay that utilizes a crystal violet dye to stain the biofilms and subsequent extraction of the dye using organic solvents or detergents [45] followed by absorbance measurement.
To improve sensitivity, robustness, and throughput, a fluorescent-dye-based assay was developed, wherein the biofilms are stained with FM1-43 fluorescent dye and fluorescence signal is measured following organic extraction of the dye [46] .
Screening of a small molecule library in this assay identified rifabutin and ethavarine, as potential inhibitors of B. pseudomallei (Bp82) and Acinetobacter baumannii biofilm production, respectively, without directly affecting the bacterial growth.
2 Phenotypic screening using high-throughput HCI.
Cells susceptible to the pathogen of interest are seeded in HCI plates.
Next day, cells are pretreated with appropriate concentration of the compounds and then infected with the pathogen of interest for optimal time wherein 70%-80% infection results.
The infected plates are then submerged in 10% formalin for 24 h to inactivate the pathogen and to fix the cells.
Immunofluorescence staining is then performed, using a primary antibody specific to a pathogen antigen, and an appropriate fluorescence-labeled secondary antibody.
Dyes such as cell mask red and Hoechst are added to detect the cell cytoplasm and nuclei, respectively.
Automated image acquisition and analysis is performed and data are analyzed using Columbus software to quantitate the percentage of inhibition of pathogen infection and loss in cell number that represents cellular toxicity, in the presence of the compound [39] .
HCI, high-content imaging.
There is a possibility that therapeutics targeting the virulence factors or other drug resistance mechanisms may not be effective by themselves and will need to be evaluated in combination with antibiotics to treat multiple drug resistance (MDR) infections.
Thus screening experiments designed to find combination therapies are warranted.
To determine the synergy of two drugs (antibiotic and nonantibiotic), conventional checkerboard assays are set up wherein the two drugs are tested in combination at varying concentrations and the MIC of each drug either alone or in combination is then used to calculate the fractional inhibitory concentration [47] .
Similarly, in the case of the biofilm assay, testing a biofilm disruptor and an antibiotic together at varying concentrations will help one to assess the effectiveness of combination therapies.
Unlike most bacteria, viruses require the mammalian host for replication.
The virus life cycle can be divided into distinct stages that include the entry, uncoating, replication, genome packaging, assembly, maturation, and budding.
Various cell-based and in vitro biochemical assays have been developed to study virus life cycles as well as to screen and identify antivirals [48] .
The conventional plaque-forming assay used to evaluate antivirals is time-consuming, not amenable for HTS, and not very robust.
Alternatively, in the absence of more sophisticated instruments or technologies, a virus-induced cytopathic effect can be used as an endpoint to test antivirals.
With advances in imaging instruments and informatics, a cell-based HCI platform ( Fig.
7 .2) that uses viral antigen-specific antibodies to detect and quantitate the viral infection is now a general approach to identify compounds that inhibit viral infection [39, 49, 50] .
However, this approach will not provide information on which steps in the viral life cycle the inhibitors are disrupting.
To help one to deconvolute the mechanism of action of identified hits ( Fig.
7.
3), cells pretreated with an inhibitor prior to virus exposure can potentially identify compounds that inhibit viral entry, while treatment of cells after exposure (i.e., after the entry step) would identify compounds that inhibit intracellular replication and/or viral spread.
Assays utilizing recombinant noninfectious viruses have been generated to screen and identify inhibitors that target different stages of the viral life cycle [48] (Fig.
7.
3).
Pseudotyped virion assays are well suited as safe alternatives for HTS, since BSL3 and BSL4 wild-type pathogens are not required to complete the screens.
These assays are based on viral vectors that harbor glycoproteins (GPs) of different enveloped viruses and a reporter gene such as green fluorescent protein (GFP) or luciferase flanked by packaging signals, are used to generate chimeric replication-deficient viruses, and then used to screen and identify entry inhibitors.
This approach has successfully identified entry inhibitors for Lassa, Ebola, and Nipah viruses [51] [52] [53] [54] .
Cell fusion assays, including cell-cell or cell-virus fusions, have been developed to screen and identify HIV-1 fusion inhibitors, but to date no such assays have been developed for biothreat viruses [55] .
Reverse genetic systems or minigenome assays have proven to be valuable models to study RNA virus replication and transcription.
This model system is used to screen and identify antivirals [56, 57] .
Replication competent minigenome systems wherein some of the viral open reading frame is replaced with a reporter gene (GFP or luciferase) and the cDNA copy cloned into a plasmid is cotransfected into mammalian cells with individual plasmids each containing a viral ribonucleoprotein (RNP).
The target genes in the expression vectors are under the control of either a mammalian RNA polymerase I or II or T7 RNA polymerase (which will require transfection of a plasmid containing the T7 RNA polymerase) promoters.
Following transcription, the resulting viral RNA is complexed with the RNP components and there is subsequent replication of the virus genome and expression of the reporter protein.
Minigenome systems have been developed for several biothreat pathogens, including filoviruses, arenaviruses, and bunyaviruses, and have been used to screen and identify small molecule inhibitors of filovirus and arenavirus replication [56] [57] [58] [59] [60] .
To study the viral assembly and budding, another surrogate model that can be used is based on virus-like particles (VLPs) that are mimics of viral protein assemblies made by reconstituting the viral recombinant structural proteins.
VLPs are noninfectious as they do not contain any viral genome, but are intrinsically immunogenic, and hence are being extensively investigated as potential vaccine candidates [61] .
In the case of the EBOV VLP-based assay, cotransfection of plasmids encoding the viral GP and the matrix protein (VP40) results in spontaneous formation of filamentous VLPs that are released into the medium and can be quantitated by ELISA [62] ; thus this model can be useful in drug discovery research [63] .
To identify inhibitors of viral genome replication, in vitro biochemical assays targeting viral enzymes such as polymerases, methyltransferases, helicases, as well as viral and host proteases such as cathepsins or kinases have been developed.
A number of antivirals that have been approved by the FDA target either the DNA or RNA polymerases.
Incorporation of radioactive nucleotide either to a DNA oligonucleotide by DNA polymerase [64] or to a homopolymeric RNA as a template by RNA polymerase are common methods to determine polymerase activity [65] .
A recent study reported the use of fluorescent dye to detect the double-stranded RNA and the feasibility of developing this assay to screen and identify inhibitors of Zika virus polymerase activity [66] .
The host lysosomal protease cathepsin L (Cat L) is necessary for the processing and cleavage of the GP of enveloped viruses, so that the virus can fuse with the host cell membrane and gain entry into the host.
Thus Cat L has been regarded as an ideal target for drug discovery.
A fluorescence resonance energy transfer (FRET)-based Cat L enzymatic assay was developed, wherein peptides derived from GPs of viruses such as Ebola, Nipah, Hendra, and severe acute respiratory syndrome and Middle East respiratory syndrome coronavirus and containing Cat L cleavage site were chemically conjugated with a quencher 5-carboxytetramethylrhodamine at the N-terminus and 5-carboxyfluorescein fluorophore at the C-terminus [67] .
The intact peptides exhibited minimal to no fluorescence, but following cleavage of the peptide by Cat L, there was an increase in fluorescence intensity.
Screening of a chemical library in this assay identified small molecules that selectively inhibited Cat L-mediated cleavage of multiple viral peptides over host proneuropeptide Y [67] .
Viral proteases are also good drug targets as they play a vital role in viral replication.
For example, the NS2B-NS3 protease is highly conserved among the flaviviruses and a FRET-based enzymatic assay using a synthetic peptide substrate [68] was developed to identify West Nile virus protease inhibitors [69] .
Functional genomic screening using gene-trapping, CRISPR's gene editing, or RNA interference (RNAi) technologies has been applied to identify host factors that are required for replication or involved in pathogenesis of several biothreat viral and bacterial agents and are summarized in Table 7 .2.
The activities of several identified host factors can be perturbed by small molecules and thus serve as potential therapeutic platforms.
For example, it was demonstrated that the novel host factor inositol-requiring enzyme 1α is required for Brucella infection in mammalian cells [70] .
Reducing the levels of either the retromer cargo-adapter complex or retromer-associated sorting nexins abrogated C. burnetii replication [71] .
Multiple host kinases such as cAMP-dependent protein kinase, protein kinase B, and protein kinase C all play a role during C. burnetii infections [72, 73] .
Zhou et al.
[74] identified TNFRSF9 and SERPINI1 that may promote activated macrophages in controlling F. tularensis replication.
Akimana et al.
[13] showed that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and that phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis.
Connor et al.
[14] revealed that 71 host proteins are required for intracellular survival of Y. pestis.
Of particular, interest was the enrichment for genes involved in endosome recycling.
Using the gene trapping approach, Carette et al.
[76] first identified several host factors that are required for EBOV infection.
These include a cholesterol transporter Niemann-Pick C1 factor involved in the fusion of endosomes and lysosomes (homotypic fusion and protein sorting complex), biogenesis of endosomes (PIKFYVE), lysosomes (BLOC1S1, BLOC1S2), and targeting of luminal cargo to the endocytic pathway [76] .
Many of these hits reoccurred in several CRISPR and small interfering RNA (siRNA)/shRNA screenings [77, 78, 81] .
In addition, lysosomal protein (BRI3) and a GTPase involved in the regulation of vesicle trafficking (RAB39B), PI3K, calcium/ calmodulin kinase-related network, and de novo pyrimidine synthesis pathway are essential for EBOV replication and transcription [78] [79] [80] .
The application of RNAi screening has been utilized for other viral pathogens such as henipavirus, JUNV, poxvirus, Vaccinia virus, and VEEV [75, [82] [83] [84] [85] [86] .
It is demonstrated that catalytic activity of fibrillarin, the enzymatic subunit of the snoRNP complex that is responsible for catalyzing the transfer of a methyl donor from a bound cofactor S-adenosyl methionine to ribose sugars of the target pre-rRNA, is required for henipavirus infection [82] .
Voltage-gated calcium channel (VGCC) subunits were shown to be important in JUNV-cell fusion and entry into cells.
Gabapentin, an FDA approved anticonvulsant drug against α 2 δ 2 subunitcontaining VGCCs, inhibited replication of the vaccine strain of JUNV in mice [75] .
Other siRNA-based screens against V. virus identified that AMP-activated protein kinase (AMPK) promotes viral entry through the control of actin dynamics, and knockdown of nuclear pore protein (Nup62) arrests virion morphogenesis [83] [84] [85] .
Lastly, an siRNA screen identified trafficking host factors that modulate VEEV infection [86] .
An alternative approach to gain an in-depth understanding of host-pathogen interactions during infection is to construct a protein-protein interaction network between host protein and bacterial virulence factors.
Using a yeast two-hybrid (Y2H) library, Memiševic´ et al.
[16] identified a molecular network that governs B. mallei infection.
Similarly, a Y2H study conducted by Yang et al.
[87] showed the involvement of focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, Toll-like receptor (TLR), and MAPK signaling pathways during Y. pestis infection.
To complement the Y2H study, reverse-phase protein microarray analysis was used to interrogate changes in protein expression and posttranslational modification.
This further revealed the roles of AMPK-α1, Src, and GSK3β in regulating B. mallei and B. pseudomallei infection [88] , and thus, as viable host targets for countermeasure development.
Prior to the initiation of medical countermeasure development against specific pathogens, a target product profile (TPP) is needed to define the required features of potential drug candidates (e.g., route of administration, prophylactic vs therapeutic, trigger to treat, and onset of action requirements).
Once a TPP is in place, a screening funnel is drafted that sets laboratory criteria and defines clear go/no-go decision points that are needed to progress countermeasures from discovery through preclinical development and into human clinical trials.
Irrespective of the types of assays used for countermeasure screening, compounds identified as having significant inhibition in primary screens are validated in subsequent dose-response experiments to determine the half maximal effective concentration (EC 50 ) and cytotoxic concentration (CC 50 ).
Potent compounds that have an adequate selectivity index (e.g., >10) that is defined as a ratio of CC 50 / EC 50 , are then often tested in orthogonal assays in appropriate cells/tissues to better understand or validate the antipathogen activity.
Ideally, compounds are further optimized for potency, selectivity, physicochemical, and pharmacokinetic (PK) properties and safety prior to in vivo evaluation to assess efficacy in appropriate animal models of infection ( Fig.
7.4) .
Many of the therapeutics that are in different stages of either preclinical or clinical development for select biothreat pathogens include small molecule antivirals (Tables 7.3 and 7.4), antibody (or antibody cocktails) against viruses or bacteria/virulence factors (Table 7 .5), and combination drug therapy (Table 7 .6).
The increased use of antivirals and antibiotics has set the stage for rapid adaptation mechanisms that microbes can use to counteract them.
The development of antimicrobial resistance is one of the biggest public health threats and hence alternative approaches to treat infectious diseases are urgently needed.
Table 7 .7 lists the resistance mechanisms identified in each biothreat bacterial pathogen and provides references for targets of resistance.
Since stand-alone antibiotics may not be sufficient to overcome resistance and/or completely clear some biothreat bacterial infections, we have also included encouraging data on host directed therapeutics, and combination therapy.
HDT is an emerging approach in the field of antiinfectives discovery.
The strategies behind HDT can include modulation of host immune responses, or interference/manipulation/targeting of host-cell factors that are required for pathogen replication [202] .
For example, in a potential bioterror scenario, where the identity of the etiological agent causing the disease is unknown, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens.
Although no FDA-approved HDT therapies are yet available for treating infectious diseases, we have summarized in this section the antimicrobial Primary screening of small molecule chemical libraries in the phenotypic HCI assay will identify compounds that inhibit pathogen infection as well as those that may contribute to cellular toxicity.
Generally, hits that show ≥50% infection inhibition and ≤20% loss in cell number are then subjected to hit triage or in silico filtering wherein compounds with optimal physicochemical properties such as solubility, Lipinski's Rule of 5, metabolism are selected for potency testing in the phenotypic screening assays.
Compounds that exhibit an EC 50 ≤ 1 µM and SI, which is a ratio of CC 50 /EC 50 > 10, are then further optimized through iterative cycles of synthesis, testing in cell-based and orthogonal assays and in in vitro ADMET studies to improve potency and physicochemical properties.
If the target of the hit molecule is identified then a target-based screen is performed and the hits identified are optimized through the iterative structureactivity relationship cycle.
The Lead series candidates are then evaluated in vivo for their pharmacokinetic properties and then for efficacy in appropriate challenge models of infection.
ADMET, absorption, distribution, metabolism, excretion, and toxicity; HCI, high-content imaging; SI, selectivity index.
Antibiotics AIGIV Combination therapy with antibiotics and AIGIV is more effective than antibiotics alone in a rabbit model of inhalational anthrax and improved survival compared to the antibiotic treatment alone [155] Ciprofloxacin Clindamycin Treatment of rabbits with systemic anthrax with clindamycin and ciprofloxacin had improved efficacy compared to monotherapy and could be used to prevent relapse of infection [156] Ciprofloxacin Combination therapy for anthrax, including antiprotective antigen (PA IgG) antibodies and ciprofloxacin in a rodent anthrax model increased survival significantly compared to ciprofloxacin treatment alone [157] Levofloxacin Raxibacumab Combination therapy with raxibacumab, an IgG1 monoclonal antibody that binds protective antigen, and the antibiotic levofloxacin provides protection in rabbits late in the disease course [158] Oligochlorophen Antibiotics Targeting cytoskeletal proteins such as FtsZ with oligochlorophen analogs is a promising new treatment method that has a 10-fold lower development of resistance compared to antibiotics used for anthrax treatment in humans [159] Penicillin, meropenem, or rifampin Linezolid Treatment of antibiotic-resistant inhalation anthrax with linezolid and penicillin, meropenem, or rifampin had the inhibitory effect on mean lethal factor levels compared to the control groups and successfully treated fluoroquinolone-resistant B. anthracis infection [160] Rifampin Clindamycin Combination therapy for anthrax with rifampin and clindamycin was shown to be synergistic in vitro [161] Brucella spp.
Rifampin Successful combination therapies used to treat pulmonary brucellosis in humans is doxycycline and rifampin for 6 weeks [162] Burkholderia mallei Antibiotic Heat-killed vaccine Combination of an antibiotic moxifloxacin, azithromycin, or sulfamethoxazole-trimethoprim, and vaccination using heat-killed B. mallei can protect BALB/c mice from lethal glanders infection, potentially by stimulating immune responses, such as gamma interferon, which acts synergistically with antibiotic therapy to inhibit bacterial growth [163] Enrofloxacin, trimethoprim, and sulfadiazine Doxycycline Successful 12-week combination treatment of parenteral administration of enrofloxacin and trimethoprim with sulfadiazine followed by oral administration of doxycycline eliminated B. mallei from glanderous horses during an outbreak [164] Burkholderia pseudomallei Combination therapy with farnesol a sesquiterpene alcohol that damages biofilm matrix and interferes with cell wall and peptidoglycan biosynthesis, facilitates antimicrobial penetration, and reduces the minimum biofilm eradication concentration for ceftazidime, amoxicillin, doxycycline, and sulfamethoxazole-trimethoprim in vitro [165, 166] Ceftazidime Avibactam Avibactam restores susceptibility to ceftazidime for genetically diverse extremely drug resistant isolates of Burkholderia from cystic fibrosis patients by binding PenA and the combination treatment significantly improved survival of larvae infected with the drug resistant isolates [166] Ceftazidime IFN-γ Interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular B. pseudomallei and markedly increases the effectiveness of antimicrobial therapy for the treatment of B. pseudomallei infection in mice [167, 168] Clostridium botulinum BoNT serotypes and subtypes differences present a significant challenge for creating monoclonal antibody treatments for neutralization, by diversifying the V-regions of mAbs and selecting cross reactivity, a combination treatment of three antibodies neutralized BoNT/F1, F2, F4, and F7 in mice and was 150 times more potent than equine antitoxin [169] Coxiella burnetii Doxycycline Chloroquine Combination therapy of doxycycline and hydroxychloroquine combination shortened the duration of therapy and reduced the number of relapses in patients with Q fever endocarditis.
And a case of Q fever endocarditis with biological prosthetic aortic valve and aortic homograft was successfully treated with doxycycline and chloroquine combination therapy [170, 171] Francisella tularensis Cytochalasin B, LY294002, wortmannin, nocodazole, MG132, and XVA143 inhibitors reduce F. tularensis update and reduce inflammatory cytokine production and can be used in combination with antibiotics to improve survival of infected mice [172] Gentamicin Membrane antigen immunization Postexposure immunization with membrane protein fraction antigens and treatment with low-dose gentamicin increased survival of mice and significantly reduced bacterial burdens in the liver and spleen [173] potential of several small molecule immunomodulators and host cell factors that have been investigated to date.
Immunomodulators directly target the host rather than the pathogen (Fig.
7.5 ).
This is accomplished by targeting pattern recognition receptors, such as TLRs that are present on innate immune cells in the host to detect features of microbes known as pathogenassociated molecular patterns.
Since immunomodulators target host immune cells, they are an attractive candidate for use against bacterial agents as they are unlikely to result in the development of antibiotic resistance even after repeated use.
In particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunomodulation an attractive complementary or alternative strategy to directly targeting bacterial biothreat agents.
For example, a synthetic TLR9 agonist, 5'-C-phosphate-G-3' oligodeoxynucleotide (CpG ODN), appears to be able to stimulate protective immunity against intracellular bacterial infection and/or eliminate chronic infections.
Indeed, studies in mice have demonstrated that the innate immune defenses activated by CpG ODNs protect against lethal challenge with B. anthracis, B. mallei, and Combination therapy, including antibiotics with an efflux pump inhibitor, would be a novel mechanism to restore the efficacy of the antibiotic in resistant strains of Y. pestis [174] Antibody therapy Corticosteroid The addition of antiinflammatory methylprednisolone, a corticosteroid, in combination with antibody therapy correlates with improved mouse survival, with reduction in neutrophil and matrix metalloproteinase 9 in the tissue, and the mitigation of tissue damage [175] Ciprofloxacin L-97-1 A novel postexposure medical countermeasure l-97-1, an A 1 adenosine receptor antagonist blocks LPS-induced activation of immunomodulatory cytotoxic substance accumulation to prevent acute lung injury, and in combination with ciprofloxacin improves survival of rats following infection with Y. pestis [176] AIGIV, anthrax immune globulin intravenous; IFN, interferon; LPS, lipopolysaccharide; PA, protective antigen.
Aminoglycosides RND-type efflux pumps, S-adenosyl-l-methioninedependent methyltransferase, amrR [186, 187] β Lactams penA a , nlpD1, dacC, FlgN, sch, TR70_0856, TR70_1911, ftsI, amrR, bpeR, bpeT, spoT, tRNA, rRNA, proteins with unknown function, SerS seryl-tRNA synthetase, and RND efflux pump AmrAB-OprA, and BpeEF-OprC [186, [188] [189] [190] [191] [192] Macrolides AmrAB-OprA efflux pump [193] Quinolones AmrAB-OprA efflux pump, BpeAB-OprB efflux pump [194] Sulfamethoxazole/ Trimethoprim RND BpeEF-OprC efflux pump, LysR-type regulator BpeT BpeS, Ptr1, FolA, AmrR TetR-type regulator, AmrAB-OprA, metF [186, 195] Quinolones gyrA a [196] Tetracyclines Putative protein secretion targets, biosynthesis of pantothenate and coenzyme A, aspartate biosynthesis, DNA replication [197] Francisella tularensis β Lactams blaB1 [198] Chloramphenicol 23S rRNA, the L4 and L22 ribosomal proteins, and overexpression of efflux pumps [199] Quinolones gyrA a and gyrB [200] F. tularensis or their surrogates [203] .
Similarly, human monocyte-derived macrophages treated with poly(I:C), a synthetic TLR3 agonist, showed significantly reduced intracellular F. tularensis [both Schu 4 and LVS (live vaccine strains)] replication.
Mice administered with poly(I:C) before or after Schu 4 or LVS infection showed reduced bacterial burden in the lungs and prolonged survival.
Mice treated with poly(I:C), challenged with F. tularensis, and then treated with levofloxacin showed 100% survival relative to no survival in animals receiving levofloxacin alone [204] .
In addition to targeting innate immune cell receptors, there is a growing interest in modulating autophagy as an immunotherapeutic intervention.
Autophagy is a dynamic process that targets cellular cytoplasmic contents for lysosomal degradation.
More specifically, xenophagy is a type of selective autophagy that specifically targets intracellular pathogens to lysosomes, retracing their replication and survival [205] .
The use of autophagy inducer rapamycin, decreased the survival of B. pseudomallei in vitro [206] .
However, several bacteria exploit autophagic machinery as part of their intracellular life cycles (i.e., Brucella abortus, C. burnetii, and F. tularensis).
Therefore infection may be exacerbated by the induction of autophagy (Fig.
7.1C ) [205] .
Research to further understand the balance between infective and protective cellular targets in the autophagy pathway may enhance its utilization as a therapeutic target.
HTS of FDA-approved drugs is another approach to identifying compounds that were previously approved for other disease indications but may have the potential to be repurposed as antiinfectives.
Trifluoperazine (an antipsychotic), amoxapine (an antidepressant), and doxapram (a breathing stimulant) mitigated fatal Y. pestis infection in a pneumonic plague murine model [207] .
At 48 h postinfection, these drugs provided animals with up to 100% protection against challenge with bubonic or pneumonic plague agents when administered in combination with levofloxacin [208] .
Multiple FDA-approved drugs targeting G-protein coupled receptors and calcium fluxes inhibited C. burnetii and B. abortus, whereas drugs targeting cholesterol traffic attenuated C. burnetii [209] .
Similarly, increasing evidence suggested statin, a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitor, possesses antibacterial activity by the inhibition of sterols, prenylation, and isoprenoids (C. burnetii), the inhibition of antiinflammatory cytokines (Y. pestis), and the modulation of phagosome maturation (C. burnetii) [210] .
It was demonstrated that a low dose of Gleevec, an anticancer drug inhibiting Abl1, c-Kit, and related protein tyrosine kinases, can increase the number of myeloid cells in the bone marrow, blood, and spleen and enhance antimicrobial responses in a mouse model of F. tularensis infection [211] .
In the case of viruses, small molecule targeting of innate immune receptors has also shown efficacy in several relevant viral models of infection.
For example, treatment with poly(IC:LC) has also been protective against EBOV infection in NHPs [212] .
Prophylactic pulmonary administration of TLR7 ligand (TMX201) significantly protected mice from lethal infection with VEEV [213] .
TLR3 and TLR9 agonists have also been shown to improve the efficacy of postexposure therapeutics against smallpox [214] .
Sometimes modulation of host pathophysiological responses can be evaluated as a target.
Hemorrhagic fever virus pathophysiology includes the stimulation of procoagulant pathways and increased permeability of the vascular endothelium; therefore these processes are being evaluated as possible targets for therapeutic intervention.
This could be accomplished by utilization of an anticoagulant, such as recombinant nematode anticoagulant protein c2 (rNAPc2) that blocks initiation of the extrinsic coagulation pathway by inhibiting the tissue factor-factor VIIa complex [215, 216] .
rNAPc2 has been shown to be highly protective in macaques infected with a lethal dose of Ebola Zaire virus, when treatment was initiated 1 day post viral challenge [216, 217] .
Whereas HDT targets the host directly, antibody therapy is the passive process of activating the immune system to respond to microbial threats.
Sources of antibodies can include individuals that survive infection or have received a prophylactic vaccine against a microbe.
Alternatively, antibodies can also be generated ex vivo using cell culture.
Historically, antibody-based serum or plasma therapy has been widely used to treat a variety of infectious diseases.
Limitations for clinical use arise however from the polyclonal nature of serum antibodies, resulting in lot-to-lot variation, approaches for determination of correct dose levels and regimens, and a risk for allergic reactions and transmission of transfusion-borne diseases.
In general, limited clinical applications for antibody therapy existed until the development of technology that allowed the production of mAbs through the use of hybridomas [218] .
Hybridomas allow for the production of homogenous antibodies with the same specificity of a single immunoglobulin class and isotype.
Further advancements made it possible to humanize or generate fully human mAbs.
Research advancements in the past 10-15 years have resulted in numerous mAB-based therapies that have been approved for inflammatory and neoplastic diseases.
Infectious diseases have not been included in approved treatments.
Although many mAb products targeting infectious diseases are in different stages of development, to date, one mAB-based product, Synagis (palivizumab), is currently approved for use in infectious diseases (RSV) [219] , while two mAbs Abthrax (raxibacumab) and Anthim (obiltoxaximab) have been approved under the FDA's Animal Efficacy Rule for treatment of inhalation anthrax [8] .
For treatment of Ebola infection, the single mAb mAB114 and Zmapp, a cocktail of three "humanized" mABs, have advanced in product development and are being tested for efficacy in the ongoing Ebola outbreak in the Democratic Republic of the Congo (NCT03719586, www.clinicaltrials.gov).
It is clear that mAbs offer a highly specific, potent, and generally safe platform for antimicrobials and may be a useful alternative to immune plasma.
It is imperative to find appropriate niches in infectious diseases, specifically those caused by biothreat agents, where new antibody-based treatments could prove to be efficacious [220] .
Table 7 .5 summarizes key research in antibody therapy across different bacterial and viral families of some current biothreat agents.
The utilization of mAB therapy for the prophylactic or therapeutic treatment of biothreat agents varies depending on the agent.
In all cases however, the challenge for the development of effective therapeutic antibodies against viruses is the viruses' heterogeneity and mutability.
A related problem is the low binding affinity of cross-reactive antibodies that are capable of neutralizing a variety of primary isolates.
Finally, the cost of large-scale production of mABs is a limiting factor for continued use.
A solution to the challenges with viral mutagenicity may be found in the identification of potent new mAbs that target highly conserved viral structures, which are critical for virus entry into cells.
Alternatively, utilization of combination therapy, whereby, a cocktail of several mAbs may be used or mAbs may be combined with other drugs, such as antiviral compounds, may overcome mutagenicity issues.
These areas of research will continue to be a major focus of biothreat agent therapeutic research [221] .
For countermeasures against lethal viral infections (i.e., category A), Table 7 .3 lists reported studies in either mice or NHPs that have shown significant benefits to survival in challenge models.
The table also includes in vitro potencies, viral strains, specific animal species, dosing regimens, routes of administration, PKs, and benefits to survivaldata necessary for the reader to relate in vitro potency to in vivo efficacy, assess/interpret results, and make comparisons.
The corresponding chemical structures are provided in Fig.
7 .6.
Table 7 .4 displays the status/results of clinical trials for therapeutics used for the treatment of infections caused by EBOV and LASV.
Noteworthy, most of these clinical trials were underpowered without appropriate controls and hence results may be speculative.
Combination therapies are an excellent approach to improve treatment outcomes, shorten treatment duration, and overcome microbial resistance mechanisms caused by biothreat pathogens.
Combination therapy may incorporate antibiotics or antivirals with HDT or antibody therapy at rationally designed treatment schedule.
In this way the usage of multiple treatment modalities can synergize to optimize the mechanism of action of biothreat-targeted therapies.
Table 7 .6 includes combination therapies that have been used to treat each biothreat bacteria.
In the case of viral infections, while combination therapy has been used for treatment of patients with human immunodeficiency virus (e.g., combination of nucleoside, nonnucleoside, protease, and/or host-targeted inhibitors) or chronic hepatitis C virus infection (e.g., combination of polymerase and RNA-binding protein NS5A inhibitors), to date there are no reported studies for biothreat viral agents.
Several β lactam antibiotic drugs have been able to overcome deactivation when delivered in combination with inhibitors that target extended-spectrum β lactamases (enzymes that are overexpressed in the MDR pathogens, inactivate the β lactam antibiotic by cleaving the β lactam ring and thus one of the major contributors of antibiotic resistance).
The β lactam/β lactamase inhibitor combination drugs that have been FDA-approved include Augmentin XR (amoxicillin/clavulanate combination), Unasyn (ampicillin/sublbactam combination), and Zosyn (piperacillin/tazobactam combination).
In the case of biothreat bacteria, the combination of Ceftolozone and tazobactam exhibited increased in vitro susceptibility to a variety of clinical, environmental, and animal strains of B. pseudomallei, but to date it has not been evaluated in in vivo efficacy studies [222] .
Challenges to developing countermeasures against biothreat agents are many, but some of the unique and key challenges are PK differences in healthy versus infected subjects, mapping the biodistribution of the countermeasure to the biodistribution of the pathogen, and limited opportunities to run randomized, controlled clinical trials.
Preclinical studies typically require a PK study in healthy animals to guide dose selection prior to testing a countermeasure in an animal model of infection.
In that regard, it is critical to understand what cells and tissues the pathogen is infecting over time so that countermeasures can be properly designed to reach infected tissue.
For example, countermeasures against pathogens causing encephalitis require drug to reach the central nervous system (CNS).
In contrast, EBOV was found to infect lymph nodes, spleen, and liver in NHPs 2-3 days following viral challenge, and by days 5-6 the virus was detected throughout the body (Fig.
7.7 ) [217] .
Thus if one was designing a countermeasure against EBOV infection, it would likely require a wide tissue distribution in order to be effective.
To complicate things further, infected animals often have altered metabolizing enzymes (e.g., cytochrome p450s) [223] , tissues, and barriers (e.g., blood-brain barrier) making drug exposure difficult to predict.
Running PK experiments in the presence of infection would eliminate many of these variables, but this is seldom done for countermeasures to biothreat agents, since it requires running these experiments in biocontainment labs.
Because biothreat pathogens cause infrequent human cases and outbreaks in generally remote areas of the world, planning a traditional human clinical trial with large numbers of participants is not feasible.
Even when the West African Ebola outbreak of more than 28,600 cases was unfolding in 2014-2016, and now that there is a large outbreak unfolding in the Democratic Republic of the Congo, the amount of clinical efficacy data that have been collected for EBOV therapeutics is quite limited.
The limitations are due to difficulty of performing clinical research in a remote outbreak setting where cultural, geographical, and political barriers may hinder or halt trial planning [225] .
The bulk of the efficacy data for Ebola published in the literature has been garnered through animal studies.
To enable product development for viral, bacterial pathogens as well as for chemical, toxin, and radiological threat agents for which outbreaks or cases are sparse, the FDA issued an Animal Rule, codified 21 CFR 314.600 in 2002, that proposes to permit consideration of product development and efficacy data obtained from animal studies for drug licensing, in lieu of human clinical trials when such trials would be unfeasible or unethical [226] .
Since introducing the Animal Rule in 2002, the FDA has approved more than a dozen products, including several therapeutics for anthrax, plague, botulinum toxin, and smallpox [8, 29] .
The Animal Rule does not provide an expedited pathway to FDA approval for drugs and can certainly be more challenging than traditional drug development pathways.
The developer must compile a significant body of data to prove efficacy of the drug against the target therapeutic indication.
First in 2009, and updated now into a formal document published in 2015 [226] , the FDA has released guidance for Industry describing critical data elements required for animal efficacy studies for drug approval under the Animal Rule: (1) the pathophysiology of disease and the mechanism of action by which the drug prevents or ameliorates disease must be reasonably well understood; (2) it is desired that the efficacy must be demonstrated in two animal species, although multiple studies in one species can be acceptable if the animal model is sufficiently well characterized and accurately predicts the human response; (3) the animal study end point must be clearly related to the desired human efficacy end point, such as enhancement of survival; and (4) PK and pharmacodynamics data must be generated in the animal studies to allow selection of an effective dose in humans.
Under the Animal Rule, efficacy studies are expected to demonstrate that drug effectiveness in animals reliably indicates efficacy in humans.
Thus while traditional human clinical efficacy studies require demonstration that the therapy is effective, the Animal Rule imposes an additional burden on investigators to establish a drug candidate's mode of action in at least one animal model that reproduces accurate human disease pathology.
Further, the Animal Rule outlines considerations for the development of the model(s), to include the use of an isolate of the etiologic agent that was known to cause human disease (e.g., agent was isolated from a fatal human case if it is a lethal disease, such as Ebola) [227] .
There is also a requirement that the infection model using the chosen pathogen strain must present the same or similar pathophysiology as the human disease.
Definitive animal model efficacy evaluations should be performed only after careful model development studies have been performed and accepted by the regulators.
These studies are known as natural history studies and are carefully designed to investigate and describe the course of the disease in the animal species, through clinical, serological, and histopathological evaluations, to compare the features of the disease in the model to the features of disease in human cases.
It is important to consider the route of pathogen exposure (nasal, oral, and aerosol routes) to the animal because this will model the natural or unnatural modes of exposure predicted for humans, where a biorelease would constitute an unnatural exposure.
A dose of challenge agent that is thought to be predictive of the human exposure level in a biorelease scenario should be used to develop the model, and that dose should be well characterized and reproducible by a quantitative measure.
The route of drug delivery, dose administration timing, and treatment regimen in response to a biorelease scenario must also be considered when designing the animal model studies for a drug under development for such an indication.
It is possible that a biorelease scenario would not be immediately known, and a period of time might pass before people begin to develop symptoms.
Studies evaluating the cutoff time for drug to still be effective, and what are the triggers for treatment should be investigated in the animal model.
Animal Rule pivotal efficacy studies are essentially performed in place of traditional phase 3 clinical studies, so they must be done in the containment laboratory under a quality system [226] .
Use of FDA Good Laboratory Practice or other comparable quality system with high levels of documentation and data integrity is paramount, so data packages can withstand regulatory review and audit [228] .
The studies must be designed so that the program will collect the same results and conclusions one would expect from a well-designed traditional phase 3 trial, but in addition a pivotal animal efficacy study must describe a mechanism of action for the treatment modality to prevent or block disease or tissue infection and damage [227] .
The pathologic mechanism needs to be consistent and well understood across both the human and animal models, such as the mechanism of pathogen entry into the target host cell, toxicological mechanism of lethal factors, or germination of spores and dissemination of bacterial infection in target cells and tissues, all of which may be mechanisms the drug under study is known to block; this must be proven in the animal model.
Products developed under the Animal Rule are subject to postmarketing or field studies when the product is actually used in the scenarios for which it was developed, and this is required to verify a product's clinical benefit [8] .
Part of the approval process is a requirement to have postmarketing study plans in place, for quick execution should an event occur in which the drug would be field tested.
Approval may also come with restrictions for off-label use, distribution, or access.
Actual use will also come with requirements to inform patients of the conditions under which the drug was approved by virtue of only animal efficacy data, making them informed consumers as to the risks of possible nonefficacy or unknown effects in cases of human disease.
With the advancement of systems and synthetic biology and the ease of genetic modification, biothreats are becoming more complex and there is a growing need for novel treatments that can have broad-spectrum activity against new, remerging, and engineered pathogens.
Developing novel countermeasures that can effectively treat and prevent massive casualties is an ongoing challenge that remains a central priority for future research.
The development of novel therapies relies on an improved understanding of the host-pathogen interactions.
Key virulence factors have been identified and targeted for potential treatment options, including biofilm and T3SS inhibitors for bacterial infections, and viral entry or polymerase inhibitors for viral infections.
Combining HTS with systems biology provides a robust, coordinated approach to identifying therapeutic targets.
Since stand-alone antibiotics or antivirals may not be sufficient to overcome resistant or engineered biothreat infections, a focus on combination therapy, antibodies, and HDTs is the key countermeasure.
Although many challenges are faced when developing novel therapies for biothreat pathogens and no FDA-approved HDTs are yet available for treatment, there is potential for novel small molecule host-targeted immunomodulators to be developed.
Screening of FDA-approved drugs is a powerful approach to possibly repurpose drugs for new disease indication and/or identify compounds that are safe and effective in humans, which can also have antibacterial or antiviral capabilities.
Three FDA-approved drugs have shown potential in mice against pneumonic plague [207] .
Serious challenges still remain with the prevalence of antibiotic resistance that jeopardizes the effectiveness of our current treatment options for bacterial threats.
In addition, the complex intracellular life cycle of many biothreat pathogens requires therapeutics that can penetrate the host cell.
There are limited FDA-approved viral countermeasures for prevention and treatment.
None of the filoviruses or henipaviruses has approved therapeutics or vaccines available for human disease.
Some vaccines exist for new world alphaviruses, but no current therapeutics are effective for treatment after infection.
Focused efforts using HTS to develop novel, effective, and broad-spectrum medical countermeasures will provide a robust response capability against rapidly evolving biothreats.
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key: cord-026653-094bk0t0 authors: Gülsen, Askin; Wedi, Bettina; Jappe, Uta title: Hypersensitivity reactions to biologics (part I): allergy as an important differential diagnosis in complex immune-derived adverse events* date: 2020-06-24 journal: Allergo J DOI: 10.1007/s15007-020-2550-1 sha: doc_id: 26653 cord_uid: 094bk0t0 Purpose: Biotechnological substances (BSs) are strongly relied upon to prevent rejection of transplanted organs, and to treat oncological, allergological, and other inflammatory diseases.
Allergic reactions to partly foreign biologics can occur due to their potential immunogenicity.
The severity of an immune response to a biological drug may range from no clinical significance to a severe, life-threatening anaphylactic reaction.
Methods: Detailed searches were performed on Pubmed, Web of Science, and Google Scholar to include all available publications.
In addition, the Food and Drug Administration, the European Medicines Agency, and British Columbia Cancer Agency Drug Manual databases were screened for hypersensitivity reaction (HSR), infusion reaction, injection site reaction, urticaria, and anaphylaxis for individual BSs.
Results: Treatment with BSs can cause various types of HSR.
These are mentioned in the literature with definitions such as allergic reactions, anaphylactoid reactions, anaphylaxis, HSR, infusion reactions, injection site reactions, cytokine release syndrome, and urticaria.
Due to the overlap in signs and symptoms in the reported descriptions, it is not always possible to differentiate these reactions properly according to their pathomechanism.
Similarly, many data reported as anaphylaxis actually describe severe anaphylactic reactions (grades III or IV).
Conclusion: There is an urgent need for a simpler symptomor system-based classification and scoring system to create an awareness for HSRs to BSs.
A better understanding of the pathophysiology of HSRs and increased clinical experience in the treatment of side effects will provide timely control of unexpected reactions.
As a result, immunotherapy with BSs will become safer in the future.
Cite this as Gülsen A, Wedi B, Jappe U. Hypersensitivity reactions to biologics (part I): allergy as an important differential diagnosis in complex immune-derived adverse events.
Allergo J Int 2020; 29:97-125 https://doi.org/10.1007/s40629-020-00126-6 Biotechnological substances (BSs), also known as biological agents, biologicals or biologics, are pro-duced by living organisms or are synthesized from a product made by a living organism.
Some BSs are directed against specific cytokines or cell surface signal receptors.
In recent years, the BS industry has experienced rapid growth and yielded products for treatment of a broad spectrum of diseases and disorders.
Biologics are strongly relied upon to prevent rejection after organ transplantation and to treat diseases related to allergology, pneumology, rheumatology, oncology, and dermatology.
BSs have revolutionized the treatment of diseases such as rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, and organ-specific cancers (lung, stomach, colon, cervix, breast, etc.).
Other conditions commonly treated or managed with biologics include several types of asthma, plaque psoriasis, chronic urticaria, and vasculitis.
Some new indications and less studied applications include interstitial lung disease, unstable angina pectoris during cardiac catheterization, paroxysmal nocturnal hemoglobinuria, neonatal bronchopulmonary dysplasia, and multiple sclerosis (Tab.
1).
Allergic reactions to partly foreign biologics can occur due to their potential immunogenicity.
Adverse drug reactions (ADRs), however, can also result from an agent's direct biological function.
For instance, neutralization of an off-target cytokine's activity can cause ADRs [1] .
The severity of an immune response to a biological drug may range from no clinical significance to a severe, life-threatening anaphylactic reaction [2] .
Thus it became evident that the nomenclature used to describe and document immunological adverse events was not always precise enough, or the cases had not been investigated in enough detail to clearly differentiate between truly allergic and other reactions.
This review will evaluate reports of allergic and substance-specific infusion reactions (IR), injection-site reactions (ISR), hypersensitivity reactions (HSR), urticaria, and anaphylaxis caused by BSs.
The most common indications for the use of biologics in lung diseases are allergic and severe uncontrolled asthma.
Biologics are used more rarely in interstitial lung diseases (Tab.
2).
Their use in lung cancer will be mentioned in the section Oncology.
Benralizumab, lebrikizumab, mepolizumab, reslizumab, and omalizumab are used in asthmatic patients.
The primary indication for nintedanib and pirfenidone is idiopathic pulmonary fibrosis.
Benralizumab is the newest anti-asthmatic and anti-inflammatory drug in the monoclonal antibody (mAb) family developed for the treatment of severe eosinophilic and allergic asthma.
It exerts its effects by binding to the α subunit of the interleukin (IL)-5 receptor on eosinophils and basophils.
In a phase IIb study conducted between 2011 and 2012, the ISR rate was 16 % and anaphylaxis was 0 % [4] .
FDA labels show a 3 % incidence of HSRs (ana-phylaxis, urticaria, rash, and angioedema) in the treatment group receiving benralizumab and placebo [5] .
The frequency of ISR (erythema, local pain, papule, or pruritus) was 2.2 % in patients treated with benralizumab [5] .
These reactions usually occurred within the first hours after s. c. administration of the drug.
The most reported ADR was upper respiratory tract infection and mild to moderate nasopharyngitis [6] .
In a recent study in which 13 patients were treated, HSR and anaphylaxis were not reported; only slight fever with chills was observed in two patients [7] .
In the meta-analysis of eight randomized controlled trials, the overall risk of ADRs and severe side effects was lower and the risk of headache and pyrexia higher in patients treated with benralizumab compared to placebo [8] .
In addition, an increased incidence of ISRs, hypersensitivity, and death was not observed compared to placebo.
The ISRs were reported in 2.6 % of patients treated with 30 mg benralizumab.
Post-marketing side effect recording is ongoing.
Lebrikizumab is a novel humanized mAb that specifically inhibits the activity of IL-13.
Clinical studies for the treatment of asthma that is uncontrolled by inhaled corticosteroids, chronic obstructive pulmonary disease (COPD), atopic dermatitis, and idiopathic pulmonary fibrosis (IPF) are ongoing.
In one placebo-controlled study using higher doses, ISRs (pain, erythema) were found to be more prevalent, 20.5 % and 20.3 % in groups receiving 125 and 250 mg doses of lebrikizumab, 11.1 % in a group receiving 37.5 mg, and 6 % in the group given placebo [9] .
In the phase III study (LAVOLTA) of lebrikizumab for asthma, ISRs occurred in 6 % of participants receiving 37.5 mg s.c. injections, 10 % of participants receiving 125 mg s.c. injections, and 8 % of participants receiving a placebo once every 4 weeks [10] .
In the same study, anaphylaxis was reported to be below 1 %.
Korenblat et al.
reported anaphylaxis in one patient (1 %) treated with lebrikizumab, and this reaction was attributed to a known peanut allergy [11] .
In another study, lebrikizumab was well tolerated, and death and anaphylactic reactions were not reported [12] .
ISRs rarely occurred (1.3 % in the lebrikizumab and 1.9 % in the placebo group).
Mepolizumab is a comparatively new anti-inflammatory, anti-asthmatic BS consisting of a mAb that binds to IL 5.
Therefore, it was also approved by the FDA in December 2017 for use in the treatment of Churg-Strauss syndrome (eosinophilic granulomatosis with polyangiitis).
In a multicenter study covering the years 2009-2011 (DREAM study), the most commonly reported drug-related adverse events were non-allergic, infusion-related reactions [13] .
Infusion-related reactions occurred in 5 % of participants receiving 75 mg of mepolizumab, 8 % of participants receiving 250 mg, 12 % of participants receiving 750 mg, and 6 % of participants receiving placebo.
HSRs thought to be associated with the study drug occurred in 0 % of participants receiving 75 mg mepolizumab, < 1 % of participants receiving 250 mg, 1 % of participants receiving 750 mg, and 2 % of participants receiving placebo.
No anaphylactic reaction was reported.
Anaphylaxis has not been reported on FDA labels [14] .
However, in one case, type-IV delayed HSR was observed in the 9th month of treatment, 3 days after subcutaneous (SC) administration of 100 mg mepolizumab.
Local ISRs (8% after 100 mg application, 3 % after 75 mg application, 3 % after placebo), and only few allergic reactions (≤ 2 %) have been reported.
Lugogo et al.
[15] conducted a study on SC administration of mepolizumab in severe eosinophilic asthma patients.
In this study, HSRs (type IV) occurred in ≤ 1 % of participants, local ISRs occurred in 3 %, and < 1 % of participants experienced injection-related reactions (non-allergic).
There was no report of mepolizumab-related anaphylaxis.
Finally, in a recent study of 347 severe eosinophilic asthma patients, local ISRs (12 %), allergic/HSRs (2 %), and non-allergic systemic reactions (< 1 %) were reported [16] .
Anaphylaxis was not observed.
Reslizumab is a biologically active humanized mAb used for adjunctive therapy of severe asthma with an eosinophilic phenotype.
This drug binds and inhibits the cytokine IL 5, which is responsible for growth, differentiation, recruitment, activation, and survival of eosinophils.
In a study conducted by Castro et al.
[17] in 2015, ISRs (hematoma, rash, and local pain) were reported to be less than 2 %, and an anaphylactic reaction was reported in two patients.
Anti-drug antibodies (ADA) for reslizumab were found to be negative in these patients, and therefore no further information could be obtained.
In FDA labels, treatment-related anaphylaxis was reported in three patients in the reslizumab 3 mg/kg treatment group [18] .
The same label states that this drug is produced in the murine cell line, which introduces the galactose α-1,3-galactose (α-gal) carbohydrate sequence as a post-translational modification into the carbohydrate side chains of the mAb.
This explains why this drug is immunogenic for humans, but it is unclear how α-gal acts and which role it plays in these reactions.
ADA were positive in approximately 6 % of patients treated with reslizumab, but there was no correlation with HSR and allergic reactions.
Murphy and colleagues [19] investigated the longterm effects of reslizumab and reported two HSRs (< 1 %), two drug eruptions (< 1 %), and very rare local infusion-related AEs (e.g., pain at injection site) (< 1 %).
They did not report anaphylaxis during the follow-up period.
In a recent study, one case of toxicoderma (the 4th dose of the drug 12 h after administration) was reported [20] .
The patient developed a symmetrical rash on the trunk and proximal limbs, and the symptoms improved after administration of systemic corticosteroids and antihistamines.
Omalizumab is a humanized mAb commonly used as an adjunctive and second-line treatment for severe allergic asthma and chronic spontaneous urticaria.
The effect is due to selective binding to immuno globulin E (IgE).
The task force report published by the American Allergy Academy in 2007 states that the rate of ana-phylaxis associated with omalizumab treatment is 0.09 % [21] .
Of the reactions observed to date, 61 % occurred within 2 h after one of the first three doses, and 14 % occurred within 30 min after the fourth or a later dose.
Omalizumab is produced in the ovarian cells of a Chinese hamster and does not express the α-1,3-galactosyl-transferase, and therefore does not contain α-gal.
As a consequence, pre-existing IgE-antibodies as have been described in patients with anaphylaxis to cetuximab were not detectable in humans [21] .
In a large-scale real life study conducted between 2007 and 2016, ISRs, immediate local reactions, urticaria, and anaphylaxis were reported to be 3.4 %, 1.1 %, 1.0 %, and 0 %, respectively [22] .
In 2011, a sequel of the task force report of the American Allergy Academy written in 2007 was published on the basis of post-marketing safety data provided by Genentech/Novartis [23] .
In this additional report, a total of 77 of the 127 patients (60.6 %) that developed anaphylaxis against omalizumab reported that these reactions occurred within the recommended hospital waiting period (especially within the first 2 h after the first three injections).
In FDA labels, anaphylaxis with symptoms such as angioedema, bronchospasm, urticaria, hypotension, and syncope has been reported in 0.1 % of patients [24] .
This can be seen after the first dose, as well as 1 year later.
ISRs of any severity (pain, burning, induration, warmth, redness, bruising, itching, granuloma formation, etc.)
were observed in 45 % of patients compared with placebo (43 %).
More severe reactions have been reported in 12 % of patients receiving omalizumab and 9 % in placebo.
Most of these reactions occur 1 h after injection and last for less than 8 days.
Nintedanib is an antiproliferative and antitumoral oral BS of the multikinase receptor inhibitor group usually used in the treatment of idiopathic pulmonary fibrosis and non-small cell lung cancer.
The Australian Public Assessment report and the FDA label did not observe an increase in the incidence of severe immunological and anaphylactic reactions related to the use of nintedanib [25, 26] .
Both reports documented hepatotoxicity, bleeding and thromboembolic events, gastrointestinal toxicity (diarrhea, nausea, and vomiting) and perforation, myocardial infarction, and hypertension as side effects.
In post-marketing studies, it is reported that pruritus and rash may be seen, but the rate is not clear (referenced in [26] ).
Nintedanib capsules contain the following excipients; (i) capsule content: triglycerides, hard fat, lecithin (soya); (ii) capsule shell: gelatin, glycerol, titanium dioxide, iron oxide red and yellow; (iii) printing ink: shellac glaze, iron oxide black, and propylene glycol [26] .
In addition, the summary of product characterics indicate that those patients with soy and peanut allergy should be treated with caution, but more detailed information as to the reason for legume allergy to be considered as a risk is lacking.
Insufficient data were found in our literature review to assess the prevalence of allergic reactions, HSR, anaphylaxis, and urticaria due to the use of this BS.
This is probably due to the fact that other side effects were considered as having higher priority.
Pirfenidone is an oral BS with antifibrotic and antiinflammatory properties.
Its only indication is the treatment of mild to moderate idiopathic pulmonary fibrosis.
It exerts its effect by inhibiting transforming growth factor (TGF)-β1.
Skin rash was reported in 32 % of patients treated with pirfenidone and in 12 % of patients treated with placebo [27] .
In addition, phototoxic burn-like skin rashes on sun-exposed body areas and erythematous (edematous or non-edematous) lesions were reported in 12 % of patients and in 2 % with placebo.
In newly published FDA labels, photosensitivity and rash were reported at a rate of 9%, but HSR and anaphylaxis were not mentioned in this report [28] .
Indications for which BSs are developed in dermatology include moderate to severe psoriasis, chronic urticaria, and atopic dermatitis (Tab.
3).
Currently prescribed BSs include alefacept, efalizumab, ixekizumab, secukinumab, ustekinumab, dupilumab, quilizumab, ligelizumab, and omalizumab.
TNF-α inhibitors such as etanercept, infliximab, and adalimumab have also been approved by the FDA for treatment of moderate to severe psoriasis and psoriatic arthritis [29] .
Off-label indications for TNF-α inhibitors include autoimmune bullous disease, pemphigus vulgaris, and pyoderma gangrenosum [30] .
Rarer indications include connective tissue disorders such as scleroderma, dermatomyositis, systemic lupus erythematosus, Sweet's syndrome, sarcoidosis, granuloma annulare, toxic epidermal necrolysis, pityriasis rubra pilaris, and Behcet's disease [29] .
BSs used in the treatment of psoriatic arthritis will be mentioned in the section Rheumatology.
Alefacept is a fully human recombinant lymphocyte function-associated antigen-3 (LFA-3) immunoglobulin G1 fusion protein with a dual action mechanism that targets T cells, and can be administered intramuscularly or intravenously on a weekly basis.
Its primary function is to interact with CD2 in the membrane of CD4+ and CD8+ T cells, inhibiting activation and thus regulating CD2/LFA 3 interaction.
A secondary mechanism of action is the induction of apoptosis in memoryeffect T lymphocytes.
According to FDA labels, four out of 1,869 patients (0.2 %) reported angioedema in clinical trials: two of these patients were hospitalized and treated [31] .
However, urticaria was seen in six patients (< 1 %) during the 24-week period.
In one patient, therapy needed to be terminated due to allergic reactions.
ISRs were reported in 16 % of patients receiving alefacept by intramuscular administration, compared with 8 % of patients treated with placebo.
Repeated doses did not change the rate of incidence.
ISRs were usually mild and were reported to manifest as pain (7 %), inflammation (4 %), bleeding (4 %), edema (2 %), local granuloma (1 %), and nonspecific reaction or skin hypersensitivity (< 1.0 %).
It has been reported that approximately 3 % of patients developed low titer antibodies to the fusion protein, but a long-term effect was not known.
FDA approval was withdrawn in September 2012 after a decision was made by the manufacturer to stop production of the drug, and the drug was never approved for the European market.
There is no information about the frequency of anaphylaxis in the literature.
Efalizumab is a humanized mAb that binds to the CD11a subunit of lymphocyte function-associated antigen 1 (LFA-1) on the surface of lymphocytes.
It is used to treat adult patients with moderate to severe psoriasis.
In a randomized controlled study involving 556 patients conducted by Gordon et al., 2 % of patients developed antibodies to efalizumab, but no anaphylaxis was observed [32] .
In controlled clinical trials involving 1,213 patients, 8 % experienced at least one HSR (e.g., asthma, dyspnea, angioedema, urticaria, maculopapular rash) and 1 % experienced urticaria.
Other reported side effects include angioedema, laryngospasm, erythema multiforme, serum sickness-like reaction and allergic drug eruption.
A low concentration of antibodies to protein components of the drug have been reported in 6.3 % of patients (referenced in [33] ).
In a retrospective cohort study conducted by Brunasso et al., ISRs (local erythema, itching, burn-ing, pain, edema, or urticaria) were observed in 4 % of participants [34] .
Ixekizumab is an immunosuppressive and anti-inflammatory mAb used to treat moderate to severe plaque psoriasis and can be administered subcutaneously.
Its mechanism of action involves binding of the cytokine IL-17A, an important factor in keratinocyte activation and proliferation.
According to FDA labels, ISRs were reported in 17 % of patients.
The most common type of ISRs were erythema and pain [35] .
Less frequently, serious HSRs such as angioedema and urticaria (≤ 0.1 % each) have also occurred.
In the post-marketing period, rare events of anaphylaxis requiring hospitalization have been reported.
In a long-term clinical investigation by Strober and colleagues [36] following 4,209 patients, no anaphylactic reactions were reported.
In this study, ISR, local erythema, and drug hypersensitivity were reported in 6.8 %, 2.1 %, and 0.1 % of patient-years, respectively.
Secukinumab is a mAb that is administered subcutaneously to treat plaque psoriasis, ankylosing spondylitis, and psoriatic arthritis.
Its anti-inflammatory and immunomodulatory properties result from its binding to and inactivation of IL-17A.
In the assessment report of the EMA in 2015, no cases of anaphylaxis or angioedema were reported [37] .
Urticaria was rarely (<1.0 %) observed.
Injection-site pain was reported in 5.6 % of cases, and general HSR occurred in 6.5-11.2 %.
The most common HSRs in any secukinumab group were reported to be dermatitis (1.2 %), eczema (1.4 %), and rash (1.8 %).
Less than 1 % of patients developed antibodies during sekukinumab treatment for up to 52 weeks.
In one study, erythema and itching at the injection site were reported in 3.0 % of participants and anaphylaxis in 2.0 % [38] .
According to the FDA label, urticaria was reported in 0.6-1.2 % of patients depending on the dose, and less than 1 % of patients developed ADA [39] .
Although this report states that anaphylactic and allergic reactions are rarely seen, the rate was not given.
In a recent study, HSR was reported to be 2.4 % per 100 patient-years and ISR in 0.8-1.3 %; in addition, ADAs were reported in < 1 % of patients receiving treatment for 52 weeks [40] .
Ustekinumab is a mAb that acts as an interleukin inhibitor and was approved for administration at 3 month intervals as a second-line treatment for patients with moderate to severe plaque psoriasis.
It has immunosuppressant and anti-inflammatory effects and neutralizes IL-12 and IL-23.
In the EMA evaluation report for 2017, ISRs were reported to occur in 3.0 %.
Intravenous infusion was not associated with anaphylaxis, IR, or serum sickness-like reactions [41] .
FDA labels show a 1-2 % incidence of ISRs (bruising, hemorrhage, induration, irritation, pain, pruritus, and swelling) [42] .
In addition, one patient (0.1 %) experienced signs and symptoms (flushing, tightness of the throat, shortness of breath) consistent with anaphylaxis after initial SC injection, and one patient (0.08 %) experienced signs and symptoms consistent with or related to an HSR ( urticaria, flushing, chest discomfort, and increased body temperature) after initial intravenous injection.
In addition, approximately 6-12.4 % of patients developed ADAs, which were generally of low titer (referenced in [42] ).
A recent study examined the side effects of ustekinumab in psoriasis, psoriatic arthritis, and Crohn's disease [43] .
No serum sickness-like reactions or serious anaphylactic events were reported.
Two patients (< 1.0 %) had temporary signs of treatment-associated hypersensitivity.
The patients developed flushing, shortness of breath, and throat tightness after the first SC administration.
In addition, fever, flushing, chest discomfort, and urticaria were observed after the initial intravenous administration.
Long-term safety and side effect studies are ongoing (C0168Z03 [PSOLAR] and CNTO1275PSO4005 [Nordic Database Initiative]).
Dupilumab is a relatively new BS in the mAb group, with anti-inflammatory and selective immunosuppressive properties.
It is used as second-line therapy to treat moderate to severe atopic dermatitis.
It exerts its effect by binding the alpha subunit of the IL-4 receptor, eliminating the biological effects of IL-4 and IL-13.
Since 2019 additional indications are severe uncontrolled asthma with type 2 inflammation and chronic rhinosinusitis with nasal polyposis.
In the FDA drug report, hypersensitivity reactions such as serum sickness, serum sickness-like reaction, and generalized urticaria occurred with a frequency of < 1 %, and ISRs were observed in 10 % of cases [44] .
ADA were detected in 2-8 % of treated patients, and two subjects developed serum sickness or serum sickness-like reactions.
In these patients, a high antibody titer for dupilumab was reported during treatment.
There is no information about anaphylaxis.
In a meta-analysis conducted by Ou et al., ISRs were reported in 13.2 % of cases [45] .
There is no mention of anaphylaxis, HSR, or urticaria, but it is stated that the high incidence of ISRs needs to be explained.
The EMA 2019 report shows an HSR in 3.0-4.3 % of adult patients and ISRs in 16.0-20.1 % (referenced in [46] ).
Severe ISRs were reported in 0.3-1.4 %.
Most ISRs were mild to moderate.
The other most commonly reported adverse events were urticaria (0.5-1.3 %) and rash (0.5-0.6 %), depending on the dose, but with a similar incidence to placebo.
Anaphylactic reaction or angioedema was reported in three patients (0.2 %), hypersensitivity in two patients (0.1 %), drug hypersensitivity in two patients (0.1 %), and anaphylactic shock in one patient (< 0.1 %).
Ligelizumab is a newly developed humanized mAb against human IgE and belongs to the IgG1/κ isotype subclass.
This BS has 50-fold greater affinity to human IgE compared with omalizumab.
A recent phase II clinical trial demonstrated that a higher percentage of patients had complete control of symptoms of chronic spontaneous urticaria with ligelizumab therapy of 72 mg or 240 mg than with omalizumab or placebo [47] .
In the 72-mg treatment group in this study, mild injection site erythema was observed in 2.0 % and mild to moderate ISRs in 4.0 % of patients, while in the 240-mg-treated group, these were reported in 6.0 % and 7.0 %, respectively.
In addition, serious side effects were reported in 2 % of patients in both treatment groups, but no mortality or anaphylaxis occurred.
Phase III studies are ongoing and expected to be finalized by 2021.
Quilizumab is a newly developed humanized mAb against the M1-prime segment of membrane-expressed IgE.
It leads to depletion of IgE-producing and memory B cells, thus blocking IgE production.
Its primary indications are chronic spontaneous urticaria and uncontrolled allergic asthma.
In one study assessing efficacy and safety, 6.9 % of patients reported ISRs (most often local pain) [48] .
Further studies were discontinued due to the lack of efficacy in adults.
The use of BSs in oncology is quite extensive.
The literature shows that these drugs are used for the treatment of bladder, breast, cervical, head and neck, gastrointestinal (stomach, bowel, colorectal), lung, and kidney cancers, as well as leukemia and lymphoma.
Details of the reactions are given in Tab.
4.
Aflibercept is a human recombinant fusion protein targeting vascular endothelial growth factor 1 (VEGF-1) receptors used in colorectal carcinoma with metastasis, as well as retinal diseases such as macular degeneration.
In the EMA 2019 assessment report and FDA 2019 label, it was stated that HSRs were observed at a rate of 0.3 % (placebo 0.5 %) [49, 50] .
Severe HSRs such as anaphylaxis, angioedema, bronchospasm, and dyspnea have been described as uncommon (≥ 1/1000 to < 1/100) [49] .
However, more detailed information has not been provided.
In addition, ADA have been reported to develop in 1.4-3.1 % of patients, and the clinical significance of their neutrali zation has not been assessed due to limited data [50] .
Maculopapular rash may develop after intravitrael administration of this drug in retinal diseases [51] .
Alemtuzumab is a monoclonal IgG1kappa antibody and a cytolytic agent used intravenously to treat leukemia and multiple sclerosis.
This drug binds the glycoprotein CD52 on the surface of lymphocytes and other immune cells and leads to cell death.
In the 2013 EMA assessment report, allergic side effects were reported with relatively high frequencies (rash in 48 %, urticaria in 17 %, and pruritus in 16.5 % of patients) [52] .
Serious and severe urticaria was seen in 0.4 % of cases.
Severe infusion-associated reactions, including pyrexia, urticaria, atrial fibrillation, nausea, chest discomfort, hypotension, and grade IV anaphylaxis were reported in 2.2-2.8 % of cases.
The British Columbia Cancer Agency (BCCA) Drug Manual reported anaphylactic reactions and angioedema in < 1 %, ISRs in 1.0 % (severe in 0 %) of intravenous users and ISRs in 90 % (severe in 2 %) of SC users.
Reactions occurred between 24 h after the first dose and the administration of the fourth dose.
In addition, SC injection site pain was reported in 61.0 % (severe in 2.0 %).
Pruritus and urticaria were reported in 21-30 % (1-5 % of these reactions were severe; 0 % of the reactions was associated with SC use) (referenced in [53] ).
In the last published FDA label, IRs and cytokine release syndrome were reported in 92 % of treated patients, but epinephrine or atropine was administered in 0.6 % of these patients [54] .
In some patients, infusion reactions occurred 24 h after infusion.
Symptoms include fatigue, dyspepsia, pruritus, flushing, urticaria, dyspnea, pain, nausea, dizziness, and pulmonary infiltrates.
Severe reactions occurred in approximately 3 % of patients.
Two patients experienced anaphylaxis (including anaphylactic shock).
Symptoms include headache, rash, chest pain, angioedema, bradycardia, tachycardia (including atrial fibrillation), bronchospasm, hypotension, hypertension, transient neurological symptoms, and pyrexia.
The most common side effects were rash (53 %), urti-caria (16 %), pruritus (14 %), and flushing (10 %).
Antialemtuzumab antibodies were detected in 62 %, 67 %, 29 %, and 75 % of the patients at 1, 3, 12, and 24 months of treatment, respectively.
In a recent study that evaluated efficacy and safety, 95.5 % of patients had IRs such as fever, shivers, rash, and headache [55] .
Atezolizumab is an IgG 1 class humanized antibody that acts by binding to programmed death ligand 1 (PD-L1) used to treat bladder, breast, and lung cancer.
PD-L1 is the immune checkpoint protein, which is expressed by tumor cells and inhibits the antitumor function of the T cell.
Inhibition of the ligand's interaction with its receptor by BS enhances the anti-tumoral activity of T cells and improves the immune system of the patients.
According to the FDA 2016 label, severe IRs were observed in 1.3-1.7 % of patients [56] .
These reactions include the following symptoms: back or neck pain, chills or shaking, dizziness, feeling like passing out, fever, flushing, itching or rash, dyspnea or wheezing, and swelling of face or lips.
However, immune-related adverse reactions (such as colitis, hepatitis, hypophysitis, meningoencephalitis, pancreatitis, and pneumonitis) have been reported that affect various organs.
In the EMA 2019 assessment report, HSR such as anaphylaxis and IRs is reported to be seen in up to 10 % of patients [57] .
In addition, itching of the skin and rash are reported to be seen in more than 10 % of patients.
According to the recent BCCA Drug Manual, it was reported that HSR including anaphylaxis can develop in ≤ 1 % (severe < 1 %), IRs in 1 % (severe ≤ 1 %), and immune-mediated rash in 8-18 % ( severe ≤ 1 %) of patients [58] .
Bevacizumab is a mAb used to treat various cancers including kidney, stomach, colon, breast, and lung cancer.
The antibody inhibits binding of VEGF to its receptors, reducing angiogenesis (blood vessel formation) and tumor growth.
According to the BCCA Drug Manual, IRs/HSRs with symptoms (dyspnea, redness, rash, hypo-or hypertension, oxygen desaturation, chest pain, tremor, and nausea/vomiting) were reported in up to 5 % of patients (referenced in [59] ).
Serious hypersensitivity reactions involving anaphylactic and anaphylactoid responses have occurred, but no rates have been reported.
In addition, rash was reported in 13 %, rhinorrhoea in 4 %, and flushing in 1 % of patients.
In the EMA assessment report, IRs occurred in 0.3-6.1 % of cases, and the most commonly reported [60] ).
In this report, HSRs and anaphylaxis were described as treatment-emergent adverse events (TEAE) and were reported to be any grade of TEAEs in 36.9 %, grade 3 or higher TEAEs in 5.9 %, and serious TEAEs in 2.5 % of cases.
Further information on reactions is not available.
IR and symptoms (chest pain, chills, grade 3 hyper sensitivity, diaphoresis, headache, hypertensive crises associated with hypertension, hypoxia, neurological signs and symptoms, and wheezing) have been reported in clinical trials and post-marketing studies, as indicated on the FDA label [61] .
Any grade of IRs are reported to be rare (< 3 %), and serious IRs have occurred in 0.2 % of the patients.
Symptoms include chest pain, diaphoresis, hypertensive crises, hypoxia, rigors, and wheezing.
Although urticaria and anaphylaxis are not mentioned in this label, exfoliative rash was seen in 23 % with the administration of this BS in combination with the chemotherapy protocol.
Blinatumomab is a bispecific mAb used for cancer immunotherapy and specific forms of acute lymphoblastic leukemia (ALL).
It exerts its effect in part by simultaneously binding CD19 on B cells and CD3 on T cells.
This BS can lead to cytokine release syndrome (CRS) in 11-12 % of patients, with 1 % of these cases being severe, to hypersensitivity in 2 % of patients, and infusion reactions in 29 % of patients, according to the BCCA's 2017 report (referenced in [62] ).
Symptoms of this syndrome include asthenia, nausea, pyrexia, headache, hypotension, and increased liver enzymes.
It was reported that CRS peaked within the first 2 days of treatment and was not easily differen-tiated from IR.
Although anaphylaxis and urticaria were not mentioned in this report, the incidence of rash was reported to be 12-21 % (severe 2-3 %).
According to the EMA assessment report, CRS occurred in 0.5-11.4 % of patients, rash occurred in 4.3 %, and IRs occurred in 67.2 % (grade ≥ 3 in 14.3 %).
In total, severe IRs were very rarely seen (4.3 %) [63] .
Fatal IRs were not reported.
Additionally, maculopapular rash and flushing have been reported in 4.3 % of patients.
According to the recent FDA label, this BS caused CRS in 7-15 % of patients (3 % of these instances were severe), skin rash in 16 % of patients (< 1 % of these instances were severe) and IRs in 77 % of patients (5 % of these instances were severe) [64] .
Symptoms of CRS included asthenia, headache, nausea, fever, hypotension, increased liver enzymes, increased total bilirubin, and diffuse intravascular coagulation.
IRs usually occurred during the first 48 h of infusion and lasted less than 2 days.
Clinical findings included hypotension, hypertension, rash, generalized pruritus, pyrexia, periorbital edema, and cytokine release syndrome.
Events defined as rash included maculopapular rash, erythema, contact dermatitis, and eczema.
Cetuximab is an intravenously administered epidermal growth factor receptor (EGFR) inhibitor used to treat advanced colorectal cancer, head and neck squamous cell carcinoma, pancreatic cancer, and less frequently, non-small cell lung cancer.
This antibody can reduce cancer cell survival, neovascularization, cell migration, and metastasis.
Cetuximab was the first anti-EGFR mAb and BS approved for cancer treatment; therefore, its immunological and allergic side effects are well studied.
In a review of acute infusion reactions induced by mAbs, acute IRs with cetuximab were in the range of 1.2-15 % (referenced in [65] ).
It is noted in that particular review that the pathogenic mechanism of mAb-related IRs has not yet been fully defined, with the exception of several suggested mechanisms by W. J. Pichler [66] .
HSRs have been reported in 7.4 % of participants in a safety study with 419 patients, with 2.6 % of reported HSRs being of grade 3 severity, and 1.4 % being of grade 4 anaphylactic reactions [67] .
The majority of reactions occurred after the first dose, and all grade 4 reactions occurred within minutes following the first infusion.
A 2015 review reported HSR to be in the 1.1-5.0 % range, and infusion reactions in the 15.0-21.0 % range in patients treated with cetuximab (referenced in [68] ).
According to the FDA label, mild IRs (fever, chills, shortness of breath, bronchospasm, angioedema, urticaria, hypertension, and hypotension) occurred in 8.4 % of patients, and severe IRs occurred in 2.2 % of patients, with 90 % of events occurring after the first infusion [69] .
In addition, 82 % of patients developed acneiform rash during treatment, 9.7 % of which were severe.
Anaphylactic reactions during treatment have been reported to be increased in association with tick bites by Amblyomma americanum, a history of red meat allergy, all associated with IgE-antibodies against α gal [69, 70, 71] .
In 2010, reactions during the first infusion of cetuximab and delayed type anaphylactic reactions to red meat were investigated in detail [70] .
In both diseases, patients were shown to have increased IgE antibodies specific for α-gal, a mammalian disaccharide, not expressed in humans but on the tissue of mammalians.
In most patients with an HSR and anaphylaxis, IgE antibodies to cetuximab were also present in serum before treatment, which prompted the investigations into the sensitization route [71] .
It was shown that α-gal is also the main cause of delayed anaphylaxis against red meat and that this was associated with the occurrence of the American tick Amblyomma americanum [72] .
Presently, tick bites are thought to be the predominant sensitization route for antiα-GAL IgE induction.
α-GAL, however, has another implication for drug-induced hypersensitivity reactions: It is present in mammalian gelatine and, therefore, may be part of vaccines, gelatine-containing infusion solutions, ovula, capsules and pills, suppositories, snake venom antisera, and animal-derived heart valves (referenced in [73] ).
However, in a study involving 92 patients, 14 (15.2 %) reported HSR, eight of these (57.1 %) reported severe HSR [74] .
Anti-cetuximab IgE levels were detected in seven of the eight patients with severe HSR.
Durvalumab is a PD-L1 binding and blocking mAb used in non-small cell lung carcinoma and metastatic urothelial carcinoma.
In the FDA 2019 labels, IRs were reported to occur in 2.2 % (grade 3 in 0.3 %), and immune-mediated rash in 11-26.0 % (grade 3-4 in 0.6-1.0 %) of patients [75] .
ADA developed in 2.9% of patients, but their clinical significance has not been established.
HSRs and anaphylaxis are not mentioned in these labels.
In the EMA 2020 assessment report, any grade of IR has been reported to occur in 1.9 % (grade 3 in 0.3 %), and skin rash in 21.7 % (grade 3 in 0.6 %) of patients [76] .
Symptoms of IR include: chills or shaking, dizziness, dyspnea or wheezing, fever, flushing, and itching or rash.
HSRs and anaphylaxis are not mentioned in these labels.
According to the BCCA's 2020 drug report, IRs can be observed in 1.0-2.0 % (severe in < 1 %) of patients, and skin rash in 14-26.0% (severe in < 1 %) of patients [77] .
Gemtuzumab (-ozogamicin) is an antibody against glycoprotein CD33, which is administered intravenously to treat acute myeloid leukemia (AML).
This antibody combined with a cell-toxic substance induces apoptosis of the targeted cancer cells.
In a prospective observational study involving 225 patients, as described in the FDA report, IRs were observed in 8.0 % of patients, and fatal reactions in 0.4 % [78] .
In addition, local reactions to the application occurred in 22.0 %.
Pruritus was described in 6.0 % and rash in 18.0 % of patients.
Infusion-related reactions usually developed within 24 h after infusion.
Signs and symptoms were similar to anaphylactic reactions.
These included chills, hypoxia, fever, tachycardia, bronchospasm, and respiratory failure.
According to the 2018 EMA assessment report, IRs, including anaphylaxis, were reported in 7.6 % of patients, and severe reactions in 3.6 % of patients [79] .
Symptoms included fever, chills, injection-site urticaria, and less frequently, hypotension, tachycardia, and respiratory symptoms.
All symptoms occurred during the first 24 h after drug administration.
In addition, rash was reported in 19.9 % ( s evere in 5.8 %), local erythema in 9.3 % (severe in 2.1 %), and pruritus in 5.4 % (severe in 0.3 %) of patients.
The literature contains one case report of HSR mortality, which was associated with platelet transfusion after gemtuzumab treatment [80] .
Ibritumomab (-tiuxetan) is used in non-Hodgkin's lymphoma patients or for cancers that are refractory to rituximab.
It acts by binding to the antigen CD20, which is present on the surface of malignant and normal B lymphocytes.
In the first FDA report, allergic reactions were reported in 2 % of patients, urticaria in 4 %, and severe life-threatening reactions such as angioedema, lung edema, tachycardia, subdural hematoma, and pulmonary embolism in < 1 % of patients [81] .
In the EMA evaluation report, allergic (hypersensitivity) reactions and infusion reactions were described [82] .
Common symptoms (up to one in 10 patients) include skin reactions, difficulty in breathing, swelling, itching, redness, chills, and dizziness (a potential marker for hypotension).
Severe hypersensitivity reactions involving anaphylaxis occurred in < 1 % of patients.
According to the BCCA's drug report, ISRs (dermatitis, desquamation, and ulcer following extravasation) occurred in 1.0 % of patients, angioedema in 5.0 % (severe angioedema in 1.0 %), pruritus in 9.0 %, rash in 8.0 %, and mucocutaneous reactions in < 1 % of patients (referenced in [83] ).
That particular monograph states that rituximab is an essential component of ibritumomab-based treatment regimens.
Infusion reactions may occur frequently after pretreatment with rituximab, or during or after ibritumomab administration.
Asthenia, cough, dizziness, nausea, pruritus, pyrexia, rash, rigors, tachycardia, and vomiting have been reported among the symptoms of IRs.
In a recent case report, patients were shown to develop human anti-murine antibodies (HAMA) after exposure to murine antibodies [84] .
The low incidence of hypersensitivity reactions in patients with high HAMA titers is particularly promising for ibritumomab.
Imatinib is an active ingredient in capsules or tablets containing a group of kinase inhibitors used to treat chronic myeloid leukemia.
Other and rarer indications include acute lymphoblastic leukemia, skin tumors, some gastrointestinal tumors, hypereosinophilic syndrome, atypical myelodysplastic or myeloproliferative disorders, as well as systemic mastocytosis.
The first FDA report does not include information on hypersensitivity and anaphylactic reactions.
This report includes only skin rash in 32-39 % of patients (severe skin rash in 3-4 %), and pruritus in 6-10 % (severe in 0.4-1.0 %) [85] .
BCCA drug manuals describe cutaneous reactions in <1 %, rash in 32-39 % (severe rash in 3-4 %), and pruritus in 6-10 % (with severe pruritus in 0-1 %) of patients.
This manual does not include information on hypersensitivity and anaphylactic reactions [86] .
Ipilimumab is a fully-human mAb used primarily for the treatment of metastatic melanoma, metastatic colorectal carcinoma, and renal cell carcinoma.
This BS shows its effect by binding and inhibiting human cytotoxic T lymphocyte antigen 4 (CTLA-4).
As a result, T cell infiltration increases in tumor cells and tissues, thereby leading to their destruction.
In addition, studies are ongoing for its use in the treatment of lung cancer, bladder cancer, and metastatic prostate cancer.
In the FDA 2019 labels, IRs were reported to occur in 4.2-5.1 % of patients and urticaria in 2.0 % of patients [87] .
Symptoms of IR include: chills or shaking, dizziness, dyspnea, feeling like passing out, fever, flushing, and itching or rash.
In the EMA 2020 assessment report, HSRs are reported as uncommon (≥ 1/1000-< 1/100), urticaria as common (≥ 1/100 to < 1/10), and anaphylactic reaction as very rare (< 1/10,000) [88] .
IRs were observed at a rate of 2.2-4.0 % depending on the dose of the drug, but grade 3-5 IRs were not reported.
According to the BCCA's 2020 drug report, IRs can be observed in 2.0-6.0 % (severe in < 1 %) of patients, pruritus in 24-26.0 % of patients, and skin rash in 19-26.0 % (severe in <1%) of patients [89] .
Additionally, a case that developed a delayed type of HSR during melanoma treatment has been reported [90] .
Necitumumab is a human mAb used in chemotherapy combinations in the treatment of non-small cell lung cancer, and shows its effect by binding to the EGFR.
EGFR plays an important role in the uncontrolled growth and proliferation of tumor cells and tissue as a result of apoptosis inhibition.
In the FDA 2015 labels, IRs of any severity have been reported to occur in 1.5% (grade 3 in 0.4 %) of patients [91] .
These reactions have been frequently observed after the first two doses.
The same data are also reported in the EMA 2016 assessment report [92] .
In addition, it is stated that skin reactions can be seen in 77.9 % (grade 3 in 6.3 %) of patients.
Symptoms of IR include: chills, dyspnea, or fever.
Urticaria and anaphylaxis are not mentioned in that report.
In addition, sudden death or cardiorespiratory arrest was reported in 2.8 % of patients, which may also be a symptom of anaphylaxis.
Nivolumab is a human mAb that is used in the treatment of advanced renal cell carcinoma, hepato-cellular carcinoma, Hodgkin's lymphoma, melanoma, non-small cell lung cancer, squamous cell cancer of the head and neck, as well as urothelial cancer and acts by binding to the programmed cell death protein (PD)-1-receptor.
In the FDA 2019 labels, IRs were reported to occur in 6.4 % of patients [93] .
The IR causing permanent discontinuation or withholding of the BS was reported in 0.5-1.4 % of patients within 48 h after infusion.
Anaphylaxis and hypersensitivity have not been reported, but it has been reported that the incidence of symptoms such as abdominal pain, arthralgia, asthenia, back pain, constipation, cough, decreased appetite, diarrhea, dyspnea, fatigue, headache, musculoskeletal pain, nausea, pruritus, pyrexia, rash, upper respiratory tract infection, and vomiting is more than 20 %.
Some of these symptoms can be seen in anaphylaxis.
In treatment with this BS, immune-mediated organopathies such as colitis, encephalitis, hepatitis, pneumonitis, endocrinopathies, and nephritis can also be observed.
The clinical significance of ADAs for these side effects is still unknown.
In the EMA 2020 assessment report, IRs and HSRs are stated as common (≥ 1/100-< 1/10), rash and prutitus as very common (≥ 1/10), and anaphylactic reaction as rare (≥ 1/10,000-< 1/1000) [94] .
According to the BCCA's 2020 drug report, IRs can be observed in 2.0-4.0 % of patients, pruritus in 7-17.0 % of patients, skin rash in 11-21.0 % (severe in 1.0 %), and urticaria in 1.0 % of patients [95] .
Allergic symptoms (eyelid angioedema, flushing, and hives on the neck and face) were reported in one patient during treatment with nivolumab [96] .
However, a case with cytokine release syndrome has been reported [97] .
In a recent case report, a patient who had recurrent infusion reactions despite premedication against nivolumab was successfully treated with pembrolizumab, another PD-L1/PD-L2inhibitor [98] .
Panitumumab is a mAb EGFR inhibitor delivered intravenously to treat metastatic colorectal cancer.
It acts by binding and inhibiting EGFR.
Panitumab appears to be a safe option for treatment and potential side effects in patients with anti-α-gal IgE antibodies [99] .
This is due to the fact that α-galactosylated glycans are absent in purely human mAbs and that the system in which it is expressed does not seem to induce posttranslational modifications like allergenic glycosylation.
The chimeric mAb cetuximab is not suitable in this sensitive patient group due to its high α gal content.
In the 2015 FDA report, IRs were reported in 4 % of patients (severe reactions in 1 %) [100] .
Severe IRs included anaphylactic reactions, bronchospasm, and hypotension.
Angioedema and fatal reactions have also been documented in post-marketing reports.
In the BCCA drug monograph, HSR was reported in 1 % of patients (occurring within 24 h), while IRs were reported in 3-4 % (severe IRs were seen in 1 %, occurring within 24 h) (referenced in [101] ).
Angioedema was seen very rarely.
Some cases were fatal, with possible late onset more than 24 h post infusion.
The symptoms of most IRs were mild and included chills, fever, or dyspnea.
In addition, pruritus was reported in 34-69 % (severe in 1-4 %) and rash in 20-78 % (severe in 1-3 %).
At the annual congress of the European Academy of Allergy and Clinical Immunology (EAACI) in 2019, a case of a successful desensitization was presented in a patient that had developed anaphylaxis against panitumumab [102] .
Pembrolizumab is a monoclonal antibody that is used to treat non-small cell lung cancer, lymphoma, melanoma, and urothelial carcinoma and which binds to the PD-1-receptor and inhibits its interaction with its ligands.
In the EMA 2019 assessment report, IRs are stated as common (≥ 1/100-< 1/10), rash and pruritus as very common (≥ 1/10), and gastrointestinal symptoms (such as abdominal pain, constipation, diarrhea, nausea, vomiting) as very common (≥ 1/10) [103] .
According to the BCCA's 2019 drug report, IRs can be observed in < 1.0 % of patients, pruritus in 12-30.0 % of patients, and skin rash in 1.0-29.0 % (severe in 1.0 %) of patients [104] .
In the FDA 2020 labels, severe or life-threatening anaphylaxis, HSRs or IRs were reported in 0.2 % of patients [105] .
Symptoms of IR include: chills, dizziness, fever, flushing, hypotension, hypoxemia, pruritus, rash, rigors, and wheezing.
However, the incidence of ADAs against pembrolizumab was 1.8 %, and it was reported that ADA had no clinical relevance [106] .
Pertuzumab is a humanized mAb that acts by blocking the human epidermal growth factor receptor 2 protein (HER2), and is used in combination with trastuzumab and docetaxel in the treatment of breast cancer.
HER2 plays an important role in the growth and differentiation of tumor cells, and its blocking results in apoptosis.
According to the BCCA's 2014 drug report, HSRs can be observed in 11.0 % (severe in 2-5 %) of patients, IRs in [13] [14] [15] [16] [17] [18] [19] .0 %, rash in 10-20 % (severe in 2 %), and pruritus in 10% of patients [107] .
Symptoms of IR include: asthenia, chills, fatigue, fever, hypersensitivity, and vomiting.
In the EMA 2020 assessment report, IRs and rash are stated as very common (≥ 1/10), HSRs as common (≥ 1/100 to < 1/10), anaphylaxis as uncommon (≥ 1/1000-< 1/100), and CRS as rare (≥ 1/10,000-< 1/1000) [108] .
The most common IRs have been reported as asthenia, chills, fatigue, headache, hypersensitivity, pyrexia, and vomiting.
In the FDA 2019 labels, IRs have been reported to occur in 13-21.0 % (severe less than 1.0 %), and hypersensitivity/anaphylaxis in 5.0-11.0 % of patients [109] .
It is stated that CRS may also develop.
Ramucirumab is a human mAb used in the treatment of gastric, breast, lung, and colorectal cancers, showing its effect by selectively inhibiting VEGF receptor 2.
VEGF plays an important role in angiogenesis, cell division, and migration in vascular endothelium.
According to the BCCA's 2017 drug report, IRs can be observed in 1.0-16.0 %, and rash in 4.0 % of patients [110] .
Symptoms of IR include: back pain, chest pain, chills, dyspnea, flushing, paresthesias, rigors, and wheezing.
Some cases have been observed with more severe symptoms such as bronchospasm, hypotension, and supraventricular tachycardia.
In the FDA 2019 labels, IRs have been reported to occur in 1.0-9.0 % (severe in < 1.0 %) of patients [111] .
Symptoms of IR include: back pain/spasms, chills, chest pain and/or tightness, dyspnea, flushing, hypoxia, paresthesia, rigors/tremors, and wheezing.
In the EMA 2019 assessment report, IRs can be observed in up to 10.0 % of patients [112] .
Symptoms of IR include: back or chest pain, chest tightness, chills, dyspnea, feeling of tingling or numbness in hands or feet, flushing, increased muscle tension, and wheezing.
Some cases have been observed with more severe symptoms such as breathing distress, feeling faint, and tachycardia.
Anaphylaxis and hypersensitivity are not mentioned in that report.
Trastuzumab is an anti-HER2-antibody combined with or conjugated to a cytotoxic drug that is used to treat local or advanced inoperable breast and gastric cancer.
In the BCCA drug assessment report (referenced in [113] ), IRs occurred in 21-40 % of cases, with severe reactions in 1 %.
Allergic reactions were reported in 3 % of cases.
IRs occurred in 40 % of patients with their first infusion.
Mild to moderate symptoms included headache, dizziness, rash, cough, nausea, vomiting, pain, chills, and asthenia.
Severe reactions and symptoms were most often respiratory distress, bronchospasm, wheezing, low oxygen saturation, and hypo-or hypertension.
[68] ) reported an HSR rate of 0.6-5.0 % and an infusion-related reaction rate of 40 %.
According to the EMA assessment report 2018, HSR was reported in 0.9-3.5 %, IRs in 8.5-37.1 %, and anaphylaxis in 0-0.9 % of patients [114] .
According to the FDA's 2019 report, IRs occur in 1.4-1.6 % of patients and include symptoms such as fever, chills, redness, shortness of breath, bronchospasm, wheezing, tachycardia, and hypotension.
In the same report, pruritus was seen in 6.0 % and drug hypersensitivity was found to occur in 2.2-2.7 % of patients.
In preliminary studies, anaphylaxis was seen in one patient [115] .
BSs are often used to treat rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel diseases (ulcerative colitis, Crohn's disease), Still's disease, and vasculitis in rheumatology.
Details on the reactions are given in Tab.
5.
Adalimumab is a recombinant human IgG1 that binds to TNF α with high affinity.
It acts by inhibiting the binding of TNF α to p55 and p75 surface receptors on target cells.
In a study on 201 patients conducted by Puxeddu et al.
[116] , hypersensitivity reactions occurred in seven patients (3.5 %), local reactions occurred in three patients (1.5 %), and urticaria and angioedema occurred in three patients (1.5 %).
Anaphylaxis was not observed in this study, but has been reported very rarely in the post-marketing period.
Another study conducted by Tarkiainen et al.
[117] reported HSR frequencies of 18.1 %, ISRs frequencies of 17.0 %, and allergic symptoms in 1.1 % of the patients.
FDA 2018 labeling showed that local ISRs are more common in patients treated with the TNF α inhibitor than in those treated with placebo [118] .
The incidence of ISRs including erythema, itching, hemorrhage, and pain or swelling is 8.0-20.0 % in patients treated with adalimumab vs 1 % in patients treated with placebo.
Anaphylaxis and angioedema have rarely been reported following the application of this BS, and in the first 48 weeks of treatment, approximately 6 % of patients had non-severe local allergic hypersensitivity reactions and allergic rashes.
Anakinra is a genetically engineered and recombinant human IL 1 receptor antagonist.
Its main effect is by neutralizing the biological activity of IL-1α and IL-1β, a proinflammatory cytokine.
As a result, it contributes to the reduction of synovial inflammation by reducing many cellular responses.
However, anakinra has the following therapeutic indications: rheumatoid arthritis (RA), cryopyrinassociated periodic syndrome (CAPS), and Still's disease.
In the EMA summary of product characteristics (SmPC) report, ISRs are generally reported to occur in the first 2 weeks of treatment (for RA and Still patients) and resolve within 4-6 weeks [119] .
In that particular report, allergic reactions such as anaphylactic reactions, angioedema, urticaria and pruritus are expected to range from 1/100 to 1/1,000.
According to the FDA's 2018 label, the most frequently reported adverse reactions were ISRs (71.0 %), which were mild to moderate in the majority of patients [120] .
Only 3.2 % of these reactions were classified as severe, with the majority includ-ing ecchymosis, erythema, inflammation, and local pain.
Reactions usually occurred during the first month.
This label states that anaphylaxis, angioedema, urticaria, and rash may occur, but no specific rate or detailed information is provided.
In the literature, it has been reported that anakinra treatment may cause anaphylaxis, cutaneous drug reactions, erythematous plaques, pruritic rash, and severe HSRs [121] .
In a pediatric case, a patient with anakinra-induced anaphylaxis was successfully treated with canakinumab, an alternative anti-IL 1 agent [122] .
However, a desensitization protocol is also available [123] .
Belimumab is a recombinant human IgG1 mAb that binds and inhibits the biological activity of the solIn the EMA 2018 assessment report, any event of ISRs (local pain, erythema, hematoma, induration, and pruritus) were reported in 6.1 % of patients, HSRs in < 10.0 % of patients, and post-injection anaphylactic reactions were reported in 0.1-1.0 % of patients (serious reactions, 0.9 %) [124] .
HSR and anaphylactic reaction are mentioned to occur mostly during the first two infusions.
In the newly published FDA report, intravenous administration was evaluated [125] .
HSRs were reported in 13.0 % (in the placebo group 11 %), anaphylaxis in 0.6 % (placebo 0.4 %) of patients.
Symptoms included angioedema, dyspnea, hypotension, pruritus, rash, or urticaria.
IRs were observed in 17 % of patients and severe IRs in 0.5 %, with placebo in 15 % and 0.4 %, respectively.
It is stated that it is not easy to differentiate between HSRs and IRs due to the overlap in signs and symptoms.
However, it has been reported that there is insufficient evidence as to how the premedication of some patients affects the frequency or severity of reactions.
Canakinumab is a human mAb against IL-1 beta and indicated in periodic fever syndromes, CAPS, tumor necrosis factor receptor-associated periodic syndrome (TRAPS), familial Mediterranean fever (FMF), Still's disease, and gouty arthritis.
According to the FDA's 2016 label, the most frequent adverse drug reactions were infections predominantly of the upper respiratory tract, and mild to moderate ISRs equal or greater than 10.0 % [126] .
No anaphylactoid or anaphylactic reactions were observed in more than 2,600 patients whose clinical development was tested for this biologic, but the risk of severe hypersensitivity reactions was mentioned.
No cases of canakinumab-related anaphylaxis have been reported in the current literature [127] .
In addition, patients with anaphylaxis to anakinra have been shown to tolerate canakinumab very well [122] .
Etanercept is a subcutaneously administered selective immunosuppressive and anti-inflammatory BS belonging to the group of TNF α inhibitors used to treat rheumatoid arthritis and other rheumatic diseases.
It exerts its effect by inhibiting the activity of TNF α. Puxeddu et al.
reported a 5.3 % rate of HSRs, a 0.8% rate of anaphylaxis, a 2.0 % rate of angioedema or urticaria, and a 1.6 % rate of local reactions in a study involving 245 patients treated with etanercept [116] .
In another study, the rate of HSRs was 11.3 %, that of ISRs was 7.5 %, and reactions at sites other than the injection site occurred at a rate of 4.2 % [117] .
The FDA drug label published in 2018 reports that allergic reactions associated with this treatment occur in < 2 % of patients, and mild to moderate ISRs (erythema, itching, pain, swelling, bleeding, bruising) occur in 15-37 % of patients during the first 3 months of treatment [128] .
ISRs were usually seen in the first month, and with decreased frequency thereafter.
Golimumab is a selective immunosuppressive and anti-inflammatory TNF-α-inhibitor applied subcutaneously to treat rheumatoid and psoriatic arthritis, as well as ankylosing spondylitis.
It is a mAb that inhibits the activity of soluble and membrane-bound forms of the proinflammatory cytokine TNF α by binding TNF α and inhibiting its r eceptor binding.
In a 3-year safety study, the frequency of ISRs was 4.7-11.6 %, depending on the drug dose.
No anaphylactic reactions or serum sickness-like reactions have been reported to date during the follow-up period of up to 160 weeks [129] .
The FDA 2018 drug label reports an ISR frequency of 3.4-6.0 % [130] .
The reactions were often mild and included erythema, urticaria, induration, and pain.
No anaphylactic reactions were observed in phase II and III studies with the drug.
Infliximab is an intravenously administered selective immunosuppressive and anti-inflammatory TNF α inhibitor used to treat rheumatoid and psoriatic arthritis, Crohn's disease, and ankylosing spondylitis.
This BS is a human-murine chimeric monoclonal IgG1κ antibody that binds TNF α and inhibits its effects.
In a review by Maggi et al.
summarizing nine studies, infusion reactions occurred in 1-27 % of patients depending on the disease being treated ( referenced in [65] ).
In a study on 225 patients, Puxeddu and colleagues reported hypersensitivity reactions in 13.8 %, anaphylaxis in 9.3 %, urticaria or angioedema in 4.4 %, and local reactions in 0 % of patients [116] .
Another study reported a 34.1 % rate of infusion-related HSRs, and a 1.9 % rate of anaphylaxis [117] .
The FDA 2019 drug label reports IRs in 18 % of patients during or within 1 h after the infusion [131] .
A total of 3 % of all patients given infliximab infusions experienced nonspecific symptoms (fever or tremor), while 1% experienced cardiopulmonary reactions (chest pain, hypotension, hypertension, or dyspnea).
Symptoms related to pruritus, urticaria, and cardiopulmonary reactions were detected in 1 % of patients.
Serious infusion reactions such as anaphylaxis, convulsions, erythematous rash, and hypotension have occurred in < 1 % of patients.
Delayed type HSR was detected in approximately 1 % of patients.
This usually occurred within 2 weeks of re-infusion, and was a combination of fever and/or rash symptoms with arthralgia and/or myalgia.
These findings have been classified and reported as serum sickness.
In a study conducted by Vultaggio et al., it was reported that IgE or IgM antibodies against infliximab could be detected and may play a role in IgEand non-IgE-mediated anaphylaxis [132] .
Skin testing and infliximab-specific antibody detection have been shown to be useful in predicting and preventing severe drug-related reactions [133] .
Certolizumab is a subcutaneously administered selective immunosuppressive and anti-inflammatory TNF-α-inhibitor used to treat rheumatoid and psoriatic arthritis, spondyloarthritis, and Crohn's disease.
It is a mAb that binds to and blocks the activity of the proinflammatory cytokine TNF α.
In the EMA 2015 assessment report, mild to moderate ISRs were reported in 6.4% of patients, injection-related acute systemic HSR (pre-syncope) was reported in 0.2%, and delayed HSR was seen in 1.1% of patients [134] .
Recently published FDA labels have reported that HSRs may rarely occur and include allergic dermatitis, rash, angioedema, dizziness, pyrexia, flush, ISRs, malaise, serum sickness, hypotension, dyspnea, and vasovagal syncope [135] .
It is noted that some of these reactions were observed after the first administration.
ISRs were reported in 1.7-3.2% of patients.
Although the literature contains no clear information on rates of hypersensitivity and anaphylaxis, it is stated that no single-drug related anaphylactic shock was observed in the one study conducted in 2013 [136] .
Rituximab is an intravenously delivered mAb that selectively binds to the CD20 antigen of B lymphocytes.
Indications for its use include non-Hodgkin's lymphoma, rheumatoid arthritis, and chronic lymphocytic leukemia.
Maggi et al.
have reported acute infusion reaction frequencies of 10-77% during the first infusion (referenced in [65] ).
The FDA drug label for subcutaneous use [137] reports a 16-26% rate of local ISRs (pain, swelling, induction, hemorrhage, rash, pru-ritus, and erythema).
Incidence rates reportedly decrease subsequent to the initial injection.
The label also states that severe cytokine release syndrome and its symptoms (fever, tremor, urticaria, angioedema, bronchospasm, and severe dyspnea) may be seen within 1-2 h of infusion.
However, the ratio is not specified in this label.
The recent FDA drug label for intravenous use [138] reports a ≥25.0% rate of IRs that typically (77%) occurred at the first infusion with the time onset between 30-120 min.
This rate decreased after each ongoing infusion.
Severe IRs may include symptoms such as acute respiratory distress syndrome, anaphylactic/anaphylactoid events, angioedema, bronchospasm, cardiogenic shock, hypoxia, hypotension, myocardial infarction, pulmonary infiltrates, urticaria, ventricular fibrillation, and death.
According to the BCCA drug assessment report (referenced in [139] ) HSRs occurred in 1-10.0 % of patients, IRs in 14-77.0 % (severe, 0-7.0 %), ISRs in 20.0 %, and urticaria in 7.0 % (severe, 1.0 %) of patients.
ISRs include local erythema, hemorrhage, induration, pain, pruritus, rash, and swelling at the injection site.
In a study conducted in 2009, the presence of serum anti-rituximab antibodies was reported in some patients in association with a less favourable treatment outcome [140] .
Vultaggio and colleagues have shown that rituximab plays a role in specific T-helper 2 cell and IgE responses in acute infusion reactions in addition to cytokine release mechanisms [141] .
A less frequent use of BSs is to minimize the rejection of transplanted organs (mostly kidneys, hearts, and livers).
Basiliximab, muromonab, and daclizumab have been developed for this purpose.
Today, only basiliximab is approved for this application.
The other two drugs have been withdrawn from the market for a variety of reasons.
Details about the reactions are given in Tab.
6.
Basiliximab is an immunosuppressive intravenously administered chimeric (75 % human, 25 % murine) mAb.
It is used in combination with other immunosuppressive drugs to prevent acute graft rejection following renal transplantation.
It acts by binding the alpha chain of the IL 2 receptor on the surface of T lymphocytes.
The FDA drug label reports a > 10.0 % rate of side effects including nausea, vomiting, fever, dyspnea, and hypertension, and 3.0-10.0 % of side effects include angina pectoris, cardiac failure, tachycardia, dizziness, bronchospasm, pulmonary edema, rhinitis, rash, and pruritus [142] .
More detailed informa-tion is not provided.
In a study conducted in 2010, infusion-related ISRs (pain, swelling, erythema) were found in 4.3 % of patients [143] .
In post-marketing surveys, HSRs have been reported in patients undergoing basiliximab therapy, including one case of IgE-mediated anaphylaxis [144] .
Data on this BS are relatively limited, but according to the reports it seems to be mainly well tolerated by the patients [145] .
Allergic reactions and anaphylaxis do not seem to be easily identified, since the patients experience intense immuno suppressive effects due to the concomitant medication.
It does not induce CRS like muromonab-CD3, and its side effects are reported to be similar to placebo.
Belatacept is a human CTLA-4/human IgG1 fusion protein that selectively inhibits T cell activity by binding to CD80 and CD86 of the antigen presenting cells.
According to the FDA 2017 labels [146] , no patient had developed anaphylaxis or HSRs at the 3 year follow-up.
However, milder IRs (5 %) were reported, similar to placebo, within 1 h of infusion.
In post-marketing experiences, a case of anaphylaxis has been reported.
In the EMA 2019 assessment report [147] , acute infusion events (flushing, headache, hypotension, hypertension) were reported in 4.4-5.5 % of patients.
Symptoms of diarrhea and pyrexia (≥ 2 %), as well as hypertension, peripheral edema, nausea, and vomiting (≥ 20 %), which may be "possible peri-infusional events," were reported in 3 year follow-up of the patients.
There was no association found between these reactions and belatacept ADAs.
Anaphylactic events have been observed in the post-marketing period, but no incidence was given.
Muromonab is an immunosuppressive mAb given to reduce acute rejection in patients with kidney, liver, or heart transplants.
It exerts its effect by targeting the CD3 receptor, a membrane protein expressed on the surface of T cells.
Developments and progress of medical treatment in transplant medicine led to its withdrawal in 2010.
The literature contains information on cytokine release syndrome and anaphylaxis.
In a book on this subject, symptoms of anaphylaxis such as pyrexia (90 %; 40 .0 °C or higher in 19 % of cases), chills (59 %), dyspnea (21 %), nausea and vomiting (19 %), chest pain (14 %), diarrhea (14 %), tremor (13 %), bronchospasm (13 %), headache (11 %), tachycardia (10 %), stiffness (8 %), and hypertension (8 %) were reported [148] .
Pilot studies of this drug have been performed orally in patients with moderate to severe ulcerative colitis [149] .
In this study, no severe side effects or CRS have been reported.
Daclizumab is a subcutaneously administered, immunosuppressive mAb used to treat relapsingremitting multiple sclerosis and to prevent rejection of transplanted kidneys.
The antibody reduces the number of circulating activated T cells by binding to the alpha subunit of the IL-2 receptor on T lymphocytes and inhibiting interaction with IL-2.
In 2018, the drug was withdrawn from the market owing to reports of severe hepatotoxic side effects and encephalopathies.
Other indications for the use of biologicals include multiple sclerosis, paroxysmal nocturnal hemoglobinuria, instable angina pectoris during insertion of cardiac catheters, congenital bronchopulmonary dysplasia, and congenital heart disease.
Abciximab is an intravenously administered chimeric mAb fragment that inhibits binding of platelet adhesion proteins (such as fibrinogen, fibronectin, von-Willebrand-Factor) to glycoprotein IIb/IIIa receptors on the surface of platelets.
It is frequently used to prevent ischemic complications associated with invasive heart procedures, as well as to prolong cardiac infarct prophylaxis in patients with unstable angina pectoris due to its antithrombotic effect.
The registry study of the drug reported hypersensitivity rates of 0.3-0.6 %, an urticaria rate of 0.1 %, and no cases of anaphylaxis or angioedema [150] .
In the same study, human anti-chimeric antibodies were found to be associated with thrombocytopenia.
The literature contains a very small number of case reports.
One developed an anaphylaxis 2-4 h after treatment [151] .
The FDA label reported bronchospasm (0.3 %), injection site pain (0.1-3.6 %), and pruritus (0.5 %) [152] .
No incidence of anaphylaxis has been reported in any of the phase III studies (clinical trials).
Eculizumab is the humanized mAb used primarily for the treatment of paroxysmal nocturnal hemo-globinuria and atypical haemolytic uremic syndrome.
It can also be used in the treatment of generalized myasthenia gravis and neuromyelitis optica spectrum disorder with antibody positivity.
This drug acts by binding to the C5 component of the complement system, inhibiting its activation, and consequently contributes to reducing erythrocyte destruction and hemolysis.
FDA labels reported that IRs, HRs, and anaphylaxis may develop during treatment, but IRs requiring discontinuation of the drug have not been reported [153] .
In the EMA 2019 assessment report, HSRs, IRs, urticaria, and anaphylaxis are stated as uncommon (≥ 1/1000-< 1/100) [154] .
Lanadelumab is a human mAb (class IgG1 kappa) that specifically inhibits the activity of plasma kallikrein, used to reduce attacks and representing a prophylaxis measure in patients with hereditary angioedema [155] .
In a study published in 2018, 52.4 % of patients reported ISRs (34.1 % in the placebo group) with symptoms such as bruising, dizziness, erythema, and local pain [156] .
Only one patient (1.2 %) developed mild-to-moderate HSRs, which included transient symptoms of oral tingling and pruritus, and resolved spontaneously within 1 day after onset without need for medication.
The EMA 2018 evaluation report [157] provides more detailed data on the study conducted by Banerji et al.
[156] .
In this report, ISRs are given as follows; pain (41.7 %), erythema (9.5 %), bruising (6.0 %), discomfort (3.6 %), hemorrhage (3.6 %), pruritus (3.6 %), swelling (3.6 %), hematoma (2.4 %), induration (2.4 %), paresthesia (2.4 %), warmth (2.4 %), edema (1.2 %), and rash (1.2 %).
In addition, no events of anaphylactoid reaction or anaphylaxis were reported (referenced in [157] ).
Natalizumab is an intravenously administered, immunosuppressive recombinant humanized IgG4 antibody that is produced in mouse cells.
It acts by binding integrin a4 and is used to treat relapsingremitting multiple sclerosis.
Maggi and colleagues (referenced in [65] ) reported IRs in 1-4 % of the patients in their review.
According to the EMA 2016 evaluation report [158] , 23.1 % of patients experienced infusion-related reactions, HSRs occurred in up to 4 % of patients, and anaphylactic/anaphylactoid reactions in less than 1 %.
HSRs usually occurred during infusion or within 1 h after completion.
Hypersensitivity symptoms included rash and urticaria, hypotension, hypertension, chest pain, chest complaints, dyspnea, and angioedema.
The FDA's 2019 label reported infusion-related reactions in 11-24 % of patients (placebo 7-18.0 %), acute urticaria in 1-2 %, acute HSRs in 1.5 %, pruritus in 4 %, and serious IRs and anaphylaxis in < 1 % [159] .
Symptoms frequently associated with these reactions were fever, chills, rash, urticaria, pruritus, dizziness, nausea, flush, hypotension, dyspnea, and chest pain.
The reactions occurred mostly within 2 h after infusion.
Palivizumab is an intramuscularly administered humanized mAb (95 % human and 5 % murine) that is used in pediatric patients to prevent respiratory syncytial virus (RSV) infections.
The antibody binds to coat proteins on the viral surface, thereby inhibiting its entry into host cells.
It is used in patients with bronchopulmonary dysplasia, cystic fibrosis, and neonatal heart disease that are considered to be at high risk of severe RSV-associated disease.
Serious acute HSRs were rare among the 400,000 patients treated to date (>2 million doses) [ In cr e a si n g R is k fo r se v e re R e a c ti o n s BSs are increasingly used today and play an important role in the treatment of allergic, pulmonary, dermatological, oncological, rheumatological, as well as various other diseases.
These drugs can be used as first-line or second-line treatment, as well as for prevention purposes.
Observed HSRs may be mild, moderate, but may also be life-threatening by severely affecting vital parameters.
All organ systems can be affected during reactions, so clinical symptoms are quite diverse.
Reactions may occur due to allergic, non-allergic, or immunological side effects.
The risk of allergic reactions decreases proportionally with the increase in human homology of the BS ( Fig.
1; [163] ).
Vultaggio and co-authors explain the reactions to BSs with two possible main mechanisms: (i) loss of immune tolerance due to side effect of the BSs; and (ii) triggering of immune responses and reactions to non-self epitopes in BSs [164] .
This comprehensive review reveals a hypersensitivity prevalence against various BSs and an inconsistency and lack of precision regarding the nomenclature used for immune reactions to BSs (hypersensitivity, anaphylaxis, anaphylactoid, and infusion reactions) in the literature.
Theoretically defined hypersensitivity and the hypersensitivity that is observed and classified in clinical practice seem to be different.
Similarly, many data reported as anaphylaxis actually describe severe anaphylactic reactions (grades III or IV).
It became evident that these definitions describe many common symptoms.
Due to the overlap in signs and symptoms in the reported descriptions, it is not always possible to differentiate these reactions properly according to their pathomechanism.
The common points of all these reactions are indicated in Tab.
7 [165, 166, 167] .
In addition, reactions such as pruritus, flush, and urticaria are also included in the definitions of IR, HSR, anaphylaxis, and CRS.
A summary view of the reactions according to severity is shown in Fig.
2.
Fig.
3 shows a synopsis of the incidences of HSRs (Fig.
3a) , acute IRs (Fig.
3b) , and anaphylaxis to BSs (Fig.
3c) as extracted by the authors from the comprehensive review of the databases.
There is an urgent need to define and classify these reactions more precisely in the future.
Furthermore, the development of a scoring system, including symptom-or system-based approaches, can make it easier for us to identify the reactions of our patients and treat them adequately.
This scoring can be evaluated according to which organ system (cardiac, gastrointestinal, hematologic, muscular, neurological, renal, respiratory, skin, vascular) is affected and the severity of symptoms.
One of the important points that may have been overlooked is the understanding of whether some symptoms (such as nasopharyngitis, conjunctivitis, rhinitis, thrombocytopenia, anemia, or gastrointestinal symptoms, etc.)
have an immunological origin.
The role of ADAs in these symptoms is not yet known.
In addition, the risk of patients at each injection/infusion should be defined according to real life.
In general, the literature gives percentages by calculation and only follow-up reactions in the treatment process.
The long-term evaluations seem to be rather small in number.
CRS, which is a form of severe IR, can also be observed during alemtuzumab, anti-CD28 monoclonal antibody TGN1412, anti-thymocyte globulin, blinatumomab, brentuximab, dacetuzumab, muro monab-CD3, nivolumab, rituximab, and obinutuzumab treatments (referenced in [167] ).
A large majority of patients may have sepsis/septic shock fragments, as well as symptoms that may also be confused with tumor lysis syndrome.
IL-6 plays an important role in the development of CRS; therefore siltuximab or tocilizumab, a chimeric and a humanized mAb against IL-6 and the Il-6 receptor, respectively, are used for the treatment of this condition [167] .
In cases where CRS developed during treatment, the relationship between the drug itself and ADA is so far unknown.
BSs are important immunotherapeutic tools used in the treatment of many diseases today.
Various side effects and reactions can be observed during c treatment, but there are deficiencies in their identification and classification.
During this comprehensive research, it became evident that a more precise nomenclature should be applied.
In order to achieve a harmonization in this regard, a simpler symptomor system-based classification and scoring system is needed.
A better understanding of the pathophysiology of hypersensitivity reactions and increased clinical experience in the treatment of side effects will provide timely control of unexpected reactions.
As a result, immunotherapy with BSs will become safer in the future.
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S2 guideline of DGAKI Cytokine release syndrome A. Gülsen: Data collection, design of tables and figures, analysis and interpretation of data, writing and drafting of the manuscript; B. Wedi: major role in revising the manuscript; U. Jappe: concept of the manuscript, acquisition of data, design of Figure 1 , writing and revising the manuscript.
A. Gülsen, B. Wedi, and U. Jappe declare that they have no competing interests.
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Gülsen A, Wedi B, Jappe U. Hypersensitivity reactions to biologics (part I): allergy as an important differential diagnosis in complex immune-derived adverse events.
Allergo J Int 2020;29:97-125 https://doi.org/10.1007/s40629-020-001266
key: cord-328471-oz99upzz authors: Ahmad, Jamshaid; Ikram, Saima; Ahmad, Fawad; Rehman, Irshad Ur; Mushtaq, Maryam title: SARS-CoV-2 RNA Dependent RNA Polymerase (RdRp) – A drug repurposing study date: 2020-07-23 journal: Heliyon DOI: 10.1016/j.heliyon.2020.e04502 sha: doc_id: 328471 cord_uid: oz99upzz The outbreak of SARS-CoV-2 in December 2019 in China subsequently lead to a pandemic.
Lack of vaccine and specific anti-viral drugs started a global health disaster.
For a sustained control and protection, development of potential anti-viral drugs is one of the targeted approach.
Although, designing and developing a panel of new drugs molecules are always encouraged.
However, in the current emergency, drug repurposing study is one of the most effective and fast track option.
The crystal structure of a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) RNA Dependent RNA Polymerase (RdRp) has recently been deciphered through X-ray crystallography.
The single-chain of core RNA Dependent RNA Polymerase relies on virus-encoded cofactors nsp7 and two units of nsp8 for its optimum function.
This study explored the FDA approved database of 7922 molecules and screened against the core polymerase along with cofactors.
Here we report a panel of FDA approved drugs that show substantial interactions with key amino acid residues of the active site.
Interestingly, some of the identified drugs (Ornipressin, Lypressin, Examorelin, Polymyxin B1) bind strongly within the binding pockets of both forms of RdRp.
Besides, we found strong candidates for the complex form as well which include Nacortocin, Cistinexine, Cisatracurium (among others).
These drugs have the potential to be considered while contriving therapeutic options.
COVID-19, an infectious disease caused by a novel strain of coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome).
It caught the attention of the world community after its outbreak in December 2019 from the city of Wuhan, China.
Since then it has spread globally, WHO declared COVID-19 as pandemic on 11 March 2020 [1, 2] .
The emergence of this new strain of coronavirus (SARS-CoV-2) which is highly contagious and caused the deaths of 444,813 (as of 17 th , June 2020, WHO) individuals worldwide, making it as one of the biggest challenge for the scientific community worldwide.
In this global health emergency, drug repurposing (or repositioning) is one of the fast track option that involves screening of existing FDA approved drugs for the identification of potential molecules that can disrupt the function of key proteins of the SARS-CoV-2 and can be used for treatment against COVID-19.
Coronaviruses are enveloped viruses belonging to family Coronoviridae, which is further subdivided into four genera i.e.
alpha, beta, gamma, and delta coronavirus [3] .
The members of the alpha and beta coronavirus causes infections in mammals however gamma and delta coronavirus causes disease in birds [4, 5] .
SARS-CoV-2 belongs to genus beta-coronavirus.
Other members of the same genus that are known to infect humans include HCoV-OC43, HCoV-HKU1, SARS-CoV, and MERS-CoV [4, 5] .
SARS-CoV-2 genome is a single strand positive-sense RNA.
The size of the genome is 29.8kb and has 14 Open Reading Frames (ORFs) which encode information for 27 structural and non-structural proteins [4] .
At the 5`-end of the genome, there are two ORFs (ORF-1a and ORF-1ab) encoding two long stretches of poly-proteins, pp-1a and pp-1ab.
These encode information for 15 non-structural proteins (nsp 1-10 and nsp12-16) [4] .
The vital nonstructural proteins are; nsp3 (multi-domain protein including PL-pro domain), nsp5 (3CL chymotrypsin-like), nsp9 (helicase, may participate in viral replication), nsp12 (RNA dependant RNA polymerase) and nsp13 (helicase).
The 3`-end of the genome encodes information for four structural and eight accessory proteins.
The structural proteins are; Spike surface glycoproteins (S), Envelope (E), Matrix (M), and Nucleocapsid (N) proteins.
The accessory proteins are; 3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14 [4] .
RNA dependent RNA polymerase (RdRp) is involved in the replication and transcription of the SARS-CoV-2 genome.
It is the cleavage product of the polyproteins 1a and 1ab from ORF1a and ORF1ab [6] .
There is a high degree of conservation among RNA dependent RNA polymerases of different RNA viruses.
The Core protein is a single chain of approximately 900 amino acids, showing minimal activity.
However, enhanced activity is attained with the attachment of other key subunits [6] [7] [8] .
The minimum complex for the proper activity of SARS-CoV-2 requires an attachment of nsp7/nsp8 and an additional nsp8 protein to the core protein [6] .
The attachment site for the second additional nsp8 is different.
Also, other nonstructural proteins (nsp (s)) are involved in this replication and transcription cascade [6] .
The core protein looks like a cupped right hand which is further sub-divided into subdomain including finger domain (amino acid residues 398-581, 628-687), palm domain (582-627, 688-815), and thumb domain (816-919) [9] (Figure 1 ).
Two additional Zn ions are also required for the structural stability of the RdRp.
One of the Zn ion is attached to four amino acids residue (His295, Cys301, Cys306, and Cys310) in the N-terminal domain while the second Zn ion is attached to four amino acid residues (Cys487, His642, Cys645, Cys646) located in finger domain [6] .
The presence of Zn in this location shows that it plays an essential role in stabilizing the overall three-dimensional structure of the protein.
It has no direct role in the activity of the polymerase as both are quite distal to the active site.
All the important sites including, template entry and binding, polymerase activity reaction site followed by the exit through the tunnel (thumb) remain highly conserved among coronaviruses including SARS-CoV-2 [6] (Figure 1 ).
To the extent charge potential is concerned, the palm (RNA template and NTP binding site) has positive electrostatic potential.
However, the other sites including attachment of nsp7/8 complex, nsp8 site, and the template exit site (thumb) is neutral [6] .
The key residues that are involved in the interaction include; Tyr618, Cys622, Asn691, Asn695, Met755, Ile756, Leu757, Leu758, Ser759, Asp760, Asp761, Ala762, Val763, Glu811, Phe812, Cys813 and Ser814 (numbering about the recently solved structure PDB ID 6M71) [10, 11] .
The active site key residues are adjacent aspartates i.e.
Asp761 and Asp762, which are involved in the actual reaction of the RdRp enzyme [10, 12] .
The panel of different antiviral molecules has already been identified by targeting key proteins involved in different stages of the SARS-CoV-2 life cycle.
Some of them are in a clinical trial stage.
Through repurposing study, the existing antiviral drugs (Ribavirin, Sofosbuvir, Remdesiver, Tenofovir) have already been screened [10, 12] .
Those in a clinical trial stage include; ASC09 and Ritonavir (both are HIV inhibitor), GS-5734 (a broadspectrum investigational antiviral), and Kaletra (inhibitor of 3-CL of SARS and MERS) [13] .
Similarly, other compounds in a clinical trial (Favipiravir, used against influenza) didn't show strong activity in clinical isolates [13] , while some others with positive results need further investigations [14, 15] .
Owing to its significance, the current study encompasses screening and identifying novel FDA approved drugs against the recently elucidated structure of SARS-CoV RNA Dependent RNA Polymerase.
The possible lead molecules can further be explored to combat this disease.
The crystal structure of RdRp was downloaded from PDB (ID 6M71) with a reported resolution of 2.90Å.
RNA Dependent RNA polymerase is a multimeric protein.
The minimum complex required for its proper functioning is completed by attachment of three additional protein peptides (nsp7-nsp8, and one additional nsp8) to the core polymerase which is chain A and contains 851 amino acids residues.
The current structure is unraveled in complex with all the required cofactors.
To investigate the binding affinity of the drugs, we prepared both the structures of RdRp i.e.
with and without cofactors.
Protein preparation wizard of Maestro was used for protein preparation.
Hydrogen atoms, missing residues, and loops were added.
The side chains were fixed during protein preparation of both forms of RdRp (with and without cofactors).
Besides, disulfide bonds were also created.
Protonation states of amino acids were generated at pH 7.4, by using PROPKA to simulate physiological conditions [16, 17] .
For the protein structures minimization, the OPLS-2005 force field was used [18] .
Grid box was generated by selecting the active site residues as mentioned [10] .
Active site residues Asp760 and 761 (DD) were treated as flexible during docking.
Grid box was generated as 30, 30, 30 centred (x, y, z) of (114.
52, 114.11, 122 .91) Å as reported [10] .
Receptor grid was generated using Maestro tools and the flexible rotation of certain amino acids residues (Tyr455, Tyr456, Thr462, Cys482, Tyr483, Ser549, Thr586, Thr591, Thr604, Tyr619, Cys622, Thr680, Ser681, Ser682, Thr686, Thr687, Tyr689, Ser692, Cys697, Thr701, Ser754, Ser759, Cys765, Ser778, Tyr788, Ser795, Cys799, Thr801, Cys813, Ser814, Thr817) were allowed.
FDA approved drug database (~7922) was downloaded from the NIH chemical genomics Centre (NCGC) pharmaceutical collection (NPC) database.
The database was prepared using the lig-prep module of Maestro [18] .
During protein preparation, different combinations of enantiomers and tautomers were generated, which ultimately enhanced the total number of drug molecules.
For the protonation states of the ligand at pH 7.4, Epik was used [19] .
To check the binding efficacy of the FDA approved drugs, accurate and fast Glide SP-protocol was used for the molecular docking simulations [20, 21] .
Glide Induced Fit Docking (IFD) was used to re-dock the top ten molecules in the binding pocket of RdRp [20] .
IFD was performed in various steps.
Initially, ligands were docked to rigid protein employing potential softened-potential glide docking with the van dar Waals radii scaling of 0.50/0.50, for the receptor and ligand respectively.
In the following stage receptor, sampling and refinement were carried out.
Residues with minimum one atom inside 5 Å of any of the 10 ligand poses were selected to conformational search and minimization whereas residues lying outside this range were fixed.
In this manner, the protein flexibility was considered in the process.
Additional redocking of the ligands were performed.
For all docking calculations, Glide-XP was analyzed.
3D structure of the protein model, 2D structures of molecules and interaction maps were generated using Maestro (Schrodinger).
For other illustration, Adobe Illustrator was used.
For the screening of novel potential inhibitors against RdRp, we selected FDA approved drugs database.
A similar type of repurposing study has already been performed, in which the homology model of RdRp was generated, validated and various existing antiviral drugs were investigated for their binding potential within the active site of RdRp without cofactors [10, 12] .
Furthermore, other proteins of the SARS-CoV-2 for which X-ray crystal structures were available have also been used as a potential drug target [22] .
The current study is the first of its type in which recently determined X-Ray crystal structure of RNA Dependent RNA polymerase was used for the screening of potential inhibitors from the FDA approved drugs database.
In the homology model, two aspartates at position 255 and 256 were reported as active site residues [12] .
However, in the crystal structure, these similar active site aspartates are represented as Asp760 and Asp761.
We have used both forms of the polymerase, a single-core chain, and along with its cofactors (holoenzyme, replication/transcription complex).
Initially, a single chain of the RdRp polymerase was selected for the screening, followed by further screening for the holoenzyme.
Based on docking scores (Glide SP, Glide IFD) and interactions with crucial residues and ligand efficiencies, the top-ranked molecules were selected and analyzed.
Table 1 explains the review of the top ten screened molecules and the existing usage of these drugs.
Antibiotic used against multidrugresistant gramnegative bacterial infection [39, 40] Based on the maximum occupancy of compounds bound to the reported binding tunnel of RdRp, top-ranked conformations were selected considering the hydrogen bonds and other non-covalent interactions.
Ornipressin binds into the RdRp binding tunnel with a docking score of -10.37kcal/mol and the ligand efficiency of -0.14.
Ligand interaction analysis of the Ornipressin/RdRp complex shows that ligand mostly made H-bonds and salt bridges.
Asp865 is involved in H-bond and salt bridge while Asp760, Thr591, Gln815, Ser814, Cys813, Glu811, Tyr619 formed H-bonds.
Ornipressin also depicted some interactions with the hydrophobic and polar residues (Figure 2 ).
Figure S2) .
The binding potential of Argiprespocin with RdRp was mainly governed by H-bonds.
Argiprespocin made six H-bonds with Lys798, Glu811, Leu758, and Ser814.
While two H-bonds and one salt bridge were observed for Asp761, one π-cation and H-bond was observed for Trp617 with a docking score of -9.87kcal/mol and ligand efficiency of -0.137 (Supplementary Figure S2) .
Whereas, Demoxytocin showed ten H-bonds with both active site Asp760 and Asp761 and other key residues e.g.
Trp617, Tyr619, Lys621, Ser682, Glu811, Lys621, Tyr619, Trp617, Ser682 and Glu811 with dock score -9.68kcal/mol and ligand efficiency of -0.142 (Supplementary Figure S3) .
Carbetocin, a synthetic analogue of oxytocin, also has the potential to inhibit RdRp (single chain) and showed stable interaction with a docking score of -9.479 kcal/mol and ligand efficiency of -0.137.
Eight H-bonds were observed with Lys551, Arg553, Thr556, Trp617, Tyr619, Asp623, and Asp760 (Supplementary Figure S3) .
Another screened drug Lypressin showed a docking score of -9.413 kcal/mol and ligand efficiency of -0.129.
Ligand interactions showed salt bridge and H-bonding with Asp761 and only H-bonding with other residues including Arg553, Arg555, Thr556, Trp617, Tyr619, Arg624, Asp760, Asp761, Ala762 and Glu811 (Supplementary Figure S4) .
Examorelin, a screened inhibitor of this study showed most of H-bonds with Asp760, Cys813, Ser814, Gln815, and Glu811.
Salt bridge was also observed with Glu811.
Arg555 showed π-cation interaction with a docking score of -9.30kcal/mol and ligand efficiency of -0.143 (Supplementary Figure S4) .
Colistin (polymyxin E, polypeptide antibiotics) showed most of the H-bonding with Lys551, Trp617, Tyr619, Asp618, Ser682, Asp684, Asn691, and both catalytic residues i.e.
Asp760, Asp761, with a docking score of -9.24kcal/mol and ligand efficiency of -0.113 (Supplementary Figure S5) .
Polymyxin B1 also bound tightly with RdRp core protein through salt bridges and H-bonding.
Catalytic Asp760 and Asp761 made four salt bridges and three H-bonds with RdRp which shows its strong binding affinity.
Moreover, H-bonds were formed with Arg-555, Trp-617, Asp681, Asn691, Cys813, and Ser814 with a docking score of -9.22kcal/mol and ligand efficiency of -0.109 (Supplementary Figure S5) .
To investigate the difference in binding energies, molecules were re-docked in the binding pocket of RdRp by using the Glide-Induced Fit Docking protocol.
No significant changes were observed for drugs (except for Colistin which didn't show any pose in IFD) in both the protocols.
A similar type of interaction was observed in Glide-IFD as found in Glide-SP (Table 1) .
Glide-IFD docking poses of selected top 10 molecules are given in Supplementary figures (Figure S6 and S7) .
All complexes showed promising binding and maximum occupancy of crucial binding pocket residues.
It has been observed that the top ten screened drugs binding affinity was stronger with RdRp core protein.
As previously reported, Sofosbuvir, Ribavirin, Tenofovir, Setrobuvir, Guanosine derivative (IDX-184) were shown with a docking score of -7.5, -7.8, -6.9, -9.3, -9.0 kcal/mol respectively against RdRp single-chain [12] .
In current study, we have also docked Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, Tenofovir, Guanosine derivative (IDX-184) and Setrobuvir with a docking score of -5.94, -5.57, -4.94, -7.20, -4.52, -8.57, -4.45 kcal/mol respectively (Supplementry Figure S8) .The difference in the docking score may be due to the use of different docking algorithms.
These antivirals were analysed on the basis of interactions with crucial residues and compared the interactions with screened molecules.
Our identified molecules showed more stable H-bonds, salt bridges, and ionic interactions as compared to reported antivirals.
For a potent drug, it is essential to interact with key amino acid residues in the active site pocket of the polymerase.
Once the drug binds to the active site of RdRP, the access of positive sense RNA of the virus which serves as a template for anti-genome is blocked.
No new viral genomes are synthesized and mature virus particles cannot be assembled and propagated further.
In this study, all the identified FDA approved drugs have shown stable interaction with key residues, along with lowest binding energies which indicates the potential for these drugs to inhibit the activity of RdRp, therefore contributing to stop viral proliferation.
The URL link of additional top 90 molecules including 2D structures, docking scores, ligand efficiencies and glide energies are given in the supplementary data.
The minimal complex of RdRp along with essential cofactors (nsp7-nsp8, nsp8) required for enhanced activity is used for this drug repurposing study.
We found a panel of molecules that showed significant binding with the RdRp complex.
Molecules were analyzed based on docking energies, ligand efficiencies, and other protein-ligand interactions.
Some of the screened molecules remained similar as found for single chain of RdRP, they therefore can be taken as common inhibitors for both single chain and in complex form of RdRp.
The results of top 14 selected molecules from Glide/SP along with their 2D structures, ligand efficiency, interacting residues and existing usage of the drugs are shown in table 2.
Top 14 molecules including common molecules among both structures were redocked through Glide IFD.
Docking score of Gide/IFD are shown in table 2.
For a complex, increased docking score was observed in Glide/SP.
This phenomenon might be due to the allosteric behaviour of the polymerase, as it is a common observation that upon binding of the molecules on an allosteric site, it brings conformational changes in the active site.
This then facilitates the efficient binding of the substrate within the active site.
The attachment of cofactors (nsp8/nsp7, nsp8) with a core protein (nsp12) might have similar effect on the activity of RdRp.
Additionally, the binding of some of the molecules in both the forms depend on the chemical structures of the screened molecules, changed interacting residues, steric hindrance, and stereo chemistry of the active site.
Ventilatorassociated pneumonia [37] , nosocomial pneumonia [38] , antibiotic against Nacartocin is one of the lead molecules with a docking score of -13.943 kcal/mol and ligand efficiency of -0.202.
Nacartocin showed H-bonds and non-covalent interactions with key residues and surface accessible residues (Figure 3 ).
π -cation interaction with Arg-553 and non-covalent interactions with crucial residues were observed for Cistinexine, with a docking score of -13.687 kcal/mol and a ligand efficiency of -0.201.
Whereas a neurotransmitter blocking agent Cisatracurium showed two H-bonds with Arg553 and one salt bridge with Glu811 with a docking score of -13.064, and a ligand efficiency of -0.195.
Other non-covalent interactions were also observed with polar, nonpolar, and hydrophobic residues (Supplementary Figure S9) .
Pegamotecan is another lead molecule that showed a strong binding affinity towards RdRp with their essential cofactors.
H-bond with Lys621 and other non-covalent interactions with key residues were observed with a docking score of -12.782 kcal/mol and ligand efficiency of -0.178 respectively.
Among the top 14 screened drugs Polymyxin B1 is one of the common drugs, identified in both forms of RdRp.
Almost similar types of interactions were observed for Polymyxin B1 in the catalytic site of the RdRP complex as found during core protein interactions.
Polymyxin B1 showed two salt bridges and one H-bond with Asp760 and Asp761 and two H-bonds with Arg-555 with a docking score of -12.646 kcal/mol and ligand efficiency of -0.149 (Supplementary Figure S9) .
Non-covalent interactions and one H-bond with Trp800 were observed for Ediratide with a docking score of -12.682 kcal/mol and with ligand efficiency of -0.18.
Asp760, Ser759, and Glu811 made H-bonds with Sulfomyxin through docking score of -12.468 kcal/mol and ligand efficiency of -0.152 (Supplementary Figure S10) .
π-cation with Arg553 and H-bonds with Tyr619, Arg86, and Asp833 were observed for Diagastrin, which is a peptide analogue of gastrin showed a docking score of -12.313 kcal/mol and ligand efficiency of -0.173.
Two salt bridges with Asp760 and Asp623 were observed for Ditercalinium chloride with a docking score of -12.308 kcal/mol and ligand efficiency of -0.228 (Supplementary Figure S10 ).
Flavonoid drug, Benzquercin showed two π-cation interactions with Arg553 and Lys798 and other non-covalent interactions with polar, non-polar and hydrophobic residues with docking score of -12.174 kcal/mol and ligand efficiency of -0.214 (Supplementary Figure S11) .
Examorelin, Lypressin, Ornipressin, and Colistin are also common drugs in both form of RdRp.
Only one H-bond with His810 and other non-covalent interactions were observed for Examorelin showed a docking score of -12.139 kcal/mol and ligand efficiency of -0.187.
Lypressin made three H-bonds with Arg555, Lys621, and Cys622 with a docking score of -11.923 kcal/mol and ligand efficiency of -0.163 followed by Ornipressin, showed a docking score of -11.717 kcal/mol (ligand efficiency of -0.163).
Salt bridge with Asp623 and H-bond with Lys621 is observed during interactions however, Colistin showed two H-bonds with Ile548 and Ser814 with a docking score of -11.077 kcal/mol (-0.135).
Colistin is also known as Polymyxin E (Supplementary Figure S11) .
IFD poses of top 14 molecules are given in supplementary figures S12A and S12B.
Pegamotecan, Cisatracurium, and colistin didn't show any pose during IFD docking simulation.
Colistin showed similar results in both structures with and without co-factors.
Based on docking analysis and its reconfirmation, we didn't find any significant difference in the docking energies, therefore our identified molecules have the potential to strongly bind in the binding tunnel of both single chain and complex from of the RdRp.
The URL link of 3D structures, docking scores, ligand efficiencies, and Glide energies of additional top 100 molecules against RdRp complex are given in supplementary data.
In a drug repurposing study, the aim is to screen existing approved drugs and suggest their reuse for new medical complications, being the most feasible approach as compared to the de-novo drug designing and development.
The benefits associated with repurposed drugs include; safety for human use, no escalating cost, and reduced timeline for its development.
For instance, Zidovudine, which was originally used for the treatment in cancer, was repurposed as the first anti-HIV drug, approved by the FDA in 1987 [50, 51] .
Similarly, Rituximab, originally used in the treatment of various cancers was approved by the FDA in 2006 as a repurposed drug against rheumatoid arthritis [52] .
Raloxifene, another drug used for osteoporosis, has also been approved for the treatment of breast cancer [52] .
Aspirin, which was primarily used as an analgesic is now recommended and approved by the FDA in 2015 against colorectal cancer and in cardiovascular diseases [52] .
In the last few years, about one-third of the FDA approved drugs relate to repurposed drugs [52, 53] .
All of the screened molecules identified in this study are FDA approved drugs.
We used fast and accurate docking algorithms Glide-SP to screen potential hits against RdRp (core and complex).
Glide-IFD was used to compare and confirm the difference between the binding energies of top screened molecules.
We have identified molecules for two forms of the RdRp individually.
Some of the molecules have shown promising interactions with both forms (core and holoenzyme) of the RdRp, e.g.
Polymyxin B1, Ornipressin, Lypressin, and Exemorelin.
Although all the top ten screened molecules for both the forms showed strong binding.
Polymyxin B1 shows strong binding with key amino acid residues in core as well as in the holoenzyme form.
Polymyxin B1 is currently used as an antibiotic for the treatment of respiratory tract infections i.e.
pneumonia (nosocomial, healthcare, or ventilator-associated), due to the multidrug-resistant gram-negative bacteria.
For the treatment of respiratory tract infection, it is generally used as oral inhalation via nebulization.
The general mechanism of action of Polymyxin involves the destabilization of the integrity of the bacterial membrane, thereby killing gram-negative bacterial cells through membrane lysis.
It interacts with the lipopolysaccharides of the Gram-negative bacteria and disrupts the outer membrane.
The other known mechanism is by vesicle-vesicle contact pathway, in which Polymyxin induces an osmotic imbalance, that ultimately leads to membrane lysis.
Based on the findings of the current study, Polymyxin B1 can be used for the treatment of COVID-19 by blocking the active site of RdRp.
Due to the current COVID-19 pandemic, to date, 444,813 people have lost their lives.
SARS-CoV-2A is spreading worldwide at an alarming rate.
To overcome this infection, the current study was designed to screen all those drugs which are available in the market or currently in a clinical trial.
For a potent drug, it is essential to interact with key amino acid residues in the active site pocket of the polymerase.
Once the drug binds to the active sites of RdRp, the access of positive sense RNA of the virus which serves as a template for anti-genome is blocked.
No new viral genomes are synthesized and mature virus particles cannot be assembled and propagated further.
In this study, all the identified FDA approved drugs have shown stable interaction with key residues, along with lowest binding energies which indicates the potential for these drugs to inhibit the activity of RdRp, therefore contributing to stop viral proliferation.
The top candidate drugs we found that interact with a single chain core RdRp include; Ornipressin, Otosiban, Lanreotide, Argiprestocin, Demoxytocin, Carbetocin, Lypressin, Examorelin, Colistin, and Polymyxin B1.
Additionally, we also identified compounds that interact with the complex form of RdRp (holoenzyme).
It includes; Nacartocin, Cistinexine, Cisatracurium, Pegamopecan, Ebiratide, Sulfomyxine, Diagastrin, Ditercalinium chloride, and Benzquercin.
Among these lead molecules, Ornipressin, Lypressin, Examorelin, Polymyxin B1 showed strong binding efficacy with both core and holoenzyme.
Although all of the screened molecules showed strong binding affinity towards RdRp (core and holoenzyme), however Polymyxin B1 and Colistin are already in use against respiratory tract infections.
Based on our findings, we, therefore, suggest that these screened drugs can be clinically tested to handle this intricate infection.
The SARS-CoV-2 outbreak: what we know Clinical Features of 69 Cases with Coronavirus Disease Structure, Function, and Evolution of Coronavirus Spike Proteins Genome Composition and Divergence of the Novel Coronavirus (2019-nCoV) Originating in China Inferring the hosts of coronavirus using dual statistical models based on nucleotide composition Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities RNA synthetic mechanisms employed by diverse families of RNA viruses Anti-HCV, nucleotide inhibitors, repurposing against COVID-19 The mechanism of action of T7 DNA polymerase Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study Coronavirus puts drug repurposing on the fast track Of chloroquine and COVID-19 Use of Hydroxychloroquine and Chloroquine During the COVID-19 Pandemic: What Every Clinician Should Know Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments Very fast prediction and rationalization of pKa values for protein-ligand complexes The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin Epik: a software program for pK( a ) prediction and protonation state generation for drug-like molecules Glide: a new approach for rapid, accurate docking and scoring.
1.
Method and assessment of docking accuracy Glide: a new approach for rapid, accurate docking and scoring.
2.
Enrichment factors in database screening Identification of Chymotrypsin-like Protease Inhibitors of SARS-CoV-2 Via Integrated Computational Approach The pharmacology of ornipressin (POR-8): a local vasoconstrictor used in surgery Ornipressin in the treatment of functional renal failure in decompensated liver cirrhosis: effects on renal hemodynamics and atrial natriuretic factor Management of preterm labor: atosiban or nifedipine?
International journal of women's health The safety of lanreotide for neuroendocrine tumor Vasopressin and its analogues in shock states: a review Use of oral oxytocics for stimulation of labor in cases of premature rupture of the membranes at term: a randomized comparative study of prostaglandin E2 tablets and demoxytocin resoriblets Carbetocin versus oxytocin in caesarean section with high risk of post-partum haemorrhage Usefulness in patients who manifest allergies to other antidiuretic hormone preparations Growth hormone-releasing activity of hexarelin in humans.
A dose-response study Effects of GHRP-2 and hexarelin, two synthetic GH-releasing peptides, on GH, prolactin, ACTH and cortisol levels in man.
Comparison with the effects of GHRH, TRH and hCRH The clinical use of colistin in patients with cystic fibrosis Effect of aerosolized colistin on multidrug-resistant Pseudomonas aeruginosa in bronchial secretions of patients without cystic fibrosis Combination therapy with intravenous colistin for management of infections due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis Treatment of multidrug-resistant Acinetobacter baumannii ventilator-associated pneumonia (VAP) with intravenous colistin: a comparison with imipenem-susceptible VAP Aerosolized colistin for the treatment of nosocomial pneumonia due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review Nacartocin--analogue of oxytocin with enhanced natriuretic properties: natriuretic and hemodynamic characteristics The effect of a new expectorant drug on mucus transport in chronic bronchitis Use of cisatracurium in critical care: a review of the literature A phase II study of pegylated-camptothecin (pegamotecan) in the treatment of locally advanced and metastatic gastric and gastro-oesophageal junction adenocarcinoma The neurotrophic effects of ebiratide, an analog of ACTH4-9, on cultured septal cells and aged rats Sulfomycins, a series of new sulfur-containing antibiotics.
I.
Isolation, purification and properties Effect of somatostatin analogs on gastric acid secretion in dogs and rats Ditercalinium chloride, a pro-anticancer drug, intimately associates with mammalian mitochondrial DNA and inhibits its replication In-silico identification and evaluation of plant flavonoids as dengue NS2B/NS3 protease inhibitors using molecular docking and simulation approach Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-associated virus in vitro Administration of 3'-azido-3'-deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS or AIDS-related complex Drug repurposing: progress, challenges and recommendations Challenges and opportunities with drug repurposing: finding strategies to find alternative uses of therapeutics We are thankful to Shelvia Malik and other Schrodinger team members for facilitating us with the licence to use Schrodinger Suite which enabled us to successfully complete this study.
key: cord-269194-b1wlr3t7 authors: Engstrom-Melnyk, Julia; Rodriguez, Pedro L.; Peraud, Olivier; Hein, Raymond C. title: Chapter 5 Clinical Applications of Quantitative Real-Time PCR in Virology date: 2015-12-31 journal: Methods in Microbiology DOI: 10.1016/bs.mim.2015.04.005 sha: doc_id: 269194 cord_uid: b1wlr3t7 Abstract Since the invention of the polymerase chain reaction (PCR) and discovery of Taq polymerase, PCR has become a staple in both research and clinical molecular laboratories.
As clinical and diagnostic needs have evolved over the last few decades, demanding greater levels of sensitivity and accuracy, so too has PCR performance.
Through optimisation, the present-day uses of real-time PCR and quantitative real-time PCR are enumerable.
The technique, combined with adoption of automated processes and reduced sample volume requirements, makes it an ideal method in a broad range of clinical applications, especially in virology.
Complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time PCR provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies.
All of these serve vital roles in the continuum of care to enhance patient management.
Beyond this, quantitative real-time PCR facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats.
processes, small sample volumes and can be utilised in a wide variety of applications, making it the method of choice in today's molecular laboratories.
Through the aid of fluorescent signalling probes to measure amplification of DNA at each PCR cycle, at the point of exponential DNA accumulation, real-time PCR is able to provide broader linear dynamic ranges and increased assay performance as determined by sensitivity, specificity, precision, and reproducibility.
Due to the consistency in signal intensity changes during the exponential growth phase of PCR, it is also easily adaptable for quantitative reporting.
However, there are three properties that are uniquely associated with quantitative real-time PCR: quantification, standardisation, and lower limit resulting.
The accumulation of fluorescence signal is measured at each PCR cycle of the reaction and the cycle at which this signal exceeds a predetermined background fluorescence threshold during the logarithmic phase of amplification is referred to as the cycle threshold (C T ).
The C T value is inversely proportional to the viral copy number in the specimen, and through comparisons of this value to an external calibration curve or an internal quantitation standard, the initial nucleic acid target concentration can be calculated (Heid, Stevens, Livak, & Williams, 1996; Livak & Schmittgen, 2001) .
However, accurate quantitation within each sample is hindered when relying solely on an external standard as amplification efficiencies for each individual sample may be variable and inconsistent.
By utilising a standard internal reference template, with the rationale that any variable influencing amplification efficiency should Example of amplification and detection of target nucleic acid by real-time PCR.
affect both template and target similarly, inhibition and amplification effects are compensated for which allows for more accurate quantitation ( Figure 2 ).
This control can be further enhanced when incorporating an internal reference that utilises the same primer sequence as the target since any potential additional effects on PCR efficiency for each of the two targets is eliminated.
Thus, the competitive real-time PCR strategy is the most reliable approach for nucleic acid quantitation (Diviacco et al., 1992; Gilliland, Perrin, & Bunn, 1990; Stieger, Demolliere, Ahlborn-Laake, & Mous, 1991; Wang, Doyle, & Mark, 1989; Zentilin & Giacca, 2007) and is the basis for the majority of present-day virology assays.
It is equally important to utilise appropriate quantitation standards, when available, to ensure accurate quantitative results, inter-laboratory correlation, and overall standardisation.
Standardisation of reported viral loads ensures not only interlaboratory consistency but also high clinical utility of viral load monitoring, sets the foundation for establishing clinical correlations and critical thresholds leading to better management of infections and treatments, and are critical for the development of clinical guidelines (Miller et al., 2011) .
With the wide availability of assay methods, viral targets, specimen type, and lack of standard reference material (Hayden et al., 2012) , viral load variability across laboratories can range significantly, as high as 4.3 log copies/mL .
Specifically, results from proficiency testing/external quality assessment programmes as well as interlaboratory specimen exchange studies have demonstrated that there is significant variability in quantitative results for assays that lack appropriate standards Quantitation of viral target using competitive quantitation standard (QS).
The QS compensates for effects of inhibition and controls the preparation and amplification processes, allowing a more accurate quantitation viral target in each specimen.
The competitive QS contains sequences with identical primer binding sites as the viral target to ensure equivalent amplification efficiency and a unique probe binding region that distinguishes the two amplicons.
The competitive QS is added to each specimen at a known copy number and is carried through the subsequent steps of specimen preparation, reverse transcription (when applicable), simultaneous PCR amplification, and detection.
Viral target concentration in the test specimens is calculated by comparing the viral target signal (solid line) to the QS signal (dashed line) for each specimen and control (A, B) .
In the presence of inhibitors, both QS and viral target are equally suppressed and yield accurate viral load calculations (C).
1 Introduction (Hayden et al., 2008; Pang et al., 2009; Preiksaitis et al., 2009; Wolff, Heaney, Neuwald, Stelrecht, & Press, 2009 ).
Findings such as these reinforce the fact that with this high degree of variability and discrepancy, clinicians are unable to compare test results between two different laboratories and, further, clinically relevant cut-offs set by one test would not apply to results of another .
Without standardisation, the quality of patient care is dramatically impacted, preventing meaningful inter-laboratory comparison of patient results and influencing disease prevention and management programmes (Kraft, Armstrong, & Caliendo, 2012) .
This is especially critical for transplant patients, who may be initially monitored at one institution and then transferred to another for longer-term follow-up receiving results that no longer correlate.
Therefore, whenever possible, viral load monitoring tests must report results in IU/mL and be fully traceable to the higherorder first WHO International standard.
They must generate highly accurate and reliable results based on a robust calibration methodology and have excellent reproducibility across the dynamic range of the test with demonstrated co-linearity to the WHO standard.
Lastly, there exist two distinct end-points with quantitative real-time PCR, which should be of consideration for result interpretation and reporting: the lower limit of detection (LLOD/LOD) and the lower limit of quantitation (LLOQ/LOQ).
These two limits are assessed differently and are not equivalent in either definition or, in some cases, their assigned values.
The LOD (also referred to as analytical sensitivity) represents the lowest viral load level at which !95% of tested samples are detected (CLSI EP17-A, 2004); theoretically, viral levels at or below the LOD are not detected !5% of the time.
It differentiates between 'detectable' and 'undetectable' results.
The LLOQ, on the other hand, is the lowest viral level that is within the linear and analytically acceptable range of the assay (CLSI EP17-A, 2004) .
In other words, the LLOQ is the lowest point at which an accurate viral load can be assigned and determines which 'detectable' sample will have a reported viral load.
A common misconception is that the LOD of the assay is the minimum viral level for a 'detected' result but 'undetectable' and 'detectable' viral levels are never differentiated by a single theoretical viral threshold as viral levels less than the LOD may still have a high probability of being detected.
This probability spans a broad range in which the lower the viral titre, the more likely the 'undetectable' result.
Ultimately, the statistical probability will favour the 'undetectable' result ( Figure 3 ).
And because the LLOQ can be equal or greater than the LOD on some viral load assays, it is not unusual for 'detectable but below the LOQ' (detectable/BLOQ) result reporting (Cobb et al., 2011) .
Further, the 'detectable/BLOQ' results should not be inferred that the actual viral concentration of the sample is between the LOD and LOQ.
The clinical demand has driven and shaped the evolution of PCR and continues to do so as we gain a greater understanding of the infections we monitor and treat.
Through the study of the natural history and disease progression attributed to specific viral infections, the need for sensitive, accurate, precise, reproducible, and reliable quantitative measurements of viral levels has become a necessity.
With the deeper understanding of the natural history of human immunodeficiency virus (HIV) infections, it is now well understood that progressive immunosuppression and the onset and development of clinical disease are strictly associated with increasing viral burden (Furtado, Kingsley, & Wolinsky, 1995; Ho, Moudgil, & Alam, 1989; Mathez et al., 1990; Nicholson et al., 1989; Schnittman et al., 1990) .
Thus, quantitative real-time PCR is critical for monitoring patients infected with HIV (Hufert et al., 1991; Mellors et al., 1995) and those undergoing antiretroviral therapy (ART) to ensure viral replication is sufficiently and effectively suppressed and to monitor potential for viral resistance to the medication (DHHS HIV, 2014) .
This monitoring and maintained viral suppression is absolutely necessary not only to maintain progression-free survival of HIV-infected patients but also to reduce subsequent HIV transmission (Cohen et al., 2011; Diffenbach, 2012) .
Due to the significance of viral load monitoring and maintaining viral suppression, the demand for Likelihoods of different test results given different viral concentration.
When the viral concentration tends to 0, the proportion of 'Target not Detected' increases to 1 (dotted line), increasing the likelihood of 'Not Detected' results.
As the concentration tends to LLOQ (dashed line), the likelihood of 'Detected but <LLOQ' results peaks.
When the concentration tends to infinity, the proportion of quantitative results tends to 1 (solid line), resulting in a continual increase in the likelihood of 'Detected: Quantitative' results.
At any concentration, the sum of the three types of reported results is always 100% and throughout the concentration continuum, variations in result reporting exist.
As the concentration of viral levels approaches the LLOQ, near equal likelihood of 'Detected: Quantitative' and 'Detected but <LLOQ' results are possible, as are 'Not detected' results.
Decreasing concentrations will further shift the likelihoods and increase the chance of 'Detected but <LLOQ' or 'Not Detected' results.
Diagram assumes LLOQ ¼ LLOD.
increasingly more sensitive assays for HIV has driven the innovation of diagnostic tests and continues to push the limits of the LOD and LOQ ever lower (Glaubitz et al., 2011; Sizmann et al., 2010) .
Additionally, the amount of viral DNA in samples from either hepatitis B virus (HBV) or cytomegalovirus (CMV) chronic carriers is indicative of active viral replication in liver cells and is correlated with liver disease progression (Chen, Lin, et al., 2006; Chen, Yang, et al., 2006; Emery et al., 2000; Humar et al., 1999; Humar, Kumar, Boivin, & Caliendo, 2002; Iloeje et al., 2006; Wursthorn, Manns, & Wedemeyer, 2008) , highlighting the need for quantitative viral levels in chronic disease monitoring.
Similarly, hepatitis C virus (HCV) levels have been linked to prognosis and treatment outcomes (Trepo, 2000) , treatment response (Pearlman & Ehleben, 2014; Zeuzem et al., 2009) , as well as assessing sustained virologic response (SVR), or 'cure', following treatment (Pearlman & Traub, 2011) .
More recently, as new antiviral therapies for HCV treatment rely on targeting specific biological steps of the viral replication cycle, reliable quantitative monitoring of the viral burden at specific time-points to ensure treatment compliance and resistance emergence during treatment is recommended (Au & Pockros, 2014 ; AASLD/IDSA/IAS-USA, 2014).
Alongside the changing clinical needs, several instrument and manufacturing innovations have been introduced to meet these newer requirements.
The first PCRbased diagnostic test and the first automated system were both introduced in the early 1990s, allowing for improved standardised technique, increased efficiency, and reduction in error and contamination.
In 1996, to meet the clinical needs of monitoring patients infected with HIV, the first 'personalised healthcare' diagnostic test was FDA approved, that monitored whether ART was working and whether a patient was on optimal treatment.
Entire systems were introduced in the early 2000s that automated the up-front sample preparation step, leading to further reduction of hands-on time, increased reliability and reproducibility.
With the introduction of real-time PCR techniques to further enhance assay performance, the first PCR tests that allowed for simultaneous amplification and detection were introduced in 2003.
Throughout the decade, improvements in assays and instrumentation began pushing the boundary for HIV-1 detection, achieving greater and greater sensitivity, a feat that has proven to be incredibly relevant in understanding risks of viral load rebound and virologic failure (Doyle et al., 2012; Estevez et al., 2013; Pascual-Pareja et al., 2010) .
More recently, shortly after the release of two new higher-order standards for CMV (first WHO International Standard and NIST CMV Standard), fully automated real-time quantitative PCR assays were FDA approved to meet laboratory and clinical demands for standardisation.
Not only does the instrumentation reduce assay design variability and inconsistency across laboratories, but also the assays are standardised to the WHO and report in international units, providing accuracy across the entire dynamic range that is only achieved by calibration and traceability to these higher-order standards.
But it is not until these FDA-approved tests gain widespread use will inter-laboratory agreement for CMV viral load results truly improve (Kraft et al., 2012) .
The objective of test innovation is to evolve and adapt to clinical and laboratory needs.
As patient outcome gaps are identified, and as we gain greater understanding and insight into the clinical progression of disease and disease management, technological achievements facilitate advancements in quality of diagnostics and patient outcomes.
Life-threatening illnesses convert to chronic/manageable disease with the aid of viral load monitoring.
And pressure exists to develop more precise, accurate, sensitive assays, which, in turn, drives the development of more efficacious drugs.
This synergy demonstrates the value of diagnostics: it is an integral part of the patient care continuum.
Applications of quantitative real-time PCR for virology are extensive.
It is especially necessary when antibody seroconversion is delayed after an acute infection and early diagnosis are essential (DiBiasi & Tyler, 2004; Thomson et al., 2009) , in immunocompromised patients that may not have an optimal antibody response (Kadmon et al., 2013) , and for the diagnosis of congenital or perinatally acquired viral infections (Park, Streicher, & Rothberg, 1987; Young, Nelson, & Good, 1990) .
Additionally, it is critical in maintaining a high level of patient care at each stage of the disease and infection (Figure 4 ).
Screening tests are designed with one key parameter in mind: exceptional sensitivity (Herman, Gill, Eng, & Fajardo, 2002) .
They must ensure that disease is not missed in a population that is generally free from risk to allow for appropriate early intervention, thereby effectively reducing mortality and morbidity.
Although traditional Applications of real-time PCR in the continuum of care and patient management.
population-based screening programmes and recommendations do not often utilise nucleic acid testing (NAT) for virology targets, relying more on immunoassays and antigen testing, NATs and real-time PCR assays are still integral components.
Donor-eligibility determination ensures that a donor is eligible to donate cells or tissues to be used as human cells, tissues, and cellular and tissue-based products (HCT/Ps), which can include haematopoietic stem/progenitor cell, organ, semen, and other types of donations.
In part, living and cadaveric HCT/P donor eligibility is granted if screening shows that the donor is free from risk factors for, and clinical evidence of, infection due to relevant communicable disease agents and diseases such as HIV type 1 and 2, HBV, HCV, and West Nile Virus (WNV) (FDA Testing, 2014) .
The FDA testing recommendations stipulate that HCT/Ps tests for HIV and HCV may include FDA-licensed NAT blood donor screening and that, specifically, an FDA-licensed NAT should be used to assess infection with WNV.
Despite the fact that these NAT tests provide qualitative as opposed to quantitative results, the molecular technology utilised is often real-time PCR due to its enhanced accurate and reliable resulting (FDA Assays, 2014) .
Additionally, pre-emptive virology screening post-transplant may reduce subsequent complications and provide a more cost-effective management strategy (Evers, 2013) .
Pre-emptive therapy utilises routine viral screening to initiate therapy at the first indications of viraemia, prior to clinical manifestation of disease.
This strategy reduces the overall morbidity and mortality post-transplant compared to strategies in which treatment is initiated at the onset of clinical disease (Sch€ onberger et al., 2010).
The pre-emptive treatment model utilising routine screening by quantitative realtime PCR of asymptomatic post-transplant patients has been shown to be especially effective for paediatric patients undergoing haematopoietic stem cell transplant, who are uniquely at a high risk of CMV and Epstein-Barr virus (EBV) infections (Evers, 2013) .
This strategy and prospective screening utilising a quantitative real-time PCR assay for BK polyoma virus (BKV) is recommended as part of routine posttransplant follow-up of all kidney transplants since early identification and management of BKV infection may prevent future incidence of polyoma virus-associated nephropathy (Hirsch et al., 2005; KDIGO, 2009) .
NATs and real-time PCR are also utilised as vital parts of certain screening algorithms.
The Center for Disease Control (CDC) currently recommends that HCV RNA testing be utilised for anyone who may have been exposed to HCV within the preceding 6 months.
In addition, NATs would identify active HCV infection among persons who have tested anti-HCV positive or those with an indeterminate antibody test indicating need for referral for further medical evaluation and care (CDC HCV, 2013).
Viral diagnostic tests are used to determine presence or absence of current or previous infection.
Because many viral infections present with similar symptoms, accurate diagnosis is critical as each requires unique and vastly different interventions and/or management strategies.
Historically, diagnosis of viral infections has relied on viral growth in cell culture, immunoassays, antigen assays (ELISA), haemagglutination testing, and electron microscopy (Krishna & Cunnion, 2012) .
The introduction of molecular methods including NATs and real-time PCR assays has vastly improved viral diagnosis with their superior sensitivity, specificity, and rapid result reporting (Emmadi et al., 2011) .
NAT-based infectious disease testing, providing rapid results, aids in outbreak detection (Ebola), genotype identification (HCV), and identification of possible drug resistance (HIV-1), which can lead to rapid clinical therapeutic decisions and early infection control to prevent spread of disease (Espy et al., 2006) .
Viral culture was traditionally considered to be the 'gold standard' for viral diagnosis because of increased sensitivity compared to rapid antigen-testing methods.
However, a significant limitation of viral culture was a long time to result, reaching 14 days for CMV (Gleaves, Smith, Shuster, & Pearson, 1985) .
Improvements in culture included the introduction of shell viral culture, which reduced the result turnaround-time (TAT) for CMV to 24 h. Although considered to be a vast improvement, certain viruses require immediate treatment intervention to prevent life-threatening infection and therefore, a 24-h TAT is simply much too long.
Further, reliance on viral culture for diagnostics testing introduces other pronounced drawbacks, the most noteworthy being that not all routine viruses grow in culture and that virus viability, and thus its culture ability, could be impacted by sample collection, transport conditions, or prior patient treatment.
With these limitations in mind, NAT testing has tremendous advantages and has replaced culture for most viruses due to its greatly reduced TAT (typically less than 8 h) while still retaining high sensitivity.
The applications of NAT testing, and more specifically, real-time quantitative PCR technology, in viral diagnostics and confirmatory testing continue to expand.
The CDC-updated recommendations for HIV testing state that specimens that are reactive on the initial antigen/antibody combination immunoassay and non-reactive or indeterminate on the HIV-1/HIV-2 antibody differentiation immunoassay should be tested with an FDA-approved HIV-1 NAT test (Branson, 2010; Branson et al., 2014) .
Additionally, NATs have demonstrated utility in high-risk populations, in which antibody testing alone might miss a considerable percentage of HIV infections that are otherwise detectable by NAT virologic tests (Priddy et al.
2007; Stekler et al., 2009) .
Particularly, immunoassays for HIV diagnosis are limited by the marked delay between infection and seroconversion, a time when HIV viral levels are at their peak ( Figure 5 ).
For this reason, NATs are the recommended method for diagnosis of HIV during the acute phase of infection (10-50 days post-infection).
HIV viral RNA is the first marker to manifest itself approximately 10 days post-infection at initiation of the acute phase of infection (Lindback et al., 2000) .
Not until 4-10 days after initial detection of HIV RNA do the HIVp24 antigen levels rise to detectable levels using fourth-generation immunoassays.
This marked delay and the need for rapid diagnosis by the identification of HIV RNA is especially critical when an early diagnosis is medically warranted.
During the acute phase of the HIV infection, patients typically present with flu-like symptoms, which can often lead to a misdiagnosis.
Misdiagnosing those at the highest risk of infection such as prison inmates, IV drug users, and men who have sex with men (MSM), also increases the risk of further disease spreading (Chu & Selwyn, 2010) .
Therefore, the use of a real-time PCR testing method that is able to identify the presence of the HIV virus at this initial stage is critical.
Additionally, NATs are critical for early diagnosis of congenital or perinatally acquired viral infections, especially infants born to HIV-infected mothers (Tang & Ou, 2012) .
The maternal antibodies against HIV can persist in exposed infants for up to 18 months of age, which, in turn, prevents the use of antibody-based assays for early diagnosis of infection.
There is a high level of morbidity and mortality during the first 2 years of life for infected infants; therefore, it is of high importance to determine infection status quickly in the exposed infant to implement the appropriate ART therapy early (van Rossum, Fraaij, & de Groot, 2002) .
In addition to HIV, additional viruses have been demonstrated to exhibit perinatal transmission including respiratory virus, CMV, herpes simplex virus (HSV), varicella zoster virus (VZV), HBV, enterovirus, rotavirus, and human papilloma virus (Prober et al., 1987) .
For all these, NAT testing may serve as a valuable tool for early intervention, especially in this critical neonate population that lacks fully developed immune systems.
In addition to their applications for HIV diagnosis, NAT testing is also utilised as a diagnostic for HCV.
Although diagnostic HCV RNA real-time PCR tests are predominately used as confirmation of a positive HCV antibody result (CDC HCV, 2012), NAT testing is recommended for the confirmation of a non-reactive HCV antibody test for patients suspected of having recent HCV exposure.
HCV RNA can be detected as early as 2 weeks post-exposure, whereas HCV antibodies are not detectable until 8-12 weeks post-infection (Ghany, Strader, Thomas, & Seeff, 2009 ).
Therefore, similar to strategies outlined for HIV, NAT testing is the most suitable for detection and diagnosis during the acute phase of HCV infection.
Further, immunocompromised patients, those with weakened immune systems can include those that are HIV positive, on immunosuppressive drugs, or undergoing chemotherapy, may lack the ability to generate an appropriate immune response required for an anti-HCV test.
In these special circumstances, the CDC recommends considering HCV RNA testing for diagnosing viral infections.
Rapid diagnostic testing is also very important for viruses with high potential to create an epidemic or outbreak.
Throughout history, influenza has caused many such outbreaks, the most notable being the 1918 Spanish flu thought to be responsible for an estimated 50 million deaths (Taubenberger & Morens, 2006) .
In response to the possibility of future outbreaks, the World Health Organization (WHO) has established that PCR-based influenza testing is now the first-choice diagnostic test for both humans and animals (WHO Influenza, 2011) .
The conversion to molecular tests was based on the fact that these assays are more sensitive and specific for detecting influenza viruses compared to other non-molecular methods.
NAT methods also have a lower likelihood of false positive or false negative results, and therefore, result interpretation is less impacted by community-based influenza prevalence (CDC Influenza, 2014) .
The introduction of rapid molecular testing, which can provide results in as little as 15 min, is extremely beneficial especially in hospitals, nursing homes, and chronic care facilities where early influenza identification can prevent outbreaks.
The number of molecular-based diagnostic tests has expanded even further with the introduction of tests for WNV, respiratory syncytial virus, HSV, rotavirus, and even more recently, Ebola.
As development and improvements continue to be made to real-time PCR technology that will reduce both cost and time to result even further, the number of NAT diagnostic tests will continue to increase and the applications for real-time PCR in diagnostics will also expand.
Once a patient is appropriately diagnosed and linked to care, partly through the aid of quantitative real-time PCR, clinicians will often consider several baseline factors, which collectively help to guide therapeutic decision.
These decisions are based on a series of questions including: Are treatment options available?
What treatment regimen should be prescribed?
For how long will the patient need to be on therapy?
Factors influencing these therapeutic decisions include disease complications, patient predisposition, prior treatment experience, viral genotype, viral resistance profile, and host genetic profile, among others.
And depending on the viral infection, a quantitative baseline viral load may also serve an important role in helping to guide treatment decision (Table 1) .
Over the past 25 years, pharmaceutical development and clinical trials investigating cutting-edge antiviral treatments have relied heavily on the data generated from PCR-based-and eventually quantitative real-time PCR-based-technology to determine safe and effective drug use (Cobb et al., 2011; MacKay, Arden, & Nitsche, 2002) .
Practice guidelines continue to reference registrational and nonregistrational studies utilising quantitative real-time PCR to guide both clinicians and laboratories in the proper implementation of treatment and testing in order to deliver the most effective personalised care to patients (AASLD/IDSA/IAS-USA, Kotton et al., 2013) .
Because of this extensive co-utilisation of real-time PCR, it is widely accepted as the gold-standard technology for measuring a patient's quantitative viral load before, during, and after the course of treatment.
Once the decision to initiate treatment for chronic HCV infection is made, several baseline factors are routinely considered (AASLD/IDSA/IAS-USA, 2014).
Among these are complications like liver fibrosis stage, co-infection with HIV, hepatocellular carcinoma, end-stage liver disease, genetic variations like HCV genotype, subtype and resistance markers, and prior treatment experience and outcome.
The association of baseline viral load, measured by quantitative real-time PCR, to chronic HCV treatment outcome has been well documented with earlier therapies (pegylated-interferon plus ribavirin) but, given the lack of alternative therapeutic options, has not been recommended as a therapeutic decision factor (Jensen et al., 2006; Pawlotsky, 2012) .
Historically, practice guidelines have recommended the measurement of baseline viral load to serve only as an initial time-point required for effective monitoring of treatment without any prognostic indication (Yee, Currie, Darling, & Wright, 2006) .
Currently, tremendous advancements in the treatment of chronic HCV infection that employ direct-acting antivirals (DAAs) reported cure rates (as determined by SVR) of >90% for even the once most difficult to treat HCV genotype-1 patients, the most predominant in the United States.
Because of this high potency of these drugs across patient populations and the greater importance of numerous other factors, including HCV genotype and prior treatment experience, in determining the appropriate course of treatment, the most recent AASLD/IDSA practice guidelines still do not recommend a baseline quantitative viral load as a therapeutic decision factor.
However, in the rapidly evolving field of HCV treatment, the recent FDA approval of a fixed-dose combination drug consisting of two DAAs (sofosbuvir and ledipasvir) for the treatment of HCV genotype-1, the manufacturer's drug label now includes a new indication for quantitative real-time PCR.
It is indicated that treatment naïve and non-cirrhotic patients with a specific baseline viral load are eligible for shortened therapy, an indication with tremendous implications.
According to the prescribing information, patients with a baseline viral load below 6 million IU/mL are eligible to have shorter therapy duration of 8 weeks, much shorter than the 12-or 24-week duration for other patient populations (HARVONI, 2014) .
This therapeutic decision practice is the first of its kind in treatment of chronic HCV infection and is likely to be a recurring theme as DAA manufacturers strive to develop high efficacy regimens requiring shorter treatment durations.
Additionally, shorter treatment durations are more favourable to patients and payers when considering the cost of achieving SVR with DAAs and may improve patient drug adherence and completion of therapy (Hep C Online, 2014) .
As much as quantitative real-time PCR helped to develop this claim for this particular regimen, this technology will also be employed by numerous laboratories to aid in this part of therapeutic decision.
In contrast to chronic infection, treatment of patients presenting in the acute phase of HCV infection, within the first 6 months after exposure, is not recommended by AASLD/IDSA for patients in whom HCV infection spontaneously clears (AASLD/ IDSA/IAS-USA, 2014).
Therefore, careful monitoring of HCV RNA by a sensitive nucleic acid test is required in order to confirm spontaneous clearance, defined as HCV RNA negative at two specific measurements.
Quantitative and qualitative real-time PCR assays are both widely used for this purpose, given their comparable sensitivity.
Factors influencing ART decision for HIV-infected patients include determination of pregnancy, AIDS-defining conditions, acute opportunistic infections, low CD4 counts, HIV-associated nephropathy, potential drug interactions, co-infection with HCV or HBV, HIV resistance testing, and prior treatment experience (DHHS HIV, 2014).
Plasma HIV RNA viral load, performed widely by quantitative real-time PCR, is also recommended as a pre-ART decision factor specifically for treatment naïve patients.
The Department of Health and Human Services (DHHS HIV) recommends that only ART-naïve patients with a plasma HIV viral load below 100,000 cp/mL can be prescribed various regimen options, which they otherwise should be restricted from taking with higher viral load.
This is primarily due to inferior virologic responses in patients with higher viral loads observed in clinical studies (Sax et al., 2009) .
These clinical trial studies employed quantitative real-time PCR in order to help determine this cut-off and many labs have utilised the same technology to help guide HIV-treating clinicians in this decision.
In the case of chronic HBV infection, several studies have shown that Hepatitis B 'e' antigen (HBeAg) and high levels of HBV DNA are independent risk factors for the subsequent development of cirrhosis and hepatocellular carcinoma (Chen, Lin, et al., 2006; Chen, Yang, et al., 2006; Iloeje et al., 2006) .
However, due to the fluctuating nature of chronic HBV infection, the prognostic utility of one high HBV DNA level at a single time-point is limited.
Thus, HBV baseline DNA viral load, along with HBeAg, alanine aminotransferase (ALT) levels, and fibrosis, collectively aids in the decision to treat with antiviral agents as well as which HBV antiviral regimen to choose and duration of treatment (Lok & McMahon, 2009 ).
Typically, patients with an HBV DNA viral load >20,000 IU/mL, signs of liver disease (i.e.
high ALT levels and/or significant fibrosis), and loss of HBeAg are considered for immediate treatment with antivirals, whereas patients <2000 IU/mL are closely monitored for viral load changes prior to treatment.
Patients who fall in between this range are monitored for persistent viraemia and signs of liver disease before deciding to treat.
Quantitative real-time PCR, therefore, plays a crucial role in the care of chronic HBV patients who, if not treated at the appropriate time with the appropriate regimen and duration, are at greater risk of liver complications.
Unlike treatment guidelines for HCV, HIV, and HBV, management of CMV after solid organ transplant is not associated with specific quantitative CMV viral load cutoffs in order to make therapeutic decisions (Kotton et al., 2013) .
This is partly due to the historical lack of an international standard and varying assay designs, which has led to poor inter-institutional correlation of quantitative NATs.
In addition, the widespread practice of universal prophylaxis, where CMV antiviral medication is administered to patients early in the post-transplant period and continued for a finite period of time, has diminished the clinical utility of baseline viral loads for making therapeutic decisions.
However, with the recent availability of the WHO CMV International Reference Standard, the establishment of viral load cut-offs that can be applied to pre-emptive monitoring of patients prior to treatment initiation may soon become more widely accepted .
Until then, institutions are required to determine their own test performance characteristics and clinical cut-offs.
Several studies have shown that a low CMV virologic threshold (e.g.
detectable viraemia) using quantitative real-time PCR should be used for starting pre-emptive therapy especially in high-risk cases where the organ donor screens positive and the receptor screens negative for CMV serology (Atabani et al., 2012; Couzi et al., 2012; Sun, Cacciarelli, Wagener, & Singh, 2010) .
Among a variety of baseline risk factors that may indicate longer CMV treatment duration, significant predictive value has been demonstrated with higher baseline viral loads where longer treatment duration may prevent CMV disease relapse (Kotton et al., 2013; Sia et al., 2000) .
Clinical trial studies supporting the recent FDA approval of a quantitative real-time PCR CMV test calibrated to the WHO International Standard also demonstrated clinical value for baseline testing of patients with CMV disease who are undergoing treatment with the anti-CMV drugs ganciclovir or valganciclovir (Razonable et al., 2013) .
Data from this study suggested that patients with a baseline CMV viral load <18,200 IU/mL are likely to resolve CMV disease more rapidly than those who have a higher baseline viral load.
Further studies are needed to determine universal thresholds for pre-emptive therapy initiation and predictive value for CMV baseline viral load in defining optimal treatment duration.
There exists a clear application for quantitative real-time PCR technology in baseline determination of patients with significant viral infections, and in fact, quantitative viral load determination plays a critical role in therapeutic decision for many other viral infections.
High baseline viral load has been shown to correlate with advanced disease during infection with numerous viruses such as BKV, HSV-1, EBV, and Adenovirus and may potentiate the need for longer duration therapies in certain scenarios (Cincinnati Children's Hospital Medical Center, 2012; Domingues, Lakeman, Mayo, & Whitley, 1998; Gustafson et al., 2008; Randhawa et al., 2004) .
After the patient's baseline assessment or pre-emptive monitoring suggests if treatment is available, which treatment regimen to choose and perhaps the duration of therapy, the patient can move on to therapeutic administration.
Quantitative realtime PCR has helped and continues to set the stage for decisions that potentially saves lives, reduces complications, decreases morbidity, and lessens the economic burden to both the patient and the healthcare system.
Serial measures of viral load serve as an individualised map of a viral infection through the estimation of the amount of virus found within an infected person.
Tracking viral load in the continuum of care is a vital tool used predominantly to monitor treatment response and its effectiveness, early signs of resistance emergence during therapy of chronic viral infections, and viral activation or reactivation in immunocompromised patients following bone marrow or solid organ transplantation.
While the goal of treatment for chronic HCV infection is SVR, patients may fail therapy due to non-response, on-treatment breakthrough, or post-treatment relapse ( Figure 6 ).
The early change in quantitative viral load over time may be predictive of treatment efficacy and a shorten therapy for patients who respond rapidly to treatment (Yee et al., 2006) .
This 'response-guided therapy' (RGT) is best exemplified during treatment of chronic HCV patients.
Specifically, the sooner a patient becomes HCV RNA undetectable during treatment, the lower the relapse rate when treatment is shortened.
Conversely, the longer it takes for a patient to become HCV RNA undetectable, the longer they need to remain on treatment to limit relapse.
However, given the poorer efficacy of earlier regimens, not all patients who received therapy achieved SVR.
For this reason, 'futility rules' or 'stopping rules' were also developed, which required that failure of a patient to respond (target not detected or viral load cutoff ) by a given time-point indicated the need to immediately discontinue therapy.
Monitoring HCV viral loads during treatment.
Despite advances in treatment for HCV patients, failure to achieve SVR is still a reality.
Patients who do not achieve SVR fall into four categories: (1) null responders (black line) achieve less than 2-log decrease in hepatitis C viral load upon treatment; (2) partial responders (red line; light grey in the print version) experiences at least a 2-log decrease in hepatitis C viral load during HCV treatment but fail to proceed to an undetectable viral load level; (3) breakthrough patients (orange line; light grey in the print version) have an undetectable HCV viral load, but the virus rebounded during treatment; (4) relapsers (blue line; dark grey in the print version) have had an undetectable HCV viral load, but the virus rebounded after they completed HCV treatment.
Although these RGT notions were originally developed from observations made during treatment with the older therapies, peg-IFN and ribavirin, RGT was also required during treatment with the much more potent first-generation DAAs, telaprevir and boceprevir, and stopping rules were put in place during treatment with the second-generation DAA, simeprevir (AASLD/IDSA/IAS-USA, 2014; Ghany et al., 2009; Yee et al., 2006) .
Newer IFN-free DAA regimens targeting HCV, which are better tolerated by patients and by virtue of the targets they inhibit, have a higher barrier to resistance, yield more rapidly declining viral kinetics, and, thus, do not contain treatment indications for RGT in their prescribing information (HARVONI, 2014; OLYSIO, 2014; SOVALDI, 2013; VIEKIRA, 2014) .
While RGT was a major driver for regular viral load monitoring during antiviral therapy, it is not the only reason to monitor HCV viral load.
In the interval between baseline measurement and assessment of SVR, the 2014 AASLD/IDSA guidelines also include recommendations for monitoring initial response (week 4 on treatment with a repeat at week 6 if detectable) and end of treatment in order to provide an assessment of drug compliance/early efficacy and predict treatment outcomes, respectively (AASLD/IDSA/IAS-USA, 2014).
In the most recent revision to these Web-based guidelines, it is recommended that an HCV viral load increase of greater than 10-fold on repeat testing at week 6 (or thereafter) should prompt a discontinuation of HCV treatment.
Many clinicians also closely monitor and report the declining viral loads to their patients in order to demonstrate treatment efficacy, motivating patients to continue treatment and remain adherent to the drug regimen until the next follow-up appointment (Fusfeld et al., 2013) .
Regardless of monitoring during HCV treatment for RGT, adherence/compliance, patient motivation, early treatment efficacy, etc., quantitative real-time PCR is widely used by laboratories due to its sensitivity, accuracy, and reproducibility of each consecutive viral load test.
For patients infected with chronic viral infections, such as HIV, the lifelong regimen of highly active ART aims to suppress HIV viral levels to near undetectable levels, ensuring progression-free survival (delay or all together prevention of the progression to AIDS) and reducing potential transmission.
Alongside monitoring immune function and immunologic efficacy through CD4 T-cell count, HIV viral levels are critical in the clinical evaluation and assessment of HIV-infected patients undergoing ART.
Determining a patient's HIV viral load is indicated prior to entry into care, at the initiation of ART, at 2-8 weeks after ART initiation, and then typically every 3-4 months while on treatment: (1) to establish a baseline level of HIV viral load; (2) to establish viral response to the therapy to assess the virologic efficacy of ART; and (3) to monitor for abnormalities that may be associated with antiretroviral drugs (DHHS HIV, 2014) .
The baseline HIV viral load is not only linked to treatment options (Sax et al., 2009) but also helps to establish the magnitude of viral load decline after initiation of ART and provides prognostic information about the probability of progression to AIDS or death (Marschner et al., 1998; Murray, Elashoff, Iacono-Connors, Cvetkovich, & Struble, 1999; Thiebaut et al., 2000) .
Once treatment is initiated, the goal is to reach and maintain suppressed HIV replication as determined by undetected viral levels utilising highly sensitive NAT tests, which is generally achieved within 8-24 weeks after ART initiation.
The need for sensitive assays to effectively assess viral suppression hinges on the need to suppress HIV replication to the extent that viral evolution and drug resistance mutations do not emerge, which typically do not occur in patients whose HIV RNA levels are maintained below the LLOD of current real-time quantitative PCR assays (Kieffer et al., 2004) .
Due to the introduction of more sensitive real-time PCR assays, which can detect as few as 20 viral copies/mL, natural variability in HIV viral levels over time, even in patients with effective suppression, is much more evident (Lima, Harrigan, & Montaner, 2009; Gatanaga et al., 2009; Willig et al., 2010) .
Although controversy exists between the clinical significance of viral loads between LLOD and <200 copies/ mL, there are reports suggesting that this low-level viraemia is predictive of virologic rebound (Doyle et al., 2012; Eron et al., 2013; Laprise, de Pokomandy, Baril, Dufresne, & Trottier, 2013) , virologic failure (Estevez et al., 2013) , and indication of drug resistance (Taiwo et al., 2010) , signifying the need for highly sensitive assays.
Viraemic blips, a single detectable HIV viral load (<500 copies/mL) in an otherwise seemingly suppressed patient (Figure 7) , however, do not indicate subsequent virologic failure or development of resistance mutations (Castro et al., 2013; Lee, Kieffer, Siliciano, & Nettles, 2006; Nettles et al., 2005) .
Blips are not unusual (Havlir et al., 2001) and appear to be more common in winter, suggesting that host-related and seasonal factors are associated with the occurrence of viraemia (van Sighem et al., 2008) .
On the other hand, persistent HIV RNA levels !200 copies/mL are often evidence of viral evolution and accumulation of drug resistance On-treatment HIV patient monitoring.
(A) HIV viral loads will fluctuate as patients are on treatment, and, in most instances, will remain 'undetectable' (at or below dotted line); viral 'blips' are not uncommon and will result in transient 'detectable' and even quantifiable results (above the dashed line).
(B) Virologic failure will lead to a sustained high-level viral titre that, without intervention, will increase with time.
mutations (Aleman, Soderbarg, Visco-Comandini, Sitbon, & Sonnerborg, 2002; Karlsson et al., 2004) .
Once treatment failure is confirmed, immediate intervention is recommended to avoid progressive accumulation of resistance mutations and effective response of new regimen (DHHS HIV, 2014), which is benefited by low HIV RNA levels and/or higher CD4 cell counts (Eron et al., 2013) , and even a brief interruption in therapy may lead to a rapid increase in HIV RNA and a decrease in CD4 cell count and increases the risk of clinical progression (Deeks et al., 2001; Lawrence et al., 2003) .
With the development and administration of newer drugs that target specific biological processes of HIV, routine and clinical monitoring of viral loads using a real-time quantitative PCR assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success.
Viral load monitoring is also essential when the recipient of a solid organ transplant is CMV seropositive and the decision is made to initiate treatment only once the CMV levels predictive of disease are reached.
This strategy, known as pre-emptive therapy, utilises intensive monitoring for CMV activity by sensitive real-time quantitative PCR methods and short-term antiviral treatment is given only to those with significant viral counts before symptoms occur.
CMV is one of the most common opportunistic pathogens that infect solid organ transplant recipients (Fishman, 2007) and is associated with increased morbidity and mortality (Sagedal et al., 2004; Schnitzler et al., 2003) .
Following primary infection, the virus establishes a lifelong latent infection in several sites of the body and may reactivate in the presence of immunosuppression, such as in transplant recipients.
Once reactivated, CMV is able to modulate the immune system and is known to be a potent upregulator of alloantigens (Razonable, 2008) , increasing the risk of chronic allograft dysfunction (Reischig, 2010; Sagedal et al., 2002; Smith et al., 2010) and acute rejection (Sagedal et al., 2004) .
Pre-emptive therapy reduces the incidence of CMV disease (Khoury et al., 2006; Reischig et al., 2008) , which has been documented as a serious problem in randomised trials upon completion of universal antiviral prophylaxis therapy (Kalil, Levitsky, Lyden, Stoner, & Freifeld, 2005; Lowance et al., 1999; Paya et al., 2004) .
Long-term studies have demonstrated that patients receiving preemptive therapy, when compared to prophylaxis therapy, were less likely to develop moderate-to-severe kidney scaring and atrophy and significantly better survival of the transplanted organ (Reischig et al., 2012) .
However, challenges still exist around defining appropriate thresholds to initiate pre-emptive therapy (Humar & Snydman, 2009) .
But with new standardised real-time PCR assays, widespread adoption, and utilisation of these tests, pre-emptive therapy relying in intensive viral load monitoring may become the standard for certain at-risk patients.
Test of cure, or end of treatment response, is assessed following a given therapeutic regimen for signs of treatment efficacy.
In few cases, a quantitative viral load measurement serves as a way to establish a cure rate, but, in others, may only be used as a confirmation of virologic suppression as clinical cure may not yet be possible with current therapies or technical limitations by real-time PCR that limits the overall sensitivity of viral detection.
Regardless of the clinical utility for measuring a virologic suppression, quantitative real-time PCRs with their current limits of detection and limits of quantitation are valuable tools in measuring low-level viraemia and establishing undetectable viral loads.
Utilisation of quantitative real-time PCR to assess virologic cure is perhaps best exemplified by treatment of patients with chronic HCV.
According to the AASLD/ IDSA guidelines, patients who have 'undetectable' HCV RNA in the serum, when assessed by a sensitive PCR assay, 12 or more weeks after completing treatment, are deemed to have achieved a sustained virologic response .
Achieving an SVR is considered a virologic cure of HCV infection since, in these patients, hepatitis C-related liver injury stops and recurrence of infection is marginal, detected in <1% of patients after 5 years post-treatment (AASLD/IDSA/IAS-USA, 2014; Manns et al., 2013) .
In agreement with these guidelines, the FDA recommendation to pharmaceutical DAA manufacturers also stipulates that viral RNA clearance at SVR-12 be measured in clinical trials using an FDA-approved sensitive and specific quantitative HCV RNA assay (FDA HCV, 2013) .
According to prescribing information accompanying the current DAAs, the threshold of SVR-12 is defined as a quantitative threshold of HCV RNA <25 IU/mL at 12 weeks after the end of treatment (Feld et al., 2014; Kowdley et al., 2014; Lawitz et al., 2013) .
This is somewhat dissimilar to the AASLD/IDSA guidelines as 'undetected' viral levels are not equivalent to 'detected but below the limit of quantitation' (Figure 3 ).
But, with the benefit of high sensitivity and reproducibility, quantitative real-time PCR has a clear established role in assessment of HCV virologic cure in both clinical trials and clinical practice and is able to meet the needs for assessing SVR.
Quantitative real-time PCR may also play a critical role in the assessment of CMV disease resolution.
The consensus guidelines recommend that two consecutive negative samples be obtained with a minimum treatment course of 2 weeks before treatment is discontinued, which is thought to minimise the risk for development of resistance and disease recurrence (Asberg et al., 2009; Chou, 2001; Sia et al., 2000) .
Still, some transplant centres may extend treatment (secondary prophylaxis) in patients with compartmentalised disease for as long as necessary to reduce the likelihood of recurrent CMV infection (Kotton et al., 2013) .
Resolving CMV disease has the long-term benefits of reducing mortality, potential allograft rejection, and the risk of bacterial, fungal, or viral opportunistic infections, among many other transplantand non-transplant-specific effects (Arthurs et al., 2008; Fishman, 2007) .
Although there is currently no cure for HIV infection, highly sensitive quantitative and qualitative real-time PCR tests targeting total HIV DNA and RNA have been used in clinical studies for both sterilisation (elimination of HIV-infected cells) and functional (controlled HIV in the absence of ART) cures (Kibirige, 2013; Lewin & Rouzioux, 2011) .
Improvements in real-time PCR technology may lead to profound increases in assay sensitivity and the ability to achieve single-copy detection (1 cp/mL) may lead us to a better understanding of HIV virology and what may be needed therapeutically to achieve a cure (Alidjinou, Bocket, & Hober, 2014) .
If therapeutic strategies are one day able to achieve an HIV cure, these highly sensitive tests will no doubt play a key role in the continuum of care for patients and, most importantly, in the confirmation of cure.
Clinical laboratories have undergone changes to become more efficient and flexible while delivering the same high-quality results.
When choosing to implement new testing, even beyond viral targets, laboratories have to consider first and foremost the performance and medical value of the test and then factors such as TAT, ease of use, and cost.
Real-time PCR with its wide dynamic range, high specificity, and high sensitivity is considered the gold standard for the quantification and identification of a variety of targets including bacteria, fungi, viruses, or oncological mutations (Klein, 2002) .
Furthermore, the multiplexing capability of real-time PCR increases the number of targets and information gathered from the same test, further improving laboratory workflow, TAT, and costs (Deshpande & White, 2012) .
While novel technologies have entered clinical laboratories including mass spectrometry and next-generation sequencing, real-time PCR remains a staple and an attractive option for clinical laboratories aiming to create molecular laboratory-developed tests (LDTs).
In addition, PCR can quickly be adapted to provide a robust test for the identification of emerging disease and molecular testing is now able to reach beyond the clinical laboratory and further enhance healthcare (Farrar & Wittwer, 2015; Foudeh, Didar, Veres, & Tabrizian, 2012) .
Most molecular tests used in clinical laboratories are FDA-approved and commercially available.
There are instances, however, when a test may not be available for a specific virus or the sample type and/or clinical indication used by the laboratory differs from those of the FDA-approved assay, typically leading a laboratory to design its own PCR-based test or modify existing assays.
FDA defines an LDT as 'a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory' and recognises that 'LDTs are important to the continued development of personalised medicine' (FDA LDT, 2014) .
Laboratory developed tests can be grouped into three categories, FDA-cleared or approved test that have been modified, tests that are not subject to FDA clearance or approval, and tests for which no performance specifications have been provided by the manufacturer (e.g.
analytespecific reagents or ASRs) (Burd, 2010; Code of Federal Regulations, 2009 ).
With alternative sample types or applications, FDA-approved tests are often modified to fit the testing needs of laboratories, including alternative collection media and sample types or expanded clinical applications.
As an example, a recent gap was created in the HCV-screening algorithm for the confirmation of a positive enzyme immunoassay result following the discontinuation of the only FDAapproved confirmatory test (Alter, Kuhnert, & Finelli, 2003) .
In response, the CDC published recommendation for the use of FDA-approved tests detecting HCV viraemia (CDC HCV, 2013), despite the fact that most of these assays did not have specific claims for confirmatory testing; as a result, several laboratories chose to validate these assays as LDTs to meet the screening needs for HCV.
Additionally, LDTs are the only option for the identification of the aetiologic agents of viral infections that can occur in transplant patients, such as EBV, adenovirus, VZV, and BK virus, that often present with non-specific clinical manifestations (Razonable, 2011) and for which FDA-approved assay options are lacking.
LDTs are an integral part of molecular laboratory testing.
Whether created from the ground-up or modified from FDA-approved assays, LDTs are answering the clinicians' needs for information as an aid for diagnosis or treatment of patients.
As with any clinical tests, LDTs have to meet the minimum standards set forth by CLIA prior to report patient results (Code of Federal Regulations, 2009).
In July 2014, FDA informed Congress of the agency's LDT regulatory oversight framework (FDA LDT, 2014) .
FDA aims to address concerns over high-risk LDTs with inadequately supported claims, lack of appropriate controls, and falsification of data that may lead to inadequate treatment, possible harm to patients, and unnecessary healthcare cost.
Presently, there is still a high degree of uncertainty as to what the final regulation scope will be and the possible impact on molecular laboratories will have to be seen.
Palaeopathology confirmed the truism that humanity, since its inception, has been exposed to genetic and infectious diseases with early documentation of trachoma (8000 B.C.E.
), tuberculosis (7000 B.C.E.
), and pneumonia (ca.
1150 B.C.E.)
(Aufderheide & Rodreguez-martin, 1998; Hershkovitz et al., 2008; Roberts & Manchester, 1995; Webb, 1990) .
Even today, emerging infectious diseases (EIDs) continue to appear unpredictably driven by changes in human demographic, land use, and population behaviour (Lederberg, Hamburg, & Smolinski, 2003; Sehgal, 2010; Taylor, Latham, & Woolhouse, 2001) .
These infections can be classified as either newly emerging/a previously unknown disease or re-emerging infectious diseases/a previously known disease, that reappears after a significant reduction in incidence or elimination (Morens & Fauci, 2013) .
EIDs are a threat not only to human health but also to global stability and economy.
Efforts to monitor these EIDs are in place both at the global level spearheaded by the WHO and at the national level.
In the United States, governmental agencies (Department of Health and Human Services, United States Agency for International development, Department of Defense) are supporting activities to detect, assess, and respond to potential outbreaks.
Specifically, PCR and real-time PCR are easily adaptable to detect nucleic acid targets that are unique to each given pathogen, and as such, they play essential roles in the identification and detection of infectious pathogens and have been routinely used by health organisation agencies during epidemic outbreaks such as severe acute respiratory syndrome (SARS), H5N1, H1N1, and Ebola (Shuaib et al., 2014; WHO Influenza, 2011) .
The SARS epidemic appeared in November 2002, in the Chinese province of Guangdong before reaching the adjacent Hong Kong in 2003 (WHO SARS, 2003 .
This SARS eventually spread to 26 countries and resulted in more than 8000 cases.
In response, the CDC triggered its emergency operations centre and issued a draft genome in April 2003, 33 days after the initial WHO global alert (CDC SARS, 2013) .
Soon after, real-time quantitative PCR assays were described and put in use for the diagnosis of SARS (Drosten et al., 2003; Peiris et al., 2003) .
A host of measures were taken in order to contain this epidemic, and the molecular identification and diagnosis of the infectious agent by PCR played a key role in providing critical information to address the situation and contributed to the care of the patients infected.
Additionally, the re-emerging 2014 Ebola epidemic (CDC Ebola, 2014; WHO Ebola, 2014) started in Guinea in March of 2014 before spreading to nearby West African countries and eventually reaching the United States and Europe (WHO Ebola, 2014) .
At the height of the epidemic, FDA issued an Emergency Use Authorisation (EUA) for the use of the first real-time RT-PCR assay (FDA EUA, 2014) and less than 4 months later, five additional realtime PCR tests were authorised under an EUA (FDA EUA, 2014) to provide an early diagnosis of the Ebola viral disease (CDC Ebola, 2014).
EIDs remain a constant and unpredictable threat to human health.
The flexibility of real-time PCR technology continues to show how promptly it can be used for the detection of infectious agents.
By providing a rapid diagnostic, real-time PCR can help in starting the appropriate treatment right away and maximise the chances of a positive outcome.
The goal of point-of-care testing (POCT) is to quickly obtain a test result that will be used to implement the appropriate treatment for an improved clinical outcome.
By definition, POCT is laboratory testing that takes place at or near the site of patients (CAP POC, 2013) .
The advantages of POCT are an improved TAT and result availability regardless of normal core laboratory hours, access to care in remote areas, and greater patient involvement.
The fight against AIDS largely contributed to the development of POCT devices with viral load capabilities (Hong, Studer, Hang, Anderson, & Quake, 2004; Lee et al., 2010; Marcus, Anderson, & Quake, 2006; Tanriverdi, Chen, & Chen, 2010; UNITAID, 2014; Vulto et al., 2009) .
Originally developed to meet the difficult conditions associated with remote places, far from any core laboratory facility often found in the developing world, the design and convenience of a portable POCT device with fast turnaround and accurate results extends the reach of healthcare.
With this in mind, these POCT systems could easily be used in developed nations at hospitals, within clinics a physicians' offices, pharmacies, correction facilities, or mobile health units, to target pathogens that benefit from immediate actionable results, for which not only accurate but also quick results are critical (Kiechle & Holland, 2009 ).
Ultimately, test menu available on these platforms will drive its implementation as a complement for the clinical laboratory core testing.
The ideal molecular POCT system that includes medical value, simplicity, fast TAT, and ruggedness remains an ongoing engineering challenge.
However, the latest advances in microfluidics are a great example of the potential of these devices and brings the real-time PCR lab-on-a-chip closer to mainstream diagnostic use.
This is an exciting time for molecular POCT and the upcoming years should bring new systems and perhaps a paradigm change in the world of healthcare.
As the needs of the clinicians, laboratory, and patients continue to evolve, so do the applications of molecular diagnostics and PCR.
Over the past decade, quantitative real-time PCR technology has been increasingly phased into clinical practice and all of the potential present-day applications of real-time PCR-based methods are enumerable.
They serve to advance experimental approaches within biological fields, pushing the boundaries of what we know and what we can learn, as well as to diminish empiric medical identification and management of viral diseases.
The high sensitivity of the technology has reduced risks of the most commonly transmitted transfusion illnesses and has become an integral part of managing a variety of viral infections by providing pretreatment prognostic information, therapeutic effectiveness through monitoring, and end of treatment response assessment.
Quantitative real-time PCR complements serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses and are able to predict therapeutic failures sooner than traditional methods, allowing for a more timely management response.
Real-time PCR assays can be rapidly developed in cases of emerging epidemic crises involving new pathogens that may result in significant health threats.
The next few years are likely to see an even further increase in the expansion of the clinical applications of nucleic acid quantification, particularly following bone marrow and solid organ transplantation for which the newest standardised assays may provide an avenue for the development of consensus management guidelines for initiating pre-emptive anti-CMV treatment.
Further, with the drive towards HIV eradication and complete elimination of the virus from within cells of infected patients, innovations in quantitative real-time PCR assay design will continue to push the boundaries of detection and introduce assays with progressively lower limits of detection.
Thus, quantitative real-time PCR has and will facilitate advancements in the quality of diagnostics and of what we can achieve in research, medicine, and patient outcomes.
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key: cord-018566-dd5gw66t authors: Armbruster, Walter J.; Roberts, Tanya title: The Political Economy of US Antibiotic Use in Animal Feed date: 2018-05-30 journal: Food Safety Economics DOI: 10.1007/978-3-319-92138-9_15 sha: doc_id: 18566 cord_uid: dd5gw66t This chapter examines the evidence for antibiotic resistance in the United States and globally, the public health implications, and the impact of—and related industry and political responses to—antibiotic use in animal feed.
In 1969, the Swann Report in the United Kingdom noted a dramatic increase in antibiotic-resistant bacteria in food animals receiving low levels of antibiotics in their feed.
While the Food and Drug Administration of the United States sought to control antibiotics in animal feed as far back as 1977, only in 2016 were such regulations fully implemented.
The farm-level costs of such controls are estimated by the US Department of Agriculture’s Economic Research Service to be minimal, while the Centers for Disease Control and Prevention’s estimates of the public health costs of antibiotic resistance without implementing controls are $7 billion annually.
The complex interactions which exist between economic interests, regulatory policy, and human and animal health are explored in this chapter.
Antibiotic resistance has been widely recognized as a serious public health problem.
Hence, there is a major public good to be realized in safeguarding the effectiveness of existing antibiotics and creating new ones.
Antibiotics are used to treat human infections and used in animal agriculture.
While many drugs are dual-use, others are animal-or human-use specific.
The production benefits of sub-therapeutic levels of antibiotics in animal agriculture have been recognized since the late 1940s (CAST 1981) .
In animal agricultural production, antibiotics are used at therapeutic levels to treat infections and at sub-therapeutic levels to prevent infections and promote animal growth (Sneeringer et al.
2015; Van Boeckel et al.
2017; WHO 2017) .
As the organizational complexity of the animal agricultural supply chain increased, the number of economic stakeholders in on-farm antibiotic use has also increased.
The major stakeholders include pharmaceutical companies, production integrators, feed suppliers, farm groups, producers, restaurants, food retailers, the public, the medical community, the scientific community, government regulators and policy makers.
Each of these stakeholders faces a different set of incentives and disincentives related to on-farm use of antibiotics in animal agriculture.
Knowledge of these incentives and disincentives has evolved with the accumulation of scientific and economic research.
To understand the regulatory outcomes governing antibiotic use in agriculture, it is important to recognize the political economy context in which they are developed.
The various stakeholders are driven by the relative benefits they receive under policies as they affect their industry segment (Zilberman et al.
2014 ).
Alexander Fleming, who discovered penicillin, warned that "…misuse of the drug could result in selection for resistant bacteria" (Rosenblatt-Farrell 2009) .
Antibiotic resistance (AR), a term sometimes used interchangeably with antimicrobial resistance, occurs when bacteria change in ways that make antibiotics less effective in treating infections, thereby allowing the bacteria to survive, multiply, and cause additional harm.
AR has been recognized as a serious public health problem among the medical and scientific communities.
Antibiotics are used to treat human infections and used in animal agriculture.
Particularly concerning is resistance for those antibiotics that are of value in treating human health issues, the so-called medically important antibiotics.
The use of antibiotics along with other advances in agricultural technology has facilitated the concentration of animal production on farms in the United States (US) and elsewhere.
For example, in 2012, 88% of all US sales of hogs and pigs were by the 13% of farms with 5,000 or more head, and 66% of all layers were produced on the less than 1% of farms that sold 100,000 or more to egg producers (NASS 2014) .
The majority of the production of hogs, broilers, and eggs occurred under contractual arrangements between growers and integrators, with the integrators prescribing certain production practices, including the use of antibiotics for treating infections, for disease prevention and for promoting growth.
Many of the antimicrobial drugs administered to food-producing animals are also important in treating humans, worldwide.
Domestic sales of medically important antimicrobial drugs for use in food-producing animals in the United States accounted for 62% of the domestic sales of all antimicrobials approved for use in food-producing animals.
And, 28% of domestic sales of all medically important antimicrobials approved for use in food-producing animals are labeled for therapeutic use only .
Importantly, animal drug sales data represent products sold or distributed by manufacturers through various outlets for intended sale to the user.
Since veterinarians and others in the supply chain may have substantial inventory on hand for possible use, these numbers do not accurately reflect the amount of product ultimately administered to animals.
Given the number of humans versus a much larger number of animals in each of the species, as well as other confounding factors, no definitive conclusions from any direct comparisons between the quantities of antimicrobial drugs sold for use in humans versus animals can be drawn (FDA 2016a) .
There are obvious situations where antibiotics are required to treat sick animals in agriculture, but the proper therapeutic use versus prophylactic use remains in question among stakeholders.
Farm groups and others in the food animal supply chain recognize that antibiotics in animal feed keep animals healthy and meat costs down.
But over 1000 medical doctors and other healthcare providers signed petitions to Congress asking for new legislation to reduce non-therapeutic antibiotic use in food animals (Miller 2011) .
The animal health industry is very concerned that needed preventative use will be threatened by the recent FDA ban on use of medically important antibiotics for growth.
FDA classifies as therapeutic those antimicrobials targeted for treatment, control, and prevention of bacteria or disease identified on the product label.
FDA explicitly states that the use of antibiotics in animal feed for growth promotion is not allowed.
Those who characterize preventative use as routine overlook the difference between treating animals versus humans.
If preventative measures are not taken and a disease outbreak occurs and spreads rapidly within a flock or herd, it risks large numbers of animals developing a deadly, high mortality disease.
Waiting until a disease is clearly evident makes successfully treating the active infections very difficult due to the large number of animals involved.
By contrast, a human patient can generally be quickly diagnosed and treated.
While some are concerned that producers will continue to use antibiotics for growth under the guise of prevention, the FDA-approved label is specific about dose and duration for a specified bacterium or disease.
Off-label use of antibiotics in animal production is illegal, and FDA only allows a veterinarian to decide whether to use or not to use a preventative treatment based on their judgment of a disease threat (Carnevale 2016) .
In an economic framework, antimicrobial resistance can be considered as an unwanted side effect, or externality, associated with the use of antibiotics.
The efficacy of antibiotics can be considered as a public good that must be managed with government involvement.
This is because the costs of overuse by any single individual are borne by society and, in the case of antibiotics, globally.
Hence, not only is there a role for government involvement with the animal agriculture industry in managing the stock and use of antibiotics as an important public good, but it must be done cooperatively across countries.
In 1969, the United Kingdom's (UK) Parliament received the Swann Report, which concluded that using antimicrobials at sub-therapeutic levels in food-producing animals created risks to human and animal health (Joint Committee on the use of Antibiotics in Animal Husbandry and Veterinary Medicine 1969).
It noted a dramatic increase in numbers of animal-origin bacteria strains which showed resistance to one or more antibiotics and that these strains could transmit resistance to other bacteria.
It recommended that only antimicrobials that are not medically important for humans should be used without prescription in animal feed and that antimicrobials should only be used for therapeutic purposes under veterinary supervision.
The primary reason that producers were using these sub-therapeutic doses of antibiotics was to promote faster weight gain in the animals.
In 1970, a US Food and Drug Administration (FDA) task force was charged to do a comprehensive review of antibiotic use in animal feed (FDA 2012).
Its report found that sub-therapeutic use of antimicrobials in food-producing animals was associated with development of resistant bacteria and that treated animals might provide a reservoir of antimicrobial-resistant pathogens capable of causing human disease.
The task force recommended that medically important antimicrobial drugs meet certain guidelines they identified or be prohibited from growth promotion or other sub-therapeutic use by certain dates.
Further, antimicrobials not meeting the guidelines should be limited to short-term therapeutic use only under veterinarian control.
In the 1970s, the Animal Health Institute (AHI), a US trade association for the animal drug industry, funded an on-farm study to determine the impact of adding low-dose antibiotics to chicken feed.
Within 1 week of adding tetracycline, the intestinal flora in the chickens "…contained almost entirely tetracycline-resistant organisms" (Levy et al.
1976 ).
The antibiotic resistance was not located in the DNA of the bacteria which is hard to transfer among bacteria but in plasmids located on the outside surface of the bacteria.
Plasmids are easily exchanged among bacteria living in the intestine.
Importantly, the tetracycline-resistant bacteria in the chicken's intestines were resistant to multiple antibiotics.
Furthermore, some members of the farm families began to harbor these same antibiotic-resistant bacteria in their intestines within 6 months.
In 1977, the FDA proposed withdrawing the new animal drug approvals for the sub-therapeutic uses of human medically important penicillin and tetracycline in animal feed based on lack of evidence to show they were safe.
However, the US Congress intervened and asked for more research first.
The AHI was one of the groups advocating in Congress to delay regulation pending additional research, then and now.
In 2010 Congressional Testimony, Richard Carnevale, vice-president at AHI, testified that while it is possible for human antibiotic resistance to be caused by antibiotic use in farm animals, "…it does not happen enough that we can find it and measure it" (Carnevale 2010) .
This statement contradicted the data produced by the AHI-funded study by Levy (Levy et al.
1976 ) that was published in the prestigious New England Journal of Medicine in 1976.
Richard Carnevale also mentioned in his 2010 testimony that prior to joining AHI he was Deputy Director of New Animal Drug Evaluation in FDA and had worked at USDA in the Food Safety and Inspection Service (FSIS).
His testimony illustrates two points in the political economy of food production: (1) how industry has an opportunity to influence regulators' decision-making via the revolving door of employment and (2) how industry carefully selects its facts to present a point of view that bolsters their profits, namely, for drug companies in this case (Oreskes and Conway 2010) .
Another example of the political economy in action involved USDA prohibiting an agency research microbiologist from talking about the significant levels of antibiotic-resistant bacteria detected in the air near Midwest hog confinement operations (Union of Concerned Scientists 2004).
A third element of the political economy is shown by industry efforts to influence policy makers through campaign contributions and strong lobbying of proposed legislation which may affect their bottom line.
Pharmaceutical companies spent at least $135 million and agribusiness companies another $70 million during 2009, in large part to fight possible limits on antibiotic use in animal feed (Mason and Mendoza 2009) .
In response to Congressional pressure in the late 1970s, FDA withdrew its proposal and instead funded three studies to determine the impact of using low levels of antibiotics in animal feed (industry won this round, obtained a delay in regulations, and funded more reports): 1.
In 1980, the National Academy of Sciences reported that there was limited epidemiological research on the topic.
Available evidence at that time did not prove nor disprove dangers of seven therapeutic antimicrobials in animal feed, but that did not preclude the existence of hazards (National Academy of Sciences 2009).
2.
In 1984, the FDA funded the Seattle-King County Department of Public Health to analyze Salmonella and Campylobacter, which were chosen as models to estimate the flow of potentially pathogenic bacteria from animals to humans through the food chain.
Their report was based on sampling retail meat and poultry and investigating Salmonella and Campylobacter enteritis cases in humans.
Isolates from human illness cases and retail foods were analyzed for antibiotic resistance of these pathogens, using plasmid analysis and serotyping.
The report found that Campylobacter was a more common cause of enteritis than Salmonella and appeared to flow from chickens to humans through consumption of poultry products, with tetracycline resistance being plasmid-mediated (Seattle-King County Department of Public Health 1984).
3.
In 1988, the Institute of Medicine (IOM) undertook a FDA-requested independent quantitative risk assessment of human health impacts from sub-therapeutic use of penicillin and tetracycline in animal feed.
Based on a risk-analysis model of Salmonella infections that resulted in human death, the IOM did not find substantial direct evidence that sub-therapeutic use in animal feed posed a human health hazard.
However, they found a considerable body of indirect evidence implicating both sub-therapeutic and therapeutic use of antimicrobials as a potential health hazard and strongly recommended additional study of the issue (Institute of Medicine 1988).
Numerous research results quantifying the extent of the antimicrobial resistance problem have been published in the scientific literature and indicate a growing and serious threat to human health.
The many channels for AR to affect humans are shown in Fig.
15 .1.
The two main channels for food animals are (1) AR bacteria in the food animal's gut can contaminate the meat or poultry eaten and (2) environmental contamination, such as manure used to fertilize fields that contain AR bacteria, may contaminate the environment and some of the food crops grown on these fields.
Consumer Reports (CR) tested products sold in US supermarkets and found resistance to multiple antibiotics in the following percent of samples: beef 14%, shrimp 14%, turkey 83%, and chicken 57% (Consumer Reports 2016).
CR also found that ground beef from conventionally raised cows was twice as likely to contain antibiotic-resistant pathogens as ground beef from cows raised without antibiotics.
Like other threats to human health, AR is best managed across national boundaries.
Increasing international trade may spread antibiotic resistance through imported food products as more trade agreements are approved.
This scenario could be exacerbated to the extent FSIS approves additional international facilities, local regulations, and inspections as "equivalent to the United States."
Future trade agreements will need to include provisions which address reduced use of medically important antibiotics in producing food animals.
Numerous trusted institutions from the United States (US) and the United Kingdom (UK) as well as international organizations such as the World Health Organization (WHO), the United Nations' Food and Agriculture Organization (FAO), and the World Organization for Animal Health (OIE) have acknowledged the threat of antibiotic resistance related to use in producing food animals.
The following excerpts from a few recent reports highlight the role that low-dose antibiotic use in animal feed plays in spreading AR.
The Centers for Disease Control and Prevention (2013b) reported that: Each year in the United States, at least 2 million people acquire serious infections with bacteria that are resistant to one or more of the antibiotics designed to treat those infections.
At least 23,000 people die each year as a direct result of these antibiotic-resistant infections.
Many more die from other conditions that are complicated by an antibiotic-resistant infection.
Antibiotic-resistant infections add considerable and avoidable costs to the already overburdened U.S. healthcare system.
In most cases, antibiotic resistant infections require prolonged and/or costlier treatments, extend hospital stays, necessitate additional doctor visits and healthcare use, and result in greater disability and death compared with infections that are easily treatable with antibiotics.
The total economic costs of antibiotic resistance to the U.S. economy has been difficult to calculate.
Estimates vary but have ranged as high as $20 billion in excess direct healthcare costs.
Adding on the costs for lost productivity brings the total societal costs (sic) for AR to $35 billion a year (2008 dollars).
(CDC 2013a, p. 11) This CDC report also indicates that foodborne cases are responsible for 20% of human AR infections ( Fig.
15 .1).
Thus, societal costs of these AR foodborne illnesses could total $7 billion annually of the $35 billion/year total costs to the US economy.
These societal costs could be prevented if the foods were free of contamination with AR pathogens.
There may be additional costs associated with environmental pathways of human contamination from use of antibiotics in meat production, such as exposure to contaminated water.
In 2014, WHO stated: "Antimicrobial resistance (AR) is an increasingly serious threat to global public health.
AR develops when a microorganism (bacteria, fungus, virus or parasite) no longer responds to a drug to which it was originally sensitive.
This means that standard treatments no longer work; infections are harder or impossible to control; the risk of the spread of infection to others is increased; illness and hospital stays are prolonged, with added economic and social costs; and the risk of death is greater-in some cases, twice that of patients who have infections caused by non-resistant bacteria.
The problem is so serious that it threatens the achievements of modern medicine.
A post-antibiotic era-in which common infections and minor injuries can kill-is a very real possibility for the 21st century" (WHO 2014, p. 3) .
In 2015, OIE noted: "Today, in many countries, including developed countries, antimicrobial agents are widely available, directly or indirectly, practically without restriction.
Of 130 countries recently evaluated by the OIE, more than 110 do not yet have relevant legislation on the appropriate conditions for the import, manufacture, distribution and use of veterinary products, including antimicrobial agents.
Consequently, these products circulate uncontrolled like ordinary goods and are often falsified."
To date, there is no harmonized system of surveillance on the worldwide use and circulation of antimicrobial agents.
That information is necessary, however, to monitor and control the origin of medicines, obtain reliable data on imports, trace their circulation, and evaluate the quality of the products in circulation.
It is in this context that the OIE was mandated by its member countries to gather that missing information and create a global database for monitoring the use of antimicrobial agents, linked to the OIE's World Animal Health Information System (WAHIS).
That mandate is also supported by FAO and the WHO within the framework of the WHO's global action plan on antimicrobial resistance.
The database will form a solid basis for the three organizations' work to combat antimicrobial resistance (OIE 2015, p. 2) .
In 2016, a UK evaluation of 139 academic, peer-reviewed research articles addressing antibiotic use in agriculture determined that only 5% found no link and 75% found a positive link between antibiotic use in animals and antibiotic resistance (AR) in humans (O'Neill 2016) .
Taken To evaluate proposals to ban the use of growth-promoting or sub-therapeutic levels of antibiotics in food animals, USDA's Economic Research Service (ERS) economists added questions on antibiotic use to the Agricultural and Resource Management Survey (ARMS).
ARMS is a nationally representative survey of farms administered jointly by ERS and USDA's National Agricultural Statistics Service (NASS).
Hog producers were surveyed in 2006 and 2009, and broiler producers were surveyed in 2006 and 2011.
ERS also drew upon their research using data in the National Animal Health Monitoring System (NAHMS) to develop a model to estimate the impacts of withdrawing antibiotics for other than therapeutic use in food animals.
Using Monte Carlo simulations, ERS estimated the impacts of eliminating antibiotic use for growth promotion of poultry and pork, not just the FDA-specified "medically important" antibiotics (Table 15 .1).
Simulation results showed less than 0.5% reduction in output and an approximate 0.75% increase in wholesale prices, netting pork producers greater total revenue of 0.29% and poultry producers 0.42%.
ERS concluded that these small effects were not statistically significant (Sneeringer et al.
2015) .
These ERS results are consistent with research studies post-2000 indicating that productivity gains from using antibiotics for growth promotion were lower than earlier research had found (Teillant and Laxminarayan 2015) .
Another report suggested that phase out of growth promotion use in food animals over a 5-year period would avoid most of the 67% projected global growth in such use and cost agricultural sectors a small portion of the costs of AR in each country.
Further, reduced infection risk and costs of medications would cover most farm-level costs of improving animal husbandry practices to offset loss of antimicrobials for production purposes (Laxminarayan and Chaudhury 2016) .
Presuming that any new antibiotic classes probably will not be made available for veterinary medicine, it is important to preserve the effectiveness of existing antibiotics which are necessary for treatment of infectious diseases to maintain animal health (Teillant and Laxminarayan 2015) .
An alternative to encourage development of new antibiotics would be to delay or not approve drugs which mimic others, but for which the applicant company has not performed antibiotic research (Amábile-Cuevas 2016).
Even better, several production practices may be used to enhance animal health in the absence of using antimicrobials for growth or for prophylactic disease prevention (Sneeringer et al.
2015; WHO 2017; MacDonald and Wang 2011) .
These include: • Improved management practices, such as more space per animal and better control of the housing environment • Tightened biosecurity to prevent diseases and improve productivity by avoiding introduction of infectious agents by wild animals, domestic pets, and nonessential workers or other humans; through increased cleanliness of production facilities; and from timely removal of dead animals • Optimized nutrition to increase growth and mitigate stress-related factors and provide vitamin and mineral supplements to reduce disease susceptibility • Improved gut microflora to improve feed efficiency by providing enzymes, organic acids, prebiotics, probiotics, and immune modulators • Vaccinations to prevent some diseases • Hazard Analysis Critical Control Point plans to improve productivity in the absence of using sub-therapeutic antibiotics in animal production Generally, these practices may raise production costs modestly at the farm level because of the need for more resources required to successfully manage them.
Since ERS found no statistically significant evidence that antibiotics reduce the costs of producing pork or broilers, we conclude that there are small or no costs to producers from withdrawing growth-promoting or prophylactic uses of antibiotics in production of food animals.
In contrast, the public health benefits of withdrawing these antibiotics from agriculture are significant.
As reported above, CDC estimates that the medical costs and productivity losses of AR illnesses attributed to agriculture are $7 billion US dollars annually.
The benefit/cost analysis becomes $7 billion in public health protection benefits vs. the very small costs to animal production from withdrawing antibiotics from non-therapeutic use.
In other words, the protection of the public health will come at little or no cost to agriculture.
Furthermore, this benefit/cost analysis provides a conservative estimate of public health protection benefits.
The CDC public health protection benefits do not include estimates for protection from an increasing number of "superbugs" that would be created if low-level antibiotics would continue to be used.
And CDC does not include the costs of long-term health outcomes caused by foodborne pathogens (see Chap.
8).
Aside from costs to agricultural producers, there are also other societal costs related to AR and connected to antimicrobial use in animal production, both in their production and use/misuse, affecting human and environmental health.
In economic terminology, these costs are considered negative externalities to society from the individual use of antibiotics.
Moreover, since the science of AR is unfolding, there may be additional unknown human health and environmental risks associated with the use of antibiotics in food animal production.
Pharmaceutical Production.
A major issue involved with manufacturing of active ingredients for antibiotics and the effluent from factories producing them is the potential to contaminate nearby water systems.
Pharmaceutical factories often contaminate the environment, since guidelines for pharmaceutical waste discharge focus on chemicals used in manufacturing, rather than active pharmaceutical ingredients.
This is a primary concern in countries outside the United States, but international trade makes it a worldwide problem.
Use and Misuse.
Worldwide, antibiotics are used heavily in animal agriculture.
This practice has created resistance problems transmissible from animals to humans.
For example, China has mrc-1 colicin resistance in pork and Salmonella resistance to cephalosporins at higher levels than in the United States (Zhang et al.
2016) .
Their practice of applying human waste on fields and the closeness of population centers to agriculture contribute to cross-mixing of pathogens in China.
Parasites are common in Chinese soil and can contaminate pork.
And low levels of chlorine in Chinese water supplies allow accumulation of biofilms containing antibiotic-resistant pathogens in water pipes.
In India, manufacturing of pharmaceuticals and waste disposal practices lead to contamination of water and soil.
Further, over-the-counter antibiotics are available and heavily used there.
Farm antibiotic use is of concern in India and China in poultry and pigs (APUA Newsletter 2016).
The threat of superbugs via food is worldwide, due to the distribution of animal food products from China (Zhang et al.
2016; Zhu et al.
2013) .
Rosenblatt-Farrell (2009) drew upon existing literature to identify additional environmental paths to exposure to antibiotic resistance.
Veterinary antibiotics are frequently excreted intact from food animals (Table 15 .2).
For the widely used tetracycline, 60-80% of the antibiotic is excreted in the feces or urine and not metabolized by the food animal.
The transfer of this animal waste to croplands may transfer antibiotics and possibly AR pathogens.
In one study, AR genes in soil increased fourfold after manure from hog and dairy farms was applied to the soil (Moyer 2016) .
Runoff from farms, feedlots, or cropland can lead to antimicrobial resistance problems in soil, surface water runoff, and groundwater.
Animal waste held in lagoons allows birds and insects to become contaminated with antibioticresistant bacteria, and flies around food animal facilities can carry antibioticresistant enteric bacteria which increases potential human exposure.
Migratory birds and seagulls which become infected with antibiotic-resistant bacteria or viruses can widely transmit resistance to other birds as well as marine life.
Others note that antibiotics should never be used to compensate for poor hygiene and husbandry practices or conditions in livestock production (Van Boeckel et al.
2017) .
Veterinary medicine should only use antibiotics to treat diagnostically determined bacterial infections not otherwise treatable and only those antibiotics authorized for the diagnosed pathogenic indication and the specific bacteria involved.
Further, given the potential for acute diseases that require immediate treatment, it is important that routine testing (surveillance) be carried out for farm-specific pathogens for all relevant antibiotic classes (Silley and Stephan 2017) .
WHO also emphasizes the need for surveillance and monitoring antimicrobial use in food-producing animals to evaluate the extent to which their guidelines are implemented.
FDA has increased regulation of antibiotic use in food animals.
As noted in Sect.
15.3 above, FDA attempted to withdraw new animal drug approvals for subtherapeutic uses of human medically important penicillins and tetracyclines in animal feed based on lack of evidence to show they were safe.
After industry opposition and Congressional intervention to require further study, this early policy response was withdrawn.
Subsequently, the US Congress gave something to each group when it enacted the Animal Drug Availability Act (ADAA) in 1996.
This Act both Table 15 .3, FDA in recent years issued three core documents to implement a policy framework for judicious use of medically important antimicrobial drugs in food animals: On January 3, 2017, FDA announced that it had completed implementation of the Guidance for Industry #213.
This means that medically important antimicrobials provided to food-producing animals may no longer be used for growth promotion purposes and may be used to treat, prevent, or control animal illnesses only under direction of a veterinarian.
FDA worked with industry participants to implement this voluntary compliance to slow development of antimicrobial resistance and preserve effectiveness of medically important antibiotics.
More than 70 percent of 292 new drug applications subject to GFI #213 were converted from over-the-counter to prescription status, 84 applications were withdrawn, and all 31 applications indicating production use withdrew that specified use.
FDA also indicated plans to work with industry stakeholders to support antimicrobial stewardship in food animal production and to evaluate the effectiveness of strategies to reduce antimicrobial resistance development under the allowed uses (FDA 2017).
Some industry stakeholders in the supply chain are actively engaged in responding to consumer and general public health concerns about AR in the food supply chain amidst mounting scientific evidence, but responses vary considerably by country, place in the supply chain, and individual company.
Aside from farm groups, stakeholders include feed companies, pharmaceutical companies, integrators or meat processors, restaurant chains and other retail outlets, and consumer and other interest groups.
Pharmaceutical Companies.
In the case of pharmaceutical companies, little evidence exists that they are responding to the AR problem yet.
As described earlier, most antibiotics are produced in India and China, and their production has resulted in significant risk, especially environmental risk.
Regulators need to set minimum standards for the treatment of manufacturing waste before it is released into the environment.
Other industries which purchase these pharmaceuticals need to establish higher standards through their supply chains to help correct this environmental pollution (O'Neill 2016).
Furthermore, the drug companies are not required to compensate victims who become ill or die from either the environmental or food exposure.
The drug companies and their trade associations have resisted more regulation to prevent misuse of antibiotics.
The companies therefore have been getting a "free ride" at the expense of the ill consumers and the general public.
Integrators and Meat Processors.
Some chains and food retailers have recently responded to customer concerns by restricting the use of antibiotics in their food supply chains.
Large meat processors committing to judicious use of antibiotics have already led many producers to eliminate the use of antibiotics for production enhancement purposes.
In a case study of voluntary labeling in the broiler industry, "Raised without Antibiotics" (RWA) label claims by Tyson Foods and by Perdue Farms in 2007, respectively, numbers one and three in total broiler production, resulted in mixed outcomes.
At that time, USDA FSIS had not published a standard for such claims, nor was a clear definition established.
Perdue and Tyson developed their own standards and submitted the label claims to FSIS for approval along with supporting documentation.
After initially approving both firms' label claims, FSIS determined in September 2007 that Tyson's claim was false and misleading and gave them the opportunity to submit a revised label claim.
However, Tyson continued their advertising of the RWA claims.
The diverse label claims in which Tyson and their competitors were using different standards for their claim resulted in consumer confusion, and eventually court challenges were filed jointly by Sanderson Farms, the fourth largest producer, and Perdue against Tyson.
The suit was upheld in court in April 2008.
Tyson was found not to have delivered the RWA attribute promised to the marketplace and to thereby have harmed competitors, while Tyson profited from introducing a false and misleading claim.
In June 2008, FSIS rescinded Tyson's qualified RWA label claim and required its removal within 2 weeks, after the claims and advertising had continued for more than a year.
The authors found no evidence that the events had any impact on Tyson's brand, suggesting that companies may have incentives to introduce misleading label claims since the size of penalties is uncertain (Bowman et al.
2016) .
Perdue Farms Inc. was the only major chicken producer to eliminate all medically important and animal-specific antibiotics from use in its chicken production as of 2016.
By replacing antibiotics with vaccines and improving its production facilities and practices, it has been able to produce chicken at virtually the same cost as when using antibiotics.
Perdue estimates that its conventional chicken sales are increasing by not more than 3% annually, while sales for product raised without antibiotics are growing 15-20% annually (Bunge 2016) .
GNP Company, a leading provider of premium natural chicken products, is adopting antibiotics-free production of chicken products.
Its Gold'n Plump brand will feature a "No Antibiotics-Ever" claim.
This will go well beyond what many companies are currently focusing on-eliminating the use of medically important antibiotics, rather than all antibiotics.
USDA regulations allow this label claim only for chicken never having received antibiotics, even inside the egg.
The company will continue to treat flocks for illness as necessary, but not market them under their premier Gold'n Plump brand.
The company plans extensive media and in-store support to educate consumers about the transition to its chicken products raised totally without antibiotics (GNP Company 2015).
Tyson Foods, a leading producer of chicken, pork, and beef and products thereof, adopted a position to eliminate the use of human-use antibiotics in broiler production by September 2017.
They stopped the human antibiotic use in their hatcheries and reduced usage in producing broilers by 80% since 2011.
They also have worked with farmers and others in the beef, pork, and turkey supply chains to explore ways to reduce human antibiotic use at the farm level.
Tyson is employing alternative husbandry strategies such as use of probiotics and essential oils, improved housing, and selective breeding to offset the potential impact of eliminating the use of the antibiotics.
They are also interacting with the food industry and other involved supply chain participants, as well as academics, to increase research on disease prevention and alternatives to replace antibiotics (Tyson Foods 2017).
Feed Companies.
The feed companies are also getting into the discussion to address public health concerns about antibiotic resistance and the relationship to livestock production uses of antibiotics.
Phibro Animal Health Corporation recently launched a website AnimalAntibiotics.org to "…provide accurate and credible information while still creating open dialogue about animal agriculture in the use of antibiotics."
It will address all issues involving animal antibiotics and changes underway within the industry to promote responsible use of antibiotics in livestock (Johansen 2016) .
This is very consistent with the historical pattern of the animal agriculture industry making its case in the political economy in reaction to the strong push to limit use of antibiotics to help quell rising antibiotic resistance of medically important drugs.
Restaurant Chains.
An interesting example of restaurant chains and poultry producers working together is provided by Panera Bread Co. and Perdue Farms Inc. Panera is one of the restaurant companies for which Perdue supplies chickens raised without antibiotics.
When Panera pioneered antibiotic-free chicken in its restaurant products over 10 years ago, they paid a 50% premium versus chicken produced using antibiotics.
With improved production practices, the cost differential has virtually disappeared (Bunge 2016) and is thus consistent with the ERS estimates cited earlier.
Consumer and Other Interest Groups.
In the process of developing these new FDA regulations, activist groups petitioned the Federal Courts.
For example, in May 2011, the Natural Resources Defense Council (NRDC), Center for Science in the Public Interest (CSPI), Food Animal Concerns Trust (FACT), Public Citizen, and Union of Concerned Scientists (UCS) filed a case against the FDA.
They charged that FDA failed to ban penicillins and tetracyclines used at low doses in animal feed for growth promotion, despite evidence FDA put forth in 1977 that penicillin and most tetracyclines were not shown to be safe and may pose a risk to human health (APUA 2016).
In 2012, the Federal Court ruled in favor of these petitioners.
In a later ruling in 2012, the Federal Court directed FDA to reexamine its decision on five other classes of "medically important drugs" used as growth promoters addressed in two citizen petitions (filed in 1999 and 2005) to ensure the safety and effectiveness of all drugs sold in interstate commerce (Ibid).
Given that most governments have neglected to acknowledge and address the problem of increasing antibiotic resistance, international organizations with a role in health issues have been stymied from doing so.
It will take more concerted action by societies around the globe to successfully address this cross-border issue (Amábile-Cuevas 2016).
US consumers, in general, have much less information about the product than does the seller (Chaps.
2 and 3).
This asymmetric information can offer opportunity for the selling firm behavior that is detrimental to the interests of the consumer, as when a product is labeled as containing or not containing certain desirable or undesirable attributes.
In the case of many products known as credence goods, it is impossible to determine whether the attributes are as stated, even when the product is used or consumed.
This market failure can be addressed either through government regulations or through voluntary steps by the sellers to assure that the stated attributes are factual.
The latter could be accomplished through advertising to build and maintain the firm's brand and reputation, and competition with other sellers could result in consumers having increased variety of product choices.
However, some consumers may not trust private companies' word about product attributes and prefer certification programs which monitor products against some standard established either by the private sector or by government agencies.
Lusk (Lusk 2013) argues that voluntary labels are dynamically efficient in responding to changes in market conditions and encouraging innovation more than mandatory labels implemented through regulations, since the latter are more subject to manipulation by those with vested interests.
Further, USDA's Agricultural Marketing Service (AMS) process-verified and certification programs are very effective in helping to assure the credibility of voluntary labeling, while accommodating innovation from the private sector.
GNP's adoption of antibiotics-free production discussed in 15.8 is an example of dynamic market efficiency through use of voluntary labeling to innovate in response to changing consumer demand USDA's Food Safety Inspection Service (FSIS) currently employs an animal production claims protocol for evaluating and allowing or denying labeling claims.
Labeling applications must provide supporting documentation such as operational protocols detailing production practices and affidavits or testimonials about production practices.
FSIS then evaluates whether protocols support the accuracy of the proposed label.
Also, feed formulations must be provided and reviewed to ensure they do not include substances not permitted by the claim.
Commonly approved claims relevant to the use of antibiotics include "raised without added hormones" (only allowed for use in beef cattle and lamb production) and "raised without antibiotics."
Claims not allowed include that animal products are antibiotic-, hormone-, or residue-"free" (FSIS 2016) .
Given the current trend among meat producers, restaurants, and retail livestock product marketers, it can be anticipated that there will be increasing attention to labeling the lack of antibiotic use for other than therapeutic purposes.
This will likely result in animals that have been raised with antibiotics to promote growth and uniformity of size consistent with processor contract agreements being diverted to marketing outlets where such promises do not exist.
The impact of labeling in this manner will vary according to how much consumers know about the use of antibiotics in livestock production and their ability to currently purchase antibiotic-free livestock products (Lusk et al.
2006) .
O'Neill and his British colleagues emphasize improving transparency as a major step in addressing antimicrobial resistance related to the livestock production.
Recent attention by companies such as food retailers, wholesale producers, and fastfood chains, as well as investors, for reducing antibiotic use in their supply chains, has been in response to consumer pressure.
Providing greater transparency through voluntary approaches is helpful in the short term, but it may be necessary to mandate transparency requirements about how antibiotics are used in the supply chains to have longer-term impacts.
Labeling that refers to antibiotic use could improve consumer knowledge to allow them to make better informed decisions.
They also argued that third-party validation of support from independent institutions to monitor progress may be beneficial (O'Neill 2016).
Improved transparency by food producers about antibiotics used in producing meat could help consumers make better informed purchasing decisions.
But there are large gaps in data needed to allow monitoring of types and quantities of antibiotics used in animal agriculture and their impacts (CFI 2016), as well as on emergence and spread of resistance in animals.
The WHO also identified major gaps in surveillance and data sharing on emergence of antibiotic resistance in bacteria and its impact on animal and human health.
WHO called for integrated surveillance systems harmonized across countries to enable better comparison of data from foodproducing animals, food products, and humans (WHO 2014).
In the United States, FDA requires drug companies to voluntarily submit data on drugs sold for use in food animals.
The publicly available data are not detailed, and 97% of the sales of medically important antimicrobials are over-the-counter (OTC).
Tetracyclines are primarily added to feed and accounted for 71% of domestic sales of animal drugs that are "medically important" to human medicine in 2015 (FDA 2016b) .
From 2009 to 2015, domestic sales and distribution of tetracycline products approved for use in food-producing animals increased by 31%.
While Levy et al.
(1976) discovered how rapidly tetracycline created antibiotic resistance in the gut of chickens, 40 years later, the public does not have access to information on what antibiotics are used in which food animals at what stage of life.
This will change somewhat in FDA implementation of GFI # 213 (FDA 2017) that will identify whether the sales are intended for use in cattle, sheep, hog, or poultry.
FDA (2016b) issued a final rule amending an existing requirement that sponsors of drug products containing antimicrobial active ingredients report annually the amount of each such ingredient in the drug products sold or distributed for use in food-producing animals.
Effective July 11, 2016, drug sponsors were required to submit species-specific estimates of product sales as a percent of their total sales.
Additional reporting requirements are expected to facilitate better understanding of antimicrobial drug sales for specific food-producing animal species and the relationship between such sales and antimicrobial resistance.
As reported above, drug sponsors have all adopted voluntary revision of FDA-approved labels for use of new medically important antimicrobial animal drugs administered through feed or water.
Under this rule, sponsors all voluntarily removed the growth promotion and feed efficiency uses and brought the remaining therapeutic uses under veterinarian oversight by the end of December 2016.
The rule makes it illegal to use medically important antibiotics for production purposes.
Despite the scientific and economic evidence, many comments to the proposed final regulation reflected ongoing resistance to the elimination of food animal production use of medically important antibiotics.
Data available on antibiotics used in the US livestock industry is derived primarily from two nationally representative surveys of farms conducted by the USDA's Economic Research Service (ERS) and National Agricultural Statistics Service (NASS).
The Agricultural and Resource Management Survey (ARMS) is designed to collect information on farm finances, production practices, and resource use focuses on three commodities annually, livestock included.
Different types of livestock are resurveyed every 5-6 years and represent commercial producers in states producing 90% of production for that livestock type.
Some questions have been included in these surveys on antibiotic use for hogs and broilers.
The hog surveys ask about use of antibiotics in feed or water for growth promotion, disease preven-tion, and/or disease treatment in breeding, nursery, and finishing hogs.
Given the widespread use of hog production contracts under which farm operators may receive feed from integrators, the surveyed operators may not know if antibiotics are included in it.
For broilers, there is only a single question about whether they were raised without antibiotics in feed or water other than for therapeutic treatment of illness.
Production contracts dominate the broiler industry, so surveyed farm operators are in a similar situation as hog producers in not necessarily knowing whether antibiotics are included in the feed provided.
A further complication is that ARMS does not separate traditional antibiotics and ionophores, which are not used in human medicine (Sneeringer et al.
2015) .
The National Animal Health Monitoring System (NAHMS) consists of national studies to provide essential information on livestock and poultry health and management.
Major food livestock species are surveyed about every 5 years to provide current and trend information important to industry participants, researchers, and policy makers.
Each study includes states that represent at least 70% of the targeted animal population and at least 70% of the farm operations involved and provides statistically sound information for decision-making.
A NAHMS study is a collaborative, voluntary, confidential, scientifically sound product.
Descriptive reports are prepared along with information sheets which briefly address very specific topics, such as biosecurity practices (APHIS 2016).
The NAHMS focuses on animal health and management, providing information on disease occurrence and disease prevention practices, as well as more detailed information on antibiotics used in production, including by specific purpose.
However, the information collected on antibiotics varies greatly across commodities, as well as over time with the same commodities.
Further, ARMS focuses on hog production operations with 25 or more head versus NAHMS focus on 100 or more head.
This complicates comparison of statistics across surveys, assuming smaller operations may have different characteristics than larger ones (Sneeringer et al.
2015) .
To track antimicrobial resistance changes over time, the National Antimicrobial Resistance Monitoring System-Enteric Bacteria (NARMS) was established by CDC in 1996.
The program is a collaboration between state and local public health departments and three federal agencies to monitor changes in antimicrobial susceptibility for certain enteric bacteria from ill people (CDC), retail meats (FDA), and food animals (USDA) in the United States.
It provides information about emergent bacterial resistance, the ways resistance is spread, and how resistant infections differ from susceptible ones (NARMS 2016).
The World Organization for Animal Health (OIE) plans to address antimicrobial resistance as a major risk to the international community, in the face of concern about agriculture's role in increased antimicrobial resistance.
The goal is to preserve effectiveness of antimicrobials used in animal medicine, protect animal welfare, and help maintain important antimicrobials for use in human medicine.
OIE has already developed international standards, most recently revised in 2015.
The new strategy introduced at the 84th OIE General Session in May 2016 outlines plans to help nations improve legal frameworks to preserve antibiotics, communicate about the AR problem, train animal health workers, and monitor antibiotic use in animals.
They are currently working to create a database of information on the use of antimicrobial agents in animals and develop performance indicators to assist countries by increasing information flow and transparency in their use of antimicrobials.
Further, the OIE expert network is working to reinforce scientific knowledge about new technologies and replacement solutions for current antimicrobials (Mitchell 2016) .
The EU has banned the use of antimicrobials in food animal production, other than by veterinarian prescription for specific therapeutic use.
Some other countries have adopted similar bans, and, as discussed above, the United States fully implemented voluntary guidelines in 2016 requiring current drug sponsors to withdraw antibiotics for growth promotion.
However, the Animal Health Institute's Carnevale has said that the new FDA guidance on antibiotics may not decrease the total quantities of antibiotics used in animal food production (Moyer 2016) .
Generally, variations among countries in implementing regulations have resulted in the spread of resistance.
There is ample evidence to support the need for global coordination to prevent continued spread of antimicrobial resistance, and elements of a framework to make such global coordination effective have been posited (So et al.
2015) .
The UK Review on Antimicrobial Resistance Final Report proposes three broad steps to deal with reducing unnecessary use of antibiotics in animals.
First, establish 10-year targets for reduction in use, with milestones to support progress consistent with countries' economic development.
This could encourage farmers to reduce non-therapeutic use to be able to allocate the resulting reduced amounts of antibiotics to treating sick animals.
Second, implement restrictions or bans on certain types of highly critical last-line antibiotics for humans from being used in agriculture.
This would require a harmonized approach to identify the most important human health antimicrobials across countries and good systems of veterinarian oversight to assure compliance.
Third, improve transparency from food producers on antibiotic use in meat production to allow consumers to make better informed buying decisions.
Voluntary industry efforts may be one of the most practical approaches to reduce antibiotic use in the near term, but third-party validation to monitor progress would be beneficial (O'Neill 2016) .
Generally, voluntary industry approaches require monitoring by an outside party to assure both industry participants and consumers that standards are being met as required.
The UK Medical Research Council (MRC) recently made three large grants focused on antimicrobial resistance through an initiative established in 2014 to address the growing AR issue.
The projects will use new technology to exploit natural compounds, develop a better and faster diagnostics tool, and study how the body's immune system can be harnessed to better fight infections.
The goal is not only to develop antibiotics but also explore alternatives to antibiotic use, working with other UK research councils to bring to bear a wide range of disciplines to tackle AR (MRC 2016).
The need to focus increased attention to developing new antibiotics is supported by CDC data which shows that many of the most widely used drugs have developed resistance.
The number of years to develop resistance varies greatly but never extends more than a couple of decades, and more recent antibiotic introductions have been resistant for only a year or two.
For example, the widely used tetracycline was introduced in 1950 and developed resistance to Shigella by 1959.
This is near the midrange of years to resistance reported (CDC 2013a).
Given this scientific fact, the slow pace of adopting policies to proscribe use of human-use antibiotics in animals and to encourage greater investment in developing newer antibiotics or alternatives is unacceptable.
Increasing detection of bacteria resistant to last-resort drugs has driven stakeholders to countenance accelerating government efforts to increase surveillance of drug use and to develop new antibiotics (FDA Week 2016).
Promising approaches which provide more rapid assessment utilizing newer technologies such as genomics are now being utilized by scientists.
Microbiologists are embracing high-throughput genomics to quickly examine individual organisms or entire microbial communities.
A project underway at the University of California, Davis, the 100 K Foodborne Pathogen Genome Sequencing Project, will sequence 100,000 foodborne isolates for the most important worldwide foodborne illness outbreak organisms.
It involves a consortium of academic, government, and industry to create a massive database of genome signatures for the most significant foodborne disease-causing microbes.
The goal is to allow public health agencies and the food industry supply chain to trace any foodborne illness outbreaks to their source.
By comparing the pathogen genome to the database which includes millions of pieces of information on previously detected strains, including their exact location, the contamination source will be positively identified.
Bioinformatics and the analytics involved can be used to turn the vast amount of genomic information into actionable knowledge.
These event sequencing approaches will enable new diagnostic and public health approaches to manage foodborne disease to facilitate improved public health.
The database will increase ability to detect and mitigate pathogenic organisms in food, the environment, and livestock.
That capacity is now constrained by continual genetic evolution of pathogens which hinders the ability to defend the food supply.
This project will facilitate speedy testing of raw ingredients and finished products from outbreak investigations with precision and accuracy unparalleled using existing methods of analysis.
Genomics enabled diagnostics with molecular tools will allow surveillance, risk assessment, and diagnosis of foodborne pathogens directly throughout the global food chain.
The result will be a genetic catalog for some of the most important outbreak organisms impacting human health.
The database will provide insights into molecular methods of infection and drug resistance for use in creating new vaccines and therapies.
And importantly, it will assist in systematic definition of biomarker gene sets associated with antibiotic resistance (Weimer 2016).
A recent innovative metagenomics study also provides new insights on possible impacts of antibiotic use in food animal production and AR in humans.
The research investigated antimicrobial resistance potential-the resistome-by tracking specific pens of intensively managed cattle from feedlot through slaughter to market-ready beef products.
Study results found no antibiotic-resistant determinants (ARDs) in the beef products beyond the slaughter facility.
This suggests that intervention during slaughter minimizes potential for antibiotic-resistant determinants passing through the food chain.
The results also highlight potential risks through indirect environmental exposures to the feedlot resistome through wastewater runoff, manure application on cropland, and wind-borne particulate matter.
The insights provided can be used to better inform future agricultural and public health policy.
However, this first of its kind study suggests the scientific community must develop a better understanding of the risk of different resistomes and resistance genes.
It also identifies a pressing need to standardize ARD nomenclature so that databases and analyses are comparable across studies (Noyes et al.
2016) .
The World Health Organization has recently developed guidelines to mitigate human health consequences from use of medically important antimicrobials in food-producing animals (WHO 2017).
The guidelines are evidence-based recommendations and include best practices for use of medically important antimicrobials in food-producing animals, especially antimicrobials deemed critically important to human medicine.
They also can help preserve effectiveness of antimicrobials for veterinary medicine.
The recommendations include: • An overall reduction in use of all classes of medically important antimicrobials in food-producing animals.
• Complete restriction for use in growth promotion.
• Complete restriction of use to prevent infectious diseases that have not yet been clinically diagnosed.
• Antimicrobials designated as critically important for human medicine should not be used to control spread of clinically diagnosed infectious disease identified within a group of food-producing animals, nor for treatment of food-producing animals with a clinically diagnosed infectious disease.
• For best practices, any new class of antimicrobials for use in humans will be considered critically important for human medicine unless otherwise categorized by WHO.
Further, medically important antimicrobials not currently used in food production should not be so used in the future.
These guidelines apply universally, and improved animal health management can be used to reduce the need for antimicrobials including improvements in disease prevention strategies, housing, and husbandry practices as noted in Sect 15.5 above.
Economic incentives in regulations were addressed in a recent article.
In some European countries, capping total antimicrobial use per animal through regulations has been successful in reducing use by more than half while maintaining competitive livestock sectors.
The second option was to impose user fees on veterinary antimicrobials, applied at the point of manufacture or wholesale purchases for imported products, which could also reduce use significantly.
As a policy option, some combination of these two strategies would significantly reduce antimicrobial use in food animal production (Van Boeckel et al.
2017) .
Finally, as discussed in Chap.
12, Sweden does not allow use of antibiotics in broiler production.
If there is the political will, strong regulations can provide strong economic incentives to control antibiotic use.
To promote the understanding and implementation of the FDA's new Veterinary Feed Directive, the Farm Foundation and the Pew Charitable Trusts sponsored a series of meetings with livestock and farming communities throughout the United States.
Twelve educational workshops provided livestock producers, feed suppliers, veterinarians, and support service organizations information and insights on the new policies.
The workshops also provided opportunity for participants to interact with FDA and USDA's Animal and Plant Health Inspection Service (APHIS) personnel about implementation challenges.
Among livestock producers attending the workshops, small-and medium-sized operators, as well as many veterinarians, were unaware of the pending requirements.
Lack of understanding about responsibilities under the revised VFD rule means that producers and veterinarians need education.
Some land grant university extension services are now offering balanced education programs to inform these audiences about their obligations going forward, rather than having interested parties in the food animal industry be the primary source of information to producers and veterinarians about the requirements.
While seeing positives of improved public perception and livestock management as result of the new rules, workshop participants were concerned about increased costs in animal health due to restrictions on access to antibiotics and lack of veterinary services.
Perhaps the biggest challenge is that many small producers do not have established relationships with veterinarians needed to establish a veterinarianclient-patient relationship (VCPR).
This may be particularly challenging in remote rural and urban fringe areas where fewer veterinarians are available to treat foodproducing animals.
In sum, workshop participants saw a need for education and outreach; continuing dialogue between industry representatives, consumers, and state or federal regulators; and the need to provide better access to veterinary services for food animals.
There is widespread agreement that the scientific evidence indicates a global human health and environmental crisis due to antibiotic resistance, in part resulting from production practices in animal agriculture.
Government action in regulating the animal agriculture industry, to date, has done little to slow the advance of AR.
Most countries still need to pass legislation to establish appropriate conditions for the import, manufacture, distribution, and use of veterinary products, including antimicrobial agents.
Continued easy access to antimicrobial drugs for use on the farm is not acceptable.
Important stakeholders in the animal production industry include pharmaceutical companies, feed companies, livestock production integrators, and some farm groups, each with their own set of incentives and supporters.
They must be engaged in the effort to reduce agriculture's role in contributing to development of AR, which CDC estimates at 20% (Fig.
15.1) .
Even so, other major industry groups must be engaged to significantly reduce their 80% contribution to resistance development.
In the United States, some progress was made with the passage of the Animal Drug Availability Act in 1996 and its very gradual implementation through various regulations over the past two decades.
However, there are serious gaps in these regulations.
Given the gridlock that has prevailed in the US Congress and the power of the pharmaceutical lobby at the national level, state actions are leading the way to responsive regulation in the public interest.
For example, California is the first US state to prohibit all human antibiotic use in food animal production.
In contrast to the halting actions of governments and industry, consumer and interest group actions are being at least partially successful in getting fast-food and retail establishments to not market animals fed human-use antibiotics for growthpromoting purposes.
This suggests that a productive approach may be finding ways to provide information to and educate consumers about the risks of antibiotic resistance to enable them to make better informed decisions.
There is an important role for educators to extend scientific information in a nontechnical way to the lay public.
The drive to use antibiotics more responsibly and in the public interest may be facilitated by recent economic results that show that reducing antibiotic use in animal production need not come at a significant economic cost to producers or consumers.
Since the benefits of using antibiotics for livestock growth promotion appear to have resulted in increasingly smaller productivity gains, independent producers where input mix is a farmer-driven choice based on farm-level economics may be better off to substitute good management practices rather than using antibiotics for prophylactic disease prevention.
However, much of meat animal production on US farms is produced under contract, where the integrator provides inputs, often including antibiotics, that the grower is required by contract to use.
Recent actions by integrators and meat processors to reduce antibiotic use and substitute alternative strategies to protect animals from diseases and maintain productivity are an important development, especially since production-purpose use of antibiotics is now prohibited.
Presuming that any new human-use antibiotic classes will probably not be made available for veterinary medicine, it is important to preserve the effectiveness of existing antibiotics.
Some policy makers and industry now recognize the urgency to identify new antibiotics.
This will require increased antibiotic research funding and judicious use of existing antibiotics.
The ban of human-use antibiotics in animals for production purposes is expected to help slow the growth of antimicrobial resistance, giving more time to discover new antibiotics for animal uses and for human health uses.
To the extent they can be developed and used separately, the potential for animal antibiotic use leading to antimicrobial resistance for important human antibiotics will be mitigated.
The ban on antibiotics used for humans also being used for animal production purposes will necessitate adopting improved cultural practices to reduce the poten-tial for disease and to increase feed efficiency.
This calls for research on best management practices to accommodate today's supply chain requirements for food safety, production efficiency, and attribute verification.
Moreover, there is a need to educate producers-for example, through the USDA-State Cooperative Extension Service-about safe production practices for managing AR.
This will allow producers to maintain efficiency in their operations and assure that they comply with current regulations to address the growing concern about antimicrobial resistance in the food supply chain.
Improved data collection and analysis to allow tracking of potential antimicrobial resistance development are essential to facilitate the food animal industry implementation of cultural practices to reduce the potential for contributing to antimicrobial resistance.
It would also allow policy makers to better understand the need for any necessary interventions.
These investments in the public good can be very cost-effective, though not without additional public investment or internal agency budget reallocation.
Increasing international trade may spread antibiotic resistance through imported food products as more trade agreements are approved.
This scenario could be exacerbated to the extent FSIS approves additional international facilities, local regulations, and inspections as "equivalent to the United States."
In many developed and developing countries, antimicrobial agents are readily available.
Policies need to be implemented establishing appropriate conditions for use of veterinary products, including antimicrobial agents.
Future trade agreements will need to include provisions which address reduced use of medically important antibiotics in producing food animals.
To date, there is no harmonized system of surveillance on the worldwide use and circulation of antimicrobial agents.
That information is necessary to monitor and control the origin of medicines, obtain reliable data on imports, trace their circulation, and evaluate the quality of the products in circulation.
The OIE initiative to create a global database for monitoring the use of antimicrobial agents is an important step that can provide valuable information for private sector and public policy leaders worldwide.
The serious implications of growing antibiotic resistance require a concerted effort across all stakeholders and society generally.
Increased attention to this issue is emerging in the medical community where overuse of existing drugs and inadequate sanitary precautions account for 80% of the resistance.
Lack of development of new antibiotics exacerbates the problem, and industry focus and perhaps government policy are needed to improve this situation.
Animal agriculture stakeholders need to improve production practices to reverse the other 20% of resistance attributable to foodborne sources.
Government policy and agencies have been slow to acknowledge the seriousness of antibiotic resistance and appropriately address it.
Public and private sector collaboration internationally is necessary to successfully deal with this critical societal issue.
Changes in the measurement of antibiotics and in evaluating their impacts in agroecosystems: a critical review Society must seize control of the antibiotics crisis Animal and Plant Health Inspection Service, United States Department of Agriculture APUA.
Major developments in U.S. policy on antibiotic use in food animals.
Alliance for the prudent use of antibiotics Farm antibiotic use remains worrisome in India.
Alliance for the prudent use of antibiotics Raised without antibiotics: lessons from voluntary labeling of antibiotic use practices in the broiler industry Perdue chickens now free of antibiotics Antibiotic resistance and the use of antibiotics in animal agriculture: hearing before the subcommittee on health of the house committee on Energy & Commerce Across the divide: the importance of antibiotics for animal health Antibiotic resistance threats in the United States estimates of foodborne illness in the United States.
Centers for disease control and prevention Tracking animal antibiotic use in food animals.
The center for foodborne illness research and prevention America's Antibiotic Crisis Council for Agricultural Science and Technology (CAST).
1981.
Antibiotics in animal feeds The judicious use of medically important antimicrobial drugs in food-producing animals.
Guidance for Industry #209 Summary report on antimicrobials sold or distributed for use in foodproducing animals Anti-microbial animal drug sales and distribution reporting.
FDA, Department of Health and Human Services FDA announces implementation of GFI #213, outlines continuing efforts to address antimicrobial resistance Stakeholders: colistin-resistance shows need for congressional action Animal production claims: outline of current process.
FSIS, U.S. Department of Agriculture GNP Company.
Gold'n Plump To Add 'No Antibiotics-Ever' and Humane Certified Attributes.
2015.
Perishable News.com Report of a study: human health risks with the subtherapeutic use of penicillin or tetracyclines in animal feed.
Committee on human health Accurate, credible info on animal antibiotics.
Phibro Animal Health Report presented to parliament by the secretary of state for social services, the secretary of state for Scotland, the Minister of Agriculture, Fisheries and Food and the Secretary of State for Wales by Command of Her Majesty Antibiotic resistance in India: drivers and opportunities for action Changes in intestinal Flora of farm personnel after introduction of a tetracycline-supplemented feed on a farm Consumer information and labeling Consumer demand for a ban on antibiotic drug use in pork production Foregoing sub-therapeutic antibiotics: the impact on broiler grow-out operations Pressure rises to stop antibiotics agriculture Antibiotic discussions intensify in WDC.
Pork Network World animal health organization sets out action on antibiotic resistance.
Poultry News The looming threat of factory-farm superbugs MRC announces cross-council awards worth nearly £10m to tackle antibiotic resistance Department of Health and Human Services, CDC United States summary and state data The effects on human health of subtherapeutic use of antimicrobial drugs in animal feeds.
Committee to study the human health effects of subtherapeutic antibiotic use in animal feeds Resistome diversity in cattle and the environment decreases during beef production Tackling drug-resistant infections globally: final report and recommendations.
The review on antimicrobial resistance World Organization for Animal Health Merchants of doubt: how a handful of scientists obscured the truth on issues from tobacco smoke to global warming The landscape of antibiotic resistance Prepared for United States Department of Health and Human Services, Public Health Service, Food and Drug Administration, Bureau of Veterinary Medicine Prudent use and regulatory guidelines for veterinary antibiotics-politics or science?
Economics of antibiotic use in U.S. livestock production An integrated systems approach is needed to ensure the sustainability of antibiotic effectiveness for both humans and animals Restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in food-producing animals and human beings: a systematic review and meta-analysis Economics of antibiotic use in U.S. swine and poultry production Position statements: antibiotic use Scientific integrity in policymaking: an investigation into the Bush Administration's misuse of science Insights: reducing antimicrobial use in food animals Veterinary Feed Directive A rule by the food and drug administration on 06/03/2015.
2015 Illustrative examples of probable transfer of resistance determinants from food animals to humans: Streptothricins, glycopeptides, and colistin Weimer BC 100 K genome project 2016.
Veterinary Medicine, UC Davis Antimicrobial resistance: Global Report on Surveillance.
2014 summary.
World Health Organization WHO guidelines on use of medically important antimicrobials in food-producing animals.
Geneva: World Health Organization CTX-M-27 Producing Salmonella enterica serotypes Typhimurium and Indiana are prevalent among food-producing animals in China Diverse and abundant antibiotic resistance genes in Chinese swine farms Political economy of biofuel Acknowledgments We deeply appreciated the conversations with and review comments from Mary Ahearn in developing and writing this chapter.
Any remaining errors, mistakes, or omissions are of course ours.
W. J. Armbruster and T. Roberts
key: cord-273099-zkk5d6gd authors: Muzumdar, Jagannath M.; Cline, Richard R. title: Vaccine supply, demand, and policy: A primer date: 2016-01-01 journal: J Am Pharm Assoc (2003) DOI: 10.1331/japha.2009.09007 sha: doc_id: 273099 cord_uid: zkk5d6gd OBJECTIVE: To provide an overview of supply and demand issues in the vaccine industry and the policy options that have been implemented to resolve these issues.
DATA SOURCES: Medline, Policy File, and International Pharmaceutical Abstracts were searched to locate academic journal articles.
Other sources reviewed included texts on the topics of vaccine history and policy, government agency reports, and reports from independent think tanks.
Keywords included vaccines, immunizations, supply, demand, and policy.
STUDY SELECTION: Search criteria were limited to English language and human studies.
Articles pertaining to vaccine demand, supply, and public policy were selected and reviewed for inclusion.
DATA EXTRACTION: By the authors.
DATA SYNTHESIS: Vaccines are biologic medications, therefore making their development and production more difficult and costly compared with “small-molecule” drugs.
Research and development costs for vaccines can exceed $800 million, and development may require 10 years or more.
Strict manufacturing regulations and facility upgrades add to these costs.
Policy options to increase and stabilize the supply of vaccines include those aimed at increasing supply, such as government subsidies for basic vaccine research, liability protection for manufacturers, and fast-track approval for new vaccines.
Options to increase vaccine demand include advance purchase commitments, government stockpiles, and government financing for select populations.
CONCLUSION: High development costs and multiple barriers to entry have led to a decline in the number of vaccine manufacturers.
Although a number of vaccine policies have met with mixed success in increasing the supply of and demand for vaccines, a variety of concerns remain, including developing vaccines for complex pathogens and increasing immunization rates with available vaccines.
New policy innovations such as advance market commitments and Medicare Part D vaccine coverage have been implemented and may aid in resolving some of the problems in the vaccine industry.
C urrently, vaccine-preventable disease levels are at near record lows.
1 This was not the case at the beginning of the 20th century, when infectious diseases were the greatest threat to public health and the leading cause of death in the United States and elsewhere.
2 During that period, few effective treatments or measures were available for preventing large numbers of deaths from these diseases, despite the fact that in 1796, Edward Jenner performed the Western world's first vaccination.
3 During the 20th century, the "Golden Age" of vaccines 3 witnessed the development and acceptance of vaccines for diphtheria (discovered in 1921, but not used widely until the 1930s), tetanus toxoids (1924), influenza vaccine (first used in 1945), polio vaccine with inactive virus (1955) and live attenuated virus (1961), measles (1963) , and combination measles-mumps-rubella (MMR) vaccine (1971) .
Estimates indicate that these vaccines have prevented more than 3 million deaths per year worldwide from infectious diseases.
4 Vaccines have been well documented as one of the greatest achievements of medicine and are among the most cost-effective interventions in public health.
For example, Zhou et al.
5 evaluated the economic impact of the routine U.S. childhood immunization schedule.
They reported that for every dollar invested in childhood vaccination against nine vaccine-preventable diseases, $5.80 was saved in direct medical care costs.
Further, when indirect benefits were taken into account, such as parental absenteeism costs incurred in caring for ill chilSynopsis: Supply and demand issues in the vaccine industry and the policy options that have been implemented to resolve these issues are reviewed in the current work.
Although vaccines have been responsible for some of the greatest successes in public health, the vaccine market is fragile and requires both supply-and demand-side interventions.
Vaccine availability has been limited by the number of suppliers, high research and development and production costs, and safety problems leading to increased regulatory requirements.
Demand for vaccines has been constrained by rapidly increasing vaccine costs, financing issues that have hindered efforts to achieve targets set for population immunization rates, and parental attitudes regarding the safety and efficacy of vaccine products.
Analysis: To date, a patchwork of policies to make the vaccine market more attractive for private firms and to increase patient access to these products has been implemented by the U.S. government and private philanthropies.
According to the authors, an integrated policy approach that preserves incentives for market entry and innovation in the vaccine industry while addressing parental vaccine concerns and increasing immunization funding and reimbursement for both providers and patients is needed.
dren, the amount saved rose to $17.70.
Salo et al.
6 assessed the cost effectiveness of influenza vaccination of children aged 6 months to 13 years and found that influenza vaccination for healthy children (all age groups) was more effective and less costly than not vaccinating children against influenza.
These findings are due in part to the fact that many vaccines result in long-term or lifelong protection of the recipient and people they contact.
Through the process of "herd immunity" and "herd effect," a vaccines protect not only those who receive them but also those who cannot or do not receive the vaccine because of medical conditions, parental indifference, or religious or philosophical objections to vaccinations.
7, 8 ( a The probability of unvaccinated individuals contracting a disease when part of a larger group with a certain seroprevalence [herd immunity] is called the herd effect.)
However, of note, if a susceptible person strays outside the herd or if the herd changes, that person is still susceptible.
Compared with pharmaceutical products, the number of lives saved per invested dollar is substantial.
Economists have reported that this increased life expectancy has made a considerable contribution to economic growth.
9, 10 In fact, it has been argued that more than one-half of the growth in real income in the first half of the 20th century is attributable to the declining mortality associated with the discovery of vaccines.
11, 12 Mass immunization programs have resulted in 100% eradication of smallpox from the world, elimination of diphtheria and polio, and 99% eradication of measles, mumps, and rubella in the United States.
1 Haemophilus influenzae type b (Hib) vaccines have been successful in reducing childhood mortality.
13 In addition, vaccination of healthy adults has resulted in decreased work absenteeism and decreased use of health care resources, including less use of antibiotics.
14, 15 Nevertheless, this success of vaccines is threatened because of several factors.
Problems related to vaccine research and development (R&D), manufacturing complexities, supply and distribution, safety issues, and financing have become areas of major concern.
Symptoms of this crisis include a decline in the number of vaccine producers from 26 in 1967 to 5 in 2004 16 and a decline in the number of licensed vaccine products from 380 in 1967 to 51 in 2005.
(Some of these are combination products.)
16, 17 Eight of these vaccine products are currently produced by five major companies: Sanofi Pasteur, Chiron (a business unit of Novartis Vaccines and Diagnostics), GlaxoSmithKline, Merck Vaccines & Infectious Diseases, and Wyeth Vaccines.
18 Should any one of these suppliers cease production, it could take years for a replacement vaccine to be licensed and become available publicly.
Beginning in late 2000, the United States faced shortages of 8 of the 11 recommended childhood vaccines.
19 Affected vaccines included diphtheria-tetanus-acellular pertussis (DTaP), MMR, varicella, and pneumococcal conjugate vaccines.
20 Suspension of production of PedvaxHIB and COMVAX by Merck and a subsequent voluntary recall of certain lots of both vaccines on December 13, 2007, led to a considerable disruption in the supply of Hib-containing vaccines.
21 Thus, the dearth of suppliers appears to have affected the stability of vaccine supply.
22 Compounding these problems, the epidemiology of several diseases is changing.
West Nile virus killed at least 98 people in the United States in 2007, 23 and cases of Dengue fever, formerly known only in tropical areas, have been reported in Texas.
23 The Centers for Disease Control and Prevention (CDC) received reports of 1,528 cases of malaria in 2005 among individuals in the United States or its territories.
This total represents an increase of 15.4% from the 1,324 cases reported for 2004.
24 With rapid intercontinental transportation and a larger global population, diseases can travel and spread to many countries in little time.
CDC issued a health advisory on April 2, 2008, regarding a measles outbreak in Arizona that was linked to importation of the measles virus from Switzerland.
25 The first case, with rash onset on February 12, 2008, occurred in an adult visitor from Switzerland who was hospitalized with measles and pneumonia.
In another dramatic example, severe acute respiratory syndrome spread from Asia to North America quickly, eventually infecting 8,098 people worldwide, of whom 774 died, 26 when a 78-year-old woman carried the infection from Hong Kong to Toronto, where it eventually caused 44 deaths.
8, 26 Objectives The current report seeks to provide an overview of the vaccine industry and public policy affecting it.
Specifically, we sought to (1) highlight issues faced by vaccine manufacturers that make the vaccine industry a unique segment of the prescription drug industry, (2) provide an overview of the vaccine market with regards to vaccine supply and demand, and (3) provide an overview and critical evaluation of policy options proposed and implemented by various parties to address vaccine supply and demand problems.
This research consists of a narrative literature review and critical analysis of the information retrieved.
Search criteria were limited to English language and human studies.
Keywords used for the search included vaccines, immunizations, supply, manufacturing, demand, policies, and push-pull solutions.
Indices such as Medline, Policy File, and International Pharmaceutical Abstracts were searched and the results augmented with reports produced by government agencies (e.g., Government Accountability Office) and independent think tanks.
A variety of sources were reviewed, including reports from academic journals and current texts on vaccine history and policy.
Articles pertaining to vaccine demand, supply, and public policy were selected and reviewed for inclusion in the current work.
Vaccines are biologics that introduce "weakened or killed disease-causing bacteria, viruses, and/or their components" 27 or toxoids into a person or animal to stimulate an immune reaction that the body will remember if exposed to the same pathogen in the future.
28 This unique property sets them apart from other segments of the pharmaceutical industry, such as "small-molecule" or products derived from traditional organic chemistry methods and from other biologically derived products used in a therapeutic capacity.
As such, when a private firm considers entering the vaccine market, they face several important barriers to entry, some of which are shared with these product segments and others that are unique to vaccines.
These are discussed in detail below.
New vaccines begin with the recognition of an infectious disease burden worth preventing.
29 Basic research regarding pathogens and immune responses, often funded by the National Institutes of Health (NIH), vaccine manufacturers, and nonprofit organizations such as the Bill & Melinda Gates Foundation, is performed mainly at universities.
1 Certain vaccines for yellow fever, typhoid, and anthrax are funded and developed in the Department of Defense.
Before entering clinical trials, prototype vaccines undergo toxicology testing that is conducted in a Good Laboratory Practice-compliant laboratory.
Private firms then build on this knowledge to develop clinically feasible vaccine products and shepherd them through clinical testing.
The vaccine's manufacturer then must submit a Biological License Application (BLA) to the Food and Drug Administration (FDA) for evaluation and approval before marketing.
The approval process tests extensively for safety and efficacy, along with purity and absence of contaminants.
If data raise serious concerns about product safety or efficacy during any phase, FDA may request additional information or studies or may halt ongoing clinical studies.
30 The entire research, development, and approval process may require 10 years or more.
31 Estimates of the cost of this process range from $110 million to $802 million ($US 2000).
1, 32 Table 1 summarizes information on the different phases in vaccine research.
Manufacturing complexities.
Although vaccine manufacturing regulation originally was controlled by the U.S. Public Health Service under the Biologics Control Act of 1902, this authority now rests with FDA.
The majority of vaccines approved by FDA are manufactured from live (attenuated) or killed (inactivated) organisms.
Some are based on partially purified components of an organism such as diphtheria and tetanus, and a handful are recombinantly produced, such as the hepatitis B vaccine.
4 Vaccines are manufactured by at least three methods: egg-based (e.g., influenza vaccine), cell-derived (e.g., polio vaccine), or recombinant (e.g., hepatitis B vaccine).
For bacterial vaccines, the bacterial pathogens are grown in bioreactors using media developed for optimizing the yield of the antigen (e.g., Hib) 31 ( Figure 1 ).
As such, small deviations in the manufacturing process can have a major impact on the potency and/or purity of these products.
Thus, FDA production facility requirements are rigorous, and these stringent regulatory hurdles add to the production costs of vaccines.
Because new vaccines generally are more complex than older products, vaccine suppliers face increasingly stringent regulation of manufacturing facilities even after a vaccine is approved.
Suppliers undergo frequent inspections of their production facilities by each country in which the vaccine product is licensed and by FDA.
Individual product batches require separate approval for release, and slight modifications Reviews VACCINE POLICY to production processes or the packaging of products may trigger expensive and time-consuming product reviews.
33 FDA also requires frequent upgrades of vaccine production facilities to reflect state-of-the-art manufacturing processes.
Recently, FDA quality control inspections led to Merck's recall of 1.2 million doses of childhood vaccines to protect against meningitis, pneumonia, and hepatitis B because of contaminated manufacturing equipment.
21, 34 The recall involved 11 lots of the Hib vaccine Pedvaxhib and two lots of a combination vaccine for both Hib and hepatitis B sold under the brand name Comvax.
34 Most vaccine manufacturers are profit-seeking firms, not public health agencies.
As such, they are not obligated to develop vaccines.
18 These manufacturers face the decision of whether to invest large amounts of capital in vaccine R&D for a small portion of the global pharmaceutical market representing approximately 1.5% of all pharmaceutical revenues.
35 Most vaccines are not "blockbuster" pharmaceuticals that yield large profits or returns on investment.
Although pharmaceuticals in the aggregate are a large market representing approximately $340 billion annually ($US 2000) worldwide, sales of vaccines are estimated at just $4.8 billion to $6 billion per year, with about one-quarter of total sales in the United States.
36 This $6-billion market is controlled primarily by the five major manufacturers.
Moreover, most vaccines are used at most several times in a lifetime, whereas therapeutic biologics and small-molecule drugs often are used every day.
Thus, markets for small-molecule and biotechnology drugs treating chronic diseases are considerably more attractive to investors than vaccines.
Safety concerns, both real and unsubstantiated, continue to be a threat to the present vaccine market.
Vaccines are biologics and therefore are more difficult to produce with consistent precision than small-molecule drugs.
They are subject to variability in the manufacturing process and require careful handling.
17 Despite intensive quality regulation, the biologic nature of vaccines, inherent uncertainties in manufacturing, and safety concerns make vaccine manufacturers targets for tort litigation for patients suffering an illness after vaccination.
A surge of lawsuits in the 1980s resulted in serious concerns regarding the supply of the DTaP combination vaccine, as well as other vaccines.
33 Concerns have been raised in the United States regarding the safety of thimerosal, which is a mercury-containing preservative used in some vaccines.
Another concern is with the false association of MMR combination vaccine and autism in children.
However, to date, studies have not shown an association between neurodevelopmental disorders and thimerosal.
37, 38 In addition, no evidence has been found demonstrating a link between vaccination with the MMR vaccine and autism in children.
39 Rotashield, a rotavirus vaccine licensed in 1998, was permanently withdrawn in 1999 when it was found to cause a rare but serious intestinal obstruction in some recipients.
40 These safety concerns have been a reason for a change in the attitudes of some parents regarding having their children immunized.
3 Taken together, liability issues and safety concerns provide important disincentives to manufacturers considering developing and manufacturing vaccines.
Immunization rates for recommended vaccines among children in the United States have been consistently high.
The immunization regimen was simple, costs incurred were small, and many (if not most) public schools required proof of immunization as a condition of attendance.
35, 41 Most children received vaccines from private practitioners with their parents paying for this service through third-party insurance or out of pocket.
Underprivileged children often received free immunizations from local health departments, with costs paid from general revenue funds at the local and state level.
In the 1990s, the cost of recommended immunizations began to increase, primarily as a result of the introduction of Table 2 displays a comparison of the costs for recommended vaccines for children 0 to 6 years of age for 1987 and 2008.
According to the 2007 National Immunization Survey (NIS) for children, although the number of children vaccinated had reached record highs, for some vaccines, coverage among children was lower and varied with poverty level.
43 Also, according to recent CDC data, substantial gaps continue to remain in vaccination coverage for adults.
44 These shortcomings may be due in part to the increasing costs of vaccines.
Given the increasing costs associated with vaccination and the increasing number of vaccine doses, financing for this service has taken on greater importance.
Currently, U.S. vaccine financing is a joint responsibility shared by the private and public sectors.
As of 2002, more than one-half of the vaccines recommended for children were purchased through federal contract, 45 whereas vaccines for adults typically are covered by private insurance.
Private health plans often have insurance coverage for vaccines.
However, some children enrolled in private health plans do not have coverage for vaccines and are considered underinsured for immunization.
46 Finally, some studies have shown that health care provid-ers have concerns regarding the costs of purchasing and administering vaccines and their level of reimbursement from public and private insurers.
47 Providers must order and purchase many vaccines (e.g., influenza) months before they are administered, resulting in substantial capital outlay coupled with delayed reimbursement.
47 Recently, Freed et al.
48 conducted a survey exploring physicians' perspectives on reimbursement for childhood immunizations.
Approximately one-half of the study respondents reported financial reasons and low profit margins from immunizations as factors affecting their purchase and administration of vaccines.
These authors concluded that physicians who provide vaccines to children and adolescents are dissatisfied with third-party reimbursement levels and the increasing financial strain on their practices from immunizations.
Thus, increasing vaccine prices, a greater number of vaccine doses, and declining provider reimbursement for these products appear to be factors constraining both patient and provider demand for these products.
Parental beliefs regarding vaccine safety and efficacy have led to a decrease in the demand for recommended vaccines.
3,49-51 For example, Kennedy et al.
50 reported that 12% of parents with a child still living at home in 2002 were opposed to compulsory vaccination laws and that this opposition was associated significantly with beliefs in the safety and efficacy of vaccines.
In an analysis of the 2003-2004 NIS, Gust et al.
51 found that more than 13% of parents had delayed their child's For recombinant vaccines, this involves many unit operations of column chromatography and ultrafiltration.
Formulated vaccine may include an adjuvant to enhance the immune response, stabilizers to prolong shelf life, and/or preservatives to allow multidose vials to be delivered.
Reviews VACCINE POLICY first vaccination and that 6% had refused vaccination.
Concerns about vaccine safety were associated significantly with both of these behaviors.
As described above, at least two important positive externalities (i.e., benefits accruing to individuals other than the original supplier and patient) 52 can be attributed to vaccines: (1) vaccination helps protect even those individuals not receiving the vaccine by reducing the transmission of a given disease and (2) reductions in the burden of infectious disease in the 20th century have been linked to considerable economic expansion during that period.
However, vaccine manufacturers cannot capture these third-party benefits.
This problem, together with other supply-side (e.g., barriers to entry) and demand-side (e.g., vaccine financing) issues have resulted in market failure (i.e., a quantity and variety of vaccine products supplied that is below the social optimum) in the vaccine market.
Thus, both government policy makers and various health philanthropies have implemented a number of proposals aimed at overcoming these issues.
These policies can be described as either "push" or "pull" strategies.
2 Push strategies seek to address supply-side issues in the vaccine market by providing direct assistance to ease the burden of research, development, and production costs, whereas pull strategies are designed to manipulate demand for vaccines, thereby improving the likelihood of a return on investment by increasing the number of immunizations administered.
Thus, push mechanisms can be thought of as funding inputs, while pull mechanisms can be thought of as paying for outputs.
Financial incentives.
Large, government-funded research and academic institutions play a vital role in basic vaccine research.
Public funding of vaccine discovery and early developmental efforts coupled with tax subsidies to private firms can reduce manufacturers' upfront financial outlays substantially and alter return on investment calculations for vaccine research favorably.
2,28 For example, NIH sponsors approximately one-third of all vaccine-related basic research.
Most of this funding is in the form of grants to academic institutions and health-related agencies.
The Bioshield Act of 2004 (P.L.
108-276) conferred more authority and leadership in the vaccine development effort on the National Institutes of Allergy and Infectious Diseases (NIAID).
53 The law increased the federal share of bioterrorism projects and allowed NIAID to hire technical experts and to award grants and contracts for advancing R&D efforts for specific vaccines.
To date, funds from the act have provided support for the R&D of new smallpox and anthrax vaccines.
54 After the Bioshield Act, in 2006, Congress enacted the Pandemic and All-Hazards Preparedness Act (P.L.
109-417).
55 This act gave authority for the advanced development and acquisitions of medical countermeasures to the Biomedical Advanced Research and Development Authority.
55 FDA fast-track mechanism.
The FDA Modernization Act of 1997 (P.L.
105-115) directed FDA to issue guidance describing its policies and procedures pertaining to fast-track products.
56 FDA's fast-track mechanism is designed to facilitate the development and expedite the review of new vaccines intended to treat serious or life-threatening conditions.
56 The mechanism emphasizes early communication between the manufacturer and FDA.
This allows the manufacturer and FDA to discuss development plans and strategies that can improve the efficiency of preclinical studies of the drug and focus efforts on the design of the major clinical efficacy studies before a formal submission of a BLA.
56 This early interaction can help clarify goals and plan early for obstacles that might delay approval decisions for a new vaccine.
Biovest International Inc.'s BiovaxID (a therapeutic vaccine focused on follicular non-Hodgkin's lymphoma) 57 and Intracel's OncoVAX vaccine (designed to prevent recurrence in stage 2 colon cancer) 58 are recent examples of vaccines that have been granted fast-track status.
FDA accelerated approval.
For certain biological products that are being tested for treatment of a serious or life-threatening illness, FDA regulations allow "accelerated approval" of the biologic product based on the biologic products' "meaningful therapeutic benefit over existing treatments."
59, 60 FDA grants this approval on the basis of adequate and well-controlled clinical trials establishing that the biological product has an effect on a surrogate endpoint that is reasonably likely to predict clinical benefit.
60 For example, in an effort to meet the increasing need for a flu vaccine, the FDA approved Fluarix, an influenza vaccine for adults that contains inactivated virus through this mechanism.
The manufacturer demonstrated that after vaccination with Fluarix, adults made levels of protective antibodies in the blood that FDA believes are likely to be effective in preventing influenza.
61 Fluarix was the first vaccine approved using the accelerated approval process.
FDA priority review.
Under the FDA Modernization Act, reviews for New Drug Applications (NDAs) or BLAs are designated as either standard or priority.
62 The review period changes depending on the designation given to the drug.
Drugs given a standard designation usually require 10 months to more than 1 year for review.
The priority designation can, however, shorten the anticipated amount of time until approval decision from 10 months to 6 months for some products.
The priority review process begins only when a manufacturer officially submits a BLA (or an NDA).
Priority review, therefore, does not alter the steps taken in a vaccine's development or testing for safety and effectiveness.
62 Merck's human papillomavirus vaccine-the first developed to prevent cervical cancer-was evaluated and approved in 6 months under the priority review process.
63 Liability protections and safety solutions.
Because pharmaceutical manufacturers have expressed liability concerns as an important reason for abstaining from vaccine development, proposals addressing these concerns have been seen as necessary incentives to participation in vaccine development.
28 In response to the safety concerns and the lawsuits against vaccine manufacturers, Congress enacted the National Childhood Vaccine Injury Act of 1986 (P.L.
99-660).
64 This legislation established the Vaccine Injury Compensation Program in 1988, which ensures that individuals or families of individuals who may have been injured as a result of a routinely recommended vaccine are quickly, easily, and appropriately compensated.
64 An individual claiming injury or death from a vaccine files a petition for compensation with the court.
The petition is reviewed to determine whether it meets the criteria for compensation.
A vaccine injury table lists and explains injuries/conditions that are presumed to be caused by vaccines.
It also lists time periods in which the first symptom of these injuries/conditions must occur after receiving the vaccine.
To qualify for compensation, a petitioner must show that an injury found in the vaccine injury table occurred or must prove that the vaccine caused the condition.
A case found eligible for compensation is scheduled for a hearing to assess the amount of compensation.
Most noncompensable claims receive awards for attorney fees and costs.
Congressional approval of this act also set in motion the Vaccine Adverse Event Reporting System for monitoring vaccine adverse events.
The Homeland Security Act of 2002 (P.L.
107-296) protects manufacturers and health care workers who administer the smallpox vaccine from tort liability and restricts the liability assumed by the United States to negligence of those parties.
65 The Smallpox Emergency Personnel Protection Act of 2003 (P.L.
108-20) created a mechanism to compensate individuals who, in response to a Secretarial request for smallpox vaccine preparedness, are injured by the vaccinia virus used in the smallpox vaccine.
Vaccine recipients and individuals contacted by them are eligible for medical care expense reimbursement, lost income benefits, and death benefits, administered through the Health Resources and Services Administration.
66 The Public Readiness and Emergency Preparedness Act of 2006, (P.L.
109-148) is a tort liability shield that immunizes vaccine manufacturers, distributors, program planners, and administrators.
67 The act protects these entities from financial risk in the event of any loss related to the manufacture, testing, development, distribution, administration, and use of countermeasures against chemical, biological, radiological, and nuclear agents of terrorism, epidemics, and pandemics.
67 Public-private partnerships.
Donors, foundations, and other partners have created a public-private partnership known as the Global Alliance for Vaccines and Immunization (GAVI), the mission of which is to save children's lives and protect people's health through the widespread use of vaccines.
68 As a GAVI partner, the Bill and Melinda Gates Foundation has invested millions of dollars in R&D for vaccines for diseases such as malaria and human immunodeficiency virus, currently the leading killers of children and adults around the world.
GAVI has established public-private partnerships to accelerate late-stage development and introduction of priority vaccines against disease such as rotavirus and pneumococcus.
69 Stockpiles.
Stockpiles are, put simply, an artificial enhancement to current market demand levels in anticipation of periods when supply will be insufficient to meet demand.
2 Government funding of vendor-managed stockpiles of childhood vaccines ensures that some excess vaccine supply is always available to buffer supply problems when they occur.
Currently, the United States has a large enough stockpile of smallpox vaccine to vaccinate every person in the country who might need it in the event of an emergency.
70 The government also expects to stockpile nearly 8 million doses of an investigational vaccine against pandemic influenza, and studies are under way to develop mechanisms that could stretch that supply to cover more than one-third of the population.
71 CDC also maintains a large anthrax vaccine stockpile.
Advance market commitments.
Advance market commitments involve donors who commit to buying yet-to-be-developed vaccines in bulk for poor nations if drug makers are able to deliver a vaccine that meets specifications and a price can be settled on in advance.
72 Supporters of advance market commitments range from the GAVI partners to Pope Benedict XVI.
Donors have agreed to test this mechanism for a vaccine for pneumococcal disease.
To date, the Gates Foundation, the United Kingdom, Italy, Canada, Norway, and Russia have committed a total of $1.5 billion for the project.
Vaccine bonds.
The United Kingdom has taken a lead in promoting an International Financing Facility for Immunization (IFFIm) 69 IFFIm has raised more than $1 billion in capital markets to immunize poor children in developing nations against Reviews VACCINE POLICY vaccine-preventable diseases.
73 IFFIm plans to invest $4 billion over the next decade to immunize 500 million people who would not otherwise be protected from diseases that no longer represent public health threats in developed countries.
73 The IFFIm mechanism concentrates on the funding for vaccine research by using long-term government commitments as security bonds issued in the capital markets.
The cash received for the bonds then can be used for research and future purchase of vaccines.
Whenever the bonds are issued, IFFIm pays bondholders a modest rate of interest.
As money pledged by donor governments becomes available gradually over 30 years, these funds will be used to repay the capital value of the bonds.
69 IFFIm was able to double the resources GAVI has been able to allocate-$945.6 million in 2007 compared with $418.3 million in 2006.
74 Vaccine financing programs.
Historically, the U.S. immunization system has been financed through public-private sector partnerships.
The public sector purchases vaccines for approximately 55% of the birth cohort.
75 Section 317 (a federal discretionary grant program to all states), the Vaccines for Children (VFC) Act of 1993 (P.L.
103-66), and state funds are major public sector sources for vaccine financing.
Private sector vaccine purchases are covered through private health insurance and account for 45% to 50% of the pediatric vaccines sold annually in the United States.
75 The federal government has played an evolving role in building the immunization structure in the United States.
The earliest legislation pertaining to vaccine financing is the Social Security Act of 1935.
Title V of this act pertains to immunization services for children and their mothers.
In 1963, Congress enacted the Vaccine Assistance Act (Section 317 of the Public Health Service Act).
This legislation provided grants to social service agencies and local health departments for immunization infrastructure and vaccine purchases.
41 However, barriers to immunization access still remained in some areas as a result of considerable variability in immunization efforts by state and local governments.
The deficiencies in this legislation were highlighted by the measles epidemic of 1989-1991, which involved more than 55,000 cases and led to 123 deaths.
35, 76 Substantial numbers of unimmunized preschool children, particularly in inner-city areas, contributed to this event.
76 To ensure that vulnerable children had more reliable access to vaccines, the government refocused their funding resources on helping individual states in building immunization infrastructure.
VFC is a state-operated federal entitlement program that provides free Advisory Committee on Immunization Practices-recommended vaccines to children 18 years of age or younger who are uninsured, Alaska Native or Native American, eligible for Medicaid, or receive their vaccines in a federally qualified health center.
77 At the state level, funds are earmarked for vaccine purchase and immunization programs.
35 State funds also have been used to purchase vaccines for children and adolescents not eligible for VFC.
A combination of VFC, state/local, and Section 317 program funds (i.e., VFC only, VFC and Underinsured, VFC and Underinsured Select, Universal, and Universal Select) has been used by a number of states to purchase all recommended vaccines for children in the state, including the privately insured.
78 Many states use universal programs that expand the eligibility for VFC vaccines by supplementing VFC purchases at federally discounted prices.
The universal purchase states have been successful in raising vaccination rates among the underinsured 79 and increasing access to newer and more expensive vaccines for children without insurance.
75 However, criticisms of the universal purchase programs have been raised, including (1) vaccine manufacturers' claims that universal purchase programs unfairly provide for the purchase of all vaccines at lower government contract prices, thus eliminating the private market for vaccines and decreasing revenue; (2) although immunization charges are reduced under this program, patients still pay for the vaccine administration fee; and (3) some contend that taxpayer money should not be spent to provide free vaccines for children whose insurance would otherwise pay for it.
79 State Medicaid and State Children's Health Insurance Program funds also are provided for vaccine purchase, although the level of Medicaid funding varies from state to state.
In contrast to vaccine coverage for children, adults are far less likely to be covered for immunization services and frequently face a problem of underinsurance.
The federal Medicare program covers some immunizations for all eligible beneficiaries through the Medicare Part B program.
80 The selected immunizations include influenza, pneumococcal, and hepatitis B vaccinations.
Certain other vaccines (e.g., tetanus toxoid) also are covered if their administration is considered necessary in the treatment of another covered illness.
80 The Part D program generally covers those vaccines not available for reimbursement under Medicare Parts A or B when administration is reasonable and necessary for the prevention of illness.
Private insurance coverage of immunizations for working-age adults varies widely by the type of health plan.
35 For example, health maintenance organizations typically have the highest coverage levels, while preferred provider organizations and indemnity plans historically have covered immunization services less frequently.
By saving millions of lives and millions of dollars, vaccines have been responsible for some of the greatest successes in public health.
However, the struggle against infectious disease is a continual process requiring new vaccines for the challenges that may confront human health in the future.
The vaccine market is fragile and requires both supply-and demandside interventions.
Vaccine availability has been limited by the number of suppliers, high R&D and production costs, and safety problems leading to increased regulatory requirements.
Demand has been constrained by rapidly increasing vaccine costs, financing issues that have constrained efforts to achieve targets set for population immunization rates, and parental attitudes regarding the safety and efficacy of vaccine products.
To date, the U.S. government, in concert with private philanthro-VACCINE POLICY Reviews pies, has implemented a patchwork of policies to make the vaccine market more attractive for private firms and to increase access to these products for individuals.
We would argue that what is needed is an integrated policy approach that preserves incentives for market entry and innovation in the vaccine industry while simultaneously addressing parental vaccine concerns and increasing immunization funding and reimbursement for both providers and patients.
Year Legislation Description 1813 The Vaccine Act The first federal law dealing with patient protection and therapeutic substances.
An agent to be appointed for preserving the genuine vaccine matter and to furnish the same to any citizen of the United States, whenever it may be applied for, through the medium of the post office.
Packets not exceeding half an ounce and relating to vaccination to go free of postage to and from the agent.
1902 Biologics Control Act In addition to the testing of the final product, this act also mandated the testing and control of manufacturing materials and establishments.
1963 Vaccine Assistance Act (Section 317 of the Public Health Service Act) Infrastructure support for preventive health services such as immunization activities, including vaccine purchase assistance, is provided under Section 317 of the Public Health Service Act.
1986 National Childhood Vaccine Injury Act Ensures that children who might be injured as a result of a routinely recommended vaccine are quickly, easily, and appropriately compensated.
This act set into motion VAERS for monitoring vaccine adverse events.
1993 Vaccines for Children Act State-operated federal entitlement program that provides free ACIP-recommended vaccines to eligible children through age 18 years.
1997 FDA Modernization Act: fast-track mechanism The mechanism is designed to facilitate the development and expedite the review of new potential vaccines intended to treat serious or life-threatening conditions.
1998 FDA Modernization Act: priority review Reviews for NDAs or BLAs are designated as either standard or priority.
The review period changes depending on the designation given to the drug.
2002 Homeland Security Act Protects manufacturers and health care workers who administer the smallpox vaccine from tort liability and restricts that liability assumed by the United States to negligence of those parties.
2003 Smallpox Emergency Personnel Protection Act Mechanism to compensate individuals who, in response to a Secretarial request for smallpox vaccine preparedness, are injured by the vaccinia virus used in smallpox vaccines.
Vaccine recipients and their contacts are eligible for medical care expense reimbursement, lost income benefit, and death benefits, administered through HRSA.
Project Bioshield Act Increased the federal share of bioterrorism projects and allowed NIAID to hire technical experts and to award grants and contracts for advancing the research and development efforts in vaccine areas.
Pandemic and All-Hazards Preparedness Act Intended to improve U.S. public health and medical preparedness and response capabilities for emergencies, whether deliberate, accidental, or natural.
2006 Public Readiness and Emergency Preparedness Act Tort liability shield intended to protect vaccine manufacturers, distributors, program planners, and administrators of vaccines from financial risk in the event of a loss from vaccines.
Abbreviations used: ACIP, Advisory Committee on Immunization Practices; BLA, Biological License Application; HRSA, Health Resources and Services Administration; NDA, New Drug Application; NIAID, National Institutes of Allergy and Infectious Diseases; VAERS, Vaccine Adverse Event Reporting System.
Immunizations in the United States: success, structure, and stress The vaccine industry: does it need a shot in the arm?
National Health Policy Forum Vaccines: the controversial story of medicine's greatest lifesaver.
Part II Vaccine manufacturing: challenges and solutions Economic evaluation of the 7-vaccine routine childhood immunization schedule in the United States Cost-effectiveness of influenza vaccination of healthy children Herd immunity and herd protection Vaccines in the public eye Demographic change and economic growth in East Asia The health and wealth of nations Vaccination greatly reduces disease, disability, death and inequity worldwide The health of nations: the contribution of improved health to living standards Impact of vaccines universally recommended for children: United States, 1990-1998.
MMWR Morb Mortal Wkly Rep Effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized control trial Vaccines for preventing influenza in healthy adults Financing vaccines: in search of solutions that work Putting markets to work in vaccine manufacturing Why are pharmaceutical companies gradually abandoning vaccines?
Centers for Disease Control and Prevention.
Vaccines & immunizations: 2002 immunization news Strengthening the supply of routinely recommended vaccines in the United States: recommendations from the National Vaccine Advisory Committee Red Book online: special alert: Hib shortage Vaccine shortages: history, impact, and prospects for the future Reuters Health Information.
Tropical Dengue fever may threaten U.S.: report.
Accessed at Malaria surveillance: United States Epidemiological and genetic analysis of severe acute respiratory syndrome Just the facts: vaccines provide effective protection and FDA makes sure they are safe CRS report for Congress: vaccine policy issues The epidemiology of rotavirus diarrhea in the United States: surveillance and estimates of disease burden Vaccine product approval process The price of innovation: new estimates of drug development costs The fragility of the US vaccine supply Merck recalls childhood vaccine Financing vaccines in the 21st century Lessons learned: new procurement strategies for vaccines: final report to the GAVI Board Early thimerosal exposure and neuropsychological outcomes at 7 to 10 years Neuropsychological performance 10 years after immunization in infancy with thimerosal-containing vaccines Autism's false prophets: bad science, risky medicine, and the search for a cure Withdrawal of rotavirus vaccine recommendation Financing immunizations in the United States Vaccine shortages: history, impact, and prospects for the future National, state, and local area vaccination coverage among children aged 19-35 months: United States New data show unacceptably low adult immunization rates and that adults unaware of infectious disease threat.
Accessed at www.reuters.com/article/pressRelease/ idUS181452+23 Epidemiology: infectious diseases: preparing for the future Gaps in vaccine financing for underinsured children in the United States Estimating medical practice expenses from administering adult influenza vaccinations Primary care physician perspectives on reimbursement for childhood immunizations Qualitative analysis of mothers' decision-making about vaccines for infants: the importance of trust Vaccine beliefs of parents who oppose compulsory vaccination Parents with doubts about vaccines: which vaccines and reasons why Cost-benefit analysis: concepts and practice CRS report for Congress: RS21507 Project Bioshield Project Bioshield: protecting Americans from terrorism Department of Health and Human Services.
Pandemic and All-Hazard Preparedness Act Guidance for industry, fast track drug development programs: designation, development, and application review Biovest moves to Fast Track with cancer vaccine FDA grants "fast track" designation for the development of OncoVAX Government Printing Office.
21 CFR 601 subpart E: accelerated approval of biological products for serious or life-threatening illnesses CFR -Code of Federal Regulations Title 21 FDA approves new influenza vaccine for upcoming flu season Fast track, priority review and accelerated approval FDA licenses new vaccine for prevention of cervical cancer and other diseases in females caused by human papillomavirus Department of Health and Human Services, Health Resources and Services Administration.
National Vaccine Injury Compensation Program (VICP).
Accessed at www.hrsa.gov/vaccinecompensation CRS report for Congress: RL31649, Homeland Security Act of 2002: tort liability provisions CRS report for Congress: RL31960, smallpox vaccine injury compensation Public Readiness and Emergency Preparedness Act questions and answers.
Accessed at www.hhs.gov/disasters/emergency/manmadedisasters/bioterorism/medication-vaccine-qa.html Accessed at www.gavialliance.org The problems and promise of vaccine markets in developing countries Department of Health and Human Services.
CDC efforts in implementing a smallpox vaccination program builds stockpile of vaccine for flu pandemic Why a market-driven vaccine plan faces big obstacles Vaccine bonds provide model for other aid projects.
Accessed at www.medscape.com/viewarticle/573432 GAVI Alliance announces dramatic funding boost for Hib vaccine Assuring vaccination of children and adolescents without financial barriers: recommendations from the National Vaccine Advisory Committee (NVAC) Measles surveillance: United States Accessed at www.
cdc.gov/vaccines/programs/vfc Childhood vaccine supply policy Impact of North Carolina's universal vaccine purchase program by children insurance status Adult immunization.
Accessed at www.cms.hhs.gov/adultImmunizations Instructions: The assessment test for this activity must be taken online; please see "CPE processing" below for further instructions.
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This CPE will be available at www.pharmacist.com no later than July 31, 2009.
1.
Which of the following is the process by which vaccines protect not only those who receive them but also those who cannot or do not receive the vaccine?
a.
Passive immunization b.
Active immunization c. Herd To obtain 2.0 contact hour of continuing pharmacy education credit (0.2 CEUs) for "Vaccine supply, demand, and policy: A primer," go to www.pharmacist.com and take your test online for instant credit.
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The ACPE Universal Activity Number assigned to the program by the accredited provider is 202-000-09-209-H04-P."Vaccine supply, demand, and policy: A primer" is a home-study continuing education activity for pharmacists developed by the American Pharmacists Association.
key: cord-022039-y0l943xg authors: Gruber, Marion F.; Marshall, Valerie B. title: Regulation and Testing of Vaccines date: 2017-07-17 journal: Plotkin's Vaccines DOI: 10.1016/b978-0-323-35761-6.00079-1 sha: doc_id: 22039 cord_uid: y0l943xg nan Vaccines are one of the most significant achievements of science and public health.
As a result of successful vaccination programs and campaigns, many vaccine-preventable diseases are now uncommon in the United States.
Vaccines for prevention of infectious diseases are regulated by the U.S. Food and Drug Administration (FDA) and the legal framework for regulation is derived from Section 351 of the Public Health Service Act and from certain sections of the federal Food, Drug, and Cosmetic Act (FD&C Act).
1, 2 The FD&C Act defines drugs, in part, by their intended use as "articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease."
2 Thus, vaccines are a unique class of pharmaceutical products that meet the statutory definition of both a drug and biological product.
Prophylactic vaccines differ from many other drugs and biologicals primarily in how they are administered to a large population, in particular, young healthy people to prevent rather than treat disease, their mechanism of action, and their risk/benefit profile.
Although subject to the same regulations as other biological products, vaccines are inherently more difficult to develop, characterize, and manufacture than most pharmaceutical products.
Current U.S. licensed vaccines are listed in Tables 79.1 and 79.2.
Regulation of biologics has historically been initiated in response to issues of safety.
Over time, legislative authorities have evolved to strengthen and modernize the regulation of vaccines and other biologics.
Prior to 1902, manufacturing and product standards for biologics were not federally mandated.
However, in 1902, the U.S. Congress passed an act to regulate the sale of viruses, serums, toxins, and analogous products (later referred to as the Biologics Control Act) following the deaths of 20 children who received contaminated products.
3 This Act authorized the Hygienic Laboratory of the Public Health and Marine Hospital Service to issue regulations that governed all aspects of commercial production of vaccines, serums, toxin, and antitoxins and similar products with the objective of ensuring their safety and purity.
The regulations under this legislation contained the primary concepts for regulation of biologicals, such as labeling, mandatory facility inspections, and batch-certification guidelines.
In 1930, the Hygienic Laboratory was reorganized, expanded, and renamed the National Institutes of Health (NIH).
In 1944, Congress recodified the 1902 Biologics Control Act as part of the U.S. Public Health Service Act (PHS Act) of 1944.
4 The PHS Act incorporated the 1902 Biologics Control Act into Section 351 of the PHS Act (42 U.S.C.
262).
As with the 1902 Act, the 1944 PHS Act focused primarily on extensive control over manufacturing methods to ensure safety and purity.
Unique to the 1944 PHS Act was Congress' explicit addition of the requirement that biologics manufacturers demonstrate potency as a measure of clinical usefulness.
The PHS Act created the Laboratory of Biologics Control to facilitate testing and licensure of biologicals products and manufacturing establishments.
After 1944, the authority of the Laboratory of Biologics Control was derived from Section 351 of the PHS Act and from certain sections of the 1938 FD&C Act.
In 1948, the Laboratory of Biologics Control joined the 79 NIH Division of Infectious Diseases and Division of Tropical Diseases to form the National Microbiological Institute (later renamed the National Institute of Allergy and Infectious Diseases).
Administrative authority for regulation of biologics was originally granted to the National Microbiological Institute.
Although important regulations had been enacted to improve product safety, by the 1950s, the only legal requirement for vaccine licensing was submission of written protocols for vaccine production and safety testing to the Laboratory of Biologics Control.
Regulations were dramatically expanded in 1955, when more than 200 cases of polio were attributed to incompletely inactivated polio vaccine manufactured by the Cutter Laboratories.
As a result of the "Cutter Incident," administrative authority for the regulation of biologicals was transferred by Congress to the Division of Biologics Standards, a newly created division within the NIH.
Regulations were strengthened that required more precise experimental testing to assess the safety of vaccines.
Congress passed the Consumer Safety Act of 1972 and transferred regulatory authority from NIH to FDA for the administration of the 1944 PHS Act.
In 1972, the Division of Biologics Standards, which was charged with administering and enforcing Section 351 of the PHS Act, was transferred by the Secretary of Health, Education and Welfare to the FDA, and became the Bureau of Biologics.
Once administrative responsibility for the regulation of biologicals was transferred from NIH to FDA, the FDA announced its intention to require that all new biologicals satisfy the additional standards of safety and efficacy mandated in the Drug Amendments Act of 1962.
This resulted in the transfer of the regulations pertaining to biologics from Part 73 of Chapter I of Title atric subpopulation for which the product is safe and effective.
The FDAAA of 2007 includes 11 titles that added many new provisions to the FD&C Act.
7 It reauthorized and amended several drug and medical device provisions, and provided the FDA with additional responsibilities and new authorities.
The provisions of FDAAA that have had a significant impact on the regulations of vaccines and the review process are contained in Title IV, the PREA, and Title IX, Enhanced Authorities Regarding Postmarket Safety of Drugs.
The FDAAA reauthorized and revised the PREA, primarily to enhance FDA oversight and applicant accountability for the agreed-upon The Pediatric Research Equity Act (PREA) of 2003 amended the FD&C Act by adding Section 505(B) to address product development for pediatric subjects from birth to 16 years of age.
6 It requires that manufacturers submit a pediatric assessment with every application submitted under Section 505 of the FD&C Act or section 351 of the PHS Act for a new active ingredient, new indication, new dosage form, new dosing regimen, or new route of administration unless the applicant has obtained a waiver or deferral from the FDA.
The pediatric assessment must contain data adequate to assess the safety and effectiveness of the drug or the biological product for the claimed indications in all relevant pediatric subpopulations and data to support dosing and administration for each pediSection 505 of the FD&C Act or Section 351 of the PHS Act (42 USC §262) .
Section 901 of the FDAAA also created new Sections 505-1 and 505(o)(4) of the FD&C Act, authorizing the FDA, under certain circumstances, to require risk evaluation and mitigation strategies and safety-related labeling changes, respectively.
The FDAAA also specifies adverse event reporting requirements for products with labeling changes that are a result of a pediatric assessment.
Specifically, during the 12 months from the date that such a labeling change is made, all adverse event reports are reviewed by the FDA Pediatric Advisory Committee.
Following review, the Pediatric Advisory Committee makes recommendations regarding whether pediatric assessments.
8 Of note, a new provision directed the FDA to establish the Pediatric Review Committee (PeRC), an internal review committee with pediatric expertise.
This committee is required to provide consultation to FDA review divisions on all pediatric plans and assessments and on all deferral and waiver requests.
Thus, early in the application review process the review team must assess whether PREA applies.
If PREA applies, then a pediatric assessment must be presented to the PeRC.
Section 901 of Title IX of the FDAAA authorizes the FDA to require certain postmarketing studies and clinical trials for prescription drug and biological products approved under the FDA should take action in response to such reports and whether the current pharmacovigilance plan is adequate.
The FDASIA was signed into law in 2012 and expanded the FDA's authority by strengthening the agency's ability to safeguard and advance public health by promoting innovation, increasing stakeholder involvement in FDA processes, and enhancing the safety of the drug supply chain.
9 In addition to reauthorizing the prescription drug and medical device user fee programs, FDASIA established new user fee programs for generic drugs and biosimilar biological products.
The provisions of FDASIA that impact the regulation of vaccines are contained in Pediatric Drugs and Devices (Title V) and Drug Approval and Patient Access (Title IX).
Title IX expands the scope of products that qualify for accelerated approval and creates a new "breakthrough therapy" program, among other things (see below).
FDASIA also revised PREA to include a provision that requires vaccine manufacturers to submit a Pediatric Study Plan early in the drug development process.
This initial Pediatric Study Plan must contain an outline of the pediatric study or studies that the sponsor plans to conduct including, to the extent practicable, study objectives and design, age groups, relevant end points, and statistical approach, as well as any request for a deferral, partial waiver, or waiver.
The FDA internal PeRC must be consulted for the review of the initial study plan, the agreed initial pediatric plan, and certain amendments to such plans.
Both sponsors and the FDA must comply with prescribed timelines regarding submission, review, responses, and agreements reached apply to vaccines, regardless of their indication or intended target population.
Section 351 of the PHS Act (42 USC §262) states that a BLA can be approved based on a demonstration that "…(a) the biological product that is the subject of the application is safe, pure, and potent; and (b) the facility in which the biological product is manufactured, processed, packed, or held meets standards designed to assure that the biological product continues to be safe, pure, and potent…" Some of the more pertinent operational definitions for biologics contained in the statutes and 21 CFR are as follows: • Section 351 of the PHS Act defines a biological product as any virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, or analogous product applicable to the prevention, treatment, or cure of diseases or conditions of human beings.
Thus, vaccines clearly are regulated as biological products.
• Safety is defined as the relative freedom from harmful effect to people affected directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the regarding the Pediatric Study Plan, which are also described in applicable FDA guidance.
10 Of note, these amendments to the FD&C Act also renewed the Prescription Drug User Fee Act (PDUFA) that was first enacted in 1992, and authorized the FDA to collect user fees from companies.
These fees enabled FDA to hire additional reviewers and support staff and upgrade its information technology systems.
In return for these additional resources, the FDA agreed to certain review performance goals, such as completing reviews of new drug applications and biologics license applications (BLAs) and taking regulatory actions on them in predictable timeframes.
These changes revolutionized the drug approval process in the United States and enabled FDA to speed the application review process for new drugs and biologics without compromising the FDA's high standards for demonstration of safety, efficacy, and quality.
The FDA's CBER is the national regulatory authority in the United States charged with the regulation of biological products including vaccines.
These regulations cover not only the methods and establishment standards pertaining to the manufacture of a biological product to assure that the product is safe and meets the quality and purity characteristics that are claimed by the manufacturer, but also requirements for performing clinical trials (i.e., 21 CFR §312).
A single set of basic regulatory requirements applies to all vaccines, regardless of the technology used to produce them.
The regulatory approval criteria contained in Title 21 CFR also recipient at the time.
Thus, the property of safety is relative and cannot be ensured in an absolute sense.
• Purity is defined as the relative freedom from extraneous matter, regardless of whether it is harmful to the recipient or deleterious to the product.
Usually, the concepts of purity and safety coincide; purity most often relates to freedom from such materials as pyrogens, adventitious agents, and chemicals used in manufacture of the product.
• Potency is defined as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through administration of the product in the manner intended, to effect a given result.
Potency, as thus defined, is equivalent to the concept that the product must be able to perform as claimed, and, if possible, this must correspond with some measurable effect in the recipient or correlate with some quantitative laboratory finding.
• Standards mean specifications and procedures applicable to an establishment or to the manufacture or release of products that are designed to ensure the continued safety, purity, and potency of biological products.
The word standard is also used with a secondary meaning, usually in the sense of a reference preparation, such as a bacterial or viral antigen that can be used in evaluating potency or, in some cases, safety and purity.
• The regulations regarding biological products, in addition, define effectiveness as the reasonable expectation that, in a significant proportion of the target population, pharmacologic or other effects of the biological product, when administered under adequate directions for use and warnings against unsafe use, will serve a clinically significant function in the diagnosis, cure, mitigation, treatment, or prevention of disease in humans.
• CGMPs define a quality system that manufacturers use as they build quality into their products.
The regulations outline the minimum manufacturing, quality control, and quality assurance requirements for the preparation of a drug or biological product for commercial distribution.
For example, approved products developed and produced according to CGMPs are safe, properly identified, of the correct strength, pure and of high quality.
The FDA also periodically publishes various guidelines and guidance documents with regard to the manufacture and clinical evaluation of biologicals.
These documents published by the FDA do not have the force of law, but are intended to provide useful and timely recommendations; Table 79 .5 lists those applicable to vaccines.
Guidance documents are particularly useful as a means for the FDA to provide recommendations that are current with areas of rapidly progressing science, and for specifying a degree of detail beyond what is included in the regulations.
In the past few years, several FDA regulations and guidance documents have had a direct impact on the review of vaccines for licensure by the FDA, such as the "Guidance for Industry: Expedited Programs for Serious Conditions-Drugs and Biologics (2014)."
12 Some of these regulations and guidance documents evolved from an effort to streamline the regulatory process, while others-such as "Guidance The FDA's CBER regulatory review staff consists of an internal multidisciplinary team of scientists, medical officers, and regulatory and public health professionals.
To stay current on scientific advances and biotechnologies, the team is involved in a dynamic exchange of information with the outside scientific community through laboratory research and collaborations, participation in workshops and seminars, and engagement with national partners.
The FDA also relies on the expertise of formal advisory committees, which include experts in the fields of vaccinology, microbiology, infectious diseases, immunology, biostatistics, epidemiology, and clinical trial design.
In addition, CBER works closely with its counterparts in other U.S. government agencies within the Department of Health and Human Services and the U.S. Public Health Service (PHS), such as the National Vaccine Program Office, the Centers for Disease Control and Prevention (CDC), the NIH, and the Health Resources and Services Administration.
The CDC is responsible, among its other duties, for epidemiologic surveillance of disease and for support of immunization programs.
Its Advisory Committee on Immunization Practices makes recommendations for vaccine use.
The Director of the National Vaccine Program Office coordinates vaccine efforts throughout the PHS and other governmental agencies.
The NIH is responsible for conducting and providing funds for a wide variety of biomedical research.
The Health Resources and Services Administration is responsible for managing the National Vaccine Injury Compensation Program.
Other important collaborators inside the U.S. government involved in vaccine activities include the U.S. Department of Defense, and the Department of Veterans Affairs.
Additionally, CBER works closely with its multilateral partners, notably the Pan American Health Organization and the World Health Organization (WHO), to provide assistance in regulatory capacity building.
CBER has been very active in supporting the WHO's Developing Countries' Vaccine Regulators Network and the WHO's African Regional Office-led African Vaccine Regulatory Forum.
As a WHO Collaborating Center, CBER contributes to a range of activities, including establishment of physical and written standards, implementation of WHO international standards, strengthening global regulatory systems, and serving as the National Regulatory Authority (NRA) of reference for WHO's vaccine prequalification program.
CBER experts also contribute as members of several WHO Advisory Committees including the Global Advisory Committee on Vaccine Safety, the Polio Research Committee, the HIV Vaccine Advisory Committee, and the Expert Committee on Biological Standardization.
CBER is an Essential Regulatory Laboratory in WHO's Global Influenza Surveillance and Response System.
In addition, the FDA has confidentiality arrangements with many NRAs around the globe, allowing it to share information as part of its regulatory processes.
These arrangements have strengthened interactions between the regulatory authorities and have contributed to improving the promotion and protection of public health globally.
The regulatory review in CBER incorporates a managed and integrated regulatory process that is continuous from The regulatory requirements for biological products cover the entire life-cycle of the product from the pre-IND stage, through the premarketing (consisting of the various IND phases and prelicensure) and postmarketing stages.
The pre-IND stage consists of laboratory development, preclinical testing of candidate vaccines, and development of the manufacturing process.
The clinical development of a new drug in the United States usually begins with a sponsor approaching the FDA for permission to conduct a clinical study with an investigational product through submission of an IND application form.
These requirements can be found in the IND regulations.
20 Sponsors are encouraged to request a pre-IND meeting with FDA to discuss preclinical studies, clinical study design, and data requirements that require resolution prior to the initiation of clinical trials.
In the application, the sponsor (a) describes the composition, source, and method of manufacture of the product and the methods used in testing its safety, purity, and potency; (b) provides a summary of all laboratory and preclinical animal testing; and (c) provides a description of the proposed clinical study and the names and qualifications of each clinical investigator.
The FDA has a maximum of 30 days to review the original IND application and determine whether study participants will be exposed to any unacceptable risks.
As part of the IND process, each clinical investigator files information describing the investigator's qualifications for performing clinical trials, details of the proposed study, and assurance that a number of conditions specified by the regulations will be met.
A signed informed consent must be obtained from each study participant.
Approval for the study must be obtained in advance from a local institutional review board.
The regulations also cover the evaluation of the preclinical laboratory animal studies undertaken to support the use of the product in humans.
Only licensed vaccines may be shipped from one state to another; however, during the premarketing phase, interstate shipment of products for investigational use is allowed under the law and regulations.
There are generally three separate phases in the clinical evaluation of experimental biologicals at the premarketing stage ( Fig.
79.
2).
These phases may overlap, and the clinical testing may be highly iterative because discovery to postmarketing, and is designated as the Managed Review Process.
18 CBER's managed review process is designed to effectively review all regulatory submissions to reach informed evidence-based regulatory decisions to ensure safe and effective biological products.
CBER uses a team-based approach with substantive involvement of discipline team leaders and management.
The Managed Review Process begins when a sponsor requests a pre-investigational new drug (IND) meeting that may result in the submission of an IND and, eventually, a BLA.
The review process in CBER begins with an initial review of a submission for scientific content and compliance with the regulations.
Members of a multidisciplinary review team are selected based on their expertise with the type of product and its method of manufacture.
It is the responsibility of CBER's review component to evaluate submissions and recommend appropriate regulatory action to facilitate the approval of safe and effective biological products.
The review includes an evaluation of chemistry, manufacturing, and controls information; the manufacturing facility and equipment; preclinical and clinical data on the safety, efficacy, pharmacology, and toxicology; the suitability of clinical trial design; and analysis of clinical data derived from such trials.
In addition, reviewers monitor for conformance with FDA regulations in all phases of biological product development, including postmarketing.
CBER scientists also perform research in the areas of statistical and epidemiologic analysis, clinical trial design, and chemistry, manufacturing and control specific to product issues, and contribute to policy development.
Surveillance activities are performed to ensure that the safety of biological products is not compromised.
These activities ensure the rapid availability and approval of safe and effective biological products.
The FDA encourages meetings with sponsors to the extent that they aid in the evaluation of the vaccine and in resolving scientific issues concerning the product.
The general principle underlying the conduct of such meetings is that there should be free, full, and open communication about any scientific or medical question that may arise.
Agreements reached at PDUFA meetings (e.g., pre-IND, IND, pre-BLA, and BLA meetings) are recorded in official minutes taken by FDA personnel and provided to the sponsor.
They serve as a permanent record of any agreements reached.
Detailed information on the conduct of regulatory meetings is described in 21 seven-valent polysaccharide conjugate vaccine (PCV7) was successful in preventing a low incidence of invasive pneumococcal disease caused by the Streptococcus pneumoniae capsular serotypes included in the vaccine enrolled close to 40,000 children who were randomized equally to receive the pneumococcal conjugate vaccine or an unrelated control vaccine.
In contrast, licensure of the pneumococcal 13-valent polysaccharide conjugate vaccine (PCV13) was based on noninferiority comparative studies to PCV7.
Effectiveness of PCV13 was inferred from measuring anti-polysaccharide binding and functional opsonophagocytic antibodies because clinical end point disease efficacy studies were no longer feasible owing to the further decline of invasive pneumococcal disease as a result of introduction of PCV7 in the United States.
In some situations, human challenge studies have been conducted during early clinical development or in lieu of clinical trials in an endemic area.
These studies served to demonstrate "proof of concept" of the vaccine early in clinical development (e.g., Plasmodium falciparum sporozoite challenge of malaria-naïve U.S. volunteers previously administered a candidate malaria vaccine).
Human challenge studies may also be considered to demonstrate the efficacy of the vaccine.
For example, the Agency convened the Vaccines and Related Biologics Products Advisory Committee (VRBPAC) to consider whether data from human challenge studies in U.S. subjects could be sufficient to demonstrate efficacy of a cholera vaccine in travelers to endemic areas who are at high risk for contracting the disease.
In 1998, the VRBPAC agreed that human challenge studies could suffice to demonstrate efficacy of a cholera vaccine provided that studies were adequate and well-controlled and conducted under the provisions of good clinical practices.
21 In 2016, the FDA approved Vaxchora, a live, attenuated vaccine for the prevention of cholera in adults traveling to cholera-affected areas.
Efficacy of Vaxchora was demonstrated in a controlled human challenge study in adult U.S. volunteers.
Safety is one of the most important considerations when evaluating new vaccines and modifications to currently multiple Phase I or Phase II trials may be performed as new data are obtained.
Phase I trials are intended primarily to provide a preliminary evaluation of safety and immunogenicity.
These trials are typically conducted in a small number (e.g., 20 to 80) of closely monitored adult volunteers.
If the ultimate target population for the vaccine is infants or young children, as is commonly the case, the product is usually evaluated in a stepwise progression from older to younger age groups down into the first year of life.
Phase II studies can involve up to several hundred participants, are often randomized and wellcontrolled, and provide further information on safety and immunogenicity and optimal dose.
In some cases, Phase II studies may provide preliminary data on the vaccine's activity against the infectious disease of interest.
Phase III studies are large-scale trials to provide a more thorough assessment of safety as well as a definite assessment of efficacy.
The general considerations for clinical studies to support licensure of a vaccine include demonstration of safety, efficacy (immunogenicity may be sufficient in some cases), and evaluation of simultaneous administration with other licensed vaccines.
Vaccine efficacy should be demonstrated, ideally in randomized, double-blind, well-controlled trials.
The end points are product specific, and may be clinical disease end points or immune response end points if efficacy against clinical disease had been previously established and there are immune correlates or surrogates of that protection.
In recent years, efficacy trials for various vaccines have involved a broad range in the number of study participants, from thousands to tens of thousands.
This broad range is related to a number of interconnected variables such as study design and the incidence of the disease to be prevented.
For example, clinical disease end point studies that are designed to demonstrate that a new vaccine is noninferior to an already existing product of the same type generally require larger numbers than one in which a new vaccine can be compared with a control that has no activity against the clinical disease.
The incidence of the disease to be prevented in the study population is also important.
As an example, a trial to show that pneumococcal submission, including methods for presenting the data; (c) information on the status of needed or ongoing studies; and (d) any other information for discussion at the meeting.
The primary purpose of this exchange is to uncover any major unresolved problems; identify those studies that the sponsor is relying on as adequate and well-controlled to establish the product's effectiveness; identify the status of ongoing studies; acquaint FDA reviewers with the general information to be submitted in the BLA (including technical information); review methods used in the statistical analysis of the data; and discuss the best approach for the presentation and formatting of data in the application.
At the time that an applicant submits a BLA to the Director of CBER's Office of Vaccines Research and Review precise production methods and procedures should be defined, and the manufacturing process should be standardized.
Critical information to be contained in the BLA include data derived from nonclinical laboratory and clinical studies that demonstrate that the manufactured product meets prescribed requirements for safety, purity, and potency.
The BLA should contain information that supports compliance with standards addressing requirements for (a) organization and personnel; (b) buildings and facilities; (c) equipment; (d) control of components, containers, and closures; (e) production and process controls; (f) packaging and labeling controls; (g) holding and distribution; (h) laboratory controls; and (i) records to be maintained.
Furthermore, a full description of manufacturing methods; data establishing stability of the product through the dating period; sample(s) representative of the product for introduction or delivery for introduction into interstate commerce; summaries of test results performed on the lot(s) represented by the submitted sample(s); specimens of the labels, enclosures, and containers; and the address of each location involved in the manufacture of the biological product should be included in the BLA.
An application for a biologics license is not considered as filed (or accepted by the agency for review) until CBER determines that it has received all pertinent information and data from the applicant.
In this regard, CBER can refuse to file a BLA if it deems the submission to be incomplete.
Additionally, the manufacturing facility must be inspection-ready at the time the BLA is submitted.
The applicant is also required to include either an environmental assessment or a claim for categorical exclusion from the requirement to submit an environmental assessment or an environmental impact statement.
Other components of the BLA review include product labeling, which describes the indications for use, contraindications, dosage and possible adverse effects; protocols for the manufacturing and testing of the number of product lots specified to establish the consistency of the process; and confirmatory testing results within CBER of samples of in-process material or product in final containers and conformance to existing regulations.
An internal CBER multidisciplinary committee performs the scientific review of the BLA.
This process occurs for each BLA or supplement to a BLA in which significant changes are proposed.
During the review, there are discussions and exchanges of correspondence between the sponsor and the CBER review committee concerning issues that may arise.
During the FDA review of the BLA an announced prior approval inspection (PAI) of the manufacturing facility is performed.
This inspection is designed as an in-depth review of the facilities, records, total production process, methods, equipment, quality control procedures, and personnel.
With the implementation of the BLA process, changes have occurred in the scope of issues reviewed during the PAI.
Instead of the manufacturer submitting detailed records with the BLA regarding studies on cleaning validation, monitoring data for licensed vaccines.
The initial responsibility for determining vaccine safety starts with clinical investigators and vaccine manufacturers.
The FDA is responsible for assuring that clinical trials are done under good clinical practices, a requirement essential for the evaluation of safety data intended to support a license application.
In general, when evaluating safety, one must compare the risk of the vaccine-preventable disease with the risk of the adverse event(s) potentially associated with the vaccine, and these may change over time.
As an example, the reported association between Rotashield (rotavirus vaccine, live, oral, tetravalent, manufactured by Wyeth) and intussusception resulted in the additional requirement for the evaluation of the safety of RotaTeq (live, oral pentavalent human-bovine reassortant rotavirus vaccine, manufactured by Merck) with respect to intussusception.
This clinical trial enrolled more than 70,000 infants divided equally between RotaTeq and placebo.
The primary safety hypothesis was that the oral RotaTeq would not increase the risk of intussusception relative to placebo within 42 days of any dose.
The intended target population should be taken into consideration in assessing the adequacy of the safety database.
For routinely administered childhood vaccines in the United States, the target population would be the birth cohort in the United States (approximately 4 million/year).
This is generally a healthy population, and a government body (e.g., state or local governments) may mandate vaccination.
Common reactions can be studied adequately in hundreds of individuals, but many thousands will be required to define low-incidence adverse reactions.
For vaccines evaluated in clinical end point efficacy trials, a large safety database likely will derive from a double-blind, randomized, well-controlled efficacy study.
However, for vaccines evaluated in immunogenicity end point studies, additional studies likely will be needed to obtain an adequate safety database.
Additional controlled safety studies are often requested when the numbers of subjects included in the efficacy studies are deemed insufficient to provide adequate safety data.
Safety studies may be unblinded if the number of injections, route of administration, or schedule differs between groups, in particular when infants and young children are involved.
Phase II safety studies should provide data on common local and systemic reactions to the study vaccine.
Phase II clinical development also should include immunogenicity and preliminary safety data on the concurrent administration of the study vaccine with other vaccines, if relevant.
Phase III safety studies are designed to evaluate less common reactions, may be unequally randomized, and may have a simplified trial design for assessing less-common adverse events in large trials.
If a vaccine is recommended on the same schedule as other routinely recommended vaccines, safety and immunogenicity data should be obtained in prelicensure studies to support simultaneous administration.
Following completion of IND studies demonstrating the safety and efficacy of the vaccine for a specific use and population the sponsor can submit a BLA to obtain a license for a new vaccine under section 351 of the PHS Act for commercial manufacture and distribution of the product.
Prior to the submission of a BLA, a pre-BLA meeting with the FDA is strongly encouraged to discuss the sponsor's product development plan.
For the FDA to provide sponsors with advice regarding the adequacy of information to support a BLA, the following information in advance of the pre-BLA meeting should be submitted, depending on the type of meeting: (a) an executive summary of the clinical studies to be submitted in the application; (b) a proposed format for organizing the strate safety and efficacy in humans are required at the time of use.
Under new Section 505(o) of the FD&C Act, the FDA is authorized to also require postmarketing studies or clinical trials at the time of approval or after approval if the FDA becomes aware of new safety information.
Section 505(o)(3) (B) states that postmarketing studies and clinical trials may be required to (a) assess a known serious risk related to the use of the drug involved, (b) assess signals of serious risk related to the use of the drug, and (c) identify an unexpected serious risk when available data indicate the potential for a serious risk and when the adverse event reporting system is not adequate.
The FDA has defined a clinical trial as any prospective investigation in which the sponsor or investigator determines the method of assigning treatment or other intervention to one or more human subjects.
A study is all other investigations, such as investigations using humans, that are not clinical trials as defined above (e.g., observational epidemiologic studies), animal studies, and laboratory experiments.
The FDA has issued guidance for industry to describe the type of studies and clinical trials that are required (Post Marketing Requirement [PMR]) under the FDAAA 2007, and those that will remain agreed-upon postmarketing commitments.
A PMR describes all required postmarketing studies or clinical trials including those required under Accelerated Approval, PREA, the Animal Rule, and FDAAA.
Examples of required studies are pharmacoepidemiologic studies designed to assess a serious risk, trials with a primary safety end point, preclinical studies investigating specific end organ toxicities, as well as pharmacokinetic studies in the indicated population at potential risk for high drug exposure that could result in toxicity.
Studies that generally would not be considered required postmarketing studies or clinical trials are agreed-upon studies (postmarketing commitments) and include biologic quality studies (such as manufacturing, stability, and immunogenicity studies that do not have a primary safety end point), trials in which the primary end point is related to further defining efficacy, and pharmacoepidemiologic studies designed to examine the natural history of disease or background rates for adverse events.
Since passage of FDAAA 2007, several new vaccines have been approved with either PMRs or postmarketing commitments.
The FDA has the authority to monitor the progress of postmarketing studies or trials by requiring the applicant to submit an annual status report.
Applicants are required to provide a timetable for study completion, a periodic status report on the status of the study including whether enrollment has begun, the number of participants enrolled, the expected completion date, and whether any difficulties in completing the study have been encountered.
The FDA is responsible not only for approving vaccines but also for monitoring their safety postlicensure.
Because of the relatively small size of most prelicensure trials, rare adverse events are unlikely to be detected.
Consequently, postlicensure or postmarketing surveillance (i.e., the continued monitoring of vaccine safety in the general population after licensure) is critical for identifying and evaluating rare or uncommon adverse events.
An adverse event refers to any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug related.
The Vaccine Adverse Event Reporting System (VAERS) is a national system for passive surveillance of adverse events following vaccination.
Established in 1990 as a result of the National Childhood Vaccine Injury Act of 1986, VAERS is administered jointly by the FDA and the CDC and, in recent years, has received more than pharmaceutical-grade water, facility support systems (e.g., clean steam, compressed air, and building management systems), and other facility-related systems, a more detailed review of this type of data is done onsite during the PAI.
PAIs tend to require longer periods of time for the FDA inspectors to be in the facility because of the increased scope of issues that are reviewed onsite.
If licensure is denied following inspection for the original license application, reinspection will occur after receipt of assurance that all deficiencies that were the basis of the denial were corrected.
With the implementation of FDAAA (2007) and FDASIA (2012), in addition to completing the discipline reviews during the PDUFA V mandated timelines, the review committee must complete numerous additional tasks.
These include, but are not limited to, review committee assignments, internal information exchange meetings, filing decisions, presentation of the application to the PeRC, midcycle review meetings and midcycle communication with the applicant, late-cycle meetings with the applicant, and presentation of planned or required postmarketing studies to an internal FDA safety committee.
After CBER reviews the entire package of information in the BLA, its advisory committee (the VRBPAC) and consultants, if needed, are asked to review and comment on the adequacy of the data to support safety and efficacy in the target population.
The standards for safety and efficacy are relative; that is, the benefit-to-risk ratio of a biological product is considered.
The VRBPAC's advice is considered in CBER's decision regarding licensure, and in developing recommendations for use to be given in the package insert.
The committee may recommend additional studies to be performed either before or after approval.
Once CBER determines that the data and information from the applicant are satisfactory and support the safety and efficacy of the product, the product is licensed.
If the manufacturer wishes to significantly modify the approved manufacturing process or directions for vaccine use, prior approval must be obtained from the FDA before these changes can be implemented.
The applicant is required to submit an account of these changes to the appropriate license applications.
Modifications to the manufacturing process may occur post licensure, such as scale-up or change in equipment to optimize the production process.
Furthermore, clinical studies with the product also may be performed after licensure as the manufacturer seeks additional indications for product use (e.g., new target populations that would benefit from vaccination).
For most new approvals, manufacturers may be asked to commit to completing specific postmarketing or so-called Phase IV studies, for example, to provide additional assessments of less-common or rare adverse events or further assess the duration of vaccine-induced immunity.
These studies may also be designed to collect additional safety data in large numbers of vaccine recipients, as well as focus on issues that were identified during the prelicensure testing.
Submission of status reports for certain postmarketing studies are required by regulation.
In particular, this requirement for status reports pertains to postmarketing studies for clinical safety, efficacy and pharmacokinetics, and nonclinical toxicology to which an applicant committed in writing prior to licensure.
22 Prior to FDAAA 2007, the FDA required postmarketing studies in the following situations (a) accelerated approvals for products approved under 505(b) of the FD&C Act or Section 351 of the PHS Act, respectively, which require postmarketing studies to demonstrate clinical benefit, (b) deferred pediatric studies, where studies are required under PREA, and (c) Animal Efficacy Rule approvals, where studies to demon-lesser importance for which the manufacturer must provide notification 30 days before distribution of product made using the change; and (c) changes for which the manufacturer need only notify the agency by submission of an annual report.
The guidance document, "Changes to an Approved Application: Biological Products" (1997) , provides examples of changes that fall into these categories.
26 Following approval, there is continued surveillance of the product and of the manufacturer's production activities.
For most licensed vaccines, samples are submitted along with protocols for each lot prepared by the firm that provide the details of production and a summary of test results.
Although not required by law or regulation, CBER often performs selected laboratory tests.
The type and extent of confirmatory testing performed by CBER depend on several factors, such as the newness of the product or the difficulties that may have arisen with manufacture or use of the product.
Release or rejection is based on a review of all test results, including those done by the manufacturer and those performed by CBER.
Alternatives to official lot release are allowable under the provisions outlined for extensively characterized products having a track record of continued safety, purity, and potency.
27 A manufacturer must be able to produce a vaccine that repeatedly meets the standards for potency, purity, and stability of bulk and final container material while using a consistent process.
Important factors to be considered are the nature of the product with respect to correlation between the measure of potency and biological activity and effectiveness.
Surveillance samples and protocols may be required to be submitted to CBER at predetermined intervals.
Licensed establishments are inspected at least every 2 years.
The purpose of the inspection is to determine whether licensed products are manufactured and tested as described in the license application and in accordance with applicable regulations.
Manufacturers who fail to meet product standards or who are not in compliance with CGMPs may have their licenses suspended or revoked, depending on the nature of the potential health hazards created.
The major issues observed during inspections can be categorized in three major areas: (1) process-related issues, (2) quality unit-related issues, and (3) facility-and production environment-related issues.
Some examples of process validation issues include lack of documentation of time limits for major steps in the production process, lack of validation of rework or reprocessing steps in the manufacturing process, and lack of data to support in-process specifications.
Quality unit-related issues include the appropriate reporting of out-of-specification results and process deviations (including adequate investigations into causes), appropriate documentation of product release, and adequate training of personnel.
Facility and production monitoring concerns include controlling production environments by appropriately monitoring heating, ventilation, and air conditioning (HVAC) system performance and microbial quality (e.g., pressure differentials, appropriate sampling sites, and frequency of sampling).
Other concerns pertaining to the facility include adequate cleaning, sanitization, storage, and changeover procedures for multiproduct areas and equipment.
If the inspection team finds CGMP deficiencies in an already licensed facility, the team may remain in the facility until they have achieved an audit that provides confidence in the ability of the firm to reproducibly manufacture a safe and potent product.
Mechanisms for providing earlier access to vaccines to prevent or treat severe and life-threatening illness have been developed.
30 ,000 reports per year.
The purpose of VAERS is to detect possible signals of adverse events associated with vaccines to help ensure the safety of U.S.-licensed vaccines.
VAERS collects and analyzes information from reports of adverse events that occur after the administration of U.S.-licensed vaccines.
Reports are submitted by healthcare providers, vaccine recipients or their parents or guardians, vaccine manufacturers, and other interested parties.
FDA medical officers review all serious reports (defined as events that are fatal, disabling, or lifethreatening; require or prolong hospitalization; result in congenital anomalies; require medical intervention to prevent such outcomes; or are deemed to be other medically important conditions).
The VAERS system is not limited to routinely recommended pediatric vaccines; voluntary reports of adverse events occurring after administration of any vaccine are also accepted.
FDA and CDC continually monitor VAERS reports for any unexpected patterns of adverse events.
Another important mechanism used by CBER to monitor adverse events is the Vaccine Safety Datalink, a collaborative effort between CDC's Immunization Safety Office and nine healthcare organizations.
The Vaccine Safety Datalink uses electronic health data from participating sites and conducts vaccine safety studies based on questions or concerns raised from the medical literature and VAERS reports.
When there are new vaccines that have been recommended for use in the United States, or if there are changes in how a vaccine is recommended, the Vaccine Safety Datalink will monitor the safety of these vaccines.
23 The FDA's Sentinel Initiative was launched in 2008 in response to a Congressional mandate in the FDAAA of 2007.
The Sentinel Initiative aims to develop and implement a proactive system that will complement existing systems that the FDA has in place to track reports of adverse events linked to the use of its regulated products.
It is a national electronic system that is transforming the FDA's ability to track the safety of drugs, biologics, and medical devices once they reach the market.
24 The Post-Licensure Rapid Immunization Safety Monitoring system (PRISM), a component of the FDA's Sentinel Initiative dedicated to vaccines, uses the FDA's Sentinel Distributed Database, which includes a population exceeding 178 million.
PRISM monitors the largest U.S. general population cohort designated for active surveillance of vaccine safety by linking data from health plans with data from state and city immunization registries.
The FDA structured PRISM as a program that includes specific vaccine evaluations.
For example, several vaccines including a human papillomavirus vaccine, Gardasil, and two rotavirus vaccines, RotaTeq and Rotarix, were chosen for surveillance because their evaluations would benefit most from PRISM's large cohort size.
In 1997, the FDA published a Final Rule, Changes to an Approved Application, which amended 21 CFR §201.12 and §314.70 to simplify and categorize manufacturing reporting requirements for changes in testing methods, equipment, facilities, or personnel.
25 Proposed changes in manufacturing methods that have a substantial potential to have an adverse effect on the safety or effectiveness of the product may not become effective until notification is given of CBER's approval.
The changed created the following categories: (a) those sufficiently significant with regard to safety, purity, potency, and effectiveness of the product to require preapproval of a supplemental application before product distribution; (b) those of Feasible.
This rule, referred to as the "Animal Rule," allows the use of animal efficacy data in lieu of human efficacy data when human challenge studies cannot be conducted ethically and field efficacy studies are not feasible because of infectious disease epidemiology (in the case of vaccines).
In these situations, certain drug and biological products (e.g., vaccines) that are intended to reduce or prevent serious or life-threatening conditions caused by lethal or permanently disabling toxic chemical, biological, radiologic, or nuclear substances may be approved for marketing based on evidence of effectiveness derived from appropriate studies in animals and additional supporting data.
Safety, pharmacokinetics, and immunogenicity data are still necessary in humans.
Under the animal rule, the FDA licensure of a product for which safety has been established and the requirements of 21 CFR §601.60 have been met is based upon adequate and well-controlled animal trials, when results of these animal studies establish that the product is reasonably likely to provide clinical benefit to humans.
The FDA can rely on the evidence from animal studies to provide substantial evidence of the efficacy of these products when: 1.
There is a reasonably well-understood pathophysiological mechanism for toxicity of the chemical, biological, radiologic, or nuclear substance and its amelioration or prevention by the product.
2.
The effect is demonstrated in more than one animal species that is expected to react with a response that is predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently wellcharacterized animal model (in other words, the model has been adequately evaluated for its responsiveness) in predicting the response in humans.
3.
The animal end point is clearly related to the desired benefit in humans, which is generally the enhancement of survival or prevention of major morbidity.
4.
The data or information on the pharmacokinetics and pharmacodynamics of the product or other relevant data or information in animals and humans is sufficiently well understood to allow selection of an effective dose in humans, and it is reasonable to expect the efficacy of the Breakthrough therapy designation provides for increased interaction with FDA to expedite the development and review of the application.
In contrast to Fast Track designation, a breakthrough therapy designation requires evidence of substantial improvement over current treatments.
Products regulated by CBER are eligible for priority review if they provide a significant improvement in the safety or effectiveness of the treatment, diagnosis, or prevention of a serious or life-threatening disease.
The FDA has 8 months to complete the review of a new BLA it designates as a priority, as opposed to 12 months for the completion of the review of a standard BLA submission.
Under the FDA's traditional approval pathway, a demonstration of vaccine effectiveness is based on a clinical disease end point (e.g., prevention of disease) or, alternatively, an accepted correlate of protection.
In addition to these programs, the FDA's regulations provide for expedited pathways for licensure.
Accelerated approval, 21 CFR §601.40, may be granted for certain biological products that have been studied for their safety and effectiveness in treating a serious or lifethreatening disease or condition and that provide meaningful therapeutic benefit over existing treatments.
Such an approval is based on adequate and well-controlled clinical trials establishing that the product has an effect on a surrogate end point that is reasonably likely to predict clinical benefit or on a clinical end point that can be measured earlier than irreversible morbidity or mortality that is reasonably likely to predict an effect on irreversible morbidity or mortality or other clinical benefit.
Approval under this pathway is subject to the requirement that the sponsor study the biological product further, to verify and describe its clinical benefit, where there is uncertainty as to the relation of the surrogate end point to clinical benefit.
Of note, the FDASIA of 2012 provided that evidence to support an end point is reasonably likely to predict clinical benefit to include "epidemiological, pathophysiological, therapeutic, pharmacologic, or other evidence developed using biomarkers, for example, or other scientific methods or tools."
In other words, FDASIA expanded the scope of available end points that can be used to demonstrate that a product qualifies for accelerated approval, but do not affect the quantity and quality of evidence needed to demonstrate substantial evidence of effectiveness or safety.
Two vaccines to protect against meningococcal B diseases were licensed using the accelerated approval provisions and were designated breakthrough therapy.
While the incidence Vaccines are tested during both the prelicensure and the postlicensure phases.
Testing procedures are developed with the goals of controlling and minimizing the potential for productrelated adverse events by taking into consideration the experience gained from the same or related products.
As an example, for inactivated vaccines, a clear understanding of the kinetics of inactivation is critical.
For live vaccines, attenuation must be stable both to avoid reversion to virulence and to avoid the vaccine becoming over attenuated and, as a consequence, less potent.
For example, during the first decade of its widespread use, Yellow Fever 17D vaccine virus was serially propagated in eggs, as required to meet demand.
However, it soon became obvious that the level of attenuation of the vaccine from one passage to the next could vary considerably.
Some lots were excessively neurovirulent, especially in infants and young children, whereas successive lots might by chance be overattenuated and, consequently, not immunogenic.
The WHO formulated a solution to this problem, which was to adopt a "seed-lot system" wherein the vaccine is prepared from a master seed virus at a specified passage number in eggs.
Working seed virus is prepared by one passage of the master seed and is, in turn, used to generate all production lots.
All 17D vaccines and all other live virus vaccines now adhere to a seed-lot system for manufacture.
The FDA requires that cell substrates and vaccine viral seeds used in production be appropriately selected and tested to ensure that they do not introduce any unintended risks.
28 This document provides manufacturers of viral vaccines with guidance for the characterization and qualification of cell substrates, viral seeds, and other biological materials used for the production of viral vaccines for human use to assure that they meet the highest safety standards achievable using modern technology.
Characterization of cell substrates should address certain general issues that might affect the safety and purity of vaccine products.
For example, in the early 1960s, exogenous and endogenous contamination product in animals to be a reliable indicator of its efficacy in humans.
The animal rule does not apply if the product can be approved based on standards described elsewhere in FDA regulations (e.g., accelerated approval based on surrogate markers or clinical end points other than survival or irreversible morbidity).
Emergency use authorization (EUA) is another regulatory mechanism by which the FDA can accelerate the availability of vaccines and other pharmaceutical products.
Under an EUA, the FDA can authorize the use of an unapproved product or the unapproved use of an approved product when an emergency or a potential emergency exists.
Section 564(b)(1) of the Federal FD&C Act was amended by the Project BioShield Act of 2004 to allow the Secretary of Health and Human Services (Secretary) to authorize the introduction into interstate commerce of a drug, device, or biological product intended for use in an actual or potential emergency.
Before an EUA may be issued by FDA, the Secretary must declare an emergency justifying the authorization based on: • A determination by the Secretary of Homeland Security that there is a domestic emergency or a significant potential for an emergency that involves a heightened risk of attack with a specified biologic, chemical, radiological, or nuclear agent or agents; or • A determination by the Secretary of Defense that there is a military emergency or a significant potential for an emergency that involves a heightened risk of attack with a specified biologic, chemical, radiological, or nuclear agent or agents; or • A determination by the Secretary of Health and Human Services of a public health emergency under section 319 of the PHS Act that affects or has the significant potential to affect national security and that involves a specified biological, chemical, radiological or nuclear agent or agents or a specified disease or condition that may be attributable to such agent(s).
Once the Secretary declares an emergency, the FDA can authorize the emergency use of a particular product if the other statutory criteria and conditions are met.
Based on the particular circumstances, the process for authorization can be expected to range in duration from hours to days.
The Secretary has delegated the authority to issue an EUA under Section 564 of the FD&C Act to the FDA Commissioner.
facture of many biotechnology-derived biological products.
Thus, the FDA has published a final rule to remove this requirement.
In addition to the tests required by regulation, other tests tailored to the specific product may be required (e.g., neurovirulence testing and cell culture and animal tests for extraneous viruses).
Once the product is licensed, the manufacturer's testing must be conducted according to the exact specifications in the manufacturer's license application, and the results of these tests must be within the specified prescribed limits.
Prescription drug labeling, also known as the package insert, package circular, or prescribing information, is the primary mechanism through which the FDA and drug manufacturers communicate essential, science-based prescribing information to healthcare professionals.
Labeling provisions contained in 21 CFR § §201.57 and 201.56 require that prescribing information must summarize the essential information on the safe and effective use of the product; that information contained in the labeling must be accurate and not false and misleading; and that there must be no implied claims or suggestions for use if evidence of safety or effectiveness is lacking.
32 Whenever possible, data contained in labeling should be derived from human experience.
In the United States, the FDA regulates the format and content of labels for product containers, cartons, and the package insert that accompanies the product.
In January 2006, the FDA issued a final drug labeling rule, commonly referred to as the Physicians' Labeling Rule, amending the content and format of prescribing information for human drug and biologic products.
The new format is intended to provide healthcare professionals with clear and concise prescribing information by reorganizing critical information into a streamlined format.
Moreover, these revisions make it simpler for healthcare professionals to access, read, and use prescribing information, and enhance the safe and effective use of prescription drug products.
New sections were added to the label, such as the Highlights section, which contains key benefit and risk information, and a table of contents for the full prescribing information.
On December 3, 2014, the FDA issued the Pregnancy Lactation and Labeling Rule, which revises the content and format of the pregnancy subsection of labeling for prescription drugs and biologics (Sections 8.1 to 8.3 of the prescribing information).
Previous regulations required that each product be classified under one of five pregnancy categories (A, B, C, D, or X) based on the risk of reproductive and developmental adverse effects or, for certain categories, such risk weighed against potential benefit.
The most significant change proposed by the rule is the replacing of these letter risk categories with a narrative summary of the risks and benefits of using a drug during pregnancy, based on the available human and/or animal data and a discussion of the data.
Additionally, the rule requires that prescription drug labeling include relevant clinical information to help healthcare providers make prescribing decisions and counsel women about the use of drugs during pregnancy and/or lactation.
The structured product labeling defines the content of human prescription drug labeling in XML format.
It is a document markup standard approved by Health Level Seven and adopted by the FDA as a mechanism for exchanging product and facility information.
33 Structured product labeling documents contain both the content of labeling (all text, tables, and figures) for a product and additional machine-readable information (drug listing data elements).
Drug listing data elements include information about the product (product and generic names, ingredients, ingredient strengths, dosage forms, of primary monkey kidney cells with simian virus 40 and chick embryo fibroblasts with avian leukosis virus were reported.
Although chick embryo fibroblasts are still used for the production of viral vaccines, these cell substrates must be well-characterized and tested to assure absence of potentially infectious agents.
Issues related to cell substrates have been discussed in a variety of forums.
[29] [30] [31] Furthermore, if a vaccine is manufactured in a cell substrate that is derived from a tumor, or that has developed a tumorigenic phenotype through an unknown mechanism, it was considered to carry a higher theoretical risk of containing oncogenic substances such as oncogenic viruses and cellular DNA, derived from that cell substrate.
This was the main reason tumorigenic cells or cells derived from human tumors had been considered unsuitable as cell substrates.
However, at a 2012 meeting of the VRBPAC, experts recognized that cell lines derived from human tumor are an important tool for manufacturing of vaccines and could provide a wider repertoire of cell substrates for vaccine production.
30 They indicated that risk-mitigation strategies are the same for vaccines generated using human tumor-derived cell lines as for other cell substrates.
A thorough characterization of the cell substrate with respect to adventitious viruses, which includes oncogenic viruses, should be done using new virus-detection technologies such as massively parallel sequencing, virus microarrays and broad-range polymerase chain reaction to complement existing assays.
The manufacturing process should lower the amount and reduce the size of the DNA.
Residual DNA for continuous nontumorigenic cells, such as low-passage Vero cells, should be limited to less than 10 ng/dose for parenteral inoculation and to less than 100 µg/dose for oral vaccines.
Cells with tumorigenic phenotypes or other characteristics that give rise to special concerns may require more stringent limitation of residual DNA quantities and size to assure product safety.
The regulation of biologicals includes requirements for testing of licensed products (21 CFR §610).
These tests include those for bacterial and fungal sterility, general safety, purity, identity, suitability of constituent materials and potency, thereby this specific testing performed may vary depending on the vaccine.
For example, tests for potency may be based on studies of immunogenicity or, for some vaccines, protection from virulent challenge in laboratory animals.
However, in vitro tests, including virus titration (e.g., live vaccines such as polio, measles, mumps, and rubella), antigen content (e.g., influenza and inactivated poliovirus vaccines), and biochemical and biophysical measurements (e.g., meningococcal conjugate vaccines) have been used.
Tests for purity are designed to determine that the product is free of extraneous material, except that which is unavoidable in the manufacturing process described in the approved license application.
Tests for residual moisture and pyrogenic substances may also be included.
Final-container material must be identified by a test specific for each product (e.g., neutralization of each of the components of live measles, mumps, and rubella vaccine with specific antisera).
With regard to constituent materials, the manufacturer must ensure that all ingredients used in the product, such as diluents, preservatives, or adjuvants, meet generally accepted standards of purity.
An adjuvant may not be used unless there is adequate proof that it does not adversely affect the safety or potency of the product.
Of note, the FDA periodically evaluates the appropriateness of testing requirements.
An example is the general safety test (required under 21 CFR §610.11) used to detect extraneous toxic contaminants that may be present in the product in the final container from every final filling of each lot of the biological product.
Technological advances have increased the ability of manufacturers to control and analyze the manu-unless there is satisfactory evidence that it does not adversely affect the safety or potency of the product."
From a regulatory perspective, adjuvants are not considered active ingredients as defined in 21 CFR §210.3(b)(7) and vaccine adjuvants are not licensed separately.
35 It is the adjuvanted vaccine formulation, in toto, that is tested in nonclinical and clinical trials and licensed.
There is a requirement that the adjuvanted vaccine formulation, as with any vaccine, must be both safe and effective, with its benefits outweighing the risks of adverse events that may occur.
However, there is no explicit requirement for demonstrating the added safety and effectiveness of the adjuvanted vaccine formulation over that of the unadjuvanted vaccine formulation in comparative clinical trials.
The regulatory considerations for the nonclinical and clinical development of preventive vaccines, as well as pathways to licensure described elsewhere in this chapter, are largely also applicable to vaccines formulated with adjuvants.
However, adjuvants can exhibit a range of properties that invoke complex immune responses, the mode of action is not always known or fully understood, and animal models that are relevant for evaluating both the safety and the efficacy of an adjuvantantigen combination are frequently not available.
Thus, there are some unique issues to be addressed during preclinical and clinical development of the adjuvanted vaccine formulation.
A WHO guideline published in 2013 describes the nonclinical, quality, pharmacological, toxicological, and other information needed to support initiation of clinical trials with a vaccine combined with a novel adjuvant.
36 In 2007, CBER published two guidance documents ("Guidance for Industry: Clinical Data Needed to Support Licensure of Pandemic Influenza Vaccines" 37 and "Guidance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines" 38 ).
Each of these documents discusses the development of adjuvanted inactivated influenza vaccines and notes that "[d]ata to support the safety of the adjuvanted formulation and added benefit over the unadjuvanted formulation must be submitted in the BLA…" 37, 38 Sponsors were advised that "at an early stage of development, clinical data supporting the value of adding the adjuvant should be provided…" 37, 38 The seasonal influenza vaccine guidance notes further that "[i]f an adjuvant is added to a licensed seasonal vaccine without antigen sparing effects…the immune response elicited by the adjuvanted formulation should be substantially better than that elicited by the unadjuvanted vaccine…" 38 As the FDA's experience with novel adjuvants has grown, the agency continues to reexamine guidance and engages with sponsors and applicants regarding the clinical development of adjuvanted vaccines.
Vaccine manufacturers should provide a rationale for the use of an adjuvant in the vaccine.
The "added benefit" of an adjuvant may be defined as evidence of enhanced immune response, antigen-sparing effect, dose sparing, increased breadth of immune response, or superior clinical efficacy.
Information to support the "added benefit" of the adjuvant may be derived from preclinical studies, for example, in vitro assays and/or proof-of-concept studies in animal models conducted prior to the initiation of clinical trials or early phase clinical trials.
There is no regulatory requirement to demonstrate the "added benefit" of an adjuvant in clinical comparative Phase III effectiveness trials unless the applicant plans to make a claim of superiority of the adjuvanted product over unadjuvanted product.
The benefits from incorporating or adding an adjuvant to any vaccine formulation need to be balanced with the risk of adverse reactions.
Adjuvants have their own pharmacological activity, which may affect both the immunogenicity and the safety of vaccines.
Adverse reactions may include local routes of administration, appearance, Drug Enforcement Agency schedule) and the packaging (package quantity and type).
During the BLA review, the agency considers the draft labeling and clinical studies submitted by the manufacturer, and the proposed indication for the licensed product, which is based on clinical data submitted by the sponsor demonstrating the safety and effectiveness of the product for its intended use and target population.
Subsequently, significant changes in labeling, including new indications for use, new dosage forms or regimens, expanded patient populations who receive the product and additional information regarding safety and effectiveness, require manufacturers to submit a supplemental filing for review and approval by CBER.
Unlike other product labeling, the promotional labeling and advertising are not subject to preclearance; however, they are monitored for misleading claims.
These documents must also meet the standard of fair balance, that is, claims of efficacy are balanced with information about the product's safety.
Labeling changes are usually initiated by the manufacturer but may be initiated by CBER.
Historically, manufacturers have had to obtain prior approval from CBER before the labeling changes were made.
The changes to 21 CFR §601.12, mentioned previously, also apply to labeling changes and allow exceptions for a change that adds or strengthens a contraindication, warning, precaution, or adverse reaction; adds or strengthens instructions about dosage and administration intended to increase safe use; or deletes false, misleading, or unsupported indications for use or effectiveness claims.
Under this regulation, a manufacturer could effect such changes and, at the same time, submit them and the supporting data to CBER without preapproval.
As described earlier, the FDAAA amended the FD&C Act by adding new Section 505(o) authorizing FDA to require and, if necessary, order labeling changes if the FDA becomes aware of new safety information that the FDA believes should be included in the labeling of the drug.
Section 505(o)(4) of the FD&C Act imposes timeframes for application holders to submit and for FDA staff to review such changes, and gives the FDA new enforcement tools to bring about timely and appropriate safety labeling changes.
Strategies and approaches for the development and delivery of vaccine antigens have expanded over the last several decades, leading to a broad range of novel products comprised of purified subunit antigens or subunit proteins.
These antigens may require the presence of adjuvants to enhance the immune response to the vaccine antigens, reduce the dosing frequency, induce cross-protective effects, direct the immune response and/or achieve antigen sparing.
The number of investigational vaccines containing novel adjuvants evaluated in clinical trials has increased in recent years and some vaccines containing novel adjuvants have been licensed by the FDA.
For example, the human papillomavirus vaccine manufactured by GlaxoS-mithKline, Cervarix, contains AS04, an adjuvant system comprised of an aluminum hydroxide and monophosphoryl lipid A. GlaxoSmithKline's pandemic influenza vaccine, Q-Pan, contains AS03, an adjuvant system comprised of an oil-inwater emulsion.
The CFR defines adjuvants, together with ingredients, preservatives, and diluents as constituent materials (21 CFR §610.15).
34 These regulations state, "All ingredients…shall meet generally accepted standards of purity and quality" and that, "An adjuvant shall not be introduced into a product infectious diseases (EIDs)-from pandemic influenza to novel pathogens like severe acute respiratory syndrome and Ebola, to biological threats that have the potential to be intentionally released into the general population by humans-also pose a threat to global public health.
Vaccines will continue to be an important medical countermeasure against a broad range of infectious diseases from anthrax, smallpox, and influenza to newly emerging infectious diseases.
Moreover, infectious diseases, such as tuberculosis and malaria present global public health challenges and increasing resistance to currently available treatments by common bacteria such as staphylococci, underscores the importance of the development of safe and effective vaccines.
The development of safe and effective vaccines to protect against global infectious diseases (e.g., tuberculosis, malaria, HIV/ AIDS), enteric diseases, and other neglected diseases of the developing world is of critical public health importance.
Development and availability of such vaccines, particularly for use in the developing countries most affected by these diseases, will benefit U.S. and global health.
In 2011, the FDA published a revised guidance document to assist sponsors in developing vaccines targeted against infectious diseases or conditions endemic in areas outside the United States.
The FDAAA of 2007 revised the FD&C Act by adding Section 524, recognizing the importance of having products to treat and prevent tropical diseases that disproportionately affect poor and marginalized populations and for which there is no significant market in developed nations.
Under Section 524, the Agency can grant priority review of applications under Section 505(b)(1) of the FD&C Act or Section 351 of the PHS Act for the treatment and prevention of specified tropical diseases, including tuberculosis, malaria, cholera, and "any other infectious disease for which there is no significant market in developed nations and that disproportionately affects poor and marginalized populations, designated by regulation by the Secretary."
Consequently, this guidance provides general recommendations for regulatory pathways to use in the development of vaccines to protect against global infectious diseases for U.S. licensure and clarifies applicable regulations: (a) the FDA can license vaccines to protect against infectious diseases or conditions that are not endemic or have not been reported to occur in the United States; (b) the regulatory pathways to U.S. licensure for the development of vaccines to protect against infectious diseases that are not endemic or have not been reported to occur in the United States are the same as for vaccines to protect against diseases that are endemic in the United States; (c) a sponsor may submit data from clinical trials conducted outside the United States to support product licensure, (d) noting that the accelerated approval regulations may be used in appropriate cases; and (e) that when pivotal studies are conducted outside the United States, in some instances, it may not be necessary to conduct studies in the United States.
This guidance also responded to the congressional mandate in Section 740 of the fiscal year 2010 Appropriation Act (Agriculture, Rural Development, Food and Drug Administration, and Related Agencies Appropriations Act, 2010, Public Law 111-80) by requiring the FDA to make recommendations on appropriate preclinical, trial design, and regulatory paradigms to prevent, diagnose, and treat rare diseases and neglected diseases.
The U.S. military has implemented vaccination programs to protect troops against several biological threats; however, the risk-to-benefit ratio for protecting civilians against agents of bioterrorism is more difficult to assess.
As of this writing, there is one licensed smallpox vaccine in the United States, Sanofi Pasteur Biologics' ACAM2000.
New smallpox vaccines are being developed under IND applications, with the goal to seek licensure, and new vaccinia immunoglobulin reactions such as pain, swelling, injection site necrosis, and granulomas.
Systemic reactions may include nausea, fever, arthritis, as well as potential immunotoxic reactions.
Unexpected, rare events may also occur.
For example, during the H1N1 influenza pandemic in 2009-10, an increased risk of narcolepsy, a chronic neurological disorder caused by the brain's inability to regulate sleep-wake cycles normally, was observed in several European countries following vaccination with an AS03 adjuvanted, monovalent 2009 H1N1 influenza vaccine, Pandemrix.
[39] [40] [41] [42] [43] The finding of narcolepsy in several European countries following vaccination with Pandemrix caused the European Medicines Agency to recommend restricting use of Pandemrix.
44 Pandemrix was not licensed for use in the United States and no adjuvanted influenza vaccines were used in the United States during the influenza pandemic.
The CDC published a study on a possible association between U.S.-licensed unadjuvanted 2009 H1N1 influenza vaccines, 2010-11 seasonal influenza vaccines, and narcolepsy.
45 The analysis included more than 650,000 people who received the pandemic influenza vaccine in 2009 and more than 870,000 people who received the seasonal influenza vaccine in 2010-11.
The study found that neither vaccine was associated with an increased risk for narcolepsy.
Additional studies including those conducted by the vaccine manufacturer will help discern whether this finding is attributable to the adjuvant, the H1N1 vaccine antigen, or both.
The safety of an adjuvanted vaccine formulation has to be demonstrated in adequate and well-controlled prelicensure safety studies.
Safety information supporting licensure of an adjuvanted vaccine may include the safety experience obtained from domestic or foreign trials conducted using the adjuvanted vaccine formulation.
In addition, safety experience with the same adjuvant formulated with other vaccine antigens may also contribute to the safety evaluation of the adjuvant.
Early in clinical development (e.g., Phase I and Phase II clinical trials), supportive safety data may be derived by comparing the adjuvanted vaccine to a placebo or the unadjuvanted vaccine antigen, if feasible.
Safety follow-up of human subjects administered vaccine with novel adjuvant is typically longer than for nonadjuvanted vaccines (e.g., 12 months rather than 6 months) and includes specific inquiries regarding symptoms consistent with autoimmune and neuro-inflammatory diseases.
Furthermore, the safety database required to support licensure of a vaccine formulated with novel adjuvant may be larger than for unadjuvanted vaccines.
In summary, regulatory pathways supporting development and approval of vaccines formulated with novel adjuvants are similar to those for unadjuvanted vaccines.
Efficient planning of the development pathway for any adjuvanted vaccine requires careful attention to preclinical testing, study design, dosing decisions, and safety monitoring.
Although manufacturers are not required to demonstrate the "added benefit" of adjuvanted versus unadjuvanted vaccines in clinical comparative Phase III studies, manufacturers should provide a justification for including an adjuvant in the vaccine.
Lastly, evaluation of safety of an adjuvanted vaccine needs to include special safety considerations.
Immunization programs in the United States have been remarkably effective at reducing morbidity and mortality from the most common, naturally transmitted infectious diseases, such as polio, measles, and diphtheria.
However, emerging conducted according to applicable regulations including the requirement for nonclinical testing and protection of human subjects.
These efforts resulted in rapid availability of safety and immunogenicity data for the leading Ebola vaccine candidates, initiation of U.S. government-sponsored Phase III clinical studies to demonstrate the safety and effectiveness of these vaccines in individuals residing in outbreak areas, and collection of data supporting licensure of these products.
To enable an initiation of these pivotal studies in an expedited manner, the FDA engaged in joint reviews with vaccine manufacturers, the WHO, regulatory agencies in Europe and Canada, and West African regulators to discuss study designs, ethical considerations, and required product information to reach international regulatory convergence, thus facilitating trial initiation.
It is not always possible to test whether a vaccine or treatment will work against a new or emerging infectious disease, or against a biothreat, because the threat may be rare or even nonexistent at the time the vaccine or therapy needs to be developed.
Moreover, many vaccines against EIDs and biothreats pose difficult challenges with regard to obtaining clinical efficacy data.
For many of these infectious agents or toxins, human efficacy trials are not feasible because natural exposure no longer occurs (e.g., smallpox), occurs at a very low incidence, or occurs in an unpredictable manner.
The animal rule described earlier (see "Accelerating Availability of Vaccines and Pathways to Licensure") is one regulatory mechanism that allows the FDA to address the challenges of obtaining clinical efficacy data for these products if the results of adequate and well-controlled animal studies establish that the product is reasonably likely to provide clinical benefits to humans.
Animal testing is often the only available option, but many diseases lack even good animal models, and animal studies are technically difficult to conduct and typically limited in size.
Consequently, regulatory science is needed to develop and validate improved predictive models.
Regulatory science can also support the identification and validation of surrogate measures of product efficacy.
Biomarkers that predict efficacy are not yet available for most terrorism threats, emerging pathogens or major global infectious diseases.
Efforts to develop, refine, and validate new biomarkers may lower development costs and improve and speed the development of safe and effective products for unmet public health needs.
In summary, for licensure, an EID vaccine product, just as for any product, must have an acceptable quality, safety, efficacy, and potency profile.
Likewise, production and quality control also must be in compliance with CGMPs.
However, vaccines against EIDs and biothreats agents present unique issues for clinical development and evaluation by the FDA.
Overall planning and coordination among the FDA and its national and international partners is necessary to move these products toward licensure and into distribution.
FDA guidance and engagement with partners is critical to make sure these products can move from the future into the present.
The primary responsibility of NRAs is to ensure the quality, safety, and effectiveness of pharmaceutical products.
The implementation of a strong regulatory system will facilitate these goals, which are especially critical for vaccines that are inherently more difficult to develop, characterize, and manufacture than most pharmaceutical products.
The FDA has developed a managed review process that provides regulatory oversight through all phases of vaccine development.
Advances across a wide range of scientific disciplines have enhanced the preparations to treat certain complications of smallpox vaccination have been approved.
There is one licensed anthrax vaccine in the United States, anthrax vaccine adsorbed (Emergent BioSolutions' BioThrax).
There are significant scientific and regulatory challenges associated with developing and testing new vaccines against EIDs and biothreat agents.
Vaccines against EIDs are more likely to use novel technologies, and the science behind these technologies may be more complex; for example, use of novel cell substrates, the need to develop alternative potency assays, and the need to identify surrogate markers in humans or animals that predict vaccine effectiveness.
To respond to this challenge, the FDA collaborates with interagency groups within the Department of Health and Human Services, such as the Biomedical Advanced Research and Development Authority, the CDC, and the NIH, as well as the Department of Defense and Department of Homeland Security to prepare for responding to an emergency.
A committed, continuous investment in regulatory science is essential to producing medical countermeasures against public health threats.
As noted in the Public Health Emergency Medical Countermeasures Review, 46 "Enhancement and ultimate application of updated regulatory science and scientific review capacity will help strengthen the MCM [medical countermeasure] regulatory process and thus streamline the MCM development process.
[The] FDA will undertake a new initiative designed to focus on augmenting the tools used to assess the safety, efficacy, and quality of medical products, with a particular focus on MCMs, and to get them from concept through the approval process efficiently."
An example of how the FDA's regulatory science efforts have assisted the agency in facilitating the licensure of vaccines against emerging diseases and biothreats is the successful public-private partnership during the 2009 H1N1 influenza pandemic, which resulted in the development and approval of safe and effective vaccines against the pandemic in record time.
This included creating vaccine strains needed for vaccine manufacturing within weeks of the very first 2009 pandemic H1N1 influenza cases appearing, developing reagents and tests through international collaborations to measure the vaccine's potency, consulting the FDA's expert vaccine advisory committee to review the Agency's approach to approval of the 2009 H1N1 vaccine s as well as extensive in process quality control and product testing.
Licensure of vaccines against the 2009 H1N1 influenza virus occurred in September 2009 based on the FDA's determination that standards to ensure the safety and potency of these vaccines had been met.
In parallel to these efforts, NIH and vaccine manufacturers initiated clinical trials to determine the optimal vaccine dosage and number of doses needed to induce a protective immune response against pandemic 2009 H1N1 virus.
Another example illustrating collaboration both within the Department of Health and Human Services and between the Department and international partners is the response to the Ebola virus disease outbreak in West Africa in 2014-15 that caused more than 25,000 cases of Ebola virus disease and claimed the lives of more than 10,000 people, representing the most widespread epidemic of Ebola virus disease in history.
To address this need, the FDA engaged with federal partners, sponsors of medical products, and international organizations to facilitate development of vaccines.
To advance promising vaccine candidates to Phase I clinical trials to obtain preliminary human safety data, the FDA worked closely with manufacturers, clinical trial sponsors, and international regulators to rapidly assess product characterization data and protocols for these first human clinical trials and granted permission for the initiation of these Phase I studies in an expedited manner.
Guidance provided by the FDA assured that these studies were References for this chapter are available at ExpertConsult.com.
prospects of developing new and better vaccines.
Novel vaccine approaches such as recombinant vaccines and novel adjuvants and delivery systems pose regulatory challenges for NRAs.
However, NRAs should be dynamic and flexible entities, as they strive to develop regulatory requirements to address the evolving science.
Further, NRAs must be prepared to address public health emergencies that will require expedited approval mechanisms, such as biological terrorist events, pandemic influenza, and other EIDs.
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Guidance for Industry: Expedited Programs for Serious Conditions-Drugs and Biologics Guidance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines Guidance for Industry: Clinical Data Needed to Support the Licensure of Pandemic Influenza Vaccines Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications Considerations for Developmental Toxicity Studies for Preventive and Therapeutic Vaccines for Infectious Disease Indications Food and Drug Administration A primer on CBER's regulatory review structure and process Code of Federal Regulations.
Title 21, Part 312.47.
Washington, DC, Office of the Federal Register, National Archives & Records Administration Office of the Federal Register, National Archives & Records Administration Vaccines and Related Biological Product Committee (VRBPAC) Meeting Transcript Postmarketing studies for approved human drugs and licensed biological products; status reports Vaccine Safety: Vaccine Safety Datalink (VSD) Food and Drug Administration Food and Drug Administration.
21 CFR Parts 314, 600, 601, 610, and 640.
Changes to an Approved Application.
Final Rule Food and Drug Administration Guidance on alternatives to lot release for licensed biological products Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications Continuous Cell Lines-An International Workshop on Current Issues Recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biological medicinal products and for the characterization of cell banks Meeting of The Vaccines and Related Biological Products Advisory Committee (VRBPAC) (Transcript) Office of the Federal Register, National Archives & Records Administration Guidance for Industry: Providing Regulatory Submissions in Electronic Format -Content of Labeling Office of the Federal Register, National Archives and Records Administration Office of the Federal Register, National Archives and Records Administration Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines Guidance for Industry: Clinical Data Needed to Support Licensure of Pandemic Influenza Vaccines Guidance for Industry: Clinical Data Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines Risk of narcolepsy in children and young people receiving AS03 adjuvanted pandemic A/H1N1 2009 influenza vaccine: retrospective analysis Increased childhood incidence of narcolepsy in western Sweden after H1N1 influenza vaccination The adjuvant component α-tocopherol triggers via modulation of Nrf2 the expression and turnover of hypocretin in vitro and its implication to the development of narcolepsy Narcolepsy in association with pandemic influenza vaccination: a multi-country European epidemiological investigation AS03 adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland European Medicines Agency recommends restricting use of Pandemrix, EMA/CHMP/568830.
Press release Vaccine Safety Datalink.
Narcolepsy and influenza A(H1N1) pandemic 2009 vaccination in the United States Department of Health and Human Services.
The Public Health Emergency Medical Countermeasures Enterprise Review: Transforming the Enterprise to Meet Long-Range National Needs I would like to acknowledge the employees in the Office of Vaccines Research and Review, Office of Biostatistics and Epidemiology, and the Office of Compliance and Biologics Quality/CBER for their assistance in the preparation of this manuscript.
key: cord-002626-jzwwses4 authors: Kaul, Karen L.; Sabatini, Linda M.; Tsongalis, Gregory J.; Caliendo, Angela M.; Olsen, Randall J.; Ashwood, Edward R.; Bale, Sherri; Benirschke, Robert; Carlow, Dean; Funke, Birgit H.; Grody, Wayne W.; Hayden, Randall T.; Hegde, Madhuri; Lyon, Elaine; Murata, Kazunori; Pessin, Melissa; Press, Richard D.; Thomson, Richard B. title: The Case for Laboratory Developed Procedures: Quality and Positive Impact on Patient Care date: 2017-07-16 journal: Acad Pathol DOI: 10.1177/2374289517708309 sha: doc_id: 2626 cord_uid: jzwwses4 An explosion of knowledge and technology is revolutionizing medicine and patient care.
Novel testing must be brought to the clinic with safety and accuracy, but also in a timely and cost-effective manner, so that patients can benefit and laboratories can offer testing consistent with current guidelines.
Under the oversight provided by the Clinical Laboratory Improvement Amendments, laboratories have been able to develop and optimize laboratory procedures for use in-house.
Quality improvement programs, interlaboratory comparisons, and the ability of laboratories to adjust assays as needed to improve results, utilize new sample types, or incorporate new mutations, information, or technologies are positive aspects of Clinical Laboratory Improvement Amendments oversight of laboratory-developed procedures.
Laboratories have a long history of successful service to patients operating under Clinical Laboratory Improvement Amendments.
A series of detailed clinical examples illustrating the quality and positive impact of laboratory-developed procedures on patient care is provided.
These examples also demonstrate how Clinical Laboratory Improvement Amendments oversight ensures accurate, reliable, and reproducible testing in clinical laboratories.
The field of pathology offers the opportunity to better understand the science behind the mechanisms of disease, to lead innovation of new diagnostic technologies, to provide quality oversight of these developments, and to have enormous impact on the lives of patients every day.
Patients benefit from laboratory medicine testing throughout their lives as every medical decision can be impacted by the result of a laboratory test.
Laboratory results constitute the majority of data in a patient's electronic medical record, and our procedures dictate the majority of downstream medical decisions for patients.
1, 2 Clinical laboratory medical professionals have the unique responsibility to patients for assessing the performance of technologies in providing the most accurate information to ensure the most appropriate and efficient course of care.
It is an understatement to say that the medical field is rapidly changing.
Technology and new genomic data developed as a result of the human genome project are leading to an explosion of knowledge applicable to individual patient care; this is the promise of precision medicine.
We must continue to innovate and integrate novel diagnostic tools, genomic information, and new treatments into clinical practice.
Considerable technologic advances allow clinical laboratory professionals to offer new testing that provides more information than ever before and often within a time frame that allows rapid patient care and better outcomes.
Next-generation sequencing (NGS) in genetics and oncology, MALDI-TOF in the clinical microbiology laboratory, and a variety of mass spectroscopy-based methods in clinical chemistry now allow precise and rapid testing with demonstrated improvements in patient care.
It is often thought that when "laboratory tests" are done to reach a diagnosis, they are done with a kit or on a machine, but in fact, most are procedures done with the direct involvement of a laboratory professional or physician.
Laboratory tests are generally not fully encompassed by a "test kit" but often start with the pathologist examining the tissue section, bone marrow aspirate, or gram stain and determining what additional information is needed to provide the clinician with the best scenario so that the patient can be treated most effectively.
Often, clinical laboratory professionals lead the development and optimization of these approaches to improve care and fill a clinical need, and their involvement in the process helps ensure that the highest quality standards are maintained.
Ongoing development of these novel methods is critical to medicine and must be done with the highest level of safety and accuracy, yet simultaneously addressing growing demands to lower the cost of medical care in the United States.
The regulatory oversight of laboratory-developed testing procedures (LDPs) has a critical impact on patients' access to testing and diagnosis.
Currently, there is national discussion regarding the optimal regulatory oversight of laboratory tests and procedures that will balance the needed accuracy and safety with ensuring that new tests are made available to patients safely and expeditiously.
Oversight provided by the Clinical Laboratory Improvement Amendments (CLIA) and the Food and Drug Administration (FDA) currently exists in the clinical laboratory.
It must be noted that a spectrum of testing activities take place in clinical laboratories.
These activities range from complex procedures, such as evaluation of a tissue biopsy, classification of a tumor, or culture of a microbe, to more automated or standardized tests for which an FDAapproved kit is utilized.
All of these activities take place under the direction of a laboratory professional and in accordance with the detailed requirements of CLIA.
The FDA review process is well suited for diagnostic assays that are commercially marketed as kits designed to operate across a spectrum of laboratory settings, in laboratories with a range of expertise.
Currently, FDA approval or clearance requires prospective clinical trial data and a lengthy review process, and thus, FDA approval takes considerable effort and time.
The investment needed for this process impacts the types of tests, and also sample types, that are submitted for approval, as manufacturers must recover their investment afterward.
Additionally, FDA approval for an in vitro diagnostic (IVD) specifies the sample type, clinical purpose, and other aspects of performance of the assay.
When a new clinical need for the IVD arises, the need to use a new sample type arises, or any needed modification that arises in response to the ongoing and sometimes rapid advancements seen in medicine, incorporation of these improvements renders the test an LDP, with the validation needs of an LDP under CLIA.
Laboratories can be caught between the regulations and the needs to best serve their patients.
The CLIA provide for oversight of clinical laboratories by defining all aspects of laboratory operation, including the quality programs required for clinical tests, personnel requirements, and the validation requirements for LDPs.
The CLIA certification of laboratories can be accomplished through several deemed agencies such as the College of American Pathologists (CAP) or the New York State Department of Health (NYS-DOH).
These organizations provide operational guidelines and perform on-site inspections based upon checklists developed via consensus of laboratory experts, which include hundreds of pages of requirements and data points.
Compliance with CLIA has been built into clinical laboratory operations, mechanisms for data collection, training, proficiency testing (PT), and test implementation.
Laboratories are subject to unannounced inspections and must demonstrate satisfactory performance characteristics for any test offered to ensure that results are accurate.
For testing not reviewed by the FDA, such as LDPs, laboratories must go through a rigorous validation process before offering the test for clinical use.
Under CLIA, the validation data collected by these laboratories are subject to ongoing peer review.
In particular, organizations with deemed status overseeing laboratories (such as CAP and NYSDOH) conduct rigorous peer-inspection using detailed criteria developed specifically for molecular pathology.
Laboratories also participate in required PT to demonstrate assay quality, with interlaboratory data sharing and assessment (most often led by the CAP) that leads to ongoing broad improvement in LDPs.
Recent public documents have presented examples of specific LDPs in which the clinical validity of the test, the interpretation or use by clinicians, or the reproducibility of the laboratory data was questioned, raising concern about the safety and efficacy of this category of tests.
3, 4 Enhanced regulatory oversight has been proposed to help ensure laboratories are delivering meaningful, high-quality results to patients.
However, additional regulations have the potential to slow innovation and to limit and delay patient access to novel testing that impacts their clinical care, as well as add redundant reporting efforts and significant cost.
Expanded oversight of laboratories through a revised CLIA process has also been proposed (http://www.cap.org/ShowProperty?nodePath=/UCMCon/Con tribution%20Folders/WebContent/pdf/2015-cap-ldt-legisla tive-proposal.pdf.
Accessed April 27, 2017).
5 In an effort to illustrate the need for and positive impact of laboratorydeveloped procedures on patient care, the following series of vignettes have been assembled.
In each case, timely and highquality laboratory-developed procedures filled a key clinical need.
The impact on patient care has been summarized.
As possible, data describing interlaboratory comparisons are provided to demonstrate the quality of these LDPs, as validated and performed in accordance with CLIA.
These examples also serve to highlight the quality efforts and interlaboratory comparisons that take place before LDPs are offered by laboratories for clinical use; these activities and data are often unknown to the clinicians who utilize these testing services.
Table 1 provides a guide to the examples included and summarizes the impact of each assay along with key points about its utility.
Encephalitis is the most serious complication of herpes simplex virus (HSV) infection.
6 While rare, HSV is commonly included among causes of viral encephalitis.
Since HSV infection can be treated effectively with acyclovir, it is critical to rapidly and accurately establish the diagnosis and initiate treatment to reduce morbidity and mortality.
Culture of cerebrospinal fluid (CSF) is insensitive for the diagnosis of HSV encephalitis, so brain biopsy, a very invasive procedure with significant morbidity, was historically required to make a diagnosis, with results not available for days afterward.
Several studies demonstrated that detection of viral DNA by polymerase chain reaction (PCR) using CSF samples performed equivalently to brain biopsy, including a landmark study in 1995.
[7] [8] [9] Given the ease of collecting CSF samples, the lower risk of complication, the lower cost, and speed of results, PCR became the standard of care for diagnosing HSV CNS infections in the mid-1990s, with LDPs used for nearly 20 years.
It was not until 2014 that the first FDA-cleared PCR test for the diagnosis of HSV encephalitis became available, with a second test cleared in 2015.
Laboratory-developed testing procedures dramatically improved the quality of care for many patients and continue to be used successfully today, as the 2 cleared tests have limitations, requiring specific instrumentation not standard in many laboratories.
Additionally, one of the tests is highly multiplexed, which may not be appropriate in all clinical situations.
Herpes simplex virus can also cause infections in neonates, with a frequency of 1:3500 to 1:5000 deliveries in the United States.
The infection is acquired by exposure to maternal genital secretions.
The presentation of neonatal HSV varies and includes skin, eye, and mouth infection, with encephalitis and disseminated disease in over 50% of cases.
Again, rapid diagnosis is needed to prevent morbidity and sequelae from encephalitis.
The diagnosis of encephalitis is made via PCR of CSF samples, while testing of plasma or serum is critical to diagnose disseminated disease.
The ability to use plasma or serum is very important as it may be difficult to obtain enough CSF from a newborn to assess all diagnoses in the differential.
Although there are 2 FDAcleared assays to test CSF for HSV, there are no assays cleared for testing serum and plasma.
Laboratory-developed testing procedures continue to play a critical role in the diagnosis and management of disseminated HSV infection in newborns.
Additionally, LDPs have performed with a high degree of accuracy and interlaboratory agreement.
Blinded PT samples were distributed to 383 laboratories as part of a CAP PT survey for analysis of HSV; 91.6% correctly identified HSV-2, and 7.8% identified HSV, but not noting the subtype; an overall accuracy of 99.4% was obtained.
Previous proficiency surveys showed similar results for samples containing HSV1.
Over 90% of the laboratories used LDPs.
10 BK virus infection is very common, with a seroprevalence in the adult population of *90%.
After primary infection, the virus colonizes the renal and urinary tracts; healthy individuals will occasionally shed virus in their urine without consequence.
However, in renal transplant recipients, BK virus is the major cause of polyomavirus-associated nephropathy (PVAN), putting 1% to 15% of kidney transplant patients at risk of premature allograft failure.
11 Given the lack of effective antiviral therapy for BK virus, the key to preventing allograft loss is to identify at-risk renal transplant recipients early and reduce immunosuppressive therapy before PVAN develops.
Reduction of immunosuppressive therapy helps control viral replication and in most cases prevents the development of PVAN.
12 There is a critical balance between too much immunosuppressive therapy, which can lead to PVAN, and too little immunosuppression causing rejection.
The ability to monitor BK virus levels in the blood allows for informed clinical decisions.
Studies have shown that monitoring patients with viral load testing during the first 2 years after transplant can dramatically reduce the development of PVAN.
Consensus guidelines recommend that screening for BK replication be performed at least every 3 months during the first 2 years posttransplant and then annually until the fifth year posttransplant.
13 When the plasma viral load value rises above a threshold (10 000 to 50 000 copies/mL), a renal biopsy may be performed to assess for PVAN, and immunosuppressive therapy is reduced based on the results of the biopsy.
Viral load testing is also done if there is an increase in serum creatinine, as the level of BK virus aids in distinguishing rejection from PVAN.
Although BK viral load testing has been the standard of care for several years and is used in transplant centers across the country, there is no FDA-cleared or FDA-approved test available.
All testing is performed using LDPs.
If these LDPs were not available, most cases of PVAN would not be identified promptly, leading to negative patient outcomes such as allograft loss or rejection.
Cytomegalovirus (CMV) can cause a wide range of complications among both solid organ transplant and hematopoietic stem cell transplant recipients, as well as in other immunocompromised patients.
Cytomegalovirus has a high seroprevalence, and large numbers of transplant patients experience reactivation or primary infection, with probability increasing based on pre-transplant serostatus, severity of pretransplant conditioning, and allograft relatedness.
14, 15 Although infection can be subclinical, high or increasing viral load signals increased the risk of symptomatic disease, which can range from relatively mild constitutional symptoms to severe end-organ infection or potentially fatal disseminated disease.
Preemptive therapy of high-risk patients based on the detection of increasing CMV load in peripheral blood was shown to be effective in the 1990s 16, 17 ; however, the first FDA-approved IVD test did not appear on the market until 2012.
18 In the intervening years, laboratory-developed assays played a critical role in bridging the gap.
The presence of such methods and their adoption across the country enabled the routine use of CMV screening for asymptomatic patients in transplant centers soon after data supported its utility.
The availability of such methods has likely saved many lives over the years, supporting early diagnosis, preemptive treatment strategies, and the assessment of therapeutic treatment efficacy.
14, [18] [19] [20] The widespread use of LDP CMV quantitative methods produced a generation of transplant physicians comfortable with the use of such methods and changed the epidemiology of posttransplant CMV disease, markedly reducing the incidence of early disease in such patients.
Over the years of LDP use, numerous studies focused on continuous improvement and optimization of methods, 21 including the development of international quantitative standards in 2010.
22 The ability to rapidly incorporate advances in technology (including the advent of real-time quantitative methods) has been demonstrated by the improvement in sensitivity and other performance characteristics of such tests over time.
The data accumulated throughout these experiences informed the development of the first commercially available CMV IVD assays.
In fact, the absence of LDPs and the vast clinical and laboratory experience that they provided likely would have delayed the availability of commercial methods, and their performance characteristics taken longer to optimize to today's levels.
Human papilloma virus (HPV), the causative agent of genital warts, has been implicated in the development of *99% of cervical cancers.
23 More than 200 HPV genotypes have been described, 24 and approximately 40 are capable of infecting the human genital tract.
25 Of these, a relative few are known to cause cervical cancer and other malignancies; these can be identified by genotyping.
Today, 4 assays are FDA approved for detecting high-risk HPV genotype strains in cervical specimens.
[26] [27] [28] [29] These tests are routinely used to confirm the presence of an HPV infection, screen for cervical cancer, and refer cases with an indeterminate cytology examination.
30 During the past decade, a rise in oropharyngeal squamous cell cancer, primarily in 40 to 55-year-old Caucasian males with limited alcohol and tobacco exposure, has been described.
31 Unexpectedly, investigators discovered the frequent presence of high-risk HPV genotype strains in these lesions.
Human papilloma virus-positive head and neck cancer is biologically distinct from HPV-negative disease.
Patients with HPV-driven tumors have a significantly better prognosis, including response to chemoradiation therapy and overall survival, compared to HPV-negative patients.
32 Therefore, testing head and neck cancer specimens for high-risk HPV genotypes has become standard of care and is used to guide treatment.
33, 34 However, the HPV tests that are FDA approved for cervical specimens have not been approved for head and neck cancer, leaving a critical gap for patient care.
Clinical laboratories have thus had to develop new assays or modified the existing FDA-approved ones to detect high-risk HPV genotypes in head and neck cancer specimens.
Inclusion of patients in several clinical trials is based on these LDPs (www.clinicaltrials.gov.
Accessed April 27, 2017).
Without laboratory-developed tests, patients with head and neck cancer cannot benefit from personalized treatment strategies.
Clinical laboratories must be highly vigilant for infectious disease outbreaks, since they are likely the first to recover the pathogen and recognize the potential for an outbreak.
Recent examples of emerging or reemerging pathogens causing substantial human morbidity and mortality include avian influenza virus ("bird flu"), chikungunya virus, Ebola virus, Middle Eastern respiratory syndrome virus, severe acute respiratory syndrome virus, and Zika virus.
35, 36 At the time of their emergence, no FDA-approved tests were available to detect any of these pathogens.
In response, many clinical laboratories developed, validated, and implemented the LDPs needed to care for patients at their institutions.
37, 38 These assays were based on extensive data sets and reported in peer-reviewed journals, supporting their quality claims.
Since performance characteristics such as analytic sensitivity (limit of detection) may vary between different assay designs, 39 as was recently discussed for several different Zika virus tests, 40 these sorts of collaborative efforts are essential.
In many cases, laboratories leading these efforts collaborated with one another to share validation materials, perform interlaboratory comparisons, and exchange blinded testing samples (http://www.usatoday.com/story/ news/2016/02/23/texas-hospitals-develop-rapid-zika-test/ 80776382/.
Accessed April 27, 2017).
While perhaps not ideal, there is an urgent need for a more integrated and coordinated mechanism for rapid diagnosis of novel infectious agents that incorporates both the public health and hospital laboratories.
The recent emergence of Zika virus, which has been linked to severe birth defects, 41 underscores the need for clinical laboratories to have rapid access to diagnostic tools.
When the Secretary of Health and Human Services declared Zika virus to be a public health emergency, the first FDA Emergency Use Authorization for Zika virus testing was granted to a test available only to the public health laboratory system, not to hospital laboratories on the front lines of patient care (https://www.fe deralregister.gov/documents/2016/03/28/2016-06888/authori zation-of-emergency-use-of-an-in-vitro-diagnostic-device-fordiagnosis-of-zika-virus.
Accessed April 27, 2017).
As a result, the public health system quickly became overwhelmed, as it has been during past outbreaks, such as influenza.
42 In many areas, turnaround times for Zika virus testing exceeded 4 to 8 weeks (http://www.nytimes.com/2016/09/13/us/zika-testdelays-florida-pregnant.html?_r¼0.
Accessed April 27, 2017, http://medicalxpress.com/news/2016-10-lab-constraints-zikaresults.html.
Accessed April 27, 2017, http://www.miamiher ald.com/news/health-care/article104856631.html.
Accessed April 27, 2017).
These delays critically impact patient care, particularly for pregnant women with possibly affected fetuses.
In comparison, many hospital-based and national reference laboratories are able to report results within hours (http:// www.usatoday.com/story/news/2016/02/23/texas-hospitalsdevelop-rapid-zika-test/80776382/.
Accessed April 27, 2017, http://www.nytimes.com/2016/09/13/us/zika-test-delays-flor ida-pregnant.html?_r¼0.
Accessed April 27, 2017).
For example, in Miami-Dade Florida, where more than 1000 people have been confirmed Zika positive and the virus is circulating in the local mosquito population, the public health laboratory system encountered a backlog of nearly 1000 untested specimens (http://www.miamiherald.com/news/health-care/arti cle104856631.html.
Accessed April 27, 2017).
Diagnostic assays for Zika and other emerging pathogens will continue to evolve, 43 but it is clear that rapid identification of pathogens during other outbreaks facilitates rapid treatment and appropriate isolation of patients, leading to improved patient outcomes and potentially slowing the spread of infections such as influenza (http://www.cdc.gov/flu/professionals/diagnosis/molecu lar-assays.htm.
Accessed April 27, 2017).
Implementation of the first rapid diagnostic tests for influenza was directly associated with reduced length of hospital stay, decreased mortality, and reduced costs.
44 Access to diagnostic tests, early in the course of an outbreak and in hospital laboratories, has a positive public health impact.
The KRAS gene encodes a GTPase critical in signal transduction that is known to be mutated in a wide range of tumor types.
45 A landmark study presented at the American Society of Clinical Oncology (ASCO) meeting in 2007 demonstrated that patients with metastatic colorectal cancer harboring a mutated KRAS failed to respond to targeted therapy with cetuximab.
46, 47 At the time, there were no clinical tests for KRAS mutations available.
Molecular pathology laboratories worked quickly to fill this need, to define the best analytic approaches, and to ensure that test results done in one laboratory matched those done in another.
[48] [49] [50] Within a few months, laboratories were able to offer fully validated KRAS assays that worked reliably and were safe for patient care.
Under CLIA, the validation data collected by these laboratories were subject to ongoing peer review, and laboratories participate in ongoing PT to demonstrate consistent assay quality.
In 2009, the ASCO and the National Comprehensive Cancer Network (NCCN) recommended mutational profiling of KRAS exons 12 and13 before institution of anti-epidermal growth factor receptor (EGFR) therapy for patients with metastatic colorectal cancer; it became standard of care to assess formalin-fixed paraffin-embedded tumor tissues from patients with metastatic colon cancer for KRAS mutation status.
51, 52 It was not until 5 years later that the FDA cleared QIAGEN's therascreen KRAS test, designed to detect the presence of 7 mutations in the KRAS gene in colorectal cancer.
53 By this time, new data demonstrated that KRAS analysis alone was not enough; mutation analysis of other genes was necessary, 54, 55 and the FDA-approved assay was already inadequate for patient testing compliant with national treatment guidelines.
Without LDPs, an estimated 10% of patients with non-exon 2 KRAS mutations would be overtreated with expensive anti-EGFR therapy.
56 The use of LDPs has thus persisted in clinical practice to provide patients with the most complete information to guide treatment.
In the CAP KRAS-B-2015 mailing, 204 laboratories reported results from testing 3 blinded proficiency-testing specimens.
The specimens contained recurring somatic mutations in KRAS exons 12 or 13 (NM_004985.3), c.35G>T (p.G12 V), and c.38G>A (p.G13D).
An acceptable response was reported by over 96% of the laboratories for both mutations (197/204 for p.G12V and 195/202 for p.G13D).
The vast majority of reporting laboratories utilized LDPs.
57 KRAS and RAS family gene mutation analysis is also critical in the management of patients with non-small-cell lung cancer (NSCLC) and other tumors, 58 for which FDA approval of kits has not occurred; LDPs or off-label use of kits is required.
BRAF belongs to a family of serine-threonine protein kinases that participate in signal transduction cascades involving RAS, RAF MEK, and ERK family members.
This pathway is important in the regulation of normal cell proliferation and differentiation.
59 Activating mutations in BRAF can lead to increased proliferation and prolonged cell survival in a variety of tumor types.
60 Laboratories will typically test for BRAF mutations in low-grade gliomas (for diagnosis), colorectal cancer (to establish the sporadic origin of MSI-H tumors and response to anti-EGFR therapy), hairy cell leukemia (for diagnosis), lung adenocarcinomas (to predict response to therapy), thyroid cancer (for preoperative detection of thyroid cancer in FNA samples and prognosis in papillary thyroid carcinoma), or melanoma (to predict response to BRAF kinase inhibitors).
For one of these indications, malignant melanoma, FDAapproved companion diagnostics are available.
Vemurafenib (ZELBORAF) is a kinase inhibitor indicated for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation.
Dabrafenib (TAFINLAR) is a kinase inhibitor indicated as a single agent for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E mutation or in combination with trametinib (MEKINIST) for the treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations (www.fda.gov.
Accessed April 27, 2017).
The Cobas 4800 BRAF V600 Mutation Test (Roche Molecular Systems, Pleasanton, CA, USA) sporadically cross reacts with BRAF V600K and BRAF V600D mutations 61,62 and thus is neither sensitive for BRAF V600 mutations nor specific for BRAF V600E mutations, confounding accurate outcome evaluations and preventing its usefulness in selecting patient for Tafinlar therapy.
The THxID BRAF kit (bioMerieux, Boston, MA, USA) does detect both BRAF V600E and BRAF V600K mutations (but not other BRAF V600 mutations) and is necessary to distinguish between alternate therapeutic options (single agent vs combination therapy).
It is important to note that increased cell proliferation has been seen in tumors treated with BRAF inhibitors with normal BRAF.
61 It is therefore important to identify all BRAF activating mutations to assist in the selection of appropriate therapy.
The FDA-approved assays are therefore not adequate for current clinical needs.
In a 2015 European multicenter study, 62 420 consecutive tumor samples of histologically proven melanoma tumor tissue were assessed for BRAF mutation status by the Cobas system and a variety of laboratory developed procedures.
Testing was concordant for 392 (93.3%) of 420 samples but discordant for 28 (6.7%).
Among the discordant cases, 11 had invalid results (8 samples with the Cobas and 3 with LDPs).
Of 10 samples with BRAF V600 mutations detected by the LDPs (but not by the Cobas Mutation Test), 5 were V600K, D, or R mutations, and 2 contained only 20% tumor cells.
For the 7 samples with BRAF V600 mutations detected by the Cobas but not by the LDPs, 4 were confirmed with retesting, 1 was not mutated, and 2 were considered invalid results.
This study documents similar results between the performance of BRAF LDPs and IVDs.
Further data can be gathered from the CAP PT program.
In the BRAF-B-2015 CAP proficiency survey, 173 laboratories reported on the detection of V600E and other BRAF mutations by a variety of analytic methods.
Two of the well-characterized specimens in the proficiency test contained BRAF V600E alleles, and 98.8% and 99.4% of laboratories correctly reported the mutation.
The majority of these laboratories used LDPs.
63 There are no currently available BRAF mutation tests approved for use in other tumor types such as those mentioned above, and use of the existing IVD tests would constitute offlabel use and hence LDPs.
Microsatellite instability is the presence of hypermutability in repetitive DNA sequences resulting from impaired DNA mismatch repair.
Microsatellite instability can be an inherited or acquired feature of tumors.
Microsatellite instability occurs in approximately 15% of all colorectal carcinomas and is a consistent feature of colorectal and other tumors in patients with Lynch syndrome.
64 Tumors are classified as showing high levels of MSI (MSI-H phenotype) if 2 or more of 5 microsatellite markers (or !30%) exhibit instability, a microsatellite-stable phenotype if none of the markers show instability, and an MSIlow phenotype if only 1 of 5 or less than 30% of the markers show instability.
65 Studies have confirmed that the appropriate cutoff for determining an MSI-H phenotype is the finding of instability in 30% or more of the markers tested.
The finding of an MSI-H phenotype is consistent with the presence of defective DNA MMR in the tumor.
66 The finding of an MSI-H phenotype in a CRC increases the likelihood that the patient has LS but is not specific for LS.
The definitive establishment of a diagnosis of LS requires the finding of a pathogenic germline mutation in one of the DNA MMR genes.
Additional testing that can be offered to determine whether a patient with an MSI-H CRC is likely to have LS includes testing the tumor for DNA MMR protein expression using IHC, BRAF V600E point mutation analysis (since BRAF-mutated MSI-H colorectal carcinomas are known to have sporadic MMR gene mutations), and MLH1 promoter hypermethylation.
Studies have shown that an MSI-H phenotype is a favorable independent prognostic indicator in patients with CRC.
67 In addition, some reports indicate that MSI-H tumors may not be responsive to 5-fluorouracil-based therapies.
68 Recent draft guidelines developed collaboratively by 4 professional societies recommend that deficient mismatch repair/microsatellite instability testing must be performed in all colorectal cancers for prognostic stratification and identification of patients with Lynch syndrome.
69 Although numerous laboratories offer MSI testing using LDPs, there are currently no FDA-approved tests for the evaluation of microsatellite instability.
A summary of CAP PT results 70 demonstrates excellent performance of laboratories participating in the MSI proficiency surveys.
This good performance of laboratories over the years may be partly due to the educational nature of the CAP PT, which provides laboratories with an external mechanism to monitor the quality status of their testing.
Epidermal growth factor receptor is a membrane-bound tyrosine kinase which activates several signaling pathways known to be altered in human cancer, including NSCLCs.
[71] [72] [73] Nonsmall cell lung cancer tumors with EGFR-activating mutations are responsive to gefitinib and erlotinib, small molecule tyrosine kinase inhibitors of EGFR.
74, 75 The FDA approval of anti-EGFR therapies based on clinical trial outcomes data resulted in the need for clinical laboratories to test tumor tissue for the EGFR-sensitizing mutations in order for patients to be eligible for treatment.
With no FDA-approved "companion" diagnostic test on the market, CLIA-licensed laboratories developed and validated LDP tests for the 2 most common EGFR mutations as early as 2004.
73 The FDA followed with approval of the Roche Cobas EGFR Mutation Test in 2013 along with the Qiagen therascreen EGFR RGQ Kit.
Both assays tested for the exon 19 deletions and the exon 20 L858R point mutation.
Of note was that each test was approved for specific therapeutic indications and specimen types.
As new drugs became available, approval for new claims was needed.
Laboratory-developed procedures continue to be the method of choice due to the limitations of claims made for FDA-approved assays and performance characteristics, including types of mutations being detected.
76 Clinical laboratories participate in twice-yearly proficiency test challenges of unknown samples that must be analyzed and reported, with results graded and compared to other laboratories performing the testing.
In the CAP EGFR-B-2015 proficiency test, 192 laboratories reported results from testing 3 unknown proficiency-testing specimens in late 2015.
The specimens conNote: Some laboratories do not test for certain mutations, hence, the denominator is often less than 192.
77 As patients being treated with these new targeted anti-EGFR therapies began to relapse, further studies revealed that EGFR harbors both sensitizing and resistance mutations.
The CLIA laboratories have demonstrated the ability to detect all mutations in the EGFR gene as well as in other genes using NGS assays to sequence panels of cancer-related genes.
78 This panel approach allows the laboratory to provide the oncologist with a more comprehensive profile of the tumor, using a costeffective technology that makes maximal use of small tissue samples and thus makes treatment strategies more effective.
In addition, the time saved by testing a broader panel of gene targets can result in better outcomes for the patient as well as fewer adverse drug reactions.
For the payer, the cost savings of such an approach versus algorithm-based testing with single gene assays is significant.
Currently, there are no FDAapproved sequencing assays for EGFR mutation status, and LDPs are solely used for the detection of these mutations that define therapy selection.
The complexity of cancer biology and the ever-evolving therapeutic approach to management of the patient with cancer more often requires expanded knowledge of the tumor beyond single-gene mutation status.
Over the past several years, the laboratory's ability to multiplex testing for several genes or genetic variants has been limited by the available technologies.
Next-generation sequencing or massively parallel sequencing has allowed the laboratory to provide a more comprehensive genomic profile of tumor cells in a single assay than previous methods.
The ability to detect numerous mutations in multiple genes results in information that can allow the oncologist to develop a more accurate treatment strategy including therapies selected based on a "responsive" tumor profile and those not selected based on the presence of resistance mutations.
[79] [80] [81] Instrumentation from Thermo Fisher (Thermo Fisher Life-Technologies, Carlsbad, CA, USA) and Illumina (Illumina, San Diego, CA, USA), the Personal Genome Machine (PGM), and MiSeq, have made NGS suitable for routine clinical laboratory testing.
[82] [83] [84] In 2015, the MiSeq Dx obtained FDA approval.
Despite this approval and the routine use of NGS in LDPs setting, there are no currently FDA-approved NGS tests for application in oncology.
Although many drug package inserts require or allude to the use of a companion diagnostic for eligibility, very few companion diagnostic tests are FDA approved and none using NGS technology.
As an LDP, clinical laboratories are required to demonstrate rigorous performance criteria for wet-bench testing and analysis pipelines to ensure the test is functioning properly for its intended clinical purpose.
73, [85] [86] [87] Most NGS testing for therapeutic selection in cancer consists of panels of genes that range from 10 to 400 or more genes.
Each test can be designed to detect hotspots of known mutations in those genes, to sequence the entire coding region of the genes, or to sequence the entire gene.
The end result is a comprehensive profile of the tumor genome that can then be used to tailor therapy for the individual patient.
Although NGS assays cost more than single-gene assays, the cost per gene sequence is dramatically reduced and results in cost savings over using multiple single-gene tests.
Furthermore, NGS panels can be applied to very small specimen samples, using as little as 10 to 250 ng of input DNA depending on the analytic platform utilized.
Since evaluation of therapeutic biomarkers is usually needed in the setting of advanced disease and such patients often have only limited tissue samples available for testing, NGS assays allow for much more extensive genomic information to be obtained compared to single-gene assays, each of which can require DNA input comparable to that of an entire NGS panel.
A total of 111 laboratories recently participated in a CAPsponsored proficiency assessment of NGS cancer panel testing (NGSST-A-2016), with data collection on 10 gene mutations (AKT1, ALK, BRAF, EGFR, FBXW7, IDH1, KIT, KRAS, NRAS, and PIK3CA).
Of 1010 genotyping calls across the spectrum of mutations tests, 993 (98.3%) were called concordantly (unpublished data).
88 No other LDP in the field of oncology has had a greater impact on patient care than has the quantitation of RNA in chronic myelogenous leukemia (CML).
One of the first (and arguably most successful) molecularly targeted cancer therapies is the BCR-ABL1-targeted tyrosine kinase inhibitor, imatinib, which was FDAapproved in 2001.
In those very early days of targeted therapy, long before the advent of FDA-approved "companion diagnostics," the ubiquitous and obvious method to determine the efficacy of novel leukemia treatments was to directly quantitate the target of the inhibitor drug, namely, the cancer cellspecific BCR-ABL1 fusion gene.
A reduction in posttreatment BCR-ABL1 RNA levels, as measured by sensitive laboratorydeveloped PCR-based methods, was shown to be the best available test for predicting therapeutic response and long-term progression-free survival in TKI-treated patients with CML.
89 Consensus oncology practice guidelines in both the United States (NCCN) 90 and Europe (ELN), 91 going back at least a decade, have universally recommended that TKI-treated patients with CML should be serially monitored with a (laboratory-developed) BCR-ABL1 RT-PCR blood test at least every 3 to 6 months during their lifelong course of TKI therapy.
The NCCN and ELN guidelines have also long recommended serial BCR-ABL1 RNA testing to directly inform not only the appropriate dose of TKI (to overcome developing resistance) but also the therapeutic switch from one TKI to another (depending on the drug's known resistance profile).
To directly support optimal therapeutic decision-making in the routine care of patients with CML, clinical laboratories have been offering accurate and sensitive PCR-based laboratory-developed procedures for BCR-ABL1 for at least the last 15 years.
Recognizing that standardization of these LDP's was necessary to promote uniform therapeutic decisionmaking, the laboratory community undertook an extensive multiyear project to create a standardized "international scale" (IS) of measurement for BCR-ABL1 messenger RNA.
92 Follow-up efforts resulted in the creation of a World Health Organization-recognized panel of reference materials directly linked to the BCR-ABL1 IS 93 and the subsequent creation of secondary IS-calibrated reference materials that could be used for routine daily QC in clinical laboratories.
94, 95 Recognizing the additional need for PT, the CAP has been offering semiannual BCR-ABL1 PT surveys for at least 10 years, with a progressive increase in the number of participating laboratories (from *100 in 2009 to *190 in 2016).
As proof of the nearuniversal recognition of assay standardization, approximately 90% of these accredited laboratories now report their PCR results using the standardized IS.
A 2016 CAP survey confirmed excellent interlaboratory precision, with over 90% of laboratories reporting a BCR-ABL1 IS result within internationally acknowledged acceptable tolerance limits (0.5 logs) for IS reporting of a sample with an approximate 1000-to 10 000-fold reduction in pretreatment BCR-ABL1 levels 96 The primary driving force behind the remarkable increase in longevity and quality of life for patients with CML over the past 15 years has no doubt been the availability of noveltargeted TKI therapies.
This has become the paradigm for personalized/precision cancer medicine programs, coupled with parallel effort of the laboratory community toward building, improving, and standardizing accurate, precise, and sensitive laboratory-developed tests for BCR-ABL1.
Of note, this 15year targeted therapy program for CML occurred entirely without the availability of FDA-approved BCR-ABL diagnostic reagents, which have only become available in 2016.
These FDA-approved assays were based upon those developed in clinical laboratories; they are not approved for diagnosis of CML nor do they cover the spectrum of breakpoints that occur in the disease.
During those ground-breaking first 15 years of the targeted cancer therapy era, if the laboratory community had been prohibited from providing high-quality, standardized LDP-based testing under existing CLIA guidelines, the negative consequences to patient care in the past and the future would have been substantial.
Fragile X (FX) syndrome is one of the most common inherited causes of intellectual disability.
The causative molecular mechanism is an expansion of a CGG repeat region of the 5 0 regulatory region of the FMRP gene.
When the CGG repeat expands beyond approximately 200 CGG repeats, the gene is methylated and silenced.
Laboratory testing for FX includes sizing the number of repeats as well as methylation analysis and has been available for clinical diagnostic and carrier status testing for over 20 years.
The American College of Medical Genetics and Genomics (ACMG) has published practice guidelines for appropriate test ordering.
97 It is a first tier test for individuals and families in which an X-linked inheritance pattern of intellectual disability is suspected.
Once an expanded FX allele has been identified, other family members can be tested to identify premutation carriers at risk of having affected offspring.
Prenatal (fetal) or preimplantation genetic diagnostic testing is available for known FX carriers.
According the Genetic Test Registry, over 50 laboratories in the United States offer testing for FX (https://www.ncbi.nlm.
nih.gov/gtr/; accessed 11-01-2016).
Currently, all FX testing is performed as LDPs: No FDA-cleared assay is available.
PCR primers and Southern blot reagents are available commercially as analyte-specific reagents (ASR) or as investigational use only.
Clinical laboratories use these commercial reagents, or design primers or probes, combine them internally to develop the assay and establish performance characteristics.
The ACMG has published standards and guidelines for clinical laboratories that perform this test.
98 Reference materials to standardize sizing were developed through the Genetic Testing Reference Material program 99 sponsored by the Centers for Disease Control and Prevention (CDC), the National Institute of Standards and Technology (https://www.nist.gov/node/ 608501, Accessed November 1, 2016), and the World Health Organization.
100 Proficiency testing through the CAP has demonstrated the excellent performance of clinical laboratories of this high-complexity LDP.
101 As the genetic basis for many human diseases became apparent, sequence-based diagnostic testing was implemented as a way to provide definitive diagnoses for patients and their families.
Many laboratories began offering sequence-based testing for heritable disorders in the 1990s, using a variety of mutationscanning techniques, such as single-strand conformation polymorphism, denaturing HPLC, MLPA, and others.
102 Sanger-based sequencing was the gold standard, despite being slow and expensive.
The multigenic nature of some of these disorders made these sequencing approaches challenging due to the sheer number of genes and size of the sequence requiring analysis.
In recent years, however, most of this testing has been converted to NGS, which offers significant advantages in terms of analytic capabilities, quality, speed, and cost.
103 The repetitive sequence reads in a single region ensure an enhanced level of quality, and NGS assays can be designed to interrogate anything from small to large gene panels, whole exomes, and beyond, depending on the clinical need being addressed.
Consensus guidelines for NGS assays have been developed by multiple professional societies and address the development, validation, and quality control of these assays.
104, 105 The CAP has developed inspection checklists for laboratories performing NGS for detection of somatic mutations in cancers as well as germline mutations that cause heritable diseases.
Progress has also been made in the planning and production of reference materials and proficiency samples.
106 Together, these practices and resources have yielded laboratory-developed assays that can be demonstrated to meet the quality levels needed for patient care.
In addition to continuous quality assessment of the "wet laboratory" procedures, ongoing national and international efforts to share data, and construct and maintain up-to-date curated databases for variant interpretation, are critical for quality care.
As new mutations and variants are detected, interpretation and assessment of clinical impact, including determination of the clinical importance of variants of undetermined significance, is critical.
Rapid generation of genomic data, disorganized data sharing, and a lack standardization have created challenges, and a more consistent approach to clinical sequence interpretation is needed, along with centralized and openly accessible databases for sequence and clinical information.
In recognition of the urgent need for up-to-date variant classification resources, the ACMG and Association for Molecular Pathology released a landmark guidance document in 2015, 105 which has been implemented by US and international laboratories.
Additional resources are outlined and include NCBI's ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/), which has quickly become a valuable centralized resource for clinically classified variants, and the Clinical Genome Resource (ClinGen, www.clinicalgenome.org), which serves as a centralized site for managing genomic knowledge surrounding genes and variants.
With exome and genome sequencing being increasingly implemented, guidance has been issued by the ACMGG on how to deal with the incidental identification of variants in the so-called "actionable" genes in patients tested for unrelated conditions.
107 Additionally, quality assessment focusing on the informatics pipeline and variant interpretation could effectively utilize sequence data sets, as has been recently outlined.
108 The genetics and pathology communities are increasingly embracing data sharing, which will lead to needed improvements in divergent interpretation of gene sequence variants.
These interpretative tasks would be beyond the scope of an FDA approval for a kit, or the CLIA oversight of an LDP, but have a critical impact on patient care based on genomic information.
Next-generation sequencing analysis of a variety of gene panels has become routine in clinical care and has had a positive impact on the diagnosis and treatment of patients and families with complex syndromes and disorders.
Examples of 3 of the most common clinical settings in which gene panels are tested for potential germline mutations are provided below, along with information on the clinical setting, potential benefits, and also challenges in utilizing these approaches.
Inherited cardiomyopathies are common disorders and include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and restrictive cardiomyopathy.
109, 110 Several characteristics of all inherited cardiomyopathies provide compelling reasons for genetic testing and include (1) a substantial genetic component with detection rates currently ranging between 30% and 50%, 111, 112 (2) long presymptomatic phases with acute disease onset typically not before adolescence, and (3) a predisposition to sudden cardiac death (SCD), which can be the first presenting sign.
Frequent and heartbreaking publicity following cases of sudden death of competitive athletes has brought these diseases to public attention, 113 and a recent study shows that 16% of SCD cases are due to an underlying unrecognized inherited cardiomyopathy.
114 The importance of these heritable abnormalities is underscored by the fact that nearly a third of the genes for which the ACMG recommends return of results, regardless of the test indication, are made up of these cardiomyopathy genes.
These disorders are heterogeneous, which can lead to clinical diagnostic uncertainty or error; hence, large multigene panels are particularly useful to cover the spectrum of genes that may cause a clinical disorder.
Importantly, the identification of pathogenic variants in affected individuals can inform medical management of family members and can identify those at risk early and also release negative individuals from clinical screening.
111 Genetic testing can also identify phenocopies such as Fabry disease, which can masquerade as isolated HCM.
While Fabry disease is rare, disease-modifying treatment is available, and therefore, genetic testing provides essential clinical information for these patients.
Epilepsy is a common disorder of the central nervous system characterized by periodic loss of consciousness with or without convulsions associated with abnormal electrical activity in the brain.
This can significantly affect the quality of life and has major psychological and socioeconomic consequences.
It is estimated that there are more than 50 million people with epilepsy worldwide, and an estimated 1 in 26 people in the United States will develop epilepsy at some point in their lifetime.
A significant proportion of cases show a familial distribution, and there is an increased risk of epilepsy with a family history.
The prime requirements for successful management of epilepsy are a complete diagnosis and selection of an optimal treatment to benefit the patient.
Development of epilepsy may involve multiple gene abnormalities or a gene abnormality in concert with an environmental trigger.
More than 100 genes have been shown to be associated with epilepsy, and a precise genetic diagnosis can help in deciding the accurate treatment and follow-up.
115, 116 Evaluation of all genes implicated in epilepsy is most efficiently accomplished using an NGS panel to provide accurate information to the physician for treatment planning.
Nextgeneration sequencing assays are performed as LDPs under CLIA.
An example of such one gene is SCN1A, which causes Dravet syndrome and can be successfully treated.
117 It is important to avoid treatment with sodium channel blockers as these can worsen seizures in Dravet syndrome.
These include phenytoin (Dilantin), fosphenytoin (Cerebyx, Prodilantin), carbamazepine (Tegretol), and other medications.
As ongoing research reveals new genes and mutations relevant to these diseases, it is important to classify newly found variant quickly and accurately.
Open access to new information and consensus efforts to define standards for classification and reporting of variants and VUSs will be critical in ensuring that patients get the most upto-date and complete information from genomic testing.
104, 105 Another example is the recently discovered gene TBCKrelated epilepsy.
118 TBCK-related intellectual disability syndrome is rare with developmental delay, hypotonia, and seizures.
Children with lower levels of the TBCK protein have slower cell mTOR signaling, which can be improved by the addition of leucine, which may provide future therapeutic options.
It is important that clinical laboratories adapt their NGS panels to incorporate new targets as clinical utility is established.
Neuromuscular diseases (NMDs) refer collectively to the many disorders that affect the peripheral nervous system, either by impairing the proper development or functioning of muscles or by damaging the associated nerves or neuromuscular junctions.
Muscular dystrophies that form the majority of inherited NMDs share clinical, genetic, and pathological characteristics, including muscle degeneration and wasting, progressive muscle weakness, hypotonia, and variably elevated serum creatine kinase levels.
Cardiac involvement is often present, accounting for high morbidity and mortality.
There are 80 different genetically defined types of muscular dystrophies categorized based on the age of onset, the specific muscles involved, and common characteristic clinical features.
[119] [120] [121] [122] Congenital muscular dystrophies and limb-girdle muscular dystrophies are the 2 major subgroups; these are genetically heterogenous, with many new genes being implicated in recent years.
Lack of pathognomonic signs or specific biochemical markers and the presence of high phenotypic overlap with other forms of NMDs make diagnosis difficult.
Molecular assessment is critical not only to establish a diagnosis but also to allow participation in clinical trials of therapeutic treatments that are designed for a specific set of variants or variant types.
An extensive diagnostic workup involving protein studies on muscle biopsy may be used to narrow the number of single genes to be tested, but many patients never are specifically diagnosed.
Comprehensive approaches to expedite molecular diagnosis now include NGS-based panel testing for sequence analysis of all disease-associated genes in a single analysis.
[123] [124] [125] Heritable Cancer Panel Genomic testing for familial cancer syndromes has become routine over the past 2 decades.
Approximately 50 heritable cancer syndromes are recognized and are causative of 5% to 10% of all cancers.
Although familial breast cancer has perhaps become the most publicly known example of testing, genes for other inherited cancer syndromes may be included in NGS multigene panels.
Patients who carry a germline mutation of BRCA1 or 2 have a lifetime risk of breast cancer of 60% to 70%.
The US Preventative Task Force recommends BRCA1 and 2 testing for women who have family members with breast, ovarian, fallopian tube, or peritoneal cancer or meet other criteria.
126 Numerous studies have demonstrated the psychosocial benefits of genetic counseling and testing.
126, 127 For patients who carry a germline mutation of BRCA1 or 2, surgical interventions may significantly reduce the risk of cancer or death.
Contralateral mastectomy has been shown to reduce the risk of death in carriers by 48%.
128 Prophylactic salpingooophorectomy has been shown to dramatically reduce the mortality due to ovarian cancer or breast cancer in BRCA1 mutation carriers.
129 Similarly, identification of Lynch syndrome mutations can permit surveillance leading to earlier detection and marked improvement in survival in patients developing colorectal, endometrial, or ovarian cancer.
130 Although more than 2 decades have passed since sequencebased analysis of high-penetrance cancer genes has been performed, only laboratory-developed procedures have been available.
Countless patients and families have benefited from the availability of these LDPs.
Epidermolysis bullosa (EB) is an inherited skin and connective tissue disease that causes skin and oral blistering with only mild trauma (http://www.niams.nih.gov/health_info/epidermolysis_ bullosa/epidermolysis_bullosa_ff.
Accessed November 4, 2016) .
The severity of the disorder depends on the layer of skin where the tissue separation occurs.
131 Approximately 99% of patients with biopsy-proven EB will have mutation(s) in 1 of 18 genes known to cause the disorder (http://www.niams.nih.gov/health_info/epider molysis_bullosa/epidermolysis_bullosa_ff.
Accessed November 4, 2016).
Knowledge of the specific gene can direct therapy and provide reproductive options for the family.
For many years, it was possible only to sequence the suspect genes one by one, taking months to years, and only on a research basis.
Recently, with the advent of NGS technology, a small number of clinical laboratories have stepped in to develop a rapid multigene NGS approach that provides answers quickly in time to make treatment decisions as well as to provide carrier and prenatal testing in at-risk families.
131, 132 As new candidate genes have been identified, NGS has been validated and offered for clinical testing.
Under CLIA, the validation data collected by the laboratory was subject to ongoing peer review, and the laboratory participates in ongoing PT to demonstrate assay quality.
As the disease is rare (200 children born with EB annually), 133 the cost of bringing such a test through FDA for approval is prohibitive.
Without an available LDP, patients and families affected by this disease would go without specific diagnoses, be unable to enroll in gene/mutation specific therapies (currently in development by at least 4 pharmaceutical companies and academic centers) (http://www.debra.org/research-trials.
Accessed November 4, 2016), or have control over their reproductive lives.
Whole-exome sequencing (WES) involves evaluating the coding regions of all *20 000 human genes at once, to search for the underlying molecular cause of an undiagnosed but presumed genetic disorder.
These tests are used for patients who already have undergone extensive genetic diagnostic testing and exhausted the (limited) FDA and LDP singlegene tests or in cases where it is more cost-and timeeffective to start with WES.
Whole-exome sequencing is used for the so-called "diagnostic odyssey" patient and has a high diagnostic yield for these patients, with 25% to 30% of studies yielding a diagnosis.
134, 135 The tests are highly complex and involve capture of the relevant DNA segments, sequencing of those segments, bioinformatics approaches to sequence alignment and identification of variants (differences between patient DNA and reference), interpretation of the identified variants, and report generation.
Under CLIA and NYSDOH, the technical validation data collected by the laboratory prior to offering WES are required, and the laboratory participates in ongoing PT (through CAP and sample exchanges) to demonstrate assay quality.
As this is cutting-edge science and medical practice, the capture and sequencing technology, bioinformatics, and interpretation tools are evolving at a very fast pace.
To provide the best service to patients, laboratories must frequently update, revalidate, and offer new services.
Whole-exome sequencing is offered as an LDP by laboratories which have extensive experience in genetic testing.
The time delay involved in bringing this test and its frequent modifications to the FDA is prohibitive.
As these tests serve the rare disease community, and reimbursement is limited by the lack of pricing for a specific CPT code, the cost of bringing WES through FDA approval would be a major deterrent.
Innovation would be slowed, and likely several laboratories would remove the test from test menus.
With 30 000 affected individuals in the United States, 136, 137 Huntington disease (HD) falls under the category of a rare (or orphan) disorder, and given its limited market, no commercial genetic testing platform has been developed or submitted to the FDA for review.
The relatively small number of laboratories offering diagnostic or predictive (presymptomatic) testing for this disorder must therefore rely entirely on LDPs, without which patients with HD and their at-risk relatives would have no access to testing and diagnosis.
Despite the characteristic clinical features (the movement disorder [chorea] along with intellectual decline), and lack of preventive or curative treatment for HD, genetic testing is widely relied upon by HD families and their physicians.
Onset of symptoms is often insidious and nonspecific, and definitive early diagnosis can only be accomplished at the DNA level.
132 Presymptomatic testing, which is offered to the adult offspring of patients with HD (who are at 50% risk of inheriting this autosomal dominant disease), can only be accomplished using LDPs and allows crucial life-planning decisions, such as educational pursuits and career choices, marriage, and whether to have children (and if so, affording the ability to pursue prenatal diagnosis) and whether to begin planning for inevitable disability.
Without this test, all of these at-risk relatives (of which there are an estimated 200 000 in the United States) 137 would lead their lives anxiously waiting for the symptoms to begin, when half of them are actually at no risk because they did not inherit the mutant gene from their affected parent.
Although there are at-risk relatives who choose not to avail themselves of the predictive test, the many who do opt to be tested credit it with freeing them of years of obsessive uncertainty.
The results can afford the affected patients the opportunity to enroll in clinical trials (involving drugs or neuronal stem cells), with the aim of preventing or delaying the onset of symptoms.
138 Although these studies are still in an early phase with no outcome data yet available, they give patients some hope for the future, perhaps for themselves but also for future patients.
Given the mechanism of gradual neuronal cell death in the basal ganglia, it stands to reason that the earlier such intervention is initiated-ideally in the presymptomatic stage-the higher the chances of success.
Huntington disease is one of the trinucleotide repeat disorders, caused by expansion of a tandem repeat of CAG in the first intron of the huntingtin (HTT or IT15) gene.
In contrast to FX syndrome and some of the other trinucleotide repeat disorders, the difference between the mutated and nonmutated repeat length can be as little as a single repeat (ie, 3 nucleotides).
Thus, extreme care is required in the sizing of the repeat, especially when it falls near the cutoff length of 40 40 repeats or higher which is diagnostic or predictive of HD, with 100% penetrance.
Fortunately, the LDPs in current use, relying on capillary electrophoresis, are very accurate in determining repeat length, as attested by the excellent performance in CAP proficiency surveys.
Busulfan is a bifunctional DNA alkylating agent typically given to patients as a conditioning agent prior to hematopoietic cell transplantation (HCT) for the treatment of hematologic malignancies.
Therapeutic drug monitoring (TDM) is crucial for the safe and efficacious use of busulfan due to a narrow therapeutic index based on area under the curve (AUC) calculations.
Too low a dose places the patient at risk of either graft failure or early relapse.
139, 140 On the other hand, too high a dose increases the risk of neurotoxicity 141 as well as a severe and life-threatening complication termed hepatic sinusoidal obstruction syndrome (SOS).
139, 142 Hepatic SOS, previously termed hepatic veno-occlusive disease (VOD), refers to the occlusion of terminal hepatic venules and hepatic sinusoids.
Severe cases of VOD can lead to hepatorenal syndrome, causing multi-organ failure, hepatic encephalopathy, and death.
Veno-occlusive disease typically occurs in the context of HCT, particularly after administration of conditioning regimens prior to HCT.
It is one of the most feared complications of HCT and accounts for a significant fraction of HCT-related mortality.
143 Severe cases, which account for approximately 25% to 30% of SOS, are almost always fatal.
142, 144, 145 Despite a clear need for busulfan TDM, there are currently no FDA-approved assays available for the quantitation of busulfan in blood.
For this reason, various bioanalytical methods have been developed 146 and are currently in use by multiple laboratories.
Data from a busulfan proficiency program organized by the University of Washington/Seattle Cancer Care Alliance show a total of 24 participating laboratories at the present time.
Of these, 6 laboratories use gas chromatography (GC) methods, 3 use HPLC methods, and 14 use liquid chromatography-tandem mass spectrometry (MS) methods.
147 All of these methods are non-FDA-approved tests independently developed and validated for clinical use by their respective clinical laboratories.
The use of these methods is also driven by the need for high precision and accuracy.
Current criteria for acceptable laboratory performance in the analysis of busulfan is +10% of the known concentrations for medium and high concentrations and within +15% of the known concentration for low concentrations.
147 This is due to the fact that dosing change decisions are based on AUC calculations, which depend on blood concentrations of busulfan measured from multiple timed blood draws.
Multiple small analytical errors can easily add up to big differences in calculated AUC values.
Busulfan testing is currently available from reference laboratories.
However, many busulfan regimens call for intravenous infusions every 6 or 24 hours for 3 to 4 days.
Bone marrow transplant teams need quicker turnaround times than can be reasonably provided by sendout testing in order to be able to make dosing adjustments within this limited timespan.
Guidelines have also been recently published by the American Society for Blood and Marrow Transplantation Guidelines Committee advocating for personalized busulfan dosing using busulfan TDM in certain busulfan regimens.
148 For these reasons, laboratory-developed methods for busulfan will continue to play key roles in the management of hematologic malignancies at cancer centers throughout the world.
Very sensitive measurements of serum androgens are important in adult and pediatric endocrinology and oncology.
Very-low-level testosterone (Te) measurements are needed for adult women, whose values are routinely <50 ng/dL, in children, and men undergoing antiandrogen therapy whose values are usually <10 ng/dL.
149 The most commonly used methods for steroid analysis are FDA-approved immunoassays because they are rapid and sensitive enough for most routine applications involving healthy adult males.
However, Te immunoassays lack the sensitivity requirements for chemically castrated males, women, and children.
Many immunoassays also lack specificity and accuracy as immunoassays may show cross-reactivity with structurally similar compounds.
[150] [151] [152] In addition, most immunoassays are not standardized against internationally recognized standards.
For these reasons, a number of sensitive and specific assays using MS have been described for Te.
[153] [154] [155] The lack of sufficient accuracy and standardization of Te assays is a major concern for the clinical and public health communities.
156 Several years ago, the Endocrine Society, in partnership with the CDC, convened a meeting with various relevant professional societies and industrial partners to create the Partnership for the Accurate Testing of Hormones (PATH) whose mission is to improve the accuracy and standardization of a variety of steroid hormone tests.
[156] [157] [158] The PATH has worked with the CDC and begun to address this concern through its HoSt program, which provides laboratories with specimens spanning their analytical measurement range that have been previously analyzed using the CDC reference method.
155 Many assays using MS have been approved by the CDC HoST program, 159 however not a single FDA-approved immunoassay has met the performance requirements.
Testosterone is a perfect example of an LDP that is indispensable for patient care and allows for accurate measurements to be made on children, women, and male patients with cancer receiving antiandrogen medication.
Ethylene glycol is a colorless, sweet-tasting liquid commonly encountered in automobile antifreeze.
Because of this widespread availability, it is also a commonly encountered toxicological agent in both accidental and self-inflicted poisonings with 6078 exposures in 2014.
160 Ethylene glycol poisoning classically presents with a metabolic acidosis caused by the production of toxic metabolites, primarily glycolic acid and oxalic acid.
This is also often accompanied by an anion gap and osmolal gap.
Untreated ethylene glycol poisoning can also progress to acute renal failure when high levels of oxalate anions combine with calcium to develop crystals in the kidneys and urinary tract.
161 Ethylene glycol poisoning is an urgent, toxicological emergency.
Once ethylene glycol is identified, the drug fomepizole is typically administered.
Fomepizole inhibits alcohol dehydrogenase, the enzyme that metabolizes ethylene glycol, to slow the accumulation of toxic metabolites.
Fomepizole and ethanol dramatically lengthen the half-life of ethylene glycol, and therefore, hemodialysis is often required to clear the poison.
162 Both the diagnosis and treatment of ethylene glycol poisoning are heavily dependent on laboratory measurements.
No FDA-approved assays for ethylene glycol are currently available, and all testing is performed by laboratory-developed procedures.
The 3 most common methods for the analysis of ethylene glycol are GC with flame ionization detector, GC with MS, and enzymatic assays.
163, 164 Gas chromatography with mass spectrometry is considered the gold standard for the analysis of ethylene glycol, as it can differentiate it from interferences that plague the other 2 methods.
163, 165 In addition to initial detection needed for diagnosis, the ethylene glycol blood concentration is used to determine when hemodialysis has cleared ethylene glycol to undetectable levels.
Measurement of thyroglobulin (Tg) in serum has proven useful for detecting recurrence of treated differentiated thyroid carcinoma (DTC).
According to the American Cancer Society, the United States has over 60 000 new thyroid cancer cases each year.
The death rate is almost 2000 per year.
Differentiated thyroid cancer accounts for over 90% of cases.
Differentiated thyroid cancer produces Tg, making its measurement useful as a tumor marker for detecting recurrence.
The NCCN and American Thyroid Association (ATA) guidelines recommend Tg testing following total thyroidectomy and radioiodine ablation treatment, including tests at baseline, 6 to 12 weeks after treatment, 6 months, 12 months, and annually thereafter.
Patients free of disease have undetectable Tg.
Older competitive Tg-RIA methods are available but produce falsely high Tg results in the presence of Tg autoantibodies (Tg-Ab).
166 Newer FDA-cleared Tg assays are immunometric immunoassays (Tg-IA) and can detect Tg at concentrations down to approximately 0.1 ng/mL.
Generally, Tg is captured with a solid-phase antibody, then quantitated using a detection anti-Tg reagent.
The signal is directly proportional to the amount of Tg.
The assay design is susceptible to interference from endogenous anti-Tg-Ab.
166 The intended use of these Tg-IA are Tg measurement in Tg-Ab-negative (Tg-Ab � ) patients.
The FDA requires Tg-Ab testing whenever Tg is measured using these cleared methods.
Depending on the method used, up to 36% of treated patients with DTC are Tg-Ab positive (Tg-Ab þ ).
Thus, many of these patients have falsely low or even falsely negative Tg results when measured by IA.
Four Tg-Ab assays are in common use and are not harmonized, detecting Tg-Ab in widely divergent numbers of treated patients with DTC.
167 In addition, the degree of Tg interference cannot be predicted from the magnitude of the Tg-Ab result.
Thus, up to 20% of patients with recurrence are missed by Tg-IA testing.
To circumvent the Tg-Ab interference, Hoofnagle and Wener developed and validated a MS Tg method (Tg-MS) using tryptic digestion and immunocapture of Tg-specific peptides followed by MS focused on those peptides.
167 They demonstrated the Tg-MS method accurately measures Tg in the presence of Tg-Ab.
162 The tryptic digestion destroys the Tg-Ab, eliminating their interference with the assay.
Four national reference laboratories have adopted versions of this method, and harmonization efforts are underway.
A recent clinical outcome study compared Tg-IA, Tg-RIA, and Tg-MS in both Tg-Ab � and Tg-Ab þ patients.
As predicted from the assay designs, all methods were equivalent for Tg-Ab � cases, but the Tg-MS was more accurate for Tg-Ab þ cases.
Tg-IA methods had more false negatives and Tg-RIA had more false positives.
168 Although Tg-MS has not yet been incorporated into the current guidelines, the ATA guideline mentions it as a promising new technique.
Without Tg-MS, thyroid cancer recurrence in Tg-Ab þ patients can be missed, thus delaying follow-up and creating patient harm.
Antimicrobial susceptibility testing, used to determine whether antibiotic treatment will be successful, is an essential component of the microbiology culture report.
Emerging resistance among pathogenic bacteria and new antimicrobial agents require frequent updates to both testing methods and interpretation of the results.
Most laboratories use automated instruments to perform minimal inhibitory concentration (MIC) testing to determine whether a patient's isolated bacteria are susceptible, susceptible dose dependent, intermediate, or resistant to a panel of antibiotics.
These interpretations are based on FDA breakpoint criteria published at the time of drug approval and periodically updated to respond to the appearance of new resistance mechanisms.
169 The FDA also clears automated instrumentation used to determine MIC values (via the 510k process).
170 Although the MIC testing process may not be changed, new breakpoints added to the instrument software require a revised 510k application.
Because the FDA does not have the authority to require manufacturers to submit data for revised breakpoints within a specified time frame, manufacturers may elect to use outdated breakpoints rather than face the expense of a 510K resubmission.
A recent example was the 3year delay between the release of updated breakpoints for diagnosing carbapenem-resistant Enterobacteriaceae in 2010 and the ability to use these breakpoints in the clinical laboratory.
This delay was used to calculate the potential for additional carriers of multidrug-resistant Enterobacteriaceae in Southern California health-care systems.
As many as 1821 additional carriers of MDRO Enterobacteriaceae were estimated to have occurred in Orange County, California because of this delay.
171 The disastrous spread of MDRO Enterobacteriaceae can be mitigated by laboratories validating testing methods that enable use of updated antimicrobial breakpoint interpretations before FDA-cleared testing is available.
Specifically, modifying a manufacturer's instructions, including interpreting MIC results using a revised breakpoint other than that listed in the product insert, is a change that renders the procedure an LDP.
Without the option of using an LDP, one is left reporting outdated interpretations that miss resistant strains leading to unacceptable patient care.
As illustrated, LDPs are an integral part of the spectrum of tests and procedures performed by clinical laboratories which fulfill a critical need for patient care, particularly in rapidly evolving areas such as testing for personalized medicine.
Laboratory testing should be consistent with national/international consensus treatment guidelines, which may require development of procedures earlier or for new clinical purposes not fulfilled by FDA-approved kits.
In such cases where clinical testing needs exist beyond the original FDA purpose, laboratories must be able to perform additional validation of new sample types or develop additional assays to include new mutations or analytes that are needed.
As illustrated by these case examples, laboratories and professional organizations often work together to broadly compare and optimize assay performance, creating consensus standards that raise the quality of testing overall.
In contrast, the current structure for FDA approval requires review of assay kits individually, or in comparison with the predicate method, rather than assessing and improving performance across the spectrum of assay options available.
The science of laboratory medicine has advanced dramatically in the almost 3 decades since CLIA was enacted, and updates and expansions to CLIA regulations could be useful.
Additional resources, such as reference materials, and consensus practice guidelines would extend the quality framework that all laboratories and manufacturers utilize.
For example, consensus guidelines that include such details as the target for percent allele frequency detectable, requirements for percent tumor cell content, what mutations and variants should be included, and sample types to be tested would be useful as a guide for assay validation as well as in standardization of the practice.
Professional expert groups are already generating assay and practice guidelines.
69, 73, [172] [173] [174] [175] Ideally, clinical laboratories and kit manufacturers would utilize appropriate reference materials to help standardize the results obtained for any particular analyte regardless of technology platform or laboratory setting.
To address the needs for reference materials to facilitate assay result standardization, a multistakeholder initiative, the Diagnostic Quality Assurance Pilot, has been launched to design, develop, and evaluate traceable reference sample materials (referred to as reference materials) to better provide molecular pathology laboratories with the means to demonstrate equivalent performance of LDPs and companion diagnostic IVDs for targeted cancer therapy.
This Quality Pilot emerged from the Sustainable Predictive Oncology Therapeutics and Diagnostics working group, launched in 2013 by Tapestry Networks (Waltham, Massachusetts), which was composed of diverse stakeholders (oncologists, pathologists, patient advocates, third party payers, and regulators) for the purpose of designing a quality pilot to advance these goals (http://www.ta pestrynetworks.com/initiatives/healthcare/oncology-therapeu tics-and-diagnostics/diagnostic-quality-assurance-pilot.cfm.
Accessed April 27, 2017).
176 The Tapestry pilot proposes that laboratories would be allowed to utilize assays that best serve the needs of their patients based upon performance, quality, clinical needs, and the test menus and volumes of that particular laboratory.
It is not critical that laboratories all use the identical assay or test platform, provided that all are able to get the correct answer.
To close, the overarching goal is the efficacy and safety of our clinical laboratory tests and procedures for patients.
Pathologists and laboratory professionals need the best and most upto-date tools to do their jobs and optimize patient care.
Some of these will be FDA approved or cleared kits, and others will be laboratory-developed procedures performed under CLIA; both have their place.
Laboratories have a long history of success performing LDPs, as illustrated by these case studies.
As much as possible, these capabilities need to be performed on-site to insure that the results can be integrated with other clinical and laboratory findings, interpreted as a whole and completed in a timely fashion.
Also important is the handson training of the next generation of physicians, for whom, we hope, maximal use of genomic and other laboratory information will be a way of life as they treat human disease.
That is the promise of personalized medicine!
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) received no financial support for the research, authorship, and/or publication of this article.
The '70% claim': what is the evidence base?
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Ms Mary Williams, Dr Barbara Zehnbauer, Dr Peter Hulick, and Dr David Klimstra provided valuable comments on the manuscript.
Ms Laura Metz provided clerical support.
key: cord-332038-icyut3xa authors: Pillaiyar, Thanigaimalai; Meenakshisundaram, Sangeetha; Manickam, Manoj; Sankaranarayanan, Murugesan title: A medicinal chemistry perspective of drug repositioning: Recent advances and challenges in drug discovery date: 2020-04-02 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2020.112275 sha: doc_id: 332038 cord_uid: icyut3xa Drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs.
Recently, it emerges as an alternative approach for the rapid identification and development of new pharmaceuticals for various rare and complex diseases for which lack the effective drug treatments.
The success rate of drugs repurposing approach accounts for approximately 30% of new FDA approved drugs and vaccines in recent years.
This review focuses on the status of drugs repurposing approach for various diseases including skin diseases, infective, inflammatory, cancer, and neurodegenerative diseases.
Efforts have been made to provide structural features and mode of actions of drugs.
Sir James Whyte Black, winner of the 1988 Nobel Prize in Medicine [1] It is reported that the Food and Drug Administration (FDA) has approved agents against about 400 human proteins [2] , 90% of which comes under the classification of enzymes, transporters, G protein-coupled receptors (GPCRs), cluster of differentiation (CD) markers, voltage-gated ion channels, and nuclear receptors.
It is a fact that the typical de novo drug discovery program takes 10 to 15 years [3, 4] from the identification of lead molecule to market the drug and the probability of success rate is less than 10% [5] .
Over five years, the number of new drugs approved by FDA has been around 40 per year, although billions of US dollars spent by various pharma industries on the research and development [6] [7] [8] .
The success rate is less than 6% of new drug discovery and development, which is far away from addressing an unmet clinical need for disease treatments.
Because, the effective therapeutics for complex diseases like Alzheimer's disease (AD), Parkinson's disease (PD), cardiovascular diseases, and neglected diseases are still lacking.
This outcome strongly suggests that new strategies, approaches, and technologies are needed to accelerate drug discovery to advance the success rate of drug development.
Drug repurposing is a strategy consisting of finding new indications for already known marketed drugs used in various clinical settings or highly characterized compounds despite they can be failed drugs [9] .
It is a drug discovery program, which is faster and safer to develop medications against diseases/disorders for which no potential treatment is available.
In recent years, the success rate of drug repurposing approach accounts for approximately 30% of the newly FDA approved drugs, and vaccines.
This is one of the main reasons for pharmaceutical companies to show their interest in drug repurposing approach.
This approach does not require the initial six to 10 years typically needed for the development of new drugs.
Additionally, many phases of de novo drug discovery and development can be by-passed, melanin protects human skin from the radiation, continuing irradiation can result in the risk of skin damage and malignant melanoma, a cancer of melanocytes.
Besides, the abnormal production of melanin leads to a serious of dermatological disorders including melasma [13] [14] [15] [16] , freckles, age spots, and post-inflammatory melanoderma.
[15, 17] .
Melanogenesis, a process of synthesis of melanin, is a complex enzymatic and biochemical catalyzed reactions, in which tyrosinase plays a rate-limiting step: hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) followed by the oxidation of L-DOPA to Ldopaquinone, which serves as a substrate for the production of melanin [18] .
Therefore, targeting tyrosinase has been recognized as a potential approach for controlling the abnormal production of melanin.
Tyrosinase is also an important target in the food industry as inhibition of tyrosinase can prevent the enzymatic browning of fruits and vegetables.
Besides, it essential for wound healing process and immune responses in many plants, sponges and some invertebrates.
Abnormal activities of tyrosinase have been linked to neurodegenerative disorders including Parkinson's [19] and Huntington's diseases [20] [21] [22] [23] [24] .
Thiourea and analogs.
Tyrosinase inhibitors can be broadly classified into two categories: (a) polyphenols, which are mostly natural products such as arbutin, hydroquinone and kojic acids, and b) thiourea and its derivatives.
Since the 1940's, phenylthiourea (PTU, 1) has been well known as a tyrosinase inhibitor [25, 26] .
Many research groups including ours have extensively studied the structure-activity relationships for PTU as tyrosinase inhibitors [27, 28] .
Choi et al.
screened the FDA approved drug library that has closed structural similarity to PTU (see Figure 1 ) [29] .
For example, ethionamide (2), a second-line antituberculosis drug used for the treatment of multi-drug resistant tuberculosis, shares a chemical similarity that led to the discovery of a new mushroom tyrosinase inhibitor with an IC 50 value of 4.0 µM.
Other commercially available analogs of 2 including, prothionamide (3), thioisonicotinamide (4), pyridine-3-carbothiomide (5), pyridine-2-carbothiomide (6) , and thiobenzamide (7) were identified as potent tyrosinase inhibitors.
In particular, compound 7 had a strong inhibitory activity than other molecules, which suggests that the pyridine ring in compound 2 can be replaced by other aromatic moieties, including a benzene ring.
However, the poor inhibitory activity of isoniazid (8), a first-line antituberculosis drug, suggests that carbothiomide group was crucial for tyrosinase inhibitory activity.
Moreover, comparing the inhibitory activities of drugs 2 and 3 implies that the additional aliphatic tail was not required for inhibiting the tyrosinase.
Inhibitory kinetic studies suggest that drug 2, and its analogs 3-7 were reversible and non-competitive.
In cellular assay, drugs 6 and 7 markedly decreased the melanin content in B16 cells to the values of 44% and 37%, respectively, without inducing any cytotoxicity up to 50 µM concentration.
Further studies suggest that the drug 7 had strong inhibition of mammalian tyrosinase.
However, the inhibition exhibited by drug 2, and its analogs were weaker than those obtained by PTU observed in enzyme, and melanin content assays.
The same research group continued to search for molecules that are in clinical usage, and contain thiourea moiety [30] .
As a result, they could retrieve some thiourea containing drugs such as thioacetazone (9) , ambazone (10) , methimazole (11) , carbimazole (12) , thiouracil (13) , methylthiouracil (14) , and propylthiouracil (15) .
Thioacetazone (9) (also called as thiacetazone) is an anti-tuberculosis drug [31] .
Ambazone (10) is an oral antiseptic drug used in Europe [32] .
The other five molecules (11) (12) (13) (14) (15) are antithyroid drugs [33] .
These drugs, except 12, exhibited remarkable inhibitory activities against mushroom tyrosinase (see IC 50 values of each in Figure 1 ), although they were comparatively weaker than 1 (1 µM) .
Kinetics studies of tyrosinase inhibition assigned these thiourea-containing drugs as non-competitive inhibitors.
In cellular assay, using B16 cells, drug 10 among other thioureas, significantly decreased the melanin content by 20% without inducing any cytotoxicity up to 20 µM.
Further, enzymatic studies with cell extracts of B16F10 cells confirmed that thioureacontaining drugs affected the function of mammalian tyrosinase.
In an extended study, the same group repositioned thiopurine drugs 16-19 as tyrosinase inhibitors [34] .
Thioguanine (16) , a drug used for the treatment of leukemia, is one of the essential medicines required for a basic health system, recommended by the world health organization (WHO).
It inhibited tyrosinase activity with a K i value of 52 µM.
In addition to that, two other thiopurine drugs such as mercaptopurine (17) and azathioprine (18) were discovered as tyrosinase inhibitors, and among them, drug 17 showed stronger K i value (K i 52 µM) than the drug 16.
Azathioprine (18) exhibited a poor tyrosinase inhibition.
These results suggest that the sulfur atom possibly plays an important role in the interaction of tyrosinase.
Interestingly, thioinosine (19) , a metabolic product of mercaptopurine through the attachment of sugar moiety exhibited an excellent tyrosinase inhibitory activity with a K i value of 8.0 µM.
Further, kinetics studies classified the drugs 16, 17, and 19 as competitive inhibitors.
In the cellular assay, these drugs inhibited the melanin content without cytotoxicity up to 50 µM.
In particular, drug 16 at 50-µM concentration, remarkably reduced the melanin content by 57%, without any apparent cytotoxicity.
The thiopurine drugs were docked into four different crystal structures complexed with inhibitors tropolone, kojic acid, hydroquinone and PTU ( Figure 2 ) [35] [36] [37] .
In the molecular It also predicted that the intramolecular hydrophilic contacts with the residue of E-256 disappeared in mercaptopurine ( Figure 2C ), whereas thioguanine ( Figure 2B ) and thioinosine possess the contacts within 3.8 Å. thioinosine (19) are represented.
This Figure was adopted from the publication of Choi, J. et al [28] .
Drugs repositioning for cancer therapy.
As cancer is one of the leading cause of death worldwide [38] , pharmaceutical companies invest billions of dollars in developing new anticancer drugs.
The drug discovery and development process for the cancer treatment takes an average of 13 years at a cost of approximately $ 1.8 billion [39] .
However, only 5% of the drugs that enter clinical trials are approved.
This prolonged duration for the drug development and enormous cost of the (pre)clinical trials for their approval emphasizes the need for a drug repurposing approach (see Figure 3 for drugs repurposing in cancer therapy).
Aspirin (20) , also known as acetylsalicylic acid, is one of the non-steroidal anti-inflammatory drugs (NSAIDs), which has been widely used as an analgesic and an antipyretic to prevent the heart attack and stroke.
For the first time, Gasic and co-workers reported the possible role of aspirin in cancer therapy.
They discovered that the antiplatelet activity of 20 in tumor-bearing mice was associated with a 50 % reduction in lung metastasis [40] .
A recent study also indicated that the daily intake of the drug 20 (75 mg) produced a significant beneficial effect against gastrointestinal, esophageal, pancreatic, brain, prostate, and lung cancer [41] .
The mode of action aspirin is reported to modulate numerous molecules, which are associated with the tumorigenesis process [42] .
Preclinical studies revealed that the anticancer activity of drug 20 has been attributed by its inhibition of cyclooxygenase (COX) enzymes that promotes carcinogenesis through the synthesis of prostaglandins (PG), including PGE2 [43] .
Besides, drug 20 was reported to inhibit the activation of transcription factor NF-κB, which is critical to regulating the expression of genes involved in apoptosis [44] .
In a study reported by Li Ling et al.
compound 20 not only inhibited proliferations and promoted apoptosis of cancer cells, but also delayed and overcame acquired resistance to targeted therapy.
The underlying mechanism could be attributed to enhanced cancer stemness and activated NF-kB signaling in acquired resistant tumors were suppressed by aspirin and rendered resistant tumors more sensitive to aspirin than parental, sensitive cells in terms of proliferation, apoptosis and cancer stemness.
On the contrary, aspirin has no effects on normal lung and mammary epithelial cell proliferation at concentrations used on lung and breast cancer cells.
Hence, aspirin could be a potential candidate for combination therapy for lung and breast cancers [45] .
Other studies suggest that the anticancer property of aspirin (20) has been linked to the phosphatidylinositol-3-kinase (PI3K) pathway, and Ras-Raf-MEK-ERK signaling cascade.
While numerous studies suggest the potent anticancer activities of drug 20, the overall benefit is limited as it is associated with serious side effects including the gastrointestinal and renal toxicities.
Therefore, there is no clear recommendation to take 20 for population-wide use.
On the other hand, as a primary cancer prevention tool, many reports revealed using 20 would have a greater benefit in the population at age 40-85.
Thus, U.S. preventive services task force recommendation statement (USPSTF), recommended for daily intake of 20 for patients above 40 years with increased risk of cardiovascular disease and colorectal cancer.
Celecoxib (21) belongs to the family of NSAID, which has been used to treat pain and inflammation associated with rheumatoid arthritis (RA) and osteoarthritis (OA) [46] .
In 1999, the FDA approved celecoxib, which is a highly selective reversible inhibitor of COX-2, a well-known inflammatory cancer target.
Because of its COX-2 inhibitory activity, the antitumor activities of drug 21 have been extensively studied and shown to have chemopreventive activities against various cancer types.
Other than COX-2, celecoxib targets glycogen synthase kinase (GSK) 3β, β-catenin, NF-κB, AKT8 virus oncogene cellular homolog (AKT), and B cell lymphoma (Bcl)-2 families [47] .
In people with familial adenomatous polyposis (FAP), a daily dosage of drug 21 (400 mg) significantly reduced the risk of colorectal adenomas.
FDA approved this compound to reduce colon and rectal polyps in people with familial adenomatous polyposis [48] .
However, it was associated with some drawbacks including gastrointestinal, renal, and cardiotoxic effects.
Ibuprofen (22) is an NSAID, which has been primarily used to treat fever, pain, and inflammation.
At the molecular level, ibuprofen inhibits COX, which converts arachidonic acid to prostaglandin.
However, it is not selective towards any isoform of COX.
The drug was marketed for the treatment of rheumatoid arthritis in the United Kingdom (1969) , and in the United States (1971) as well.
The anticancer activity of drug 22 has been investigated in various cancer cell types.
Ibuprofen has been shown to inhibit the growth of prostate cancer cells [49] .
In adenocarcinoma gastric cells, drug 22 showed antitumor effects, which have been mediated by the anti-angiogenesis, induction of apoptosis, and reduction of cell proliferation [50] .
The administration of drug 22 induces apoptosis in metastatic melanoma cell lines [51] .
Ibuprofen also reported to increases the chemosensitivity of cisplatin by decreasing the levels of heat shock protein 70s (Hsp70s) in lung cancer cells.
Hsp70s are an important part of the cell's machinery for protein folding, and their function was associated with resistance to apoptosis [52] .
Therefore, by blocking Hsp70s, ibuprofen increased the apoptosis by increasing the sensitivity of cisplatin.
Thalidomide (23) is an immunomodulatory drug, which was originally developed as a sedative-hypnotic for the treatment of nausea during pregnancy.
However, it was withdrawn due to its teratogenic effects.
The drug was demonstrated whether it could be used for treating patients with refractory myeloma, because of its anti-angiogenic activity.
After successful clinical evaluations, FDA approved the drug 23 for treating multiple myeloma.
Additionally, molecule 23 also showed efficacy against several malignancies, including acute myeloid leukemia [53] , myelodysplasia [54] , and myelodysplastic syndrome [55] .
Mechanistically, thalidomide binds to cereblon, which forms an E3 ubiquitin ligase complex, resulting in the rapid ubiquitination and proteasomal degradation of transcription factors, Ikaros and Aiolos [56] .
These two transcription factors are transcriptional regulators of B and T cell development [57] .
Metformin (24) is an orally available first-line drug and has been widely used for the treatment of type 2 diabetes.
The molecular mechanism of metformin involves the activation of adenosine monophosphate (AMP)-induced protein kinase (AMPK), a key enzyme regulating cellular metabolism.
Rapamycin (mTOR), a gene that is involved in the survival of cancer cells is negatively regulated by AMPK.
Metformin is also able to reduce the signals of mTOR by inhibiting Rag-mediated activation of mTOR [58] .
In general, diabetic patients have an increased risk of several cancer types; especially diabetic women have a 20% risk of developing breast cancer.
Several studies suggested the anticancer property of drug 24.
The drug 24 at the dose of 250-500 mg/day has been shown to reduce the risk of cancer as well as its daily dose reduces the incidence of gastrological cancer in patients with diabetes [59, 60] .
Several reviews and meta-analysis suggests that taking drug 24 was associated with reduced risk of all cancer mortality in patients with diabetes [61] .
In a recent meta-analysis of several anti-diabetic drugs, it was found that the patients using drug 24 had an overall reduced risk of cancer and decreased mortality rate by 14 and 30%, respectively.
Whereas, the use of insulin was associated with an increased risk of cancer and mortality.
The recent phase 3 clinical trial studies using the occurrence of colorectal adenomas as a biomarker for cancer as a primary endpoint at 1 year after intervention revealed that metformin reduced both occurrence and number of adenomas/polyps in the patients at low dosage level.
Methotrexate (25) is a competitive inhibitor of dihydrofolate reductase (DHFR); a critical enzyme that involved in the synthesis of DNA, RNA, thymidylates, and proteins.
The hydrofolate inhibitory activity of drug 25 was responsible for its anti-leukemia activity.
Besides, drug 25 was effective against a wide range of malignancies including breast, head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, and trophoblastic neoplasms [62] .
FDA approved this compound in 1988 for the treatment of osteosarcoma, breast cancer, acute lymphoblastic leukemia, and Hodgkin lymphoma.
Several reports suggest that the antitumor activity of methotrexate is also due to its ability to target inflammatory pathways.
For instance, methotrexate was reported to suppress the NF-κB through the release of adenosine in cancer cells [63] .
Rapamycin (26) , also known as sirolimus, which was originally developed as an anti-fungal agent.
However, drug 26 was withdrawn due to its potent immunosuppressant and antitumor activities.
Mechanistically, the drug inhibits T cells and B cells by decreasing their sensitivity to IL-2 through inhibition of mTOR, which is highly upregulated in many tumor cells [64] .
In the year 1999, FDA, approved rapamycin for the prevention of allograft rejection.
After that, this drug has been investigated for its anticancer properties.
In recent studies, drug 26 was reported to reduce the colony formation of leukemia progenitor cells in patients with acute myeloid leukemia [64] .
In addition to that, drug 26 also showed efficacious in patients with imatinib-resistant chronic myelogenous leukemia through the suppression of vascular endothelial growth factor (VEGF) mRNA levels in leukemia cells with mild side effects [65] .
Diclofenac (27) is an acetic acid derivative of NSAID class, which has been used to treat pain and inflammatory diseases such as gout.
Its mode of action was believed to suppress the [73] .
In the Phase II clinical trial, it has been investigated in combination with calcitriol for recurrent prostate cancer.
The results showed that the combination was well tolerated.
Statin family of drugs are lipid-lowering agents that inhibit the rate-limiting 3-hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) reductase in the cholesterol biosynthesis pathway.
Statins are commonly prescribed to reduce cholesterol synthesis in patients with a high risk of cardiovascular disease.
In addition to that, statins inhibit the mevalonate pathway that provides mevalonate, farnesyl, and geranyl pyrophosphate.
These molecules are important for the cell cycle progression and cell proliferation, and therefore statins represent promising candidates in cancer therapeutics.
In chronic myeloid leukemia cells, simvastatin (29) [74] .
and other natural statins including mevastatin (30) , lovastatin (31) , and pravastatin (32) displayed TNF-induced apoptosis through the downregulation of NF-κB mediated antiapoptotic gene products [75] .
The anti-cancer activity of statins was also investigated in animal studies, in which statins were effective in reducing the incidence and growth of tumors [76] .
Several observational studies and meta-analysis supports the positive correlation of using statins with their chemopreventive effect in humans.
Meta-analyses revealed that the use of statins reduced the risk of patients with gastric cancer [77] .
as well as esophageal [78] , and hepatocarcinoma cancer types [79] .
In a case-control study, drug 29 with a dosage of 40 mg/day for 2-5 years significantly reduced the incidence of colorectal cancer [80] .
Depakine (33) or valproic acid (VPA) is a short-chain free fatty acid mainly used to treat epilepsy, bipolar disorders, and migraine.
Its anticonvulsant activity has been attributed to the blockade of voltage-gated sodium channels and increased levels of gamma-aminobutyric acid (GABA) in the brain.
Anti-cancer activity of drug 33 was first established in leukemia cells, in which the drug 33 was shown to inhibit histone deacetylase (HDAC) [81] .
Depakine has also been found to suppress the production of cytokine and to modulate inflammatory signaling cascade.
In human leukemia and human glioma cells, drug 33 was able to suppress the production of IL-6 and TNF-α [82] .
In prostate cancer cells, drug 33 suppressed the IL-6 through inhibition of NF-κB activity [83] .
Some of the clinical trials of drug 33 have advanced to Phase II for sarcomas, thyroid cancers, acute myelogenous leukemia, B cell lymphoma, breast cancer, melanoma, non-small, and small-cell lung cancers, prostate cancer, recurrent glioblastoma, and relapsed/refractory leukemia (www.clinicaltrials.gov.)
Recently, Abdullah et al [84] has shown that pitavastatin (34) antagonizing the PRC2 catalytic activity [86] .
Treatment for acute myeloid leukemia (AML) has not significantly changed in the last decades and new therapeutic approaches are needed to achieve prolonged survival rates [87] .
Bromocriptine (38) is an ergoline derivative and dopamine agonist that is used in the treatment of Parkinson's disease, acromegaly, hyperprolactinemia and galactorrhoea, and recently repositioned for diabetes mellitus [88, 89] .
Repurposing strategy handled by Lara-Castillo, María Carmen, et al has shown bromocriptine as a potent anti-leukemia drug that mainly targets leukemia stem cells [90] .
[95] [96] [97] .
Typically, the drug-discovery program to develop new potent anti-viral agents and to obtain approval for clinical use takes more than 10 years.
Until now, no effective vaccines or drugs are approved treat these infections, although many are in (pre)clinical development.
Hence, the drugs that have been used for the treatment of other diseases or disorders that may inhibit the replication of coronaviruses (CoVs) might be useful in an attempt to save the life of several affected patients.
In a search of potential anti-viral agents against MERS-CoV, de Wilde et al identified four drugs such as chloroquine (39) , chlorpromazine (40) , loperamide (41) , and lopinavir (42) from the screening of FDA approved drugs library ( Figure 4 ) [98] .
They all were able to inhibit the replication of MERS-CoV in the low micromolar range.
In addition to that, all four drugs inhibited SARS-CoV as well as human coronavirus (HCoV)-229E, which suggests that they could be used for broad-spectral anti-viral activity.
As a mode of action, compounds [109] .
This yielded a series of hit compounds, primarily categorized as, anticancer (41, 42) , antipsychotics (43) , antidepressent (44) and antipsychotic (45) pathways.
Interestingly, co-treatment of the drug 46 with gemcitabine, a deoxycytidine analog that is commonly used for the treatment of cancers [110, 111] , showed a synergistic anti-viral effect with a minimal cytotoxic effect.
This supports the hypothesis of using them in a combination therapy to treat CoV diseases.
Dengue fever is a life-threatening disease caused by four antigenically distinct dengue virus serotypes.
It became a global burden, which causes approximately 390 million infections each year, of which around 10,000 to 20,000 people die.
Even though a vaccine against dengue is available, its long-term protective action against each of the serotypes of dengue virus remains yet to be determined.
Besides, currently, no clinically approved antiviral therapy is available to combat this virus.
Among the many approaches applied to identify novel drugs for dengue fever, drug repurposing gained much attention to the scientific community.
Several antiviral, antimalarial, antidiabetic, antihistamine, anticancer, antipsychotic, antiparasite, and anticholesteremic drugs have been repurposed to combat dengue virus infection.
A recent publication by Botta et al.
discussed each class of drugs and its repositioning in detail [112] .
To identify novel and potent drug candidates, approved drugs such as lovastatin (31), chloroquine (39) , prednisolone (51), balapiravir (52) , and celgosivir (53) were investigated for the proof-off concept clinical trials for dengue viral infection ( Figure 5 ).
Although the results showed that they were safe in patients with acute dengue, drugs failed to meet prior-defined trial endpoints [112] [113] [114] [115] [116] .
Besides, two clinical trials conducted in Thailand and Singapore involving ivermectin (54) and ketotifen (55), the preliminary result was quite promising as phase 2 study of the drug 54 suggested a reduction in serum NS1 levels and body temperature [117] .
Recently, Malakar et al.
screened various classes of FDA approved drugs including aminolevulinic acid (56) , azelaic acid (57), mitoxantrone hydrochloride (58), quinine sulfate (59) and tested their ability to inhibit dengue virus (DENV) replication [118, 119] .
Figure 5 .
Representative example of drugs repurposed for dengue infections.
Aminolevulinic acid (56), an endogenous non-proteinogenic amino acid, and azelaic acid (57) are used to treat skin diseases [120, 121] , while mitoxantrone hydrochloride (58) , an anthracenedione antineoplastic agent used for the treatment of leukemia [122] .
Quinine sulfate (59) is a natural compound extracted from Cinchona bark, which is commercially available in 324-mg tablets under the brand name qualaquin.
It has been widely used to treat chloroquine-resistant plasmodium falciparum [123] .
Its ability to reduce the viral replication was also demonstrated against herpes simplex virus (HSV) [124] , and influenza virus [125] .
Among these four drugs investigated, the drug 59 was found to be very effective in inhibiting the replication of DENV by about 80% compared to untreated controls, while the others showed only moderate reduction of about 50%.
It was very impressive that drug 59 was able to reduce virus replication of all four serotypes of DENV in three different cell lines of human origin.
At the molecular level, it inhibited DENV replication by reducing viral protein and RNA synthesis in a dose-dependent manner.
Moreover, drug 59 enhanced the expression of genes related to innate immune response.
These findings suggest that the efficacy of drug 59 for stimulating antiviral genes, which led to reduce DENV replication.
From another set of prescribed drug candidates (63-70) were tested against blood-stage P. falciparum cultures ( Figure 6 ) and liver-stage P. berghei [129, 130] .
They all showed promising antimalarial activity with IC 50 values ranged from 2.8 µM to 1.7 nM.
Drugs raloxifene hydrochloride (63) Salirasib (72, Figure 6 ) is a promising cancer drug candidate inhibits isoprenylcysteine carboxyl methyltransferase (ICMT), a validated target for cancer drug development.
Recently, Salirasib and its analogs with 1,2,3-triazole were repuposed for their pontential antimalarial activity [133] .
In general, triazole derivartives are known to have potent antimalarial activity [134] .
Compound were investigated the in vitro toxicity to P. falciparum in the asexual stages and in Vero cells.
An antiplasmodial activity assay was performed using a simple, highsensitivity methodology based on nanoluciferase (NLuc)-transfected P. falciparum parasites.
The results showed that some of the analogs were active at low micromolar concentration, Alzheimer's is the most common type of dementia.
It affects around 5% of people over the age of 65, 20% over the age of 80, and more than a third of those over the age of 90 [136] .
In the year 2015, there were approximately 29.8 million people worldwide with AD of which dementia resulted in about 1.9 million deaths.
It is estimated that there will be more than 115 million people with dementia worldwide by 2050.
Therefore, AD represents a major and rising public health concern, and there is an urgent need to develop more therapies that are effective.
AD refers to a devastating condition leading to progressive cognitive decline, functional impairment, and loss of independence.
Accumulation of amyloid-β peptide (Aβ)-enriched neuritic plaques, and neurofibrillary tangles, synaptic, and neuronal dysfunction, as well as loss in combination with the associated neurochemical changes in the brain, are the crucial pathological event for AD [137] .
Recent work suggests that higher levels of total tau may potentiate the toxic effects of Aβ [138] .
Other factors such as inflammatory processes and mitochondrial function are also likely to have an important role [139] .
Three acetylcholinesterase inhibitors including donepezil (76), rivastigmine (77) and are cost-effective [140] .
For example, acetylcholinesterase inhibitors improve cognition to above pre-treatment performance for ~6-12 months.
The availability of these drugs has substantially advanced the treatment of patients with AD, but there is a persistent need to build on our increasing understanding of disease pathogenesis to develop more effective symptomatic treatments and disease-modifying therapies.
drugs.
The MD simulation results exposed that these five drugs (Risperidone, Domperidone, Verapamil, Tamsulosin and Cinitapride) showed better profiles with respect to their RMSD, RMSF, SASA and Rg evaluations graphs and steady stable behavior in all docking complexes.
In-vitro AChE inhibition assay of the above best-screened drugs were performed by spectrophotometric method using acetylthiocholine iodide as substrate.
The enzyme inhibition and kinetic mechanism of these drugs showed that Cinitapride (79) has good therapeutic potential with respect to standard and other drugs.
It is well known that, Cintapride (79) is a gastroprokinetic agent and antiulcer agent of the benzamide class [142] .
It is an agonist of 5-HT 1 and 5-HT 4 receptors and as an antagonist of the 5-HT 2 receptors [143] .
Based on aforementioned results, it is justified that Cinitapride has better repositioning profile which may be used in the treatment of AD after clinical assessment.
Efforts to develop more effective therapies have so far been unsuccessful with several highprofile clinical trial fails to demonstrate the benefit.
The reasons for this are probably multifactorial.
The majority of putative disease-modifying therapies that have been evaluated have targeted amyloid pathology.
This lack of breadth in treatment approaches has been criticized, and some commentators have argued that a more sophisticated knowledge of disease pathways is needed before we can develop more effective candidate therapies.
For example, Phase II trial of tarenflurbil (80, Figure 7 ) only provided a suggestion of benefit in a posthoc subgroup analysis [144, 145] ; the putative mechanism of action via γ-secretase modulation and its related impact on amyloid pathology was never confirmed in patients with AD.
Subsequent Phase III trial had negative outcomes.
Although, the results of a Phase II trial of dimebon (81) were much more favorable than of analog 80 (Figure 7 ).
It seems that the significant benefit seen in the treatment group was driven by a larger-than-expected deterioration in the group receiving placebo treatment, and the mechanism of action was not well characterized.
In the central nervous system (CNS), angiotensin II mediates key processes including, the release of inflammatory mediators, vasoconstriction, mitochondrial dysfunction, and inhibition of acetylcholine release at central synapses.
All of which are proposed to be relevant to AD and are potential targets for therapeutic intervention [146, 147] .
Based on this background, it has been proposed that angiotensin receptor blockers (ARBs) may confer symptomatic benefits on cognition.
A large-scale screen of 55 antihypertensive drugs by Want et al.
[148] , identified the ARB valsartan (82, Figure 7 ) as the only compound that was able to reduce Aβ accumulation in cultured neurons and inhibit Aβ aggregation in vitro.
The same group was demonstrated that the reduced plaque burden as well as the improved learning and memory in cognitive tests (including the Morris water maze task) following 5 months of treatment with valsartan in 6month-old Tg2576 transgenic mice.
As a result, the greatest benefits were seen at a dose of 40 mg/ kg/day, which is equivalent to 1.5 times the maximum recommended dose for treating patients with hypertension.
Using another ARB, olmesartan (83, Figure 7 ) by Takeda et al.
[149] , demonstrated that daily treatment of young APP23 mice for one month improved cerebral blood flow without affecting Aβ1-40 and Aβ1-42 levels.
In the Aβ1-40-injected mouse model, in which Aβ fragments are injected intracranially to generate deficits, pre-treatment with telmisartan (84, Figure 7 ) increased cerebral blood flow and inhibited the plaque deposition [150] .
However, the physiological relevance of this model is unclear.
Perhaps, the most striking preclinical evidence comes from a study in which losartan (85, Figure 7 ) was administered intranasally to APP/PSEN1 mice, at a dose much lower than that mediating hypotensive effects; drug 80 led to a 3.7-fold reduction in Aβ plaques compared to vehicle-treated mice but also reduced the levels of pro-inflammatory mediators and increased the levels of anti-inflammatory mediator IL-10 in the serum of these animals [151, 152] .
Overall, there was significant reduction in the incidence of dementia in patients taking ARBs compared to those taking the comparator cardiovascular drugs (hazard ratio: 0.76; 95% confidence interval: 0.69-0.84) and those taking angiotensin-converting enzyme (ACE) inhibitor lisinopril (hazard ratio: 0.81; 95% confidence interval: 0.73-0.90).
Although the evidence for the potential of ARBs in AD is conflicting, and difficult to interpret, in our view, there is a sufficient indication of potential benefit to merit further in vivo work to clarify the relative importance of different mechanisms, the optimal dose, and the optimal agent, which could lead to proof-of-concept study in patients with AD.
The best evidence from in vitro and in vivo studies points to either drug 85 or 84 as a preferred ARB, with some animal studies also highlighting drug 83 as a potential candidate.
However, there is an unfortunate disconnect between in vivo and clinical studies, as no formal studies to provide direct clinical evidence have so far been conducted on any of the most promising candidates.
Calcium channel blockers (CCBs) of the dihydropyridine class are widely used to treat hypertension and angina through their vasodilatory activity on smooth muscle vasculature.
Most of the drugs in this class have good blood-brain barrier penetration and induces cerebral vasodilatation, increased cerebral blood flow in animals and humans [153] .
In vitro studies have revealed that certain CCBs reduce Aβ production, oligomerization, and accumulation, rescue Aβ-induced neurotoxicity, and improve cell survival in the presence of Aβ [154] [155] [156] .
CCBs have also been shown to reduce glutamate-induced cell death and levels of intracellular calcium [157] .
The ability of CCBs to prevent Aβ1-40 and Aβ1-42 production was investigated in Chinese hamster ovary cells (CHO) by Paris et al.
and Iwasaki et al.
In that study, amlodipine (86) and nilvadipine (87) (Figure 7) were identified as the only agents that inhibited Aβ production [158] .
However, the concentrations studied were several-fold higher than can be achieved therapeutically.
Isradipine (88, Figure 7 ) was shown to have a neuroprotective effect against Aβ-induced apoptosis in neuroblastoma MG65 cell lines.
A protective effect in a Drosophila melanogaster model of Aβ-induced neurotoxicity and it's brain-penetrant in the 3xTg-AD mouse model of AD was also reported [156, 159] .
The differential effects of CCBs indicate that their potential benefits in AD are probably independent of their anti-hypertensive activity and may be specific to individual drugs within this class.
Dihydropyridines seem to be more effective than compounds with different chemical structures (such as verapamil or diltiazem), with evidence from preclinical studies that highlighted nilvadipine (87) as the best therapeutic candidate.
There was some clinical evidence regarding the potential benefit of the CCB nimodipine (89, Figure 7 ) in patients with clinically significant dementia.
The evidence has been summarized in a Cochrane review 51 of 15 nimodipine trials in more than 3,000 patients with dementia.
The review reported that the treatment showed efficacy in improving cognition, but not activities of daily living, at doses of 90 mg/day [160] .
However, the evidence was limited by small size and duration (mostly 12 weeks) of trials as well as the lack of operational diagnostic criteria for AD or vascular dementia.
Tetracycline antibiotics are widely used to treat bacterial infections and are well tolerated in older people, but most of the treatment data pertain to short-term periods of exposure.
Studies related to AD have predominantly focused on minocycline (90, Figure 7) because it is the most lipophilic tetracycline, with greater blood-brain barrier penetration than other agents in this class.
Concerning preclinical studies, the drug 90 has been shown to reduce Aβ1-42 aggregation, and promote disassembly of pre-formed fibrils in vitro studies.186 Various groups have independently shown that drug 90 reduces the levels of proinflammatory mediators and microglial activation in a range of mouse models of AD [161] [162] [163] [164] [165] .
Besides, two of the three studies using transgenic mouse models of AD for 28 days or more of treatment have shown a significant decrease in cerebral Aβ accumulation and improvements in behavioral outcomes as well [165] .
One of these studies used 8-month-old 3xTg-AD mice and reported reductions in cortical amyloid levels following 4 months of minocycline treatment, but no changes in tau pathology was observed.
190 The third study reported that no benefit concerning amyloid accumulation or behavior over a treatment period of 12 months.194 An additional study in a rat model of diabetes demonstrated a reduction in Aβ1-40 and Aβ1-42 levels and associated improvements in behavioral outcomes over 8 weeks of treatment [165] .
Doxycycline (91) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies.
Compound 91 is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in AD mice.
Balducci, Claudia, et al.
showed that 15-to 16month-old APP/PS1dE9 (APP/PS1) AD mice receiving 96 under different treatment regimens recovered their memory without plaque reduction.
An acute 96 treatment was, also, sufficient to improve APP/PS1 mouse memory, suggesting an action against soluble AbOs.
This was confirmed in an AbO-induced mouse model, where the AbO-mediated memory impairment was abolished by a its pretreatment.
Although AbOs induce memory impairment through glial activation, assessing the anti-inflammatory action of 96, we found that in both the AbOtreated and APP/PS1 mice, the memory recovery was associated with a lower neuroinflammation.
Our data promote 96 as a hopeful repositioned drug counteracting crucial neuropathological AD targets [166] .
Drugs that activate retinoic acid receptors (RARs) are used to treat several skin-related conditions such as acne and psoriasis.
Retinoic acid is also vital for normal nerve function and repair.
There is genomic and epidemiological evidence suggest that impaired retinoic acid signaling may contribute to the etiology of AD [167] .
Chronic deprivation of retinoic acid in rats leads to deposition of Aβ in the vasculature [168] and dysregulation of amyloid processing in the cortex [169] .
It has been shown that treatment with retinoid X receptor (RXR) agonist bexarotene (92, Figure 7) , which is approved for the treatment of cutaneous T cell lymphoma, leads to pathological and behavioral improvements in transgenic mouse models of AD.
Acute treatment with drug 92 (lasting less than 14 days) caused a rapid reduction (25%) in Aβ1-40 and Aβ1-42 levels and Aβ plaque burden in both young and old mice [170] .
Chronic 90-day treatment resulted in a sustained reduction (30%) insoluble Aβ levels.
Mechanistically, drug 93 resulted in the upregulation of components of the high-density lipoprotein (HDL) pathway such as apolipoprotein E (APOE), which promotes the proteolytic degradation of Aβ [171] .
Further, potential mechanism of action of retinoids may include the upregulation of enzymes involved in amyloid clearance such as insulin-degrading enzyme [172] and components of the APOE pathway [170] .
Retinoids may also induce potentially beneficial changes related to insulin signaling and increased neurogenesis and promote neuronal differentiation of progenitor cells [173, 174] .
Retinoids also act as antioxidants (by regulating SOD and inhibiting glutathione depletion to reduce mitochondrial damage) as well as anti-inflammatory agents (by reducing the production of IL-6) in vitro.
Both of these activities have potential importance in AD pathology [175, 176] .
Therefore, there was a strong mechanistic rationale for the potential benefit of retinoid therapies beyond amyloid modulation, but there is a need to further clarify the impact of treatment on these pathways through in vivo studies.
Overall, studies in the literature indicate that retinoids have strong potential mechanistic plausibility as therapies for AD owing to their effects on APP processing, Aβ clearance, insulin signaling, and neurogenesis.
Out of the approved drugs, data for bexarotene have provided proof of concept as potential candidate for the treatment of Alzheimer's disease as noted above, whereas acitretin (93, Figure 7) , which is known to penetrate tissues including brain may also be a promising candidate for AD [177] .
It is a long-term degenerative disorder of the CNS, which mainly affects the motor system.
As of 2015, PD affected 6.2 million individuals, of which 117400 people died.
It usually occurs people over the age of 60, of whom about one percent are affected.
In addition to the classic motor symptoms caused by the death of dopaminergic neurons, Parkinson's disease encompasses a wide range of nonmotor symptoms.
Although novel disease-modifying medications that slow or stop Parkinson's disease progression are being developed, drug repurposing, which is the use of existing drugs that have passed numerous toxicity and clinical safety tests for new indications, can be used to identify treatment compounds.
This strategy has revealed that tetracyclines (96) are promising candidates for the treatment of Parkinson's disease [178] .
Tetracyclines, which are neuroprotective, inhibit proinflammatory molecule production, matrix metalloproteinase activity, mitochondrial dysfunction, protein misfolding/aggregation, and microglial activation.
Two commonly used semisynthetic second-generation tetracycline derivatives, minocycline (90) and doxycycline (91) , exhibit effective neuroprotective activity in experimental models of neurodegenerative/ neuropsychiatric diseases and no substantial toxicity.
Moreover, novel synthetic tetracyclines with different biological properties due to chemical tuning are now available.
In this review, we discuss the multiple effects and clinical properties of tetracyclines and their potential use in Parkinson's disease treatment.
In addition, we examine the hypothesis that the anti-inflammatory activities of tetracyclines regulate inflammasome signaling.
Based on their excellent safety profiles in humans from their use for over 50 years as antibiotics, we propose the repurposing of tetracyclines, a multitarget antibiotic, to treat Parkinson's disease.
Isradipine (88) is an L-type calcium channel blocker of the dihydropyridine class, which has been widely used for the treatment of high blood pressure to reduce the risk of heart attack and stroke.
This drug 88 came into medical use in the year 1989.
Epidemiological data support that calcium channel blockers may have the potential to reduce the risk of developing PD.
Among dihydropyridines, drug 88 has attracted very much as it inhibits the subtype Cav1 Ca2+ channels Cav1.2 and Cav1.3, which are most likely mediate the risk in PD.
Moreover, the good brain bioavailability of drug 88 has made it the most promising candidate for repurposing [178, 179] .
Studies have shown that drug 88 dose repentantly protect the dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP) and 6hydroxydopamine (6-OHDA)-induced toxicity by reverting dopaminergic neurons to a latent juvenile pacemaking mechanism independent of calcium [180, 181] .
An open-label, doseescalation study assessing safety (STEADY-PD) of drug 88 controlled release 5-20 mg/day in patients with early PD suggested that the acceptable tolerability at doses of ≤ 10 mg per day; however at higher doses caused leg edema and dizziness [182] .
Isradipine (88) with 10 mg being the highest dosage was again confirmed in another STEADY-PD-II, a randomized, double-blinded trial, which was undertaken in 99 subjects with early PD not requiring dopaminergic therapy [183] .
Results suggest that the most common adverse effect was peripheral edema, which occurred in 34% of patients receiving drug 88 (10 mg).
Placebocontrolled phase III clinical study to assess the efficacy of drug 88 ( Inosine (94, Figure 7) is a purine nucleoside, which has been used as a dietary supplement by athletes for improving aerobic performance.
It has been shown to have neuroprotective roles by elevating the level of serum urate, a natural antioxidant and peroxynitrite scavenging property with potential benefits to patients with multiple sclerosis.
Many studies suggest that individuals with increased levels of urate in serum have a reduced risk of developing PD, as well as in patients with PD are associated with a reduced rate of disease progression.
Moreover, in toxin-based models of PD, increased urate levels have conferred protection against dopaminergic cell death stimulated by MPTP, 6-OHDA, and rotenone.
Akt-GSK-3B signaling and nuclear factor (erythroid-derived-2)-like 2 (Nrf2) were thought to involve in these effects.
Therefore, given the data supporting a neuroprotective role of urate, inosine has been repurposed in the pathogenesis of PD.
The ability to raise urate level of drug 88 in serum was demonstrated in SURE-PD, a randomized, double blind, placebo-controlled in 75 patients with early PD not yet requiring any medication.
The results showed that inosine raised the mild urate level (6.1-7.0 mg/dl) or moderate urate elevation (7.1-8.0 mg/dl) after 25 months and well-tolerated with favorable progression rate in UPDRS score, which amounted to ~1 point per year on the total UPDRS scale.
On the other hand, the elevated level of urate in serum have the risk of hypertension, coronary heart disease, and stroke over the long term.
These side effects are potentially limiting its utility in older patients with PD.
However, in patients of Asian origin with PD, inosine elevated urate levels (6.1-7.0 mg/dl) without side effects after 1 year of treatment was reported.
The multicenter SURE-PD3 trial, which involves a large number of patients (240 patients), is currently underway intending to elevate the urate level to 7.1-8.0 mg/dl.
The result of the study will be expected in the year 2020.
Simvastatin (29), Since statins are also known to modulate various biological processes relevant to the pathogenesis of PD [185, 186] .
Simvastatin has been repurposed for treating this disease.
Pretreatment with simvastatin preserved dopaminergic cells and motor behavior in rodents treated with 6-hydroxydopamine (6-OHDA), promoting antioxidant protein expression or via modulation of NMDA receptor and pro-inflammatory cytokine expression [187/189] .
Similarly, in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models, pretreatment with simvastatin suppressed activation of NF-κB, protected dopaminergic neurons, and improved the motor function [190, 191] .
Although, stains, in general, have given encouraging preclinical studies, epidemiological data regarding the association between usage of statins and the risk of PD are unclear.
Moreover, the modest protective effect of statins that disappeared when adjusted for cholesterol level [192] .
However, due to the promising biochemical and pharmacological properties of simvastatin, it is currently being examined with 235 patients having moderate-stage PD in phase II double-blind, randomized, controlled, multicenter trial.
Nilotinib (95, Figure 7) is a selective c-Abl tyrosine kinase inhibitor approved for the treatment of imatinib-resistant chronic myelogenous leukemia (CML).
Accumulating evidence suggests that c-Abl activation has been linked in the pathogenesis of PD and other synucleinopathies.
Activated (phosphorylated) c-Abl has been found in a high level in postmortem studies of patients with PD [193, 194] .
It was also reported that activation of c-Abl in mice induces neurodegeneration in the hippocampal and striatal brain areas [195] .
Continuous work has demonstrated that c-Abl phosphorylation occurs as a result of mitochondrial dysfunction, and oxidative stress [196] , which can promote the accumulation of α-synuclein in through effects on autophagy mechanisms [195] and can further promote phosphorylation of parkin, causing inhibition of its ubiquitin E3 ligase activity, inducing mitochondrial dysfunction and dopaminergic neuronal death [194] .
All the above evidences propose that c-Abl may be a promising therapeutic target in the management of PD.
The CNS penetration of nilotinib over other Abl inhibitors has favored it to fetch more data in PD related studies.
Indeed, in preclinical models of PD, drug 92 has been shown to cross the blood-brain barrier and reduces c-Abl activity, ameliorating autophagic clearance of α-synuclein in transgenic and lentiviral gene-transfer models [195] .
More importantly, these effects were seen at doses far lower (1-10 mg/kg/day) than those used to treat CML, which is considered as the key characteristics for potential drug repurposing.
Furthermore, nilotinib prevented dopaminergic cell loss and motor impairments induced by MPTP in mice, which were associated with inhibition of parkin phosphorylation and reduced accumulation of parkin substrate PARIS, thus hinting at another potential mechanism of action [197, 198] .
Based on these preclinical data, a small, open-label proof-of-concept study was recently conducted to evaluate the safety and tolerability of nilotinib in 12 patients with PD dementia or dementia with Lewy bodies followed-up for 24 weeks, followed by a final assessment 12 weeks later [199] .
The necessary lowest choice of dose for the clinical study was taken as 150-300 mg.
The authors reported that nilotinib was well tolerated, though one patient receiving 300 mg was diagnosed with myocardial infarction, and two had transient QTc prolongations.
There was also evidence of CNS penetration with nilotinib CSF plasma ratio of 12 and 15% with 300 and 150 mg, respectively.
Due to numerous methodological limitations, these findings should be interpreted with caution [200] .
Due to unwanted off-target nonselective tyrosine kinase inhibition; side effects of nilotinib at doses used to treat CML include cardiac conduction abnormalities.
Therefore, claims of tolerability should be interpreted with caution.
Indeed, the effects on efficacy was also highly impossible to be concluded.
Besides, it was also reported in the CSF, none of the markers used were validated as biomarkers in PD; they can also vary greatly between patients and track poorly with disease stage and progression.
This situation again raises questions about the optimum dose of nilotinib, its brain penetrance, assessments of cardiovascular effects in patients.
Parkinson's disease (PD) is a neurodegenerative disorder for which a greater prevalence and incidence is described in men.
This suggests a protective effect of sex hormones in the brain.
Intellectual property related issues hinder the commercialization of repositioned molecule.
In conclusion, repurposing of drugs has the potential to find and improve treatments for various diseases.
The drugs listed above are only a few of plenty drugs that can be repurposed in various therapies.
The drugs discussed in the perspective are summarized in Table 1 and compared to the previous uses of each drug with the repurposed use.
The opportunities for drug repurposing are diverse, but a lot still has to be done for its exploration.
Drug repurposing or drug repositioning offers a new strategy to academic centers, research council programs, and not-for-profit organizations, as well as pharmaceutical and biotechnology companies for drug development.
The main advantage of drug repurposing is the established safety of the known candidate compounds when compared to that of the development of novel therapeutic compounds.
The time and cost required to advance a candidate into clinical trials can be substantially reduced because in vitro and in vivo screening, chemical optimization, toxicity studies, bulk manufacturing, and formulation development have already been completed in many cases, and can, therefore, be bypassed.
Moreover, these drugs have been on the market for many years, and consequently, the side effects are already known, and safety is therefore very high.
For medicinal chemists, the repurposing of drugs on cancer is a rewarding job for the global healthcare system and possibly a step forward for people to get cheaper and safer medicines rather than afford for cancer therapy.
However, time and minimum investment have to be spent to conduct further studies to improve the safety and success of the repurposed drugs.
Drug repurposing also offers the possibility to develop multi-target drugs that can interact with more than one pathway/protein at the same time.
For complex diseases such as (neuro) inflammatory and degenerative disorders, the development of multi-target drugs will be an emerging area for the treatment having multiple advantages such as i) synergistic effect ii) Reduced drug-resistance iii) better compliance iv) simplified pharmacokinetic and pharmacodynamic profile and a reduced risk of drug-drug interactions.
On the other hand, the limitations of drug repurposing should also be considered.
They involve a) technical challenges and legal requirements such as intellectual property rights which could hamper the whole process and are often hard to overcome b) serious problem on the development of resistant germs on account of consuming a drug for a variety of diseases c) owing to target selectivity, it is difficult to identify a drug that can cure or treat two different diseases on its own.
Nevertheless, drug repurposing is only a part of the solution for the sinking numbers of new diseases and its treatment.
However, it is quite hard to replace the common way of identifying new drug candidates.
In furuter, this approach could be improved in many differents ways: Integrating data about repositioning drugs which are available in many public platforms, such as PubChem), The Nobel chronicles A comprehensive map of molecular drug targets Trends in development and approval times for new therapeutics in the United States Drug repositioning identifying and developing new uses for existing drugs Rebuilding big pharma's business model FDA drug approvals Genetic and rare diseases information centre Drug Repositioning: Bringing new life to shelved assets and existing drugs Drug repurposing: progress, challenges and recommendations A review of computational drug repurposing Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells Melanins and melanosomes: biosynthesis, biogenesis, physiological, and pathological functions Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors Melanogenesis-inhibitory and cytotoxic activities of triterpene glycoside constituents from the bark of Albiziaprocera Inhibitors of melanogenesis: a patent review Recent updates in melanocyte function: the use of promising bioactive compounds for the treatment of hypopigmentary disorders Inhibitors of melanogenesis: an updated review Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson's disease pathogenesis Novel tyrosinase inhibitory peptide with free radical scavenging ability Tyrosinase-expressing neuronal cell line as in vitro model of Parkinson's disease The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease Skaltsounis, Design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease Degradation of tyrosinase induced by phenylthiourea occurs following Golgi maturation Inhibition of L-tyrosine-induced micronuclei production by phenylthiourea in human melanoma cells Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase Structural requirement(s) of N-phenylthioureas and benzaldehyde thiosemicarbazones as inhibitors of melanogenesis in melanoma B 16 cells Analogues of ethionamide, a drug used for multi drug resistant tuberculosis, exhibit potent inhibition of tyrosinase Repositioning of thiourea-containing drugs as tyrosinase inhibitors Pharmacovigilance and tuberculosis: Applying the lessons of thioacetazone Antithyroid drugs Thiopurine drugs repositioned as tyrosinase inhibitors Crystal structure of agaricusbisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone The unravelling of the complex pattern of tyrosinase inhibition Exploring the interaction of N/S compounds with a dicopper center: tyrosinase inhibition and model studies United States cancer statistics: 1999-2011 incidence and mortality web based report Cancer drug discovery by repurposing: teaching new tricks to old dogs Systematic review update of observational studies further supports aspirin role in cancer treatment: Time to share evidence and decision-making with patients?
Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials Aspirin, salicylates, and cancer The biology of prostaglandin synthesis and inhibition Nonsteroidal antiinflammatory agents differ in their ability to suppress NF-kappa B activation, inhibition of expression of cyclooxygenase-2 and cyclin D1 and abrogation of tumor cell proliferation Repositioning Aspirin to Treat Lung and Breast Cancers and Overcome Acquired Resistance to Targeted Therapy American society of health-system pharmacists Targeting apoptosis pathways by celecoxib in cancer An international randomised trial of celecoxib versus celecoxib plus difluoromethylornithine in patients with familial adenomatous polyposis Molecular Mechanisms and Bioavailability of Polyphenols in Prostate Cancer Inhibitory effect of ibuprofen on tumor survival and angiogenesis in gastric cancer cell Ibuprofen and hydrogel-released ibuprofen in the reduction of inflammation induced migration in melanoma cells Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70 Single-agent lenalidomide induces complete remission of acute myeloid leukemia in patients with isolated trisomy13 Thalidomide potentiates etoposide-induced apoptosis in murine neuroblastoma through suppression of NF-κB activation A combination of thalidomide and arsenic trioxideis effective and well tolerated in patients with myelodysplastic syndromes How thalidomide works against cancer Suppression of Aiolos and Ikaros expression by lenalidomide reduces human ILC3-ILC1/NK cell transdifferentiation Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner Type 2 diabetes increases and metformin reducestotal, colorectal, liver and pancreatic cancer incidences in taiwanese: a representative population prospective cohort study of 800,000 individuals The potential effect of metformin on cancer: an umbrella review Cancer risk in diabetic patients treated with metformin: a systematic review and meta-analysis miR-770-5p modulates resistance to methotrexate in human colorectal adenocarcinoma cells by downregulating HIPK1 Anti-inflammatory therapy in chronic disease: challenges and opportunities Clinical activity of mammalian target of rapamycin inhibitors in solid tumors Evaluation of Combination of atorvastatin with sulindac or naproxen profoundly inhibits colonic adenocarcinomas by suppressing the p65/beta-catenin/cyclin D1signaling pathway in rats Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGFbeta-activated kinase-1 Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements Statin uses and mortality in colorectal cancer patients: an updated systematic review and meta-analysis Statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study Statin use and the risk of colorectal cancer in a population-based electronic health records study The role of statins for primary prevention in non-elderly colorectal cancer patients Statin use is associated with a reduced incidence of colorectal cancer: a colonoscopy-controlled case-control study Sodium valproate and 5-aza-2'-deoxycytidine differentially modulate DNA demethylation in G1 phase-arrested and proliferative HeLa cells Exploring the drug repurposing versatility of valproic acid as a multifunctional regulator of innate and adaptive immune cells Therapeutic value of voltage-gated sodium channel inhibitors in breast, colorectal, and prostate cancer: a systematic review Screening a library of approved drugs reveals that prednisolone synergizes with pitavastatin to induce ovarian cancer cell death Towards the first targeted therapy for triplenegative breast cancer: repositioning of clofazimine as a chemotherapy-compatible selective Wnt pathway inhibitor, Cancer letters A drug repurposing screening reveals a novel epigenetic activity of hydroxychloroquine Acute myeloid leukaemia in adults Drug reformulations and repositioning in pharmaceutical industry and its impact on market access: reassessment of nomenclature Differential actions of antiparkinson agents at multiple classes of mono-aminergic receptor I. a multivariate analysis of the binding profiles of 14 drugs at 21 native and cloned human receptor subtypes Repositioning of bromocriptine for treatment of acute myeloid leukemia The genome sequence of the SARS-associated coronavirus Severe acute respiratory syndrome An Overview of Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) 3CL Protease Inhibitors: Peptidomimetics and Small Molecule Chemotherapy Middle East respiratory syndrome-coronavirus (MERS-CoV): An updated overview and pharmacotherapeutics Mers: South Korea closes 700 schools after third death Severe respiratory disease associated with Middle East respiratory syndrome coronavirus Interaction of ethambutol with human organic cation transporters of the SLC22 family indicates potential for drug-drug interactions during antituberculosis therapy Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome corona virus replication in cell culture Effects of chloroquine on viral infections: an old drug against today's diseases?
A systematic screen of FDA-approved drugs for inhibitors of biological threat agents Simulating henipa virus multi cycle replication in a screening assay leads to identification of a promising candidate for therapy Pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents Clathrin mediates infectious hepatitis C virus particle egress Inhibitors of alpha virus entry and replication identified with as table Chikungunya replicon cell line and virus-based assays Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15 Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted Small molecules targeting severe acute respiratory syndrome human coronavirus Saracatinib inhibits Middle East respiratory syndrome-coronavirus replication invitro Gemcitabine: a critical nucleoside for cancer therapy Synergistic antiviral activity of gemcitabine and ribavirin against enteroviruses Drug repurposing approaches to fight Dengue virus infection and related diseases Drug repositioning for dengue haemorrhagic fever by integrating multiple omics analyses Effects of short-course oral corticosteroid therapy in early dengue infection in Vietnamese patients: a randomized, placebo-controlled trial Efficacy and safety of celgosivir in patients with dengue fever (CELADEN): a phase 1b, randomised, double-blind, placebo-controlled, proof-of-concept trial Lovastatin for the treatment of adult patients with dengue: a randomized, double-blind, placebo-controlled trial Ivermectin: a promising anti-dengue replication treatment Pesented at 26th European congress of clinical microbiology and infectious diseases Current status of dengue therapeutics research and development Repurposing approved drugs on the pathway tonovel therapies.
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Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: a randomized controlled trial Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease The renin-angiotensin system and antihypertensive drugs in Alzheimer's disease: Current standing of the angiotensin hypothesis?
Valsartan lowers brain β-amyloid protein levels and improves spatial learning in a mouse model of Alzheimer disease Angiotensin receptor blocker prevented β-amyloid-induced cognitive impairment associated with recovery of neurovascular coupling Telmisartan prevented cognitive decline partly due to PPAR-γ activation Protective effects of intranasal losartan in the APP/PS1 transgenic mouse model of Alzheimer disease Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis Favourable effects of nilvadipine on cognitive function and regional cerebral blood flow on SPECT in hypertensive patients with mild cognitive impairment Identification of antihypertensive drugs, which inhibit amyloid-β protein oligomerization Selective dihydropyiridine compounds facilitate the clearance of β-amyloid across the blood-brain barrier L-type voltage-gated calcium channel blockade with isradipine as a therapeutic strategy for Alzheimer's disease Protective effects of ginsenoside Rg2 against glutamate-induced neurotoxicity in PC12 cells Selective antihypertensive dihydropyridines lower Aβ accumulation by targeting both the production and the clearance of Aβ across the blood-brain barrier A translational continuum of model systems for evaluating treatment strategies in Alzheimer's disease: Isradipine as a candidate drug Nimodipine for primary degenerative, mixed and vascular dementia Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid Matrix metalloproteinase inhibition reduces oxidative stress associated with cerebral amyloid angiopathyin vivo in transgenic mice Minocycline as a potential therapeutic agent in neurodegenerative disorders characterised by protein misfolding Reductions in amyloid-β-derived neuroinflammation, with minocycline, restore cognition but do not significantly affect tau hyperphosphorylation Increases in β-amyloid protein in the hippocampus caused by diabetic metabolic disorder are blocked by minocycline through inhibition of NF-κB pathway activation Doxycycline counteracts neuroinflammation restoring memory in Alzheimer's disease mouse models Evidence for defective retinoid transport and function in late onset Alzheimer's disease Disruption of the retinoid signalling pathway causes a deposition of amyloid β in the adult rat brain Retinoic acid normalizes nuclear receptor mediated hypo-expression of proteins involved in β-amyloid deposits in the cerebral cortex of vitamin A deprived rats ApoEdirected therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models Human apoE isoforms differentially regulate brain amyloid-β peptide clearance Regulation by retinoic acid of insulin degrading enzyme and of a related endoprotease in human neuroblastoma cell lines Interactions between retinoic acid, nerve growth factor and sonic hedgehog signalling pathways in neurite outgrowth Sequential RARβ and α signallingin vivo can induce adult forebrain neural progenitor cells to differentiate into neurons through Shh and FGF signalling pathways All-trans retinoic acid as a novel therapeutic strategy for Alzheimer's disease Towards retinoid therapy for Alzheimer's disease Kinetics of tissue distribution and elimination of retinoid drugs in the rat Tetracycline repurposing in neurodegeneration: focus on Parkinson's disease Expression and 1,4-dihydropyridine binding properties of brain L-type calcium channel isoforms Rejuvenation protects neurons in mouse models of Parkinson's disease The L-type channel antagonist isradipine is neuroprotective in a mouse model of Parkinson'sdisease Tolerability of isradipine in early Parkinson's disease: a pilot dose escalation study Phase II safety, tolerability, and dose selection study of isradipine as a potential disease-modifying intervention in early Parkinson's disease (STEADY-PD) Simvastatin as a potential disease modifying therapy for patients with Parkinson's disease: rationale for clinical trial, and current progress Prospects of statins in Parkinson disease Simvastatin inhibits activation of NADPH oxidase/p38 MAPK pathway and enhances expression of antioxidant protein in Parkinson disease models Simvastatin prevents neuroinflammation by inhibiting N-methyl-D-aspartic acid receptor 1 in 6-hydroxydopamine-treated PC12 cells Neuroprotective potential of atorvastatin and simvastatin (HMG-CoA reductase inhibitors) against 6-hydroxydopamine (6-OHDA) induced Parkinson-like symptoms Simvastatin inhibits the activation of p21ras and prevents the loss of dopaminergic neurons in a mouse model of Parkinson's disease Simvastatin prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineinduced striatal dopamine depletion and protein tyrosine nitration in mice Confounding of the association between statins and Parkinson disease: systematic review and meta-analysis Novel regulation of parkin function through c-Abl-mediated tyrosine 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Repositioning is a promising approach for the rapid identification and development of new pharmaceuticals for rare and complex diseases
key: cord-322915-zrjx31ev authors: Demain, Arnold L; Sanchez, Sergio title: Microbial drug discovery: 80 years of progress date: 2009-01-09 journal: J Antibiot (Tokyo) DOI: 10.1038/ja.2008.16 sha: doc_id: 322915 cord_uid: zrjx31ev Microbes have made a phenomenal contribution to the health and well-being of people throughout the world.
In addition to producing many primary metabolites, such as amino acids, vitamins and nucleotides, they are capable of making secondary metabolites, which constitute half of the pharmaceuticals on the market today and provide agriculture with many essential products.
This review centers on these beneficial secondary metabolites, the discovery of which goes back 80 years to the time when penicillin was discovered by Alexander Fleming.
Back in 1928, Alexander Fleming 1 began the microbial drug era when he discovered in a Petri dish seeded with Staphylococcus aureus that a compound produced by a mold killed the bacteria.
The mold, identified as Penicillium notatum, produced an active agent that was named penicillin.
Later, penicillin was isolated as a yellow powder and used as a potent antibacterial compound during World War II.
By using Fleming's method, other naturally occurring substances, such as chloramphenicol and streptomycin, were isolated.
Naturally occurring antibiotics are produced by fermentation, an old technique that can be traced back almost 8000 years, initially for beverages and food production.
Beer is one of the world's oldest beverages, produced from barley by fermentation, possibly dating back to the sixth millennium BC and recorded in the written history of ancient Egypt and Mesopotamia.
Another old fermentation, used to initiate the koji process, was that of rice by Aspergillus oryzae.
During the past 4000 years, Penicillium roqueforti has been utilized for cheese production, and for the past 3000 years soy sauce in Asia and bread in Egypt has represented examples of traditional fermentations.
2 Natural products with industrial applications can be produced from primary or secondary metabolism of living organisms (plants, animals or microorganisms).
Owing to technical improvements in screening programs, and separation and isolation techniques, the number of natural compounds discovered exceeds 1 million.
3 Among them, 50-60% are produced by plants (alkaloids, flavonoids, terpenoids, steroids, carbohydrates, etc.)
and 5% have a microbial origin.
Of all the reported natural products, approximately 20-25% show biological activity, and of these approximately 10% have been obtained from microbes.
Furthermore, from the 22 500 biologically active compounds that have been obtained so far from microbes, 45% are produced by actinomycetes, 38% by fungi and 17% by unicellular bacteria.
3 The increasing role of microorganisms in the production of antibiotics and other drugs for treatment of serious diseases has been dramatic.
However, the development of resistance in microbes and tumor cells has become a major problem and requires much research effort to combat it.
Drugs of natural origin have been classified as (i) original natural products, (ii) products derived or chemically synthesized from natural products or (iii) synthetic products based on natural product structures.
Evidence of the importance of natural products in the discovery of leads for the development of drugs for the treatment of human diseases is provided by the fact that close to half of the best selling pharmaceuticals in 1991 were either natural products or their derivatives.
4 In this regard, of the 25 top-selling drugs reported in 1997, 42% were natural products or their derivatives and of these, 67% were antibiotics.
Today, the structures of around 140 000 secondary metabolites have been elucidated.
It is important to understand that many chemically synthesized drugs owe their origin to natural sources.
Applications of chemically synthesized natural metabolites include the use of a natural product derived from plant salicyclic acid derivatives present in white willow, wintergreen and meadowsweet to relieve pain and suffering.
Concoctions of these plants were administered by Hippocrates back in the year 500 BC, and even earlier in Egypt and Babylonia, for fever, pain and childbirth.
Synthetic salicylates were produced initially by Bayer in 1874, and later in 1897, Arthur Eichengrun at Bayer discovered that an acetyl derivative (aspirin), reduced acidity, bad taste and stomach irritation.
These plant-based systems continue to play an essential role in health care, and it has been estimated by the World Health Organization (WHO) that approximately 80% of the world's inhabitants rely mainly on traditional medicines for their primary health care.
5 Other synthesized compounds originating from natural products include a nonapeptide, designated teprotide, which was isolated from the venom of the Brazilian pit viper Bothrops jararaca.
6 This led to the design and synthesis of angiotensin-converting enzyme (ACE) inhibitors such as captopril, which was the first marketed, orally active ACE inhibitor.
7 Enalapril, another ACE inhibitor used in the treatment of cardiovascular disease, was approved for marketing by the Food and Drug Administration (FDA) in 1985.
6 The alkaloid quinine, the active constituent of the 'fever tree' Cinchona succirubra, has been known for centuries by South American Indians to control malaria.
During the twentieth century, massive programs to synthesize quinoline derivatives, based on the quinine prototype, were carried out.
The first of the new quinolones to be used clinically as an antibacterial agent was nalidixic acid, which emerged as part of a large chemical synthesis program developed at the Sterling Winthrop Research Institute.
8, 9 The program was begun when 7-chloro-1,4-dihydro-1-ethyl-4-oxoquinolone-3-carboxylic acid was obtained as a side product during purification of chloroquine and found to have antibacterial activity.
The best compound found in the program was nalidixic acid, which had remarkable activity against Gram-negative bacteria and was shown to be an inhibitor of DNA gyrase.
Its discovery led to a whole series of synthetic quinolone and fluoroquinolone antibiotics (pefloxacin, norfloxacin, ciprofloxacin, levofloxacin, ofloxacin, lomefloxacin, sparfloxacin, etc.
), which have been very successful in medicine and have achieved major commercial success (Table 1) .
It is important to appreciate that all quinolones, though synthetic, are based on the structure of the natural plant product quinine.
Secondary metabolites have exerted a major impact on the control of infectious diseases and other medical conditions, and the development of pharmaceutical industry.
Their use has contributed to an increase in the average life expectancy in the USA, which increased from 47 years in 1900 to 74 years (in men) and 80 years (in women) in 2000.
11 Probably, the most important use of secondary metabolites has been as anti-infective drugs.
In 2000, the market for such antiinfectives was US$55 billion (Table 1 ) and in 2007 it was US$66 billion.
Table 1 shows that, among the anti-infective drugs, antivirals represent more than 20% of the market.
Two antivirals that are chemically synthesized today were originally isolated from marine organisms.
They are acyclovir (active against the herpes virus by inhibition and inactivation of DNA polymerase) and cytarabine (active against non-Hodgkin's lymphoma).
Both compounds are nucleoside analog drugs, originally isolated from sponges.
12 Other antiviral applications of natural compounds are related to human immunodeficiency virus (HIV) treatment.
In the pathogenesis of this disease, HIV-1, similar to other retroviruses, depends on its stable integration into the host genome to facilitate efficient replication of the viral RNA and maintenance of the infected state.
Therefore, de novo viral DNA synthesized during reverse transcription is immediately integrated into the host cell DNA (through the integration step), allowing for further transcription of viral RNA.
In the late phase of HIV viral replication, the large precursor polyprotein (gag-pol precursor, Pr 160) must be appropriately cleaved by a viral protease.
The cleavage of the gag precursor protein of HIV is critical for the maturation and infectivity of the viral particle.
Without the appropriate cleavage of the precursor polyproteins, non-infectious viral particles are generally produced.
To confront this problem, a tremendous effort has been made at the US National Cancer Institute (NCI), in search of natural metabolites capable of inhibiting HIV reverse transcriptase and HIV protease.
Chemically synthesized derivatives of these compounds are the main agents now used against HIV.
Furthermore, reports have been published on natural product inhibitors of HIV integrase obtained from among the marine ascidian alkaloids; that is, the lamellarins (produced by the mollusk Lamellaria sp.
), and from terrestrial plants (Baccharis genistelloides and Achyrocline satureioides).
The most consistent anti-HIV activity was observed with extracts prepared from several Baccharis species.
13 In addition, NCI has been evaluating the HIV-1 inhibitory activity of pepstatin A, a small pentapeptide produced by several Streptomyces species.
It contains a unique hydroxyamino acid, statine, that sterically blocks the active site of HIV-1 protease.
14, 15 REASONS FOR DEVELOPING NEW ANTIBIOTICS New antibiotics that are active against resistant bacteria are required.
Bacteria have lived on the Earth for several billion years.
During this time, they encountered in nature a wide range of naturally occurring antibiotics.
To survive, bacteria developed antibiotic resistance mechanisms.
Therefore, it is not surprising that they have become resistant to most of the natural antimicrobial agents that have been developed over the past 50 years.
16 This resistance increasingly limits the effectiveness of current antimicrobial drugs.
The problem is not just antibiotic resistance but also multidrug resistance.
In 2004, more than 70% of pathogenic bacteria were estimated to be resistant to at least one of the currently available antibiotics.
17 The so-called 'superbugs' (organisms that are resistant to most of the clinically used antibiotics) are emerging at a rapid rate.
S. aureus, which is resistant to methicillin, is responsible for many cases of infections each year.
The incidence of multidrug-resistant pathogenic bacteria is increasing.
The Infectious Disease Society of America (IDSA) reported in 2004 that in US hospitals alone, around 2 million people acquire bacterial infections each year (http://www.idsociety.org/Content.aspx?id¼4682).
S. aureus is responsible for half of the hospital-associated infections and takes the lives of approximately 100 000 patients each year in the USA alone.
18 The bacteria produce a biofilm in which they are encased and protected from the environment.
Biofilms can grow on wounds, scar tissues and medical implants or devices, such as joint prostheses, spinal instrumentations, catheters, vascular prosthetic grafts and heart valves.
More than 70% of the bacterial species producing such biofilms are likely to be resistant to at least one of the drugs commonly used in anti-infectious therapy.
14 called 'nosocomial bacteria.'
More than 60% of sepsis cases in hospitals are caused by Gram-negative bacteria.
14 Among them, Pseudomonas aeruginosa accounts for almost 80% of these opportunistic infections.
They represent a serious problem in patients hospitalized with cancer, cystic fibrosis and burns, causing death in 50% of cases.
Other infections caused by Pseudomonas species include endocarditis, pneumonia and infections of the urinary tract, central nervous system, wounds, eyes, ears, skin and musculoskeletal system.
This bacterium is another example of a natural multidrug-resistant microorganism.
Although many strains are susceptible to gentamicin, tobramycin and amikacin, resistant forms have also developed.
These multidrug-resistant bacteria make hospitals ''dangerous places to be, especially if you are sick, but even if not.''
19 Although we are seeing a steady increase in resistance in almost every pathogen to most of the current antibiotics over time, not all the antibacterial agents show the same rate of resistance development.
For example, antimicrobials such as rifampicin, which targets single enzymes, are most susceptible to the development of resistance, whereas agents that inactivate several targets irreversibly generate resistance more slowly.
In addition to the antibiotic-resistance problem, new families of anti-infective compounds are needed to enter the marketplace at regular intervals to tackle the new diseases caused by evolving pathogens.
At least 30 new diseases emerged in the 1980s and 1990s and they are growing in incidence.
Emerging infectious organisms often encounter hosts with no prior exposure to them and thus represent a novel challenge to the host's immune system.
Several viruses responsible for human epidemics have made a transition from animal host to humans and are now transmitted from human to human.
HIV, responsible for the acquired immunodeficiency syndrome (AIDS) epidemic, is one example.
Although it has not been proven, it is suspected that severe acute respiratory syndrome (SARS), caused by the SARS coronavirus, also evolved from a different species.
20 In the early 1990s, after decades of decline, the incidence of tuberculosis began to increase.
The epidemic took place owing to inadequate treatment regimens, a diminished public health system and the onset of the HIV/AIDS epidemic.
The WHO has predicted that between 2000 and 2020, nearly 1 billion people will become infected with Mycobacterium tuberculosis and that this disease will cost the lives of 35 million people.
Sexually transmitted diseases have also increased during these decades, especially in young people (aged 15-24 years).
The human papillomavirus, chlamydia, genital herpes, gonorrhea and HIV/AIDS are examples.
HIV/AIDS has infected more than 40 million people in the world.
Together with other diseases such as tuberculosis and malaria, HIV/AIDS accounts for over 300 million illnesses and more than 5 million deaths each year.
Additional evolving pathogens include the (i) Ebola virus, which causes the viral hemorrhagic fever syndrome with a resultant mortality rate of 88%; (ii) the bacterium Legionella pneumophila, a ubiquitous aquatic organism that lives in warm environments, which causes Legionnaire's disease, a pulmonary infection; (iii) the Hantavirus, which can infect humans with two serious illnesses: hemorrhagic fever with renal syndrome and Hantavirus pulmonary syndrome; (iv) at least three species of bacteria from the genus Borrelia, which cause Lyme disease, an emerging infection.
In this case, the infection is acquired from the bite of ticks belonging to several species of the genus Ixodes.
Borrelia burgdorferi is the predominant cause of Lyme disease in the US, whereas Borrelia afzelii and Borrelia garinii are implicated in most European cases.
The disease presentation varies widely, and may include a rash and flu-like symptoms in its initial stage, followed by musculoskeletal, arthritic, neurologic, psychiatric and cardiac manifestations.
In the majority of cases, symptoms can be eliminated with antibiotics, especially if the treatment begins early in the course of illness.
However, late or inadequate treatment can lead to 'late-stage' Lyme disease that can be disabling and difficult to treat.
21 (v) Other evolving pathogens include the Escherichia coli 0157:H7 (enterohemorrhagic E. coli), a strain that causes colitis and bloody diarrhea by producing a toxin called Shiga toxin, which damages the intestines.
It is estimated that this bacterium causes infection in more than 70 000 patients a year in the USA.
Another example is (vi) Cryptosporidium, an obligate intracellular parasite commonly found in lakes and rivers.
Cryptosporidium parvum is one of the common species affecting the digestive and respiratory organs.
Intestinal cryptosporidiosis is characterized by severe watery diarrhea.
Pulmonary and tracheal cryptosporidiosis in humans is associated with coughing and is frequently a low-grade fever.
People with severely weakened immune systems are likely to have more severe and more persistent symptoms than healthy individuals.
In the developing world, nearly 90% of the infectious disease deaths are caused by six diseases or disease processes: acute respiratory infections, diarrhea, tuberculosis, HIV, measles and malaria.
In both the developing and developed nations, the leading cause of death by a wide margin is acute respiratory disease.
In the developing world, acute respiratory infections are attributed primarily to seven bacteria: Bordetella pertussis, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Chlamydia trachomatis.
In addition, the major viral causes of respiratory infections include respiratory syncytial virus, human parainfluenza viruses 1 and 3, influenza viruses A and B, as well as some adenoviruses.
These diseases are highly destructive in economic and social as well as in human terms and cause approximately 17 million deaths per year, and innumerable serious illnesses besides affecting the economic growth, development and prosperity of human societies.
22 Morse 23 identified six general factors in the emergence of infectious diseases: ecological changes, human demographics and behavior, international travel, technology and industry, microbial adaptation and change, and breakdown in public health measures.
24 One additional reason for developing new antibiotics is related to their own toxicity.
As with other therapeutic agents, the use of antibiotics may also cause side effects in patients.
These include mild reactions such as upset stomach, vomiting and diarrhea (cephalosporins, macrolides, penicillins and tetracyclines), rash and other mild and severe allergic reactions (cephalosporins and penicillins), sensitivity to sunlight (tetracyclines), nervousness, tremors and seizures (quinolones).
Some side effects are more severe and, depending on the antibiotic, may disrupt the hearing function (aminoglycosides), kidneys (aminoglycosides and polypeptides) or liver (rifampin).
During recent decades, we have seen an increasing number of reports on the progressive development of bacterial resistance to almost all available antimicrobial agents.
In the 1970s, the major problem was the multidrug resistance of Gram-negative bacteria, but later in the 1980s the Gram-positive bacteria became important, including methicillin-resistant staphylococci, penicillin-resistant pneumococci and vancomycin-resistant enterococci.
25 In the past, the solution to the problem has depended primarily on the development of novel antimicrobial agents.
However, the number of new classes of antimicrobial agents being developed has decreased dramatically in recent years.
The advent of resistant Gram-positive bacteria has been noticed by the pharmaceutical, biotechnology and academic communities.
Some of these groups are making concerted efforts to find novel antimicrobial agents to meet this need.
A new glycopeptide antibiotic, teicoplanin, was developed against infections with resistant Gram-positive bacteria, especially bacteria resistant to the glycopeptide vancomycin.
In another instance, the approach involved the redesign of a mixture of two compounds, called streptogramin, into a new mixture, called pristinamycin, to allow administration of the drug parenterally and in higher doses than the earlier oral preparation.
26 The two components of streptogramin, quinupristin and dalfopristin, were chemically modified to allow intravenous administration.
The new combination, pristinamycin, was approved by the FDA for use against infections caused by vancomycin-resistant Enterococcus faecium.
Additional moves against resistant microorganisms are the glycylcyclines developed to treat tetracycline-resistant bacteria.
These modified tetracyclines show potent activity against a broad spectrum of Gram-positive and Gram-negative bacteria, including strains that carry the two major tetracycline-resistance determinants, involving efflux and ribosomal protection.
Two of the glycylcyline derivatives, DMG-MINO and DMG-DMDOT, have been tested against a large number of clinical pathogens isolated from various sources.
The spectrum of activity of these compounds includes organisms with resistance to antibiotics other than tetracyclines; for example, methicillin-resistant staphylococci, penicillin-resistant S. pneumoniae and vancomycin-resistant enterococci.
27 Tigecycline was approved by the FDA in 2005 as an injectable antibiotic.
28 Among the novel class of antimicrobial agents used in treating resistance to Gram-positive infections, we can also mention the cyclic lipopeptide antibiotic daptomycin produced by Streptomyces roseosporus.
This compound was approved in 2003 by the FDA for skin infections resulting from complications following surgery, diabetic foot ulcers and burns.
It represents the first new natural antibiotic approved in many years.
Its mode of action is distinct from any other approved antibiotic: it rapidly kills Gram-positive bacteria by disrupting multiple aspects of bacterial membrane function (by binding irreversibly to the bacterial cell membrane, causing membrane depolarization, destroying the ion concentration gradient and provoking the efflux of K + ).
It acts against most clinically relevant Gram-positive bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp.
equisimilis and Enterococcus faecalis), and retains in vitro potency against isolates resistant to methicillin, vancomycin and linezolid.
Traditionally, these infections were treated with penicillin and cephalosporins, but resistance to these agents became widespread.
[29] [30] [31] [32] Daptomycin seems to have a favorable side effect profile, and it might be used to treat patients who cannot tolerate other antibiotics.
Telithromycin, a macrolide antibiotic, is the first orally active compound of a new family of antibacterials named the ketolides.
It shows potent activity against pathogens implicated in communityacquired respiratory tract infections, irrespective of their b-lactam, macrolide or fluoroquinolone susceptibility.
Some of the microorganisms susceptible to this antibiotic are pneumococci, H. influenzae and Moraxella catarrhalis, including b-lactamase-positive strains.
In addition, telithromycin has a very low potential for selection of resistant isolates or induction of cross-resistance found with other macrolides.
33 Clavulanic acid, first detected in Streptomyces clavuligerus, contains a bicyclic b-lactam ring fused to an oxazolidine ring with an oxygen in place of a sulfur, a b-hydroxyethylidene substituent at C-2 and no acylamino group at C-6.
It was first described in 1976 and shown to be a potent inhibitor of the b-lactamases produced by staphylococci and plasmid-mediated b-lactamases of E. coli, Klebsiella, Proteus, Shigella, Pseudomonas and Haemophilus.
Although it is a broad-spectrum antibiotic, clavulanic acid possesses only very low antibacterial activity.
Therefore, the molecule has been combined, as a b-lactamase inhibitor, with a variety of broad-spectrum semisynthetic penicillins.
For example, when administered with amoxicillin, it is used for the treatment of infections caused by b-lactamase-producing pathogenic bacteria.
34 It has world sales of over US$1 billion, and in 1995 it was the second largest selling antibacterial drug.
Clavulanic acid can also be combined with ticarcillin, which is a penicillin effective against organisms such as E. coli, Proteus, Salmonella, Haemophilus, Pseudomonas and S. aureus.
It is normally used in hospitals for treating severe infections affecting blood or internal organs, bones and joints, upper or lower airways or skin and soft tissue.
The combination extends ticarcillin antimicrobial activity by inhibiting the action of the b-lactamases produced by certain bacteria.
Mycosis is a condition in which fungi pass the resistance barriers of the human or animal body and establish infections.
These organisms are harmless most of the time, but sometimes they can cause fungal infections.
In most cases, these infections are not life threatening.
However, when they are deeply invasive and disseminated, they lead to more serious infections, particularly in critically ill patients, elderly people and those who have conditions that affect the immune system (by disease or through the use of immunosuppressive agents).
In addition, the use of antineoplastic and broad-spectrum antibiotics, prosthetic devices and grafts, and more aggressive surgery has increased invasive fungal infections.
Patients with burns, neutropenia, pancreatitis or after organ transplantation (40% of liver transplants, 15-35% of heart transplants and 5% of kidney transplants) are also predisposed to fungal infection.
35 Approximately 40% of death from nosocomial infections are caused by fungi, and 80% of these are caused by Candida and Aspergillus, although Cryptococcus spp., Fusarium spp., Scedosporium spp., Penicillium spp.
and zygomycetes are increasingly involved.
36 Pulmonary aspergillosis is the main factor involved in the death of recipients of bone marrow transplants, and Pneumocystis carinii is the leading cause of death in AIDS patients from Europe and North America.
37 The rising incidence of invasive fungal infections and the emergence of broader fungal resistance have led to the need for novel antifungal agents.
Amphotericin B is the first-line therapy for systemic infection because of its broad spectrum and fungicidal activity.
However, considerable side effects limit its clinical utility.
Echinocandins are large lipopeptide molecules that inhibit the synthesis of 1,3-b-Dglucan, a key component of the fungal cell wall.
Three echinocandins (caspofungin, micafungin and anidulafungin) have reached the market.
Caspofungin is also known as pneumocandin or MK-0991.
This compound was the first cell-wall-active antifungal approved as a new injectable antifungal; this was in 2000.
38 It irreversibly inhibits 1,3-b-D-glucan synthase, preventing the formation of glucan polymers and disrupting the integrity of fungal cell walls.
39 It is more active and less toxic than amphotericin B and shows a broad spectrum of activity against Candida (including fluconozole resistance), Aspergillus, Histoplasma and P. carinii, the major cause of HIV death.
Micafungin is licensed for clinical use in Asian countries and in the US.
This compound exhibits extremely potent antifungal activity against clinically important fungi, including Aspergillus and azole-resistant strains of Candida.
In animal studies, micafungin is as efficacious as amphotericin B with respect to improvement of survival rate.
It is characterized by a linear pharmacokinetic profile and substantially fewer toxic effects.
Anidulafungin is currently licensed in the US.
40 Although several new antifungal drugs have been developed in the past 6 years, some patients remain resistant to treatments.
The main reasons for this include intrinsic or acquired antifungal resistance, organ dysfunction preventing the use of some agents and drug interactions.
In addition, some drugs penetrate poorly into sanctuary sites, including the eye and urine, and others are associated with considerable adverse events.
However, there has been some progress.
Posaconazole is a new member of the triazole class of antifungals.
It has shown clinical efficacy in the treatment of oropharyngeal candidiasis and has potential as a salvage therapy for invasive aspergillosis, zygomycosis, cryptococcal meningitis and a variety of other fungal infections.
It is available as an oral suspension and has a favorable toxicity profile.
The wide spectrum of posaconazole activity in in vitro studies, animal models and preliminary clinical studies suggests that it represents an important addition to the antifungal armamentarium.
41 In addition to the screening programs for antibacterial activity, the pharmaceutical industry has extended these programs to other disease areas.
42, 43 Microorganisms are a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important products of the pharmaceutical industry.
Microbial secondary metabolites are now being used for applications other than antibacterial, antifungal and antiviral infections.
For example, immunosuppressants have revolutionized medicine by facilitating organ transplantation.
44 Other applications include antitumor drugs, enzyme inhibitors, gastrointestinal motor stimulator agents, hypocholesterolemic drugs, ruminant growth stimulants, insecticides, herbicides, coccidiostats, antiparasitics vs coccidia, helminths and other pharmacological activities.
Further applications are possible in various areas of pharmacology and agriculture, developments catalyzed by the use of simple enzyme assays for screening before testing in intact animals or in the field.
In the year 2000, approximately 10 million new cases of cancer were diagnosed in the world, resulting in 6 million cancer-related deaths.
The tumor types with the highest incidence were lung (12.3%), breast (10.4%) and colorectal (9.4%).
45 Microbial metabolites are among the most important of the cancer chemotherapeutic agents.
They started to appear around 1940 with the discovery of actinomycin and since then many compounds with anticancer properties have been isolated from natural sources.
More than 60% of the current compounds with antineoplasic activity were originally isolated as natural products or are their derivatives.
Among the approved products deserving special attention are actinomycin D, anthracyclines (daunorubicin, doxorubicin, epirubicin, pirirubicin and valrubicin), bleomycin, mitosanes (mitomycin C), anthracenones (mithramycin, streptozotocin and pentostatin), enediynes (calcheamycin), taxol and epothilones.
Actinomycin D is the oldest microbial metabolite used in cancer therapy.
Its relative, actinomycin A, was the first antibiotic isolated from actinomycetes.
The latter was obtained from Actinomyces antibioticus (now Streptomyces antibioticus) by Waksman and Woodruff.
46 As it binds DNA at the transcription initiation complex, it prevents elongation by RNA polymerase.
This property, however, confers some human toxicity and it has been used primarily as an investigative tool in the development of molecular biology.
Despite the toxicity, however, it has served well against Wilms tumor in children.
The anthracyclines are some of the most effective antitumor compounds developed, and are effective against more types of cancer than any other class of chemotherapy agents.
47 They are used to treat a wide range of cancers, including leukemias, lymphomas, and breast, uterine, ovarian and lung cancers.
Anthracyclines act by intercalating DNA strands, which result in a complex formation that inhibits the synthesis of DNA and RNA.
It also triggers DNA cleavage by topoisomerase II, resulting in mechanisms that lead to cell death.
In their cytotoxic effects, the binding to cell membranes and plasma proteins plays an important role.
Their main adverse effects are heart damage (cardiotoxicity), which considerably limits their usefulness, and vomiting.
The first anthracycline discovered was daunorubicin (daunomycin) in 1966, which is produced naturally by Streptomyces peucetius.
Doxorubicin (adriamycin) was developed in 1967.
Another anthracycline is epirubicin.
This compound, approved by the FDA in 1999, is favored over doxorubicin in some chemotherapy regimens as it appears to cause fewer side effects.
Epirubicin has a different spatial orientation of the hydroxyl group at the 4¢ carbon of the sugar, which may account for its faster elimination and reduced toxicity.
Epirubicin is primarily used against breast and ovarian cancer, gastric cancer, lung cancer and lymphomas.
Valrubicin is a semisynthetic analog of doxorubicin approved as a chemotherapeutic drug in 1999 and used to treat bladder cancer.
Bleomycin is a non-ribosomal glycopeptide microbial metabolite produced as a family of structurally related compounds by the bacterium Streptomyces verticillus.
First reported by Umezawa et al.
48 in 1966, bleomycin obtained FDA approval in 1973.
When used as an anticancer agent (inducing DNA strand breaks), the chemotherapeutic forms are primarily bleomycins A2 and B2.
Mitosanes are composed of several mitomycins that are formed during the cultivation of Streptomyces caespitosus.
Although the mitosanes are excellent antitumor agents, they have limited utility owing to their toxicity.
Mitomycin C was approved by the FDA in 1974, showing activity against several types of cancer (lung, breast, bladder, anal, colorectal, head and neck), including melanomas and gastric or pancreatic neoplasms.
49 Recently, mitomycin dimers have been explored as potential alternatives for lowering toxicity and increasing efficiency.
50 Mithramycin (plicamycin) is an antitumor aromatic polyketide produced by Streptomyces argillaceous that shows antibacterial and antitumor activity.
51 It is one of the older chemotherapy drugs used in the treatment of testicular cancer, disseminated neoplasms and hypercalcemia.
It binds to G-C-rich DNA sequences, inhibiting the binding of transcription factors such as Sp1, which is believed to affect neuronal survival/death pathways.
It may also indirectly regulate gene transcription by altering histone methylation.
With repeated use, organotoxicity (kidney, liver and hematopoietic system) can become a problem.
Streptozotocin is a microbial metabolite with antitumor properties, produced by Streptomyces achromogenes.
Chemically, it is a glucosamine-nitroso-urea compound.
As with other alkylating agents in the nitroso-urea class, it is toxic to cells by causing damage to DNA, although other mechanisms may also contribute.
The compound is selectively toxic to the b-cells of the pancreatic islets.
It is similar enough to glucose to be transported into the cell by the glucose transport protein of these cells, but it is not recognized by the other glucose transporters.
As b-cells have relatively high levels of glucose permease, the relative streptozotocin toxicity for these b-cells can be explained.
52 In 1982, FDA granted approval for streptozotocin as a treatment for pancreatic islet cell cancer.
Pentostatin (deoxycoformycin) is an anticancer chemotherapeutic drug produced by S. antibioticus.
It is classified as a purine analog, which mimics the nucleoside adenosine and thus tightly binds and inhibits adenosine deaminase (K i of 2.5Â10 À12 M).
It interferes with the cell's ability to process DNA.
53 Pentostatin is commonly used to treat hairy cell leukemia, acute lymphocytic leukemia, prolymphocytic leukemia (B-and T-cell origin), T-cell leukemia and lymphoma.
However, it can cause kidney, liver, lung and neurological toxicity.
54 The FDA granted approval for pentostatin in 1993.
Calicheamicins are highly potent antitumor microbial metabolites of the enediyne family produced by Micromonospora echinospora.
Their antitumor activity is apparently due to the cleavage of double-stranded DNA.
55 They are highly toxic, but it was possible to introduce one such compound into the clinic by attaching it to an antibody that delivered it to certain cancer types selectively.
This ingenious idea of the Wyeth Laboratories avoided the side effects of calicheamicin.
In this regard, gemtuzumab is effective against acute myelogenous leukemia (AML).
Calicheamicin is bound to a monoclonal antibody against a transmembrane receptor (CD33) expressed on cells of monocytic/myeloid lineage.
CD33 is expressed in most leukemic blast cells, but in normal hematopoietic cells the intensity diminishes with maturation.
It was approved by the FDA for use in patients over the age of 60 years with relapsed AML who are not considered candidates for standard chemotherapy.
56 A successful non-actinomycete molecule is taxol (paclitaxel), which was first isolated from the Pacific yew tree, Taxus brevifolia, but is also produced by the endophytic fungi Taxomyces andreanae and Nodulisporium sylviforme.
57 This compound inhibits rapidly dividing mammalian cancer cells by promoting tubulin polymerization and interfering with normal microtubule breakdown during cell division.
The drug also inhibits several fungi (Pythium, Phytophthora and Aphanomyces) by the same mechanism.
In 1992, taxol was approved for refractory ovarian cancer, and today it is used against breast and advanced forms of Kaposi's sarcoma.
58 A new formulation is available in which paclitaxel is bound to albumin.
Taxol sales amounted to US$1.6 billion in 2006 for Bristol Myers-Squibb, representing 10% of the company's pharmaceutical sales and its third largest selling product.
Currently, taxol production uses plant cell fermentation technology.
The epothilones (a name derived from its molecular features: epoxide, thiazole and ketone) are macrolides originally isolated from the broth of the soil myxobacterium Sorangium cellulosum as weak agents against rust fungi.
59 They were identified as microtubulestabilizing drugs, acting in a similar manner to taxol.
60,61 However, they are generally 5-25 times more potent than taxol in inhibiting cell growth in cultures.
Five analogs are now undergoing investigation as candidate anticancer drugs, and their preclinical studies have indicated a broad spectrum of antitumor activity, including taxol-resistant tumor cells.
With the best currently available therapies, the median survival time for patients with metastatic breast cancer is only 2-3 years, and many patients develop resistance to taxanes or other chemotherapy drugs.
One epothilone, ixabepilone, was approved in October 2007 by the FDA for use in the treatment of aggressive metastatic or locally advanced breast cancer no longer responding to currently available chemotherapies.
62 In tumor cells, p-glycoprotein reduces intracellular antitumor drug concentrations, thereby limiting access of chemotherapeutic substrates to the site of action.
The epothilones are attractive because they are active against p-glycoprotein-producing tumors and have good solubility.
62 Epothilone B is a 16-membered polyketide macrolactone with a methylthiazole group connected to the macrocycle by an olefinic bond.
Testicular cancer is the most common cancer diagnosis in men between the ages of 15 and 35 years, with approximately 8000 cases detected in the United States annually.
63 The majority (95%) of testicular neoplasms are germ cell tumors, which are relatively uncommon carcinomas, accounting for only 1% of all male malignancies.
Remarkable progress has been made in the medical treatment of advanced testicular cancer, with a substantial increase in cure rates from approximately 5% in the early 1970s to almost 90% today.
64, 65 This cure rate is the highest of any solid tumor, and improved survival is primarily due to effective chemotherapy.
A major advance in chemotherapy for testicular germ cell tumors was the introduction of cisplatin in the mid-1970s.
Two chemotherapy regimens are effective for patients with a good testicular germ cell tumor prognosis: four cycles of etoposide and cisplatin or three cycles of bleomycin, etoposide and cisplatin.
66 Of the latter three agents, bleomycin and etoposide are natural products.
Enzyme inhibitors have received increasing attention as useful tools, not only for the study of enzyme structures and reaction mechanisms but also for potential utilization in medicine and agriculture.
Several enzyme inhibitors with various industrial uses have been isolated from microbes.
67 The most important are (1) clavulanic acid, the inhibitor of b-lactamases discussed above in the section 'Moves against antibiotic resistance development in bacteria,' and the statins, hypocholesterolemic drugs presented below in the section 'Hypocholesterolemic drugs.'
Some of the common targets for other inhibitors are glucosidases, amylases, lipases, proteases and xanthine oxidase (XO).
Acarbose is a pseudotetrasaccharide made by Actinoplanes sp.
SE50.
It contains an aminocyclitol moiety, valienamine, which inhibits intestinal a-glucosidase and sucrase.
This results in a decrease in starch breakdown in the intestine, which is useful in combating diabetes in humans.
68 Amylase inhibitors are useful for the control of carbohydratedependent diseases, such as diabetes, obesity and hyperlipemia.
69, 70 Amylase inhibitors are also known as starch blockers because they contain substances that prevent dietary starches from being absorbed by the body.
The inhibitors may also be useful for weight loss, as some versions of amylase inhibitors do show potential for reducing carbohydrate absorption in humans.
71, 72 The use of amylase inhibitors for the treatment of rumen acidosis has also been reported.
73 Examples of microbial a-amylase inhibitors are paim, obtained from culture filtrates of Streptomyces corchorushii, 74 and TAI-A, TAI-B, oligosaccharide compounds from Streptomyces calvus TM-521.
75 Lipstatin is a pancreatic lipase inhibitor produced by Streptomyces toxytricini that is used to combat obesity and diabetes.
It interferes with the gastrointestinal absorption of fat.
76 The commercial product is tetrahydrolipstatin, which is also known as orlistat.
In the pathogenic processes of some diseases, such as emphysema, arthritis, pancreatitis, cancer and AIDS, protease inhibitors are potentially powerful tools for inactivating target proteases.
Examples of microbial products include antipain, produced by Streptomyces yokosukaensis, leupeptin from Streptomyces roseochromogenes and chymostatin from Streptomyces hygroscopicus.
70 Leupeptin is produced by more than 17 species of actinomycetes.
67 XO catalyzes the oxidation of hypoxanthine to uric acid through xanthine.
An excessive accumulation of uric acid in the blood, called hyperuricemia, causes gout.
77 The inhibitors of XO decrease the uric acid levels, which result in an antihyperuricemic effect.
A potent inhibitor of XO, hydroxyakalone, was purified from the fermentation broth of Agrobacterium aurantiacum sp.
nov., a marine bacterial strain.
78 Fungal products are also used as enzyme inhibitors against cancer, diabetes, poisonings, Alzheimer's disease, etc.
The enzymes inhibited include acetylcholinesterase, protein kinase, tyrosine kinase, glycosidases and others.
79 Immunosuppresants Suppressor cells are critical in the regulation of the normal immune response.
An individual's immune system is capable of distinguishing between native and foreign antigens and of mounting a response only against the latter.
A major role has been established for suppressor T lymphocytes in this phenomenon.
Suppressor cells also play a role in regulating the magnitude and duration of the specific antibody response to an antigenic challenge.
Suppression of the immune response either by drugs or by radiation, to prevent the rejection of grafts or transplants or to control autoimmune diseases, is called immunosuppression.
A number of microbial compounds capable of suppressing the immune response have been discovered.
Cyclosporin A was originally introduced as a narrow-spectrum antifungal peptide produced by the mold, Tolypocladium nivenum (originally classified as Trichoderma polysporum and later as Tolypocladium inflatum), by aerobic fermentation.
Cyclosporins are a family of neutral, highly lipophilic, cyclic undecapeptides containing some unusual amino acids, synthesized by a non-ribosomal peptide synthetase, cyclosporin synthetase.
Discovery of the immunosuppressive activity led to its use in heart, liver and kidney transplants and to the overwhelming success of the organ transplant field.
80 Cyclosporin was approved for use in 1983.
It is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes, especially T lymphocytes.
This complex of cyclosporin and cyclophilin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin-2.
It also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector T cells.
Sales of cyclosporin A have reached US$1.5 billion per year.
Other important transplant agents include sirolimus (rapamycin) and tacrolimus (FK506), which are produced by actinomycetes.
Rapamycin is especially useful in kidney transplants as it lacks the nephrotoxicity seen with cyclosporin A and tacrolimus.
It is a macrolide, first discovered in 1975 as a product of S. hygroscopicus, and was initially proposed as an antifungal agent.
However, this was abandoned when it was discovered that it had potent immunosuppressive and antiproliferative properties.
This compound binds to the immunophilin FK506-binding protein (FKBP12), and this binary complex interacts with the rapamycin-binding domain and inactivates a serine-threonine kinase termed the mammalian target of rapamycin.
The latter is known to control proteins that regulate mRNA translation initiation and G1 progression.
81 The antiproliferative effect of rapamycin has also been used in conjunction with coronary stents to prevent restenosis, which usually occurs after the treatment of coronary artery disease by balloon angioplasty.
Rapamycin also shows promise in treating tuberous sclerosis complex (TSC), a congenital disorder that leaves sufferers prone to benign tumor growth in the brain, heart, kidneys, skin and other organs.
In a study of rapamycin as a treatment for TSC, University of California, Los Angeles (UCLA) researchers observed a major improvement in mice regarding retardation related to autism.
82 As rapamycin has poor aqueous solubility, some of its analogs, RAD001 (everolimus), CCI-799 (tensirolimus) and AP23573 (ARIAD), have been developed with improved pharmaceutical properties.
Everolimus is currently used as an immunosuppressant to prevent the rejection of organ transplants.
Although it does not have FDA approval in the USA, it is approved for use in Europe and Australia, and phase III trials are being conducted in the US.
Everolimus may have a role in heart transplantation as it has been shown to reduce chronic allograft vasculopathy in such transplants.
83 Everolimus is also used in drug-eluting coronary stents as an immunosuppressant to prevent rejection.
CCI-779 is a rapamycin ester that can be converted to rapamycin in vivo.
RAD001 is a rapamycin analog currently being investigated in phase II trials for recurrent endometrial cancer as a single agent, and in phase I/II trials for the treatment of glioblastoma in combination with the inhibitor of certain epidermal growth factor receptor and vascular endothelial growth factor receptor family members.
84 AP23573 is a novel non-prodrug rapamycin analog with a nonlinear pharmacokinetic behavior that has demonstrated antiproliferative activity against several human tumor cell lines in vitro and against experimental tumors in vivo.
85 This agent is currently under evaluation in phase I-II trials, including patients with different tumors.
Two additional small-molecule rapamycin analogs, AP23841 and AP23675, are currently in preclinical development for the treatment of bone metastases and primary bone cancer.
86 Tacrolimus (FK506) was discovered in 1987 in Japan.
87 It is produced by Streptomyces tsukubaensis.
However, its use was almost abandoned because of dose-associated toxicity.
Dr Thomas Starzl (University of Pittsburgh) rescued it by using lower doses, realizing that it was approximately 100 times more active as an immunosuppressive than cyclosporin A.
88 It was introduced in Japan in 1993, and in 1994 it was approved by the FDA for use as an immunosuppressant in liver transplantation.
Furthermore, its use has been extended to include bone marrow, cornea, heart, intestines, kidney, lung, pancreas, trachea, small bowel, skin and limb transplants, and for the prevention of graft-vs-host disease.
Topically, it is also used against atopic dermatitis, a widespread skin disease.
In the laboratory, tacrolimus inhibits the mixed lymphocyte reaction, the formation of interleukin-2 by T lymphocytes, and the formation of other soluble mediators, including interleukin-3 and interferon g. Recently, it has been reported that tacrolimus inhibits tumor growth factor-b-induced signaling and collagen synthesis in human lung fibroblastic cells.
This factor plays a pivotal role in tissue fibrosis, including pulmonary fibrosis.
Therefore, tacrolimus may be useful for the treatment of pulmonary fibrosis, although its use in the acute inflammatory phase may exacerbate lung injury.
89 Hypocholesterolemic drugs Atherosclerosis is generally viewed as a chronic, progressive disease characterized by the continuous accumulation of atheromatous plaque within the arterial wall.
The past two decades have witnessed the introduction of a variety of anti-atherosclerotic therapies.
The statins form a class of hypolipidemic drugs used to lower cholesterol by inhibiting the enzyme HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway of cholesterol biosynthesis.
Inhibition of this enzyme in the liver stimulates low-density lipoprotein (LDL) receptors, resulting in an increased clearance of LDL from the bloodstream and a decrease in blood cholesterol levels.
Through their cholesterol-lowering effect, they reduce the risk of cardiovascular disease, prevent stroke and reduce the development of peripheral vascular disease.
90 In addition, they are anti-thrombotic and antiinflammatory.
Currently there are a number of statins in clinical use.
The entire group of statins reached an annual market of nearly US$30 billion before it became a generic pharmaceutical.
The first member of the group (compactin; mevastatin) was isolated as an antibiotic product of Penicillium brevicompactum and later from Penicillium citrinum.
Although not of commercial importance, compactin's derivatives achieved overwhelming medical and commercial success.
An ethylated form, known as lovastatin (monacolin K; mevinolin), was isolated in the 1970s in the broths of Monascus ruber and Aspergillus terreus.
91 Lovastatin, the first commercially marketed statin, was approved by the FDA in 1987.
A semisynthetic derivative of lovastatin is simvastatin, a major hypocholesterolemic drug, selling for US$7 billion per year before becoming generic.
Another statin, pravastatin (US$3.6 billion per year), is made through different biotransformation processes from compactin by Streptomyces carbophilus 92 and Actinomadura sp.
93 Other genera involved in the production of statins are Doratomyces, Eupenicillium, Gymnoascus, Hypomyces, Paecilomyces, Phoma, Trichoderma and Pleurotus.
94 A synthetic compound, modeled from the structure of the natural statins, is atorvastin, which has been the leading drug of the entire pharmaceutical industry in terms of market share (approximately US$14 billion per year) for many years.
An insecticide is a pesticide used against insects in all developmental forms.
They include ovicides and larvicides used against the eggs and larvae of insects, respectively.
Insecticides are used in agriculture, medicine, industry and households.
The use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity in the twentieth century.
Synthetic insecticides pose some hazards, whereas natural insecticides offer adequate levels of pest control and pose fewer hazards.
Microbially produced insecticides are especially valuable because their toxicity to non-target animals and humans is extremely low.
Compared with other commonly used insecticides, they are safe for both the pesticide users and consumers of treated crops.
The action of microbial insecticides is often specific to a single group or species of insects, and this specificity means that most microbial insecticides do not naturally affect beneficial insects (including predators or parasites of pests) in treated areas.
The spinosyns (A83543 group) are a group of natural products produced by Saccharopolyspora spinosa that were discovered in 1989.
The researchers isolated spinosyn A and D, as well as 21 minor analogs.
They are active on a wide variety of insect pests, especially lepidopterans and dipterans, but do not have antibiotic activity.
95 The compounds attack the nervous system of insects by targeting two key neurotransmitter receptors, with no cross-resistance to other known insecticides.
The spinosyns are a family of macrolides with 21 carbon atoms, containing four connected rings of carbon atoms at their core to which two deoxysugars (forosamine and 2,3,4, tri-O-methylrhamnose, which are required for bioactivity) are attached.
Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea.
96 A mixture of spinosyn A (85%) and D (15%) (spinosad) is being produced through fermentation and was introduced to the market in 1997 for the control of chewing insects on a variety of crops.
Spinosyn formulations were recently approved for use on organic crops and for animal health applications.
Recently, a new naturally occurring series of insect-active compounds was discovered from a novel soil isolate, Saccharopolyspora pogona NRRL30141.
97 The culture produced a unique family of over 30 new spinosyns.
They have a butenyl substitution at the 21 position on the spinosyn lactone and are named butenyl-spinosyns or pogonins.
Herbicides are chemicals marketed to inhibit or interrupt normal plant growth and development.
They are widely used in agriculture, industry and urban areas for weed management.
Approximately 30 000 kinds of weeds are widely distributed in the world; yield losses caused by 1800 kinds of weeds are approximately 9.7% of total crop production every year.
98 Herbicides provide cost-effective weed control with a minimum of labor.
Most are used on crops planted in large acreages, such as soy, cotton, corn and canola.
99 There are numerous classes of herbicides with different modes of action, as well as different potentials for adverse effects on health and the environment.
Over the past century, chemical herbicides, used to control various weeds, may have caused many serious side effects, such as injured crops, threat to the applicator and others exposed to the chemicals, herbicide-resistant weed populations, reduction of soil and water quality, herbicide residues and detrimental effects on non-target organisms.
100 For example, alachlor and atrazine were reported to cause cancer in animal tests.
With increasing global environmental consciousness, bioherbicides, which are highly effective for weed control and environmentally friendly as well, are very attractive both for research and for application.
Microbial herbicides can be divided into microbial preparations (microorganisms that control weeds) and microbially derived herbicides.
The first microbial herbicide was independently discovered in Germany and Japan.
In 1972, the Zähner group in Germany isolated phosphinothricin tripeptide, a peptide antibiotic consisting of two molecules of L-alanine and one molecule of the unusual amino acid L-phosphinothricin; that is, N(4[hydroxyl(methyl)phosphinoyl]homoalanyl)alanylalanine.
They isolated it from Streptomyces viridochromogenes as a broad-spectrum antibacterial including activity against Botrytis cinerea.
101 In Japan, it was discovered at the Meiji Seiki laboratories in 1973 from S. hygroscopicus and named bialaphos.
102 The bioactive L-phosphinothricin is a structural analog of glutamic acid, acting as a competitive inhibitor of glutamine synthetase, and has bactericidal (Gram-positive and Gram-negative bacteria), fungicidal (B. cinerea) and herbicidal properties.
103 Glufosinate (DL-phosphinothricin) (without Ala-Ala) was developed as a herbicide.
Therefore, the agent acts as a herbicide with or without Ala-Ala. Bialaphos has no influence on microorganisms in the soil and is easily degraded in the environment, having a half-life of only 2 h. This low level of environmental impact is of great interest to environmentalists.
In 2006, the global animal health market was valued at US$16 billion, of which 29% was derived from parasiticides.
Parasites are organisms that inhabit the body and benefit from a prolonged, close association with the host.
Antiparasitics are compounds that inhibit the growth or reproduction of a parasite; some antiparasitics directly kill parasites.
In general, parasites are much smaller than their hosts, show a high degree of specialization for their mode of life and reproduce more quickly and in greater numbers than their hosts.
Classic examples of parasitism include the interactions between vertebrate hosts and such diverse animals as tapeworms, flukes, Plasmodium species and fleas.
Parasitic infections can cause potentially serious health problems and even kill the host.
Parasites mainly enter the body through the mouth, usually through ingestion of tainted food or drink.
This is a very common problem in tropical areas, but is not limited to those regions.
There are 3200 varieties of parasites in four major categories: Protozoa, Trematoda, Cestoda and Nematoda.
The major groups include protozoans (organisms having only one cell) and parasitic worms (helminths).
Each of these can infect the digestive tract, and sometimes two or more can cause infection at the same time.
The WHO reported that approximately 25% of the world's population is infected with roundworms.
In addition, a major agricultural problem has been the infection of farm animals by worms.
The predominant type of antiparasitic screening effort over the years was the testing of synthetic compounds against nematodes, and some commercial products did result.
Certain antibiotics were also shown to possess antihelmintic activity against nematodes or cestodes, but these failed to compete with the synthetic compounds.
Although Merck had earlier developed a commercially useful synthetic product, thiabendazole, they had enough foresight to examine microbial broths for antihelmintic activity, and found a non-toxic fermentation broth that killed the intestinal nematode Nematosporoides dubius in mice.
The Streptomyces avermitilis culture, isolated by Ō mura and coworkers at the Kitasato Institute in Japan, 104 produced a family of secondary metabolites (eight compounds) with both antihelmintic and insecticidal activities.
These compounds, named 'avermectins,' are pentacyclic, 16-membered macrocyclic lactones, that harbor a disaccharide of the methylated sugar, oleandrose, with exceptional activity against parasites, especially Nemathelminthes (nematodes) and arthropod parasites (10 times higher than any known synthetic antihelmintic agent).
Surprisingly, avermectins lack activity against bacteria and fungi, do not inhibit protein synthesis and are not ionophores.
Instead, they interfere with neurotransmission in many invertebrates, causing paralysis and death by neuromuscular attacks.
105 The annual market for avermectins surpasses US$1 billion.
They are used against both nematode and arthropod parasites in sheep, cattle, dogs, horses and swine.
A semisynthetic derivative, 22,23-dihydroavermectin B1 ('ivermectin') is 1000 times more active than thiabendazole and is a commercial veterinary product.
The efficacy of ivermectin has made it a promising candidate for the control of human onchocerciasis and human strongyloidiasis.
106 Another avermectin, called doramectin (or cyclohexyl avermectin B1), produced by 'mutational biosynthesis' was commercialized for use by food animals.
107 A semisynthetic monosaccharide derivative of doramectin called selamectin is the most recently commercialized avermectin, and is active against heartworms (Dirofilaria immitis) and fleas in companion animals.
Although the macrocyclic backbone of each of these molecules (ivermectin, doramectin and selamectin) is identical, there are different substitutions at pharmacologically relevant sites such as C-5, C-13, C-22,23 and C-25.
108 The avermectins are closely related to the milbemycins, a group of non-glycosidated macrolides produced by S. hygroscopicus subsp.
Aureolacrimosus.
109 These compounds possess activity against worms and insects.
Coccidiostats are used for the prevention of coccidiosis in both extensively and intensively reared poultry.
Coccidiosis is the name given to a common intestinal disease caused by the invading protozoan parasites of the genus Eimeria that affects several different animal species (cattle, dogs, cats, poultry, etc.).
The major damage is caused by the rapid multiplication of the parasite in the intestinal wall and the subsequent rupture of the cells of the intestinal lining, leading to high mortality and severe loss of productivity.
Coccidia are obligate intracellular parasites that show host specificity; only cattle coccidia will cause disease in cattle; other species-specific coccidia will not.
For many years, synthetic compounds were used to combat coccidiosis in poultry; however, resistance developed rapidly.
A solution came on the scene with the discovery of the narrow-spectrum polyether antibiotic monensin, which had extreme potency against the coccidian.
110 Made by Streptomyces cinnamonensis, monensin led the way for additional microbial ionophoric antibiotics, such as lasalocid, narasin and salinomycin.
All are produced by various Streptomyces species.
They form complexes with the polar cations K + , Na + , Ca 2+ and Mg 2+ , severely affecting the osmotic balance in the parasitic cells and thus causing their death.
111 The widespread use of anticoccidials has revolutionized the poultry industry by reducing the mortality and production losses caused by coccidiosis.
Of great interest was another extremely valuable application of monensin; that is, growth promotion in ruminants.
Synthetic chemicals had been tested for years to inhibit wasteful methane production by cattle and sheep and increase fatty acid formation (especially propionate) to improve feed efficiency; however, they failed.
The solution was monensin, which became a major success as a ruminant growth enhancer.
110 For more than 40 years, certain antibiotics have been used in foodanimal production to enhance feed utilization and weight gain.
112 From a production standpoint, feed antibiotics have been consistently shown to improve animal weight gain and feed efficiency, especially in younger animals.
These responses are probably derived from an inhibitory effect on the normal microbiota, which can lead to reduced intestinal inflammation and improved nutrient utilization.
113 Pigs in the USA are exposed to a great variety of antibiotics.
These include b-lactam antibiotics (including penicillins), lincosamides and macrolides (including erythromycin and tetracyclines).
All these groups have members that are used to treat infections in humans.
In addition, bacitracin, flavophospholipol, pleuromutilins, quinoxalines and virginiamycin are utilized as growth stimulants.
Flavophospholipol and virginiamycin are also used as growth promoters in poultry.
As described above, cattle are also exposed to ionophores such as monensin to promote growth.
The Animal Health Institute of America 114 has estimated that without the use of growth-promoting antibiotics, the USA would require an additional 452 million chickens, 23 million more cattle and 12 million more pigs to reach the levels of production attained by the current practices.
Considering that animal health research and the development of new anti-infective product discovery have decreased, the discovery of new antibiotics has decreased over the past 15 years, with few new drug approvals.
115 Therefore, it will be incumbent on veterinary practitioners to use the existing products in a responsible manner to ensure their longevity.
It remains to be seen what effects the dearth of new antibiotics for veterinary medicine will have on the future practice of veterinary medicine, production agriculture, food safety and public health.
116 Since the 1999 EU decision to prohibit antibiotic use for foodanimal growth promotion, four antibiotic growth promoters have been banned, including the macrolide drugs tylosin and spiramycin.
117 Although macrolides are no longer formally used as 'growth promoters,' their use under veterinary prescription has risen from 23 tons in 1998 to 55 tons in 2001, which suggests that more of them are being used now than before the prohibition.
It is well known that the most effective route for feeding is via the gastrointestinal tract.
Many critically ill patients who accept early feeding improve their health.
In some post-operative patients, gastric stasis and excessive volumes in the stomach increase the risk of aspiration and subsequent pneumonia.
On account of the importance of achieving early and adequate nutritional intake, it is common practice in many intensive care units to use drugs to improve gastrointestinal motility.
Erythromycin is a macrolide antibiotic with a broad spectrum of activity.
It is well recognized that when prescribed, either intravenously or orally, it causes side effects, such as diarrhea, nausea and vomiting.
These side effects are, in part, due to the action of erythromycin at motilin receptors in the gut.
This makes this antibiotic very attractive to be used in ill patients with gastrointestinal motility problems.
There have been some developments on erythromycin analogs that lack antibiotic action but retain action at motilin receptors.
These have been named 'motilides.'
118, 119 Recently, an orally active erythromycinderived motilin receptor agonist (mitemcinal) has been tested in patients with idiopathic and diabetic gastroparesis.
In both cases, an improvement of gastroparetic symptoms was observed.
120 The 80-year contribution of microorganisms to medicine and agriculture has been overwhelming.
However, antibiotic resistance in microbes has created a dangerous situation and the need for new antibiotics is clear.
Unfortunately, most of the large pharmaceutical companies have abandoned the search for new antimicrobial compounds.
Owing to the economics, they have concluded that drugs directed against chronic diseases offer a better revenue stream than do antimicrobial agents, as for the latter the length of treatment is short and government restriction is likely.
Some small pharmaceutical and biotechnology companies are developing antibiotics, but most depend on venture capital rather than sales income, and with the present regulations, they face huge barriers to enter the market.
These barriers were raised with the best intentions of ensuring public safety, but will have the opposite effect if they terminate antibiotic development while resistance continues to increase.
121 However, there are some bright possibilities.
One of the most promising is the utilization of uncultivated microorganisms.
Considering that 99% of the bacteria and 95% of the fungi have not been cultivated in the laboratory, putting efforts into finding means to grow such microorganisms are proceeding and succeeding.
122 Furthermore, researchers are now extracting bacterial DNA from soil and marine habitats, cloning large fragments into, for example, bacterial artificial chromosomes, expressing in a host bacterium and screening the library for new antibiotics.
This metagenomic effort is allowing access to a vast untapped reservoir of genetic and metabolic diversity, 123, 124 which could result in the discovery of new and useful natural products.
125 In addition to these two relatively new techniques, the chemical and biological modification of old antibiotics could still supply new and powerful drugs.
These comments also apply to non-antibiotics such as antitumor agents and other microbial products.
Bioactive microbial metabolites.
A personal view Natural products in drug discovery and development Natural products and design: interrelated approaches in drug discovery Design of angiotensin converting enzyme inhibitors Antibiotics: where did we go wrong?
Natural and synthetic substances related to human health The future of cephalosporins business Infectious history Oceans: medicine chests of the future?
Inhibitors of HIV-1 replication that inhibit HIV integrase Medicinals for the millennia.
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cells converting compactin to pravastatin Production and purification of statins from Pleurotus ostreatus (Basidiomycetes) strains Evaluation and development of spinosyns to control ectoparasites on cattle and sheep Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes Research progress on microbial herbicides Weed control for the preservation of biological diversity Control of problem vegetation: a key to ecosystem management Metabolic products of microorganisms.
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key: cord-017208-7oew461e authors: Aurigemma, Rosemarie; Tomaszewski, Joseph E.; Ruppel, Sheryl; Creekmore, Stephen; Sausville, Edward A. title: Regulatory Aspects in the Development of Gene Therapies date: 2005 journal: Cancer Gene Therapy DOI: 10.1007/978-1-59259-785-7_29 sha: doc_id: 17208 cord_uid: 7oew461e Preclinical therapeutics development research is directed toward fulfilling two overlapping sets of goals.
A set of scientific goals includes defining the best molecule or biologic construct for the task at hand, and proving the case for its development.
The second set of goals addresses regulatory requirements necessary to introduce the agent into human subjects.
In the case of “small molecule” drugs, in most cases the identity of the molecule and appropriate safety studies are straightforward.
In contrast, the development of biologic agents, including gene therapies discussed here, presents distinct challenges.
The nature of the “drug” may be an organism subject to mutation or selection of variants through recombination.
Its properties may vary depending on the scale and method of its preparation, purification, and storage.
How to test adequately for its safety prior to first introduction in humans may not be straightforward owing to intrinsic differences in response to the agent expected in humans as compared to animals.
Preclinical therapeutics development research is directed toward fulfilling two overlapping sets of goals.
A set of scientific goals includes defining the best molecule or biologic construct for the task at hand, and proving the case for its development.
The second set of goals addresses regulatory requirements necessary to introduce the agent into human subjects.
In the case of "small molecule" drugs, in most cases the identity of the molecule and appropriate safety studies are straightforward.
In contrast, the development of biologic agents, including gene therapies discussed here, presents distinct challenges.
The nature of the "drug" may be an organism subject to mutation or selection of variants through recombination.
Its properties may vary depending on the scale and method of its preparation, purification, and storage.
How to test adequately for its safety prior to first introduction in humans may not be straightforward owing to intrinsic differences in response to the agent expected in humans as compared to animals.
The general principles, however, in allowing "first-in-human" experiences are similar for both small molecules and biologics.
The ethical conduct of clinical trials in patients with a dire or lifethreatening disease demands an understanding of the identity and dose of an agent that has the possibility of causing clinical benefit with adverse events expected at worst to be easily reversible and well predicted by the preclinical experience.
In normal volunteers or patients who are otherwise well, evidence should be gathered that would support an initial range of doses of the test agent expected to be without substantial toxicity or long-term effects.
Thus, the successful clinical introduction of a novel therapeutic concept requires an organized approach to integrate scientific, technical, and regulatory requirements.
This integration should begin in the research laboratory, as the concept becomes a candidate for the clinic, to prevent avoidable and expensive delays in clinical development.
For example, if a product is created using a mammalian cell line for which viral or other contamination has not been ruled out, costly rederivation will be required before that product can be manufactured for clinical trials using current good manufacturing practices (cGMP).
On the other hand, during the discovery phase, an excessive and premature concern over cGMP compliance can impede research.
Therefore, a clear strategic understanding of the principles underlying regulatory issues is desirable and is the goal of this chapter.
We proceed from the experience of the Developmental Therapeutics Program (DTP) of the National Cancer Institute in the manufacture of biologicals, including gene therapy constructs for preclinical and clinical use.
We outline the basis for our approach to safety testing studies to be included in an Investigational New Drug (IND) application to the Food and Drug Administration (FDA).
We focus on studies that would allow phase I and perhaps early phase II clinical trials.
In 2002, DTP contributed to over 40 different cGMP biological projects.
Most of these activities were selected competitively from applications received from academic researchers or from the intramural laboratories of the National Institutes of Health (NIH).
DTP products include viruses for vaccines or gene therapy, plasmids, monoclonal antibodies, recombinant proteins, synthetic peptides, natural product fermentations, and oligonucleotides.
During the 2002 fiscal year, over 30 different lots were manufactured and released under cGMP for clinical use or further cGMP manufacturing.
Most products are destined for phase I or phase II clinical trials in cancer.
Beyond early (phase I/II) clinical trials, technology transfer for some products has occurred, with further development through phase III now addressed by pharmaceutical companies.
Based on experience accumulated over several years, we abstracted the initial profiles of the more successful concepts (Table 1) , as well as some early project characteristics that can impede clinical development ( Table 2) .
We note correlations between the thoroughness of the early research, attention to "the rules," outlined in the references cited here, and the development of commercial interest in the product.
Gene therapy and other biologic therapeutics are regulated within the FDA by the Center for Biologics Evaluation and Research (CBER), which was created in 1972 to address products emerging from the new biotechnology.
Reorganization at the FDA is currently under way that will result in regulation of many biotherapeutics by the Center for Drug Evaluation and Research, which has oversight of small molecule drugs.
It is anticipated, however, that blood products, vaccines, and gene therapy products will remain with CBER.
The Biological Response Modifiers Advisory Committee is a chartered advisory group with the role of advising the FDA to ensure the safety and effectiveness of biological products, including gene therapy.
The Recombinant DNA Advisory Committee (RAC) also oversees gene therapy research through the NIH Office of Biotechnology Activities.
The RAC was established in 1974 in response to public concerns regarding the safety of recombinant deoxyribonucleic acid (DNA) technology.
Human gene transfer trials in which NIH funding is involved (either directly or indirectly) are to be submitted to the RAC for review.
Table 1 Beyond a Good Idea: What the Successful Investigator Has Already Done With a Project Leading to Commercial Development Defined candidate biologic (or molecule) Made comparisons with similar products Characteristics of product are consistent with pharmaceutical requirements Production scale is adequate Product characterization is adequate Laboratory reference standard exists In vitro potency assay has been developed Stability studies develop confidence product is a "drug" Reproducible model systems have confirmed in vivo activity with clinical product Early animal work includes some toxicology Scale-up requirements practical for initial clinical trials In general, reflects experience and scientific maturity of investigator In addition to the US agencies that develop the regulations that govern drug development and licensing, the International Conference on Harmonization (ICH) was formed in April 1990 involving the United States, the European Union, and Japan to address the issue of globalizing such regulations.
The ICH Steering Committee meets at least twice a year to continue their agenda of updating and harmonizing regulations for medicinal products; they emphasize safety, quality, and efficacy.
Expert Working Groups were formed within ICH to address specific topics related to these basic areas.
Although the FDA has not formally adopted all of the ICH guidelines, these guidelines should be followed when they exist in preliminary form.
For investigators planning to conduct investigational drug trials in foreign countries, it is imperative that they be familiar with, and adhere to, the regulations set forth by ICH.
In 1996-2001, a series of FDA and ICH guidance documents on characterization and preclinical safety evaluation of biotechnology-derived pharmaceuticals was developed (1) (2) (3) (4) (5) (6) (7) (8) .
These guidances represent the FDA's current thinking on preclinical safety evaluation of biotechnology-derived pharmaceuticals.
These are defined as products derived from characterized cells using a variety of expression systems, including bacteria, yeast, insect, plant, and mammalian cells.
The active substances may include proteins and peptides, their derivatives, and products of which they are components.
These materials could be derived from cell cultures or produced using recombinant DNA technology, including production by transgenic plants and animals.
Examples include cytokines, enzymes, fusion proteins, growth factors, hormones, monoclonal antibodies, plasminogen activators, recombinant plasma factors, and receptors.
The intended indications for use in humans may include in vivo diagnostic, therapeutic, or prophylactic uses.
The principles outlined in these guidance documents may also be applicable to recombinant DNA protein vaccines, chemically synthesized peptides, plasmaderived products, endogenous proteins extracted from human tissue, and oligonucleotide drugs.
The FDA defines gene therapy as "a medical intervention based on modification of the genetic material of living cells" (9) .
Cells may be modified ex vivo for subsequent administration to humans or may be altered in vivo by gene therapy given directly to the patient.
When the genetic manipulation is performed ex vivo on cells that are then administered to the patient, this is also considered a form of somatic cell therapy (9) .
"The genetic manipulation may be intended to have a therapeutic or prophylactic Table 2 Issues Requiring Attention at the Outset of a Project Inappropriate antibiotic selection markers (e.g., ampicillin for recombinant proteins) Lab-scale affinity purification Solubility problems Low yield Errors in genetic sequence Extraneous genetic material Poorly defined production systems Inadequate purification schemes Unvalidated or nonexistent in vitro potency assay(s) Lack of key reagents (e.g., antibodies to desired product) Poor biochemical characterization Inappropriate raw materials Raw material qualification problems Inappropriate cell banks Difficult or unidentified toxicology systems Failed vendor qualification Intellectual property concerns effect or may provide a way of marking cells for later identification.
Recombinant DNA materials used to transfer genetic material for such therapy are considered components of gene therapy and as such are subject to regulatory oversight".
Specific information related to gene therapy issues is contained in the 1998 "FDA Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy" (9) .
This guidance document updates and replaces the 1991 FDA "Points to Consider" on this subject (9a).
New information was intended to provide manufacturers with current information regarding regulatory concerns for production, quality control testing, and administration of recombinant vectors for gene therapy and of preclinical testing of both cellular therapies and vectors.
The FDA defines somatic cell therapy as "the administration to humans of autologous, allogeneic, or xenogeneic living non-germline cells, other than transfusable blood products, for therapeutic, diagnostic, or preventive purposes."
The evaluation of the safety of gene therapy products is perhaps one of the more difficult tasks that faces toxicologists in the drug development arena today.
Because many of the agents, like other biologicals, are species specific and because these agents integrate into the host genome, the choice of animal models and study designs is fraught with uncertainty, and each product frequently breaks new regulatory ground.
Until recently, many investigators working in this field were probably lulled into a false sense of security because of the close scrutiny that preclinical studies and clinical protocols received from the NIH RAC and the FDA.
With the death of Jesse Gelsinger, a patient enrolled in a gene therapy clinical trial to correct a metabolic disease, in 1999 and the recent reports of a leukemialike disease produced in children who received gene therapy treatments to correct severe combined immunodeficiency disease (SCID) (10) (11) (12) (13) , the safety of these agents is called into question more than ever.
As a result, the toxicologist is under even more pressure to design more rigorous safety evaluation programs.
There have been a number of reviews in this area in recent years by toxicologists from the FDA (14) , industry (15, 16) , and international workshops (17) that cover many of the fundamentals regarding safety evaluation of gene therapy products.
These resources, in conjunction with this chapter and the various guidance documents from the FDA and the ICH (7, 9, 18) , can be used by toxicologists to develop sound safety programs.
These issues are discussed in greater detail in the latter half of this chapter.
The basic foundation of regulations for drug development can be found in the Code of Federal Regulations, Title 21 Food and Drugs (21 CFR; (19) (20) (21) (22) (23) (24) (25) (26) .
In addition to Title 21, FDA maintains an extensive number of Web sites containing regulatory information that should be consulted during the development of a novel biotherapeutic.
The collection of available regulatory information includes Points to Consider, Guidance Documents, Drafts, and reports from public forums and symposia as well as information on the meetings of the Biological Response Modifiers Advisory Committee.
ICH also sponsors a Web site for obtaining the most recent guidelines.
A free subscription to an e-mail advisory update service is also available ( Table 3) .
In addition to the regulatory guidelines provided by the FDA, the NIH has published, and frequently updates, the NIH Guidelines for Research Involving Recombinant DNA Molecules, which can also be found electronically ( Table 3) .
Although published documents disseminated by the FDA and NIH are essential starting points for planning a cGMP development strategy, it is important to realize that, in this rapidly evolving field, some requirements may be reflected in public comments or a growing consensus among industry long before they are formally adopted.
Furthermore, it is not unusual during the development of a new biologic to have also developed alternatives to conventional practices that are based on sound scientific data and are then implemented after discussions with the FDA.
Product-specific factors can influence the regulatory requirements for an investigational agent.
These issues should be explored in detail with the FDA in a pre-IND meeting at which the IND sponsor presents relevant preclinical data and manufacturing and animal safety testing to support the proposed approach to clinical development.
The types of further studies pertinent to the particular agent can then be proposed, and input from the agency help shape the final development plan.
Interactions should take place with regulatory authorities at intervals that will facilitate the development of a product (Table 4) .
A key issue frequently not understood is that regulatory demands become more stringent as a product moves from phase I (safety), through phase II (activity), to phase III (comparative efficacy) trials and licensure (6, (27) (28) (29) (30) (31) .
This philosophy reflects the conscious effort not to stifle innovation in early phase clinical testing, but to ensure that, by the time registrationoriented late-stage trials are contemplated, issues related to production variability, assay, and assurance of safety are mature and well-substantiated because the results of such trials could be the basis for sale of the agent to the public.
Another factor that affects the level of regulatory compliance is the nature of the study population.
Products manufactured for Phase I trials in healthy normal volunteers typically must meet much stricter requirements than those studied in patients with dire, life-threatening conditions (e.g., cancer or end-stage acquired immunodeficiency syndrome).
As improved technology becomes available, requirements also tend to increase (27-31).
The level of regulatory compliance to be followed during different stages of development is dependent on the type of biologic product and the technology available for supporting its development.
Assays, methods, and technologies for monoclonal antibody development (32) , for instance, are better defined than the techniques available for some of the new virus vectors that are emerging.
Furthermore, new technologies to support product development are also constantly evolving.
The number of specific viral contaminant tests required of cGMP human cell lines, for example, has increased steadily as new pathogens are identified and assays become available.
As new scientific knowledge accumulates, novel regulatory challenges can appear.
The issue of transmissible spongiform encephalopathy, for example, has resulted in stringent requirements in raw material qualifications and traceability (33) .
To minimize the impact of regulatory changes, careful record keeping of all processes and materials involved in deriving the product is highly recommended.
Finally, because of the availability of improved techniques for characterizing certain biologicals, the FDA is reorganizing its regulatory approach in ways that are analogous to the regulation of small molecule drugs.
Technical demands will rise as regulatory requirements become more standardized.
Beyond identification and confirmation of an interesting novel concept, a major challenge in the preclinical development of biologicals is the optimal allocation of research and development resources.
Key to this is proper assessment of a candidate concept's readiness for clinical development.
All applicants for the National Cancer Institute's biologicals production resources now receive a list of "generic questions" corresponding to the appropriate product type.
At the beginning of a project, it is not always reasonable to expect all issues to be resolved, but the assumption is that, for a successful candidate, these issues should be in hand by the time the project is completing phase I clinical testing.
Table 5 lists the generic questions for cGMP production of recombinant virus vectors.
Because it is not possible to provide a complete guide to cGMP development in a few pages, we highlight some concerns common to many projects arising from academic laboratories.
These are based on DTP's experience (both successes and failures) with projects making the transition from the preclinical research phase to pilot clinical studies.
Our discussion is organized primarily around the concepts of identity, purity, potency, and safety that underlie development, manufacture, and release.
From the viewpoint of regulatory compliance, it is essential to establish the identity of the product and the components used to generate it during manufacturing (9, 22) .
We have noted that, frequently in proposed gene therapy or recombinant DNA-derived products submitted to us, DNA sequencing shows some deviation from the sequence published and/or submitted by the investigator, sometimes with major consequences for the project.
When the DNA product, such as plasmid vaccines, will be administered to the patient, full plasmid sequencing has occasionally revealed unacceptable genetic sequences outside the open reading frame, as passengers from previous experiments, spurious promoters, frame shifts resulting in translation of nonsense sequences beyond the intended termination, and so on.
DNA sequencing and repair are available at relatively low cost compared to the cost of repeating critical preclinical experiments.
The FDA now requires complete sequencing of vectors of sizes up to 40 kilobases (kb) (34) .
For viral vectors, genetic stability is a major concern, particularly with respect to the possible issues of recombination with generation of replication-competent viruses.
Specific guidelines are provided for adenovirus, retrovirus, and lentivirus vectors (9, 35, 36) .
For other virus vectors, specific assays (e.g., neurovirulence testing of recombinant poliovirus and herpes virus vectors) are required to ensure that an attenuated phenotype is preserved after scale-up.
If possible, the investigator should attempt to assess the genetic stability of the vector during preclinical studies, after administration in vivo or propagation in vitro.
In addition to the gene therapy product itself, the cells used to produce the product must be similarly identified and qualified for cGMP manufacturing.
Excellent guidance documents are available for the production of master cell banks, working cell banks, and master virus banks (9, 32, 37) .
At minimum, the complete cell history should be known and documented, and the cells should be tested to verify their origin.
Peptide sequencing or mapping employing liquid chromatography-mass spectrometry is typically used to provide critical information for synthetic peptides and recombinant proteins.
For recombinant vectors containing transgenes, the expression of the desired gene product should be verified, for example, by immunoassay using a specific antibody against the product.
A. Non-GMP for additional preclinical development B. cGMP (clinical grade) 2.
Provide details of molecular construct(s), including starting materials (e.g., plasmid, relevant vector maps, detailed vector construction scheme, and so on) 3.
Does the construct contain an antibiotic resistance gene or other selectable marker?
Are alternative methods of selection available?
Why was the proposed selection chosen?
4.
Is the vector replication competent or replication defective?
(For replication-selective vectors, what is the molecular basis of the selectivity and the conditions under which the vector would replicate?)
5.
Does the vector have an altered cell tropism?
Define.
What is the effect of altered tropism on anticipated host toxicity?
6.
Has this construct been sequenced?
Provide a sequence in an electronic format.
7.
Are data available evaluating the genetic stability of the recombinant vector?
Have mutation rates been established and/or rates of reversion to either wild-type or alternate viral genomes?
8.
Are data available evaluating the potential for genetic recombination with other organisms in the patient or in the environment?
9.
Is the organism currently grown in a qualified cGMP cell line?
If not, is there a qualified cell line available for propagation of this vector?
Was the cell line genetically modified to support this vector?
Provide details of its construction and any information regarding the stability of the genetic alteration in the cell line.
10.
Is there a cGMP-qualified virus seed bank?
11.
Provide details of the proposed production method.
12.
Has this material ever been produced for laboratory or clinical studies using this production system?
13.
Has this material been produced in a related or other production system?
If so, please provide the details.
14.
Please provide details of existing purification methods.
15 .
What is the average yield of the production system before and after purification?
What is the largest amount of material that you have produced in your laboratory in a single production batch?
Please provide average ratios obtained by this production method for virus particle/infectious unit and infectious units/cell.
How does this scale to anticipated quantities for clinical trial?
16.
How much material is available as a reference standard?
17.
Is material available as bulk biological substance for preliminary pharmacology and toxicology studies?
18.
Are there reproducible assays for the product?
Please provide the following assays, if available: A.
Identity B.
Purity C. Potency 19.
What are to be the release criteria for the product?
How does one know that a lot of product is qualified for use?
20.
In what form (lyophilized, formulated product, and so on) and fill size is the desired final product?
What is the desired final product formulation?
21.
Are there issues of formulation that must be resolved?
22 .
What is known about the product stability with respect to physical integrity and activity?
23.
Do you have any information regarding the estimated costs of this production project?
24.
Have you identified any possible sources of production with any commercial firms?
Please provide details.
25 .
Are there any safety issues connected with the production, purification, and/or handling of the product?
26.
What is the status of the product(s) regarding intellectual property issues?
27.
Sometimes, proposed projects are an improvement or modification of an existing approach.
In these cases, this information may significantly affect the analysis of feasibility, cost, and other production issues.
Please provide a brief summary of the nature of any such antecedents or other approaches that appear closely related to the proposed project.
28 .
Have there been any meetings scheduled with regulatory agencies, such as a pre-IND meeting with the FDA or a presentation to the NIH RAC?
If so, please indicate the type of meeting, the regulatory agency, and the date or proposed date.
29.
If you have had a pre-IND or RAC meeting, were any issues concerning manufacturing, safety, or stability raised that will have an impact on producing your product?
30. Who will sponsor the IND for the proposed study?
31.
Has a source of funding been identified for performing the clinical trial with this product?
Purification strategies depend on the nature of the biologic agent and expected impurities.
These approaches are guided by the early development of reliable analytical techniques appropriate to the product and to the manufacturing approach.
For example, purification of recombinant proteins and monoclonal antibodies for cGMP manufacture typically involve large-scale chromatography based on multiple isolation principles (e.g., charge, hydrophobicity, size, and so on).
Specific contaminants, such as DNA, endotoxin, viruses from mammalian cell production systems, contaminants introduced in the manufacturing process, and the like must be quantified and may require additional specific purification measures to remove or inactivate them.
Problems in refolding or solubility, tendencies to aggregate, and product stability at intermediate holding points can be significant issues in process development for scale-up.
These represent key challenges in scale-up from investigator laboratory-generated lots to a potentially suitable scale to allow clinical testing.
Additional concerns include subtle degradations of proteins that can lead to undesirable immunogenicity.
A major concern is the impact of each additional step on the downstream product, which should be reassessed using in vitro potency assays as well as physicochemical characterization.
At major development milestones, selected in vivo models should be reexamined using purified product.
Production cells must be cGMP qualified and tested for adventitious agents and other contaminants, before initiation of production as well as at end of production.
A number of cGMP-qualified cell lines and starting vectors are available commercially at relatively low cost and should be considered for use as raw materials to initiate cGMP seed banks in preference to shared materials of uncertain provenance despite the good intentions of the original provider.
In the handling of cell lines, care should also be taken to avoid contamination (e.g., from media components, trypsin, or activities taking place in nearby laboratory space).
Postproduction cells can be tested for specific contaminants in the presence of a viral product (e.g., using polymerase chain reaction [PCR]).
In the presence of virus product, however, it is unlikely that the full set of cGMP tests (e.g., cocultivation) for adventitious agents can be performed on postproduction cells.
Before initiating cGMP production, therefore, investigators should consider the parallel propagation of a mock-infected control to provide a surrogate postproduction test article.
In addition to the usual tests for sterility and purity of purified investigational product (9, 20, 21) , it is important to have an assay for residual host cell DNA.
Assays for host cell proteins are not always required for all phase I products, but are required for phase II and beyond.
This consideration is another reason to start with cGMP-qualified cell lines from a commercial source because host cell DNA and protein assays may already be available.
The general requirement for adventitious agent testing is given in guidance documents (e.g., ref.
9).
It is important to note that some specific assays are not yet described in published FDA documents, but can enter widespread cGMP practice by sponsor-based industry consensus, liability considerations, or other factors.
Endotoxin assays are available as kits, which are useful to guide laboratory process development.
A qualified good laboratory practice (GLP) laboratory, however, should perform endotoxin assays for clinical product release.
Specific assays may also be required to quantify process residuals from production and purification components (CsCl, antibiotics, and so on).
In production facilities, particularly those where different types of gene therapy products may be produced, assays are necessary to support decontamination and cleaning, product changeover, environmental monitoring, and raw material qualification (25, 38) .
In general, all equipment that has contact with the investigational product should be verified free of contaminants before use.
Special consideration must also be given to assays to qualify virus seeds and end product for the presence of defective particles, replication-competent viruses, or defective genomes.
In addition to monitoring for replication-competent and/or pathogenic vectors during manufacture, suitable assays may also be required to monitor patients receiving the therapeutic agent.
In this case, levels of sensitivity for detection must be suitable for different types of patient specimens (serum, urine, sputum, and so on).
Evolving requirements for long-term follow-up of gene therapy patients (39) should be consulted to ensure that proper assay support is maintained beyond the duration of the planned clinical trial.
The FDA regulations governing the performance of assays that support the production of biologics for human use can be found in 21 CFR 211, subpart I, "Laboratory Controls" (25) .
GLP regulations only per-tain to the performance of preclinical animal and in vitro studies (23).
The measured activity of a biological candidate depends on the hypothesized molecular mechanism of therapeutic action (21 CFR 600.
3; 9, 19) .
Although it is most reassuring to see in vivo demonstration of efficacy in appropriate animal models, the efficient development of a cGMP process will strongly benefit from the availability of rapid, reliable, and reproducible in vitro assays relating to the mechanism of action in addition to assays for purity and identity.
Assays based on the basic therapeutic mechanism, therefore, are critical goals of early research and development.
Formulation development should begin as early as possible as suitable assays become available and experience with real-time stability accumulates.
It is preferable to choose formulations from candidates likely to be acceptable to the FDA, such as those whose components are already used for licensed products.
As production reaches larger scales, handling and storage considerations become increasingly important.
Stability studies incorporate assays for identity, purity (including aggregation), and potency.
Although they can provide some useful information, accelerated stability studies are typically not reliable for predicting real-time stability of biologics.
Therefore, there is a need for real-time stability studies to be initiated as soon as possible.
Suitability of formulated product should also be assessed in the identical administration and handling conditions expected in the clinic.
This may include transient exposure to conditions expected during transit to the study site and storage in an environment that closely mimics study site conditions.
These may result in markedly different product behavior at the study site from that expected from the behavior of vouchered specimens at a central repository site.
The results of ongoing stability studies are useful to support process development; to evaluate product at intermediate hold points in scaleup production and at product release; and to support formulation development, product storage, shipment, and handling during toxicology studies and clinical applications.
As development proceeds, master specifications for release of intermediate and final product should be established and refined.
The IND must indicate a schedule for real-time stability studies to be performed throughout the duration of the clinical trial.
Some key early milestones common to all product areas include the attainment of an adequate scale of high-quality, single-batch production, the availability of adequate amounts of high-quality laboratory reference standard, and the development of reliable assays for identity, purity, and potency of the product.
These milestones are necessary in addition to the exploration of animal models showing safety and promising evidence of efficacy.
At early stages in a project, investigators should expect substantial variability in product quality, assays, and animal models.
Ideally, therefore, a single high-quality batch should be used to establish laboratory standards, support multiple assay qualification runs, and perform replicate animal model experiments.
Multiple production runs could then be performed to explore process development issues, including scale-up.
In this way, fundamental issues could be explored at the research stage to prepare for development required for cGMP manufacture.
Following this reasoning, our facility often manufactures high-quality GLP lots to provide a uniform supply of material for additional preclinical research and development for selected biologics of interest before making the decision to undertake cGMP production.
The early establishment of certain aspects of GLP (21 CFR 58) is crucial to the advancement of a drug development project.
By following simple rules of laboratory cleanliness, documentation, and segregation of materials and activities at the start of development, time can be saved by avoiding the necessity to duplicate results that were not properly performed or documented from the outset.
At the discovery phase, development of reliable assays to explore basic therapeutic mechanisms of action are just as important as the performance of animal models in laying the groundwork for future cGMP product development.
Laboratory facilities and staff should be adequate to perform necessary studies.
Assay protocols should be specific and reproducible.
Research documentation should be kept at a GLP level with complete and secure laboratory notebooks.
Records of all reagents (i.e., manufacturer, catalog number, lot number, Certificates of Analysis [COAs] , and expiration dates) should be routinely archived.
Even if cGMP production or testing is not contemplated in the development laboratory, fluctuations in product activity are not unusual during later scale-up, and these materials and information may be useful in resolving such issues.
Key assays for product or reagent identity should be repeated at appropriate intervals.
Access to critical raw materials and reagents, reference standards, and cell banks should be limited.
Staff should avoid comingling of research-grade, GLP, and GMP activities or reagents by labeling reagent containers and sequestering them as much as possible.
Similarly, signs should be posted on dedicated equipment, and access should be limited as appropriate.
If common equipment must be used, standard operating procedures should be developed to define the use and control of such equipment, to clean equipment before and after use to avoid cross-contamination, and to document the use, cleaning, and calibration of the equipment.
Critical raw materials (e.g., those used in seed development or pilot product manufacture) must be traceable to their source and obtained from reliable vendors.
It is beneficial when possible to use vendors subjected to commercial audits.
Animal-derived reagents should be avoided; reagents such as glycerol, detergents, proteins, amino acids, and the like should preferably come from vegetable sources.
If this is not possible, animal-derived reagents should come from acceptable herds in countries without endemic or questionable transmissible spongiform encephalopathy (33) .
It is important to ensure that raw materials are stored under appropriate conditions and not used beyond their expiration date.
Inventories and logbooks should be used to track use of important reagents.
Critical materials requiring special storage conditions should be stored at more than one location to prevent loss in the event of equipment failure.
cGMP-qualified cell lines should be purchased from vendors if possible, but if cGMP-qualified cell lines do not exist, cell lines should be obtained directly from a reliable repository source such as the American Type Culture Collection (ATCC) and documentation should be archived.
Incoming cell lines must be tested for sterility and mycoplasma contamination.
Thorough records should be kept on cell passages, observations, frozen storage, and the COAs from media and other components used to grow, freeze, and otherwise manipulate the cells.
Critical cell lines should be segregated to prevent cross-contamination.
Stock cells should not be cultured in incubators containing virus-infected cell lines.
Vectors should be purchased from a reliable vendor, and documentation should be kept on the propagation, storage, and use, including COAs, lot numbers, and so on of the reagents used to propagate the vector.
If the vector is acquired from another laboratory (i.e., is unavailable for purchase from a vendor), a detailed history should be obtained of the generation of the vector, and detailed records should be kept from that time.
All genetic manipulations of the vectors should be well documented and verified by sequencing.
A lab-generated reference standard is a critical raw material for a biologic.
It is ideal to have a large enough stock of this reference standard to use for the duration of the development work.
It is often not possible, however, to produce sufficient quantities or material of sufficient stability at the early development stages.
For this reason, it may be necessary to produce fresh batches and to test them thoroughly against independent standards or the current standard before that standard is depleted or loses potency.
The same consideration should be given to other critical reagents, such as cell lines and compounds obtained from outside sources.
Reference standards and key reagents should be made or obtained in adequate amounts, characterized as well as possible, and stored under conditions that will maintain stability for at least the duration of the development process.
As improved manufacturing and assay processes are developed, improved reference standards will be required, but quantities of the original standards should also be preserved to provide material for later comparisons as required (40) .
In some cases, such as for retrovirus and adenovirus vector development, the FDA has made available reference material against which all sponsors can standardize their own reference reagents.
To avoid future questions about data reliability, investigators should consider outsourcing of difficult but common technologies, such as transmission electron microscopy, tandem liquid chromatography mass spectroscopy, peptide mapping, or DNA sequencing, if these are not adequate in their facility.
Product-specific assays, such as potency studies and pilot animal efficacy and toxicity studies, are likely to be performed best at the researcher's own facility, early in development.
Preclinical assay protocol development and record keeping must ensure that data are useful for later IND submission.
At some point in cGMP process development, consideration should be given to technology transfer of critical assays to a GLP-compliant laboratory prepared to support the repetitive studies required during cGMP process development, manufacture, release, and postrelease stability studies for the duration of the clinical trial.
Toxicology material should ideally be manufactured using the same process used to manufacture the cGMP clinical material.
Therefore, a toxicology lot is produced late in process development.
If there are concerns over batch-to-batch variability, production of a single lot for both toxicology and the initial phase I trial is recommended.
Typically, a toxicology lot can be available several months before the clinical lot is ready for release.
For studies involving autologous cells, the handling of cells must be under GMP conditions to preserve sterility and prevent cross-contamination with other cells.
For allogeneic cells, it is important to use a cGMP-tested cell line with adequate traceability, including its origin, passage history, and exposure to media products that may have been derived from animal sources.
Starting material must be routinely checked for sequence accuracy; therefore, the complete plasmid should be sequenced.
It is also preferable to examine genetic stability that can lead to the introduction of coding errors and changes in protein expression.
The use of penicillin-like antibiotics (b-lactams) for selection is unacceptable because of the possibility of allergic reactions in patients administered products produced using this selection system.
Other antibiotics, such as kanamycin, are substituted, or alternative methods of selection are employed.
Measures of DNA quality include supercoiled content, as well as assays for endotoxin, genomic DNA, and other contaminants from the production system.
More in-depth guidance is available through guidance documents (9).
It is generally recommended by the FDA that a vector smaller than 40 kb must be completely sequenced (34) .
As technology improves, this criterion may well be expanded to include larger vector genomes.
Those vectors with genomes larger than 40 kb (e.g., herpes viruses, poxviruses) must have the transgene sequenced along with 5' and 3' flanking regions and any significant modifications to the vector backbone or sites vulnerable to alteration during molecular manipulation.
When qualified vaccine strains exist for the vector of interest (e.g., vaccinia, poliovirus) it is preferable for cGMP manufacture to derive investigational constructs using the vaccine strain if available from the NIH, ATCC, or commercial sources.
For adenovirus vectors, the recent availability of an adenovirus reference standard allows for the normalization of dosing based on virus particle concentration and infectious unit (IU) titer.
Current recommendations by the FDA are for a ratio of viral particle to infectious unit of less than or equal to 30:1 (35) .
For replication-defective adenoviruses, generation of replication-competent adenoviruses (RCA) must be measured in lots produced for clinical use.
The current target requirement is fewer than 1 RCA per 3 ´ 10 10 virus particles as measured by a cell culture/cytopathic effect method (35) .
For viruses that are replication selective, different testing strategies (e.g., quantitative PCR) may be called for and should be discussed with the FDA.
Similarly, the agency may have special considerations for viruses with altered tropism to ensure appropriate containment and prevent the generation of a replicationcompetent adenovirus with an expanded cell tropism.
It should be noted that RCA assays must be optimized regarding the presence of defective particles and other factors that may affect the sensitivity of the assay.
Retroviruses are of special concern because of the possibility of insertional carcinogenesis.
This potential safety problem is amplified if replication-competent retroviruses (RCRs) are generated (9, 36) .
The general guideline is to test at least 5% of the total virus vector supernatant produced by amplifying any RCR on a permissive cell line.
In addition, 1% of the producer cells or 10 8 (whichever is less) must also be tested at the end of production by the method of coculturing on permissive cells (36) .
As with adenovirus vectors, retrovirus vectors with tropism modifications are of special concern and may require more stringent containment and patient follow-up (39) .
Promoter modifications may also affect the safety profile of these virus vectors.
Lentiviruses generally have the same safety concerns as retroviruses, particularly because they can replicate in a broader variety of cells (dormant as well as actively dividing cells).
Although there is a retrovirus standard available through ATCC to investigators who are developing retrovirus vectors, there is no lentivirus standard currently offered.
A lentivirus standard is not planned for the future primarily because of the great variability in lentivirus backbones currently under development for clinical investigations (e.g., equine, murine, human).
Herpes viruses under development for clinical use either must be replication defective or, if replication competent, must be shown to be nonneurovirulent.
Neurovirulence is an issue for poliovirus as well.
Adeno-associated virus (AAV) vectors are of concern because, although these vectors are designed to be maintained episomally, there can be reversion to wild type, resulting in integration into the host chromosome, or the vector could be rescued in a patient with a concurrent adenovirus infection (41) .
Several interesting concepts seek to employ modified bacteria as the therapeutic agent.
As with recombinant viruses, general issues of safety as well as specific issues of genetic stability and exchange should be considered.
Stabilization of the new genetic material may be required by incorporation into the bacterial genome rather than through a plasmid that can be lost or exchanged.
Strategies to incorporate new genetic material into bacterial DNA will depend on confirming the sequence accuracy of the target bacterial sequences as well as the novel genetic material.
Introduction of an antibiotic resis-tance gene through a manufacturing step raises special concerns and can be avoided using alternative selection approaches.
Whether evaluating small molecules or biologically derived materials such as gene therapy products, the basic intent of nonclinical toxicity studies is to define the pharmacological and toxicological effects predictive of the human response, not only prior to initiation of phase I clinical trials in humans, but also throughout the entire drug development process leading ultimately to Biologics License Application (BLA).
The goals of these studies include, first, to define an initial safe starting dose and dose escalation schemes for first-in-human clinical trials; second, to identify potential target organs for toxicity, biomarkers or other parameters that can be monitored in patients receiving these therapies, and to determine if this toxicity is reversible; and finally, to determine which patient populations may be at greater risk for developing toxicity to a given cellular or gene therapeutic product (42) .
These nonclinical studies should be designed with the following points in mind: whether the product is transduced cells, the population of cells to be administered, or the class of vector used; the most appropriate animal species and physiological state of that model most relevant for the clinical indication and product class; and the intended doses, route of administration, and treatment regimens that will be used in the clinic.
Many of the questions that need to be taken into consideration and addressed during the design phase for safety studies include what is already known about the most likely toxicities related to the agent's biodistribution, local as well as systemic toxicity, immune responses (immunogenicity and immunotoxicity), the potential for insertional mutagenesis, and biological activities of the transgene product.
Then specific questions that arise with the new product or use are addressed.
For example, are the safety issues primarily related to the vector, the transgene product, the method of administration, the formulation/excipient, or some combination of the above?
How might existing published or unpublished nonclinical or clinical data address the questions mentioned above?
Safety issues that should be addressed in these studies include evaluation of the toxicity of the vector alone (irrespective of the transgene), including its potential toxicity and/or tumorigenicity (in some cases, this is apparent from previous evaluations with the same vector); toxicity of transgene expression in vivo that may not be evident from in vitro studies; occurrence and consequences of ectopic transgene expression in nontargeted tissues; occurrence and consequences of immune responses to transgene or vector proteins such as autoimmunity; and finally the possibility of germline transduction (34) .
Because conventional pharmacology and toxicity testing as typically used for the evaluation of small molecules may not always be appropriate to determine the safety and biologic activity of cellular and gene therapy products, issues such as species specificity of the transduced gene, permissiveness for infection by viral vectors, and comparative animal to human physiology should be considered in the design of these studies.
Available animal models mimicking the disease indication may be useful in obtaining both sufficient safety and efficacy data prior to entry of these agents into clinical trials.
Early pre-IND discussions with the FDA during development of a toxicology plan may prevent delays and added expenses because of inadequate data or the use of inappropriate species.
Some of the questions that should be answered by preclinical pharmacology/toxicology studies are the following (43) : What is the relationship of the dose to the biologic activity?
What is the relationship of the dose to the toxicity?
Does the route and/or schedule affect activity and/or toxicity?
What risks can be identified for the clinical trial?
For ex vivo gene transfer, the product is considered to be the transduced cells.
The general safety test (21 CFR 610.11) must be performed on the final product.
When appropriate, modified procedures may be developed according to 21 CFR 610.9.
The FDA is considering proposed rule making to amend the general safety test rules and scope of applicability, especially for cell therapy products (9) .
Finally, it is expected that these nonclinical toxicity studies will be conducted in compliance with GLP regulations.
However, there will be situations in which highly specialized assays will be required because of the nature of biotechnology-derived products, and it will not be possible to conduct these assays in full compliance with GLPs (e.g., in university or other discovery laboratories).
It will be important that these areas be identified for any impact that they may have on the interpretation of toxicity data.
In most cases, carefully performed studies such as this can be used to support INDs and BLAs (7).
When selecting the animal model that will be used in the various preclinical biodistribution, pharmacology, and toxicology studies, consideration should be given to the scientific rationale for the animal species used.
For example, would there be an advantage to performing the studies in rodents when larger numbers of animals might be more practical, or is there a necessity for a large animal model, such as a canine or nonhuman primate?
If nonhuman primate studies are proposed, is it clear that another large animal or rodent model would not provide the same information?
Would there be any utility in a genetically deficient model, and would this deficient model be more relevant to the proposed study either because of the potential for adverse immunologic consequences or because of the biological effects in the deficient condition?
Animal models of disease may not be available for every cellular or gene therapy system proposed for development.
This makes species selection an even more difficult process.
Preclinical pharmacological and safety testing of these agents should employ the most appropriate, pharmacologically relevant animal model available.
A relevant animal species might be one in which the biological response to the therapy mimics the human response.
This entails some knowledge of the pathophysiology of the disease in humans and of how faithfully it is reproduced in the animal model.
The species of animal chosen for preclinical toxicity evaluations of viral preparations should be selected for its sensitivity to infection and production of pathologic sequelae induced by the wild-type virus related to the chosen vector, as well as its utility as a model of biologic activity of the vector construct.
There should be a reasonable expectation of a similar distribution of receptors or permissivity in the animal model as there is in humans.
Thus, the species utilized may vary with the vector administered, the transgene expressed, the route of administration, the patient population treated, and the disease studied.
Rodent models rather than nonhuman primates may be useful if they are susceptible to pathology induced by the virus class (e.g., cotton rats are semipermissive hosts for adenovirus infections) (44) ; the use of the SCID mouse (45) or the cotton rat (46) may be suitable for the evaluation of herpes simplex virus (HSV) rather than the Aotus monkey.
Some investigators have also suggested the use of miniature swine for evaluation of adenoviral vectors (47, 48) .
When evaluating the activity of a vector in an animal model for the clinical indication, safety data can be gathered from the same model to assess the contribution of disease-related changes in physiology or underlying pathology to the response to the vector.
Some specialized circumstances illustrating these points follow.
The inbred cotton rat (Sigmodon hispidus) has been used extensively as an animal model for research since the 1940s, when it was first used in poliomyelitis research.
Since that time, it has been shown to be a semipermissive host for adenoviral infection (44, 49) .
In those studies, it was shown that the pulmonary lesions and replication pattern of the virus seen in the cotton rat paralleled those seen in humans.
Virus persisted in the nasal mucosa and lung for up to 21 and 28 days, respectively, after inoculation.
This was even in the presence of high-titer neutralizing antibody that was detected by day 7.
Although cotton rats have readily adapted to the laboratory environment, they have retained a number of the characteristics of their wild counterparts.
These animals have a tendency to bite, panic when handled, jump out of their cages, and have a large fight-or-flight zone.
Care and handling of these animals have been described by other investigators (50,51) .
The cotton rat has been used for the evaluation of numerous adenovirus vectors by many routes of administration, and some of these studies are described here.
When early E3-deleted adenoviral vectors were evaluated in the cotton rat, it was discovered that the E3 region was not required for replication, but that this region plays a critical role in the pathogenesis of the disease in that these mutants induced a markedly greater lymphocyte and macrophage/monocyte inflammatory response in the lungs (52) .
E3 replacement recombinants were significantly less pathogenic than E3-deleted viruses after intranasal administration (53) .
This study also demonstrated that adenovirus replicated in BALB/c and CBA mice and produced results that were similar to those seen in cotton rats.
The intracranial administration of a replication-defective adenovirus expressing the herpes simplex virus thymidine kinase (HSVtk) gene at a dose of 1.0 ´ 10 9 pfu into both adenoviral immune and adenoviral naïve cotton rats resulted in only mild gliosis and trace meningitis along the injection tract and approximated a "no toxic effect" dose (54) .
When this same vector was administered to either Wistar rats or rhesus monkeys, direct neuronal injury or a dose-related inflammatory response was seen at the injection site and in the surrounding parenchyma.
There was no apparent injury to tissues not of the central nervous system in any of the three models, and all cerebral spinal fluid, blood, urine, and stool samples failed to culture for adenovirus.
In a study with a similar HSVtk adenovirus inoculated into cotton rats via intracardiac injection at doses up to 3 ´ 10 10 viral particles per animal with and without ganciclovir (GCV), the only significant microscopic lesions observed were epicardial inflammation and splenic hemosiderosis (55) .
Vector sequences persisted throughout the 14-day assay period in the heart, lung, and lymphoid organs.
Infectious virions were detected for 24 hours, but these virions were only detected at the site of injection of two animals in the highest dose group.
When a similar vector was administered as either one or two subcutaneous injection cycles with 2.3 ´ 10 12 viral particles/kg each or as a single course with 6.9 ´ 10 13 viral particles/kg, the only significant treatment-related histopathological finding was dermatitis with mild acanthosis at the site of vector injection (56) .
In addition to these local effects, mild hyperamylasemia, lymphocytosis, and granulocytosis were seen clinically, but no other clinical signs of toxicity or death were observed.
Vector sequences were detected in the skin at the injection site and to a lesser extent in the liver, spleen, and lungs, and small amounts of vector DNA were detected in the ovaries.
These were cleared rapidly, and the absence of viral sequences in the excreta and swabs of the majority of animals suggested that there was no significant replication of this adenovirus vector in this host and little shedding.
The owl monkey (Aotus trivirgatus or nancymae) has been an excellent model for oncogenic and nononcogenic viruses such as HSV type 1 (HSV-1) and others (57) , and the herpes virus that infects these animals is a strain of HSV-1 (58).
These animals have been routinely used to test vaccines against HSV-1 and found to mimic the course of the disease in humans (59,60).
As a result, it was only natural that these animals be used to evaluate the safety of gene therapy vectors produced from HSV-1.
However, these animals tend to be more fragile to use than other species and as a result must be handled with greater care.
G207, an attenuated, replication-competent HSV-1 recombinant, was tested for safety after intracerebral inoculation in the Aotus (61).
These animals received doses of either 10 7 or 10 9 pfu of G207 or 10 3 pfu of the wild-type HSV-1 strain F. Wild-type HSV-1 caused rapid mortality and symptoms consistent with HSV encephalitis, including fever, hemiparesis, meningitis, and hemorrhage in the basal ganglia.
For up to 1 year after G207 inoculation, seven of the treated animals were alive and exhibited no evidence of clinical complications, indicating that this form of HSV was considerably attenuated in comparison to wild-type virus.
Two animals were reinoculated with 10 7 pfu of G207 at the same stereotactic coordinates 1 year after the initial dose.
These animals were alive and healthy 2 years after the second inoculation.
As a further, more comprehensive clinical evaluation, animals were subjected to cerebral magnetic resonance imaging (MRI) studies both before and after G207 inoculation.
These studies failed to reveal radiographic evidence of the typical HSV-related sequelae in the brain seen in the animals treated with the wild-type virus.
Microscopic examination of multiple tissues found no evidence of HSV-induced histopathology or dissemination in spite of the fact that measurable increases in serum anti-HSV titers were detected.
Viral shedding and biodistribution in the Aotus were also evaluated using PCR analyses and viral cultures of tear, saliva, or vaginal secretion samples (62) .
Neither infectious virus nor viral DNA was detected at any time-point up to 1 month postinoculation.
Analyses of tissues obtained at necropsy at 1 month or 2 years after inoculation showed the distribution of G207 DNA was restricted to the brain, although infectious virus was not isolated in these samples.
The safety of this construct was also evaluated in the Aotus after intraprostatic injections (63) .
Safety was assessed on the basis of clinical observations, viral biodistribution, virus shedding, and histopathology.
None of the injected monkeys displayed evidence of clinical disease, shedding of infectious virus, or spread of the virus into other organs.
No significant microscopic abnormalities were observed in the organs evaluated.
The results of these studies demonstrated that G207 can be safely inoculated into either the brain or the prostate, and that the Aotus monkey could be successfully used in preclinical toxicological evaluations.
In addition to the studies performed with this vector in Aotus monkeys, BALB/c mice were also used to evaluate the safety of G207.
Mice were inoculated in the same manner as the Aotus either intracerebrally or intraventricularly with 10 7 PFU of G207 and survived for over 20 weeks with no apparent symptoms of disease.
In contrast, over 80% of animals inoculated intracerebrally with 1.5 1 0 3 pfu of HSV-1 wild-type strain KOS and 50% of animals inoculated intraventricularly with 10 4 pfu of wild-type strain F died within 10 days.
When mice were inoculated intrahepatically with G207 (3 ´ 10 7 pfu), all animals survived for over 10 weeks, whereas no animals survived for even 1 week after inoculation with 10 6 pfu of wild-type KOS (64) .
Mice were also injected in the prostate with either G207 or wild-type HSV-1 strain F and observed for 5 months (63) .
None of the G207-injected animals exhibited any clinical signs of disease or died.
However, 50% of mice injected with strain F displayed sluggishness and hunched behavior and were dead by day 13.
On microscopic examination, the prostates injected with G207 were normal, whereas those injected with strain F showed epithelial flattening, sloughing, and stromal edema.
These studies and those described by Whitley with the SCID mouse (45) point to the fact that rodents can be used in place of the owl monkey and produce adequate safety data for the evaluation of HSV-1 vectors for gene therapy.
Finally, safety data can also be obtained in well-designed efficacy studies.
In many cases, mouse studies can provide similar information as studies conducted in nonhuman primates, so smaller species should not be automatically rejected.
The nonhuman primate should not be relied on for use as a model simply because of the comfort of going into studies in humans only after evaluation of the toxicity of the agent in nonhuman primates.
Experience has repeatedly shown for numerous classes of agents, both small molecules as well as biologicals, that no one species may be predictive of all human toxicities, and that not all human toxicities may be seen in other animal species (65) .
Finally, certain human populations may not be predictive of all other human populations.
This last fact makes predicting each and every toxicity almost impossible.
The doses of vectors used in nonclinical studies should be selected based on preliminary efficacy/ activity data from both in vitro and in vivo studies.
A no-effect dose level, an overtly toxic dose, and several intermediate doses should be evaluated, along with appropriate controls, such as naïve or vehicle-treated animals.
For new formulations, it is very important to include this last group to distinguish formulation-related effects from those of the agent of interest.
When products are difficult to produce in large quantities and as a result are in limited supply or for products with an inherently low toxicity, a maximum tolerated dose may not be achievable; as a result, a maximum feasible dose may be administered as the highest level tested in the preclinical studies and should be so designated in appropriate reports.
Although this may not be intellectually or scientifically satisfying, the data derived from such a study should at least establish the safety of the clinical starting dose.
Preclinical safety/ toxicity studies should include at least one dose that is equivalent to and at least one dose escalation level exceeding those proposed for the clinical trial.
The multiples of the human dose required to determine adequate safety margins may vary with each class of vector employed, and the relevance of the animal model to humans and the rationale for dose selection should be provided.
Scaling of doses based on either body weight or total body surface area as appropriate facilitates comparisons across the animal species used and humans.
Although most small molecule cancer therapeutics are scaled based on body surface area (66), body surface area may not be appropriate for gene therapeutics.
Information generated in the preclinical studies can be used to determine the margin of safety of the vector for use in the clinical trial, as well as gage an acceptable dose escalation scheme depending on the steepness of the toxicity curve.
In a cross comparison of doses for an adenoviral vector for cystic fibrosis (14), very similar toxicities were seen in cotton rats, mice, hamsters, rhesus monkeys, and baboons when the agent was directly instilled into the lungs of the animals.
When the doses were scaled for body surface area, the no observable adverse effect levels for the various species were remarkably similar to one another and to the first human dose at which toxicity was observed, 0.4-2.4 ´ 10 9 IU/m 2 vs 1.2 ´ 10 9 IU/m 2 for humans.
The only notable exception was the rhesus monkey at 4.6 ´ 10 7 IU/m 2 .
Studies like this enable other investigators to make wiser choices in the selection of doses and species to evaluate.
The route of administration of vectors can have an obvious influence on toxicity in vivo because of the distribution and concentrations of the agent that are produced.
For example, intravenous bolus doses can produce very high concentrations for short durations; other routes of administration, such as subcutaneous, may produce much lower concentrations and more prolonged exposure.
Current practice recommends that safety evaluations in preclinical studies should be conducted by the identical route and method of administration as that proposed for the phase I clinical trial whenever possible.
When this is difficult to achieve in a small animal species, a method of administration similar to that planned for use in the clinic is advised.
For example, intrapulmonary instillation of adenoviral vectors by intranasal administration in cotton rats or mice is an acceptable alternative to direct intrapulmonary administration through a bronchoscope because the latter procedure is simply not feasible in rodents.
If the proposed clinical route is a nonintravenous (e.g., intratumoral), it may be wise also to conduct an intravenous study to provide perhaps "worse-case" data for what may happen in the event of an accidental injection directly into a patient's blood vessel.
When possible, the schedule of administration in the animal studies should also be identical to that intended in the phase I clinical trial.
This may not be feasible in certain instances because of the production of neutralizing antibodies in the animal model that might preclude repeated administration; that may not be a factor in humans.
In studies in which additional agents will be administered in combination with the gene therapy agent (e.g., in suicide therapy using HSVtk and GCV or HSV cytosine deaminase and 5-fluorocytosine), the route and schedule should also be identical to that planned for the clinic.
Evaluating the vector alone in animal models would not provide sufficient data for predicting additional toxicity that may be produced by the combination, but should be at least one arm of the planned preclinical animal studies.
At a minimum, treated animals should be monitored for general health status (clinical observations, body weight and temperature changes, changes in food and water consumption), serum biochemistry, and hematological profiles.
Target organs and other critical tissues should be examined for gross and microscopic changes.
The addition of other parameters to be evaluated will depend on the nature of the product studied, the species used, and the route of administration.
There is no set of all-inclusive parameters that is sufficient for each and every new agent.
Studies should be designed specifically for each agent, utilizing the most appropriate tests to capture as much relevant data as possible.
Because many biotechnology-derived pharmaceuticals intended for human use will be immunogenic in animals, the use of animal-derived proteins/products, if available, should be considered to define the intrinsic toxicity of the new agent.
This may entail parallel development processes in which a construct relevant to the species in the safety test is developed to a point to allow a most relevant safety test to proceed.
The analogous human construct then may actually be brought into the clinic supported by these results.
If human material is used, measurement of antibodies associated with administration of products should always be performed when conducting repeated dose toxicity studies.
These data will assist the investigator in the interpretation of the results of these studies.
Antibody responses produced in animals should be fully characterized (e.g., titer, number of responding animals, neutralizing or nonneutralizing), and their appearance should be correlated with any pharmacological and/or toxicological changes observed.
More specifically, the effects of antibody formation on pharmacokinetics and/or pharmacodynamics, incidence and/or severity of adverse effects, complement activation, or the emergence of new toxic effects should be considered when interpreting the data.
Attention should also be paid to the evaluation of possible pathological changes related to immune complex formation and deposition, especially in the kidney of treated animals.
The detection of antibodies in animals should not be the sole criterion for the early termination of a preclinical safety study or modification of the duration of the study unless the immune response neutralizes the pharmacological and/or toxicological effects of the biopharmaceutical in a large proportion of the animals.
In most cases, the immune response to biopharmaceuticals in animals will be variable, similar to such responses in humans.
If these issues do not compromise the interpretation of the data from the safety study, then no special significance should be ascribed to the antibody response.
The induction of antibody formation in animals is not necessarily predictive of a potential for antibody formation in humans.
By the same token, humans may also develop serum antibodies against humanized proteins, and frequently the therapeutic response persists in their presence.
The same may happen in animals if a purified protein is administered via a gene therapy viral vector.
In the case of human factor IX, when the purified protein was administered to rhesus macaques, the monkeys did not make antibodies (67) .
However, when factor IX was administered in a first-generation adenoviral vector, the animals mounted an acute phase response that produced neutralizing antibody that eliminated factor IX from the circulation (68) .
Finally, the occurrence of severe anaphylactic responses to recombinant proteins is rare in humans.
The results of guinea pig anaphylaxis tests, which are generally positive for protein products, are not considered predictive for reactions in humans; therefore, studies such as this are considered of little value for the routine evaluation of these types of products even though they are frequently performed.
Inflammatory, immune, or autoimmune responses induced by the gene product may be of concern.
Animal studies should be conducted over a sufficient duration of time to allow development of such responses.
Host immune responses against viral or transgene proteins may limit their usefulness for repeated administration in the clinic.
The immune status of the intended recipients of a gene therapy should be considered in the risk-benefit analysis of a product, particularly for viral vectors.
If exclusion of immunocompromised patients would unduly restrict a clinical protocol, immune-suppressed, genetically immunodeficient, or newborn animals may be used in preclinical studies to evaluate any potential safety risks.
It is extremely important to investigate the potential for undesirable pharmacological activity in appropriate animal models and, when necessary, to incorporate particular monitoring for these activ-ities in nonclinical toxicity studies and/or clinical studies.
Safety pharmacology studies are designed to measure functional indices of potential toxicity.
These indices may be investigated in separate studies or may be carefully incorporated into the design of nonclinical toxicity studies.
The aim of these studies should be to reveal any functional effects on the major physiological systems of the body (e.g., cardiovascular, respiratory, renal, and central nervous systems) that will have a major impact on whether or how the agent is administered in the clinic.
Some of these investigations may include the use of isolated organs or other test systems that do not involve the use of intact animals, such as the use of a perfused rabbit heart model for the evaluation of torsade de pointes and QT prolongation (69, 70) .
The results from all of these safety pharmacology studies may allow a mechanistically based explanation of specific organ toxicities, which should be considered carefully with respect to human use and intended indications.
The use of additional biomarkers, exemplified by cardiac troponin T or I (71,72) for agents with potential cardiac toxicity, may be warranted in additional nonclinical animal studies and/or in clinical studies in humans.
Pharmacology studies can be divided into three main categories, depending on the nature of the effect: primary and secondary pharmacodynamic studies and safety pharmacology studies.
Safety pharmacology studies are defined in the ICH guidance document (S7A) on this subject (18) "as those studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above."
This last point is particularly important in that these studies should be conducted at dose levels or serum concentrations that are therapeutic targets based on prior efficacy/activity studies.
Simply conducting these studies at low doses does not provide much useful information or adequately assess the safety of the agent.
The objectives of these studies are to identify undesirable pharmacodynamic properties of a drug substance that may have relevance to its human safety and toxicity; to evaluate more fully adverse pharmacodynamic and/or pathophysiological effects of a drug substance that were previously observed in nonclinical toxicology and/or clinical studies; and to investigate the mechanism of action of the adverse pharmacodynamic effects that were either previously observed or suspected.
The investigational plan developed to meet these objectives should be clearly identified and delineated by the drug development team.
For biotechnology-derived products that achieve highly specific receptor targeting, it is often sufficient to evaluate safety pharmacology end-points as a part of well-designed toxicology and/or pharmacodynamic studies; therefore, the need for separate safety pharmacology studies can be reduced or eliminated.
For those bioproducts that represent a new therapeutic class and/or those products that do not achieve highly specific receptor targeting, a more extensive evaluation in separate safety pharmacology studies should be considered.
Biodistribution studies are generally performed for gene therapy products, and typical pharmacokinetic studies used for most types of drugs that measure serum or plasma levels, half-life, clearance, and the like are generally not performed.
These preclinical animal biodistribution studies are designed to determine the distribution of the vector to sites other than the intended therapeutic site as an indicator of potential toxicity.
The goals of these studies are generally twofold: determination of dissemination of the vector to the germline and distribution of vector to nontarget tissues.
The first has been routinely accomplished by assaying total gonadal tissue.
The second provides information on potential target organs of toxicity.
Both may be addressed in the same preclinical study.
Studies may use normal, intact animals or animal models of disease.
The latter study may be more representative of the clinical setting.
Whenever possible, the intended route of administration should be employed, again with the consideration that groups of animals might also be treated intravenously as a worst-case scenario.
Transfer of the gene to normal, surrounding, and distal tissues as well as the target site should be evaluated using the most sensitive detection methods possible, such as reverse transcriptase PCR, and should include evaluation of gene persistence.
When aberrant or unexpected localization is observed, additional studies should be conducted to determine whether the gene is expressed and whether its presence is associated with any pathologic effects.
Biodistribution studies may not be necessary for all new agents (73) .
With "previously defined" vectors, if there is previous experience with a similar vector, route of administration, formulation, and schedule (e.g., adenovirus type 5 vectors), if the transgene product is considered "innocuous" if expressed ectopically, and when the size of the new vector is not essentially different, biodistribution aspects of the prior agent may be referenced.
On the other hand, studies may not be postponed if a new class of vector is used (i.e., there is little or no previous experience; e.g., AAV, lentivirus, others); if there is a change in the formulation (i.e., lipid carrier instead of an aqueous formulation); if the route of administration is changed to an intentional systemic route from local administration of the "established" vector; and finally, if the transgene has the potential to induce toxicity if it is aberrantly expressed in nontarget organs.
As with toxicity studies, there are a number of factors that should be taken into consideration when designing vector biodistribution studies.
Regarding species selection, nonhuman primates are not always needed.
Rodents may be perfectly acceptable.
The animal gender should reflect the intended patient population.
At least 3-5 animals per sex and group should be used as a minimum.
The use of smaller animals (i.e., mice or rats) allows the inclusion of larger numbers of animals and the easy evaluation of more time points.
As in other studies, the following dose groups should be included when practical: controls, the maximally feasible/clinically relevant dose, and a lower dose for establishment of the no observable adverse effect level.
The route of administration should mimic intended clinical route to the greatest extent possible.
Regarding animal sacrifice and/or sampling time points, an early point that reflects peak vector transduction/expression should be included, as should a later timepoint determined by intended clinical use and a time-point that should reflect clearance from the gonads and nontarget organs to determine persistence.
The following tissues are generally recommended: peripheral blood; gonads; injection site; highly perfused organs (to assist in determination of toxicity) such as brain, liver, lung, kidneys, heart, spleen; other tissues based on toxicity/pathology as determined by transgene (e.g., bone marrow); and those based on the route of administration, such as draining, contralateral lymph nodes.
The methodology used to detect the agent should detect a sequence of the vector DNA (or ribonucleic acid) that is unique to that product and should be appropriate to detect the vector sequence adequately in tissue samples from both preclinical animal studies and samples obtained during the initial clinical trials.
Many of the points presented and discussed in this section are elaborated in publicly accessible FDA documents (43,73).
Shedding of viral vectors through the skin or in excreta is of obvious concern with highly infectious viruses.
To measure the dissemination, persistence, and shedding of these vectors, multiple tissues (e.g., brain, heart, lungs, spleen, liver, kidneys, ovaries, and skin) as well as bodily fluids such as urine, feces, tears, saliva, vaginal secretions, and skin swabs are taken at multiple time-points throughout the study and analyzed by real-time quantitative PCR for the presence of vector sequences.
If the vector sequences are rapidly cleared and viral sequences are absent in the excreta and swabs of the majority of animals, this suggests that there was no significant replication of the vector in the host (56,62,74).
Even if the intended clinical schedule involves repeated doses, single-dose studies may generate useful data to describe the relationship of dose to systemic and/or local toxicity and the steepness of the dose/toxicity curve.
Data from these studies can be used to select doses for repeated-dose toxicity studies.
Information on dose-response relationships may be gathered through the conduct of a single-dose toxicity study or as a component of pharmacology or animal model efficacy studies.
The incorporation of safety pharmacology parameters in the design of these studies should be considered, which will reduce the number of animals used, the amount of product required, and the number of studies that must be performed.
The route and dosing regimen for these studies (e.g., daily vs intermittent dosing) should reflect the intended clinical use or exposure (e.g., once a week for 3 weeks, every other day, etc.).
A recovery period should be included to determine the reversal or potential worsening of pharmacological/ toxicological effects and/or the potential for delayed toxic effects.
For biopharmaceuticals that induce prolonged pharmacological/toxicological effects, recovery group animals should be monitored until reversibility is demonstrated.
This may not be fundamentally obvious at the outset of the study.
The duration of repeated dose studies should be based on the intended duration of clinical exposure and disease indication.
This duration of animal dosing has generally been 1-3 months for most biotechnology-derived pharmaceuticals, but this probably will not be the case for most gene therapy studies.
However, in the case of life-threatening diseases such as cancer, longer term studies are generally not required to support phase I trials.
One aspect of immunotoxicological evaluation includes the assessment of potential immunogenicity as described in Section 8.4.
Many biotechnology-derived pharmaceuticals are intended to stimulate or suppress the immune system and therefore may affect not only humoral, but also cell-mediated immunity.
Inflammatory reactions at the injection site may be indicative of a stimulatory response.
It is important, however, to recognize that simple injection trauma or specific toxic effects caused by the formulation vehicle may also result in toxic changes at the injection site.
In addition, the expression of surface antigens on target cells may be altered, which has implications for autoimmune potential.
For conventional small molecule drugs, reproductive toxicity is usually assessed in rats and rabbits.
The species specificity and potential immunogenicity of biologicals has led to the increased use of nonhuman primates for this purpose.
The need for reproductive and developmental toxicity studies will depend on the product, clinical indication, and intended patient population.
The specific study design and dosing schedule may be modified based on issues related to species specificity, immunogenicity, biological activity, and/or a long elimination half-life.
The issue of germline integration has prompted considerable public discussion (75) .
For gene therapy products directly administered to patients, the risk of vector transfer to germ cells should be seriously considered.
Animal testicular or ovarian samples should be analyzed for vector sequences by the most sensitive method available.
If a signal is detected in the gonads, further studies should be conducted to determine if the sequences are present in germ cells as opposed to stromal tissues; techniques used may include, but are not limited to, cell separations, in situ PCR, or other techniques.
Semen samples for analysis can be collected from mature animals, including mice, by well-established methods (76, 77) for determination of vector incorporation into germ cells.
Evaluation of biodistribution to the gonads may not be needed prior to all phase I clinical trials, and this issue should be considered carefully in pre-IND meetings with the FDA.
The Informed Consent Form should address the lack of data and the unknown risks.
Genotoxicity studies, such as the Ames salmonella assay, the micronucleus test, and the mouse lymphoma assay, which are routinely conducted for small molecule pharmaceuticals, are not appli-cable to biotechnology-derived pharmaceuticals, especially gene therapy products, and therefore are not needed.
The administration of large quantities of peptides, proteins, or viruses may yield uninterpretable results.
When there is cause for concern about the product, genotoxicity studies should be performed in available and relevant systems, including newly developed systems.
The use of standard genotoxicity studies as indicated for assessing the genotoxic potential of process contaminants is not considered appropriate.
If standard assays are performed for this purpose, the rationale should be provided.
Standard 2-year carcinogenicity bioassays in normal mice and rats are generally inappropriate for biotechnology-derived pharmaceuticals and probably more so for gene therapy products.
This issue has received additional attention owing to the emergence of a lymphoproliferative syndrome in a potentially significant fraction of recipients of a vector designed to treat SCID syndrome (10, 11) .
This clinical result actually recapitulates to a certain degree toxicities anticipated from experience in animal models (78) .
Thus, product-specific assessment of carcinogenic potential will still be needed for biopharmaceuticals, and studies utilized must be refined after consideration of the duration of anticipated clinical dosing, the patient population, or the biological activity of the product (e.g., retrovirus vectors, growth factors, immunosuppressive agents, etc.).
When there is a concern about carcinogenic potential, a variety of new approaches may be considered to evaluate this risk.
When the potential to support or induce proliferation of transformed cells and clonal expansion leading to neoplasia is considered possible, the product should be evaluated with respect to receptor expression for the biopharmaceutical or for the transgene's expressed form in various malignant and normal human cells, especially those potentially relevant to the patient population under study.
The ability of the biopharmaceutical to stimulate growth of normal and/or malignant cells expressing the relevant receptors should be determined.
When in vitro studies such as this give cause for concern about carcinogenic potential, further studies in relevant animal models may be needed if these are available and relevant.
As stated in this section, when gene transfer agents must be evaluated, the standard rodent models (mice and rats) and the 2-year carcinogenicity bioassay are probably not appropriate.
Daily administration of vector as is usually performed in these studies is not feasible; however, several of these vectors, including AAV, continue to express over the lifetime of the animal.
The other factor that may be limiting is that the host immune response to the vector or to the transgene may either limit the toxicity, perhaps because of the development of neutralizing antibodies, or may have effects on tumor development.
It will be necessary to consult with the FDA to develop product-specific studies on an individualized basis or to determine whether and which carcinogenicity studies are needed.
Local tolerance to administration of the new agent should be evaluated.
The formulation intended for the clinical trial should be tested unless there is a cogent reason why this would not be feasible or biologically meaningful.
In most cases, the potential adverse effects of the product at the site of administration can be evaluated in the single-or repeated-dose toxicity studies that are usually conducted in the normal course of development, thus eliminating the need for separate studies.
Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types both in vitro and in vivo, resulting in a high level of transient gene expression.
Considerable modifications have been made in the wild-type virus to reduce infectivity and toxicity in normal tissues or to improve transduction or tropism for tumor cells.
The death of Jesse Gelsinger because of several complications, including liver failure, coupled with the fact that adenovirus infections in immunocompromised oncology patients can lead to fatal hepatotoxicity (79, 80) , and reports of serious hepatotoxicity and death in nonhuman primates treated with different adenoviral vectors make the safety evaluation of these vectors for cancer treatment paramount.
When a first-generation adenovirus vector expressing human factor IX was intravenously injected into rhesus macaques at doses from 1 ´ 10 10 to 1 ´ 10 11 pfu/kg, no toxicity was seen at the lower dose level, but substantial, dose-limiting liver toxicity was observed at the higher dose (68) .
This hepatotoxicity was manifested as elevated serum transaminase levels, hyperbilirubinemia, hypoalbuminemia, and prolongation of clotting times.
All evidence of liver toxicity resolved except for persistent hypofibrinogenemia in the high-dose recipient, indicating possible permanent liver damage.
These data suggested a very narrow therapeutic window for this first-generation adenovirus-mediated gene transfer vector.
In follow-up studies (81) , it was concluded that these abnormalities may be caused by direct toxic effects of the adenovirus vector itself, or may result indirectly from the accompanying acute inflammatory response marked by elevations in interleukin 6.
When another first-generation adenoviral vector expressing b-galactosidase was intravenously injected into two baboons at doses of 1.2 ´ 10 12 or 1.2 ´ 10 13 particles/kg, the baboon receiving the high dose developed acute symptoms, decreased platelet counts, and increased liver enzymes and became moribund at 48 hours after injection; the baboon receiving the lower dose developed no symptoms (82) .
Again, a very narrow therapeutic index was demonstrated.
Recombinant adenoviruses infused into the portal vein of adult rhesus monkeys at a dose of 10 13 particles/kg resulted in the formation of neutralizing antibody, severe liver toxicity, and death.
Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response (83) .
The administration of several gene transfer vehicles and routes was studied in rhesus monkeys to develop a model for adenovirusmediated gene transfer for liver.
Vectors administered via the portal vein or saphenous vein were efficient, but this resulted in transient gene expression and was accompanied by an immune response to both vector and transgene products and acute hepatitis (84) .
Turning to models of intracerebral administration, baboons received intracerebral injections of either a high dose of a replication-defective adenoviral vector expressing HSVtk (1.5 ´ 10 9 pfu) with or without GCV or a low dose of ADV/RSVtk (7.5 ´ 10 7 pfu) with GCV to evaluate the safety of this regimen.
Animals receiving the high-dose vector and GCV either died or became moribund and were sacrificed during the first 8 days of treatment.
Necropsy of these animals revealed cavities of coagulative necrosis at the injection sites.
Animals that received only the high-dose vector were clinically normal; however, lesions were detected with MRI at the injection sites corresponding to cystic cavities at necropsy.
Animals that received the low-dose vector and GCV were clinically normal and exhibited small MRI abnormalities, and although no gross lesions were present at necropsy, microscopic foci of necrosis were present.
Neutralizing antibodies were produced in the animals, but no shedding of the vector was found in urine, feces, or serum 7 days after intracerebral injection (74) .
Intrapulmonary administration uses are exemplified through the use of recombinant adenovirus vectors containing expression cassettes for human cystic fibrosis transmembrane conductance regulator, which were instilled through a bronchoscope into limited regions of lung in baboons.
The only adverse effect noted was a mononuclear cell inflammatory response within the alveolar compartment of animals receiving doses of virus that were required to induce detectable gene expression.
Minimal inflammation was seen at 10 7 and 10 8 pfu/mL, but at 10 9 and, more prominently, at 10 10 pfu/mL, a perivascular lymphocytic and histiocytic infiltrate was seen (85).
Host immune elimination of infected cells often limits gene expression in vivo to 1-2 weeks after infection (86, 87) .
In addition to a cell-mediated immune response to the adenovirus infection, a humoral response to the injected virus is often generated (88) .
Although this humoral response may prevent the use of adenoviral vectors for repeated dosing, it may be blocked or reduced by coadministration of immunosuppressive agents or cytokines.
Alternatively, the use of adenoviruses of different serotypes may allow for repeated administration, even in the presence of neutralizing antibodies (88) .
Harvey et al.
(89) reported on 6 years of experience with the local administration of low (<10 9 particle units) and intermediate (10 9 to 10 11 particle units) doses of E1 -/E3adenovirus vectors to six different sites.
With a group incidence of only 0.7% for major adverse events and no deaths related to administration of the adenovirus vectors, local administration of low and intermediate doses of adenovirus vectors was well tolerated.
Second-generation adenoviral vectors, mutated in E2a, have been proposed to decrease host immune responses against transduced cells, reduce toxicity, and increase duration of expression as compared with first-generation vectors deleted only in E1.
The safety of and E1-, E2a-, E3-deleted adenoviral vector (Av3H82) encoding an epitope-tagged B-domain-deleted human factor VIII complementary DNA was evaluated in cynomolgus monkeys.
Animals received intravenous administration of either 6 ´ 10 11 or 3 ´ 10 12 particles/kg.
Vector distribution was widespread, with the highest levels observed in liver and spleen.
Histopathology, hematology, and serum chemistry analysis identified the liver and blood as major sites of toxicity.
Transient mild serum elevations of liver enzymes were observed, along with a dose-dependent inflammatory response in the liver.
In addition, mild lymphoid hyperplasia was observed in the spleen.
Mild anemia and a transient decrease in platelet count were observed, as was marrow hyperplasia and extramedullary hematopoiesis (90) .
When vectors deleted in E1 and containing either a temperature-sensitive mutation in the E2a gene or a deletion of the E4 region were infused into the hepatic artery of nonhuman primates, minimal toxicity was seen.
Histopathology showed that portal inflammation was present throughout both livers in the animals receiving the high dose.
No differences were seen in the level of portal inflammation in targeted and untargeted lobes.
PCR analysis detected viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector.
In baboons treated with lower doses of an E1-E4-deleted vector expressing the human ornithine transcarbamylase gene, DNA was detectable by nested PCR in liver, but not gonads, at days 29 and 61.
The data suggested that intraarterial administration of recombinant adenoviral E1-E4-deleted vector is feasible and safe.
(91) .
Toxicity of first-generation and E2a-deleted vectors expressing human a1-antitrypsin was evaluated in C3H mice after administration of increasing doses starting at 1 ´ 10 12 particles/kg.
Both vectors induced dose-dependent toxicity, including transient thrombocytopenia, elevated alanine aminotransferase, and increased hepatocyte proliferation, followed by inflammation and then hypertrophy.
There were no differences in toxicity between the two vectors when measured at matching levels of human a1-antitrypsin expression.
However, the E2a-deleted vector had slightly reduced hepatocyte toxicity at an intermediate particle dose (92) .
Although these vectors are purported to be less toxic, the fact remains that the human fatality that occurred in the ornithine transcarbamylase deficiency trial at the University of Pennsylvania was an E1, E4-deleted construct (93).
The current E1-deleted adenoviruses can infect a wide variety of cells through a specific interaction between the viral fiber protein and at least one cell surface receptor.
Entry of the virus into the cell is further enhanced through a specific interaction of the fiber with an integrin "coreceptor."
The host's range of tissue susceptibilities to the virus can therefore be altered by various strategies so that it can bind more efficiently to the target cell surface (94) (95) (96) .
Antibodies against tissue-specific cell surface proteins can also be coupled to the fiber protein to facilitate partial targeting of the virus (97) .
Another approach to achieve "targeting" of the virus is the use of cell-specific promoters to drive expression of a therapeutic gene in the context of the recombinant virus (98) .
Enhanced uptake strategies through fiber modification may present special concerns for toxicity, especially regarding hepatotoxicity when administered by an intravenous or direct hepatic artery injection.
Careful comparison of a tropism-modified adenoviral vector to the nontropism-modified vector in mouse toxicity and biodistribution studies as well as nonhuman primate and toxicity studies might be desirable.
Members of the family Parvoviridae AAVs are among the smallest of the DNA viruses (99) .
Unlike autonomous parvoviruses, AAVs or dependo-viruses require coinfection with unrelated helper viruses for a productive infection to occur (100, 101) .
As recombinant vectors for gene therapy, they seem to have several advantages compared to other vectors, such as the transduction of terminally differentiated and nondividing cells (102, 103) , relatively high stability of transgene expression (104) , and the potential for targeted integration (105, 106) .
From a safety point of view, AAV vectors show a lack of pathogenicity (107-109), low immunogenicity (104, 110) , and low risk of insertional mutagenesis (111) .
Also, there did not appear to be any evidence of transduction in the gonads of rhesus monkeys (112) .
However, AAV has a limited DNA capacity.
HSV vectors can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression are obvious obstacles to their use.
They also have the advantages of the abilities to infect nondividing cells and to establish latency in some cell types.
The ability to establish latency in neuronal cells makes HSV an attractive vector for treating neurological disorders such as Parkinson's and Alzheimer's diseases.
In addition, the ability of HSV to infect efficiently a number of different cell types, such as muscle and liver, may make it an excellent vector for treating nonneurological diseases.
One problem associated with HSV-based vectors has been the toxicity of the vector in many different cell types.
The generation of HSV vectors with deletions in many of the immediate early gene products, which is similar to the strategy used for adenovirus, has resulted in vectors with reduced toxicity and antigenicity as well as prolonged expression in vivo (60) (61) (62) (63) (64) (65) .
No clinical study has been reported in detail with these vectors.
Section 8.1.2. details a summary of preclinical safety considerations pertaining to use of the Aotus monkey in comparison to rodent species.
Retrovirus vectors are replication-defective and are primarily based on the Moloney murine leukemia virus (MMLV), which is a well-studied and well-characterized retrovirus (113, 114) with numerous advantages.
They have been extensively studied, produce stable integration into the host genome, and are very efficient at gene transfer.
Disadvantages include an infection that is limited to dividing cells, which makes gene transfer into nondividing cells such as hematopoietic stem cells, hepatocytes, myoblasts, and neurons an impossibility, and low titer of products.
There are four theoretical concerns that exist for retroviral-mediated gene transfer that relate to two potential delayed toxicities.
These are insertional mutagenesis, recombination with endogenous retroviral sequences, transfer of exogenous genetic material, and accidental exposure to replicationcompetent murine retroviruses (115) .
Because retroviral vectors can permanently integrate into the genome of the infected cell, there is a serious concern regarding insertional mutagenesis causing the development of a secondary malignancy.
The presence of RCRs is of major concern because of the fact that RCRs have produced lethal malignant T-cell lymphomas in 3/10 rhesus monkeys (78) .
These concerns resulted in a publication concerning the FDA considerations on these issues (116) and the issuance of a new FDA guidance on this subject in October 2000 (36) .
Some of these con-cerns are no longer theoretical.
The elation that this type of retroviral-mediated therapy was successful in curing a number of children with SCID (117) has been severely dampened by the reports of a leukemialike disease produced in two of these children (10-13).
Unlike oncoretrovirusus such as Moloney murine leukemia virus, one subclass of retroviruses, the lentiviruses, can infect nondividing cells.
This makes these viruses attractive for gene transfer.
One of these viruses, human immunodeficiency virus (HIV), has been the subject of investigation by a number of groups.
The most obvious concerns with using HIV for gene therapy is safety and the possible generation of replication-competent virus during vector production.
This involves engineering the vector so that it is replication defective.
This has been done in a number of cases by eliminating all accessory genes, such as tat, vif, vpr, vpu, and nef, from a packaging construct that still has the ability to transduce cells (118) .
Concern about the possibility of insertional activation of cellular oncogenes by a random integration of the vector provirus into the host genome has led to the development of self-inactivating vectors (119) (120) (121) (122) .
The use of self-inactivating viruses significantly improves the biosafety of HIV-derived vectors because it reduces the likelihood that RCRs will originate during vector production and target cells and hampers recombination with wild-type HIV in an infected host.
In an attempt to make even safer constructs, other groups are working on the development of lentiviral vectors from HIV-2 (123), simian immunodeficiency virus (124), bovine immunodeficiency virus (125, 126) , and feline immunodeficiency virus (127) .
These last vectors may be inherently more acceptable because they are not based on HIV-1.
None of these newer constructs has moved toward the clinic, so there is little animal safety data and no human data on these vectors yet.
This chapter presents a range of issues that might be considered in contemplating the development of a gene therapy agent to the point of an early phase clinical trial.
As no gene therapy product has yet been recognized as "safe and effective," the standard approach to these issues should be regarded very much as a "work in progress."
Indeed, the nature of these agents would suggest that each new opportunity would call for its own unique set of requirements, so that a single approach will probably never "standardly" exist.
Rather, the principles that underlie regulatory policy should be woven into the approach to each new agent.
In broad strokes, these involve approaches to answering the following questions: Is the identity of the agent clearly defined?
Can successive batches of the material be made reproducibly in the quantity to support clinical development, and how is this known?
Are the biological features of the vector, and its transgene when applicable, clearly similar in the animal species used for safety studies and in the human, at least as far as this can be ascertained?
What dose is likely to be required for therapeutic effect?
What level of gene expression or replication is necessary to attain a therapeutic effect?
When toxicity occurs because of the agent, what is the evidence the toxicity will be reversible?
Is toxicity after repeated doses of agent likely to be attenuated or magnified by immunological response to the agent?
What are the consequences of long-term presence of the therapeutic agent in the recipient?
Is there a danger of producing directly (as the therapeutic agent itself) or indirectly (through recombination and/or replication) an infectious agent that acts horizontally in the population or vertically across generations?
How will the presence and distribution of the gene therapy agent be followed in the patient?
Sponsors are above all encouraged to see the regulatory process as a collaborative interaction with the regulatory agencies with the end not only of protecting the patient, but also of advancing the most scientifically defensible and rigorous questions to clinical trial.
Far more costly than the conduct of experiments designed to be compliant with regulatory requirements is a failed or overtly injurious clinical trial.
A clear understanding and a proactive approach in addressing regulatory issues outlined here will maximally ensure the likelihood of an interpretable clinical outcome.
The regulatory issues outlined here must be approached with continuing appreciation of the evolving science associated with the gene therapy field.
As such, requirements may evolve with the state of the science, and careful sustained contact with the regulatory agencies is important in incorporating the best and most current science into the design, conduct, and interpretation of regulatory studies.
ICH guidance on quality of biotechnology/biological products: derivation and characterization of cell substrates used for production of biotechnical/biological products ICH: final guideline on quality of biotechnological products: analysis of the expression construct in cells used for production of r-DNA derived protein products ICH guidance on specifications: test procedures and acceptance criteria for biotechnological/biological products ICH Guidance for Industry Q1A (R2), stability testing of new drug substances and products ICH guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin ICH guidance: Q7A good manufacturing practice guidance for active pharmaceutical ingredients ICH guidance for industry: S6 preclinical safety evaluation of biotechnology-derived pharmaceuticals ICH: final guidance on stability testing of biotechnological/biological products FDA guidance for industry: guidance for human somatic cell therapy and gene therapy Gene therapy: a tragic setback A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency Regulators split on gene therapy as patient shows signs of cancer Second cancer case halts gene-therapy trials Preclinical development strategies for novel gene therapy products The role of the toxicologic pathologist in the preclinical safety evaluation of biotechnologyderived pharmaceuticals Nonclinical safety evaluation of biotechnologically derived pharmaceuticals Non-clinical safety studies for biotechnology-derived pharmaceuticals: conclusions from an international workshop ICH guidance to industry: S7A safety pharmacology studies for human pharmaceuticals Code of Federal Regulations, Title 21, Food and Drugs, Part 610.10, Subpart B, General biological products standards; general provisions; potency Code of Federal Regulations, Title 21, Food and Drugs, Part 610.12, Subpart B, General biological products standards Code of Federal Regulations, Title 21, Food and Drugs, Part 610.13, Subpart B, General biological products standards Code of Federal Regulations, Title 21, Food and Drugs, Part 610.14, Subpart B, General biological products standards Code of Federal Regulations, Title 21, Food and Drugs, Part 58, Good laboratory practice for nonclinical laboratory studies Current good manufacturing practice in manufacturing, processing, packing, or holding of drugs; general Code of Federal Regulations, Title 21, Food and Drugs, Part 211, Current good manufacturing practice for finished pharmaceuticals Code of Federal Regulations, Title 21, Food and Drugs, Part 312, Investigational new drug application INDs) for phase I studies of drugs, including well-characterized, therapeutic, biotechnology-derived products FDA guidance for industry: IND's for phases 2 and 3 studies of drugs, including specified therapeutic biotechnology-derived products, chemistry, manufacturing and controls content and format FDA guidance for industry: content and format of chemistry, manufacturing, and controls information and establishment description information for a vaccine or related product FDA guidance for industry for the submission of chemistry, manufacturing, and controls information for a therapeutic recombinant DNA-derived product or a monoclonal antibody product for in vivo use FDA guidance for industry: formal meetings with sponsors and applicants for PDUFA products FDA points to consider in the manufacturing and testing of monoclonal antibody products for human use FDA letter to manufacturers of biological products: recommendations regarding bovine spongiform encephalopathy (BSE) FDA Biological Response Modifiers Advisory Committee: current policy on sequence characterization of gene transfer products FDA Biological Response Modifiers Advisory Committee: adenovirus titer measurements and RCA levels FDA guidance for industry: supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients in clinical trials using retroviral vectors FDA points to consider in the characterization of cell lines used to produce biologicals FDA gene therapy patient tracking system final document FDA guidance concerning demonstration of comparability of human biological products, including therapeutic biotechnology-derived products Third national NIH gene transfer safety symposium: safety considerations in the use of AAV vectors in gene transfer clinical trials Basic principles of gene therapy: basic principles and safety considerations Preclinical animal models in gene therapy research A new animal model for human respiratory tract disease due to adenovirus Use of Aotus monkey to assess neurovirulence of replication-selective herpes vectors Herpes simplex type 1 infects and establishes latency in the brain and trigeminal ganglia during primary infection of the lip in cotton rats and mice Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus Porcine toxicology studies of SCH 58500, an adenoviral vector for the p53 gene Pathogenesis of adenovirus type 5 pneumonia in cotton rats (Sigmodon hispidus) The cotton rat in biomedical research Handling the cotton rat for research Role of early region 3 (E3) in pathogenesis of adenovirus disease Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self limiting inflammatory response Distribution, persistency, toxicity, and lack of replication of an E1A-deficient adenoviral vector after intracardiac delivery in the cotton rat Subcutaneous administration of a replication-competent adenovirus expressing HSV-tk to cotton rats: dissemination, dersistence, shedding, and pathogenicity.
Hum The owl monkey (Aotus trivirgatus) as an animal model for viral diseases and oncologic studies Characterization of four herpesviruses isolated from owl monkeys and their comparison with Herpesvirus saimiri type 1 (Herpesvirus tamarinus) and herpes simplex virus type 1 Immunization of experimental animals with reconstituted glycoprotein mixtures of herpes simplex virus 1 and 2: protection against challenge with virulent virus In vivo behavior of genetically engineered herpes simplex viruses R7017 and R7020.
II.
Studies in immunocompetent and immunosuppressed owl monkeys (Aotus trivirgatus) Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation of intracerebral injection in nonhuman primates Viral shedding and biodistribution of G207, a multimutated, conditionally replicating herpes simplex virus type 1, after intracerebral inoculation in Aotus Preclinical safety evaluation of G207, a replication-competent herpes simplex virus type 1, inoculated intraprostatically in mice and nonhuman primates Attenuated, replication-competent herpes simplex virus type 1 mutant G207: safety evaluation in mice Concordance of the toxicity of pharmaceuticals in humans and in animals Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man The rhesus macaque as an animal model for hemophilia B gene therapy Adenovirus-mediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity Experimental models of torsade de pointes Drug-related torsade de pointes in the isolated rabbit heart: comparison of clofilium, d,l-sotalol and erythromycin Elevations in cardiac troponin measurements: false false-positives: the real truth Predicting cancer therapy-induced cardiotoxicity: the role of troponins and other markers Preclinical considerations for gene transfer clinical trials: vector biodistribution Adenoviral-mediated thymidine kinase gene transfer into the primate brain followed by systemic ganciclovir: pathologic, radiologic, and molecular studies Meeting summary (available on-line at: www4.od.nih.gov/oba/rac/summaries/3-99sum.htm.)
and meeting minutes RAC Minutes-03/11-12/99.
Available on-line at: www4.od A technique for the artificial insemination of mice An improved method for the artificial insemination of mice Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer Adenovirus infection in the immunocompromised patient Fulminant hepatic failure due to disseminated adenovirus infection in a patient with chronic lymphocytic leukemia Toxicity of a first-generation adenoviral vector in rhesus macaques Lethal toxicity, severe endothelial injury, and a threshold effect with high doses of an adenoviral vector in baboons Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration Liver-directed gene transfer in non-human primates Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: toxicity study Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4 + CTLs in vivo Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype Safety of local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions Adenoviral vector-mediated expression of physiologic levels of human factor VIII in nonhuman primates Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing alpha1-antitrypsin after systemic delivery Recombinant adenovirus gene transfer in adults with partial ornithine transcarbamylase deficiency (OTCD).
Hum Targeting of adenovirus penton base to new receptors through replacement of its RGD motif with other receptor-specific peptide motifs Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism Towards the use of replicative adenoviral vectors for cancer gene therapy Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and b-galactosidase Characteristics and taxonomy of Parvoviridae Adenovirus-associated defective virus particles Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication Prospects for the use of adeno-associated virus as a vector for human gene therapy Adeno-associated virus vectors for gene therapy Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector Site-specific integration by adeno-associated virus Mapping and direct visualisation of regionspecific viral DNA integration site on chromosome 19q13-qter Epidemiology of adenoassociated virus infection in a nursery population A seroepidemiologic study of adenovirus-associated virus infection in infants and children Adeno-associated viruses of humans High efficiency transfer of the T cell co-stimulatory molecule B7-2 to lymphoid cells using high-titer recombinant adeno-associated virus vectors.
Hum Regulated high level expression of a human g-globin gene introduced into erythroid cells by an adeno-associated virus vector Preclinical evaluation of AAV vectors expressing the human CTFR cDNA Retroviral vectors for use in human gene therapy for cancer, Gaucher disease, and arthritis Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells Safety aspects of gene therapy Evaluation of recommendations for replication competent retrovirus testing associated with use of retroviral vectors Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1 Development of a self-inactivating lentivirus vector Advanced modular self-inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction Transduction of acute myeloid leukemia cells with third generation self-inactivating lentiviral vectors expressing CD80 and GM-CSF: effects on proliferation, differentiation, and stimulation of allogeneic and autologous anti-leukemia immune responses Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery Human immunodeficiency virus type 2 (HIV-2) vectormediated in vivo gene transfer into adult rabbit retina Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus Mapping of the bovine immunodeficiency virus packaging signal and RRE and incorporation into a minimal gene transfer vector Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors
key: cord-294108-uvnh0s9r authors: Dube, Taru; Ghosh, Amrito; Mishra, Jibanananda; Kompella, Uday B.; Panda, Jiban Jyoti title: Repurposed Drugs, Molecular Vaccines, Immune‐Modulators, and Nanotherapeutics to Treat and Prevent COVID‐19 Associated with SARS‐CoV‐2, a Deadly Nanovector date: 2020-10-25 journal: Adv Ther (Weinh) DOI: 10.1002/adtp.202000172 sha: doc_id: 294108 cord_uid: uvnh0s9r The deadly pandemic, coronavirus disease 2019 (COVID‐19), caused due to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has paralyzed the world.
Although significant methodological advances have been made in the field of viral detection/diagnosis with 251 in vitro diagnostic tests receiving emergency use approval by the US‐FDA, little progress has been made in identifying curative or preventive therapies.
This review discusses the current trends and potential future approaches for developing COVID‐19 therapeutics, including repurposed drugs, vaccine candidates, immune‐modulators, convalescent plasma therapy, and antiviral nanoparticles/nanovaccines/combinatorial nanotherapeutics to surmount the pandemic viral strain.
Many potent therapeutic candidates emerging via drug‐repurposing could significantly reduce the cost and duration of anti‐COVID‐19 drug development.
Gene/protein‐based vaccine candidates that could elicit both humoral and cell‐based immunity would be on the frontlines to prevent the disease.
Many emerging nanotechnology‐based interventions will be critical in the fight against the deadly virus by facilitating early detection and enabling target oriented multidrug therapeutics.
The therapeutic candidates discussed in this article include remdesivir, dexamethasone, hydroxychloroquine, favilavir, lopinavir/ritonavir, antibody therapeutics like gimsilumab and TJM2, anti‐viral nanoparticles, and nanoparticle‐based DNA and mRNA vaccines.
The entire world is facing the pandemic coronavirus disease of 2019 (COVID19) due to the outbreak of 2019 novel coronavirus (2019-nCoV; SARS-CoV-2) and an international health emergency has been declared.
The disease has touched nearly every corner of the world.
2019-nCoV infection is highly contagious and containment efforts mostly deal with identification and quarantine of exposed and asymptomatic suspects, contact tracing, detection, and strict isolation of infected patients to limit its spread.
[1] [2] [3] [4] World Health Organization (WHO), on 30th January 2020, declared the disease caused by 2019-nCoV as the sixth international public health emergency.
Due to its rapid escalation across the globe, the WHO declared the 2019-nCoV outbreak a global pandemic on the 11th of March 2020.
So far, i.e., from 31st December 2019 to 21st September 2020, more than 30 949 804 individuals were afflicted by 2019-nCoV, and 959 116 deaths have occurred worldwide.
[3] On the 11th of February 2020, the WHO officially declared a name for the ongoing 2019-nCoV related disease pandemic as coronavirus disease 2019 whereas, the International Committee on Taxonomy of Viruses (ICTV) has renamed 2019-nCoV as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on its close genetic resemblance with another coronavirus SARS-CoV.
[2, 5] SARS-CoV-2 can transfer from an infected person with or without symptoms to another individual, with a high basic reproduction number (R 0 ) value.
Although the initial R 0 was estimated to be between 2.2 and 2.7 for SARS-CoV-2, one estimate published in February 2020 indicated that the R 0 value is as high as 4.7 to 6.6, with the infected individual doubling time of 2.4 d. [6, 7] The R 0 value indicates that each individual can spread the infection to 4.7-6.6 individuals on average.
More recent studies published in August 2020 indicated that COVID-19 has a higher transmission rate than severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS) with a basic reproduction number of 3.32 and an incubation period of 5.24 d. [7] Globalization and the convenience of country to country travel had additionally fueled the worldwide spread of the disease.
[2, [8] [9] [10] This article discusses SARS-CoV-2 nanostructure, the virus biology in connection to its epidemiology, clinical manifestations, and potential and future therapeutic options including repurposed drugs, vaccine/protein therapies, immune therapies, and nanotherapeutics.
Additionally, the recommended preventive and protective measures are presented (Figure 1 ).
Coronaviruses (CoVs) are a large family of viruses and are known causative agents for respiratory, hepatic, intestinal, and neurological diseases of altering severity in several animal species.
Tyrell and Bynoe in 1966 were the first to cultivate and describe the CoVs from patients having common colds.
[11] CoVs are single-stranded ribonucleic acid (RNA) viruses.
Morphologically, they are spherical virions, having a core of RNA that is being surrounded by a membrane decorated with protein projections (called spike proteins) like a ring/crown (Latin: corona = crown).
nanomaterials with very complex structures.
They are efficient nanovectors capable of transferring their genetic materials to the infected host cells, followed by hijacking host-cell machinery to express their proteins.
[28] Many viruses have proven their worth in the field of drug, gene, and vaccine delivery.
[29, 30] Each SARS-CoV-2 virion has an average diameter of 50-150 nm (Figure 2) .
[14, 31, 32] Similar to other CoVs, SARS-CoV-2 carries four major structural genes encoding major structural proteins, known as the spike protein (S), an envelope protein (E), the membrane protein (M), and the nucleocapsid protein (N); N protein embraces the genome consisting of ssRNA and the proteins S, E, and M constitute the envelope of the virus (Figure 3) .
The S protein enables the virus to bind to and amalgamate with the host cell membrane.
It is the key immunogenic antigen and has a decisive role in defining virulence, tissue tropism, host range, and protective immunity.
[25] The trimeric S protein belongs to a class I fusion protein consisting of S1 (receptor binding) and S2 (membrane fusion) subunits.
The subunit S1 consists of a C domain carrying the RBD, and an N domain.
S2 contains fusion peptide, the heptad repeat (HR-1 and HR-2), transmembrane, and cytoplasmic domains.
The locking of the RBD to the ACE2 receptor of the host cell initiates conformational modifications in the S2 subunit that further facilitates fusion among viral and host cell membranes.
The S1/S2 juncture is the cleavage site for proteases and is also required to trigger membrane fusion, viral entry, and in the formation of syncytium.
[16, 27] Other structural proteins, like E, M, and N, are involved in the viral assembly.
Alternatively, nonstructural proteins constituting viral cysteine proteases such as papain-like protease (PL pro), 3chymotrypsin like protease (3CL pro), RNA-dependent RNA polymerase (RdRp), helicase, and other accessory proteins, participate in the viral transcription followed by replication.
To counter the host immune response, the M protein and PL pro, antagonize the host interferon (IFN) response and help the virus to control in vivo replication efficacy and pathogenesis.
[16] Alike SARS-CoV, SARS-CoV-2 ingress into target cells is also aided by the S protein.
Viral entry depends on i) the locking of S1 unit of S protein to host cell ACE2 receptors; ii) the S protein priming by host cellular protease TMPRSS2, which facilitates S protein cleavage right at the S1/S2 juncture and the S2' site, facilitating the merger between viral and cellular membranes.
Following cellular entry, CoVs disassemble to release their genome (nucleocapsid and viral RNA) into the cytoplasm of the infected host cell for initiating the translation of viral polyproteins and subsequent replication of genomic RNA (Figure 4) .
[33] Replicase polyproteins of SARS-CoV-2 are processed by PL pro and 3CL pro to form nonstructural proteins (helicase/RdRp).
[16] Various 3CL pro inhibitors can be expected to repress viral replication by obstructing the cleavage function of the protein.
RdRp is a fundamental enzyme of the RNA synthesizing machinery of RNA viruses, and it is highly conserved in all HCoVs.
Therefore, it served as a prime target in various viral infections to halt genome replication and can be inhibited by various polymerase inhibitors (favilavir, remdesivir).
[16, [33] [34] SARS-CoV-2 utilizes the ACE2 receptors to gain entry into the host cells.
[35] ACE2 is a counteractive component of the renin angiotensin system (RAS) that is responsible for maintaining a balance between fluid volume and pressure by utilizing the cleavage products of angiotensin (AGT) and their receptors.
[36] [37] [38] Among these peptides, angiotensin II (Ang-II) causes vasoconstriction and helps in sodium retention by means of AGTR1 receptor and results in vasodilation and natriuresis by binding to the AGTR2 receptor.
The enzyme ACE is responsible for producing Ang-II.
After infecting host cells, the virus also uses its protease 3CL pro to suppress NFkappaB by degrading the activating factor IKKgamma.
[39] Since, NFkappaB is involved in the induction of ACE by attaching to its promoter and enhancing transcription.
[40] The virus thus reduces the expression of ACE.
ACE further downregulates the expression of ACE2 in-part by Ang-II, which is its catalytic product.
[41] Thus, the viral infection results in upregulation of ACE2 and downregulation of ACE.
It has been shown that, bradykinin, another important molecule that forms an important component of vasosuppressor system involved in inducing hypotension and vasodilation, is being regulated by ACEs.
Bradykinin is degraded by ACE and its concentration is enhanced in presence of angiotensin1-9 a peptide produced by ACE2.
Thus, the enhanced ACE2 expression caused by the virus leads to increased levels of bradykinin causing "bradykinin storm."
This is because SARS-CoV-2 increases the amount of bradykinin in the infected tissues.
Bradykinin causes vasodilation leading to swelling and inflammation of the tissue and induces pain.
It also increases the production of hyaluronic acid.
The bradykinin storm induced leakage of fluid and hyaluronic acid induced formation of hydrogel into the lungs may be the reason behind low oxygen uptake in severe COVID-19 patients and generation of severe symptoms like hypokalemia associated with arrhythmia and sudden cardiac death.
[42] [43] [44] [45] Hence, the drugs which can take care of the bradykinin storm can also be considered as potential therapeutics for COVID-19.
SARS-CoV-2 phylogenetic analysis showed that it is meticulously related to two bat-derived SARS-like CoVs (bat-SL-CoVZC45 and bat-SL-CoVZXC21) found in horseshoe bats (Rhinolophus) with ≈89% nucleotide sequence resemblance with bat-SL-CoVZC45.
It exhibits ≈79% nucleotide sequence resemblance with SARS-CoV and ≈50% with MERS-CoV.
Further, a 98.7% nucleotide sequence resemblance to the partial RdRp gene of the bat-CoV strain BtCoV/4991 was also observed.
[2, 10] Gaining in-depth insight into the SARS-CoV-2 virus epidemiology, and characterizing its possible impact, is a pressing need of the current time in order to expand health care measures to tackle the pandemic.
The overall impact of a pandemic is governed by the total number of infected persons, transmissibility, and its medical severity (asymptomatic, mild-to-severe symptomatic, requiring hospitalization, or fatal).
[46] COVID-19 pandemic is continuously evolving, with massive number of cases and deaths each day.
Based on data from the initial outbreak and considering worldwide incidence, the trend of an increasing incidence of COVID-19 principally follows exponential growth in the number of reporting cases.
[2, 47] The present mean incubation time for COVID-19 is 5.5 d, and the median incubation time is 5.1 d, with the potential asymptomatic transmission.
Following SARS-CoV-2 infection, most patients (≈97.5%) develop symptoms within 11.5 d and almost every patient shows symptoms within 14 d. Very few (≈2.5%) SARS-CoV-2 infected patients develop symptoms within 2.2 d. [1, 2, 31, 48] As of 21st September 2020, 216 countries/areas/territories/ regions have reported confirmed cases of the disease.
USA has reported the largest number of confirmed COVID-19 patients (6 703 698), followed by India (5 487 580), Brazil (4 528 240), (136 532).
[49] SARS-CoV-2 can affect all age groups especially the higher risk groups which include: i) children <59 months, ii) pregnant women, iii) elderly, iv) people with chronic medical ailments (cardiac, pulmonary, hepatic, renal, metabolic, hematologic, neurodevelopmental diseases, or weak im-mune system), v) people with immunosuppressive conditions (HIV/AIDS, under chemotherapy or steroids), and vi) health care professionals.
[4, 47, 50] Lower risk groups should self-isolate themselves at home, drink plenty of fluids, and follow general good-health guidelines to keep their immune system strong and healthy.
SARS-CoV-2 infection is highly contagious, and containment efforts mostly emphasize on quarantine of exposed and asymptomatic suspects and strict isolation of infected patients during the incubation period to limit the disease spread.
Preliminary clinical feature of the SARS-CoV-2 infection in humans is pneumonia, which formed the basis of identification, detection as well as isolation of patients.
SARS-CoV-2 infection manifests varied clinical features, extending from asymptomatic to a condition with acute respiratory distress syndrome (ARDS).
[51] In symptomatic patients so far, the clinical manifestations consist of fever >39.1°C (most common symptom), dry cough, nasal congestion, sore throat, dyspnea, headache, myalgia, fatigue, upper respiratory tract infections, smell/taste dysfunctions, and diarrhea.
Very few patients are reported with rhinorrhea.
Pneumonia mostly occurs by 2-3 weeks following the initial infection with prominent signs of hypoxemia and deviations in blood gas.
[1, 2, 47, 50, 52] Visible changes observed in chest X-rays and computed tomography demonstrating deterioration in lung tissue are observed to be the ground glass irregularities of the disease.
Pulmonary consolidation, alveolar exudates, and interlobular involvements have also been observed.
Patients also show low white blood cell count (leukopenia), and elevation in inflammatory markers (C-reactive proteins, proinflammatory cytokines, etc.)
and formation of blood clots in some cases.
COVID-19 patients seeking intensive care unit (ICU) are particularly older and more likely to carry pre-existing comorbid conditions like hypertension and related heart diseases followed by diabetes.
Although, certain epidemiological features were identified, additional studies are still warranted.
[1, 2, 46] Recent reports have also revealed the existence of asymptomatic infections and gastrointestinal symptoms, specifically among youngsters.
[53] The occurrence of asymptomatic individuals with the ability to spread the infection may be a great hurdle in the control and containment of the disease and are posing significant public health threats.
However, detailed studies on the extent of asymptomatic persons, their viral loads, and share in transmission need further evaluation.
This allows a better understanding of the viral pathogenesis and will aid policy makers to develop scientifically sound guidelines.
[8] Currently, there is no specific approved therapeutic agent available to treat SARS-CoV-2.
Treatment is supportive and based on the patient's medical condition to alleviate symptoms.
Proper public health measures to check the viral infections are of an urgent need to tackle the growing pandemic.
Additionally, stringent measures must be taken to check the human-to-human transmission to minimize secondary infections and prevent community transmission among near contacts, and health care professionals, and to prevent further spread.
Based on earlier knowledge of the MERS and SARS outbreak, the WHO recommends adaptation of various infection control measures to lessen the overall risk of the disease transmission and prevent overall spread.
This includes avoiding close proximity with people showing symptoms of acute respiratory infections, washing hands with soap frequently (minimum for a span of 20 s), using 60-95% alcohol-based hand sanitizers after contacting infected people, and avoiding contact with infected inanimate surfaces and pet/wild animals without having proper protection.
[54] The practice of cough etiquettes by people suffering from acute respiratory tract infection, maintaining distance from these patients, proper covering of cough or sneezes, and washing hands frequently are few other disease preventive and control measures being recommended.
The WHO further recommends proper and consistent adoption of environmental disinfection and cleaning methods like cleaning of supposedly infected surfaces with water, soaps, disinfectants, and detergents.
People are recommended to stay at home, avoiding social contact (stay home stay safe).
While the governments have imposed lockdown measures, travel bans, and ban on people gathering in parks and public places.
People should leave their homes putting facemasks and only for essential/basic commodities or medical needs.
People should avoid unnecessary journeys and travel to or from work only when they cannot work from home.
People going outside should maintain a social distancing of more than 2 m apart from anyone other than members of their own family.
A person showing SARS-CoV-2 symptoms (fever of ≥37.8°C, a persistent cough or breathing problems) should take extra precautions and self-isolate (quarantine) himself/herself for 10-14 d including his family members.
[55] Although, significant methodological advances have been made in the field of viral detection of COVID-19 with many companies receiving emergency use approvals for their in vitro diagnostic tests, molecular-based high complexity laboratory tests, or antibody tests as mentioned by the US-FDA website (a total of 251 as of September 21, 2020), little progress has been made in identifying curative or preventive therapies.
[56] Thus, there is a definite need to find therapeutic agents directly targeting SARS-CoV-2, and several scientists around the world are focusing on the development of fruitful prevention/treatment strategies and finding new drugs/antivirals/vaccines against SARS-CoV-2, to halt the ongoing outbreak.
The US-FDA website lists dozens (a total of 100 companies as of September 21, 2020) of companies involved in developing antiviral or vaccine therapies for COVID-19.
[57] Antivirals can be broadly categorized into two classes, i.e., 1) virus targeting antivirals, which target the viral life cycle, machinery and pathways or directly inactivate viral structural proteins; and 2) host targeting antivirals, which either target the host cellular machinery important for viral infection or target the host's immune response pathways and cascades elicited toward the viral infection.
[12, 58] In the course of viral infection, various events in the viral life cycle and virus-host protein-protein interactions have been identified as the potential targets for antivirals.
[59] Cellular events such as virion adsorption, intracellular transport, uncoating, genome and protein synthesis, and assembly inside infected host cells play a decisive role in the viral pathogenesis and targeting these can be a good strategy in the development of current therapies for tackling viral infections.
The existence of a potentially small number of viral targets and fast mutating genes sometimes make the business of finding and developing novel and potent antivirals a challenging task.
Viruses offer limited intrinsic targets for engineering antivirals.
[60] As viruses are greatly dependent on the host cellular machinery for replication, [61] targeting various host factor(s) [62] and molecular pathways hijacked by the virus opens a pandora of opportunities to construct novel antivirals.
[12, 62, 63] Potential therapeutic targets in SARS-CoV-2: Mostly various genes and their encoded proteins of SARS-CoV-2 can act as favorable diagnostic, therapeutic, or vaccine targets for the virus (Figures 4 and 5) .
Mechanisms such as inhibition of viral enzymes (DNA and RNA polymerases, 3CL pro, TMPRSS2, reverse transcriptase, neuraminidase, endonucleases, and other proteases) or processes such as ACE2 cellular receptor inhibitors and endosomal acidification mediators prohibiting viral fusion; molecules interfering with glycosylation of the viral protein, viral assembly, new viral particle transport, and release, and immunomodulation of cytokine release can be potential targets in developing various antiviral drugs for the SARS-CoV-2.
[64] [65] [66] TMPRSS2 inhibitors (camostat, nafamostat) and furin inhibitors can abrogate the S1/S2 proteolytic cleavage, thereby blocking the viral entry.
[35] In addition, the viral entry can be curbed by using carbohydrate-binding protein inhibitors such as griffithsin, which bind to the spike glycoprotein.
[67] Endosomal cell entry and S protein activation inside endosomes depend on the pH-dependent endosomal protease cathepsins and might be clogged using lysosomotropic agents like ammonium chloride, chloroquine, and cathepsin inhibitors.
[68] Antibodies against the RBD and other viral proteins can effectively inhibit virus entry into the host cell.
The enzyme 3CL pro, one of the essential proteases of the SARS-CoV life cycle, plays a crucial role in the proteolysis of various viral polyproteins, controlling viral replication.
The 3CL pro enzyme is considered as a key drug target in the case of SARS-CoV and MERS.
[69] Recent studies revealed that SARS-CoV-2 3CL pro is conserved and carries 99% nucleotide sequence identity with SARS-CoV 3CL pro.
Hence, 3CL pro inhibitors that impede the cleaving ability of 3CL pro might repress virus replication, rendering this enzyme an attractive therapeutic target against COVID-19.
[69, 70] However, most of the potential drugs/vaccines in the spotlight as potential COVID-19 therapeutics are only experimental candidates, and vigorous clinical studies are warranted to verify their efficacy and safety.
The antivirals being investigated may be toxic and many of them can merely alleviate certain conditions.
To date, apart from the emergency use approval of the antiviral drug favilavir in China, India, Russia, and parts of the Middle East and the emergency use approval of remdesivir by the US-FDA and Japan in COVID-19 patients, there are no approved therapeutic molecules to treat the COVID-19 pandemic.
[56, 71] Thus far, therapeutic modalities including various western, [72] natural product-based (NCT04382040), and traditional Chinese medicines [73, 74] with some potential activity against COVID-19 have been briskly tested clinically, and they exhibited preliminary efficacy against COVID-19.
[65] Drug repurposing (repositioning, or retasking) expedites drug product development by identifying new uses for existing approved or experimental drugs in the current global crisis.
This www.advancedsciencenews.com www.advtherap.com strategy could significantly reduce the cost and duration of drug development compared to discovering and developing entirely new therapeutics.
[75] The following are the lists of various drugs/therapeutics under consideration and testing to be repositioned against COVID-19.
Favilavir (also known as favipiravir, T-705) a guanosine nucleotide analogue, is an antiviral with broad-spectrum activity.
It is a pyrazine carboxamide derivative and is currently being mar-keted in China and Japan as Avigan for treating influenza.
It became the first antiviral drug to gain approval from the National Medical Products Administration of China for clinical trials in treating SARS-CoV-2.
Favilavir is highly effective in treating RNA virus infections by inhibiting the RdRp.
Favilavir, originally was formulated to battle catarrhal (inflammation in nose and throat), has shown efficacy in clinical trials carried out in COVID-19 patients.
ChiCTR2000029600 and ChiCTR2000029544 are ongoing clinical studies evaluating the safety profile and efficacy of favilavir.
It has been approved in Italy by Italian Medicines Agency (AIFA) for experimental use against COVID-19 and clinical trials are underway (NCT04336904).
It is being administered to www.advancedsciencenews.com www.advtherap.com moderate COVID-19 patients at 1800 mg twice a day on the first day, and thereafter 600 mg thrice a day up to 14 d; however, the dose may vary based on indications.
Favilavir is one of the potential treatment options currently being tried for possible use in the treatment of patients suffering from COVID-19.
Despite its potential effectiveness and mass production in China, favilavir is not yet approved as a drug product for COVID-19.
NCT04310228 is another ongoing clinical trial assessing the usefulness and safety of favilavir in combination with tocilizumab.
Vero E6 cells infected with nCoV2019BetaCoV/Wuhan/WIV04/2019 exhibited a half-maximal effective concentration value (EC 50 ) of 61.9 × 10 −6 m against favilavir.
[72] Remdesivir has been labeled as the most promising antiviral by the WHO for the ongoing SARS-CoV-2 caused COVID-19 pandemic.
[76] Remdesivir (development code GS-5734), an adenosine nucleotide analogue, is a broad-spectrum antiviral drug.
A study reported the EC 50 value of remdesivir against Vero E6 cells infected with nCoV2019BetaCoV/Wuhan/WIV04/2019 to be around 0.77 × 10 −6 m and also showed its antiviral effect against 2019-nCoV infected human liver cancer Huh-7 cell line.
[72] It is a monophosphoramidate prodrug and is metabolized to the active form, GS-441524, which disguises and gets incorporated in new RNA strand by viral RNA polymerase, thereby escapes proofreading by viral exonuclease, halting genome replication.
[77] [78] [79] Originally, remdesivir was developed and synthesized to battle Ebola and was reported to have treated an American COVID-19 patient, who has now fully recovered after receiving the drug.
[80] However, more clinical data are required before the drug can be approved and is considered as an effective and official drug to treat either SARS-CoV-2 or Ebola virus.
Warren and group reported the EC 50 of remdesivir against Ebola virus infected macrophages, human endothelial and liver cells to be around 0.01 × 10 −6 to 0.20 × 10 −6 m, and demonstrated its therapeutic efficacy in an Ebola virus infected monkey model of the disease.
[81] Initially China planned Phase III clinical studies to estimate the safety profile and efficacy of remdesivir in COVID-19 patients based on its promising preclinical data in SARS-CoV and MERS-CoV infections, [82, 83] evaluating remdesivir in severe COVID-19 (NCT04257656) and in mild/moderate COVID-19 patients (NCT04252664).
However, these trials were either terminated or suspended due to the unavailabilty of eligible COVID-19 patients.
Similarly, in February 2020, the U.S. National Institute of Allergy and Infectious Diseases (NIAID) carried out a Phase III adaptive COVID-19 treatment trial (ACTT) globally to assess the efficacy of various investigational molecules compared to the control arm (NCT04280705).
Preliminary results from the trial indicated that remdesivir was superior as compared to placebo in shortening the recovery time in hospitalized COVID-19 patients.
[84] Another multicentric trial for remdesivir compared the drug with the standard of care treatment, which includes supplementary oxygen and ventilator support when indicated, in severe COVID-19 (NCT04292899) patients and patients with moderate COVID-19 (NCT04292730).
In the trial, along with the standard care, a 200 mg loading dose of remdesivir was administered on day 1, followed by 100 mg dose administered as intravenous (IV) infusions every 24 h for 2-5 or 2-10 d. [85] Results of the trials indicated no significant difference in clinical status of COVID-19 patients compared to the standard of care.
[86, 87] Other drugs like chloroquine or hydroxychloroquine are also under consideration in the global hunt to discover an effective COVID-19 therapy [88] after their approval for limited and emergency use for COVID-19 by the US-FDA.
Chloroquine a "4-aminoquinoline" is an old drug used in the prevention and therapy of malaria.
It is further prescribed in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) due to its anti-inflammatory activity.
In preliminary studies, the drug has shown efficacy and tolerable safety profile against COVID-19 associated pneumonia.
[89] Wang and group reported effective in vitro inhibition of the virus, using chloroquine.
Vero E6 cells infected with nCoV2019BetaCoV/Wuhan/WIV04/2019 and treated with chloroquine exhibited an EC 50 of 1.13 × 10 −6 m. [72] Chloroquine also acts as zinc ionophore, and allows passage of extracellular zinc to the cytoplasm and inhibits viral RdRp.
[90] [91] [92] It also interferes with viral entry by inhibiting host receptor glycosylation.
Administered to a patient orally at a dose of 500 mg in 12 to 24 h for 5 to 10 d. [85] Hydroxychloroquine, a chloroquine derivative carrying a similar mechanism of action as well as therapeutic activity as chloroquine [93] but with minimal adverse effects, has also been evaluated as an anti-COVID-19 therapeutic.
[94] In a study by Yao and group, hydroxychloroquine inhibited SARS-CoV-2 infected Vero cells in vitro after 48 h of growth with a lower EC 50 value (0.72 × 10 −6 m) as compared to chloroquine (5.47 × 10 −6 m).
[93] Multicentric clinical trials in China have also revealed that the drug shows a potent broad-spectrum antiviral effect.
The mechanism of action may be increased endosomal pH, thereby interfering with the fusion process of the virus with the cell membrane.
Thus, the virus is incapable of releasing its genetic payload inside the cell and replicate further.
Unlike the US-FDA, European regulators restricted the general use of chloroquine for COVID-19 without significant data and limited their use to clinical trials only.
Therefore, clinical research at a large-scale is still looked-for to elucidate its mode of action and potential prophylactic/therapeutic efficacy against COVID-19.
A study (ChiCTR2000029559) in China has tested the drug in 62 COVID-19 patients showing mild to moderate symptoms.
All the patients received treatments like antiviral and antibacterial drugs, oxygen therapy, immunoglobulin as standard care, whereas only half of them received hydroxychloroquine (400 mg per day) together with standard care up to 5 d. Clinical symptoms like the rate of recovery of the body temperature, the time required for cough remission were significantly shortened in patients receiving hydroxychloroquine.
Larger percentage of patients (80.6%) with improved pneumonia symptoms in the treatment group compared to the control was reported (54.8%).
[95] Another Phase II clinical study (NCT04335084) testing the efficacy of hydroxychloroquine in combination with vitamin C, D, www.advancedsciencenews.com www.advtherap.com and zinc toward the prevention of COVID-19 infection is underway.
A report by Dr. Raoult from France further demonstrated significant improvements in COVID-19 patients administered with hydroxychloroquine at a dose of 600 mg kg -1 for 6 d along with azithromycin.
[88] However, WHO has pulled out the drugs from their Solidarity trial due to lack of efficacy in treating COVID-19.
[96] 6.1.4.
NP-120 NP-120 (Ifenprodil-brand name Cerocal), a potential therapeutic option for idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), and persistent coughs may be repurposed for COVID-19.
NP-120 is N-methyl-d-aspartate (NDMA) receptor glutamate receptor antagonist, which targets the NMDA-type subunit 2B (Glu2NB).
[97] Ifenprodil is a vasodilator, originally developed by Sanofi as an oral medication to treat blood circulation disorders in French and Japanese markets.
It is no longer sold in France but is still marketed in Japan.
Ifenprodil is an approved drug in countries like Japan and South Korea to treat certain neurological conditions.
An independent animal study showing a considerable reduction in ALI and enhanced survivability in Avian H5N1 infected mice encourages researchers to expand clinical programs to ALI and ARDS associated with COVID-19 infection.
[97] An adaptive Phase IIb/III study assessing the safety and efficacy of ifenprodil (20/40 mg three times a day) together with standard of care in comparison to standard of care alone in the treatment of hospitalized COVID-19 patients is underway (NCT04382924).
Ifenprodil is already being tested in clinical trials in patients suffering from IPF and its associated cough (NCT04318704).
A repurposed drug combination treatment against SARS-CoV-2, lopinavir and ritonavir (protease inhibitor combination; Kaletra), is currently used as both first-and second-line antiretroviral medication for HIV.
Lopinavir is given in conjunction with ritonavir to increase its half-life.
[98, 99] Several clinical trials of lopinavir/ritonavir, either alone or with various combinations, are underway.
China launched a controlled trial (ChiCTR2000029308) to test the efficacy of lopinavir/ritonavir and IFN -2b combination in patients hospitalized with COVID-19.
The most commonly studied dosing regimen of lopinavir/ritonavir for COVID-19 infection is 200 mg/50 mg orally twice daily for 14 d. [98] Some other antiretrovirals were also screened for anti-SARS-CoV-2 activity.
[85] A randomized controlled Phase III trial (NCT04252274) assessing the efficacy and safety of darunavir/cobicistat combination (PREZCOBIX) for treatment of COVID-19 is underway.
However, due to the lack of efficacy in hospitalized COVID-19 patients, WHO withdrew lopinavir/ritonavir from their Solidarity trial.
[96] 6.1.
6 Hoffmann and co-workers showed that SARS-CoV-2 infection relies on the host cellular factors, such as ACE2 and TMPRSS2, and could be successfully blocked by using protease inhibitors.
[35] ACE2 enzyme is a protein recognized by various CoVs (SARS-CoV and SARS-CoV-2) to gain cell entry.
[27, 100] ACE2 receptor is expressed by the epithelial cells covering organs like lung, intestine, and blood vessels.
[101] In addition to ACE2 as the entry receptor, the virus also requires cellular proteases for the priming of S protein to enter the host cells.
TMPRSS2 is a serine protease employed by CoVs for S protein priming.
[35] Therefore, it is anticipated that the viral entry inside host cells can be obstructed by serine protease TMPRSS2 inhibitors.
The marketed TMPRSS2 inhibitors, nafamostat and camostat have been demonstrated to be effective in blocking the SARS-CoV-2 cellular entry.
[102] A Phase IIa trial studying the impact of camostat mesilate (Foipan) on 180 COVID-19 patients was initiated during March 2020 (NCT04321096).
Nafamostat exhibits inhibitory effect against nCoV2019BetaCoV/Wuhan/WIV04/2019 infected Vero E6 cells with an EC 50 value of 22.5 × 10 −6 m. [72] Leronlimab (PRO 140) is used to treat breast cancer and HIV.
Leronlimab is a humanized monoclonal antibody (MAb) capable of blocking CCR5 (CCR5 antagonist).
It is in clinical trials for mild to moderate COVID-19 patients (NCT04343651) and it is expected to benefit patients showing respiratory complications by diminishing the cytokine storm.
Currently, leronlimab has received fast track approval by the US-FDA as a combination therapy with antiretroviral therapy for HIV and triple-negative metastatic breast cancer and has completed nine clinical trials, including a Phase III trial in HIVinfected patients.
[103] 6.1.
8 Another promising repurposed antiviral drug is umifenovir, also known as Arbidol which inhibits the virion membrane fusion to host cell by targeting the interaction between S protein/ACE2 receptors.
Arbidol is currently approved in China and Russia as prophylactic and treatment drug against influenza.
In vitro cellular models showed antiviral activity against SARS and therefore it is anticipated to have promising results against SARS-CoV-2.
The most studied dosing regimen of umifenovir for COVID-19 infection is 200 mg orally every 8 h. Ongoing clinical trials are further evaluating the efficacy of umifenovir.
[85] A randomized, placebo-controlled, Phase IV clinical trial assessing the safety and efficacy of umifenovir as an adjuvant therapy to the combined therapeutic regimen of IFN 1a, lopinavir/ritonavir and hydroxychloroquine in moderate to severe COVID-19 patients (NCT04350684) is underway.
Our immune system relies on two vital pillars: 1) the innate/general immunity; and 2) the adaptive/specialized immunity.
Recent literature suggests that innate immunity can also influence the nature of adaptive responses apart from Adv.
Therap.
2020, 2000172 Figure 6 .
The immune responses generated against SARS-CoV-2.
Mainly two types of immune responses are generated in the host against the virus.
One is the normal or positive immune response, which leads to virus neutralization and ceasing of the disease progression and the other one is the abnormal or aggressive immune response that gives rise to disease associated complications like the ARDS during a severe COVID-19 infection.
protecting the host against viruses during early infection when the initial adaptive immune responses take effect.
[104] The synchronized activities of innate and adaptive immunity are vital for gaining overall protection against viruses.
Innate immunity gives early reactions by enabling the detection and destruction of pathogens quickly within hours and consists of the skin and mucous membranes in the body openings forming external barriers, phagocytic cells, natural killer (NK) cells, various substances in the blood and body fluids.
[105] Adaptive immunity takes more time in the detection and destruction of pathogens, but it targets the pathogen more precisely and can patrol the body against antigens for months/years by producing memory cells.
Adaptive immunity consists of T cells, B cells, antibodies, and cytokines as soluble proteins in the blood and tissue.
[105] Upon virus entry inside host cells, here most specifically the airway epithelial cells for SARS-CoV-2, two types of immune responses are being observed (Figure 6) .
One is the positive and healthy immune response and the other is the negative and defective immune response.
In the case of RNA viruses, after the virus entry and replication and further release inside the host cells, the infection is detected by a set of pattern/pathogen recognition receptors (PRRs) of innate immune system comprising first-line of defense against viral infection.
Inside the cell, PRRs like toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) sense the viral ss/dsRNA genome and its replication intermediates.
Upon cellular entry, the virus is recognized by the endosomal ssRNA sensor (TLR7/8), the cytosolic dsRNA sensor (RIG-I, MDA-5), and the cytosolic inflammasome sensor (NLRP3).
Subsequently, these different sensors recruit adaptor proteins including myeloid differentiation primary response 88 (MyD88) and mitochondrial antiviral signaling protein (MAVS) respectively to further activate downstream signaling pathways.
This causes activation of the transcription factors like nuclear factor kappa B (NF-B) and IFN regulatory factors (i.e., IRF3 and IRF7), and subsequently triggers the production of type I/III interferons (IFN-and IFN-) and proinflammatory cytokines like interleukin 1 beta (IL-1 ), IL-6, and tumor necrosis factor alpha (TNF-), respectively.
[106] The antiviral activity of IFN-and IFN-is further amplified by the expression of IFN stimulated genes (ISGs) such as ribonuclease L (RNAse L) and the proinflammatory chemokine (CXCL10) and is vital in limiting the spread and replication of the virus and modulating the innate/adaptive immune responses.
Cytokines released by infected cells further modulate the adaptive immune response by recruiting and activating immune cells in eliminating the virus.
This comprises the positive immune response, wherein the infection attracts T cells specific to the virus, and destroys the virus checking the infection.
Neutralizing antibodies are also produced against the viruses, which block the viral infection, and alveolar macrophages further clear the viruses by phagocytosis.
[107] The virus additionally activates the inflammasome sensor, NLRP3, that lead to the secretion of highly inflammatory cytokine IL-1 and the initiation of pyroptosis (highly Reproduced with permission.
[107] Copyright 2020, InvivoGen.
inflammatory form of programmed cell death).
This comprises the negative immune response, wherein the host cells undergo pyroptosis, elicited against cytopathic viruses, in response to the viral release.
Then pathogen-associated molecular patterns (PAMPs) like viral m-RNA and damage-associated molecular patterns (DAMPs) like ATP, nucleic acids, and ASC oligomers are released from the host cells in reaction to the virus.
These molecular patterns are then recognized by other neighboring cells, which include macrophages, epithelial and endothelial cells causing them to release various chemokines and proinflammatory cytokines like IFN gamma-induced protein 10 (IP-10), macrophage inflammatory protein 1 (MIP-1 ), MIP-1 , and MIP-1 , and IL-10.
These chemokines and cytokines attract immune cells like macrophages, monocytes, and T-cells to the infection site leading to further inflammation and additional production of IFN by T cells.
In a defective response, higher accumulation of immune cells causes overproduction of the proinflammatory cytokines causing a cytokine storm that damages the organ being infected, and then it circulates to other organs causing multiorgan damage.
It has also been observed that non-neutralizing antibodies produced against the virus by the activated B cells further enhance the damage by SARS-CoV-2 infection by a phenomenon called antibody-dependent enhancement.
[108] Downregulation of the host IFN response can cause an unbalanced immune response producing high levels of proinflammatory cytokines and infiltration of inflammatory cells leading to hyperinflammation causing more severe clinical symptoms of COVID-19.
CoVs have evolved many mechanisms to stop type I IFN induction and signaling.
Severe COVID-19 patients demonstrated unusually impaired type I IFN signatures in comparison to mild or moderate cases.
[107] Therefore the category of host immune response generated against the virus (Figure 7) defines the disease severity in the SARS-CoV-2 infection, and aggressive immune response against the virus causes significant damage to the lung airways.
[109] So, the adoption of immune therapeutic strategy against the virus could be either based on fortifying the positive immune response or minimizing the dysfunction immune response by means of immune-suppressive therapies.
The process of vaccine development against the virus should be dealt with caution.
Following are some of the list of therapeutic molecules/drug candidates that are meant to target the immune response generated against the virus and under consideration in the present scenario.
One promising vaccine candidate for COVID-19 is the Fusogenix DNA vaccine (Covigenix).
Fusogenix is a proteo-lipid vehicle (PLV), formulated with well-tolerated neutral lipids and Entos proprietary fusion-associated small trans-membrane www.advancedsciencenews.com www.advtherap.com proteins (FAST proteins), which have a novel fusion mechanism to deliver therapeutic nucleic acid payloads directly into target cells, intact and unmodified.
Fusogenix DNA vaccine utilizes plasmid DNA, which encodes several protein epitopes (antigen) from key immunogenic SARS-CoV-2 proteins to develop an advanced therapeutic payload with maximum protection.
These protein epitopes will help activate the body's inherent antibody production and elicitation of a protective immune response against COVID-19.
Further, in comparison to traditional vaccines, the DNA-based vaccine avoids the use of the infectious agent, shows enhanced stability, easy large-scale production, and is expected to stimulate both B-and T-cell responses.
In preclinical in vivo studies, Covigenix showed high immunogenicity, efficacy, and safety.
In Phase I/II human clinical trials, Covigenix will be further evaluated for its safety, tolerability, immunogenicity, and efficacy.
[103] Another vaccine candidate developed against SARS-CoV-2 is mRNA-1273.
It is a lipid nanoparticle (LNP) loaded with an mRNA that encodes for a complete, prefusion stabilized form of the S protein of the SARS-CoV-2.
mRNA-1273 is in Phase I human clinical trial to examine the safety and immunogenicity of its five dose levels (10, 25, 50, 100, and 250 µg) on a double-dose vaccination schedule (NCT04283461).
On 12th May 2020, mRNA-1273 was granted fast track designation by US-FDA.
Data from the Phase 1 study (first clinical batch), with 25 µg and 100 µg dose levels, have shown the presence of well-tolerated neutralizing antibodies titers at or above convalescent sera.
Safety data obtained from the Phase 1 study led to the discontinuation of the 250 µg dose level in the subsequent Phase II studies.
[110] Phase III trial was next launched to assess the efficacy, safety, as well as immunogenicity of the vaccine at a dose of 100 µg in adult participants (NCT04470427).
Another vaccine for COVID-19 is based on an adenovirus and is named as Ad5-nCOV.
This vaccine is being developed by the Chinese company CanSino Biologics in partnership with China's Institute of Biology at the country's Academy of Military Medical Sciences.
Results from a Phase I safety (NCT04313127) and Phase II (NCT04341389) trials conducted for the vaccine, demonstrated strong immune response against SARS-CoV-2.
Therefore, in an unprecedented move, the Chinese military approved the vaccine as a "specially needed drug."
[111, 112] Its Phase III trial in Saudi Arabia is underway (NCT04526990).
Another RNA based vaccine candidate developed against SARS-CoV-2 is BNT162b2.
It is based on LNPs loaded with nucleosidemodified messenger RNA (modRNA) that encodes for an optimized SARS-CoV-2 full length S protein and RBD.
Based on preclinical and Phase I/II studies (NCT04380701) data and in consultation with the US-FDA's Center for Biologics Evaluation and Research (CBER), BNT162b2 has received fasttrack designation by the US-FDA and will be tested at a 30 µg dose level (in a two dose regimen) in a global Phase II/III study (NCT04368728).
[113] Another vaccine candidate against COVID-19 is a purified inactivated SARS-CoV-2 virus vaccine called CoronaVac (formerly called as PiCoVacc).
Preclinical studies for the vaccine showed the induction of specific neutralizing antibodies against SARS-CoV-2 in rats, mice, and macaques (nonhuman primates) those were able to neutralize 10 representative SARS-CoV-2 strains.
Three immunizations with the vaccine at two different doses (3/6 µg per dose) provided partial or complete protection in non-human primates (macaques) against SARS-CoV-2 infection.
[114] Preliminary results of Phase I/II clinical trials (NCT04383574 and NCT04352608), which was tested at three/two different doses in a two doses regimen either scheduled on day 0/28 or on day 0/14 or both.
(antigen content of 300, 600, 1200 SU/0.5 mL) showed promising immunogenicity and no severe adverse events.
CoronaVac got an emergency approval for limited use by the Chinese government.
Phase III clinical trial (NCT04456595) is underway to assess its safety and tolerability.
The immunization schedule is two doses IM (600 SU/0.5ml) with a 14 d interval.
INO-4800 DNA vaccine is designed in such a way to directly deliver optimized DNA plasmids into cells using Inovio's trademarked device called CELLECTRA.
CELLECTRA utilizes a transitory electric pulse to reversibly open tiny cellular pores, enabling the plasmids to enter.
Once inside the cell, the plasmids begin replicating along with the cell's DNA and are translated into proteins (MAbs), producing a specific immune response.
This methodology has the potential to generate therapeutic MAbs in vivo.
Clinical trials are underway to assess its safety, tolerability, and immunogenicity (NCT04336410).
Smith and co-workers in a preclinical study testing humoral immunogenicity in both mice and guinea pigs have shown the successful generation of neutralizing antibodies and T cell immune responses against SARS-CoV-2.
These encouraging preclinical results further support its translation to large randomized clinical trials.
[115] An adenoviral vaccine candidate AZD1222 (provisionally named ChAdOx1 nCoV-19) based on the SARS-CoV-2 S protein that was originally developed to target MERS is also a potential vaccine candidate for COVID-19.
It is a weakened and safer form of a common cold adenovirus (ChAdOx1) that cannot reproduce within the body but can produce CoV S protein after vaccination resulting in the formation of antibodies against the S proteins.
The vaccine's seed stock was in production at Oxford University's Biomanufacturing Facility, UK, and Phase I/II clinical trials (NCT04324606/ISRCTN15281137 and NCT04444674) assessing its safety, efficacy, and immunogenicity against SARS-CoV-2 are underway.
ChAdOx1 nCoV-19 is being administered intramuscularly (IM) either at a single dose of 5 × 10 10 viral particles (vp) or a single dose of 5 × 10 10 vp followed by a booster dose.
The preliminary results from Phase I/II trial (NCT04324606) showed an acceptable safety profile, and a second vaccine dose (boosting dose) further enhanced the antibody responses inducing both humoral and cellular immune responses.
[116] Doremalen and co-workers in a study successfully demonstrated a robust humoral and cell-mediated response from a single dose of an investigational vaccine, AZD1222.
A single vaccination has prevented pneumonia caused by SARS-CoV-2 in six rhesus macaques by halting SARS-CoV-2 replication.
[117] The vaccine has progressed to Phase II/III (NCT04400838) as well as Phase III trials (ISRCTN89951424).
On September 6, 2020 AstraZeneca stopped global trials of the vaccine after finding a volunteer, who developed a type of inflammation called transverse myelitis.
The British trial resumed on September 12, 2020, but trials in other countries are still on hold.
Another vaccine in progress is based on virus-like particles (VLP).
Benthamiana.
VLP mimic viruses without genetic material and enables the body's immune system to generate an immune response.
VLPs cannot replicate and are noninfectious.
In this plant-based approach, the genetic sequence of a virus is inserted into agrobacterium, which then enters into the plant tissues.
The plant begins to produce the protein that is used as a vaccine.
In case the virus begins to mutate, the production may be updated in new plants.
Using plants and genetically engineered agrobacteria, the mass-production of vaccines is easy, faster, and inexpensive.
The Phase I trial (NCT04450004) evaluating safety, tolerability, and immunogenicinity at dosages of 3.75, 7.5, or 15 µg of the vaccine candidate alone or followed by a booster dose in healthy human volunteers is underway.
Medicago is also planning to initiate its Phase II/III trials.
[118] A genetically modified infectious bronchitis virus (IBV) vaccine is also in the pipeline to treat COVID-19.
IBV was originally developed to treat avian coronavirus.
It is available in oral form and has shown efficacy in preclinical trials.
The new vaccine is anticipated to turn SARS-CoV-2 infection into a very mild cold.
[119] Another potential protein-based vaccine candidate in the pipeline is COVID-19 S-Trimer (SCB-2019).
It is a recombinant subunit vaccine developed using Clover's patented Trimer-Tag technology via a prompt mammalian cell expression system.
S-Trimer vaccine is based on the S protein of the SARS-CoV-2.
The company also identified the antibodies against the trimeric S protein in the serum of patients fully recovered from COVID-19.
A randomized, placebo-controlled Phase I trial of SCB-2019 has been launched and will be evaluating the safety, reactogenicity, as well as immunogenicity at multiple dose levels (3, 9, and 30 µg) with and without the adjuvant (NCT04405908).
[103, 118] VAAST, oral recombinant vaccine tablets based on the genome COVID-19 causative virus using Vaxart's proprietary oral vaccine platform, are in preclinical trials.
Each vaccine constructs is a nonreplicating viral vector based on a different SARS-CoV-2 antigen combination.
[103, 118] AdCOVID is another potent single dose, intranasal vaccine candidate in preclinical trials to protect against COVID-19.
It is an adenovirus-based vaccine expressing SARS-CoV-2 S protein.
[103] 6.2.13.
Sputnik V Sputnik V a COVID-19 vaccine was registered on 11th August 2020 and approved for early use by the Russian Ministry of Health under the adopted emergency rules during the pandemic.
It is a dual adenovirus vectors (rAd26 and rAd5) based vaccine loaded with a fragment of gene coding S protein of the SARS-CoV-2.
The use of two dissimilar forms of adenovirus vectors, for the first and second vaccination doses is a unique technology of the Gamaleya National Center for ensuring long lasting immunity.
On 1st August 2020, Phase I/II clinical trials of Sputnik V (NCT04437875 and NCT04436471) have been completed and results suggested the induction of strong antibody and cellular immune responses.
Postregistration Phase III clinical trial comprising ≥ 40000 people in Russia was expected to get started 31st August, 2020 onwards (NCT04530396).
Other countries (UAE, Saudi Arabia, Philippines, India, and Brazil) may also join the Phase III clinical trial locally.
[118] The key focus of developing therapeutics against the virus has been revolving around finding antivirals and vaccines.
However, many reports point toward various patients associated complications such as cytokine storm syndrome (CRS) and macrophage activation syndrome (MAS), which lead to ARDS during severe COVID-19 infection.
COVID-19 patients developing CRS secondary to COVID-19 pneumonia show increased production of various proinflammatory cytokines, chemokines, and other growth factors.
Therefore, treatment of hyperinflammation or the aggressive immune dysfunctions raised in response to the viral infection using immunosuppression mechanism can be advantageous and aid in reducing the mortality.
[52, 120] www.advancedsciencenews.com Although immunomodulatory therapy is not recommended in COVID-19 pneumonia in general, the compassionate use of immunomodulators might be advantageous in COVID-19 patients exhibiting CRS complications, decreasing the high levels of proinflammatory cytokines, and thereby preventing multiorgan failure.
[121] The following are some of the immunomodulators under consideration as anti-COVID-19 therapeutics.
Gimsilumab (KIN 1901) , a human MAb, working by targeting granulocyte macrophage colony stimulating factor (GM-CSF), is under clinical trials to prevent and treat ARDS in COVID-19 patients.
As GM-CSF elevated levels in blood further augment the expression of other proinflammatory cytokines causing the progression of ARDS and serum from COVID-19 patients have shown high levels of GM-CSF; therefore, gimsilumab is anticipated to reduce the mortality rate by reducing lung damage.
A multicenter, Phase II trial (NCT04351243) is underway to assess gimsilumab's efficacy and safety profile in patients with ARDS/lung injury secondary to COVID-19.
[103] TJM2 (TJ003234) is a neutralizing antibody against human GM-CSF.
TJM2 shows a high affinity towards human GM-CSF, thereby blocking GM-CSF binding to its receptor.
This will further prevent downstream signaling cascades resulting in response to GM-CSF binding, halt target cell activation, resulting in inhibition of inflammatory responses causing reduced tissue inflammation and death.
TJM2 is in clinical trials to study its efficacy in reducing the severity of complications associated with COVID-19.
[103] A Phase I/II, randomized, multicenter trial (NCT04341116) will assess the efficacy of TJM2 under supportive care when administered as an IV infusion (3 mg kg -1 or 6 mg kg -1 ) in subjects with severe COVID-19.
Lenzilumab, another GM-CSF neutralizing immunotherapy, is being studied in minimizing and treating the cytokine storm and associated lung dysfunction/ARDS in COVID-19 patients.
Lenzilumab is Humanigen's proprietary Humaneered MAb targeting GM-CSF and is being approved by US-FDA for sympathetic use in COVID-19 patients.
[103] Currently, it is being studied in a multicentric, Phase III trial in comparison to the current standard of care for taking care of respiratory failure and prevention of death in hospitalized COVID-19 patients (NCT04351152).
Namilumab (IZN-101, AMG203) is another fully human immunoglobulin G1 MAb targeting GM-CSF, which has received compassionate use approval from US-FDA in the treatment of critical as well as hospitalized COVID-19 patients before their admission to ICU.
103 The double center compassionate use study of the drug will be conducted in Bergamo and Milan cities in Italy.
It is currently in later stages of clinical development programs against RA and ankylosing spondylitis.
[122] TZLS-501 is another antibody in progress as a potential treatment for COVID-19.
TZLS-501 is a humanized MAb targeting interleukin-6 receptor (anti-IL-6R).
Early clinical studies channeled in China suggest that anti-IL-6R MAbs might be used clinically for treating COVID-19 patients.
TZLS-501 binds to both forms of IL-6R (membrane-bound or soluble), thereby speedily depleting the levels of IL-6 in blood.
Overproduction of IL-6 leads to the elicitation of long-lasting inflammation, causing lung damage following SARS-CoV-2 infection.
[103] 6.3.6.
AT-100 Another immunotherapeutic candidate explored for COVID-19 is AT-100.
AT-100 is a human recombinant surfactant protein D (rhSP-D).
Initially, AT-100 was developed for broncho-pulmonary dysplasia (BPD) in premature infants who requisite mechanical ventilation and oxygen therapy.
[103] Tocilizumab (Actemra) is a recombinant version of a humanized MAb targeting the IL-6 receptor, which binds to IL-6R, thereby inhibiting signal transduction.
US-FDA has approved it for the treatment of CRS, rheumatoid arthritis, giant cell arthritis, polyarticular juvenile idiopathic arthritis, and systematic juvenile idiopathic arthritis.
[121, 123] Xu and group from China in their pilot study used tocilizumab (single dose of 400 mg IV) to treat a total of 21 COVID-19 patients.
Tocilizumab treated patients showed a significant reduction in fever and IL-6 levels.
Lymphocyte counts in the patients returned to normal after treatment and they demonstrated an improvement in lung function.
[123] A multicentered randomized Phase III trial of tocilizumab is underway in China to assess its efficacy and safety in COVID-19 patients with pneumonia and elevated IL-6 (ChiCTR2000029765).
Another proof of concept study (TOSCA) in Italy is underway for its efficacy and safety profile assessment in the treatment of patients with ARDS and CRS secondary to COVID-19 (NCT04332913).
Another multicentric Phase III placebo-controlled clinical trial, called COVACTA (NCT04320615), is underway to evaluate tocilizumab (maximum single dose of 800 mg IV and one more dose only if clinical symptoms deteriorate/or exhibits no improvements) along with standard of care versus placebo along with standard of care in severe COVID-19 patients globally.
Sarilumab (Kevzara) is another IL-6 receptor blocker under clinical trials for its ability to lessen the effect of the inflammatory Adv.
Therap.
2020, 2000172 www.advancedsciencenews.com www.advtherap.com immune response against SARS-CoV-2 infection.
It is a fully human MAb against the IL-6 receptor.
It has been approved by US-FDA toward the treatment of rheumatoid arthritis, but its use to treat COVID-19 patients is investigational.
Sarilumab was developed using proprietary VelocImmune technology (Regeneron's) using a genetically engineered mouse equipped with a genetically humanized immune system to produce fully human antibodies.
A multicenter, double blind, Phase II/III trial (NCT04327388) is underway to assess the efficacy and safety of a single IV dose of sarilumab compared to supportive care plus placebo in severe/critical COVID-19 patients.
In Phase II, patients were distributed into three groups at random, with two doses of sarilumab (200 and 400 mg) and placebo.
Preliminary data from the trial led to its immediate amendment to enroll only critically ill COVID-19 patients (either on ICU or requiring mechanical ventilation/high flow oxygenation) receiving a higher dose of the drug (400 mg) or placebo.
[103] CytoSorb is an extracorporeal blood purification technology based on hemo-adsorbent polymer to treat uncontrolled inflammation in patients those have undergone cardiac surgery.
It consists of polymer beads with high porosity those can successfully adsorb and remove toxic substances and inflammatory mediators from blood and other bodily fluids.
CytoSorb has received US-FDA's emergency use authorization in the US for use in severely ill COVID-19 patients approaching or confirmed respiratory/multiple organ failure and are admitted to ICU.
It is approved in the European regions as an adjuvant therapy designed to lessen the complications associated with CRS by absorbing a wide range of cytokines, PAMPs and DAMPs, thereby reducing their levels in circulation.
[124] The use of convalescent sera is a promising treatment strategy against SARS-CoV-2.
It consists of patient derived serum enriched with passive neutralizing antibodies against the virus, obtained from COVID-19 recovered patients.
[125] Many reports from China suggest the use of convalescent serum as therapy for patients with COVID-19.
China has offered convalescent plasma therapy to 200 COVID-19 patients (ChiCTR2000029757).
Convalescent plasma therapy is promising, but its safety and efficacy as a treatment modality for COVID-19 are yet to be established.
Therefore, it is important to study and establish the safety as well as efficacy of convalescent plasma therapy in COVID-19 patients in clinical trials.
The US-FDA based on encouraging results from early trials has given it the emergency use authorization against COVID-19.
Glucocorticoids may modulate inflammation-mediated lung injury in COVID-19 patients and thereby reduce progression to respiratory failure and the resulting death.
However, there is uncertainty about the effectiveness of glucocorticoids in COVID-19.
Dexamethasone is a corticosteroid used in a wide range of conditions for its anti-inflammatory along with immunosuppressant effects.
Preliminary findings of the Phase II/III RECOVERY trial (NCT04381936), a randomized, controlled, open-label study conducted in the UK, noted a survival benefit with the use of dexamethasone (oral or IV at a dose of 6 mg per day for up to 10 d) as compared to standard care alone (invasive mechanical ventilation or oxygen) in hospitalized COVID-19 patients.
Patients on ventilators showed one third and patients requiring only oxygen showed one fifth reductions in the 28 day mortality.
Dexamethasone can also be used in both children and elderly, but in pregnancy/breast-feeding cases, the recovery trial used prednisolone (40 mg orally) or hydrocortisone (80 mg twice daily IV) instead of dexamethasone.
[126] Among several drugs employed for the treatment of COVID-19, N-acetylcysteine (NAC) is a potential therapeutic/preventive/adjuvant against SARS-CoV-2.
NAC is a precursor of reduced glutathione (GSH).
It has antiinflammatory, antioxidant, antiviral, and anticoagulant/plateletinhibiting properties.
It is approved as an over-the-counter dietary supplement and is in the WHO list of essential medications.
The US-FDA has approved the usage of NAC to treat acetaminophen overdose associated liver side effects, and also as mucolytic agent (due to its free sulfhydryl group) in cystic fibrosis or chronic obstructive pulmonary disease (COPD).
It has been also used as an antioxidant in a variety of disorders involving GSH depletion and oxidative stress.
Thiols can block ACE2 receptors thereby blocking penetration of SARS-CoV-2 into cells.
NAC can act as a potential therapeutic agent in the treatment and/or attenuating the risk associated with COVID-19 through a variety of potential mechanisms, including increasing glutathione level, improving T cell response, and modulating inflammation thereby preventing COVID-19 patients from moving to ICUs.
[127] An ongoing Phase II clinical trial (NCT04374461) is assessing the number of successful COVID-19 patients discharged/transferred from critical care units after receiving NAC (6 g per day IV) in addition to supportive or other treatments as prescribed against COVID-19.
Other Phase III (NCT04455243) and Phase IV (NCT04419025) clinical trials of NAC are assessing NAC's anti-inflammatory effect and its efficacy in minimizing the severity associated with COVID-19, respectively.
[128] Cyclosporine A (CsA) is a calcineurin inhibitor and is used to avert organ rejection and for treating T-cell associated autoimmune diseases.
Anti-inflammatory and immunosuppressive effects of CsA are exerted due to its bonding to cyclophilin-A (Cyp-A), thereby preventing the activation of nuclear factor of activated T-cell (NFAT) and production of IL-2 (a cytokine crucial for the proliferation of T-cells).
CsA has shown potent antiviral activity at noncytotoxic micromolar concentrations against many CoVs including SARS-CoV in cell cultures.
CsA may be successful in COVID-19 due to the close resemblance between SARS-CoV-2 and SARS-CoV.
This antiviral property might have resulted due to the inhibition of Cyp-A-dependent viral assembly and inhibition of the NFAT pathway.
[129] [130] [131] Ongoing Phase I safety study of CsA (oral or IV) is assessing the tolerability and clinical effects in COVID-19 patients requiring oxygen supplementation (NCT04412785).
A Phase IIa randomized clinical trial (NCT04492891) is underway for the treatment of non-ICU hospitalized COVID-19 patients.
Patients will receive CsA orally for up to 7 d at 2.5 mg kg -1 body weight twice a day along with standard of care.
Another ongoing Phase IV clinical trial (NCT04392531) is assessing the efficacy and safety of CsA plus standard treatment in comparison to standard treatment in hospitalized COVID-19 patients.
Another corticosteroid in pipeline is inhaled budesonide.
It may directly inhibit the viral replication or may exert the inflammatory effect.
Steroids reduce the effect of cytokines and thereby modulate immune response.
Budesonide may prevent the ARDS associated with COVID-19.
Inhaled corticosteroid's therapeutic effect against COVID-19 is limited and still need to be explored.
Various clinical trials assessing their efficacy in COVID-19 are ongoing (Phase IV: NCT04355637).
Vitamin D is a steroid hormone.
Vitamin D is being evaluated as an adjuvant therapy for COVID-19 based on its immunomodulatory, anti-inflammatory, antifibrotic, and antioxidant effects.
Vitamin D exerts direct effect on immune cell proliferation and activity, and ACE2 expression.
[132] The aim of one of its ongoing randomized Phase III trial (NCT04385940) in COVID-19 patients is to assess its efficacy at daily low dose (1000 IU) versus weekly high dose (50000 IU) for 3 weeks and to determine the correlation between vitamin D deficiency and clinical characteristics.
Another Phase III trial (NCT04344041) assessing the efficacy of vitamin D3 at high dose (400000 IU) in comparison to standard dose (50000 IU) is underway.
Apart from the currently available drugs, immunomodulators, and vaccine candidates, there are many other potent molecules like novel protein or peptide therapeutics and even nanoparticulate based strategies yet to be tested against the virus.
These new therapeutics, once proven, can be the game changers in tackling the pandemic.
Below we list some of the potential future therapeutics for treating COVID-19 patients.
In the last decades, peptides as therapeutics and diagnostic molecules have gained massive interest in drug development due to the dynamic research conducted by pharmaceutical and biotech companies.
[133] Currently, a large number of both natural as well as synthetic therapeutic peptides are in clinical development for various diseases ranging from cancer, neurodegenerative, cardiovascular, metabolic disorders, autoimmune disorders, hormonal deficiencies, to infectious diseases.
[133, 134] Peptide based therapeutics offer numerous benefits over other molecules such as high selectivity and specificity, high efficacy and safety, tolerability, less immunogenicity, biocompatibility, low toxicity, easy, and cost-effective production.
According to their source and method of production, peptide based therapeutics can be classified as: 1) natural peptides (derived from peptide hormones, plant proteins, animals, and human-derived peptides); 2) recombinant peptides; and 3) synthetic peptides.
[133] [134] [135] Peptides are relatively easy to modify structurally.
Improved therapeutic performances can be achieved by utilization of modified amino acids, cyclization, etc.
Many antiviral peptides are being explored for viral diseases like hepatitis, influenza, and HIV.
[136] Peptides exhibit a reduced possibility of developing resistance during the treatment.
Antiviral activities of the peptides can be attributed to three major mechanisms: 1) inhibition of viral attachment and penetration into host cells; 2) inhibition of replication by interacting with viral polymerases, and 3) disruption of the viral envelope.
They can be presented as a new generation of antiviral agents with broad-spectrum activities.
[136, 137] Potential peptide-based therapeutics which are in the pipeline against COVID-19 include FlowVax peptide vaccine from Flow Pharma, [103] Corvax signal peptide vaccine from Vaxil Bio, (Vax-Hit platform), [103] epitope-based peptide vaccine from Onco-Gen (Vaccinomics strategy), [103] Ii-Key peptide vaccine from Generex Biotechnology (EpiVax's computational tools and li-Key technology), [118, 138] etc.
Peptide-based therapeutics can be effective alternatives for COVID-19 prophylaxis.
Nanotechnology, as one of the ground-breaking approaches, can be really promising in offering remedial solutions toward fighting the on-going COVID-19 outbreak and future pandemics.
Nanotechnology allows a number of tactics to fabricate highly advanced therapeutic options to cope with SARS-CoV-2 both outside and inside the host.
The scope of nanotechnology against COVID-19 includes conventional as well as advanced biomimetic approaches and engineered nanomaterials with versatile chemical functionalities.
Therefore, nano intervention will be extremely relevant in advancing COVID-19 prophylaxis, diagnosis, and treatment (Figure 8) .
In view of what we know so far about the SARS-CoV-2 structure, its pathophysiology, life cycle, and related immunological response, we envisage key steps where nanotechnologist and nanotechnology could play a pivotal part.
Herein, nanotechnology is discussed in terms of fabricating engineered nanocarriers to overcome the limitations associated with conventional antiviral and other biological therapeutics, nanomaterials as novel therapeutics, risk free immunization techniques, engineered nanomaterials in the development of point-of-care diagnostics, and alternative advanced disinfection methodologies.
Nanotechnology aims toward safe, effective, and targeted delivery of existing as well as future therapeutics, fabricating risk-free vaccines for safe and effective immunization, curbing the interactions between viruses and host cells, and permanent disruption of viral particles.
[139] [140] [141] In the recent past, many nanoparticulate systems have been tested for their efficacy and made their way to preclinical studies against several pathogenic viruses such as HIV, herpes simplex, human papilloma virus, and respiratory viruses.
[140] [141] [142] In the dearth of a specified/broad spectrum antiviral agent against SARS-CoV-2, current therapy focusses on repurposing existing molecules.
Therefore, to make the repurposed therapeutics more effective and safer, an appropriate nanocarrier delivery system might be useful.
Successful delivery of therapeutics especially peptide/protein, DNA/RNA, and other biologicals to the target site is most often hampered due to their poor aqueous solubility, degradation, fast-clearance, low-bioavailability leading to subtherapeutic concentrations.
Nanomaterials with large surface area to volume ratios, versatile functionalities and surface modification, unique physicochemical properties, and suitable for various routes of administration can easily address the challenges associated with conventional therapeutics.
Biocompatible organic/inorganic nanoparticles can be potentially tuned to encapsulate therapeutics with large payloads, control release, minimize drug resistance, and improve pharmacokinetic/pharmacodynamic properties and patient compliance.
Further actively targeted nanocarriers can cross the biological barriers thereby reaching the protected target sites with higher specificity excluding the unwanted exposure to nontargeted areas resulting in dosage reduction, reduced systemic toxicity, improved bioavailability, and enhanced efficacy.
Nanotechnology further makes it possible to tailor the therapy according to individual needs.
[140] Itani and group have demonstrated the potential role of different nanoparticles as efficient delivery agents for potential therapeutics or immunomodulators against COVID-19.
[142] Nanoparticle based delivery of therapeutics (drugs, vaccines, RNA/DNA, and peptides) ensures targeted and optimized concentration in the desired site while minimizing adverse effects and unwanted degradation enhancing efficacy.
Donalisio et al.
demonstrated nanotechnological approach for the tropical treatment of herpes simplex virus (HSV-1 and HSV-2) by encapsulating acyclovir in chitosan nanoparticles.
The obtained gel formulation showed enhanced penetration across the porcine skin as compared to the commercial cream product, and revealed increased efficacy as compared to its free form.
[143] LaBauve and group used a lipid-coated mesoporous silica nanocarrier system to deliver antiviral molecule ML336 for inhibiting Venezuelan equine encephalitis virus (VEEV), in order to improve its solubility and stability.
The designed ML336 loaded nanosystem showed enhanced circulation time, biocompatibility, and viral inhibition in vivo in a murine model of VEEV infection.
[144] Kumar et al.
demonstrated the fabrication of zidovudine loaded lactoferrin nanoparticles.
Zidovudine is a highly bioavailable antiretroviral drug, which shows serious side effects and toxicity.
Results revealed higher efficacy and reduced toxicity of nanocarrier based formulation as compared to their free soluble counterparts.
[145] Liu et al.
demonstrated the fabrication of liposomes encapsulating cholesterol modified hydroxychloroquine for the treatment of bleomycin induced pulmonary fibrosis.
The engineered nanosystem inhibited the proliferation of rat lung fibroblasts and reduced any toxicity associated with hydroxychloroquine thereby, providing evidence as a possible therapeutic agent for pulmonary fibrosis.
[146] Encapsulation in a nanosystem enhances nucleic acid-based vaccine stability and offers a newer fusion mechanism to transport the genetic material loaded inside them directly to the cells.
Similarly, the efficacy of other mRNA/siRNA vaccines can be significantly enhanced via complexing them with lipid/polymerbased nanoparticles.
These lipid/polymer-based nanoparticles are based on ionizable monomers, which complex negatively charged RNA/DNA and thus facilitate escape of the entrapped RNA/DNA from endosomes into the cytosol where the genetic material can be translated.
[30] The successful inhibition of viruses achieved with nanoplatforms could have extensive advantage in combatting various viral infections including SARS-CoV-2.
Combinatorial therapy aims in suppressing more than one pathway simultaneously, thereby, producing synergistic effect.
Therefore, combinatorial therapy may represent another therapeutic approach against SARS-CoV-2.
It displays numerous advantages such as dose reduction of the individual therapeutics, leading to fewer adverse events.
It may also overcome drug resistance.
Nanocarriers are very promising systems for the co-delivery of multiple drugs with different physicochemical properties.
They enable encapsulation and sequential release of both hydrophobic and hydrophilic drugs in a single platform.
Nanoparticles can also be instrumental in attaining combinatorial multidrug therapeutics for controlling uncontrolled inflammation and manage disease severity.
A very recent work by Dormont et al.
developed targeted multidrug nanoparticles composed of an en-dogenous lipid squalene, an immunomodulator adenosine and an antioxidant -tocopherol for treating acute viral inflammation (Figure 9 ).
[147] Freeling et al.
fabricated a lipid-drug nanoparticle system comprising of three antiretroviral drugs (lopinavir, ritonavir, and tenofovir) to overcome lymphatic drug insufficiency and reported 50-fold higher and sustained intracellular concentrations in lymph nodes compared to current oral therapy of free drugs.
This combinatorial nanotherapy can be used to target SARS-CoV-2.
[148] Bearing in mind the risk of random mutations in SARS-CoV-2 genome, which may result in variation in antigenic protein, nanoparticle functionalization with a varied number of antigenic moieties/proteins concurrently will enhance the efficacy of vaccines against viral infections.
These next-generation nanocarrier-based vaccines will not only help in guarding the antigenic proteins against metabolism/degradation but will also help in greater exposure of antigenic proteins to antigen-presenting cells, thereby improving their delivery and efficacy.
Nanomaterials have emerged as a favorable tool both as immune stimulators and immune suppressors for immune modulation.
Nanomaterial based nonviral vectored vaccines can successfully overcome two major limitations associated with conventional vaccines of weak immunogenicity and reversion risk associated with live/attenuated viral vectors, and can be a reliable alternative to viral vaccines.
Nanosystems can imitate the genome transfer ability of viral vaccines which show high pathogenicity and can also deliver molecular adjuvants simultaneously.
[139] Recombinant protein and inactivated vaccines in general necessitate the use of adjuvants to enhance their immunogenicity.
Many nanomaterials depending on their functionalization possess an intrinsic adjuvant property and can amplify the immune response in a host.
Therefore, fabricating nanomaterials with intrinsic ability to amplify host's immune response will be very helpful in enhancing the efficacy of vaccines especially in patients with impaired immune system.
Nanosystems can also enhance the targeted delivery of immunosuppressants to both adaptive and innate immune systems.
[139] Various nanoparticles like graphene oxide, silver, gold, copper, and zinc nanoparticles show intrinsic antiviral/antibacterial properties and can act as potential therapeutics against the disease.
[149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] [160] They may offer alternative methods to traditional disinfection protocols used in healthcare sites.
Nanoparticles can potentially sustain release, enhance cell entry, and reduce efflux of toxic metal ions.
The use of metal nanoparticles would be particularly advantageous as antiviral agents because metals may attack a wide range of viral targets.
Also, there is a lower risk of developing drug resistance in the case of metals as compared to conventional antivirals.
[149] Silver nanoparticles (AgNPs) have been investigated for their antimicrobial potency against bacteria, but many reports [147] Copyright 2020, The Authors.
point toward their antiviral activities against viruses, which includes HIV, herpes simplex virus, respiratory syncytial virus, hepatitis B virus, etc.
[150] [151] [152] Li and group have demonstrated efficient in vitro inhibition of H1N1 infection utilizing AgNPs based codelivery of oseltamivir (neuraminidase inhibitor) by inhibiting viral attachment and release.
[153] The fact that AgNPs are less toxic and are nontoxic at lower concentrations to humans, vouch for their possible usage for therapeutic purposes.
Given the fact that silver ion itself attacks a broad range of targets in the microbial entry, microorganisms generally do not develop resistance against silver.
[152] Nanocolloid AgNPs have also been shown to be effective in controlling viral proliferation in the respiratory tract after inhalation delivery, thus controlling the disease progression.
[154] Another study by Levina and group demonstrated in vitro antiviral activity of DNA decorated titanium dioxide (TiO 2 ) nanosystem against different influenza A virus subtypes infected MDCK cells.
The nanosystem was found to be highly efficient in delivering nucleic acids directly inside the cells without the aid of any transfection agent with an IC 50 value of 1.5 µg mL -1 (30 × 10 −9 m for DNA).
[155] Another nanostructured material, graphene, has shown promising antimicrobial/antiviral properties.
[156] [157] [158] Recently, the efficacy of Cu to inactivate HuCoV-229E was demonstrated.
It was found that the HuCoV-229E (10 3 plaque forming units) was inactivated irreversibly in less than 60 min on brasses/alloys containing at least 70-90% Cu, whereas the virus was shown to persist on surfaces like glass, rubber, and stainless steel in an infectious state for at least 5 d. [159] Dhanasezhian et al.
fabricated biogenic gold and AgNPs using the seaweed Sargassum wightii.
Their cytotoxic and antiviral activity against HSV-1 and HSV-2 strains on Vero cells showed that functionalized metallic nanoparticles act as antiviral agents by hindering virion attachment and cellular entry, depending on their particle size.
[160] Figure 10 .
Schematic illustration for the selective naked-eye detection of SARS-CoV-2 RNA facilitated by the suitably designed ASO-capped Au nanoparticles.
Reproduced with permission.
[163] Copyright 2020, American Chemical Society (ACS).
https://pubs.acs.org/doi/10.1021/acsnano.0c03822 technologies will accelerate patient management and identification and containment of infection hotspots.
Nanoparticle based disease monitoring mediated by various metallic nanoparticles carrying magnetic, fluorescence, and surface-enhanced Raman scattering features would aid in tracking treatment response.
[161, 162] Moitra and group reported the development of gold nanoparticles (AuNPs) based colorimetric assay for rapid, selective, and visual diagnosis of positive COVID-19 patients within 10 min after the isolation of viral RNA.
In the presence of targeted RNA from the virus, thiol-modified antisense oligonucleotides against N protein of SARS-CoV-2 capped AuNPs exhibited selective agglomeration leading to altered surface plasmon resonance.
Subsequent addition of RNaseH further cleaved the RNA from the RNA-antisense oligonucleotides complex leading to the formation of a precipitate (Figure 10) .
[163] Graphene sheets conjugated to antibodies can rapidly detect targeted viral proteins and viral targeting antibody functionalized graphenes can further be useful in the development of biosensors against COVID-19.
[164] Similarly, utilizing theranostic nanoparticles, both therapy and diagnosis can be combined in order to detect and neutralize the virus in a single go, halting the virus to survive and reproduce within the host.
The theranostic nanoparticles can efficiently complex with SARS-CoV-2, detecting and disrupting their structure.
[142] Potential nanoparticle based therapeutics which are in the pipeline against the current COVID-19 outbreak include proteo-lipid nanovehicle based fusogenix DNA vaccine, lipid nanoparticle-loaded mRNA vaccine (mRNA-1273), plantbased virus-like particles (VLP), recombinant protein nanoparticle vaccine (NVX-CoV2373), [103, 165] and nanomicelles based antiviral.
[138] Analogous to cancer nanotherapeutics, COVID-19 can also be managed successfully by exercising 3D nanotechnologybased approaches that include efficient detection, diagnosis, and delivery.
The tissue and cell targeting principle achieved by targeting antibodies and peptides in handling other dreaded diseases like cancer and neurodegeneration can be adopted in the case of the antiviral nanoparticles for achieving targeted and effective therapy of infected patients.
Furthermore, theranostic nanoparticles can be developed to detect and kill the virus simultaneously.
Nanoparticle based kits can be developed to detect the virus at an early stage in asymptomatic patients with lesser viral loads.
Nanoparticle based antiviral material can be developed for fabricating superfine filters for face masks, personal protective equipment, and surface coatings.
Further, nanotechnology may assist in large-scale production of therapeutics and equipment addressing COVID-19 and similar pandemics.
Thus, there are varieties of therapeutic molecules against COVID-19.
The list of these potential therapeutic molecules under consideration for COVID-19 is summarized in Table 1 .
The exceptional speed, with which the SARS-CoV-2 pandemic is escalating across the world, is causing global anxiety to attain new heights.
COVID-19 has become the reason for the globally prevalent fear, stress, and concern.
All these are natural and common reactions to the fast-changing and undefined situation that all of us find ourselves in.
Further, there is a noticeable degree of fear, stress, and apprehension in certain groups with the higher risk factor, such as children, older people, health care professionals, frontline workers, and people with pre-existing maladies (such as asthma, diabetes, heart diseases, and hypertension).
The impact Phase III NCT04530396 [ 118] Linear DNA vaccine DNA Based on SARS-CoV-2 spike (S) protein Applied DNA Sciences/ Takis Biotech Preclinical [ 118] ZyCoV-D DNA Plasmid DNA vaccine Zydus Cadila Phase I/II CTRI/2020/07/026352 [ 103, 118] Bacillus Calmette-Guerin (BCG) vaccine Phase III ChiCTR2000034780 [ 118] (Continued) Adv.
Therap.
2020, 2000172 Phase III [ 103, 118] Covaxin Inactivated coronavirus Whole-Virion inactivated SARS-CoV-2 Vaccine (BBV152) Phase I/II NCT04471519 [ 118] NVX-CoV2373 Protein Recombinant SARS-CoV-2 nanoparticle vaccine consisting of trimeric full-length SARS-CoV-2 spike glycoproteins and saponin based Matrix-M1 adjuvant Novavax Phase I/II NCT04368988 [ 103, 165] FlowVax peptide vaccine Adjuvanted microsphere peptide vaccine targeting SARS-CoV-2 nucleocapsid Adjuvanted microsphere peptide vaccine targeting SARS-CoV-2 nucleocapsid Flow Pharma Preclinical Ebola, Marburg, HIV, Zika, Influenza, HPV [ 103] Signal peptide vaccine (VXL-301, 302, 303) Signal peptide vaccine using VaxHit bioinformatics platform Vaxil Bio Preclinical [ 103] Epitope-based peptide vaccine Peptide Synthetic long peptide vaccine candidate for S and M proteins (Vaccinomics strategy) OncoGen Preclinical [ 103] Ii-Key peptide Peptide Synthetic peptides that mimic the epitopes of the SARS-CoV-2 using EpiVax's computational tools and li-Key technology Generex/ EpiVax Preclinical Influenza, HIV, SARS-CoV [ 138] Monoclonal antibodies Gimsilumab (KIN 1901) Human Mab Targets GM-CSF Roivant Sciences Phase II NCT04351243 [ 103] TJM2 (TJ003234) Neutralizing antibody Neutralizing antibody against human GM-CSF Phase I/II NCT04341116 [ 103] Lenzilumab Neutralizing antibody Neutralizing antibody against human GM-CSF Humanigen Phase III NCT04351152 Compassionate use [ 103] Namilumab (IZN-101, AMG203) Human Mab Targets GM-CSF Izana Bioscience Compassionate use RA and ankylosing spondylitis [ 103, 122] TZLS-501 Human monoclonal antibody Mab against IL-6 receptor Tiziana Life Sciences Preclinical Autoimmune diseases [ 103] AT-100 Protein Human recombinant surfactant protein D (rhSP-D) Airway Therapeutics Preclinical Bronchopulmonary dysplasia (BPD) in premature infants [ 103] Tocilizumab (Actemra) Human Mab Mab against IL-6 receptor Roche Phase III NCT04320615 ChiCTR2000029765 RA, giant cell arthritis [ 121, 123] Sarilumab (Kevzara) Human Mab IL-6 receptor blocker Sanofi and Regeneron Phase II/III NCT04327388 RA [ 103] Convalescent sera Passive neutralizing antibody Passive neutralizing antibody developed in COVID-19 recovered patients -P h a s e 0 ChiCTR2000029757 Phase II NCT04343755 [ 125] Anti-inflammatory molecules Glucocorticoid Anti-inflammatory and immunosuppressant -Phase II/III NCT04381936 [ 126] (Continued) Adv.
Therap.
2020, 2000172 Phase IV NCT04419025 [ 127, 128] Cyclosporine A Calcineurin inhibitor Anti-inflammatory and immunosuppressant Phase IIa NCT04492891 Phase IV NCT04392531 [129] [130] [131] Budesonide Corticosteroid Anti-inflammatory -Phase IV NCT04355637 Vitamin D Steroid Anti-inflammatory, antioxidant, immunomodulatory, -Phase III NCT04385940 NCT04344041 [ 132] of the crisis on people's mental health is also of major concern.
As the new measures to prevent the infection are being introduced, such as quarantine (social isolation), cases of depression and loneliness are expected to rise.
The disruptive effects of the COVID-19 outbreak are dominating our daily lives; therefore, it is of prime importance that we should manage and react properly to this challenging situation unfolding fast in our lives.
The world is in total chaos, lives have frozen, and everything around us has come to a standstill situation.
Nature has forced us to stay indoors.
Let us hope, together; we will be able to find a cure and curb the grave epidemic as soon as possible.
Many drug candidates are under investigation for the disease.
Initial reports suggest some of them are promising at this juncture.
However, whether these drugs will act following virus dynamics and host response is a big question.
How they will behave in a clinical trial is yet another long-term debate.
Besides, their affordability in developing countries is a significant concern.
Antiviral therapy shows a wide margin of ineffectiveness as many viruses possess extraordinary genetic adaptability, which endows them with the ability to escape antiviral repression.
In some instances, the viruses reclaim advantage over the host by online mutagenesis, which creates novel strains with additional acquired resistance to the existing antivirals.
Therefore, finding new and multifaceted antiviral agents is the unmet need of the hour.
An approach analogous to cancer nanotechnology, including early diagnosis, targeted delivery, and combinatorial approaches can be followed to tackle this deadly viral nanovector.
US-FDA's extraordinary speed in reviewing emergency use authorization (EUAs) and investigational new drug (IND) applications and their prompt approval to expedite anti-COVID-19 therapeutics into the clinical development is highly appreciable.
WHO and partners had launched an international clinical trial named Solidarity, for comparing potential options against standard of care to find an effective treatment for COVID-19.
The trial's aim is to expedite the discovery of any potential drugs that can slow down the disease progression or improve patient's survival rate.
On 4th July 2020, WHO has withdrawn hydroxychloroquine and lopinavir/ritonavir from their Solidarity trial.
The Solidarity Trial's committee found that these drugs were ineffective in treating the infection.
Similar findings were also reported from the UK's Recovery trial.
Further, US-FDA's approval for the emergency use of remdesivir and other drugs, and other medical countermeasures (MCMs) and facilitating their availability is another noteworthy move in combating COVID-19 and is an example of using science and technology at its best in protecting and rescuing the world from emerging chemical, biological, radiological and nuclear (CBRN) threats.
With the current focused, rapid, worldwide discoveries in basic science, translational research, and clinical studies, and the expedited regulatory scrutiny, it is anticipated that preventive and curative therapies for COVID-19 will be a reality in the near future.
Coronavirus disease (COVID-19) weekly epidemiological update and weekly operational update World Health Organization Naming the coronavirus disease (COVID-19) and the virus that causes it Proc.
Natl.
Acad.
Sci Proc.
Natl.
Acad.
Sci Summary of probable SARS cases with onset of illness from 1 Epidemic and pandemicprone diseases (MERS situation update SARS-CoV-2 life cycle: stages and inhibition targets WHO Coronavirus Disease (COVID-19) Dashboard Infection control guidance for healthcare professionals about coronavirus (COVID-19 Coronavirus disease (COVID-19) advice for the public Emergency Use Authorization (EUA) information, and list of all current EUAs COVID-19 Company Directory Clinical Drug Information Drug Discovery Today Favilavir: First approved drug to possibly treat coronavirus Outline of trial designs for experimental therapeutics Solidarity" clinical trial for COVID-19 treatments Biopharma products in development for COVID-19 Immunobiology: The Immune System in Health and Disease Predicted immune responses to SARS-CoV-2 Coronavirus Vaccine Tracker Israeli-made oral vaccine for coronavirus on track, but testing will take months Proc.
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USA 2020 Catching Up to Coronavirus: Top 60 Treatments in Development Therapeutic advances in infectious disease 2017 Theranostics 2020 IEEE 15th Int.
Conf.
on Nanotechnology Antibacterial and Antiviral Graphene-based Coating for Public Settings
key: cord-354445-lnvc7mmf authors: Lichtenstein, David; Alfa, Michelle J. title: 4 Cleaning and Disinfecting Gastrointestinal Endoscopy Equipment date: 2019-12-31 journal: Clinical Gastrointestinal Endoscopy DOI: 10.1016/b978-0-323-41509-5.00004-9 sha: doc_id: 354445 cord_uid: lnvc7mmf Abstract Outbreaks of infection transmission due to contaminated flexible endoscopes have focused the attention of health care personnel, senior management, device manufacturers, and regulators on the need to improve the approach used to offer this valuable service.
This chapter presents the principles of flexible endoscope reprocessing along with a pragmatic approach to the judicious selection and proper reprocessing of endoscopic equipment, as well as guidance for prevention and management of infection transmission inclusive of newer sterilization (e.g., hydrogen peroxide vapor) and disinfection (e.g., improved hydrogen peroxide) technologies.
It also provides an outline of the Quality Systems approach that is applicable to flexible endoscope reprocessing and the need for ongoing staff competency and audits of endoscope cleaning, disinfection, and storage practices.
Furthermore, the most current regulatory, expert organization, and manufacturer's recommendations are reviewed.
The field of gastrointestinal (GI) endoscopy has expanded dramatically as new procedures, instruments, and accessories have been introduced into the medical community; more than 20 million GI endoscopies are performed annually in the United States.
1, 2 Although GI endoscopes are used as a diagnostic and therapeutic tool for a broad spectrum of GI disorders, more health care-associated infectious outbreaks and patient exposures have been linked to contaminated endoscopes than to any other reusable medical device.
[3] [4] [5] [6] Failure to adhere to established reprocessing guidelines or the use of defective reprocessing equipment accounts for the majority of these cases.
[7] [8] [9] [10] [11] [12] [13] In addition, complex endoscopes such as the duodenoscope and linear echoendoscope with elevator mechanisms can transmit bacterial infections even when reprocessing protocols are reportedly followed in accordance with manufacturer and societal guidelines.
[14] [15] [16] The topic of endoscope reprocessing has largely been taken for granted by many endoscopists; however, standardized cleaning and disinfection protocols have been available for some time, and, with few exceptions, changes have been gradual.
This slow evolution with a high safety profile may have engendered some complacency on the part of endoscopists, to the point that many endoscopists are only vaguely aware of what goes on "behind the curtain" of the endoscope reprocessing room.
Instruments are used on patients, taken away by GI nurses or other health care personnel, reprocessed, and returned ready for patient use.
As the complexity of reprocessing and recognition of its importance become a concern to the medical community and our patients, endoscopists must become more educated on these issues and thereby able to participate in informed discussions with their patients.
This chapter presents a pragmatic approach to proper reprocessing of endoscopic equipment, with guidance for prevention and management of infection transmission, and includes newer sterilization and disinfection technologies.
2.
Semicritical: semicritical devices contact intact mucous membranes and do not ordinarily penetrate sterile tissue.
These instruments include endoscopes, bronchoscopes, transesophageal echocardiography probes, and anesthesia equipment.
Reprocessing of these instruments requires a minimum of HLD.
3.
Noncritical: noncritical devices contact intact skin (e.g., stethoscopes or blood pressure cuffs).
These items should be cleaned by low-level disinfection.
Endoscopes GI endoscopes are considered semicritical devices, and the resultant minimal standard for reprocessing is HLD.
This standard is endorsed by governmental agencies including the Joint Commission (JC), the Centers for Disease Control and Prevention (CDC), 37 and the FDA.
38 It is also endorsed by gastroenterology societies such as the American Society for Gastrointestinal Endoscopy (ASGE), American College of Gastroenterology (ACG), and American Gastroenterological Association (AGA), as well as medical organizations, including the Association of Perioperative Registered Nurses (AORN), Society of Gastroenterology Nurses and Associates (SGNA), Association for Professionals in Infection Control and Epidemiology (APIC), and American Society for Testing and Materials (ASTM).
[39] [40] [41] [42] HLD of endoscopes eliminates all viable microorganisms, but not necessarily all are alike, however.
Steam is the most extensively utilized process and is routinely monitored by the use of biologic indicators (e.g., spore test strips) to show that sterilization has been achieved.
When liquid chemical germicides (LCGs) are used to eradicate all microorganisms, they can be called chemical sterilants; however, the US Food and Drug Administration (FDA) and other authorities have stated that these processes do not convey the same level of assurance as other sterilization methods.
[28] [29] [30] Other commonly used sterilization processes include low-temperature gas such as ethylene oxide (ETO), liquid chemicals, and hydrogen peroxide gas plasma.
31 Disinfection is defined broadly as the destruction of microorganisms, except bacterial spores, on inanimate objects (e.g., medical devices such as endoscopes).
Three levels of disinfection are achievable depending on the amount and kind of microbial killing involved.
These levels of disinfection are as follows: 1.
High-level disinfection (HLD): the destruction of all viruses, vegetative bacteria, fungi, mycobacterium, and some, but not all, bacterial spores.
32, 33 For LCGs, HLD is operationally defined as the ability to kill 10 6 mycobacteria (a six-log reduction).
The efficacy of HLD is dependent on several factors and includes the type and level of microbial contamination; effective precleaning of the endoscope; presence of biofilm; physical properties of the object; concentration, temperature, pH, and exposure time to the germicide; and drying after rinsing to avoid diluting the disinfectant.
32 2.
Intermediate-level disinfection: the destruction of all mycobacteria, vegetative bacteria, fungal spores, and some nonlipid viruses, but not bacterial spores.
3.
Low-level disinfection: a process that can kill most bacteria (except mycobacteria or bacterial spores), most viruses (except some nonlipid viruses), and some fungi.
Although this categorization for disinfection levels generally remains valid, there are examples of disinfection issues with prions, viruses, mycobacteria, and protozoa that challenge these definitions.
34 Antiseptics are chemicals intended to reduce or destroy microorganisms on living tissue (e.g., skin), as opposed to disinfectants, which are used on inanimate objects (e.g., medical devices such as endoscopes).
The difference in the way the same chemical is used to achieve different levels of disinfection and sterilization is important for endoscopy because the contact times for sterilization with any given LCG are generally much longer (hours) than for high-level disinfection (minutes) and may be detrimental to the endoscope.
The relative resistance of various microorganisms to LCGs is shown in Box 4.1.
More than 40 years ago, Earle H. Spaulding developed a rational approach to disinfection and sterilization of medical equipment based on the risk of infection involved with the use of these instruments.
35, 36 The classification scheme defined these categories of medical devices and their associated level of disinfection as follows: 1.
Critical: critical devices or instruments come into contact with sterile tissue or the vascular system.
These devices confer a high risk for infection if they are contaminated.
This category includes biopsy forceps, sphincterotomes, surgical instruments, and implants, when used in sterile anatomic locations.
Reprocessing of these instruments requires sterilization.
disinfection at least once daily.
The water bottle should be filled with sterile water.
[49] [50] [51] [52] Because accessory items often do not have unique identification numbers, it is critical to ensure they are dedicated to and stored with the endoscope that they are used with.
This is necessary to ensure that if there is an outbreak, it is possible to identify which accessory components were used.
This may require the use of disposable accessory holders or holders such as mesh bags that are also reprocessed along with the accessories.
Most accessory instruments used during endoscopy either contact the bloodstream (e.g., biopsy forceps, snares, and sphincterotomes) or enter sterile tissue spaces (e.g., biliary tract) and are classified as critical devices.
As such, these devices require sterilization.
49, 50 These accessories may be available as disposable "single-use" or "reusable" instruments.
Reuse of devices labeled single-use only remains controversial but has been commonly employed in many practices, primarily for economic benefits.
44, [53] [54] [55] [56] The FDA 57 considers reprocessing a used single-use device into a ready-for-patient-use device as "manufacturing," and as a result, hospitals or third-party reprocessing 58, 59 companies that reprocess these devices are required to follow the same regulations as the original equipment manufacturers (i.e., obtain 510[k] and premarket approval application; submit adverse event reports; demonstrate sterility and integrity of the reprocessed devices; and implement detailed quality assurance monitoring protocols).
This includes the development of standards and policies to determine the maximum number of uses for the devices and the training of staff in the reprocessing procedures.
[59] [60] [61] [62] The regulatory burden imposed by these requirements essentially eliminated the practice of the reprocessing of single-use devices by most hospitals.
AERs were developed to replace some of the manual disinfection processes and standardize several important reprocessing steps, thereby eliminating the possibility of human error and minimizing exposure of reprocessing department personnel to chemical bacterial spores.
43 Although spores are more resistant to HLD than other bacteria and viruses, they are likely to be killed when endoscopes undergo thorough manual cleaning.
In addition, survival of small numbers of bacterial spores with HLD is considered acceptable because the intact mucosa of the GI tract is resistant to bacterial spore infection.
Endoscope sterilization, as opposed to HLD, is not required for "standard" GI endoscopy, as a reprocessing endpoint of sterilization has not been demonstrated to further reduce the risk of infectious pathogen transmission from endoscopes.
44 Sterilization of endoscopes is indicated when they are used as "critical" medical devices, such as intraoperative endoscopy when there is potential for contamination of an open surgical field.
45, 46 In addition, individual institutional policies may dictate sterilization of duodenoscopes and linear endoscopic ultrasound instruments due to elevator mechanisms that have been difficult to clean and eradicate all bacterial contaminants with HLD alone (see the later section on Duodenoscope-Related Infections).
Despite the complex internal design (Fig.
4 .1) of endoscopes, HLD is not difficult to achieve with rigorous adherence to currently accepted reprocessing guidelines.
47 Endoscope features that challenge the reprocessing procedures include: • Complex endoscope design with several long, narrow internal channels and bends that make it difficult to remove all organic debris and microorganisms (e.g., elevator channel and elevator lever cavity of duodenoscopes).
• A large variety of endoscope vendors and models require different cleaning procedures and devices and materials.
• Occult damage (e.g., scratches, crevices) to the endoscope can sequester microorganisms and promote biofilm formation.
All valves, caps, connectors, and flushing tubes need to be adequately cleaned, rinsed, and disinfected or sterilized at the same time the patient-used endoscope is being reprocessed.
48 The water bottle used to provide intraprocedural flush solution and its connecting tubing should be sterilized or receive high-level LCGs have inherent limitations; however, they are universally used to reprocess flexible endoscopes and accessories due to their relative convenience, safety, and rapid action.
LCGs used as HLDs should ideally have the following properties: broad antimicrobial spectrum, rapid onset of action, activity in the presence of organic material, lack of toxicity for patients and endoscopy personnel, long reuse life, low cost, odorless, ability to monitor concentration, and nondamaging to the endoscope or the environment.
18, 32 HLD solutions can act as sterilants if an increased exposure time is used 28, 48, 78 ; however, the exposure time required to achieve sterilization with most LCG solutions is far longer than is practical, and therefore these formulations are only used for HLD.
48, 79 The efficacy of chemical disinfectants and sterilants is dependent on their physical properties including concentration and temperature; the length of exposure of the endoscope to the chemical solutions; the type and amount of microbial debris on the endoscope; and the mechanical components of the endoscope such as channels and crevices.
Because the chemicals are toxic to humans and the environment, proper handling, thorough rinsing, and appropriate disposal are essential for human safety.
71 When selecting a HLD product, institutional requirements need to be taken into consideration with important variables including the number of endoscopes processed per day, training requirements, turnaround time, cost information, and regulatory issues regarding safe use of the HLD products.
Health care workers who use HLDs need to be familiar with and have readily accessible, product/brand-specific Material Safety Data Sheets (MSDS) and keep current with regulatory changes and new product developments.
18 Users should consult with manufacturers of endoscopes and AERs for compatibility before selecting an LCG.
The most commonly used FDA approved LCGs for disinfection of flexible endoscopes include glutaraldehyde, ortho-phthalaldehyde (OPA), peracetic acid, and hydrogen peroxide (Table 4 .1) 71,80,81 based chemicals in varying combinations and concentrations.
Some formulations contain combinations of microbicidal agents, including glutaraldehyde and phenol/phenate, peracetic acid and hydrogen peroxide, and glutaraldehyde and isopropyl alcohol.
The FDA periodically updates a list of approved HLD solutions along with some of their attributes, such as contact time and temperature required for HLD.
82 Sterilization of endoscopes is indicated on occasions when they are used as critical medical devices during open surgical procedures.
The risk for contamination of the operative field exists when a nonsterile endoscope enters the abdomen through sterilants.
[63] [64] [65] [66] [67] [68] [69] [70] AERs continuously bathe the exterior surface of the endoscope and circulate the LCG under pressure through the endoscope channels.
The AER manufacturer identifies each endoscope (brand and model) that is compatible with the AER and specifies limitations of reprocessing models of endoscopes and accessories.
Variations in AERs may require customization of the facility design to accommodate requirements for ventilation; water pressure, temperature, and filtration; plumbing; power delivery; and space.
All models of AERs have disinfection and rinse cycles.
In addition, the AERs may also have one or more of the following automated capabilities: 32,68,71 1.
Some AERs utilize and discard small quantities of LCG per HLD cycle, whereas others have a reservoir of LCG that is reused over multiple cycles.
The latter design results in gradual dilution of the LCG and requires intermittent testing to verify maintenance of the minimum effective concentration (MEC).
Product-specific test strips need to be used regularly to monitor these solutions, 48 which should be discarded whenever they fall below the MEC or when the use-life expires, whichever comes first.
2.
The temperature and cycle length can be altered to ensure HLD or sterilization based on the LCG and type of endoscope.
3.
The AER should ensure circulation of LCGs through all endoscope channels at an equal pressure with flow sensors for automated detection of channel obstruction.
4.
The AER should be self-disinfecting.
5.
Vapor recovery systems are available.
6.
Low intensity ultrasound waves are an option.
7.
Variable number of endoscopes per cycle.
8.
Some AERs flush the endoscope channels with forced air or with 70% to 80% ethyl or isopropyl alcohol followed by forced air to aid in drying the endoscope channels, thereby eliminating residual water, which reduces microbial growth during storage.
9.
The AER should incorporate a self-contained or external water filtration system.
LCGs and AERs must meet specified performance levels for HLD to receive FDA clearance.
This is defined as a reduction in residual organic loads and a 6-log 10 killing of resistant indicator organisms (typically Mycobacterium bovis).
All AERs marketed in the United States meet these criteria.
The ASGE has published a summary of vendor-specific AERs and their compatible LCGs.
65 The FDA has approved labeling some AERs as washer-disinfectors due to the introduction of automated, brushless washing of endoscope channels prior to the disinfection cycle.
Utilization of this AER washing cycle provides an extra margin of safety by providing redundancy of cleaning; however, the existing multisociety guideline 45 and other international standards emphasize that manual cleaning is still necessary when a washer-disinfector is used to assure the overall efficacy of HLD.
65, 72 One AER (Steris System 1E [SS1E]; Steris Corp, Mentor, OH) has received FDA approval for liquid chemical sterilization, as opposed to HLD, for heat-sensitive devices that cannot be sterilized by traditional means.
73 This system uses filtered, ultraviolettreated water that enters the AER and mixes with a peracetic acid-based formulation that is subsequently heated to 46°C to 55°C for liquid chemical sterilization.
74 This system is designed for "point of use" sterilization, as sterile storage is not possible.
For flexible endoscopes processed through the SS1E, there is still a requirement for an alcohol rinse and drying prior to placing the endoscope into a storage cabinet.
The FDA also requested that AER manufacturers conduct additional validation testing to evaluate AER reprocessing Agent/Action ever more complex GI flexible endoscopes.
The combination of ultrasonic capability with flexible endoscopes has opened up a new tool to use for the diagnosis and staging of cancers.
However, along with these improvements that enhance diagnostic capabilities comes the increasing complexity of the endoscope channels.
These complexities include double instrument channels with connector bridges, ultrasound probe channels, auxiliary channels, and elevator lever wire channels (sealed and unsealed).
These complexities in endoscopes have far-reaching impacts in terms of reprocessing of reusable flexible endoscopes.
This has been painfully highlighted by the recent outbreaks of antibiotic resistant bacteria associated with fully reprocessed endoscopes that remain contaminated 15,28,93-104 and act as fomites that transmit bacteria to a high percentage of subsequent patients who are exposed to the contaminated endoscope (see later section on Infection Control Issues for more detailed information on infection transmission).
Such outbreaks have focused attention on the cleaning and disinfection of flexible endoscopes.
There has been a paradigm change in that it is now recognized that reprocessing of GI flexible endoscopes is an extremely complex process that requires a quality systems approach, which includes specific training for reprocessing personnel, adequate monitoring of various stages in the reprocessing cycle, and ongoing documentation of staff competency.
48, 95, [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] Human factors play a critical role in compliance with reprocessing of GI endoscopes.
115 Ofstead et al (2010) demonstrated that compliance with all the reprocessing steps occurred for only an incision, as occurs with selected methods of intraoperative enteroscopy or postsurgical anatomy endoscopic retrograde cholangiopancreatography (ERCP).
84, 85 Endoscopes, when sterilized, require low-temperature methods because they are heat labile and therefore, unlike most other medical or surgical devices, they cannot undergo steam sterilization.
ETO is the most commonly employed low-temperature sterilization process and a valuable method of sterilizing flexible endoscopes.
However, a lengthy aeration time is required following ETO sterilization to allow desorption of all residual toxic gas from the endoscope.
Additional steps must be taken, such as the application of a venting valve or the removal of the water-resistant cap to ensure proper perfusion with the gas and to prevent damage to the endoscope due to excessive pressure build-up.
In addition, there are potential hazards to staff, patients, and the environment related to ETO toxicities (Table 4 .2).
86 The International Agency for Research on Cancer has classified ETO as a known (group 1) human carcinogen.
Within the past two decades, several new, low-temperature (< 60°C) sterilization systems have been developed, including hydrogen peroxide gas plasma, vaporized hydrogen peroxide, peracetic acid immersion, and ozone 87-92 (see Table 4 .2).
endoscopes and are not trained on specific cleaning requirements.
The use of different sizes and types of channel brushes for the various different channel sizes, the fact that some channels cannot be brushed, and the multitude of different types of cleaning brushes available makes duodenoscope reprocessing a confusing process prone to human error.
Major changes in GI endoscope reprocessing over the past five years have occurred and include new regulatory requirements, 1.7% of flexible endoscopes reprocessed when cleaning steps were performed manually and disinfection was automated, compared to 75.4% compliance when both cleaning and disinfection were automated.
115 Fig.
4 .2 outlines the basic steps in reprocessing of a GI flexible endoscope.
Until recently, the only aspect of this process that was monitored was to test the MEC of the high-level disinfectant to ensure it contained a sufficient concentration of the active ingredient.
It is easy to see from the outline provided in Fig.
4 identified.
97, 98, 108, 110, 113, [118] [119] [120] [121] If biofilm forms, the ability of disinfectants to reliably kill microorganisms within biofilm is dramatically reduced.
118, 122 Although most published reports of infectious outbreaks related to flexible endoscopes have involved duodenoscopes, other endoscopes including colonoscopes, gastroscopes, bronchoscopes, cystoscopes, and ureteroscopes (see later sections on Reprocessing Errors and Outbreak Management and Infection Control Issues), have been shown to be contaminated and involved in infectious outbreaks.
As such, every site offering flexible endoscopy procedures should ensure they have an established quality system for reprocessing these complex devices as recommended by the FDA 110 and CDC.
123 Table 4 .3 provides an overview of what components are needed for such a system.
As recommended by the CDC, 123 the first step that all health care facilities should undertake is to perform an audit by reviewing their current endoscopy services to ensure they meet all aspects of a quality system approach.
This requires input from administration, risk management, endoscopy staff, and infection prevention and control to ensure that all components are adequately assessed and the appropriate policies and procedures documented and implemented.
Table 4 .4 provides an overview of the key steps in reprocessing and outlines where mistakes are frequently made, as well as the impact of such mistakes.
It is important that audits done for endoscope reprocessing are observational and based on a specific checklist of critical components, as well as data to substantiate processes (e.g., time data to show contact time with detergent during cleaning, as well as transport times to determine how frequently it exceeds 1 hour).
Table 4 .4 is a useful aid to review during initial training of staff, as well as during discussion of audit data on a yearly basis.
The need to monitor the adequacy of cleaning 48 is a critical step in the Total Quality System approach (see Table 4 .3) to endoscope reprocessing.
Appropriate benchmarks for cleaning markers have been established.
48, 54, 109, 118, 121, [124] [125] [126] There are a number of rapid cleaning tests (RCT) available for monitoring organic residuals such as hemoglobin, carbohydrate, and protein, as well as those that monitor ATP residuals.
There are published data for some of these rapid test methods, 112,125-132 but when selecting a rapid cleaning monitor, it is important to request that the rapid test manufacturer provide their validation data.
Review of the pros and cons of the testing method based on the validation data provided by the manufacturer is an important step when selecting the RCT.
Examples of the considerations for selecting the RCT are shown in Table 4 .5.
Once the RCT has been selected, a way to document the RCT results is needed (e.g., a record sheet that identifies the scope tested, the person doing the testing, date and time of testing, result of testing, and the result of retesting for those scopes that fail the RCT and require recleaning).
Regardless of the method selected, the site should do initial testing for ALL endoscopes to determine the current baseline status.
This should be performed on the next patient use of each endoscope after completion of manual cleaning and rinsing.
If the initial RCT fails for the majority of the endoscopes, this device-reprocessing guidelines, and endoscope manufacturers' instructions for use.
The key "domino" in this chain of changes was the 2015 FDA Guide to Manufacturers of Reusable Medical Devices (FDA, March 2015) that required manufacturers to validate that their cleaning instructions were effective and could achieve predetermined benchmarks.
Cleaning validation for medical device reprocessing was not previously required; the focus was on validation of the disinfection or sterilization protocols recommended by manufacturers for their medical devices.
When transmission of carbapenem-resistant Enterobacteriaceae (CRE) associated with contaminated duodenoscopes was recognized and first investigated and reported in the United States, 14 it was unclear why reprocessing of duodenoscopes was failing.
However, it was clear that transmission rates from contaminated duodenoscopes were high (up to 45%) and that, in addition to causing infections, patients often became colonized with CRE and remained colonized long after the exposure to the CRE contaminated duodenoscope.
Some health care facilities continued to report CRE transmission and suggested that the manufacturers' instructions for use (MIFUs) for endoscope reprocessing were inadequate 15, 103, 116 and that was why endoscope contamination was ongoing.
Although there had been recent design changes by the three main duodenoscope manufacturers whereby the elevator wire channel was sealed, the transmission of CRE was reported for duodenoscopes with both sealed and unsealed elevator wire channels.
14,15,103 However, one thing was clear; it was very difficult to adequately clean the lever cavity in duodenoscopes, and visible patient debris under the elevator lever was detected in one published outbreak.
95 This has prompted new validated cleaning instructions for the lever cavity of duodenoscopes.
117 The CRE outbreaks linked to contaminated duodenoscopes prompted the FDA to convene an advisory panel meeting in May 2015, and on August 4, 2015, the FDA issued a Safety Communication 110 with the recommendations from the advisory panel meeting that included: 1.
Establishing and implementing a comprehensive quality control program for endoscopy reprocessing to ensure meticulous adherence to MIFUs for duodenoscope reprocessing, adequate training of reprocessing personnel, and audits to ensure ongoing compliance.
2.
Supplemental measures to be considered by sites offering ERCP procedures, including: • Microbiological culture with quarantine of contaminated endoscopes until culture results become available; • Meticulous cleaning and HLD followed by one of: • ETO sterilization, • Liquid chemical sterilization, or • Repeat HLD.
The FDA safety alert was followed by a CDC Health Advisory in September 2015, that indicated an immediate need for sites utilizing duodenoscopes to undertake audits of the reprocessing protocol, as well as staff training and longitudinal audits to document ongoing staff competency.
These events and actions have led to a "paradigm shift" regarding the reprocessing of flexible endoscopes, 113 whereby the need for a total quality systems approach has been recognized.
It is no longer adequate to accept that endoscope cleaning is being done properly just because the MIFUs are available to staff; rather, there needs to be evidence of monitoring and ongoing audits of cleaning compliance for all staff who reprocess endoscopes.
In addition, the need to ensure the endoscopes are truly dry during storage to prevent biofilm formation has been Another stage where monitoring of the endoscope can be done is post-HLD.
A Rapid Post-Disinfection Test (RPDT) that could be completed just prior to the next patient use of the scope to confirm that the endoscope does not contain viable microorganisms would be ideal.
However, there is little published data for indicates that there may already be build-up biofilm (BBF) in the scopes being used.
Remedial action would be needed for the endoscopes, which may include a longer soak time in detergent followed by extended brushing and flushing (as per scope manufacturer's input).
If the endoscope fails the RCT after the remedial action, then it should be sent to the manufacturer for further remedial action (e.g., change of channels, etc.).
There must be documentation of all reprocessing components to ensure there is a way to link which endoscopes (and corresponding accessories) were used on which patient.
• Document which endoscope was used in the patient's medical record • Document in the reprocessing area which personnel cleaned each endoscope, which patient the endoscope was used on, the date/time it was cleaned, and which AER (automated endoscope reprocessor) was used to disinfect the endoscope (or which sterilizer was used).
• Ensure the MIFU for reprocessing is available in the reprocessing area • Create a site-specific set of instructions that are based on the MIFU but indicate specific detergent, brushes, etc.
that are used for reprocessing of each make/model of endoscope used at that site • If pump-assisted flushing is used during manual cleaning, ensure instructions for use are available • For manual cleaning, a succinct visual "aide" consisting of a summary of the process posted above the reprocessing sink, counters, etc., is useful.
Table 4 .2 for additional information) • Traceability of each endoscope and reusable accessories used • Documentation of all monitoring performed for cleaning and minimum effective concentration (MEC) testing • Timely reprocessing • Routine cleaning and decontamination protocol for AER, flushing pump, sinks, connector tubing, endoscope storage cabinets • Policy on disposable and reusable ancillary items (e.g., water bottles, connector tubing, etc.)
• Preventative maintenance (PM) program for endoscopes, AERs, flushing pump with repair history records • Record keeping of preventative maintenance on all equipment • Regular audits to ensure ongoing adequacy of all stages of the program • Involvement of Infection Prevention and Control (IPAC) and workplace safety in all components of the endoscopy reprocessing is crucial • A structured management scheme with regular review of the endoscopy program that includes reprocessing considerations.
• There should be regular review and reporting of monitoring data at appropriate management meetings to identify any potential issues The bedside clean reduces the load of organic material and microbes for the full manual cleaning step.
Detergent or water solution (as per endoscope manufacturer's instructions) is used to: • Wipe exterior using sponge or lint-free cloth • Suction or flush ALL channels (Note: this may require use of specific endoscope flushing adaptors as per MIFU) • Place all accessory components in detergent or water to keep moist during transport.
1.
Forget to wipe exterior or forget to suction or flush some channels after patient procedure.
1.
Dismantle reusable components such as valves, caps, flushing connectors etc.
and process along with endoscope.
Ensure endoscope is totally immersed in detergent solution.
3.
Ideally suction detergent through all channels and discard this material (not in the cleaning basin).
Brush all openings including suction cylinder, air/water cylinder, instrument port, and outlets on umbilical end.
5.
Adequate contact time with detergent (as per detergent manufacturer's instructions) to loosen debris.
6.
Rinse away detergent using tap water.
1.
Reusable components not properly cleaned.
Performing brushing while endoscope is not immersed.
into the detergent solution.
4.
Inadequate brushing of channels due to improper brush size or confusion regarding which brush to use.
Inadequate contact time with detergent.
6.
Many detergents are protein solutions that need to be removed before the disinfection/sterilization step.
1.
Increased risk of accumulation of organic material and biofilm forming.
Increased risk to personnel and environment due to aerosols of infectious debris.
Contamination of detergent solution with high levels of patient secretions increases the risk of high levels of organic material and microbes on exterior and in channels.
Inadequate brushing leads to improper cleaning and risk of excessive organic and microbial load for HLD.
of organic material and microbes.
6.
If detergent is not adequately rinsed away this could lead to accumulation of protein and failure of HLD.
1.
Once manual cleaning has been completed collect a sample from the channel(s) of the endoscope.
Minimally a sample from the instrument port to the distal end should be collected and tested.
Follow the MIFU for the rapid cleaning monitor.
If the test result is below the manufacturer's cut-off for adequate cleaning, then the scope can be transferred to the AER for HLD (or manual HLD done) or sent for sterilization.
If the test result is above the MIFU cut-off, then the endoscope needs to be fully recleaned and tested after the second manual clean.
NOTE: For duodenoscopes there should also be a sample from the elevator lever recess.
If the endoscope fails the rapid cleaning test after three rounds of cleaning it should be sent to the endoscope manufacturer for evaluation.
Breach of any of the manual cleaning steps shown in Fig.
4 .1 can lead to patient-derived material remaining in the endoscope that is sent for HLD or sterilization.
HLD or sterilization processes fix the organic residues to the channel surface leading to build-up biofilm formation (BBF).
1.
Accumulation of BBF in the endoscope increases the risk of microbes in the BBF matrix surviving HLD and possibly being transmitted to subsequent patients.
Disinfection or Sterilization HLD and sterilization are intended to kill any remaining microbes left after the cleaning step.
HLD and sterilization can achieve 6 and 12 Log 10 microbial reductions, respectively.
All reusable accessory items must also be exposed to HLD or sterilization along with the endoscope they are dedicated to.
Ideally an automated endoscope reprocessor (AER) should be used for HLD or sterilization.
Manual HLD instructions are available from manufacturers but the manual process is fraught with concerns including ensuring adequate vapor control to prevent staff exposure to toxic fumes, adequate channel perfusion when using manual syringe to inject HLD, dilution of HLD from water remaining in channels after manual cleaning, etc.
When using an AER, it is necessary to change the sub-micron filter inside the AER as per MIFU.
3.
Fully reprocessed endoscopes and accessory components need to be handled using clean gloves to ensure skin organisms from bare hands are not transmitted to the endoscope post-HLD.
1.
Inadequate HLD contact due to poor manual perfusion of channels (e.g., bubbles or inadequate immersion).
Breach in sub-micron filter inside AER leads to contaminated rinse water.
Handling of endoscope post-HLD with un-gloved hands 1.
Increased risk of microbes surviving the HLD process due to inadequate contact with HLD.
Microbes on the endoscope when put into storage can facilitate biofilm formation.
3.
Microbes on hands transferred to disinfected endoscope can survive and subsequently be transmitted to next patient that the endoscope is used for.
Microbes transferred to endoscope from hands also increase the risk of biofilm formation if there is sufficient moisture.
1.
Through drying of endoscope exterior and ALL channels is critical prior to storage.
This can be done using appropriately filtered forced air.
If a "channel-purge" storage cabinet is used the manual drying only needs to be done briefly to remove excess water prior to placing the endoscope in the cabinet (ongoing air purging once the endoscope is placed in the cabinet is preferred to ensure dryness is maintained as additional wet endoscopes are sequentially placed into the storage cabinet).
There are also small air-flushing pumps that can be used to ensure channel drying prior to placing the endoscope in a regular storage cabinet.
Ensure adequate time is used for air-flushing to ensure scope is totally dry before being placed in a regular storage cabinet.
2.
Ensure fully reprocessed endoscopes are somehow labeled or identified so they can be easily differentiated from patient-used endoscopes that are contaminated.
1.
The volume of alcohol flushed and the time for manual forced air drying is not specified in MIFU and is often sub-optimal when performed manually.
2.
Contaminated endoscope may be mistaken for a fully reprocessed endoscope and accidentally used on a patient.
1.
Inadequate drying during storage is one of the key factors that leads to biofilm formation.
Once formed, the biofilm protects microbes from being adequately killed by HLD or sterilization and increases the risk of organisms being transmitted to patients exposed to this contaminated endoscope.
Use of a contaminated endoscope can lead to transmission of infectious agents that result in colonization or infection.
2.
Contamination of endoscope may lead to microbial replication and biofilm formation if moisture is also present.
Valves inserted into the valve cylinders can lead to moisture retention that facilitates microbial replication and can lead to biofilm formation.
endoscope reprocessing.
As outlined by recent guidelines, 48, 107, 108, 123 initial training and ongoing competency assessment are critical to ensuring that staff can effectively reprocess flexible endoscopes.
The compliance of reprocessing personnel with endoscope reprocessing protocols should be reviewed at least annually to document ongoing competency.
48 It is clear from some outbreaks that despite having adequate written protocols, staff may create breaches by not following some steps in the protocol.
95, 98, 115 As such, observational audits are a useful approach to determining if staff are fully compliant in following the site protocol.
If ongoing cleaning monitoring is performed, the results of these tests can be included as part of documentation of ongoing competency.
Transmission of exogenous pathogens (i.e., not derived from the patient) can be categorized as "nonendoscopic," which is related to care of intravenous lines and administration of medications and anesthesia, or "endoscopic," which is related to transmission by the endoscope, water bottles, and its accessories.
Outbreaks of infection have been traced to process failures, including endoscopes that are damaged or difficult to clean; AER design problems or failures such as breakdowns in AER water filtration systems; and lack of adherence to reprocessing guidelines for endoscopes and accessories.
There are also data that demonstrate that all the steps associated with manual endoscope reprocessing are rarely performed and some essential steps, such as brushing all endoscope channels and adequate drying prior to storage, are frequently deficient.
115 These deficient reprocessing practices can be summarized as follows: 32,96,115 1.
Inadequate or absent mechanical cleaning of the endoscope and channels before disinfection.
the two RPDT tests that are currently available (Table 4 .6).
In the absence of validated rapid test methods, culture is the only well-studied method for detection of microbial contamination of flexible endoscopes postdisinfection/-sterilization.
However, there are a number of considerations when culture is used.
There have been a variety of published studies on culture results from endoscopes, but there is little data on the recovery efficiency of the various endoscope extraction methods that have been used.
137 If a patient-ready endoscope is extracted by flushing the channel with bacterial culture media or other harvesting fluids containing various proteins or buffers containing salt, then the endoscope requires recleaning and disinfection prior to being used on a patient.
If sterile, high-quality water (i.e., reverse osmosis or deionized water) is used to flush endoscope channels, the endoscope can be dried and then still be safely used on the next patient.
Personnel who reprocess flexible endoscopes must have thorough initial training regarding the reprocessing of all makes and models of endoscopes that they will be responsible for reprocessing (see Tables 4.3 and 4.4 ).
The training process should be documented and new staff not allowed to reprocess endoscopes on their own until they have demonstrated, under supervision, that they are competent to perform reprocessing independently.
The use of rapid cleaning monitor (RCM) tests for each endoscope reprocessed during training is an excellent way to document the adequacy of the trainee's ability to perform the cleaning process.
Reprocessing errors are a common underlying problem for many of the reported outbreaks.
95, 97, 98, 123 The "human factors" study done by 115 showed that inadequate cleaning of channels related to the lack of adequate channel brushing (43% of scopes) and inadequate drying (45% of scopes) prior to storage of endoscopes were the two most common breaches in The CDC guidelines for safe injection practices include the following recommendations: 150 • Use aseptic technique when preparing and administering medications and fluids.
• A sterile, single-use, disposable needle and syringe should be used for each injection on a single patient.
• Do not administer medications from single-dose vials or use IV solutions as a common source of supply for multiple patients.
• Do not keep multidose vials in the immediate patient treatment area.
• Do not reuse a syringe to access or administer medications from a vial that may be used on multiple patients, even if the needle is changed.
• In times of critical need, medications from unopened singledose/single-use vials can be subdivided for multiple patients.
However, this should only be performed by qualified health care personnel in accordance with standards in the United States Pharmacopeia chapter on Pharmaceutical Compounding.
3.
An inadequate disinfectant was used or used improperly at an incorrect concentration, temperature, or exposure period.
4.
Flawed or malfunctioning AER units or use of incorrect connectors.
20,101,142 5.
Failure to disinfect or sterilize the irrigation bottle of the endoscope regularly.
143,144 6.
Endoscopic accessory instruments were not sterilized.
7.
The endoscope and all channels were not dried adequately before storage.
8.
Unrecognized problems with water supply.
Outbreaks of hepatitis B and C viruses have occurred due to failure to follow fundamental principles of aseptic technique and safe injection practices.
145, 146 These included improper handling of intravenous sedation tubing, reuse of syringes and needles, and use of single-dose or single-use medical vials on multiple patients.
146 • Detects viable organisms of high concern including Gram negatives and Gram positives.
• Allows assessment of whether the bacteria detected are multi-antibiotic resistant • For outbreak investigation allows for genetic typing methods (e.g., PFGE) to help identify a point-source outbreak • Requires 48 to 72 hrs before results of culture are reported.
• During outbreak investigation, quarantine of the endoscope pending culture results is necessary.
• For routine surveillance (i.e., not an outbreak investigation) the endoscope is often not quarantined and may be used on multiple patients before culture results are available.
Sites need to have a response plan in place regarding notification if culture shows organisms of concern on an endoscope that has been used on multiple patients (i.e., notify the patient, the doctor or both?)
not be detected after HLD as these organisms commonly result in a clinically significant infection including gram-negative bacteria (e.g., Escherichia coli, Klebsiella spp., Shigella spp., Salmonella spp., Pseudomonas aeruginosa, other Enterobacteriaceae) as well as Staphylococcus aureus, and Enterococcus spp.
159 LCO are less often associated with disease; these bacteria typically include coagulase-negative staphylococci, micrococci, diptheroids, and Bacillus spp.
The levels of LCO on a surveillance endoscope culture can vary depending on the reprocessing, handling, and culturing practices in a facility.
Typically, fewer than 10 colony forming units (CFU) of LCO does not require intervention as this most likely represents collection process contamination rather than a significant problem with the disinfection or cleaning process.
159 Interpretation of culture results with 10 or greater CFU of LCO should be considered in the context of typical culture results at the facility.
111 Any endoscope found to be contaminated with a HCO or unacceptable CFU of LCO should cause concern and lead to repeat endoscope reprocessing followed by post-reprocessing cultures.
The endoscope should be quarantined until it has been demonstrated to be free of HCO and has an acceptable level of LCO.
Positive cultures should also prompt a review of the endoscopy unit reprocessing procedures to ensure adherence to the manufacturer's reprocessing instructions and to ensure proper culture methodology.
If a reprocessing breach is identified, appropriate facility personnel should be notified and corrective actions should be immediately implemented.
When bacteria are persistently recovered by surveillance cultures, refer to the manufacturer's instructions 111, 159 for evaluating the endoscope for mechanical defects and consider having the endoscope evaluated by the manufacturer.
In addition, when ineffective reprocessing is suspected based on surveillance cultures, it might be helpful to review positive cultures among affected patients to determine whether transmission of relevant pathogens could have occurred.
111, 159 The vast majority of exogenously acquired endoscope-related infections have been caused by bacterial transmission.
The bacteria involved have been true pathogens, which always have the potential to cause infection (e.g., Salmonella spp.
), or opportunistic pathogens that cause infection if the microbial load is sufficient and/or host-factors are permissive (e.g., Pseudomonas aeruginosa).
In the hierarchy of relative resistance to HLD, vegetative bacteria such as Pseudomonas spp.
and Salmonella spp.
are the most susceptible to disinfectants, whereas the mycobacteria are less susceptible and bacterial spores (e.g., Bacillus subtilis and Clostridium difficile) are the most difficult to eliminate (see Box 4.1).
Nevertheless, as previously stated, all bacteria with the exception of a few bacterial spores are highly sensitive and eliminated by HLD.
Salmonella is a serious primary pathogen, and Pseudomonas is ubiquitous in many water sources, and although both these pathogens have been associated most frequently with endoscopic transmission, they are both sensitive to multiple agents, including glutaraldehyde, and other HLDs.
Transmission of bacterial pathogens from flexible endoscopes has been rare since the adoption of the current 2011 multisociety reprocessing guideline, 45, 160 with the exception of duodenoscope-related infections (discussed later).
The most commonly reported infectious agents transmitted during GI endoscopy have been Pseudomonas aeruginosa (45 cases) [161] [162] [163] and Salmonella spp.
(84 cases).
17 Isolated reports of More health care-associated outbreaks and patient exposures have been linked to contaminated endoscopes than to any other reusable medical device.
32, 102 Nevertheless, endoscopy-related transmission of infection is very low and was originally estimated to have an incidence of approximately 1 infection per 1.8 million procedures.
3, 17 This is very likely an underestimate, as many endoscopy-related infections go unrecognized because of inadequate or nonexistent surveillance programs, the absence of clinical symptoms in many patients who are colonized, a long lag time between colonization and clinical infection, and the fact that the pathogens transmitted by endoscopy are often normal enteric flora.
151 Endoscope-related transmission of bacterial infection has been rare since the adoption of the current multisociety reprocessing guidelines.
45, 151 However, recent outbreaks have occurred with duodenoscopes even when the manufacturers and societal guidelines were reportedly followed correctly (see later section on Duodenoscope-Related Infections).
152 The primary concern raised by infectious outbreaks is that current reprocessing guidelines are not adequate to ensure patient safety when undergoing endoscopic procedures.
Endoscopes can harbor between 10 9 and 10 12 enteric organisms at the completion of some patient procedures.
This bioburden is reduced by cleaning (i.e., bedside precleaning followed by manual cleaning) by a factor of 2 to 6 log 10 and the HLD step is expected to provide a 6 log 10 reduction of any microbes remaining after cleaning.
124, 153 Therefore the margin of safety associated with cleaning and HLD of GI endoscopes is low, and any deviation from proper reprocessing could lead to failure to eliminate contamination, with a possibility of subsequent patient-to-patient transmission.
Biofilms can contribute to reprocessing failure and endoscoperelated infectious outbreaks.
154 Biofilms form in endoscope channels, in AERs, and within municipal and hospital water supplies as multilayered bacteria within exopolysaccharide.
These biofilms protect the bacteria against physical (e.g., brushing, fluid flow) and chemical (e.g., disinfectant) forces, making the microorganisms more difficult to remove or completely kill by HLD.
151, 155 There is evidence that accumulation of fixed material within endoscope channels occurs over repeated usage.
Biofilms that develop in endoscopes and AERs may not be detectable by surveillance culture, as cleaning and disinfection may have destroyed bacteria within the superficial layers but not those within the deeper layers.
Prompt, meticulous, manual cleaning to remove biologic material and strict adherence to reprocessing protocols is the optimal approach to reduce biofilm formation/accumulation.
[155] [156] [157] [158] Better biofilm removal protocols are needed to address this issue.
Pathogens of concern to the GI endoscopy community and general public include Clostridium difficile, Helicobacter pylori, Escherichia coli, norovirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), and multidrug resistant organisms (MDROs) such as M. tuberculosis, vancomycin-resistant enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and CRE.
All these established pathogens are susceptible to currently available chemical disinfectants and sterilants.
17, 32 Low Concern Organisms (LCO) versus High Concern Organisms (HCO) Surveillance cultures of endoscopes are assessed for two general categories of microbial growth, LCO and HCO.
HCO should be changed to protect our patients from CJD, citing no reported cases of CJD transmission by endoscopy and the lack of exposure to high-risk CNS tissue during endoscopic procedures.
17, 183 vCJD is a rare but fatal condition caused by the consumption of beef contaminated with a bovine spongiform encephalopathy agent.
It differs from CJD in that the mutated prion protein can be found in lymphoid tissue throughout the body, including the gut and tonsils.
182, 184 Only three cases of vCJD have been reported in the United States, and all three patients contracted the disease elsewhere.
184 As vCJD is resistant to conventional disinfectants and sterilants, endoscopy should be avoided, if at all possible, in patients known to harbor this agent.
17, 184 Endoscopes used in individuals with definite, probable, or possible vCJD should be destroyed after use or quarantined to be reused exclusively on that same individual if required.
182, 184 Between 2012 and 2015, duodenoscopes resulted in 25 international outbreaks (at least eight in the United States) of antibiotic-resistant infections with CRE and other MDROs that sickened a reported 250 patients and resulted in 20 deaths.
16, 97, 103, 138, [185] [186] [187] In addition, transmission resulting in a long-term carrier state has been recognized as a risk of exposure to contaminated duodenoscopes.
Long-term carriage has important clinical implications due to the development of a delayed infectious complication weeks to months later or patient-topatient transmission of pathogens when these carriers are subsequently admitted to health care or chronic care facilities.
Investigative cultures identified persistent contamination of duodenoscopes as the cause for patient infections with MDROs in most of the outbreaks.
185, 188 Furthermore, these duodenoscopeassociated infections occurred even though the sites reported strict adherence to reprocessing procedures according to manufacturer's instructions and professional guidelines.
14, 16, 97, 142, 189 It is likely that MDROs are acting as a marker for ineffective reprocessing due to the complex design of duodenoscopes that have difficultly reaching crevices and channels involving the elevator mechanism where persistent colonization was identified.
186, 190 Duodenoscopes that persistently yield positive cultures likely harbor biofilms that cannot be eradicated with standard reprocessing.
155 In October 2015 the FDA and the CDC released an official health advisory alerting health care facilities to review their reprocessing procedures.
[191] [192] [193] [194] [195] [196] [197] [198] [199] [200] [201] [202] In response to the problems with duodenoscope reprocessing, the FDA requested all three duodenoscope manufacturers to revise and validate their reprocessing instructions with provisions for additional duodenoscope reprocessing measures.
191 This led to the modification of manufacturers' reprocessing protocols with a larger emphasis on precleaning and manual cleaning before HLD.
117, [192] [193] [194] One duodenoscope manufacturer (Olympus; Center Valley, PA) subsequently modified the design of the closed elevator channel to create a tighter seal.
195, 196 In addition, the FDA has recommended that health care facilities performing ERCP consider employing supplemental measures for duodenoscope reprocessing when facilities have the resources to do so.
110 Most sites where outbreaks have occurred have chosen per procedure ETO sterilization after HLD as its primary reprocessing method, and in all reported instances, ETO has prevented further MDRO transmission.
110, 185 However, one site reported failure to eliminate MDRO contamination of a duodenoscope after HLD and ETO.
116 Alternative reprocessing endoscopic transmission of other enteric bacteria include Klebsiella spp., 164 Enterobacter spp., 165 Serratia spp., 166 and Staphylococcus aureus.
164 The few reports of endoscopic transmission of Helicobacter pylori were related to inadequate reprocessing of endoscopes and biopsy forceps.
[167] [168] [169] Current reprocessing guidelines are shown to inactivate Clostridium difficile spores, 170 and no cases of endoscopic transmission of this infection or mycobacteria have been reported.
In summary, there have not been any observed GI endoscopy-related transmission of bacterial pathogens since introduction of the currently accepted reprocessing standards with the exception of duodenoscope-related outbreaks (discussed later).
Much greater anxiety is associated with the possibility of transmission of viral infections.
This anxiety is surprising because the viruses of greatest concern (HBV, HCV, and HIV) are among the easiest microorganisms destroyed with standard reprocessing.
171 Transmission of viral pathogens by GI endoscopy procedures is rare because these microorganisms are obligate intracellular microorganisms that cannot replicate outside living tissue.
Thus, even when a flexible endoscope is contaminated with viral pathogens, the burden of virus cannot increase, as they are not capable of ex vivo replication.
Enveloped viruses (e.g., HIV, HBV, HCV) die readily once dried and are more readily killed by HLD compared to nonenveloped viruses (e.g., enteroviruses, rotavirus), which can survive in dry conditions.
There has been concern about the possibility of HIV transmission by flexible GI endoscopy; however, no cases have been reported to date.
[171] [172] [173] There is only one well-documented case of HBV transmission by GI endoscopy that occurred in the setting of inadequate endoscope reprocessing.
174 However, transmission of HBV is very rare or does not occur when accepted reprocessing guidelines are followed.
175 The presence of fungi is associated with prolonged storage of flexible endoscopes.
Although transmission of Trichosporon beigelii and Trichosporon asahii occurred in the 1980s, 176, 177 there are no documented cases of fungal infections by GI endoscopy when updated reprocessing guidelines are followed.
A single publication in the 1970s reported circumstantial evidence of Strongyloides stercoralis transfer to four patients from a contaminated upper endoscope.
No subsequent reports of parasite transmission by GI endoscopes have been identified.
178 Creutzfeldt Jacob Disease (CJD) and variant CJD (vCJD) are degenerative neurologic disorders transmitted by proteinaceous infectious agents called prions.
All prions remain infectious for years in a dried state, and resist all routine sterilization and disinfection procedures commonly used by health care facilities.
17, [178] [179] [180] [181] CJD is confined to the central nervous system (CNS) and is transmitted by exposure to infectious tissues from the brain, pituitary, or eye, whereas tissues and secretions that come into contact with the endoscope during procedures, such as saliva, gingival tissue, intestinal tissue, feces, and blood, are considered noninfectious by the World Health Organization.
17, [179] [180] [181] [182] The CDC and other infection control experts conclude that current guidelines for cleaning and disinfecting medical devices need not will help with the investigation and lead to an expeditious correction of any deficiencies that are identified.
• A user facility is not required to report a device malfunction, but it can voluntarily advise the FDA of such product problems using the voluntary MedWatch Form FDA 3500 under FDA's Safety Information and Adverse Event Reporting Program.
201 However, if a device failure leads to a death or serious injury, the FDA and the manufacturer must be contacted, as outlined in facility policies, by the designated individual or department at the facility.
202 The FDA encourages health care professionals, patients, caregivers, and consumers to submit voluntary reports of significant adverse events or product problems with medical products to MedWatch (https://www.accessdata.fda.-gov/scripts/ medwatch/), the FDA's Safety Information and Adverse Event Reporting Program • Manufacturers are required to report to the FDA when they learn that any of their devices may have caused or contributed to a death or serious injury or when they become aware that their device has malfunctioned and would be likely to cause or contribute to a death or serious injury if the malfunction were to recur.
201,202 6.
Patient notification and counseling: 200, 203 in instances where a breach in the reprocessing protocol or damaged equipment poses a risk to patients for adverse events, it becomes the institution's ethical obligation to notify patients in a timely manner.
Notification may be accomplished by a direct meeting, telephone call, and letter sent by registered mail.
The content should include an assessment of the risk, possible adverse events that may occur, symptoms and signs of the adverse event, time range for the adverse event, risk to other contacts, possible prophylactic therapy (including benefits and risks), and recommended medical follow-up.
Prompt notification allows patients to take precautions to minimize the risk of transmitting infection to others and allows for early serologic testing.
This may help distinguish chronic infections from those potentially acquired at the time of endoscopy and to permit earlier initiation of treatment for newly acquired infections.
On the other hand, adverse publicity associated with the reporting of a reprocessing error might lead patients to avoid potentially life-saving endoscopic procedures because of an unwarranted fear of infection.
Personal counseling should be offered to all patients.
The risk of infection should be discussed and placed in context to minimize patient anxiety.
Patients should be advised against donating blood and tissue products and engaging in sexual contact without barrier protection until all serologic testing is complete.
A toll-free helpline should be established to provide information to all patients at risk.
7.
Develop a long-term follow-up plan (e.g., long-term surveillance, changes in current policies and procedures) and prepare an after action report.
There are risks related to infection transmission to personnel who handle patient-used endoscopes as well as to patients.
Sites offering endoscopy procedures need to ensure the risk to personnel and patients is minimized.
Flexible GI endoscopes are expensive and easily damaged.
Unlike surgical instruments where the microbial load is less than 100 methods employed have included double HLD after each procedure 110, 185 or HLD with duodenoscope quarantine until negative culture results are obtained.
15 Another supplemental option for reprocessing endorsed by the FDA includes the use of a liquid chemical sterilant processing system.
110, 197 Surveillance microbiological culturing should be considered in addition to these supplemental reprocessing measures.
This involves sampling the duodenoscope channels and the distal end of the scope to identify any bacterial contamination that may be present on the scope after reprocessing.
159, 198 It must be recognized that the sensitivity of surveillance culturing of the elevator channel, the elevator lever cavity, or other scope channels is unknown.
Until there are evidence-based guidelines, individual hospitals should choose from these different options based on available information and feasibility for their medical practice.
However, at a minimum, there should be an audit of all facilities offering duodenoscope procedures to ensure the site has a quality system in place and is compliant with current MIFUs and guidelines.
Breaches of disinfection guidelines and device failures (e.g., endoscopes or AERs) are common in health care settings, resulting in potential patient injury or infection transmission.
31 The identification of such a problem may stem from the result of microbiologic surveillance cultures, an infectious outbreak within an institution or isolation of a pathogen from individuals having a recent endoscopic procedure, identification of a break in reprocessing protocol, or a visibly faulty device.
Endoscopy facilities should have written policies on the roles and responsibilities within the organization to identify, report, and analyze these failures.
81 The investigation of a breach in reprocessing or resultant outbreak should be undertaken using a standardized approach.
It should focus on the identification of factor(s) that led to the exposure and protect patients from potential adverse events.
The investigation should not be punitive and not attempt to assign blame to any particular individual.
Rutala et al (2007) 199 described a process for exposure investigation, and the ASGE has published guidelines for patient assessment and notification when there is a suspected failure in the disinfection or sterilization protocol.
200 These can be summarized as follows: 199, 200 1.
Confirm that the reprocessing failure occurred and assess the duration of exposure (e.g., review sterilization methods and AER records of biological parameters).
2.
Quarantine any endoscopes or associated accessories that malfunctioned or are at risk for inadequate reprocessing.
3.
Do not use the devices in question, such as the endoscope or AER, until proper functioning is confirmed.
4.
Prepare a list of potentially exposed patients, dates of exposure, and inadequately reprocessed or malfunctioning devices used.
5.
Reporting: • Inform facility leadership: breaches in patient safety with serious potential infection risks should be reported to facility leadership, including infection control, risk management, public relations, legal department, and selectively to local/state public health agencies, the FDA, CDC, and the manufacturers of the involved equipment.
200, 203 This The workflow should proceed from "dirtiest to cleanest" in the reprocessing area, and there should be physical separation of "dirty" reprocessing areas and "clean" areas.
48, 107, 108 This requires appropriate removal of PPE and hand hygiene when leaving the dirty reprocessing area to enter any of the clean areas.
Staff should take every precaution to reduce the generation of aerosols during reprocessing of GI endoscopes.
This includes total immersion of the endoscope during cleaning.
48, 108 This ensures that any patient material removed from the channels during cleaning is contained within the detergent cleaning solution.
Care is needed to ensure all brushing steps are done underneath the water surface to reduce aerosols.
Holding the control head above water to insert the channel brush and then pulling the brush out of the channel while the control head is above the water generates significant aerosols of the contaminated detergent solution.
In addition, during the air-flushing process after cleaning is completed, a piece of gauze should be placed over the distal end of the endoscope channel prior to placing it in an AER to prevent creation of aerosols when flushing out residual rinse water.
A final, often overlooked step, is rinsing and decontamination of the sink after EACH endoscope is cleaned.
This ensures that the sink does not accumulate microbial contamination over time and act as a reservoir within the reprocessing area to contaminate reprocessing personnel or other endoscopes.
If flushing pumps are used as part of the manual cleaning step, they also require routine (usually daily) decontamination as per MIFU to ensure they do not become a reservoir of microbes that develop biofilm and subsequently contaminate endoscopes that they are used on.
Any single-use disposable sharps used in the procedure room should be disposed of in appropriate sharps containers in the procedure room.
There should be no single-use disposable sharps transported to the reprocessing area.
If there are reusable sharps (e.g., biopsy forceps) used for patient procedures, these should be appropriately transported to the reprocessing area in a labeled, rigid, sealed container that ensures separation from the endoscope.
This reduces the risk that the biopsy forceps (or other sharp accessory device) could damage the endoscope during transit.
Reprocessing of reusable sharps requires specific MIFU and adequate staff training to reduce the risk of sharps injuries to reprocessing personnel.
Single-use, disposable accessories are preferred to eliminate the risks associated with reprocessing of reusable sharps.
14 bacteria for 75% of instruments, 139, 204 the load of microorganisms in channels of flexible endoscopes can be as high as 10 10 bacteria 124 per instrument channel (e.g., for colonoscopes).
During transport from the procedure room to the reprocessing area, 48 flexible GI endoscopes require a rigid, sealed container that is appropriately labeled as biohazardous.
This protects the endoscope from accidental damage and also ensures that any patient-derived secretions and microorganisms are adequately contained and cannot drip out and contaminate the environment.
All reusable accessory items (valves, flushing adaptors, cleaning valves, etc.)
should be transported along with the associated endoscope.
During transport, the endoscope and all accessory items should be kept moist to prevent drying of patient-derived material.
If endoscopes are transported to a central reprocessing facility, evaluation of the time of transport should be conducted to determine the frequency of excessive transit times.
There are risks to reprocessing personnel being exposed to patient-derived infectious materials.
Endoscopes contacting the GI tract can have very high levels of infectious organisms (including bacteria, viruses, fungi, etc.)
in channels or on the endoscope surface.
To mitigate these risks, reprocessing personnel need to be trained regarding standard precautions, personal protective equipment (PPE), hand hygiene, disposal of sharps, and dealing with chemical and/or infectious material spills.
Standard precautions are required when reprocessing any patient-used medical device.
This means that staff treat all patient-used endoscopes as potentially infectious regardless of the underlying known illnesses that patients might have (e.g.
Clostridium difficile infection, VRE colonization, human papilloma virus infection, candidiasis, etc.).
Any handling of GI endoscopes should be done with due consideration to the potential to transmit infectious microorganisms to reprocessing personnel.
Staff must be trained in appropriate PPE and reprocessing considerations aimed at reducing the generation of aerosols.
It is critical that appropriate PPE be available 48,107,108 and include a gown (preferably a water-resistant gown), gloves (appropriate to the task), and a face shield/mask.
Reprocessing personnel must be adequately trained in the proper donning and doffing of all PPE.
Gowns, gloves, and a full-face shield (or combined face shield/mask) are required for cleaning of flexible endoscopes.
The reprocessing staff needs to be trained in the appropriate use of protective gloves, as well as hand hygiene after removing gloves.
Utility gloves used for cleaning of endoscopes should never be used at other stages in endoscope reprocessing (i.e., they are dedicated to the cleaning sinks).
Disposable examination gloves must be available for handling cleaned endoscopes during connection to the AER.
Fresh disposable gloves are needed for removing and handling fully reprocessed endoscopes from the AER and during manual channel drying and placing the endoscope into the clean storage cabinet.
Fresh disposable gloves should also be used whenever an endoscope is removed from the clean storage cabinet.
The use of gloves helps protect both the reprocessing personnel from contamination with patientderived microorganisms and the fully reprocessed endoscope from contamination with skin-derived microorganisms from reprocessing personnel.
Staff should always perform hand hygiene immediately after removing any type of glove.
Handwashing sinks with appropriate soap, as well as waterless hand hygiene agent dispensers, must be available in the reprocessing area.
Endoscope reprocessing methods: a prospective study on the impact of human factors and automation Automated endoscope reprocessors Society of Gastroenterology Nurses and Associates: Guideline for use of high level disinfectants & sterilants for reprocessing flexible gastrointestinal endoscopes Food and Drug Administration: FDA-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices Food and Drug Administration: Reprocessing medical devices in health care settings: validation methods and labeling.
Guidance for Industry and Food and Drug Administration Staff Food and Drug Administration: Supplemental measures to enhance reprocessing: FDA Safety Communication Current issues result in a paradigm shift in reprocessing medical and surgical instruments Society of Gastroenterology Nurses and Associates: Standards of infection prevention in reprocessing flexible gastrointestinal endoscopes Impact of ethylene oxide gas sterilization of duodenoscopes after a carbapenem-resistant Enterobacteriaceae outbreak The use of channel-purge storage for gastrointestinal endoscopes reduces microbial contamination Comparison of clinically relevant benchmarks and channel sampling methods used to assess manual cleaning compliance for flexible gastrointestinal endoscopes The ATP test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels The ATP test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels Control and Prevention: Interim sampling method for the duodenoscope -distal end and instrument channel Control and Prevention: Interim protocol for healthcare facilities regarding surveillance for bacterial contamination of duodenoscopes after reprocessing United States Senate; Health, Education, Labor, and Pensions Committee: Preventable tragedies: superbugs and how ineffective monitoring of medical device safety fails patients.
Minority staff report Risk of transmission of carbapenem-resistant Enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy Food and Drug Administration: Design of endoscopic retrograde cholangiopancreatography (ERCP) duodenoscopes may impede effective cleaning: FDA Safety Communication Reprocessing failure Antimicrobial efficacy of endoscopic disinfection procedures: a controlled, multifactorial investigation High-level disinfection of gastrointestinal endoscopes: are current guidelines adequate?
Microbial bioburden in endoscope reprocessing and an in-use evaluation of the high-level disinfection capabilities of Cidex PA Efficacy of high-level disinfectants for reprocessing GI endoscopes in simulated in-use testing What is disinfection, sterilization?
(letter) Food and Drug Administration: Guidance for industry and FDA reviewers: content and format of premarket notification [510(k)] submissions for liquid chemical sterilants/high level disinfectants Are all sterilization processes alike?
Low-temperature sterilization technologies: do we need to redefine sterilization?
Disinfection and sterilization of patient-care items Centers for Disease Control and Prevention: Guideline for disinfection and sterilization in healthcare facilities Guideline for disinfection and sterilization of prion contaminated medical instruments Disinfection: is it time to reconsider Spaulding?
Chemical disinfection and antisepsis in the hospital Disinfection of medical and surgical materials Food and Drug Administration: Draft guidance for the content of premarket notifications for endoscopes used in gastroenterology and urology CDC guideline for handwashing and hospital environmental control Reprocessing of flexible gastrointestinal endoscopes APIC guidelines for infection prevention and control in flexible endoscopy American Society for Testing and Materials: Standard practice for cleaning and disinfection of flexible fiberoptic and video endoscopes used in the examination of the hollow viscera Guideline implementation: processing flexible endoscopes New developments in reprocessing semicritical items In vitro evaluation of integrity and sterilization of single-use argon beam plasma coagulation probes Multisociety guideline on reprocessing flexible gastrointestinal endoscopes Department of Health and Human Services Burden of gastrointestinal disease in the United States: 2012 update Overview of infection control problems: principles in gastrointestinal endoscopy Infectious disease complications of GI endoscopy: part II, exogenous infections ERCP scopes: what can we do to prevent infections?
American Society for Gastrointestinal Endoscopy: Guidelines for safety in the gastrointestinal endoscopy unit Transmission of infection by gastrointestinal endoscopy and bronchoscopy Lessons from outbreaks associated with bronchoscopy Jr: The prevention of infection following gastrointestinal endoscopy: the importance of prophylaxis and reprocessing An outbreak of Mycobacterium chelonae infection following liposuction Mycobacterium chelonae causing otitis media in an ear-nose and-throat practice Centers for Disease Control and Prevention: Pseudomonas aeruginosa infections associated with transrectal ultrasound-guided prostate biopsies-Georgia Prevention of flexible bronchoscopy-associated infection New Delhi metallo-betalactamase-producing carbapenem-resistant Escherichia coli associated with exposure to duodenoscopes A quarantine process for the resolution of duodenoscope-associated transmission of multi drug-resistant Escherichia coli Withdrawal of a novel design duodenoscope ends outbreak of a VIM-2-producing Pseudomonas aeruginosa Disinfection of endoscopes: review of new chemical sterilants used for high level disinfection Significant factors in the disinfection and sterilization of flexible endoscopes Endoscope reprocessing: where do we go from here?
Mycobacteria and glutaraldehyde: is high-level disinfection of endoscopes possible?
Natural bioburden levels detected on flexible gastrointestinal endoscopes after clinical use and manual cleaning Endoscope reprocessing methods: a prospective study on the impact of human factors and automation Automated endoscope reprocessors Automatic flexible endoscope reprocessors Society of Gastroenterology Nurses and Associates: Guideline for use of high level disinfectants & sterilants for reprocessing flexible gastrointestinal endoscopes Automated washing with the Reliance endoscope processing system and its equivalence to optimal manual cleaning Food and Drug Administration: 510(k) Summary for SYSTEM lE Liquid Chemical Sterilant Processing System Steris Corporation: System 1E TM liquid chemical sterilant processing system Food and Drug Administration: Information about automated endoscope reprocessors (AERs) and FDA's evaluation FDA News Release: FDA orders recall under consent decree for all Custom Ultrasonics automated endoscope reprocessors Food and Drug Administration: FDA recommends health care facilities stop using Custom Ultrasonics' System 83 Plus automated endoscope reprocessors (AERs) for reprocessing duodenoscopes; these reprocessors remain available to reprocess other flexible endoscopes: FDA Safety Communication Module 2: Cleaning, disinfection and sterilization Effectiveness of the SYSTEM 1E Liquid Chemical Sterilant Processing System for reprocessing duodenoscopes Public Health Agency of Canada: Infection prevention and control guideline for flexible gastrointestinal endoscopy and flexible bronchoscopy.
PART IV.
Issues related to reprocessing flexible endoscopes Food and Drug Administration: FDA-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices Food and Drug Administration: FDA-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices Sterilization, high level disinfection and environmental cleaning Public Health Agency of Canada (formerly Health Canada): Infection control guidelines: hand washing, cleaning, disinfection and sterilization in health care APIC guideline for selection and use of disinfectants.
1994, 1995, and 1996 APIC Guidelines Committee High-level disinfection or "sterilization" of endoscopes?
Flexible and semi-rigid endoscope processing in health care facilities Canadian Society of Gastroenterology Nurses and Associations: Infection control: recommended guidelines in endoscopy settings Multi-society guideline for reprocessing flexible gastrointestinal endoscopes.
Society for Healthcare Epidemiology of America APIC guideline for infection prevention and control in flexible endoscopy Reprocessing endoscopes: United States perspective Methodology of reprocessing reusable accessories In vitro evaluation of wire integrity and ability to reprocess single-use sphincterotomes A prospective study of the repeated use of sterilized papillotomes and retrieval baskets for ERCP: quality and cost analysis Reuse of disposable sphincterotomes for diagnostic and therapeutic ERCP: a one-year prospective study Food and Drug Administration: Enforcement priorities for single-use devices reprocessed by third parties and hospitals Third-party reprocessing of endoscopic accessories Methodology of reprocessing one-time use accessories Decontaminated single-use devices: an oxymoron that may be placing patients at risk for cross-contamination A performance, safety and cost comparison of reusable and disposable endoscopic biopsy forceps: a prospective, randomized trial Risk associated with reprocessed reusable endoscopic instruments Society of Gastroenterology Nurses and Associates: Standards of infection control in reprocessing of flexible gastrointestinal endoscopes.
Chicago, SGNA Advantages and limitations of automatic flexible endoscope reprocessors Automated endoscope reprocessors Endoscope disinfection a resourcesensitive approach.
World Gastroenterology Organization Practice Guideline -Endoscope Disinfection -a Resource-Sensitive Approach British Society of Gastroenterology: BSG guidelines for decontamination of equipment for gastrointestinal endoscopy: the report of a working party of the British Society of Gastroenterology Endoscopy Committee ESGE-ESGENA guideline for quality assurance in reprocessing: microbiological surveillance testing in endoscopy Canadian Standards Association (CSA): Z314.8-14 Decontamination of reusable medical devices Food and Drug Administration: Reprocessing medical devices in health care settings: validation methods and labeling.
Guidance for Industry and Food and Drug Administration Staff Food and Drug Administration: Supplemental measures to enhance reprocessing: FDA Safety Communication Real-time monitoring in managing the decontamination of flexible gastrointestinal endoscopes Current issues result in a paradigm shift in reprocessing medical and surgical instruments Society of Gastroenterology Nurses and Associates: Standards of infection prevention in reprocessing flexible gastrointestinal endoscopes Endoscope reprocessing methods: a prospective study on the impact of human factors and automation Impact of ethylene oxide gas sterilization of duodenoscopes after a carbapenem-resistant enterobacteriaceae outbreak Urgent safety notification: Important updated labeling information: new reprocessing instructions for the Olympus TJF-Q180V Duodenoscope The role of biofilms in reprocessing of medical devices Evaluation of the quality of reprocessing of gastrointestinal endoscopes The use of channel-purge storage for gastrointestinal endoscopes reduces microbial contamination Evaluation of endoscope cleanliness after reprocessing: a clinical-use study Dynamics of action of biocides in Pseudomonas aeruginosa biofilm Prevention: Health Advisory: Immediate need for healthcare facilities to review procedures for cleaning, disinfecting, and sterilizing reusable medical devices Worse-case soiling levels for patient-used flexible endoscopes before and after cleaning Comparison of clinically relevant benchmarks and channel sampling methods used to assess manual cleaning compliance for flexible gastrointestinal endoscopes Studies on the chemical sterilization of surgical instruments.
I.
A bacteriological evaluation Ethylene oxide sterilization of medical devices: a review Validation of low-temperature steam with formaldehyde sterilization for endoscopes, using validation device Comparison of ion plasma, vaporized hydrogen peroxide, and 100% ethylene oxide sterilizers to the 12/88 ethylene oxide gas sterilizer Comparison of liquid chemical sterilization with peracetic acid and ethylene oxide sterilization for long narrow lumens Comparative evaluation of the sporicidal activity of new low temperature sterilization technologies: ethylene oxide, 2 plasma sterilization systems, and liquid peracetic acid A comparative study of ethylene oxide gas, hydrogen peroxide gas plasma, and low-temperature steam formaldehyde sterilization Ethylene oxide sterilization of medical devices: a review Current practice of duodenoscope reprocessing Carbapenem-resistant Enterobacteriaceae and endoscopy: an evolving threat Early identification and control of carbapenemase-producing Klebsiella pneumoniae, originating from contaminated endoscopic equipment Reported gastrointestinal endoscope reprocessing lapses: the tip of the iceberg Multidrug-resistant Klebsiella pneumoniae outbreak after endoscopic retrograde cholangiopancreatography Control of a multi-hospital outbreak of KPC-producing Klebsiella pneumonia type 2 in France Outbreak of ertapenem resistant Enterobacter cloacae urinary tract infections due to a contaminated ureteroscope Duodenoscope-associated bacterial infections: a review and update Infectious diseases linked to cross-contamination of flexible endoscopes Transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy Endoscopic retrograde cholangiopancreatography-associated AmpC Escherichia coli outbreak A carbapenem-resistant Klebsiella pneumoniae outbreak following bronchoscopy AMMI TIR34 Water for reprocessing of medical devices TIR30 A compendium of processes, materials, test methods and acceptance 147.
Muscarella LF: Recommendations for preventing hepatitis C virus infection: analysis of a Brooklyn endoscopy clinic's outbreak Prevention: Transmission of hepatitis B and C viruses in outpatient settings Viral hepatitis transmission in ambulatory health care settings Guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings Evaluation of the risk of transmission of bacterial biofilms and clostridium difficile during gastrointestinal endoscopy ERCP scopes: what can we do to prevent infections?
Disinfection, sterilization and antisepsis: principles and practices in healthcare facilities Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination Effectiveness of current disinfection procedures against biofilm on contaminated GI endoscopes Modeling microbial survival in buildup biofilm for complex medical devices A study of the efficacy of bacterial biofilm cleanout for gastrointestinal endoscopes Evaluation of detergents and contact time on biofilm removal from flexible endoscopes Control and Prevention: Interim protocol for healthcare facilities regarding surveillance for bacterial contamination of duodenoscopes after reprocessing Antimicrobial efficacy of endoscopic disinfection procedures: a controlled, multifactorial investigation Pseudomonas aeruginosa cross-infection following endoscopic retrograde cholangiopancreatography Pseudomonas aeruginosa and Enterobacteriaceae bacteremia after biliary endoscopy: an outbreak investigation using DNA macrorestriction analysis Nosocomial infections from contaminated endoscopes: a flawed automated endoscope washer.
An investigation using molecular epidemiology A prospective analysis of fever and bacteremia following ERCP Polymicrobial sepsis following endoscopic retrograde cholangiopancreatography Infection control practices in gastrointestinal endoscopy in the United States: a national survey Iatrogenic Campylobacter pylori infection is a cause of epidemic achlorhydria Development and validation of rapid use scope test strips (RUST) to determine the efficacy of manual cleaning for flexible endoscope channels Rapid method for the sensitive detection of protein contamination on surgical instruments The ATP test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels Validation of ATP to audit manual cleaning of flexible endoscope channels Persistent contamination on colonoscopes and gastroscopes detected by biologic cultures and rapid indicators despite reprocessing performed in accordance with guidelines Assessing residual contamination and damage inside flexible endoscopes over time Simulated-use validation of a sponge ATP method for determining the adequacy of manual cleaning of endoscope channels Validation and comparison of three adenosine triphosphate luminometers for monitoring hospital surface sanitization: a Rosetta Stone for adenosine triphosphate testing The use of rapid indicators for the detection of organic residues on clinically used gastrointestinal endoscopes with and without visually apparent debris The ATP test is a rapid and reliable audit tool to assess manual cleaning adequacy of flexible endoscope channels Hemoglobin assay for validation and quality control of medical device reprocessing Control and Prevention: Interim sampling method for the duodenoscope -distal end and instrument channel Detection of persistent vegetative bacteria and amplified viral nucleic acid from in-use testing of gastrointestinal endoscopes Natural bioburden levels detected on rigid lumened medical devices before and after cleaning Microbiological monitoring of endoscopes: 5-year review Is bacteriologic surveillance in endoscope reprocessing stringent enough?
Surveillance cultures of samples obtained from biopsy channels and automated endoscope reprocessors after high-level disinfection of gastrointestinal endoscopes Pseudomonas aeruginosa sepsis following retrograde cholangiopancreatography (ERCP) Pseudomonas septicaemia after endoscopic retrograde cholangiopancreatography: an unresolved problem Acute hepatitis C virus infections attributed to unsafe injection practices at an endoscopy clinic-Nevada Nonhospital health care-associated hepatitis B and C virus transmission: United States News Release: FDA orders duodenoscope manufacturers to conduct postmarket surveillance studies in health care facilities USA: Safety Communication -FUJIFILM Medical Systems validates revised reprocessing instructions Food and Drug Administration: PENTAX validates reprocessing instructions for ED-3490TK video duodenoscopes: FDA Safety Communication Olympus validates new reprocessing instructions for model TJF-Q180V duodenoscopes: FDA Safety Communication Food and Drug Administration: FDA clears Olympus TJF-Q180V duodenoscope with design modifications intended to reduce infection risk.
FDA News Release Urgent medical device removal and corrective action: elevator mechanism replacement, updated operation manual, and new reprocessing instructions for the Olympus TJF-Q180V duodenoscope Outbreaks of carbapenem-resistant Enterobacteriaceae infections associated with duodenoscopes: what can we do to prevent infections?
Surveillance of guideline practices for duodenoscope and linear echoendoscope reprocessing in a large healthcare system How to assess risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines Reprocessing failure Food and Drug Administration: Medical device reporting (MDR) Food and Drug Administration: Mandatory reporting requirements: manufacturers, importers and device user facilities Assessing the risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines Analysis of microbial load on surgical instruments after clinical use and following manual and automated cleaning Patient-to-patient transmission of Campylobacter pylori infection by fiberoptic gastroduodenoscopy and biopsy Conventional cleaning and disinfection techniques eliminate the risk of endoscopic transmission of Helicobacter pylori Inactivation of Clostridium difficile spores by disinfectants Inactivation of hepatitis B virus by intermediate-to-high level disinfectant chemicals Viral transmission and fibreoptic endoscopy Elimination of high titre HIV from fiberoptic endoscopes Endoscopic transmission of hepatitis B virus Absence of transmission of hepatitis B by fiberoptic upper gastrointestinal endoscopy Contamination of an endoscope due to Trichosporon beigelli Transmission of trichosporon asahii oesophagitis by a contaminated endoscope Complications associated with esophagogastroduodenoscopy and with esophageal dilation Creutzfeldt-Jakob disease: recommendations for disinfection and sterilization Managing the risk of nosocomial transmission of prion diseases Creutzfeldt-Jakob disease: implications for gastroenterology Current issues in endoscope reprocessing and infection control during gastrointestinal endoscopy Guideline for disinfection and sterilization of prion-contaminated medical instruments Variant Creutzfeldt-Jakob disease (vCJD) and gastrointestinal endoscopy United States Senate; Health, Education, Labor, and Pensions Committee: Preventable tragedies: superbugs and how ineffective monitoring of medical device safety fails patients.
Minority staff report Food and Drug Administration: Infections associated with reprocessed duodenoscopes Risk of transmission of carbapenem-resistant Enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy Infection using ERCP endoscopes Multisociety guideline on reprocessing flexible GI endoscopes: 2016 update Food and Drug Administration: Design of endoscopic retrograde cholangiopancreatography (ERCP) duodenoscopes may impede A complete reference list can be found online at ExpertConsult .com
key: cord-016293-pyb00pt5 authors: Newell-McGloughlin, Martina; Re, Edward title: The flowering of the age of Biotechnology 1990–2000 date: 2006 journal: The Evolution of Biotechnology DOI: 10.1007/1-4020-5149-2_4 sha: doc_id: 16293 cord_uid: pyb00pt5 nan the significance of developing genetic and physical maps of the genome, and the importance of comparing the human genome with those of other species.
It also suggested a preliminary focus on improving current technology.
At the request of the U.S. Congress, the Office of Technology Assessment (OTA) also studied the issue, and issued a document in 1987 -within days of the NRC report -that was similarly supportive.
The OTA report discussed, in addition to scientific issues, social and ethical implications of a genome program together with problems of managing funding, negotiating policy and coordinating research efforts.
Prompted by advisers at a 1988 meeting in Reston, Virginia, James Wyngaarden, then director of the National Institutes of Health (NIH) , decided that the agency should be a major player in the HGP, effectively seizing the lead from DOE.
The start of the joint effort was in May 1990 (with an "official" start in October) when a 5-year plan detailing the goals of the U.S. Human Genome Project was presented to members of congressional appropriations committees in mid-February.
This document co-authored by DOE and NIH and titled "Understanding Our Genetic Inheritance, the U.S. Human Genome Project: The First Five Years" examined the then current state of genome science.
The plan also set forth complementary approaches of the two agencies for attaining scientific goals and presented plans for administering research agenda; it described collaboration between U.S. and international agencies and presented budget projections for the project.
According to the document, "a centrally coordinated project, focused on specific objectives, is believed to be the most efficient and least expensive way" to obtain the 3-billion base pair map of the human genome.
In the course of the project, especially in the early years, the plan stated that "much new technology will be developed that will facilitate biomedical and a broad range of biological research, bring down the cost of many experiments (mapping and sequencing), and finding applications in numerous other fields."
The plan built upon the 1988 reports of the Office of Technology Assessment and the National Research Council on mapping and sequencing the human genome.
"In the intervening two years," the document said, "improvements in technology for almost every aspect of genomics research have taken place.
As a result, more specific goals can now be set for the project."
The document describes objectives in the following areas mapping and sequencing the human genome and the genomes of model organisms; data collection and distribution; ethical, legal, and social considerations; research training; technology development; and technology transfer.
These goals were to be reviewed each year and updated as further advances occured in the underlying technologies.
They identified the overall budget needs to be the same as those identified by OTA and NRC, namely about $200 million per year for approximately 15 years.
This came to $13 billion over the entire period of the project.
Considering that in July 1990, the DNA databases contained only seven sequences greater than 0.1 Mb this was a major leap of faith.
This approach was a major departure from the single-investigator-based gene of interest focus that research took hitherto.
This sparked much controversy both before and after its inception.
Critics questioned the usefulness of genomic sequencing, they objected to the high cost and suggested it might divert funds from other, more focused, basic research.
The prime argument to support the latter position is that there appeared to be are far less genes than accounted for by the mass of DNA which would suggest that the major part of the sequencing effort would be of long stretches of base pairs with no known function, the so-called "junk DNA."
And that was in the days when the number of genes was presumed to be 80-100,000.
If, at that stage, the estimated number was guessed to be closer to the actual estimate of 35-40,000 (later reduced to 20-25,000) this would have made the task seem even more foolhardy and less worthwhile to some.
However, the ever-powerful incentive of new diagnostics and treatments for human disease beyond what could be gleaned from the gene-by-gene approach and the rapidly evolving technologies, especially that of automated sequencing, made it both an attractive and plausible aim.
Charles Cantor (1990) , a principal scientist for the Department of Energy's genome project contended that DOE and NIH were cooperating effectively to develop organizational structures and scientific priorities that would keep the project on schedule and within its budget.
He noted that there would be small short-term costs to traditional biology, but that the long-term benefits would be immeasurable.
Genome projects were also discussed and developed in other countries and sequencing efforts began in Japan, France, Italy, the United Kingdom, and Canada.
Even as the Soviet Union collapsed, a genome project survived as part of the Russian science program.
The scale of the venture and the manageable prospect for pooling data via computer made sequencing the human genome a truly international initiative.
In an effort to include developing countries in the project UNESCO assembled an advisory committee in 1988 to examine UNESCO's role in facilitating international dialogue and cooperation.
A privately-funded Human Genome Organization (HUGO) had been founded in 1988 to coordinate international efforts and serve as a clearinghouse for data.
In that same year the European Commission (EC) introduced a proposal entitled the "Predictive Medicine Programme."
A few EC countries, notably Germany and Denmark, claimed the proposal lacked ethical sensitivity; objections to the possible eugenic implications of the program were especially strong in Germany (Dickson 1989) .
The initial proposal was dropped but later modified and adopted in 1990 as the "Human Genome Analysis Programme" (Dickman and Aldhous 1991) .
This program committed substantial resources to the study of ethical issues.
The need for an organization to coordinate these multiple international efforts quickly became apparent.
Thus the Human Genome Organization (HUGO), which has been called the "U.N. for the human genome," was born in the spring of 1988.
Composed of a founding council of scientists from seventeen countries, HUGO's goal was to encourage international collaboration through coordination of research, exchange of data and research techniques, training, and debates on the implications of the projects (Bodmer 1991) .
In August 1990 NIH began large-scale sequencing trials on four model organisms: the parasitic, cell-wall lacking pathogenic microbe Mycoplasma capricolum, the prokaryotic microbial lab rat Escherichia coli, the most simple animal Caenorhabditis elegans, and the eukaryotic microbial lab rat Saccharomyces cerevisiae.
Each research group agreed to sequence 3 megabases (Mb) at 75 cents a base within 3 years.
A sub living organism was actually fully sequenced and the complete sequence of that genome, the human cytomegalovirus (HCMV) genome was 0.23 Mb.
That year also saw the casting of the first salvo in the protracted debate on "ownership" of genetic information beginning with the more tangible question of ownership of cells.
And, as with the debates of the early eighties, which were to be revisited later in the nineties, the respondent was the University of California.
Moore v. Regents of the University of California was the first case in the United States to address the issue of who owns the rights to an individual's cells.
Diagnosed with leukemia, John Moore had blood and bone marrow withdrawn for medical tests.
Suspicious of repeated requests to give samples because he had already been cured, Moore discovered that his doctors had patented a cell line derived from his cells and so he sued.
The California Supreme Court found that Moore's doctor did not obtain proper informed consent, but, however, they also found that Moore cannot claim property rights over his body.
The quest for the holy grail of the human genome was both inspired by the rapidly evolving technologies for mapping and sequencing and subsequently spurred on the development of ever more efficient tools and techniques.
Advances in analytical tools, automation, and chemistries as well as computational power and algorithms revolutionized the ability to generate and analyze immense amounts of DNA sequence and genotype information.
In addition to leading to the determination of the complete sequences of a variety of microorganisms and a rapidly increasing number of model organisms, these technologies have provided insights into the repertoire of genes that are required for life, and their allelic diversity as well as their organization in the genome.
But back in 1990 many of these were still nascent technologies.
The technologies required to achieve this end could be broadly divided into three categories: equipment, techniques, and computational analysis.
These are not truly discrete divisions and there was much overlap in their influence on each other.
As noted, Lloyd Smith, Michael and Tim Hunkapiller, and Leroy Hood conceived the automated sequencer and Applied Biosystems Inc. brought it to market in June 1986.
There is no much doubt that when Applied Biosystems Inc. put it on the market that which had been a dream became decidedly closer to an achievable reality.
In automating Sangers chain termination sequencing system, Hood modified both the chemistry and the data-gathering processes.
In the sequencing reaction itself, he replaced radioactive labels, which were unstable, posed a health hazard, and required separate gels for each of the four bases.
Hood developed chemistry that used fluorescent dyes of different colors for each of the four DNA bases.
This system of "color-coding" eliminated the need to run several reactions in overlapping gels.
The fluorescent labels addressed another issue which contributed to one of the major concerns of sequencing -data gathering.
Hood integrated laser and computer technology, eliminating the tedious process of information-gathering by hand.
As the fragments of DNA passed a laser beam on their way through the gel the fluorescent labels were stimulated to emit light.
The emitted light was transmitted by a lens and the intensity and spectral characteristics of the fluorescence are measured by a photomultiplier tube and converted to a digital format that could be read directly into a computer.
During the next thirteen years, the machine was constantly improved, and by 1999 a fully automated instrument could sequence up to 150,000,000 base pairs per year.
In 1990 three groups came up with a variation on this approach.
They developed what is termed capillary electrophoresis, one team was led by Lloyd Smith (Luckey, 1990) , the second by Barry Karger , and the third by Norman Dovichi.
In 1997 Molecular Dynamics introduced the MegaBACE, a capillary sequencing machine.
And not to be outdone the following year in 1998, the original of the species came up with the ABI Prism 3700 sequencing machine.
The 3700 is also a capillary-based machine designed to run about eight sets of 96 sequence reactions per day.
On the biology side, one of the biggest challenges was the construction of a physical map to be compiled from many diverse sources and approaches in such a way as to insure continuity of physical mapping data over long stretches of DNA.
The development of DNA Sequence Tagged Sites (STSs) to correlate diverse types of DNA clones aided this standardization of the mapping component by providing mappers with a common language and a system of landmarks for all the libraries from such varied sources as cosmids, yeast artificial chromosomes (YACs) and other rDNAs clones.
This way each mapped element (individual clone, contig, or sequenced region) would be defined by a unique STS.
A crude map of the entire genome, showing the order and spacing of STSs, could then be constructed.
The order and spacing of these unique identifier sequences composing an STS map was made possible by development of Mullis' polymerase chain reaction (PCR), which allows rapid production of multiple copies of a specific DNA fragment, for example, an STS fragment.
Sequence information generated in this way could be recalled easily and, once reported to a database, would be available to other investigators.
With the STS sequence stored electronically, there would be no need to obtain a probe or any other reagents from the original investigator.
No longer would it be necessary to exchange and store hundreds of thousands of clones for full-scale sequencing of the human genome-a significant saving of money, effort, and time.
By providing a common language and landmarks for mapping, STS's allowed genetic and physical maps to be cross-referenced.
With a refinement on this technique to go after actual genes, Sydney Brenner proposed sequencing human cDNAs to provide rapid access to the genes stating that 'One obvious way of finding at least a large part of the important [fraction] of the human genome is to look at the sequences of the messenger RNA's of expressed genes' (Brenner, 1990) .
The following year the man who was to play a pivotal role on the world stage that became the human genome project suggested a way to implement Sydney's approach.
That player, NIH biologist J. Craig Venter announced a strategy to find expressed genes, using ESTs (Expressed Sequence Tag) (Adams, 1991) .
These so called ESTs represent a unique stretch of DNA within a coding region of a gene, which as Sydney suggested would be useful for identifying full-length genes and as a landmark for mapping.
So using this approach projects were begun to mark gene sites on chromosome maps as sites of mRNA expression.
To help with this a more efficient method of handling large chunks of sequences was needed and two approaches were developed.
Yeast artificial chromosomes, which were developed by David Burke, Maynard Olson, and George Carle, increased insert size 10-fold (David T. Burke et al., 1987) .
Caltech's second major contribution to the genome project was developed by Melvin Simon, and Hiroaki Shizuya.
Their approach to handling large DNA segments was to develop "bacterial artificial chromosomes" (BACs), which basically allow bacteria to replicate chunks greater than 100,000 base pairs in length.
This efficient production of more stable, large-insert BACs made the latter an even more attractive option, as they had greater flexibility than YACs.
In 1994 in a collaboration that presages the SNP Consortium, Washington University, St Louis MO, was funded by the pharmaceutical company Merck and the National Cancer Institute to provide sequence from those ESTs.
More than half a million ESTs were submitted during the project (Murr L et al., 1996) .
On the analysis side was the major challenge to manage and mine the vast amount of DNA sequence data being generated.
A rate-limiting step was the need to develop semi-intelligent algorithms to achieve this Herculean task.
This is where the discipline of bioinformatics came into play.
It had been evolving as a discipline since Margaret Oakley Dayhoff used her knowledge of chemistry, mathematics, biology and computer science to develop this entirely new field in the early sixties.
She is in fact credited today as a founder of the field of bioinformatics in which biology, computer science, and information technology merge into a single discipline.
The ultimate goal of the field is to enable the discovery of new biological insights as well as to create a global perspective from which unifying principles in biology can be discerned.
There are three important sub-disciplines within bioinformatics: the development of new algorithms and statistics with which to assess relationships among members of large data sets; the analysis and interpretation of various types of data including nucleotide and amino acid sequences, protein domains, and protein structures; and the development and implementation of tools that enable efficient access and management of different types of information.
Paralleling the rapid and very public ascent of recombinant DNA technology during the previous two decades, the analytic and management tools of the discipline that was to become bioinformatics evolved at a more subdued but equally impressive pace.
Some of the key developments included tools such as the Needleman-Wunsch algorithm for sequence comparison which appeared even before recombinant DNA technology had been demonstrated as early as 1970; the Smith-Waterman algorithm for sequence alignment (1974); the FASTP algorithm (1985) and the FASTA algorithm for sequence comparison by Pearson and Lupman in 1988 and Perl (Practical Extraction Report Language) released by Larry Wall in 1987.
On the data management side several databases with ever more effective storage and mining capabilities were developed over the same period.
The first bioinformatic/biological databases were constructed a few years after the first protein sequences began to become available.
The first protein sequence reported was that of bovine insulin in 1956, consisting of 51 residues.
Nearly a decade later, the first nucleic acid sequence was reported, that of yeast alanine tRNA with 77 bases.
Just one year later, Dayhoff gathered all the available sequence data to create the first bioinformatic database.
One of the first dedicated databases was the Brookhaven Protein DataBank whose collection consisted of ten X-ray crystallographic protein structures (Acta.
Cryst.
B, 1973) .
The year 1982 saw the creation of the Genetics Computer Group (GCG) as a part of the University of Wisconsin Biotechnology Center.
The group's primary and much used product was the Wisconsin Suite of molecular biology tools.
It was spun off as a private company in 1989.
The SWISS-PROT database made its debut in 1986 in Europe at the Department of Medical Biochemistry of the University of Geneva and the European Molecular Biology Laboratory (EMBL).
The first dedicated "bioinformatics" company IntelliGenetics, Inc. was founded in California in 1980.
Their primary product was the IntelliGenetics Suite of programs for DNA and protein sequence analysis.
The first unified federal effort, the National Center for Biotechnology Information (NCBI) was created at NIH/NLM in 1988 and it was to play a crucial part in coordinating public databases, developing software tools for analyzing genome data, and disseminating information.
And on the other side of the Atlantic, Oxford Molecular Group, Ltd. (OMG) was founded in Oxford, UK by Anthony Marchington, David Ricketts, James Hiddleston, Anthony Rees, and W. Graham Richards.
Their primary focus was on rational drug design and their products such as Anaconda, Asp, and Chameleon obviously reflected this as they were applied in molecular modeling, and protein design engineering.
Within two years NCBI were making their mark when David Lipman, Eugene Myers, and colleagues at the NCBI published the Basic Local Alignment Search Tool BLAST algorithm for aligning sequences (Altschul et al., 1990) .
It is used to compare a novel sequence with those contained in nucleotide and protein databases by aligning the novel sequence with previously characterized genes.
The emphasis of this tool is to find regions of sequence similarity, which will yield functional and evolutionary clues about the structure and function of this novel sequence.
Regions of similarity detected via this type of alignment tool can be either local, where the region of similarity is based in one location, or global, where regions of similarity can be detected across otherwise unrelated genetic code.
The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
An HSP consists of two sequence fragments of arbitrary but equal length whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score.
This system has been refined and modified over the years the two principal variants presently in use being the NCBI BLAST and WU-BLAST (WU signifying Washington University).
The same year that BLAST was launched two other bioinformatics companies were launched.
One was InforMax in Bethesda, MD whose products addressed sequence analysis, database and data management, searching, publication graphics, clone construction, mapping and primer design.
The second, Molecular Applications Group in California, was to play a bigger part on the proteomics end (Michael Levitt and Chris Lee).
Their primary products were Look and SegMod which are used for molecular modeling and protein design.
The following year in 1991 the Human chromosome mapping data repository, Genome Data Base (GDB) was established.
On a more global level, the development of computational capabilities in general and the Internet in specific was also to play a considerable part in the sharing of data and access to databases that rendered the rapidity of the forward momentum of the HGP possible.
Also in 1991 Edward Uberbacher of Oak Ridge National Laboratory in Tennessee developed GRAIL, the first of many gene-finding programs.
In 1992 the first two "genomics" companies made their appearance.
Incyte Pharmaceuticals, a genomics company headquartered in Palo Alto, California, was formed and Myriad Genetics, Inc. was founded in Utah.
Incyte's stated goal was to lead in the discovery of major common human disease genes and their related pathways.
The company discovered and sequenced, with its academic collaborators (originally Synteni from Pat Brown's lab at Stanford), a number of important genes including BRCA1 and BRCA2, with Mary Claire King, epidemiologist at UC-Berkeley, the genes linked to breast cancer in families with a high degree of incidence before age 45.
By 1992 a low-resolution genetic linkage map of the entire human genome was published and U.S. and French teams completed genetic maps of both mouse and man.
The mouse with an average marker spacing of 4.3 cM as determined by Eric Lander and colleagues at Whitehead and the human, with an average marker spacing of 5 cM by Jean Weissenbach and colleagues at CEPH (Centre d'Etude du Polymorphisme Humaine).
The latter institute was the subject of a rather scathing book by Paul Rabinow (1999) based on what they did with this genome map.
In 1993, an American biotechnology company, Millennium Pharmaceuticals, and the CEPH, developed plans for a collaborative effort to discover diabetes genes.
The results of this collaboration could have been medically significant and financially lucrative.
The two parties had agreed that CEPH would supply Millennium with germplasm collected from a large coterie of French families, and Millennium would supply funding and expertise in new technologies to accelerate the identification of the genes, terms to which the French government had agreed.
But in early 1994, just as the collaboration was to begin, the French government cried halt!
The government explained that the CEPH could not be permitted to give the Americans that most precious of substances for which there was no precedent in law -French DNA.
Rabinow's book discusses the tangled relations and conceptions such as, can a country be said to have its own genetic material, the first but hardly the last Franco-American disavowal of détente (Paul Rabinow, 1999) .
The latest facilities such as the Joint Genome Institute (JGI), Walnut Creek, CA are now able to sequence up to 10Mb per day which makes it possible to sequence whole microbial genomes within a day.
Technologies currently under development will probably increase this capacity yet further through massively parallel sequencing and/or microfluidic processing making it possible to sequence multiple genotypes from several species.
Nineteen ninety-two saw one of the first shakeups in the progress of the HGP.
That was the year that the first major outsider entered the race when Britain's Wellcome Trust plunked down $95 million to join the HGP.
This caused a mere ripple while the principal shake-ups occurred stateside.
Much of the debate and subsequently the direction all the way through the HGP process was shaped by the personalities involved.
As noted the application of one of the innovative techniques, namely ESTs, to do an end run on patenting introduced one of those major players to the fray, Craig Venter.
Venter, the high school drop out who reached the age of majority in the killing fields of Vietnam was to play a pivotal role in a more "civilized" but no less combative field of human endeavor.
He came onto the world stage through his initial work on ESTs while at the National Institute of Neurological Disorders and Stroke (NINDS) from 1984 to 1992.
He noted in an interview with The Scientist magazine in 1995, that there was a degree of ambiguity at NINDS about his venturing into the field of genomics, while they liked the prestige of hosting one of the leaders and innovators in his newly emerging field, they were concerned about him moving outside the NIND purview of the human brain and nervous system.
Ultimately, while he proclaimed to like the security and service infrastructure this institute afforded him, that same system became too restrictive for his interests and talent.
He wanted the whole canvas of human-gene expression to be his universe, not just what was confined to the central nervous system.
He was becoming more interested in taking a whole genome approach to understanding the overall structure of genomes and genome evolution, which was much broader than the mission of NINDS.
He noted, with some irony, in later years that the then current NIH director Harold Varmus had wished in hindsight that NIH had pushed to do a similar database in the public domain, clearly in Venter's opinion Varmus was in need of a refresher course in history!
Bernadine Healy, NIH director in 1994, was one of the few in a leadership role who saw the technical and fiscal promise of Venter's work and, like all good administrators, it also presented an opportunity to resolve a thorny "personnel" issue.
She appointed him head of the ad hoc committee to have an intramural genome program at NIH to give the head of the HGP (that other larger than life personality Jim Watson) notice that he was not the sole arbitrator of the direction for the Human Genome Project.
However Venter very soon established himself as an equally non-conformist character and with the tacit consent of his erstwhile benefactor.
He initially assumed the mantle of a non-conformist through guilt by association rather than direct actions when it was revealed that NIH was filing patent applications on thousands of these partial genes based on his ESTs catalyzing the first HGP fight at a congressional hearing.
NIH's move was widely criticized by the scientific community because, at the time, the function of genes associated with the partial sequences was unknown.
Critics charged that patent protection for the gene segments would forestall future research on them.
The Patent Office eventually rejected the patents, but the applications sparked an international controversy over patenting genes whose functions were still unknown.
Interestingly enough despite NIH's reliance on the EST/cDNA technique, Venter, who was now clearly venturing outside the NINDS mandated rubric, could not obtain government funding to expand his research, prompting him to leave NIH in 1992.
He moved on to become president and director of The Institute for Genomic Research (TIGR), a nonprofit research center based in Gaithersburg, Md.
At the same time William Haseltine formed a sister company, Human Genome Sciences (HGS), to commercialize TIGR products.
Venter continued EST work at TIGR, but also began thinking about sequencing entire genomes.
Again, he came up with a quicker and faster method: whole genome shotgun sequencing.
He applied for an NIH grant to use the method on Hemophilus influenzae, but started the project before the funding decision was returned.
When the genome was nearly complete, NIH rejected his proposal saying the method would not work.
In a triumphal flurry in late May 1995 and with a metaphorical nose-thumbing at his recently rejected "unworkable" grant Venter announced that TIGR and collaborators had fully sequenced the first free-living organism -Haemophilus influenzae.
In November 1994, controversy surrounding Venter's research escalated.
Access restrictions associated with a cDNA database developed by TIGR and its Rockville, Md.-based biotech associate, Human Genome Sciences (HGS) Inc. -including HGS's right to preview papers on resulting discoveries and for first options to license products -prompted Merck and Co. Inc. to fund a rival database project.
In that year also Britain "officially" entered the HGP race when the Wellcome Trust trumped down $95 million (as mentioned earlier).
The following year HGS was involved in yet another patenting debacle forced by the rapid march of technology into uncharted patent law territory.
On June 5, 1995 HGS applied for a patent on a gene that produces a "receptor" protein that is later called CCR5.
At that time HGS has no idea that CCR5 is an HIV receptor.
In December 1995, U.S. researcher Robert Gallo, the co-discoverer of HIV, and colleagues found three chemicals that inhibit the AIDS virus but they did not know how the chemicals work.
In February 1996, Edward Berger at the NIH discovered that Gallo's inhibitors work in late-stage AIDS by blocking a receptor on the surface of T-cells.
In June of that year in a period of just 10 days, five groups of scientists published papers saying CCR5 is the receptor for virtually all strains of HIV.
In January 2000, Schering-Plough researchers told a San Francisco AIDS conference that they have discovered new inhibitors.
They knew that Merck researchers had made similar discoveries.
As a significant Valentine in 2000 the U.S. Patent and Trademark Office (USPTO) grants HGS a patent on the gene that makes CCR5 and on techniques for producing CCR5 artificially.
The decision sent HGS stock flying and dismayed researchers.
It also caused the USPTO to revise its definition of a "patentable" drug target.
In the meantime Haseltine's partner in rewriting patenting history, Venter turned his focus to the human genome.
He left TIGR and started the for-profit company Celera, a division of PE Biosystems, the company that at times, thanks to Hood and Hunkapillar, led the world in the production of sequencing machines.
Using these machines, and the world's largest civilian supercomputer, Venter finished assembling the human genome in just three years.
Following the debacle with the then NIH director Bernine Healy over patenting the partial genes that resulted from EST analysis, another major personality-driven event in that same year occurred.
Watson strongly opposed the idea of patenting gene fragments fearing that it would discourage research, and commented that "the automated sequencing machines 'could be run by monkeys.'
" (Nature June 29, 2000) with this dismissal Watson resigned his NIH NCHGR post in 1992 to devote his full-time effort to directing Cold Spring Harbor Laboratory.
His replacement was of a rather more pragmatic, less flamboyant nature.
While Venter maybe was described as an idiosyncratic Shogun of the shotgun, Francis Collins was once described as the King Arthur of the Holy Grail that is the Human Genome Project.
Collins became the Director of the National Human Genome Research Institute in 1993.
He was considered the right man for the job following his 1989 success (along with Lap-Chee Tsui) in identifying the gene for the cystic fibrosis transmembrane (CFTR) chloride channel receptor that, when mutated, can lead to the onset of cystic fibrosis.
Although now indelibly connected with the topic non-plus tout in biology, like many great innovators in this field before him, Francis Collins had little interest in biology as he grew up on a farm in the Shenandoah Valley of Virginia.
From his childhood he seemed destined to be at the center of drama, his father was professor of dramatic arts at Mary Baldwin College and the early stage management of career was performed on a stage he built on the farm.
While the physical and mathematical sciences held appeal for him, being possessed of a highly logical mind, Collins found the format in which biology was taught in the high school of his day mind-numbingly boring, filled with dissections and rote memorization.
He found the contemplation of the infinite outcomes of dividing by zero (done deliberately rather than by accident as in Einstein's case) far more appealing than contemplating the innards of a frog.
That biology could be gloriously logical only became clear to Collins when, in 1970, he entered Yale with a degree in chemistry from the University of Virginia and was first exposed to the nascent field of molecular biology.
Anecdotally it was the tome, the Book of Life, penned by the theoretical physicist father of molecular biology, Edwin Schrodinger, while exiled in Trinity College Dublin in 1942 that was the catalyst for his conversion.
Like Schrodinger he wanted to do something more obviously meaningful (for less than hardcore physicists at least!)
than theoretical physics, so he went to medical school at UNC-Chapel Hill after completing his chemistry doctorate in Yale, and returned to the site of his road to Damascus for post-doctoral study in the application of his newfound interest in human genetics.
During this sojourn at Yale, Collins began working on developing novel tools to search the genome for genes that cause human disease.
He continued this work, which he dubbed "positional cloning," after moving to the University of Michigan as a professor in 1984.
He placed himself on the genetic map when he succeeded in using this method to put the gene that causes cystic fibrosis on the physical map.
While a less colorful-in-your-face character than Venter he has his own personality quirks, for example, he pastes a new sticker onto the back of his motorcycle helmet every time he finds a new disease gene.
One imagines that particular piece of really estate is getting rather crowded.
Interestingly it was not these four hundred pound US gorillas who proposed the eventually prescient timeline for a working draft but two from the old power base.
In meetings in the US in 1994, John Sulston and Bob Waterston proposed to produce a 'draft' sequence of the human genome by 2000, a full five years ahead of schedule.
While agreed by most to be feasible it meant a rethinking of strategy and involved focusing resources on larger centers and emphasizing sequence acquisition.
Just as important, it asserts the value of draft quality sequence to biomedical research.
Discussion started with the British based Wellcome Trust as possible sponsors (Marshall E. 1995) .
By 1995 a rough draft of the human genome map was produced showing the locations of more than 30,000 genes.
The map was produced using yeast artificial chromosomes and some chromosomes -notably the littlest 22 -were mapped in finer detail.
These maps marked an important step toward clone-based sequencing.
The importance was illustrated in the devotion of an entire edition of the journal Nature to the subject.
(Nature 377: 175-379 1995) The duel between the public and private face of the HGP progressed at a pace over the next five years.
Following release of the mapping data some level of international agreement was decided on sequence data release and databases.
They agreed on the release of sequence data, specifically, that Primary Genomic Sequence should be in the Public Domain to encourage research and development to maximize its benefit to society.
Also that it be rapidly released on a daily basis with assemblies of greater than 1 Kb and that the finished annotated sequence should be submitted immediately to the public databases.
In 1996 an international consortium completed the sequence of the genome of the workhorse yeast Saccharomyces cerevisiae.
Data had been released as the individual chromosomes were completed.
The Saccharomyces Genome Database (SGD) was created to curate this information.
The project collects information and maintains a database of the molecular biology of S. cerevisiae.
This database includes a variety of genomic and biological information and is maintained and updated by SGD curators.
The SGD also maintains the S. cerevisiae Gene Name Registry, a complete list of all gene names used in S. cerevisiae.
In 1997 a new more powerful diagnostic tool termed SNPs (Single Nucleotide Polymorphisms) was developed.
SNPs are changes in single letters in our DNA code that can act as markers in the DNA landscape.
Some SNPs are associated closely with susceptibility to genetic disease, our response to drugs or our ability to remove toxins.
The SNP Consortium although designated a limited company is a nonprofit foundation organized for the purpose of providing public genomic data.
It is a collaborative effort between pharmaceutical companies and the Wellcome Trust with the idea of making available widely accepted, high-quality, extensive, and publicly accessible SNP map.
Its mission was to develop up to 300,000 SNPs distributed evenly throughout the human genome and to make the information related to these SNPs available to the public without intellectual property restrictions.
The project started in April 1999 and was anticipated to continue until the end of 2001.
In the end, many more SNPs, about 1.5 million total, were discovered than was originally planned.
By 1998 the complete genome sequence of Mycobacterium tuberculosis was published by teams from the UK, France, US and Denmark in June 1998.
The ABI Prism 3700 sequencing machine, a capillary-based machine designed to run about eight sets of 96 sequence reactions per day also reached the market that year.
That same year the genome sequence of the first multicellular organism, C. elegans was completed.
C. elegans has a genome of about 100 Mb and, as noted, is a primitive animal model organism used in a range of biological disciplines.
By November 1999 the human genome draft sequence reached 1000 Mb and the first complete human chromosome was sequenced -this first was reached on the East side of the Atlantic by the HGP team led by the Sanger Centre, producing a finished sequence for chromosome 22, which is about 34 million base-pairs and includes at least 550 genes.
According to anecdotal evidence when visiting his namesake centre, Sanger asked: "What does this machine do then?"
"Dideoxy sequencing" came the reply, to which Fred retorted: "Haven't they come up with anything better yet?"
As will be elaborated in the final chapter the real highlight of 2000 was production of a 'working draft' sequence of the human genome, which was announced simultaneously in the US and the UK.
In a joint event, Celera Genomics announced completion of their 'first assembly' of the genome.
In a remarkable special issue, Nature included a 60-page article by the Human Genome Project partners, studies of mapping and variation, as well as analysis of the sequence by experts in different areas of biology.
Science published the article by Celera on their assembly of HGP and Celera data as well as analyses of the use of the sequence.
However to demonstrate the sensitivity of the market place to presidential utterances the joint appearances by Bill Clinton and Tony Blair touting this major milestone turned into a major cold shower when Clinton's reassurance of access of the people to their genetic information caused a precipitous drop in Celera's share value overnight.
Clinton's assurance that, "The effort to decipher the human genome will be the scientific breakthrough of the century -perhaps of all time.
We have a profound responsibility to ensure that the life-saving benefits of any cutting-edge research are available to all human beings."
(President Bill Clinton, Wednesday, March 14, 2000) stands in sharp contrast to the statement from Venter's Colleague that " Any company that wants to be in the business of using genes, proteins, or antibodies as drugs has a very high probability of running afoul of our patents.
From a commercial point of view, they are severely constrained -and far more than they realize."
(William A. Haseltine, Chairman and CEO, Human Genome Sciences).
The huge sell-off in stocks ended weeks of biotech buying in which those same stocks soared to unprecedented highs.
By the next day, however, the genomic company spin doctors began to recover ground in a brilliant move which turned the Clinton announcement into a public relations coup.
All major genomics companies issued press releases applauding President Clinton's announcement.
The real news they argued, was that "for the first time a President strongly affirmed the importance of gene based patents."
And the same Bill Haseltine of Human Genome Sciences positively gushed as he happily pointed out that he "could begin his next annual report with the [President's] monumental statement, and quote today as a monumental day."
As distinguished Harvard biologist Richard Lewontin notes: "No prominent molecular biologist of my acquaintance is without a financial stake in the biotechnology business.
As a result, serious conflicts of interest have emerged in universities and in government service (Lewontin, 2000) .
Away from the spin doctors perhaps Eric Lander may have best summed up the Herculean effort when he opined that for him "the Human Genome Project has been the ultimate fulfilment: the chance to share common purpose with hundreds of wonderful colleagues towards a goal larger than ourselves.
In the long run, the Human Genome Project's greatest impact might not be the three billion nucleotides of the human chromosomes, but its model of scientific community."
(Ridley, 2000) 6.
GENE THERAPY The year 1990 also marked the passing of another milestone that was intimately connected to one of the fundamental drivers of the HGP.
The California Hereditary Disorders Act came into force and with it one of the potential solutions for human hereditary disorders.
W. French Anderson in the USA reported the first successful application of gene therapy in humans.
The first successful gene therapy for a human disease was successfully achieved for Severe Combined Immune Deficiency (SCID) by introducing the missing gene, adenosine deaminase deficiency (ADA) into the peripheral lymphocytes of a 4-year-old girl and returning modified lymphocytes to her.
Although the results are difficult to interpret because of the concurrent use of polyethylene glycol-conjugated ADA commonly referred to as pegylated ADA (PGLA) in all patients, strong evidence for in vivo efficacy was demonstrated.
ADA-modified T cells persisted in vivo for up to three years and were associated with increases in T-cell number and ADA enzyme levels, T cells derived from transduced PGLA were progressively replaced by marrow-derived T cells, confirming successful gene transfer into long-lived progenitor cells.
Ashanthi DeSilva, the girl who received the first credible gene therapy, continues to do well more than a decade later.
Cynthia Cutshall, the second child to receive gene therapy for the same disorder as DeSilva, also continues to do well.
Within 10 years (by January 2000), more than 350 gene therapy protocols had been approved in the US and worldwide, researchers launched more than 400 clinical trials to test gene therapy against a wide array of illnesses.
Surprisingly, a disease not typically heading the charts of heritable disorders, cancer has dominated the research.
In 1994 cancer patients were treated with the tumor necrosis factor gene, a natural tumor fighting protein which worked to a limited extent.
Even more surprisingly, after the initial flurry of success little has worked.
Gene therapy, the promising miracle of 1990 failed to deliver on its early promise over the decade.
Apart from those examples, there are many diseases whose molecular pathology is, or soon will be, well understood, but for which no satisfactory treatments have yet been developed.
At the beginning of the nineties it appeared that gene therapy did offer new opportunities to treat these disorders both by restoring gene functions that have been lost through mutation and by introducing genes that can inhibit the replication of infectious agents, render cells resistant to cytotoxic drugs, or cause the elimination of aberrant cells.
From this "genomic" viewpoint genes could be said to be viewed as medicines, and their development as therapeutics should embrace the issues facing the development of small-molecule and protein therapeutics such as bioavailability, specificity, toxicity, potency, and the ability to be manufactured at large scale in a cost-effective manner.
Of course for such a radical approach certain basal level criteria needed to be established for selecting disease candidates for human gene therapy.
These include, such factors as the disease is an incurable, life-threatening disease; organ, tissue, and cell types affected by the disease have been identified; the normal counterpart of the defective gene has been isolated and cloned; either the normal gene can be introduced into a substantial subfraction of the cells from the affected tissue, or the introduction of the gene into the available target tissue, such as bone marrow, will somehow alter the disease process in the tissue affected by the disease; the gene can be expressed adequately (it will direct the production of enough normal protein to make a difference); and techniques are available to verify the safety of the procedure.
An ideal gene therapeutic should, therefore, be stably formulated at room temperature and amenable to administration either as an injectable or aerosol or by oral delivery in liquid or capsule form.
The therapeutic should also be suitable for repeat therapy, and when delivered, it should neither generate an immune response nor be destroyed by tissue-scavenging mechanisms.
When delivered to the target cell, the therapeutic gene should then be transported to the nucleus, where it should be maintained as a stable plasmid or chromosomal integrant, and be expressed in a predictable, controlled fashion at the desired potency in a cell-specific or tissue-specific manner.
In addition to the ADA gene transfer in children with severe combined immunodeficiency syndrome, a gene-marking study of Epstein-Barr virus-specific cytotoxic T cells, and trials of gene-modified T cells expressing suicide or viral resistance genes in patients infected with HIV were studied in the early nineties.
Additional strategies for T-cell gene therapy which were pursued later in the decade involve the engineering of novel T-cell receptors that impart antigen specificity for virally infected or malignant cells.
Issues which still are not resolved include nuclear transport, integration, regulated gene expression and immune surveillance.
This knowledge, when finally understood and applied to the design of delivery vehicles of either viral or non-viral origin, will assist in the realization of gene therapeutics as safe and beneficial medicines that are suited to the routine management of human health.
Scientists are also working on using gene therapy to generate antibodies directly inside cells to block the production of harmful viruses such as HIV or even cancer-inducing proteins.
There is a specific connection with Francis Collins, as his motivation for pursuing the HGP was his pursuit of defective genes beginning with the cystic fibrosis gene.
This gene, called the CF transmembrane conductance regulator, codes for an ion channel protein that regulates salts in the lung tissue.
The faulty gene prevents cells from excreting salt properly causing a thick sticky mucus to build up and destroy lung tissue.
Scientists have spliced copies of the normal genes into disabled adeno viruses that target lung tissues and have used bronchioscopes to deliver them to the lungs.
The procedure worked well in animal studies however clinical trials in humans were not an unmitigated success.
Because the cells lining the lungs are continuously being replaced the effect is not permanent and must be repeated.
Studies are underway to develop gene therapy techniques to replace other faulty genes.
For example, to replace the genes responsible for factor VIII and factor IX production whose malfunctioning causes hemophilia A and B respectively; and to alleviate the effects of the faulty gene in dopamine production that results in Parkinson's disease.
Apart from technical challenges such a radical therapy also engenders ethical debate.
Many persons who voice concerns about somatic-cell gene therapy use a "slippery slope" argument.
It sounds good in theory but where does one draw the line.
There are many issues yet to be resolved in this field of thorny ethics "good" and "bad" uses of the gene modification, difficulty of following patients in long-term clinical research and such.
Many gene therapy candidates are children who are too young to understand the ramifications of this treatment: Conflict of interest -pits individuals' reproductive liberties and privacy interests against the interests of insurance companies or society.
One issue that is unlikely to ever gain acceptance is germline therapy, the removal of deleterious genes from the population.
Issues of justice and resource allocation also have been raised: In a time of strain on our health care system, can we afford such expensive therapy?
Who should receive gene therapy?
If it is made available only to those who can afford it, then a number of civil rights groups claim that the distribution of desirable biological traits among different socioeconomic and ethnic groups would become badly skewed adding a new and disturbing layer of discriminatory behavior.
Indeed a major setback occurred before the end of the decade in 1999.
Jesse Gelsinger was the first person to die from gene therapy, on September 17, 1999, and his death created another unprecedented situation when his family sued not only the research team involved in the experiment (U Penn), the company Genovo Inc., but also the ethicist who offered moral advice on the controversial project.
This inclusion of the ethicist as a defendant alongside the scientists and school was a surprising legal move that puts this specialty on notice, as will no doubt be the case with other evolving technologies such as stem cells and therapeutic cloning, that its members could be vulnerable to litigation over the philosophical guidance they provide to researchers.
The Penn group principal investigator James Wilson approached ethicist Arthur Caplan about their plans to test the safety of a genetically engineered virus on babies with a deadly form of the liver disorder, ornithine transcarbamylase deficiency.
The disorder allows poisonous levels of ammonia to build up in the blood system.
Caplan steered the researchers away from sick infants, arguing that desperate parents could not provide true informed consent.
He said it would be better to experiment on adults with a less lethal form of the disease who were relatively healthy.
Gelsinger fell into that category.
Although he had suffered serious bouts of ammonia buildup, he was doing well on a special drug and diet regimen.
The decision to use relatively healthy adults was controversial because risky, unproven experimental protocols generally use very ill people who have exhausted more traditional treatments, so have little to lose.
In this case, the virus used to deliver the genes was known to cause liver damage, so some scientists were concerned it might trigger an ammonia crisis in the adults.
Wilson underestimated the risk of the experiment, omitted the disclosure about possible liver damage in earlier volunteers in the experiment and failed to mention the deaths of monkeys given a similar treatment during pre-clinical studies.
A Food and Drug Administration investigation after Gelsinger's death found numerous regulatory violations by Wilson's team, including the failure to stop the experiment and inform the FDA after four successive volunteers suffered serious liver damage prior to the teen's treatment.
In addition, the FDA said Gelsinger did not qualify for the experiment, because his blood ammonia levels were too high just before he underwent the infusion of genetic material.
The FDA suspended all human gene experiments by Wilson and the University of Penn subsequently restricting him solely to animal studies.
A follow-up FDA investigation subsequently alleged he improperly tested the experimental treatment on animals.
Financial conflicts of interest also surrounded James Wilson, who stood to personally profit from the experiment through Genovo his biotechnology company.
The lawsuit was settled out of court for undisclosed terms in November 2000.
The FDA also suspended gene therapy trials at St. Elizabeth's Medical Center in Boston, a major teaching affiliate of Tufts University School of Medicine, which sought to use gene therapy to reverse heart disease, because scientists there failed to follow protocols and may have contributed to at least one patient death.
In addition, the FDA temporarily suspended two liver cancer studies sponsored by the Schering-Plough Corporation because of technical similarities to the University of Pennsylvania study.
Some research groups voluntarily suspended gene therapy studies, including two experiments sponsored by the Cystic Fibrosis Foundation and studies at Beth Israel Deaconess Medical Center in Boston aimed at hemophilia.
The scientists paused to make sure they learned from the mistakes.
The nineties also saw the development of another "high-thoughput" breakthrough, a derivative of the other high tech revolution namely DNA chips.
In 1991 Biochips were developed for commercial use under the guidance of Affymetrix.
DNA chips or microarrays represent a "massively parallel" genomic technology.
They facilitate high throughput analysis of thousands of genes simultaneously, and are thus potentially very powerful tools for gaining insight into the complexities of higher organisms including analysis of gene expression, detecting genetic variation, making new gene discoveries, fingerprinting strains and developing new diagnostic tools.
These technologies permit scientists to conduct large scale surveys of gene expression in organisms, thus adding to our knowledge of how they develop over time or respond to various environmental stimuli.
These techniques are especially useful in gaining an integrated view of how multiple genes are expressed in a coordinated manner.
These DNA chips have broad commercial applications and are now used in many areas of basic and clinical research including the detection of drug resistance mutations in infectious organisms, direct DNA sequence comparison of large segments of the human genome, the monitoring of multiple human genes for disease associated mutations, the quantitative and parallel measurement of mRNA expression for thousands of human genes, and the physical and genetic mapping of genomes.
However the initial technologies, or more accurately the algorithms used to extract information, were far from robust and reproducible.
The erstwhile serial entrepreneur, Al Zaffaroni (the rebel who in 1968 founded Alza when Syntex ignored his interest in developing new ways to deliver drugs) founded yet another company, Affymetrix, under the stewardship of Stephen Fodor, which was subject to much abuse for providing final extracted data and not allowing access to raw data.
As with other personalities of this high through put era, Seattle-bred Steve Fodor was also somewhat of a polymath having contributed to two major technologies, microarrays and combinatorial chemistry, the former has delivered on it's, promise while the latter, like gene therapy, is still in a somewhat extended gestation.
And despite the limitations of being an industrial scientist he has had a rather prolific portfolio of publications.
His seminal manuscripts describing this work have been published in all the journals of note, Science, Nature and PNAS and was recognized in 1992 by the AAAS by receiving the Newcomb-Cleveland Award for an outstanding paper published in Science.
Fodor began his industrial career in yet another Zaffaroni firm.
In 1989 he was recruited to the Affymax Research Institute in Palo Alto where he spearheaded the effort to develop high-density arrays of biological compounds.
His initial interest was in the broad area of what came to be called combinatorial chemistry.
Of the techniques developed, one approach permitted high resolution chemical synthesis in a light-directed, spatially-defined format.
In the days before positive selection vectors, a researcher might have screened thousands of clones by hand with an oligonucleotide probe just to find one elusive insert.
Fodor's (and his successors) DNA array technology reverses that approach.
Instead of screening an array of unknowns with a defined probe -a cloned gene, PCR product, or synthetic oligonucleotide -each position or "probe cell" in the array is occupied by a defined DNA fragment, and the array is probed with the unknown sample.
Fodor used his chemistry and biophysics background to develop very dense arrays of these biomolecules by combining photolithographic methods with traditional chemical techniques.
The typical array may contain all possible combinations of all possible oligonucleotides (8-mers, for example) that occur as a "window" which is tracked along a DNA sequence.
It might contain longer oligonucleotides designed from all the open reading frames identified from a complete genome sequence.
Or it might contain cDNAs -of known or unknown sequence -or PCR products.
Of course it is one thing to produce data it is quite another to extract it in a meaningful manner.
Fodor's group also developed techniques to read these arrays, employing fluorescent labeling methods and confocal laser scanning to measure each individual binding event on the surface of the chip with extraordinary sensitivity and precision.
This general platform of microarray based analysis coupled to confocal laser scanning has become the standard in industry and academia for large-scale genomics studies.
In 1993, Fodor co-founded Affymetrix where the chip technology has been used to synthesize many varieties of high density oligonucleotide arrays containing hundreds of thousands of DNA probes.
In 2001, Steve Fodor founded Perlegen, Inc., a new venture that applied the chip technology towards uncovering the basic patterns of human diversity.
His company's stated goals are to analyze more than one million genetic variations in clinical trial participants to explain and predict the efficacy and adverse effect profiles of prescription drugs.
In addition, Perlegen also applies this expertise to discovering genetic variants associated with disease in order to pave the way for new therapeutics and diagnostics.
Fodor's former company diversified into plant applications by developing a chip of the archetypal model of plant systems Arabidopsis and supplied Pioneer Hi Bred with custom DNA chips for monitoring maize gene expression.
They (Affymetrix) have established programs where academic scientists can use company facilities at a reduced price and set up 'user centers' at selected universities.
A related but less complex technology called 'spotted' DNA chips involves precisely spotting very small droplets of genomic or cDNA clones or PCR samples on a microscope slide.
The process uses a robotic device with a print head bearing fine "repeatograph" tips that work like fountain pens to draw up DNA samples from a 96-well plate and spot tiny amounts on a slide.
Up to 10,000 individual clones can be spotted in a dense array within one square centimeter on a glass slide.
After hybridization with a fluorescent target mRNA, signals are detected by a custom scanner.
This is the basis of the systems used by Molecular Dynamics and Incyte (who acquired this technology when it took over Synteni).
In 1997, Incyte was looking to gather more data for its library and perform experiments for corporate subscribers.
The company considered buying Affymetrix GeneChips but opted instead to purchase the smaller Synteni, which had sprung out of Pat Brown's Stanford array effort.
Synteni's contact printing technology resulted in dense -and cheaper -arrays.
Though Incyte used the chips only internally, Affymetrix sued, claiming Synteni/Incyte was infringing on its chip density patents.
The suit argued that dense biochips -regardless of whether they use photolithography -cannot be made without a license from Affymetrix!
And in a litigious Congo line endemic of this hi-tech era Incyte countersued and for good measure also filed against genetic database competitor Gene Logic for infringing Incyte's patents on database building.
Meanwhile, Hyseq sued Affymetrix, claiming infringement of nucleotide hybridization patents obtained by its CSO.
Affymetrix, in turn, filed a countersuit, claiming Hyseq infringed the spotted array patents.
Hyseq then reached back and found an additional hybridization patent it claimed that Affymetrix had infringed.
And so on into the next millennium!
In part to avoid all of this another California company Nanogen, Inc. took a different approach to single nucleotide polymorphism discrimination technology.
In an article in the April 2000 edition of Nature Biotechnology, entitled "Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips," Nanogen describes the use of microchips to identify variants of the mannose binding protein gene that differ from one another by only a single DNA base.
The mannose binding protein (MBP) is a key component of the innate immune system in children who have not yet developed immunity to a variety of pathogens.
To date, four distinct variants (alleles) of this gene have been identified, all differing by only a single nucleotide of DNA.
MBP was selected for this study because of its potential clinical relevance and its genetic complexity.
The samples were assembled at the NCI laboratory in conjunction with the National Institutes of Health and transferred to Nanogen for analysis.
However, from a high throughput perspective there is a question mark over microarrays.
Mark Benjamin, senior director of business development at Rosetta Inpharmatics (Kirkland, WA), is skeptical about the long-term prospects for standard DNA arrays in high-throughput screening as the first steps require exposing cells and then isolating RNA, which is something that is very hard to do in a high-throughput format.
Another drawback is that most of the useful targets are likely to be unknown (particularly in the agricultural sciences where genome sequencing is still in its infancy), and DNA arrays that are currently available test only for previously sequenced genes.
Indeed, some argue that current DNA arrays may not be sufficiently sensitive to detect the low expression levels of genes encoding targets of particular interest.
And the added complication of the companies' reluctance to provide "raw data" means that derived data sets may be created with less than optimum algorithims thereby irretrievably losing potentially valuable information from the starting material.
Reverse engineering is a possible approach but this is laborious and time consuming and being prohibited by many contracts may arouse the interest of the ever-vigilant corporate lawyers.
Over the course of the nineties, outgrowths of functional genomics have been termed proteomics and metabolomics, which are the global studies of gene expression at the protein and metabolite levels respectively.
The study of the integration of information flow within an organism is emerging as the field of systems biology.
In the area of proteomics, the methods for global analysis of protein profiles and cataloging protein-protein interactions on a genome-wide scale are technically more difficult but improving rapidly, especially for microbes.
These approaches generate vast amounts of quantitative data.
The amount of expression data becoming available in the public and private sectors is already increasing exponentially.
Gene and protein expression data rapidly dwarfed the DNA sequence data and is considerably more difficult to manage and exploit.
In microbes, the small sizes of the genomes and the ease of handling microbial cultures, will enable high throughput, targeted deletion of every gene in a genome, individually and in combinations.
This is already available on a moderate throughput scale in model microbes such as E. coli and yeast.
Combining targeted gene deletions and modifications with genome-wide assay of mRNA and protein levels will enable intricate inter-dependencies among genes to be unraveled.
Simultaneous measurement of many metabolites, particularly in microbes, is beginning to allow the comprehensive modeling and regulation of fluxes through interdependent pathways.
Metabolomics can be defined as the quantitative measurement of all low molecular weight metabolites in an organism's cells at a specified time under specific environmental conditions.
Combining information from metabolomics, proteomics and genomics will help us to obtain an integrated understanding of cell biology.
The next hierarchical level of phenotype considers how the proteome within and among cells cooperates to produce the biochemistry and physiology of individual cells and organisms.
Several authors have tentatively offered "physiomics" as a descriptor for this approach.
The final hierarchical levels of phenotype include anatomy and function for cells and whole organisms.
The term "phenomics" has been applied to this level of study and unquestionably the more well known omics namely economics, has application across all those fields.
And, coming slightly out of left field this time, the spectre of eugenics needless to say was raised in the omics era.
In the year 1992 American and British scientists unveiled a technique which has come to be known as pre-implantation genetic diagnosis (PID) for testing embryos in vitro for genetic abnormalities such as cystic fibrosis, hemophilia, and Down's Syndrome (Wald, 1992) .
This might be seen by most as a step forward, but it led ethicist David S. King (1999) to decry PID as a technology that could exacerbate the eugenic features of prenatal testing and make possible an expanded form of free-market eugenics.
He further argues that due to social pressures and eugenic attitudes held by clinical geneticists in most countries, it results in eugenic outcomes even though no state coercion is involved and that, as abortion is not involved, and multiple embryos are available, PID is radically more effective as a tool of genetic selection.
The first regulatory approval of a recombinant DNA technology in the U.S. food supply was not a plant but an industrial enzyme that has become the hallmark of food biotechnology success.
Enzymes were important agents in food production long before modern biotechnology was developed.
They were used, for instance, in the clotting of milk to prepare cheese, the production of bread and the production of alcoholic beverages.
Nowadays, enzymes are indispensable to modern food processing technology and have a great variety of functions.
They are used in almost all areas of food production including grain processing, milk products, beer, juices, wine, sugar and meat.
Chymosin, known also as rennin, is a proteolytic enzyme whose role in digestion is to curdle or coagulate milk in the stomach, efficiently converting liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in a neonate's stomach.
The dairy industry takes advantage of this property to conduct the first step in cheese production.
Chy-Max™, an artificially produced form of the chymosin enzyme for cheese-making, was approved in 1990.
In some instances they replace less acceptable "older" technology, for example the enzyme chymosin.
Unlike crops industrial enzymes have had relatively easy passage to acceptance for a number of reasons.
As noted they are part of the processing system and theoretically do not appear in the final product.
Today about 90% of the hard cheese in the US and UK is made using chymosin from geneticallymodified microbes.
It is easier to purify, more active (95% as compared to 5%) and less expensive to produce (Microbes are more prolific, more productive and cheaper to keep than calves).
Like all enzymes it is required only in very small quantities and because it is a relatively unstable protein it breaks down as the cheese matures.
Indeed, if the enzyme remained active for too long it would adversely affect the development of the cheese, as it would degrade the milk proteins to too great a degree.
Such enzymes have gained the support of vegetarian organizations and of some religious authorities.
For plants the nineties was the era of the first widespread commercialization of what came to be known in often deprecating and literally inaccurate terms as GMOs (Genetically Modified Organisms).
When the nineties dawned dicotyledonous plants were relatively easily transformed with Agrobacterium tumefaciens but many economically important plants, including the cereals, remained inaccessible for genetic manipulation because of lack of effective transformation techniques.
In 1990 this changed with the technology that overcame this limitation.
Michael Fromm, a molecular biologist at the Plant Gene Expression Center, reported the stable transformation of corn using a high-speed gene gun.
The method known as biolistics uses a "particle gun" to shoot metal particles coated with DNA into cells.
Initially a gunpowder charge subsequently replaced by helium gas was used to accelerate the particles in the gun.
There is a minimal disruption of tissue and the success rate has been extremely high for applications in several plant species.
The technology rights are now owned by DuPont.
In 1990 some of the first of the field trials of the crops that would dominate the second half of the nineties began, including Bt corn (with the Bacillus thuriengenesis Cry protein discussed in chapter three).
In 1992 the FDA declared that genetically engineered foods are "not inherently dangerous" and do not require special regulation.
Since 1992, researchers have pinpointed and cloned several of the genes that make selected plants resistant to certain bacterial and fungal infections; some of these genes have been successfully inserted into crop plants that lack them.
Many more infection-resistant crops are expected in the near future, as scientists find more plant genes in nature that make plants resistant to pests.
Plant genes, however, are just a portion of the arsenal; microorganisms other than Bt also are being mined for genes that could help plants fend off invaders that cause crop damage.
The major milestone of the decade in crop biotechnology was approval of the first bioengineered crop plant in 1994.
It represented a double first not just of the first approved food crop but also of the first commercial validation of a technology which was to be surpassed later in the decade.
That technology, antisense technology works because nucleic acids have a natural affinity for each other.
When a gene coding for the target in the genome is introduced in the opposite orientation, the reverse RNA strand anneals and effectively blocks expression of the enzyme.
This technology was patented by Calgene for plant applications and was the technology behind the famous FLAVR SAVR tomatoes.
The first success for antisense in medicine was in 1998 when the U.S. Food and Drug Administration gave the go-ahead to the cytomegalovirus (CMV) inhibitor fomivirsen, a phosphorothionate antiviral for the AIDS-related condition CMV retinitis making it the first drug belonging to Isis, and the first antisense drug ever, to be approved.
Another technology, although not apparent at the time was behind the second approval and also the first and only successful to date in a commercial tree fruit biotech application.
The former was a virus resistant squash the second the papaya ringspot resistant papaya.
Both owed their existence as much to historic experience as modern technology.
Genetically engineered virus-resistant strains of squash and cantaloupe, for example, would never have made it to farmers' fields if plant breeders in the 1930's had not noticed that plants infected with a mild strain of a virus do not succumb to more destructive strains of the same virus.
That finding led plant pathologist Roger Beachy, then at Washington University in Saint Louis, to wonder exactly how such "cross-protection" worked -did part of the virus prompt it?
In collaboration with researchers at Monsanto, Beachy used an A. tumefaciens vector to insert into tomato plants a gene that produces one of the proteins that makes up the protein coat of the tobacco mosaic virus.
He then inoculated these plants with the virus and was pleased to discover, as reported in 1986, that the vast majority of plants did not succumb to the virus.
Eight years later, in 1994, virus-resistant squash seeds created with Beachy's method reached the market, to be followed soon by bioengineered virus-resistant seeds for cantaloupes, potatoes, and papayas.
(Breeders had already created virusresistant tomato seeds by using traditional techniques.)
And the method of protection still remained a mystery when the first approvals were given in 1994 and 1996.
Gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants.
It now seems that it is the consequence of accidentally triggering the plant's adaptive defense mechanism against viruses and transposable elements.
This recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals.
How this system worked was not elucidated until later in the decade by a researcher who was seeking a very different holy grail -the black rose!
Rick Jorgensen, at that time at DNA Plant Technologies in Oakland, CA and subsequently of, of the University of California Davis attempted to overexpress the chalcone synthase gene by introducing a modified copy under a strong promoter.Surprisingly he obtained white flowers, and many strange variegated purple and white variations in between.
This was the first demonstration of what has come to be known as post-transcriptional gene silencing (PTGS).
While initially it was considered a strange phenomenon limited to petunias and a few other plant species, it is now one of the hottest topics in molecular biology.
RNA interference (RNAi) in animals and basal eukaryotes, quelling in fungi, and PTGS in plants are examples of a broad family of phenomena collectively called RNA silencing (Hannon 2002; Plasterk 2002) .
In addition to its occurrence in these species it has roles in viral defense (as demonstrated by Beachy) and transposon silencing mechanisms among other things.
Perhaps most exciting, however, is the emerging use of PTGS and, in particular, RNAi -PTGS initiated by the introduction of double-stranded RNA (dsRNA) -as a tool to knock out expression of specific genes in a variety of organisms.
Nineteen ninety one also heralded yet another first.
The February 1, 1991 issue of Science reported the patenting of "molecular scissors": the Nobel-prize winning discovery of enzymatic RNA, or "ribozymes," by Thomas Czech of the University of Colorado.
It was noted that the U.S. Patent and Trademark Office had awarded an "unusually broad" patent for ribozymes.
The patent is U.S. Patent No.
4,987,071, claim 1 of which reads as follows: "An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, and causing cleavage at said cleavage site by a transesterification reaction."
Although enzymes made of protein are the dominant form of biocatalyst in modern cells, there are at least eight natural RNA enzymes, or ribozymes, that catalyze fundamental biological processes.
One of which was yet another discovery by plant virologists, in this instance the hairpin ribozyme was discovered by George Bruening at UC Davis.
The self-cleavage structure was originally called a paperclip, by the Bruening laboratory which discovered the reactions.
As mentioned in chapter 3, it is believed that these ribozymes might be the remnants of an ancient form of life that was guided entirely by RNA.
Since a ribozyme is a catalytic RNA molecule capable of cleaving itself and other target RNAs it therefore can be useful as a control system for turning off genes or targeting viruses.
The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
In the therapeutics area, they have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders.
Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials.
More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation.
However, targeting ribozymes to the cellular compartment containing their target RNAs has proved a challenge.
At the other bookend of the decade in 2000, Samarsky et al.
reported that a family of small RNAs in the nucleolus (snoRNAs) can readily transport ribozymes into this subcellular organelle.
In addition to the already extensive panoply of RNA entities yet another has potential for mischief.
Viroids are small, single-stranded, circular RNAs containing 246-463 nucleotides arranged in a rod-like secondary structure and are the smallest pathogenic agents yet described.
The smallest viroid characterized to date is rice yellow mottle sobemovirus (RYMV), at 220 nucleotides.
In comparison, the genome of the smallest known viruses capable of causing an infection by themselves, the single-stranded circular DNA of circoviruses, is around 2 kilobases in size.
The first viroid to be identified was the Potato spindle tuber viroid (PSTVd).
Some 33 species have been identified to date.
Unlike the many satellite or defective interfering RNAs associated with plant viruses, viroids replicate autonomously on inoculation of a susceptible host.
The absence of a protein capsid and of detectable messenger RNA activity implies that the information necessary for replication and pathogenesis resides within the unusual structure of the viroid genome.
The replication mechanism actually involves interaction with RNA polymerase II, an enzyme normally associated with synthesis of messenger RNA, and "rolling circle" synthesis of new RNA.
Some viroids have ribozyme activity which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates.
It has been proposed that viroids are "escaped introns".
Viroids are usually transmitted by seed or pollen.
Infected plants can show distorted growth.
From its earliest years, biotechnology attracted interest outside scientific circles.
Initially the main focus of public interest was on the safety of recombinant DNA technology, and of the possible risks of creating uncontrollable and harmful novel organisms (Berg , 1975) .
The debate on the deliberate release of genetically modified organisms, and on consumer products containing or comprising them, followed some years later (NAS, 1987) .
It is interesting to note that within the broad ethical tableau of potential issues within the science and products of biotechnology, the seemingly innocuous field of plant modification has been one of the major players of the 1990's.
The success of agricultural biotechnology is heavily dependent on its acceptance by the public, and the regulatory framework in which the industry operates is also influenced by public opinion.
As the focus for molecular biology research shifted from the basic pursuit of knowledge to the pursuit of lucrative applications, once again as in the previous two decades the specter of risk arose as the potential of new products and applications had to be evaluated outside the confines of a laboratory.
However, the specter now became far more global as the implications of commercial applications brought not just worker safety into the loop but also, the environment, agricultural and industrial products and the safety and well being of all living things.
Beyond "deliberate" release, the RAC guidelines were not designed to address these issues, so the matter moved into the realm of the federal agencies who had regulatory authority which could be interpreted to oversee biotechnology issues.
This adaptation of oversight is very much a dynamic process as the various agencies wrestle with the task of applying existing regulations and developing new ones for oversight of this technology in transition.
As the decade progressed focus shifted from basic biotic stress resistance to more complex modifications The next generation of plants will focus on value added traits in which valuable genes and metabolites will be identified and isolated, with some of the later compounds being produced in mass quantities for niche markets.
Two of the more promising markets are nutraceuticals or so-called "Functional Foods" and plants developed as bioreactors for the production of valuable proteins and compounds, a field known as Plant Molecular Farming.
Developing plants with improved quality traits involves overcoming a variety of technical challenges inherent to metabolic engineering programs.
Both traditional plant breeding and biotechnology techniques are needed to produce plants carrying the desired quality traits.
Continuing improvements in molecular and genomic technologies are contributing to the acceleration of product development in this space.
By the end of the decade in 1999, applying nutritional genomics, Della Penna (1999) isolated a gene, which converts the lower activity precursors to the highest activity vitamin E compound, alpha-tocopherol.
With this technology, the vitamin E content of Arabidopsis seed oil has been increased nearly 10-fold and progress has been made to move the technology to crops such as soybean, maize, and canola.
This has also been done for folates in rice.
Omega three fatty acids play a significant role in human health, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are present in the retina of the eye and cerebral cortex of the brain, respectively, are some of the most well documented from a clinical perspective.
It is believed that EPA and DHA play an important role in the regulation of inflammatory immune reactions and blood pressure, treatment of conditions such as cardiovascular disease and cystic fibrosis, brain development in utero, and, in early postnatal life, the development of cognitive function.
They are mainly found in fish oil and the supply is limited.
By the end of the decade Ursin (2000) had succeeded in engineering canola to produce these fatty acids.
From a global perspective another value-added development had far greater impact both technologically and socio-economically.
A team led by Ingo Potrykus (1999) engineered rice to produce pro-Vitamin A, which is an essential micronutrient.
Widespread dietary deficiency of this vitamin in rice-eating Asian countries, which predisposes children to diseases such as blindness and measles, has tragic consequences.
Improved vitamin A nutrition would alleviate serious health problems and, according to UNICEF, could also prevent up to two million infant deaths due to vitamin A deficiency.
Adoption of the next stage of GM crops may proceed more slowly, as the market confronts issues of how to determine price, share value, and adjust marketing and handling to accommodate specialized end-use characteristics.
Furthermore, competition from existing products will not evaporate.
Challenges that have accompanied GM crops with improved agronomic traits, such as the stalled regulatory processes in Europe, will also affect adoption of nutritionally improved GM products.
Beyond all of this, credible scientific research is still needed to confirm the benefits of any particular food or component.
For functional foods to deliver their potential public health benefits, consumers must have a clear understanding of, and a strong confidence level in, the scientific criteria that are used to document health effects and claims.
Because these decisions will require an understanding of plant biochemistry, mammalian physiology, and food chemistry, strong interdisciplinary collaborations will be needed among plant scientists, nutritionists, and food scientists to ensure a safe and healthful food supply.
In addition to being a source of nutrition, plants have been a valuable wellspring of therapeutics for centuries.
During the nineties, however, intensive research has focused on expanding this source through rDNA biotechnology and essentially using plants and animals as living factories for the commercial production of vaccines, therapeutics and other valuable products such as industrial enzymes and biosynthetic feedstocks.
Possibilities in the medical field include a wide variety of compounds, ranging from edible vaccine antigens against hepatitis B and Norwalk viruses (Arntzen, 1997) and Pseudomonas aeruginosa and Staphylococcus aureus to vaccines against cancer and diabetes, enzymes, hormones, cytokines, interleukins, plasma proteins, and human alpha-1-antitrypsin.
Thus, plant cells are capable of expressing a large variety of recombinant proteins and protein complexes.
Therapeutics produced in this way are termed plant made pharmaceuticals (PMPs).
And non-therapeutics are termed plant made industrial products (PMIPs) (Newell-McGloughlin, 2006) .
The first reported results of successful human clinical trials with their transgenic plant-derived pharmaceuticals were published in 1998.
They were an edible vaccine against E. coli-induced diarrhea and a secretory monoclonal antibody directed against Streptococcus mutans, for preventative immunotherapy to reduce incidence of dental caries.
Haq et al.
(1995) reported the expression in potato plants of a vaccine against E. coli enterotoxin (ETEC) that provided an immune response against the toxin in mice.
Human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of Vibrio cholerae and E. coli induces production of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells.
Similar results were found for Norwalk virus oral vaccines in potatoes.
For developing countries, the intention is to deliver them in bananas or tomatoes (Newell-McGloughlin, 2006) .
Plants are also faster, cheaper, more convenient and more efficient than the principal eukaryotic production system, namely Chinese Hamster Ovary (CHO) cells for the production of pharmaceuticals.
Hundreds of acres of protein-containing seeds could inexpensively double the production of a CHO bioreactor factory.
In addition, proteins can be expressed at the highest levels in the harvestable seed and plant-made proteins and enzymes formulated in seeds have been found to be extremely stable, reducing storage and shipping costs.
Pharming may also enable research on drugs that cannot currently be produced.
For example, CropTech in Blacksburg, Va., is investigating a protein that seems to be a very effective anticancer agent.
The problem is that this protein is difficult to produce in mammalian cell culture systems as it inhibits cell growth.
This should not be a problem in plants.
Furthermore, production size is flexible and easily adjustable to the needs of changing markets.
Making pharmaceuticals from plants is also a sustainable process, because the plants and crops used as raw materials are renewable.
The system also has the potential to address problems associated with provision of vaccines to people in developing countries.
Products from these alternative sources do not require a so-called "cold chain" for refrigerated transport and storage.
Those being developed for oral delivery obviates the need for needles and aspectic conditions which often are a problem in those areas.
Apart from those specific applications where the plant system is optimum there are many other advantages to using plant production.
Many new pharmaceuticals based on recombinant proteins will receive regulatory approval from the United States Food and Drug Administration (FDA) in the next few years.
As these therapeutics make their way through clinical trials and evaluation, the pharmaceutical industry faces a production capacity challenge.
Pharmaceutical discovery companies are exploring plant-based production to overcome capacity limitations, enable production of complex therapeutic proteins, and fully realize the commercial potential of their biopharmaceuticals (Newell-McGloughlin, 2006) .
Nineteen ninety also marked a major milestone in the animal biotech world when Herman made his appearance on the world's stage.
Since the Palmiter's mouse, transgenic technology has been applied to several species including agricultural species such as sheep, cattle, goats, pigs, rabbits, poultry, and fish.
Herman was the first transgenic bovine created by GenPharm International, Inc., in a laboratory in the Netherlands at the early embryo stage.
Scientist's microinjected recently fertilized eggs with the gene coding for human lactoferrin.
The scientists then cultured the cells in vitro to the embryo stage and transferred them to recipient cattle.
Lactoferrin, an iron-containing anti-bacterial protein is essential for infant growth.
Since cow's milk doesn't contain lactoferrin, infants must be fed from other sources that are rich in iron -formula or mother's milk (Newell-McGloughlin, 2001) .
As Herman was a boy he would be unable to provide the source, that would require the production of daughters which was not necessarily a straightforward process.
The Dutch parliments permission was required.
In 1992 they finally approved a measure that permitted the world's first genetically engineered bull to reproduce.
The Leiden-based Gene Pharming proceeded to artificially inseminate 60 cows with Herman's sperm.
With a promise that the protein, lactoferrin, would be the first in a new generation of inexpensive, high-tech drugs derived from cows' milk to treat complex diseases like AIDS and cancer.
Herman, became the father of at least eight female calves in 1994, and each one inherited the gene for lactoferrin production.
While their birth was initially greeted as a scientific advancement that could have far-reaching effects for children in developing nations, the levels of expression were too low to be commercially viable.
By 2002, Herman, who likes to listen to rap music to relax, had sired 55 calves and outlived them all.
His offspring were all killed and destroyed after the end of the experiment, in line with Dutch health legislation.
Herman was also slated for the abattoir, but the Dutch public -proud of making history with Herman -rose up in protest, especially after a television program screened footage showing the amiable bull licking a kitten.
Herman won a bill of clemency from parliament.
However, instead of retirement on a comfortable bed of straw, listening to rap music, Herman was pressed into service again.
He now stars at a permanent biotech exhibit in Naturalis, a natural history museum in the Dutch city of Leiden.
After his death, he will be stuffed and remain in the museum in perpetuity (A fate similar to what awaited an even more famous mammalian first born later in the decade).
The applications for transgenic animal research fall broadly into two distinct areas, namely medical and agricultural applications.
The recent focus on developing animals as bioreactors to produce valuable proteins in their milk can be catalogued under both areas.
Underlying each of these, of course, is a more fundamental application, that is the use of those techniques as tools to ascertain the molecular and physiological bases of gene expression and animal development.
This understanding can then lead to the creation of techniques to modify development pathways.
In 1992 a European decision with rather more far-reaching implications than Hermans sex life was made.
The first European patent on a transgenic animal was issued for a transgenic mouse sensitive to carcinogens -Harvard's "Oncomouse".
The oncomouse patent application was refused in Europe in 1989 due primarily to an established ban on animal patenting.
The application was revised to make narrower claims, and the patent was granted in 1992.
This has since been repeatedly challenged, primarily by groups objecting to the judgement that benefits to humans outweigh the suffering of the animal.
Currently, the patent applicant is awaiting protestors' responses to a series of possible modifications to the application.
Predictions are that agreement will not likely be forthcoming and that the legal wrangling will continue into the future.
Bringing animals into the field of controversy starting to swirl around GMOs and preceding the latter's commercialization, was the approval by the FDA of bovine somatotropin (BST) for increased milk production in dairy cows.
The FDA's Center for Veterinary Medicine (CVM) regulates the manufacture and distribution of food additives and drugs that will be given to animals.
Biotechnology products are a growing proportion of the animal health products and feed components regulated by the CVM.
The Center requires that food products from treated animals must be shown to be safe for human consumption.
Applicants must show that the drug is effective and safe for the animal and that its manufacture will not affect the environment.
They must also conduct geographically dispersed clinical trials under an Investigational New Animal Drug application with the FDA through which the agency controls the use of the unapproved compound in food animals.
Unlike within the EU, possible economic and social issues cannot be taken into consideration by the FDA in the premarket drug approval process.
Under these considerations the safety and efficacy of rBST was determined.
It was also determined that special labeling for milk derived from cows that had been treated with rBST is not required under FDA food labeling laws because the use of rBST does not effect the quality or the composition of the milk.
Work with fish proceeded a pace throughout the decade.
Gene transfer techniques have been applied to a large number of aquatic organisms, both vertebrates and invertebrates.
Gene transfer experiments have targeted a wide variety of applications, including the study of gene structure and function, aquaculture production, and use in fisheries management programs.
Because fish have high fecundity, large eggs, and do not require reimplantation of embryos, transgenic fish prove attractive model systems in which to study gene expression.
Transgenic zebrafish have found utility in studies of embryogenesis, with expression of transgenes marking cell lineages or providing the basis for study of promoter or structural gene function.
Although not as widely used as zebrafish, transgenic medaka and goldfish have been used for studies of promoter function.
This body of research indicates that transgenic fish provide useful models of gene expression, reliably modeling that in "higher" vertebrates.
Perhaps the largest number of gene transfer experiments address the goal of genetic improvement for aquaculture production purposes.
The principal area of research has focused on growth performance, and initial transgenic growth hormone (GH) fish models have demonstrated accelerated and beneficial phenotypes.
DNA microinjection methods have propelled the many studies reported and have been most effective due to the relative ease of working with fish embryos.
Bob Devlins' group in Vancouver has demonstrated extraordinary growth rate in coho salmon which were transformed with a growth hormone from sockeye salmon.
The transgenics achieve up to eleven times the size of their littermates within six months, reaching maturity in about half the time.
Interestingly this dramatic effect is only observed in feeding pins where the transgenics' ferocious appetites demands constant feeding.
If the fish are left to their own devices and must forage for themselves, they appear to be out-competed by their smarter siblings.
However most studies, such as those involving transgenic Atlantic salmon and channel catfish, report growth rate enhancement on the order of 30-60%.
In addition to the species mentioned, GH genes also have been transferred into striped bass, tilapia, rainbow trout, gilthead sea bream, common carp, bluntnose bream, loach, and other fishes.
Shellfish also are subject to gene transfer toward the goal of intensifying aquaculture production.
Growth of abalone expressing an introduced GH gene is being evaluated; accelerated growth would prove a boon for culture of the slowgrowing mollusk.
A marker gene was introduced successfully into giant prawn, demonstrating feasibility of gene transfer in crustaceans, and opening the possibility of work involving genes affecting economically important traits.
In the ornamental fish sector of aquaculture, ongoing work addresses the development of fish with unique coloring or patterning.
A number of companies have been founded to pursue commercialization of transgenics for aquaculture.
As most aquaculture species mature at 2-3 years of age, most transgenic lines are still in development and have yet to be tested for performance under culture conditions.
Extending earlier research that identified methylfarnesoate (MF) as a juvenile hormone in crustaceans and determined its role in reproduction, researchers at the University of Connecticut have developed technology to synchronize shrimp egg production and to increase the number and quality of eggs produced.
Females injected with MF are stimulated to produce eggs ready for fertilization.
The procedure produces 180 percent more eggs than the traditional crude method of removing the eyestalk gland.
This will increase aquaculture efficiency.
A number of experiments utilize gene transfer to develop genetic lines of potential utility in fisheries management.
Transfer of GH genes into northern pike, walleye, and largemouth bass are aimed at improving the growth rate of sport fishes.
Gene transfer has been posed as an option for reducing losses of rainbow trout to whirling disease, although suitable candidate genes have yet to be identified.
Richard Winn of the University of Georgia is developing transgenic killifish and medaka as biomonitors for environmental mutagens, which carry the bacteriophage phi X 174 as a target for mutation detection.
Development of transgenic lines for fisheries management applications generally is at an early stage, often at the founder or F1 generation.
Broad application of transgenic aquatic organisms in aquaculture and fisheries management will depend on showing that particular GMOs can be used in the environment both effectively and safely.
Although our base of knowledge for assessing ecological and genetic safety of aquatic GMOs currently is limited, some early studies supported by the USDA biotechnology risk assessment program have yielded results.
Data from outdoor pond-based studies on transgenic catfish reported by Rex Dunham of Auburn University show that transgenic and non-transgenic individuals interbreed freely, that survival and growth of transgenics in unfed ponds was equal to or less than that of non-transgenics, and that predator avoidance is not affected by expression of the transgene.
However, unquestionably the seminal event for animal biotech in the nineties was Ian Wilmut's landmark work using nuclear transfer technology to generate the lambs Morag and Megan reported in 1996 (from an embryonic cell nuclei) and the truly ground-breaking work of creating Dolly from an adult somatic cell nucleus, reported in February, 1997 (Wilmut, 1997) .
Wilmut and his colleagues at the Roslin Institute demonstrated for the first time with the birth of Dolly the sheep that the nucleus of an adult somatic cell can be transferred to an enucleated egg to create cloned offspring.
It had been assumed for some time that only embryonic cells could be used as the cellular source for nuclear transfer.
This assumption was shattered with the birth of Dolly.
This example of cloning an animal using the nucleus of an adult cell was significant because it demonstrated the ability of egg cell cytoplasm to "reprogram" an adult nucleus.
When cells differentiate, that is, evolve from primitive embryonic cells to functionally defined adult cells, they lose the ability to express most genes and can only express those genes necessary for the cell's differentiated function.
For example, skin cells only express genes necessary for skin function, and brain cells only express genes necessary for brain function.
The procedure that produced Dolly demonstrated that egg cytoplasm is capable of reprogramming an adult differentiated cell (which is only expressing genes related to the function of that cell type).
This reprogramming enables the differentiated cell nucleus to once again express all the genes required for the full embryonic development of the adult animal.
Since Dolly was cloned, similar techniques have been used to clone a veritable zoo of vertebrates including mice, cattle, rabbitts, mules, horses, fish, cats and dogs from donor cells obtained from adult animals.
These spectacular examples of cloning normal animals from fully differentiated adult cells demonstrate the universality of nuclear reprogramming although the next decade called some of these assumptions into question.
This technology supports the production of genetically identical and genetically modified animals.
Thus, the successful "cloning" of Dolly has captured the imagination of researchers around the world.
This technological breakthrough should play a significant role in the development of new procedures for genetic engineering in a number of mammalian species.
It should be noted that nuclear cloning, with nuclei obtained from either mammalian stem cells or differentiated "adult" cells, is an especially important development for transgenic animal research.
As the decade reached its end the clones began arriving rapidly with specific advances made by a Japanese group who used cumulus cells rather than fibroblasts to clone calves.
They found that the percentage of cultured, reconstructed eggs that developed into blastocysts was 49% for cumulus cells and 23% for oviductal cells.
These rates are higher than the 12% previously reported for transfer of nuclei from bovine fetal fibroblasts.
Following on the heels of Dolly, Polly and Molly became the first genetically engineered transgenic sheep produced through nuclear transfer technology.
Polly and Molly were engineered to produce human factor IX (for hemophiliacs) by transfer of nuclei from transfected fetal fibroblasts.
Until then germline competent transgenics had only been produced in mammalian species, other than mice, using DNA microinjection.
Researchers at the University of Massachusetts and Advanced Cell Technology (Worcester, MA) teamed up to produce genetically identical calves utilizing a strategy similar to that used to produce transgenic sheep.
In contrast to the sheep cloning experiment, the bovine experiment involved the transfer of nuclei from an actively dividing population of cells.
Previous results from the sheep experiments suggested that induction of quiescence by serum starvation was required to reprogram the donor nuclei for successful nuclear transfer.
The current bovine experiments indicate that this step may not be necessary.
Typically about 500 embryos needed to be microinjected to obtain one transgenic cow, whereas nuclear transfer produced three transgenic calves from 276 reconstructed embryos.
This efficiency is comparable to the previous sheep research where six transgenic lambs were produced from 425 reconstructed embryos.
The ability to select for genetically modified cells in culture prior to nuclear transfer opens up the possibility of applying the powerful gene targeting techniques that have been developed for mice.
One of the limitations of using primary cells, however, is their limited lifespan in culture.
Primary cell cultures such as the fetal fibroblasts can only undergo about 30 population doublings before they senesce.
This limited lifespan would preclude the ability to perform multiple rounds of selection.
To overcome this problem of cell senescence, these researchers showed that fibroblast lifespan could be prolonged by nuclear transfer.
A fetus, which was developed by nuclear transfer from genetically modified cells, could in turn be used to establish a second generation of fetal fibroblasts.
These fetal cells would then be capable of undergoing another 30 population doublings, which would provide sufficient time for selection of a second genetic modification.
As noted, there is still some uncertainty over whether quiescent cells are required for successful nuclear transfer.
Induction into quiescence was originally thought to be necessary for successful nuclear reprogramming of the donor nucleus.
However, cloned calves have been previously produced using non-quiescent fetal cells.
Furthermore, transfer of nuclei from Sertoli and neuronal cells, which do not normally divide in adults, did not produce a liveborn mouse; whereas nuclei transferred from actively dividing cumulus cells did produce cloned mice.
The fetuses used for establishing fetal cell lines in a Tufts goat study were generated by mating nontransgenic females to a transgenic male containing a human antithrombin (AT) III transgene.
This AT transgene directs high level expression of human AT into milk of lactating transgenic females.
As expected, all three offspring derived from female fetal cells were females.
One of these cloned goats was hormonally induced to lactate.
This goat secreted 3.7-5.8 grams per liter of AT in her milk.
This level of AT expression was comparable to that detected in the milk of transgenic goats from the same line obtained by natural breeding.
The successful secretion of AT in milk was a key result because it showed that a cloned animal could still synthesize and secrete a foreign protein at the expected level.
It will be interesting to see if all three cloned goats secrete human AT at the identical level.
If so, then the goal of creating a herd identical transgenic animals, which secrete identical levels of an important pharmaceutical, would become a reality.
No longer would variable production levels exist in subsequent generations due to genetically similar but not identical animals.
This homogeneity would greatly aid in the production and processing of a uniform product.
As nuclear transfer technology continues to be refined and applied to other species, it may eventually replace microinjection as the method of choice for generating transgenic livestock.
Nuclear transfer has a number of advantages: 1) nuclear transfer is more efficient than microinjection at producing a transgenic animal, 2) the fate of the integrated foreign DNA can be examined prior to production of the transgenic animal, 3) the sex of the transgenic animal can be predetermined, and 4) the problem of mosaicism in first generation transgenic animals can be eliminated.
DNA microinjection has not been a very efficient mechanism to produce transgenic mammals.
However, in November, 1998, a team of Wisconsin researchers reported a nearly 100% efficient method for generating transgenic cattle.
The established method of cattle transgenes involves injecting DNA into the pronuclei of a fertilized egg or zygote.
In contrast, the Wisconsin team injected a replication-defective retroviral vector into the perivitelline space of an unfertilized oocyte.
The perivitelline space is the region between the oocyte membrane and the protective coating surrounding the oocyte known as the zona pellucida.
In addition to ES (embryonic stem) cells other sources of donor nuclei for nuclear transfer might be used such as embryonic cell lines, primordial germ cells, or spermatogonia to produce offspring.
The utility of ES cells or related methodologies to provide efficient and targeted in vivo genetic manipulations offer the prospects of profoundly useful animal models for biomedical, biological and agricultural applications.
The road to such success has been most challenging, but recent developments in this field are extremely encouraging.
With the May 1999 announcement of Geron buying out Ian Wilmuts company Roslin BioMed, they declared it the dawn of an new era in biomedical research.
Geron's technologies for deriving transplantable cells from human pluripotent stem cells (hPSCs) and extending their replicative capacity with telomerase was combined with the Roslin Institute nuclear transfer technology, the technology that produced Dolly the cloned sheep.
The goal was to produce transplantable, tissue-matched cells that provide extended therapeutic benefits without triggering immune rejection.
Such cells could be used to treat numerous major chronic degenerative diseases and conditions such as heart disease, stroke, Parkinson's disease, Alzheimer's disease, spinal cord injury, diabetes, osteoarthritis, bone marrow failure and burns.
The stem cell is a unique and essential cell type found in animals.
Many kinds of stem cells are found in the body, with some more differentiated, or committed, to a particular function than others.
In other words, when stem cells divide, some of the progeny mature into cells of a specific type (heart, muscle, blood, or brain cells), while others remain stem cells, ready to repair some of the everyday wear and tear undergone by our bodies.
These stem cells are capable of continually reproducing themselves and serve to renew tissue throughout an individual's life.
For example, they continually regenerate the lining of the gut, revitalize skin, and produce a whole range of blood cells.
Although the term "stem cell" commonly is used to refer to the cells within the adult organism that renew tissue (e.g., hematopoietic stem cells, a type of cell found in the blood), the most fundamental and extraordinary of the stem cells are found in the early-stage embryo.
These embryonic stem (ES) cells, unlike the more differentiated adult stem cells or other cell types, retain the special ability to develop into nearly any cell type.
Embryonic germ (EG) cells, which originate from the primordial reproductive cells of the developing fetus, have properties similar to ES cells.
It is the potentially unique versatility of the ES and EG cells derived, respectively, from the early-stage embryo and cadaveric fetal tissue that presents such unusual scientific and therapeutic promise.
Indeed, scientists have long recognized the possibility of using such cells to generate more specialized cells or tissue, which could allow the generation of new cells to be used to treat injuries or diseases, such as Alzheimer's disease, Parkinson's disease, heart disease, and kidney failure.
Likewise, scientists regard these cells as an important -perhaps essential -means for understanding the earliest stages of human development and as an important tool in the development of life-saving drugs and cell-replacement therapies to treat disorders caused by early cell death or impairment.
Geron Corporation and its collaborators at the University of Wisconsin -Madison (Dr. James A. Thomson) and Johns Hopkins University (Dr. John D. Gearhart) announced in November 1998 the first successful derivation of hPSCs from two sources: (i) human embryonic stem (hES) cells derived from in vitro fertilized blastocysts (Thomson 1998 ) and (ii) human embryonic germ (hEG) cells derived from fetal material obtained from medically terminated pregnancies (Shamblott et al.
1998) .
Although derived from different sources by different laboratory processes, these two cell types share certain characteristics but are referred to collectively as human pluripotent stem cells (hPSCs).
Because hES cells have been more thoroughly studied, the characteristics of hPSCs most closely describe the known properties of hES cells.
Stem cells represent a tremendous scientific advancement in two ways: first, as a tool to study developmental and cell biology; and second, as the starting point for therapies to develop medications to treat some of the most deadly diseases.
The derivation of stem cells is fundamental to scientific research in understanding basic cellular and embryonic development.
Observing the development of stem cells as they differentiate into a number of cell types will enable scientists to better understand cellular processes and ways to repair cells when they malfunction.
It also holds great potential to yield revolutionary treatments by transplanting new tissue to treat heart disease, atherosclerosis, blood disorders, diabetes, Parkinson's, Alzheimer's, stroke, spinal cord injuries, rheumatoid arthritis, and many other diseases.
By using stem cells, scientists may be able to grow human skin cells to treat wounds and burns.
And, it will aid the understanding of fertility disorders.
Many patient and scientific organizations recognize the vast potential of stem cell research.
Another possible therapeutic technique is the generation of "customized" stem cells.
A researcher or doctor might need to develop a special cell line that contains the DNA of a person living with a disease.
By using a technique called "somatic cell nuclear transfer" the researcher can transfer a nucleus from the patient into an enucleated human egg cell.
This reformed cell can then be activated to form a blastocyst from which customized stem cell lines can be derived to treat the individual from whom the nucleus was extracted.
By using the individual's own DNA, the stem cell line would be fully compatible and not be rejected by the person when the stem cells are transferred back to that person for the treatment.
Preliminary research is occurring on other approaches to produce pluripotent human ES cells without the need to use human oocytes.
Human oocytes may not be available in quantities that would meet the needs of millions of potential patients.
However, no peer-reviewed papers have yet appeared from which to judge whether animal oocytes could be used to manufacture "customized" human ES cells and whether they can be developed on a realistic timescale.
Additional approaches under consideration include early experimental studies on the use of cytoplasmic-like media that might allow a viable approach in laboratory cultures.
On a much longer timeline, it may be possible to use sophisticated genetic modification techniques to eliminate the major histocompatibility complexes and other cell-surface antigens from foreign cells to prepare master stem cell lines with less likelihood of rejection.
This could lead to the development of a bank of universal donor cells or multiple types of compatible donor cells of invaluable benefit to treat all patients.
However, the human immune system is sensitive to many minor histocompatibility complexes and immunosuppressive therapy carries life-threatening complications.
Stem cells also show great potential to aid research and development of new drugs and biologics.
Now, stem cells can serve as a source for normal human differentiated cells to be used for drug screening and testing, drug toxicology studies and to identify new drug targets.
The ability to evaluate drug toxicity in human cell lines grown from stem cells could significantly reduce the need to test a drug's safety in animal models.
There are other sources of stem cells, including stem cells that are found in blood.
Recent reports note the possible isolation of stem cells for the brain from the lining of the spinal cord.
Other reports indicate that some stem cells that were thought to have differentiated into one type of cell can also become other types of cells, in particular brain stem cells with the potential to become blood cells.
However, since these reports reflect very early cellular research about which little is known, we should continue to pursue basic research on all types of stem cells.
Some religious leaders will advocate that researchers should only use certain types of stem cells.
However, because human embryonic stem cells hold the potential to differentiate into any type of cell in the human body, no avenue of research should be foreclosed.
Rather, we must find ways to facilitate the pursuit of all research using stem cells while addressing the ethical concerns that may be raised.
Another seminal and intimately related event at the end of the nineties occurred in Madison Wisconsin.
Up until November of 1998, isolating ES cells in mammals other than mice proved elusive, but in a milestone paper in the November 5, 1998 issue of Science, James A. Thomson, (1998) a developmental biologist at UW-Madison reported the first successful isolation, derivation and maintenance of a culture of human embryonic stem cells (hES cells).
It is interesting to note that this leap was made from mouse to man.
As Thomson himself put it, these cells are different from all other human stem cells isolated to date and as the source of all cell types, they hold great promise for use in transplantation medicine, drug discovery and development, and the study of human developmental biology.
The new century is rapidly exploiting this vision.
When Steve Fodor was asked in 2003 "How do you really take the Human genome sequence and transform it into knowledge?"
he answered from Affymetrix's perspective, it is a technology development task.
He sees the colloquially named affychips being the equivalent of a CD-ROM of the genome.
They take information from the genome and write it down.
The company has come a long way from the early days of Venter's ESTs and less than robust algorithms as described earlier.
One surprising fact unearthed by the newer more sophisticated generation of chips is that 30 to 35 percent of the non-repetitive DNA is being expressed as accepted knowledge was that only 1.5 to 2 percent of the genome would be expressed.
Since much of that sequence has no protein-coding capacity it is most likely coding for regulatory functions.
In a parallel to astrophysics this is often referred to in common parlance as the "dark matter of the genome" and like dark matter for many it is the most exciting and challenging aspect of uncovering the occult genome.
It could be, and most probably is, involved in regulatory functions, networks, or development.
And like physical dark matter it may change our whole concept of what exactly a gene is or is not!
Since Beadle and Tatum's circumspect view of the protein world no longer holds true it adds a layer of complexity to organizing chip design.
Depending on which sequences are present in a particular transcript, you can, theoretically, design a set of probes to uniquely distinguish that variant.
At the DNA level itself there is much potential for looking at variants either expressed or not at a very basic level as a diagnostic system, but ultimately the real paydirt is the information that can be gained from looking at the consequence of non-coding sequence variation on the transcriptome itself.
And fine tuning when this matters and when it is irrelevant as a predicative model is the auspices of the Affymetrix spin-off Perlegen.
Perlegen came into being in late 2000 to accelerate the development of high-resolution, whole genome scanning.
And they have stuck to that purity of purpose.
To paraphrase Dragnet's Sergeant Joe Friday, they focus on the facts of DNA just the DNA.
Perlegen owes its true genesis to the desire of one of its cofounders to use DNA chips to help understand the dynamics underlying genetic diseases.
Brad Margus' two sons have the rare disease "ataxia telangiectasia" (A-T).
A-T is a progressive, neurodegenerative childhood disease that affects the brain and other body systems.
The first signs of the disease, which include delayed development of motor skills, poor balance, and slurred speech, usually occur during the first decade of life.
Telangiectasias (tiny, red "spider" veins), which appear in the corners of the eyes or on the surface of the ears and cheeks, are characteristic of the disease, but are not always present.
Many individuals with A-T have a weakened immune system, making them susceptible to recurrent respiratory infections.
About 20% of those with A-T develop cancer, most frequently acute lymphocytic leukemia or lymphoma suggesting that the sentinel competence of the immune system is compromised.
Having a focus so close to home is a powerful driver for any scientist.
His co-founder David Cox is a polymath pediatrician whose training in the latter informs his application of the former in the development of patient-centered tools.
From that perspective, Perlegen's stated mission is to collaborate with partners to rescue or improve drugs and to uncover the genetic bases of diseases.
They have created a whole genome association approach that enables them to genotype millions of unique SNPs in thousands of cases and controls in a timeframe of months rather than years.
As mentioned previously, SNP (single nucleotide polymorphism) markers are preferred over microsatellite markers for association studies because of their abundance along the human genome, the low mutation rate, and accessibilities to high-throughput genotyping.
Since most diseases, and indeed responses to drug interventions, are the products of multiple genetic and environmental factors it is a challenge to develop discriminating diagnostics and, even more so, targetedtherapeutics.
Because mutations involved in complex diseases act probabilisticallythat is, the clinical outcome depends on many factors in addition to variation in the sequence of a single gene -the effect of any specific mutation is smaller.
Thus, such effects can only be revealed by searching for variants that differ in frequency among large numbers of patients and controls drawn from the general population.
Analysis of these SNP patterns provides a powerful tool to help achieve this goal.
Although most bi-alleic SNPs are rare, it has been estimated that just over 5 million common SNPs, each with a frequency of between 10 and 50%, account for the bulk of the DNA sequence difference between humans.
Such SNPs are present in the human genome once every 600 base pairs or so.
As is to be expected from linkage disequilibrium studies, alleles making up blocks of such SNPs in close physical proximity are often correlated, resulting in reduced genetic variability and defining a limited number of "SNP haplotypes," each of which reflects descent from a single, ancient ancestral chromosome.
In 2001 Cox's group, using high level scanning with some old-fashioned somatic cell genetics, constructed the SNP map of Chromosome 21.The surprising findings were blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes (interestingly enough the prevalence of each hapolytype in the examined population was in the ratio 50:25:12.5).From this the conclusion could be drawn that by comparing the frequency of genetic variants in unrelated cases and controls, genetic association studies could potentially identify specific haplotypes in the human genome that play important roles in disease, without need of knowledge of the history or source of the underlying sequence, which hypothesis they subsequently went on to prove.
Following Cox et al.
pioneering work on "blocking" Chromosome 21 into characteristic haplotypes, Tien Chen came to visit him from University of Southern California and following the visit his group developed discriminating algorithms which took advantage of the fact that the haplotype block structure can be decomposed into large blocks with high linkage disequilibrium and relatively limited haplotype diversity, separated by short regions of low disequilibrium.
One of the practical implications of this observation is as suggested by Cox that only a small fraction of all the SNPs they refer to as "tag" SNPs can be chosen for mapping genes responsible for complex human diseases, which can significantly reduce genotyping effort, without much loss of power.
They developed algorithms to partition haplotypes into blocks with the minimum number of tag SNPs for an entire chromosome.
In 2005 they reported that they had developed an optimized suite of programs to analyze these block linkage disequilibrium patterns and to select the corresponding tag SNPs that will pick the minimum number of tags for the given criteria.
In addition the updated suite allows haplotype data and genotype data from unrelated individuals and from general pedigrees to be analyzed.
Using an approach similar to Richard Michelmore's bulk segregant analysis in plants of more than a decade previously, Perlegen subsequently made use of these SNP haplotype and statistical probability tools to estimate total genetic variability of a particular complex trait coded for by many genes, with any single gene accounting for no more than a few percent of the overall variability of the trait.
Cox's group have determined that fewer than 1000 total individuals provide adequate power to identify genes accounting for only a few percent of the overall genetic variability of a complex trait, even using the very stringent significance levels required when testing large numbers of DNA variants.
From this it is possible to identify the set of major genetic risk factors contributing to the variability of a complex disease and/or treatment response.
So, while a single genetic risk factor is not a good predictor of treatment outcome, the sum of a large fraction of risk factors contributing to a treatment response or common disease can be used to optimize personalized treatments without requiring knowledge of the underlying mechanisms of the disease.They feel that a saturating level of coverage is required to produce repeatable prediction of response to medication or predisposition to disease and that taking shortcuts will for the most part lead to incomplete, clinically-irrelevant results.
In 2005 Hinds et al.
in Science describe even more dramatic progresss.
They describe a publicly available, genome-wide data set of 1.58 million common singlenucleotide polymorphisms (SNPs) that have been accurately genotyped in each of 71 people from three population samples.
A second public data set of more than 1 million SNPs typed in each of 270 people has been generated by the International Haplotype Map (HapMap) Project.
These two public data sets, combined with multiple new technologies for rapid and inexpensive SNP genotyping, are paving the way for comprehensive association studies involving common human genetic variations.
Perlegen basically is taking to the next level Fodor's stated reason for the creation of Affymetrix, the belief that understanding the correlation between genetic variability and its role in health and disease would be the next step in the genomics revolution.
The other interesting aspect of this level of coverage is, of course, the notion of discrete identifiable groups based on ethnicity, centers of origin and such breaks down and a spectrum of variation arises across all populations which makes the Perlegen chip, at one level, a true unifier of humanity but at another adds a whole layer of complexity for HMOs!
At the turn of the century, this personalized chip approach to medicine received some validation at a simpler level in a closely related disease area to the one to which one fifth of A-T patients ultimately succumb when researchers at the Whitehead Institute used DNA chips to distinguish different forms of leukemia based on patterns of gene expression in different populations of cells.
Moving cancer diagnosis away from visually based systems to such molecular based systems is a major goal of the National Cancer Institute.
In the study, scientists used a DNA chip to examine gene activity in bone marrow samples from patients with two different types of acute leukemia -acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).
Then, using an algorithm, developed at the Whitehead, they identified signature patterns that could distinguish the two types.
When they cross-checked the diagnoses made by the chip against known differences in the two types of leukemia, they found that the chip method could automatically make the distinction between AML and ALL without previous knowledge of these classes.
Taking it to a level beyond where Perlegen are initially aiming, Eric Lander, leader of the study said, mapping not only what is in the genome, but also what the things in the genome do, is the real secret to comprehending and ultimately curing cancer and other diseases.
Chips gained recognition on the world stage in 2003 when they played a key role in the search for the cause of Severe Acute Respiratory Syndrome (SARS) and probably won a McArthur genius award for their creator.
UCSF Assistant Professor Joseph DeRisi, already famous in the scientific community as the wunderkind originator of the online DIY chip maker in Pat Brown's lab at Stanford, built a gene microarray containing all known completely sequenced viruses (12,000 of them) and, using a robot arm that he also customized, in a three day period used it to classify a pathogen isolated from SARS patients as a novel coronavirus.
When a whole galaxy of dots lit up across the spectrum of known vertebrate cornoviruses DeRisis knew this was a new variant.
Interestingly the sequence had the hottest signal with Avian Infectious Bronchitis Virus.
His work subsequently led epidemiologists to target the masked palm civet, a tree-dwelling animal with a weasel-like face and a catlike body as the probable primary host.
The role that DeRisi's team at UCSF played in identifying a coronavirus as a suspected cause of SARS came to the attention of the national media when CDC Director Dr. Julie Gerberding recognized Joe in March 24, 2003 press conference and in 2004 when Joe was honored with one of the coveted McArthur genius awards.
This and other tools arising from information gathered from the human genome sequence and complementary discoveries in cell and molecular biology, new tools such as gene-expression profiling, and proteomics analysis are converging to finally show that rapid robust diagnostics and "rational" drug design has a future in disease research.
Another virus that puts SARS deaths in perspective benefitted from rational drug design at the turn of the century.
Influenza, or flu, is an acute respiratory infection caused by a variety of influenza viruses.
Each year, up to 40 million Americans develop the flu, with an average of about 150,000 being hospitalized and 10,000 to 40,000 people dying from influenza and its complications.
The use of current influenza treatments has been limited due to a lack of activity against all influenza strains, adverse side effects, and rapid development of viral resistance.
Influenza costs the United States an annual $14.6 billion in physician visits, lost productivity and lost wages.
And least we still dismiss it as a nuisance we are well to remember that the "Spanish" influenza pandemic killed over 20 million people in 1918 and 1919, making it the worst infectious pandemic in history beating out even the more notorious black death of the Middle Ages.
This fear has been rekindled as the dreaded H5N1 (H for haemaglutenin and N for neuraminidase as described below) strain of bird flu has the potential to mutate and recognise homo sapiens as a desirable host.
Since RNA viruses are notoriously faulty in their replication this accelerated evolutionary process gives then a distinct advantage when adapting to new environments and therefore finding more amenable hosts.
Although inactivated influenza vaccines are available, their efficacy is suboptimal partly because of their limited ability to elicit local IgA and cytotoxic T cell responses.
The choices of treatments and preventions for influenza hold much more promise in this millennium.
Clinical trials of cold-adapted live influenza vaccines now under way suggest that such vaccines are optimally attenuated, so that they will not cause influenza symptoms but will still induce protective immunity.
Aviron (Mountain View, CA), BioChem Pharma (Laval, Quebec, Canada), Merck (Whitehouse Station, NJ), Chiron (Emeryville, CA), and Cortecs (London), all had influenza vaccines in the clinic at the turn of the century, with some of them given intra-nasally or orally.
Meanwhile, the team of Gilead Sciences (Foster City, CA) and Hoffmann-La Roche (Basel, Switzerland) and also GlaxoWellcome (London) in 2000 put on the market neuraminidase inhibitors that block the replication of the influenza virus.
Gilead was one of the first biotechnology companies to come out with an anti-flu therapeutic.
Tamiflu™ (oseltamivir phosphate) was the first flu pill from this new class of drugs called neuraminidase inhibitors (NI) that are designed to be active against all common strains of the influenza virus.
Neuraminidase inhibitors block viral replication by targeting a site on one of the two main surface structures of the influenza virus, preventing the virus from infecting new cells.
Neuraminidase is found protruding from the surface of the two main types of influenza virus, type A and type B.
It enables newly formed viral particles to travel from one cell to another in the body.
Tamiflu is designed to prevent all common strains of the influenza virus from replicating.
The replication process is what contributes to the worsening of symptoms in a person infected with the influenza virus.
By inactivating neuraminidase, viral replication is stopped, halting the influenza virus in its tracks.
In marked contrast to the usual protracted process of clinical trials for new therapeutics, the road from conception to application for Tamiflu was remarkably expeditious.
In 1996, Gilead and Hoffmann-La Roche entered into a collaborative agreement to develop and market therapies that treat and prevent viral influenza.
In 1999, as Gilead's worldwide development and marketing partner, Roche led the final development of Tamiflu, 26 months after the first patient was dosed in clinical trials in April 1999, Roche and Gilead announced the submission of a New Drug Application to the U.S. Food and Drug Administration (FDA) for the treatment of influenza.
Additionally, Roche filed a Marketing Authorisation Application (MAA) in the European Union under the centralized procedure in early May 1999.
Six months later in October 1999, Gilead and Roche announced that the FDA approved Tamiflu for the treatment of influenza A and B in adults.
These accelerated efforts allowed Tamiflu to reach the U.S. market in time for the 1999-2000 flu season.
One of Gilead's studies showed an increase in efficacy from 60% when the vaccine was used alone to 92% when the vaccine was used in conjunction with a neuraminidase inhibitor.
Outside of the U.S., Tamiflu also has been approved for the treatment of influenza A and B in Argentina, Brazil, Canada, Mexico, Peru and Switzerland.
Regulatory review of the Tamiflu MAA by European authorities is ongoing.
With the H5N1 birdflu strain's relentless march (or rather flight) across Asia, in 2006 through Eastern Europe to a French farmyard, an unwelcome stowaway on a winged migration, and no vaccine in sight, Tamiflu, although untested for this species, seen as the last line of defense is now being horded and its patented production right's fought over like an alchemist's formula.
Tamiflu's main competitor, Zanamivir marketed as Relenza™ was one of a group of molecules developed by GlaxoWellcome and academic collaborators using structure-based drug design methods targeted, like Tamiflu, at a region of the neuraminidase surface glycoprotein of influenza viruses that is highly conserved from strain to strain.
Glaxo filed for marketing approval for Relenza in Europe and Canada.
The Food and Drug Administration's accelerated drug-approval timetable began to show results by 2001, its evaluation of Novartis's Gleevec took just three months compared with the standard 10-12 months.
Another factor in improving biotherapeutic fortunes in the new century was the staggering profits of early successes.
In 2003, $1.9 billion of the $3.3 billion in revenue collected by Genentech in South San Francisco came from oncology products, mostly the monoclonal antibody-based drugs Rituxan, used to treat non-Hodgkin's lymphoma, and Herceptin for breast cancer.
In fact two of the first cancer drugs to use the new tools for 'rational' design Herceptin and Gleevec, a small-molecule chemotherapeutic for some forms of leukemia are proving successful, and others such as Avastin (an anti-vascular endothelial growth factor) for colon cancer and Erbitux are already following in their footsteps.
Gleevec led the way in exploiting signal-transduction pathways to treat cancer as it blocks a mutant form of tyrosine kinase (termed the Philadelphia translocation recognized in 1960's) that can help to trigger out-of-control cell division.
About 25% of biotech companies raising venture capital during the third quarter of 2003 listed cancer as their primary focus, according to online newsletter VentureReporter.
By 2002 according to the Pharmaceutical Research and Manufacturers of America, 402 medicines were in development for cancer up from 215 in 1996.
Another new avenue in cancer research is to combine drugs.
Wyeth's Mylotarg, for instance, links an antibody to a chemotherapeutic, and homes in on CD33 receptors on acute myeloid leukemia cells.
Expertise in biochemistry, cell biology and immunology is required to develop such a drug.
This trend has created some bright spots in cancer research and development, even though drug discovery in general has been adversely affected by mergers, a few high-profile failures and a shaky US economy in the early 2000's.
As the millennium approached observers as diverse as Microsoft's Bill Gates and President Bill Clinton predicted the 21st century wiould be the "biology century".
By 1999 the many programs and initiatives underway at major research institutions and leading companies were already giving shape to this assertion.
These initiatives have ushered in a new era of biological research anticipated to generate technological changes of the magnitude associated with the industrial revolution and the computerbased information revolution.
Complementary DNA sequencing: expressed sequence tags and human genome project Basic local alignment search tool High-tech herbal medicine: Plant-Based Vaccines Asilomar conference on recombinant DNA molecules Potential biohazards of recombinant DNA molecules HUGO: The Human Genome Organization Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice The human genome: the nature of the enterprise Orchestrating the Human Genome Project Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis Nutritional genomics: Manipulating plant micronutrients to improve human health Helping Europe compete in human genome research Genome project gets rough ride in Europe Construction of a linkage map of the human genome, and its application to mapping genetic diseases Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields Preimplantation and the 'new' genetics A history human genome project It aint necessarily so: The dream of the human genome and other illusions High speed DNA sequencing by capillary electrophoresis A strategy for sequencing the genome 5 years early Expression of norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants Generation and analysis of 280,000 human expressed sequence tags National Academy of Sciences.
Introduction of recombinant DNA-engineered organisms into the environment: key issues Functional Foods and Biopharmaceuticals: The Next Generation of the GM Revolution in Let Them Eat Precaution Biotechnology: A Review of Technological Developments, Publishers Forfas Vitamin-A and iron-enriched rices may hold key to combating blindness and malnutrition: a biotechnology advance French DNA: Trouble in Purgatory Genome: The Autobiography of a Species in 23 Chapters Harper Collins Derivation of pluripotent stem cells from cultured human primordial germ cells Production of correctly processed human serum albumin in transgenic plants High-yield production of a human therapeutic protein in tobacco chloroplasts The common thread: A story of science, politics, ethics and the human genome Capillary gel electrophoresis for DNA sequencing.
Laser-induced fluorescence detection with the sheath flow cuvette Production of functional human alpha 1-antitrypsin by plant cell culture Genetic modification of oils for improved health benefits, Presentation at conference, Dietary Fatty Acids and Cardiovascular Health: Dietary Recommendations for Fatty Acids: Is There Ample Evidence?
Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves Antenatal maternal serum screening for Down's syndrome: results of a demonstration project Viable offspring derived from fetal and adult mammalian cells
key: cord-287758-da11ypiy authors: Mônica Vitalino de Almeida, Sinara; Cleberson Santos Soares, José; Lima dos Santos, Keriolaine; Emanuel Ferreira Alves, Josival; Galdino Ribeiro, Amélia; Trindade Tenório Jacob, Íris; Juliane da Silva Ferreira, Cindy; Celerino dos Santos, Jéssica; Ferreira de Oliveira, Jamerson; Bezerra de Carvalho Junior, Luiz; do Carmo Alves de Lima, Maria title: COVID-19 therapy: what weapons do we bring into battle?
date: 2020-09-10 journal: Bioorg Med Chem DOI: 10.1016/j.bmc.2020.115757 sha: doc_id: 287758 cord_uid: da11ypiy Urgent treatments, in any modality, to fight SARS-CoV-2 infections are desired by society in general, by health professionals, by Estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding COVID-19: knowledge is the means to discover or to produce an effective treatment against this global disease.
Scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of 2020 with over 25,000 published articles related to the new coronavirus.
Three great lines of publications related to COVID-19 were identified for building this article: The first refers to knowledge production concerning the virus and pathophysiology of COVID-19; the second regards efforts to produce vaccines against SARS-CoV-2 at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat COVID-19 by drug repurposing.
In this review, the drugs that have been repurposed so far are grouped according to their chemical class.
Their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions.
This can help identifying anti-SARS-CoV-2 promising therapeutic agents.
The world is facing a huge challenge in the coronavirus disease (COVID-19) pandemic: How to fight an enemy without weapons in terms of therapy?
Unfortunately, even before the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) worldwide spread, there were no clinical treatments nor prevention strategies available for any human coronavirus.
1 It is understandable that both society and researchers urge the discovery of new compounds or even of a drug that is commercially available that can be employed by physicians mainly for patients with the extreme presentation of COVID-19.
There is also urgency in the discovery of medicine with prophylactic action to prevent the entry of the virus in host cells after exposure.
Vaccine research experts already indicate that rescue from SARS-CoV-2 will come from a long but effective journey to produce a vaccine.
2 While this is not a reality, the scientific community, including medicinal chemists and doctors who accompany patients, are trying to identify therapeutic alternatives.
This is a meritorious attitude: The commitment with the protection of humanity.
Nevertheless, the rigorous feature of science in the discovery of a new drug cannot be disregarded, even during a pandemic and in the face of the urgent demand for a treatment, to avoid eventual mistakes and spurious hope.
The increase in studies related to SARS-CoV-2 during the first semester in 2020 has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs.
It is noteworthy the increase in outbreaks of SARS-CoV (2002) and MERS-CoV (2013), with accelerated production of knowledge on these HCoVs, which has been very useful for ongoing investigations on SARS-CoV-2.
One example is the availability of technological devices that allowed the fast sequencing of SARS-CoV-2 genome and the elucidation of a promising antigen target, the S glycoprotein.
Nonetheless, the development of a human vaccine can take years, especially because employing emergent technologies requires extensive safety tests and expansion to large scale production in order to assist the world population, as demanded in the case of the COVID-19 pandemic.
3 The development of new medicine also demands many years of research that involve stages of reasonable planning, synthesis, structural characterization, formulation of prototypes, preclinical and clinical trials.
Therefore, the literature highlights, as alternative treatments for COVID-19, the repurposing of drugs, which is fast and useful in emergencies such as the one experienced today.
The repurposing of drugs means the use of broad-spectrum medicine for a new disease, once its metabolic characteristics, doses, potential efficacy and adverse effects are pre-established due to drug studies extracellular liquid volume and arterial pressure of the human body.
It is largely expressed in fifteen human tissues, including ciliated bronchial epithelial cells and type II pneumocytes form pulmonary alveoli, the main location of lesions caused by SARS-CoV-2.
36, 37 After ACE2 receptor-binding, a conformational alteration occurs in protein S allowing the fusion between the viral envelope and the host cell membrane via endosomal.
Then, SARS-CoV-2 releases the RNA into the cytoplasm to be translated into viral replicase polyproteins pp1a and pp1ab, which are processed by 3CL pro and PL pro proteases, respectively.
The cleavage products are 16 Nsps that form the transcription and replication complex.
38 Next, the positive RNA strand is translated into a template of negative strand that allows the synthesis of new genomics and sub genomics mRNAs.
These mRNAs are translated and transcribed producing structural and accessory proteins.
Viral proteins and RNA genomic are put together in virions at endoplasmic reticulum and Golgi complex, finally transported through vesicles and released from the cell host for infecting new cells.
38, 39 The COVID-19 symptomatology starts after the virus is installed in host cells.
In general, the symptoms include lasting and high fever, dry cough, shortness of breath, muscles aches or tiredness, sputum production, headaches, and a small percentage of individuals presented gastrointestinal symptoms such as diarrhoea and vomit.
40 The incubation period of SARS-CoV-2, from exposure to first symptoms, lasts 2 to 14 days.
The pre-symptomatic stage lasts from 1-3 days (possibly more) before the beginning of symptoms.
The post-symptomatic stage lasts at least 7 days after the beginning of symptoms and 3 days after lowering of fever and improvement of respiratory symptoms.
41 There are many unanswered questions such as the duration of potential immunity of both symptomatic and asymptomatic individuals when infected with SARS-CoV-2.
31 It is noteworthy that efficient strategies to fight the disease should not depend on the symptoms of patients, once asymptomatic or pre-symptomatic subjects can play an important role in the direct and indirect transmission to others, as demonstrated by Arons et al.
42 This investigation reports that half the residents in a nursing facility, who tested positive, were asymptomatic when tested and probably contributed to the transmission to other residents.
Thus, control strategies focused on symptomatic residents were not sufficient to prevent transmission once SARS-CoV-2 had been introduced in the facility.
Laboratory exams of infected patients showed alterations in haematology and biochemistry.
It was verified the increase of leukocytes and the reduction of lymphocytes; increased D-dimer and erythrocyte sedimentation rate (ESR), prolongation in prothrombin times (PT), followed by increase in bilirubin levels, aspartate transaminase (AST), alanine transaminase (ALT), creatinine, lactate dehydrogenase (LDH), protein C reactive (PCR), hypoalbuminemia (low albumin), microcytosis and thrombocytopenia.
43 In addition, inflammatory factors that indicate the immune condition of patients, such as interleukins (IL) IL-2, IL-6, IL-7, and IL-10 and the tumoral necrosis factor-α (TNF-α) become elevated.
Plasma levels of Granulocyte-colony stimulating factor (GCSF), protein induced by interferon gamma, Monocyte Chemoattractant Protein-1 (MCP-1), macrophages inflammatory protein 1α and TNF-α also display significant increase.
44 Potential risk factors or comorbidities that can lead to complications of COVID-19 include elderly individuals (specially above 65 years of age), cardiovascular issues, cerebrovascular, chronic pulmonary diseases, immunocompromising, renal problems, hepatic disease, hypertension, diabetes and obesity.
[44] [45] [46] [47] [48] [49] There is a notorious concern regard the medicine administered to fight these comorbidities because some of them can lead to greater expression of ACE2, such as treatments for diabetes 48 or hypertension.
50 This may favour or even aggravate COVID-19 infection.
These facts justify the urgency of research that contemplate alternative therapeutic targets such as calcium channels blockers for hypertensive individuals as suggested by Fang et al.
48 However, there is little clinical evidence on the risk of treating COVID-19 patients with therapies that induce greater expression of ACE2.
Further investigation is necessary to explore whether these medicines inhibit or trigger the viral entry into the cells of an infected host.
51 A frequent report in epidemiological data regarding the mortality of COVID-19 concerns the sex of individuals, as men are the predominant fatal victims of the disease.
Therefore, being of the male sex is considered a bad prognostic factor for infection.
52 ,53 A possible explanation lies in the relation between gonadal hormones and the expression of ACE2 enzymes or even an alleged Vitamin D deficiency, according to Vignera et al.
54 The latter suggest monitoring of serum levels of testosterone and Vitamin D in infected patients for a better understanding of the different fatality rates between sexes, including the hypothesis that women's hygiene justify a lesser rate of infection.
Understanding the pathogenic effects of SARS-COV-2 for the different organs affected by the disease has also been object of investigation, such as gut-lung crosstalk.
55 Data from research conducted thus far indicate that the infection caused by SARS-COV-2 is not only capable of causing pneumonia, but it can also damage other organs such as the heart, the liver, the kidneys and organic systems such as the blood and the immune system.
44, 56, 57 Patients with the extreme form of the disease frequently manifest lymphopenia, 30, 57 hepatic insufficiency 58 and viral sepsis diagnostic, 51 whose complications can be related to the severity of the cases and the mortality of patients.
56, 57 There are reports that the eventual death of such patients is due to multiple organ insufficiency, acute respiratory distress syndrome (ARDS), cardiac insufficiency, arrhythmia and renal insufficiency.
56, 59 Therefore, great attention is necessary to the disease's potential damage to multiple organs and to therapeutic alternatives to fight COVID-19, 60 given that some of these alternatives can have side effects on organs initially unrelated to the respiratory system, but that may be susceptible to a systemic compromise prompted by the virus once the treatment has begun.
Hence, it is possible to observe the existence of different forms of aggravating the disease.
41 In this regard, Wang et al.
60 recommend the creation of a system to categorize patients with the severe form of COVID-19.
Several investigations report that all HCoVs, SARS-CoV, MERS-CoV and SARS-CoV-2 induce exaggerated immune responses in the host, which are associated to the severity of pulmonary pathology and might lead to the development of acute respiratory distress syndrome (ARDS) or death.
57 The incidence of the extreme form of the infection is associated with cytokine storm syndrome, characterized by high plasma concentration of several interleukin, inflammatory cytokine, inflammatory chemokines, among other factors that cause infiltrated inflammatory in the organs.
44, 61, 62 Survivors of this excessive response by the immune system can develop long-term fibrosis and pulmonary damages that might culminate in functional injuries to these organs, thus reducing the patient's quality of life.
63 During the development of drugs to fight microorganisms, the adoption of strategies that allow the design of molecules to act against specific biological targets of bacteria, parasites or viruses is preferred.
Therapies for CoVs can be divided into several categories, based on specific paths: (1) CoVs proteins or functional enzymes that are essential for viral replication; (2) structural proteins of the virus that prevent its binding to the respective receptors in human cells or its assembly process; (3) some viral factor that restores the host's inherent immunity and; (4) host-specific enzymes or receptors, that prevent the entry of the virus in the host cells.
5, 64 So far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-CoVs therapies (FIG.
1) .
Investigations by Wu et al.
5 through bioinformatics, analysed possible SARS-CoV-2 therapeutic targets.
The proteins coded by this virus were verified and compared to proteins coded by other CoVs.
The results enabled the detection of structural similarities to SARS-CoV, from which it was possible to conduct homology modelling to build 19 proteins for SARS-CoV-2.
Among the targets were spike (S) glycoprotein, Nsp (RNA-dependent RNA polymerase -RdRp), enzyme helicase (3CL pro and PL pro ), TMPRSS, ORF7a factor and ACE2 presents in the host cells.
5 These targets have pivotal roles in the development of the virus and have great influence on its pathogenicity, hence, some details are provided next.
Molecular modelling showed that spike (S) glycoprotein is a transmembrane protein of approximately 180 to 200 kDa type I whose N-terminal turns to the exterior of the virus and its Cterminal segment turns to the interior of the virus.
The typical structure of CoVs is given by the assemble of a bulbous projection of a corolla as trimers of protein S and it is cleaved into two important subunits from the pathogenic perspective: S1 and S2.
SARS-CoV and SARS-CoV-2 (S) glycoprotein share about 76% of amino acid identity and enable the entry of the virus in the host cells.
Therefore, S glycoprotein present in CoVs has been considered a promising biological target for antiviral mechanisms.
35, 65 The moment when the virus approximates the target cell prompts the recognition by the receptor-binding domain (RBD) in the S glycoprotein of its receptor, which leads to the binding to subunit S1.
Next, the subunit S2 allows fusion of viral and cellular membranes, which enables entry in the cell and the release of viral RNA genome.
35, 66, 67 Some investigations suggest that the strong binding affinity between S protein and ACE2 is essential for viral entry, hence, ACE2 is also relevant for the development of drugs.
5, 68 Molecules that bind to the surface of the virus can destabilize the formation of S glycoproteins and interfere both with the trimerization of the protein and with the continuity of the life cycle of CoVs.
69 Several studies have been conducted on S protein to clarify its SARS-CoV-2 structure and its binding process as well as to evaluate its relevance as target for in-silico and in-vitro assays on molecules for anti-SARS-CoV-2 therapies.
One study conducted by Hoffman et al.
35 investigated how the SARS-CoV-2 S protein facilitates viral entry in the target cells and how this process could be blocked.
Results showed that ACE2 is used as receptor for the entry of SARS-CoV-2 in host cells and that the spread of this CoV in the infected host depends on the activity of TMPRSS2 (a cellular serine protease responsible for initiating the binding process between S protein and ACE2).
This process can be blocked with clinically approved TMPRSS2 inhibitor.
Prior to this, the relevance of TMPRSS2 was highlighted in the dissemination of several types of viruses such as Influenza A and other CoVs, which also makes it a relevant target for COVID-19 therapeutic intervention.
[70] [71] [72] [73] [74] [75] [76] Binding between S proteins and ACE2 receptors was corroborated through X-ray crystallography conducted by Lan et al.
67 to elucidate the interaction between the SARS-CoV-2 RBD and ACE2 at a higher resolution.
In spite of different interactions with ACE2, the SARS-CoV-2 RBD /ACE2 and SARS-CoV RBD /ACE2 interfaces share a substantial similarity regarding the surface area, the number of interacting residues and the networks of hydrophilic interactions.
Such similarity strongly points to a convergent evolution of both SARS-CoV-2 and SARS-CoV RBD structure which improves the binding affinity for the same receptor, the ACE2.
The non-conserved RBD regions in S protein, such as subunit S2, could be potential targets for cross-reactive antibodies.
Considering RBD as a critical region for receptor binding, antibodies that target the conserved epitopes in the RBD are also good candidates for the development of highly potent cross-reactive therapeutic agents against several species of CoVs, including SARS-CoV-2.
67 Investigations on ligands obtained from DrugBank 5.1 used molecular Docking to identify target regions in the pockets of the quaternary structure of SARS-CoV-2 S glycoprotein (from Protein Data Bank-PDB).
Six pockets present in S glycoprotein deserve further investigation in medicinal chemistry due to suitable features for small molecule binding.
Among the six pockets, the eight best ligand candidates from DrugBank were all binding pocket #1, which contained residues of amino acids Proline, Leucine, Lysine, Asparagine, Phenylalanine, Glycine, Threonine, Glutamine, Alanine, Methionine and Tyrosine.
One of the best ligands was the drug Saquinavir, an antiviral from the class of protease inhibitors, used in anti-HIV therapy.
69 Nsps are involved in the RNA transcription, translation, protein synthesis, processing and modification, viral replication and infection of the host.
Significant functional proteins, 3CL pro , PL pro , helicases and RdRp are important targets for the development of small-molecule inhibitors, due to their biological function and vital enzyme active site.
77 Factors Nsp1, Nsp3c and ORF7a are related to assistance to the immune evasion of SARS-CoV-2.
Interaction between Nsp 1 and the host ribosomal subunit induce the degradation of mRNA, allowing the virus to develop resistance to the host innate immunity.
Binding between ORF7a and Bone marrow matrix antigen 2 (BST-2) inhibits activity and blocks BST-2 glycosylation.
These results suggest that all three structures are potential targets for antiviral medicine.
5 Proteases PL pro and 3CL pro mediate the proteolytic cleavage of polypeptides produced by βcoronavirus SARS after genome transcription, thus generating other proteins.
The 3CL pro , known as Nsp5, cleavages several non-structural proteins of importance for viral replication and the maturation of Nsps, which is essential in the life cycle of the virus.
Therefore, it is an attractive biological target for that has been inhibited in-silico by several antiviral, anti-inflammatory and anti-hypertensive drugs from the database ZINC (FDA).
5 In addition, docking and molecular dynamic studies conducted by Qamar et al.
78 showed that non-toxic natural products formed strong bonds with SARS-CoV-2 catalytic dyad Cis145-His41 of 3CL pro .
Moreover, the proteinase PL pro is responsible for cleavages of N-terminus in the replicase polyprotein to release Nsp1, Nsp2 and Nsp3, which are essential for correcting virus replication significant to antagonize the host's innate immunity.
Analysis of the docking model showed that ribavirin formed Hydrogen bonds with residues Gly164, Gln270, Tyr274, Asp303 as well as hydrophobic interactions between Tyr265 and the PL pro residue.
These results indicate ribavirin as a powerful PL pro enzyme inhibitor, which means it has promising features for anti-COVID-19 therapy given the inhibition of a likely PL pro therapeutic target.
5 Helicase (Nsp13) has been identified as a promising target for antiviral drug discovery, particularly against SARS-CoV-2.
It is a multifunctional protein necessary for a wide range of biological processes, such as genome replication, recombination and dislocation of proteins related to chromatin and nucleic acid remodelling.
For CoVs, helicase is indispensable for viral replication.
In studies on molecular modelling, several antibacterial, antifungal and antiviral drugs were analysed and presented elevated affinity to helicase, suggesting it as a good target for SARS-CoV-2 therapy.
5 RNA-dependent RNA polymerase (RdRp -also nominated Nsp12) catalyses the viral RNA, which performs a key role in the replication/transcription complex of SARS-CoV-2, possibly aided by Nsp7 and Nsp8 complex as cofactor.
5, 79 Nsp12 has been studied as potential target for several SARS-CoV and MERS-CoV inhibitors, due to its importance for viral control.
Satisfactory results of RdRp inhibition by several ligands were presented in the modelling studies by Gao et al.
79 Yin et al.
80 Those ligands included antiviral analogous to nucleotides, such as Remdesivir, which already shows great potential in the treatment of COVID-19 infections.
In addition, some nonstructural proteins, including Nsp3b, Nsp3e, Nsp7, Nsp8, Nsp9, Nsp10, Nsp14, Nsp15 and Nsp16, also stood out as useful targets due to their significant role in the synthesis and replication of viral RNA.
5 3CL pro is key enzyme for CoVs, also called Main protease (M pro ), that plays a pivotal role in mediating viral replication and transcription, making it an attractive target for anti-SARS-CoV-2 drugs.
Such claim is reinforced by studies by Jin et al.
81 after the virtual screening of N3 inhibitor.
Results show that N3 (1) is a time-dependent irreversible inhibitor of this enzyme and that a stable covalent bond is formed between N3 and 3CL pro .
High-throughput screening (HTS) was applied to 10,000 drugs and drug candidates, demonstrating that Ebselen (2), PX-12 (3) and Carmofur (4) are all able to covalently bind to 3CL pro do SARS-CoV-2, with IC 50 that varied from 0.67 to 21.4 µM (FIG.
2) .
It is likely that a part of the hits identified by HTS are bonded to the catalytic cysteine of 3CL pro through their sulfhydryl groups.
In-vitro studies on antiviral activity were performed to corroborate the results.
Real Time Quantitative PCR (qRT-PCR) demonstrated that Ebselen and N3 had the strongest antiviral effects at a concentration of 10 μM treatment in SARS-CoV-2 infected Vero cells.
After plaque-reduction assay, the dose-response curves suggested that both could penetrate cellular membrane to access their targets.
This result strongly supports the hypothesis that developing a single antiviral agent targeting 3CL pro or in combination with other therapies could provide an effective first line of defence against all CoVs related diseases.
In relation to SARS-CoV-2 therapy, some of the aforementioned targets have been explored for both new drug proposition as well as for SARS-CoV-2 drug repurposing.
Our focus is on this last type, and for each medicine, the putative mechanism of action and viral target will be described trying to find an understandable rational therapy even for an immediate illness situation like COVID-19 pandemic.
Carmofur (4).
As previously mentioned, SARS-CoV-2 is an enveloped virus, whose nucleocapsid consists of a positive RNA genome surrounded by multiple copies of nucleocapsid protein.
This virus, after entry in the host cell, replicates fast the viral genome with new virion production.
The RNA replication into the cell host depends on enzymes and substrates for RNA synthesis, such as ribonucleotides (adenine, guanine, cytosine or uracil) that have nitrogenous bases in the purine or pyrimidine classes.
Compounds can mimic these chemical structures and interfere with the formation or use of one of these essential normal organism metabolites.
The interference is generally prompted by enzyme inhibition in the biosynthetic pathway of the metabolite or by incorporation, as a false building block, into vital macromolecules such as proteins and polynucleotides.
So, this class of therapeutic agents is called antimetabolites.
82, 83 Diverse antimetabolites have been indicated as promising anti-SARS-CoV-2.
They are described next.
Pyrimidine derivatives are aromatic organic compounds necessary for all life forms.
Examples of pyrimidine derivatives are nitrogenous bases cytosine (5), uracil (6) and thymine (7) (FIG.
3) .
They are found in DNA and RNA and participate in the metabolic process that involves carbohydrate and lipids.
84, 85 These heterocyclic rings share two nitrogen atoms at 1 and 3 positions, but display variations between themselves, such as an amine group at 4-position in the cytosine and a methyl at 5-position in the thymine.
From the pharmacologic perspective, nitrogenous bases are investigated as pharmacophores and are found in the structure of many drugs and experimental substances with various activities, 86 such as antitumoral, 87 antibacterial, 88 antiparasitic, 89 and antiviral.
90, 91 Regarding antiviral activity, there are several approved drugs that are classified as pyrimidine nucleotide biosynthesis inhibitors (PNBI) because, after phosphorylation, they are incorporated either into the DNA or into the RNA and inhibit hosts or pathogenic enzymes, such as polymerases.
85 Therefore, the likely mechanism of action of some pyrimidine derivative drugs has been considered for repurposing.
Some pyrimidine derivatives with antiviral activity are often formulated as prodrugs.
This format solves issues of high polarity in its final structure prompted by the phosphonic acid, which interferes with pharmacological properties and causes low cellular permeability and low oral bioavailability.
91 One compound appointed as potential anti-SARS-CoV-2 is the 5-Fluorouracil (8) (5-FU) (FIG.
3) , a heterocyclic aromatic amine similar to uracil (U) that presents a fluorine-carbon bond at 5-position.
This compound is used in the treatment of oesophageal cancer, 83 stomach cancer, 92 breast and colon cancer.
93 The similarity between 5-FU and uracil allows the direct action on nuclei acid as it is incorporated into the genetic material and inhibits replication.
83 Tests with 5-FU as monotherapy confirmed its failure against any coronaviruses.
The reason proposed to such failure relied on the fact that coronaviruses RNA proofreading activities involve a 3' → 5' exoribonuclease in the Nsp14, which removes 5-FU during replication and metabolism.
Hence, the combination between 5-FU and deoxyribonucleoside and deoxyribose was suggested so that, after its insertion in the RNA, it escapes RNA proofreading and prompt lethality and/or lethal mutagenesis in the virus.
Despite the proposition of using a widely marketed drug to treat several types of cancer, which means it has well-established efficiency and safety, no other type of test has been made to confirm its efficacy against SARS-CoV-2.
Therefore, further experiments are necessary to explore 5-FU potentialities.
94 Another antitumoral drug considered for its anti-SARS-CoV-2 potential is gemcitabine (GCT) (9) (FIG.
3) , an analogue of deoxycytidine whose pharmacological action is triggered after the intracellular transformation into triphosphate gemcitabine.
The latter competes with endogenous nucleoside triphosphates by incorporation into the genetic material, thus inhibiting DNA synthesis.
95 Initially, GCT was developed for antiviral activity, however, initial results caused it to be redirected for anticancer therapy.
It became, then, widely used against non-small cell lung cancer, pancreas, bladder and breast cancers as well.
[96] [97] [98] In-vitro analyses of gemcitabine hydrochloride inhibited MERS-CoV and SARS-CoV, with a CE 50 of 1.2 μM and 4.9 μM, respectively, in addition to low cell toxicity for VERO E6 cells.
99, 100 These data are indicative of a possible activity against SARS-CoV-2, but complementary preclinical investigations are necessary before clinical trials.
Albeit considered a safe drug under predetermined doses, GCT adverse effects are noteworthy and include myelosuppression and disruption of liver functions.
In February 2017, the European Union (EU) approved Baricitinib (10) as second-line oral treatment for mild to severe active rheumatoid arthritis in adults.
9 A differential feature of Baricitinib structure is the azetidine ring bearing an ethylsulfonyl, beyond an acetonitrile group at 3-position.
The same ring binds to the N atom at 1-position in the pyrazole, which, in its turn, binds to the pyrimidine conjugated to a pyrrole ring.
101 This medicine can modulate human innate and adaptive immune system.
Based on this property, presumably, one of the important mechanisms of action of baricitinib in the treatment of rheumatoid arthritis is the inhibition of the IL-6 / JAK1 / JAK2 pathway.
102 The promising nature of Baricitinib and other small molecule inhibitors against SARS-CoV-2 was pointed by Richardson et al.
9 through in-silico tests using Benevolent AI.
The authors evaluated 378 compounds to show that sunitinib (11) and erlotinib (12) inhibit AP2-associated protein kinase 1 (AAK1) interrupting the virus entry to the cells and the intracellular assembly of new viral particles (FIG.
3) .
Regarding these two antitumor drugs, it is known that sunitinib is an oral oxindole multitargeted kinase inhibitor that inhibits certain tyrosine kinases including vascular endothelial growth factor receptors (VEGFR types 1 and 2), platelet-derived growth factor receptors (PDGFR-α and PDGFR-β), stem cell factor receptor (KIT), FMS-like tyrosine kinase-3 (FLT3), glial cell-line derived neurotrophic factor receptor (RET) and the receptor of macrophage-colony stimulating factor (CSF1R).
103 Concerning erlotinib, it was developed as reversible and highly specific small-molecule tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of epidermal growth factor receptor (EGFR), thereby inhibiting autophosphorylation and blocking downstream signalling.
104 However, these oncological drugs have serious adverse effects such as diarrhoea, loss of appetite and skin rashes.
In addition, high doses of these medications can aggravate those effects.
In relation to baricitinib, its anti-SARS-CoV-2 potential was explained in three ways: AAK1 inhibition like sunitinib and erlotinib; the kinase associated to cyclin G, which is another endocytosis regulator; and the Janus kinase, that inhibits the action of cytosines that triggers the inflammatory process.
Because Baricitinib can inhibit AAK1 at the therapeutic dose (2 or 4 mg/day), the drug is indicated for clinical trials.
It is highlighted that Baricitinib is not indicated for patients with neutropenia or lymphopenia, once it lowers rates of neutrocytes and lymphocytes, which can lead the disease to progress and increase anaemia.
Furthermore, treatment with Baricitinib can reactivate varicella-zoster, herpes simplex and Epstein-Barr viruses.
This implicates in a conflict between the potential effect and the adverse effects of Baricitinib against COVID-19 to prevent aggravating the disease and the mortality of patients.
105 An analogue of adenosine, Galidesivir (GSV) (13) is a broad-spectrum antiviral drug that blocks viral RNA polymerase by replacing a natural nucleotide with galidesivir triphosphate.
This alteration prompts changes in electrostatic interactions and prevents the formation of the RNA elongated strand.
106, 107 Adenosine and GSV differ in that galidesivir has one Carbon at 7-position in the pyrimidine ring and Nitrogen in the ribose ring, whereas adenosine has one Nitrogen in the former and Oxygen in the latter (FIG.
2) .
106 It is noteworthy that GSV has not been approved for clinical trial and is an experimental drug in advanced stages of development.
108 GSV was first developed against hepatitis C (HCV) but first clinical trials were conducted to ensure its safety (in healthy individuals) and efficacy against yellow fever.
Furthermore, GSV displayed in-vitro and in-vivo antiviral activity against Filoviridae, Alphavirus, bunyavirus, arenavirus, paramyxovirus, flavivirus, orthomyxovirus, picornavirus and SARS and MERS coronaviruses.
11, 106, 109 Recent in-silico studies have shown the existence of a strong bond between GSV and SARS-CoV-RdRp to demonstrate the capacity of alterations in RNA polymerase, which can eradicate the virus.
Although, preclinical and clinical trials are necessary to either confirm or deny this hypothesis.
7 It is noteworthy that investigations have pointed the inactivity of GSV against SARS-CoV-2 at concentrations lower than 100 mΜ.
110 The existence of antiviral activity against other coronaviruses indicates that more investigations on GSV against SARS-CoV-2 are required to elucidate its potential activity in advanced testing.
Next, sofosbuvir (SBV) (14) is an example of successful nucleotide prodrug, approved by the Food Drug Administration (FDA) since 2013, against chronic hepatitis C infections.
SBV is also combined with other antiviral drugs, such as ledipasvir, velpatasvir and voxilaprevir.
111, 112 The structural similarity between SBV (FIG.
2) and uridine allows that drug to act on HCV RdRp, incorporate itself into the viral RNA and terminate the synthesis of the nucleotide sequence.
82 Structural analysis of SBV revealed that its elevated potential is partly due to the presence of the 5'phosphate, which terminates the primary enzyme transformation monophosphate inhibitor.
113 The antiviral activity has been explored against other viruses through in-vitro and in-silico studies and shown potential for inhibiting the dengue virus, 114 yellow fever, 115 Telbivudine (TBV) (15) (FIG.
3) is a thymidine nucleoside analogue used with specific activity against the hepatitis B virus (HBV).
It starts acting after phosphorylation by cellular kinases, which results in the active metabolite, Telbivudine 5'-triphosphate, enabling DNA polymerase and inhibiting viral replication.
The hydroxyl at 3-position in the sugar β-L-2'-desoxirribose provides specificity to HBV polymerase.
120 Suggesting repurposing TBV to fight COVID-19 was prompted by virtual screening to find drugs that act on viral M pro .
Among other results were Ribavirin, TBV and two vitamins, cyanocobalamin (B12) and nicotinamide (B3).
Researchers suggest that these four drugs can be combined and used against COVID-19, once they are safe, marketed and approved by the authorities.
8 Notwithstanding, the suggestion of repurposing these drugs requires more information, including on drug interaction parameters.
In spite of well-tolerated and safe for monotherapy, associating TBV and ribavirin, another antiviral drug, can increase hepatotoxic activities of TBV.
121, 122 It is also important to consider the elevated risk of resistance to TBV, which and pyrimidine derivatives drugs: 5-Fluorouracil (8); Gemcitabine (9); Baricitinib (10); Sunitinib (11); Erlotinib (12); Galidesivir (13); Sofosbuvir (14) ; Telbivudine (15).
Purine is a 5 and 6-membered bicyclic ring.
Similar to pyrimidines, purine derivatives are essential to life.
They are basic constituents of nitrogenous bases adenine (A) (16) and guanine (G) (17) (FIG.
4) .
84 The safety of RDV for humans infected with EBOV was evaluated in the Democratic Republic of Congo.
Results confirmed its safety but did not point RDV as the best therapeutic option, once its mortality rate reached 53% of treated group.
133 It has been proved that GS-5734 inhibits epidemic and zoonotic HCoV.
134 of viral loads and weight loss in murine.
135 Therefore, RDV is a potential drug to treat MERS-CoV infections.
Regarding COVID-19, RDV was used to treat the first US case.
The patient was 35 years old, had slight cough, low fever and no evidence of pneumonia at day 4 of the disease.
When the clinical symptoms became worse, the patient was given vancomycin and cefepime.
As the symptoms worsened, intravenous treatment with RDV was administered at day 7, and vancomycin and cefepime were no longer administered.
At day 8, the patient displayed clinical improvement, unfortunately details on the doses and duration of treatment were not provided.
136 After this first case, a clinical trial with a larger number of COVID-19 patients was conducted.
137 This study of efficacy involved 53 patients infected with SARS-CoV-2 who displayed saturation equal or inferior to 94% while they were breathing ambient air or receiving oxygen support.
The treatment lasted 10 days, patients were given 200 mg intravenously on day 1, followed by 100 mg daily for the remaining 9 days of treatment.
Follow up of patients treated for 18 days indicated that, after the first dose of RDV, 68% improved oxygen support whereas 15% of patients got sicker, 47% were discharged and mortality rate was 13%.
The most common adverse events (60% of patients) were increased hepatic enzymes, diarrhoea, rash, renal impairment, and hypotension.
Some limitations were noted in the study, such as the small size of the cohort, the short duration of follow-up and the lack of information on the patients.
Hence, the efficacy of RDV requires validation by the ongoing randomized, placebo-controlled trials.
One advantage of repurposing RDV is the availability of data on safety and pharmacokinetics, which were obtained previously at phase 1 clinical trial.
In addition to the promising results shown by RDV, other purine analogues have been investigated for SARS-CoV2.
Ganciclovir (GCV) (19) also named, according to its chemical structure, 9-(1,3-dihydroxy-2-propoxymethyl) guanine (FIG.
4) , is a guanine analogue, similar to acyclovir, except for the bond between the methyl group and one hydroxyl.
GCV inhibits the Human Herpesvirus and is also indicated in the treatment of cytomegalovirus infections related to Acquired Immunodeficiency Syndrome (AIDS).
139 GCV is converted into ganciclovir triphosphate by cellular kinase, which inhibits dGTP and disrupts viral DNA synthesis due to substitution of various adenosine bases in the DNA chain.
140 Recently, GCV was used to treat COVID-19 patients in China.
141 The drug was administered with other antivirals, such as oseltamivir and Kaletra.
As a descriptive study, the relation between GCV as key factor in clinical outcome of the 99 patients (31% of which were discharges) was not possible.
141 Therefore, GCV efficacy as monotherapy or part of combined therapy is yet necessary for more robust investigations.
Valganciclovir (20) (FIG.
4) is the antiviral prodrug of GCV taken by mouth.
It is indicated for the same treatments as GCV (cytomegalovirus in people who have acquired immunodeficiency syndrome, gastrointestinal disorders related to AIDS).
The drug has great bioavailability and is converted by hydrolysis into ganciclovir.
Using valganciclovir in its oral form enables clinical treatment and makes patients more comfortable.
[142] [143] [144] The mechanism of action is the same of GCV.
145 Valganciclovir was computationally evaluated for COVID-19.
5 The assay with the main proteins coded for SARS-CoV-2 allowed the determination of 21 possible binding targets, of which 19 were proteins and 2 host targets.
Valganciclovir, one of the drugs used in the study, was presented as a possible anti-SARS-CoV-2 therapeutic drug due to its high binding affinity to two wellestablished viral targets.
The first target was PL pro , indispensable enzyme in viral replication; the second, RdRp, conserved Nsp12 in coronavirus, which is vital for its replication/transcription.
Therefore, valganciclovir could be a significant antiviral drug to treat SARS-CoV-2.
But there are no clinical reports on valganciclovir used to treat COVID-19 in addition to what has been reported about GCV.
141 Hence, its efficacy is yet to be confirmed as anti-SARS-CoV-2 therapeutic.
Tenofovir (TFV) (19) is another adenine analogue pointed as promising COVID-19 therapeutic (FIG.
4) , it is also called Tenofovir disoproxil fumarate or alafenamide Tenofovir (TAF).
Approved by the FDA in 2001, TFV is a prodrug used to treat HIV and cases of nucleoside resistance.
146 TFV is an analogue reverse-transcriptase inhibitor (NtRTI).
Inside cells, TFV is phosphorylated and competes with deoxyadenosine 5'-monophosphate (d-AMP), thus preventing the formation of DNA.
Once incorporated into a growing DNA strand, it causes premature termination of DNA transcription and prevents viral replication.
146, 147 Modelling and docking studies evaluated the antiviral effects of TFV and verified a strong bond to SARS-CoV-RdRp, which can disrupt this polymerase and terminate the viral infection.
7 However, in-vitro tests showed that TFV lacks apparent antiviral effect at concentrations inferior to 100 μM for SARS-CoV-2.
110 In spite of lukewarm in-vitro and in-silico outcomes, an ongoing clinical study on TFV (ChiCTR2000029468), expected to end in June 2020, aims at assessing the effect of the combination Tenofovir + emtricitabine (cytidine analogue) related to LPV/r in COVID-19 patients.
131 In addition to the efficacy of treatment, clinical trials can validate the prevalence of adverse effects related to the toxicity of TFV in patients.
TFV is also a powerful nephrotoxic drug causing damage to proximal tubular cells.
In spite of that, interrupting the treatment is sufficient to improve adverse effects, which makes monitoring of patients essential.
145 Heterocyclic compounds with different heteroatoms such as Nitrogen, Sulphur and Oxygen can present different pharmacological properties.
One such property is to serve as analogue of nitrogenous bases of nucleic acids, such as triazoles, which have a five-membered ring of two carbon atoms and three nitrogen atoms.
This aromatic ring can assume two isometric forms, 1,2,3-triazole and 1,2,4-triazole.
The former is stable under acid and basic conditions and becomes more reactive when binding to electronegative elements.
148, 149 Triazoles are important and stand out for their various biological activities, such as anticancer, 150 antituberculosis, 151 anti-inflammatory, 152 antimicrobial, 153 and antiviral.
154 Specifically for the latter action, triazole-based derivatives have shown promising invitro activity against coronavirus, probably by 3CL Pro inhibition.
155 Ribavirin (22) (FIG.
4) is a powerful triazole-based antiviral analogue to guanosine.
It presents a wide range of pharmacological activities related to several viruses, for instance: herpes simplex virus, human immunodeficiency (HVI-1), influenza, respiratory syncytial (RSV) and hepatitis C. 149, 156 The drug was initially used in 1980 to treat syncytial virus in children, generally combined with Interferon (INF).
However, ribavirin treatment presents undesirable adverse effects, like lowering of haemoglobin, which limit clinical use.
157 Its action mechanism relies on the inhibition of enzyme inosine monophosphate dehydrogenase, necessary in the synthesis of guanosine triphosphate, which prevents viral DNA and, mainly, RNA replication.
The necessary concentration for in-vitro inhibition of RSV and influenza ranges from 3-10 μg/mL.
158 site similar to other SARS-PL pro inhibitors.
The formation of hydrogen bonds and π-π stacking were also predicted.
These findings suggest that ribavirin as a powerful PL pro inhibitor.
Nonetheless, investigation on triazole derivatives for anti-SARS-CoV-2 therapy are still preliminary.
On the other hand, Favipiravir (FPV) (23) (FIG.
4) is a prodrug, approved in 2014 in Japan, to treat cases of influenza A and B that displayed resistance to first line drugs.
It has provided results that indicate a promising character and is currently undergoing clinical trials against COVID-19.
Its antiviral efficacy has also been investigated in different countries to fight Ebola and Lassa, for example.
The molecular structure of the drug consists of a pyrazine heterocyclic ring with fluorine at 5-position, carboxamide at 3-position and a double bond between oxygen and Carbon 2, which renders its analogue to guanine (17).
162, 163 The metabolization of the prodrug into its active form, favipiravir-ribofuranosyl-5'-triphosphate, requires intracellular ribosylation and phosphorylation.
163 FPV therapeutic targets are RdRp enzymes, necessary in viral transcription and replication, and its inhibition blocks synthesis of viral RNA for a spectrum of viruses, including human coronavirus.
A different investigation compared patients treated with FPV plus interferon inhalation to LPV/RTV.
Patients under FPV therapy responded better to the progression of the disease with accentuated viral depuration.
Also, the incidence of nausea and vomit was higher for LPV/RTV.
166 In addition to these clinical trials, the antiviral activity of FPV against SARS-CoV-2 was also evaluated but no clear antiviral effect was noted for doses lower than 100 μM.
110 An in-vitro study using molecular docking focused on the binding properties of SARS-CoV-2 protein structures to 61 antiviral agents, including oseltamivir.
The study showed that 37 molecules form bonds to SARS-CoV-2 crystal proteins.
However, data did not show oseltamivir as the best structure because Lopinavir, Asunaprevir and Remdesivir interacted with more than two protein structures in the virus.
Hence, they are likely more promising than oseltamivir.
178 Notwithstanding, we suggest further look into oseltamivir against other enzyme targets since different studies achieved positive results regarding its use as anti-SARS-CoV-2.
Nelfinavir (27) (FIG.
5) is a safe anti-retroviral drug largely used for HIV-1 protease inhibition with strong in-vivo activity.
179 Generally, Nelfinavir is combined with other anti-retroviral medication as part of a highly active antiretroviral therapy (HAART) that reduces significantly the viral load by increasing cell number to 200 mm -3 CD4(+) lymphocytes.
The drug is prescribed for children, young individuals, adults and pregnant women.
180, 181 Nelfinavir and its active metabolite M8 strongly bind to serum proteins, displaying optimal tissue distribution.
A frequent adverse effect is light to moderate diarrhoea, reported for 15 to 20% of patients.
180 The SARS-CoV outbreak in several countries triggered the search for antiviral drugs active against the disease.
Among the 24 drugs likely to inhibit SARS-CoV, nelfinavir stands out in all assays.
181 The mechanism of action suggested for nelfinavir involves preventing SARS-CoV replication after its entry in the host cell and disrupting virion production.
Based on results from previous studies as well, nelfinavir was considered a likely therapy for COVID-19 after its indication for clinical trials as a promising anti-SARS drug.
Recently, 1,903 drugs were evaluated for their binding affinity to SARS-CoV-2 M pro .
182 Among the compounds, 15 drugs were selected based on the docking score and three-dimensional Atazanavir (28) (FIG.
5) is an antiretroviral drug protease inhibitor used to treat HIV infections with in-vitro inhibitory concentration of 2,6-5,3 nmol.
Compared to other protease inhibitors, atazanavir has the advantage of allowing a daily posology regimen with a favourable metabolic profile and low frequency of adverse effects.
183, 184 Several HIV-1 resistant to protease inhibitors are still sensitive to in-vitro atazanavir, which is considered safe and well tolerated.
185 The atazanavir acts to inhibit HIV-1 protease, which is indispensable in the processing of polyproteins precursors of viral structures and prevents the formation of infectious and mature viral particles.
183 The good activities reported for this drug as well as the search for safe and fast therapy for Captopril (29) (FIG.
5) is an angiotensin-converting enzyme inhibitor (ACEi).
It is a zinc metallopeptidases inhibitor that converts angiotensin-I into angiotensin-II, an essential function that regulates arterial pressure.
It is predominantly indicated as vasodilator in patients with cardiac insufficiency.
This drug was suggested as potential antibiotic capable of inhibiting zinc succinyls/dipeptidase by blocking its zinc catalytic center.
189, 190 Tolerance to captopril has been largely investigated; its single dose by mouth is well-established and confirms the pharmacological activity in the short term (10-30 minutes) at the cellular level.
This capacity is related to captopril transport mainly through plasma proteins such as albumins with absorption rate between 70-75%.
Reported adverse effects are neutropenia, proteinuria, dysgeusia and cough, but less frequent for low doses.
189, 190 Some investigations have suggested captopril as possible COVID-19 treatment.
Serafin et al.
100 indicated captopril as potential for inhibiting the bond between human SARS-CoV-2 and ACE-2 and reduce severe pneumonia symptoms.
In-silico studies using molecular docking were conducted with FDA-approved drugs capable of binding to the main active site in proteinase 3CL pro .
189 Two drugs were identified as ligands for the enzyme active site: captopril and disulfiram.
The former binds to the active site at the same position of N3 inhibitor (a standard inhibitor that reacts irreversibly in the same site with 3CL pro Cys145).
It is, thus, suggested that captopril binds to the same site of N3, obstructing the function of Cys145-His41 catalytic dyad.
Captopril probably inhibits the enzyme in two stages.
Initially, it establishes non-covalent bonds to sites in the enzyme targets, then, a reaction takes place between the critic groups, which results in a more stable inhibitor complex.
The hypothesis is that captopril can bind covalently to 3CL pro Cys145.
Although the potential of captopril on the enzyme has been demonstrated, therapeutic use against COVID-19 is controversial, once the drug induces overexpression of ACE-2 -the main receptor used by SARS-CoV-2 to entry the cells.
Therefore, combination with other drugs, such as angiotensin-II receptor blockers, needs analysis to clarify the effects of captopril in COVID-19 treatment.
The cyclosporin A (CsA) (30) (FIG.
5) is isolated from the fungus Beauveria nivea and was approved for use by the FDA in 1983.
This drug has been used for decades to prevent organ rejection and to treat T cell-associated autoimmune diseases such as Behcet's disease, psoriatic arthritis, lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus or interstitial lung disease.
Such drug exerts its immunosuppressive function and anti-inflammatory effects by inhibiting the transcription of genes required for T cell proliferation, notably interleukin-2.
[191] [192] [193] Due to the severity of COVID-19, CsA can be potential to prevent hyperinflammation-induced lung injury.
194 In this regard, it is known SARS-CoV Nps1 induces the expression of interleukin-2 via nuclear factor of activated T cell (NF-AT) activation, 195 which can trigger the cytokine storm seen in patients with severe COVID-19 status.
138 Another advantage presented by CsA in relation to other antiinflammatory drugs is its already known anti-CoV action against all genus, including SARS-CoV, [195] [196] [197] at low and non-cytotoxic micromolar concentrations verified in cell culture assays.
This antiviral property is thought to be mediated by the inhibition of cyclophilin-A-dependent viral assembly as well as inhibition of the NF-AT pathway or even by genetic or pharmacological specific inhibition of cyclophilin-D, hindering the viral replication.
195, 198 As already reported, SARS-CoV and SARS-CoV-2 are very similar (79.5% sequence identity).
17 Teicoplanin (31) (FIG.
6) is an antibiotic used against gram-positive bacteria with 5 major compounds at different side chains.
It prevents polymerization of peptidoglycans and inhibits the development of the cell-wall, thus prompting cell death.
203 It is a big molecule that has displayed antiviral activity on an early stage of the viral life cycle by inhibiting the low-pH cleavage of the viral spike protein by cathepsin L in the late endosomes thereby preventing release of viral RNA and replication.
This compound has already shown inhibitory activity against Ebola virus, MERS-CoV and SARS-CoV.
203 Recent investigations have suggested teicoplanin as alternative treatment for COVID-19 after an in-vitro assay achieved IC 50 value of 1.66 µM, thus proving its efficacy against SARS-CoV-2.
These results need to be confirmed through randomized clinical trials, which are still to be conducted.
100, 204, 205 Using antibiotics to fight viruses, albeit completely ignored, can become useful to treat COVID-19.
206, 207 Some studies report the possibility of repurposing drugs like terconazole, which displayed good in-vitro results against MERS-CoV and SARS-CoV, 100 Dasabuvir (32) (FIG.
6) is a drug from the naphthalene class and phenyl-naphthalene subclass due to the bond between its naphthalene ring and a phenyl group.
Dasabuvir is a first line drug used as combined therapy for chronic hepatitis C. 208 Dasabuvir is a non-nucleoside inhibitor that binds to Nps5B (non-structural protein 5B -RdRp) and induces conformational change that makes RdRp incapable of elongating the viral RNA.
209 Repurposing of this drug can be useful as SARS-CoV-2 therapy due to its antiviral activity.
210 Dasabuvir was subjected to docking studies against SARS Briefly, Dasabuvir forms π-cation interactions with Lys31a (present in the ACE2), π-π interactions with Phe170b (S Protein residue) and hydrogen bonds to ACE2 residues Glu35a and Asp38a and Gly176b and Ser174b (S Protein residue).
The authors highlight the importance of repurposing drugs as new therapeutic alternatives not only for the new coronavirus but for the next viral outbreaks.
Darunavir (33) (FIG.
6) is a benzene derivative that has been evaluated for repurposing against COVID-19.
This drug is an antiviral used in the treatment of HIV-1 infections.
It provides a great genetic barrier to resistance and is highly active against resistant strains of HIV-1 that are not susceptible to other protease inhibitors.
211 Darunavir is administered orally as pills or suspension and is often used with low doses of ritonavir as part of a combined ART protocol.
212 Its mechanism of actions works by protease inhibition.
Darunavir establishes high affinity bonds to HIV-1 protease forming a stable complex, thereby selectively inhibiting polyprotein gag-pol coded by the virus.
This prevents the formation of mature viral particles.
211 FDA-approved drugs against 3CL pro , RdRp, helicase, exonuclease 3′ a 5′, endoRNAse e 2′-O-ribose methyltransferase.
Among the best drugs in the assay, darunavir was a surprise because, despite inhibiting viral proteinase, the study showed that it binds to the replication complex components of SARS-COV-2 with inhibitory potency Kd < 1000 nM.
One example is RdRp, whose Kd value was 148.74 nM and exonuclease 3′ to 5′ with K d value of 195.73 nM.
A docking study was conducted by Sang et al.
215 as in-silico evaluation of anti-HIV drugs in their interaction capacity to proteinase 3CL pro .
Results suggest that all drugs have higher binding affinity to SARS-CoV-2 3CL pro than to the homolog SARS-CoV proteinase.
Among the evaluated drugs, indinavir and darunavir displayed the highest docking scores, therefore, they were subjected to molecular dynamic (MD) simulations, free binding energy calculations and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) to detail molecular interactions between inhibitors and proteinase.
The data suggest darunavir had better binding affinity to SARS-CoV-2 3CL pro with binding affinity of -10,24 kJ / mol.
In addition, darunavir bind to SARS-CoV-2 3CL pro via 19 contact residues and to SARS-CoV 3CL pro via 17 residues.
This difference explains the lower binding energy values between darunavir and SARS-CoV 3CL pro .
It was also noted 5 hydrogen bonds between darunavir and SARS-CoV-2 3CL pro but none for indinavir and this proteinase.
Because hydrogen bonds are important in the stability of the inhibitor-enzyme complex, darunavir is probably more promising against COVID-19.
Finally, a different in-silico assay was conducted by Pant at al.
216 to assess a large variety of compounds (300) from several data banks and 66 potential compounds from FDA-approved drugs.
The compounds were tested against SARS-COV-2 3CL pro .
Darunavir was among the 20 best FDA-approved drugs, with a score of -7.208.
All data collected from in-silico studies still require experimental studies to validate the anti-SARS-CoV-2 activity of darunavir.
A research conducted by Dyall et al.
99 performed a robust in-vitro assay and showed that the drug Dasatinib (34) (FIG.
6) , a kinase signalling inhibitor developed to treat human cancers, inhibited MERS-CoV and SARS-CoV, exhibiting EC 50 values 5.4 and 2.1, respectively.
This study also revealed that kinase signalling may also be important for replication of this HCoVs.
Nevertheless, the authors reported that dasatinib may be valuable against coronaviruses infections if a dosing regimen that minimizes immunotoxicity while still blocking viral replication can be defined.
Results indicated this drug as a likely therapeutic alternative against SARS-CoV-2 infection.
An in-silico study carried out by Qiao et al.
217 showed that dasatinib, among others, is one of the most promising drugs for the inhibition of SARS-CoV-2 3CL pro .
More preclinical and clinical studies are required to prove whether dasatinib is really promising for COVID-19 patient treatment.
Imatinib (35) (FIG.
6) is an oral anticancer agent that inhibits the activity of some tyrosine kinases, most prominently the BCR-ABL fusion oncoprotein (whose overactivation can lead to chronic myeloid leukaemia, CML), c-kit (involved in gastrointestinal stromal tumours development), platelet-derived growth factor receptor (PDGFR), and the native ABL kinase, which has a ubiquitous expression and plays important roles in several biological processes.
218 In addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during CoV infection), by interfering with virus-cell fusion, 219 and other RNA viruses including coxsackie virus, 220 hepatitis C virus, 221 Ebola, 222 among others, mainly by blocking viral entry or egress from the host cell.
Besides, this drug showed activity against SARS-CoV and MERS-CoV, 223 both phylogenetically related to SARS-CoV-2.
24 In this regard, it is reported that imatinib has anti-CoV activity in two points of the virus life cycle.
In the early phases of infection, it inhibits virion fusion with the endosome and subsequent release into the cytoplasm, thus preventing viral entry and viral replication via ABL-mediated cytoskeletal rearrangement.
In a later phase of the infection, ABL2 protein expression, which is inhibited by this drug, enables SARS-CoV and MERS-CoV replication, which suggests that ABL2 is a new host cell protein required for viral growth.
223 Furthermore, evidences suggest that imatinib can modulate the immune response by sundry mechanisms, [224] [225] [226] for several diseases, such as rheumatoid arthritis, 227 asthma, 228 and Crohn's disease.
229 This information insinuates that such drug might perform its potentially beneficial immunomodulatory role as a treatment alternative for COVID-19 pneumonia.
In addition, the use of imatinib as treatment appears to be reasonable from an economic point of view and its high availability in hospitals, 230 since this drug is well tolerated and the risk of severe adverse effects is relatively low, especially in short-term administration.
231 It is also recognized that adverse effects, mostly mild to moderate in intensity, will be easily controlled by dose reduction or discontinuation.
232 In light of this information, Tatar and Turhan 233 used the docking methodology to better understand the mechanism of inhibition of the SARS-CoV-2 N protein with 34 antiviral compounds.
Based on this study results, imatinib was one of the highly binding affinities performed against the aforementioned target, with the lowest micromolar Ki values among the compounds evaluated.
In line with this study, an in-vitro research carried out by Weston et al.
234 found 17 FDA approved drugs that inhibited SARS-CoV-2 at non-cytotoxic concentrations.
The authors indicate imatinib as one of the hits, since it exhibited IC 50 value of 3.24 μM.
They subsequently determined the mechanism of action, demonstrating this drug inhibits fusion of CoVs with cellular membranes, precluding their entry.
This result indicates imatinib use against SARS-CoV-2.
However, its efficacy and safety need to be better confirmed in further preclinical and clinical trials in order of elect him as candidate drug in the treatment of COVID-19.
Synthesized for the first time by Jean Francois Rossignol in the beginning of the 1970s, the 2acetyloxy-N-(5-nitro-2-thiazolyl) benzamide, sold under the name nitazoxanide (NTZ) (36) (FIG.
6) , is the result of a structural modification in the antiparasitic niclosamide when a benzene ring is replaced with a nitrothiazole.
235, 236 Developed and sold as antiparasitic, NTZ is also a first line broad spectrum antiviral with good results against parainfluenza, coronavirus, rotavirus, hepatitis and other respiratory infections.
[236] [237] [238] [239] Following oral administration, NTZ is absorbed in the intestine, where it is rapidly hydrolysed by plasma esterase into the active metabolite tizoxanide.
236 The mechanism of action of NTZ varies according to the pathogen.
In relation to antiviral activity, NTZ blocks the maturation of the viral hemagglutinin at the post-translation stage in treatments against influenza.
In treatments against HCV (hepatitis C), it activates protein kinase R (PKR), which leads to phosphorylation of the eukaryotic initiation factor-2α thereby preventing translation.
240 Cao et al.
241 conducted an in-vitro evaluation of NTZ against recombinant murine coronavirus expressing the firefly luciferase (MHV-2aFLS).
The strand was pivotal to triage the 727 drugs with likely anti-CoV activity.
The first assay resulted in 84 molecules among which was NTZ.
The antiviral effect of NTZ verified for mouse astrocytoma (DBT) and fibroblasts (17Cl-1).
241 The DBT cells infected with MHV-2aFLS were treated with NTZ at 5 µM for 12 hours, after which the viral titer (TCID 50 ) was determined and viral N protein was subjected to Western blot.
Results show the strong inhibitory effect of NTZ on the viral titer.
241 In-vitro studies on NTZ or its metabolite tizoxanide were also conducted to verify efficacy against different coronaviruses.
The replication of Canine coronavirus (strain K378) in A72 cells, for instance, was blocked by the tizoxanide with IC 50 of 1 µg/mL.
On the other hand, NTZ inhibited the viral N protein in bovine coronavirus L9 (βCoV-L9) and human enteric coronavirus 4408 (HECoV-4408) with approximate values of 0.3 µg/mL.
239, 241 NTZ is also responsible for inhibiting pro-inflammatory cytokines, such as TNF-α, IL-2, ILNitazoxanide (36) drugs.
More recently, molecules with a quinoline group have been widely investigated as treatment for the new coronavirus (SARS-CoV-2), such as Chloroquine (CQ) (37) and Hydroxychloroquine (HCQ) (38) (FIG.
7) that belong to the quinoline class and aminoquinoline subclass.
Both are quickabsorption synthetic drugs approved to treat malaria (Plasmodium falciparum) by several regulating agencies in the world.
CQ and HCQ are water soluble; the latter is more soluble due to presence of hydroxyl group.
They are currently used to treat autoimmune diseases such as lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis as they have immunomodulatory and antithrombosis properties.
[243] [244] [245] Therefore, these drugs could be useful against COVID-19 due to the elevated levels of cytokine caused by CoV infections in humans.
246 The mechanism of action of CQ for anti-malarial treatment is not entirely clear, but the interference with the digestion of haemoglobin by the parasite has been suggested.
245 HCQ has a similar mechanism, however, in regard to SARS-CoV-2, clinical trials showed it to be safer.
[246] [247] [248] Therefore, HCQ, rather than CQ, is used against SARS-CoV-2.
Recent studies report antiviral activity of CQ and HCQ as they impair viral entry and release in different in-vitro and in-vivo models.
244, 249 A factor that can also justify viral mechanisms is the aminoquinoline bioaccumulation in the tissues, as defended by Patil, Singhal & Masand.
243 A factor that facilitates viral replication is the acidic pH of endosomes, lysosomes and Golgi complex of the host.
Thus, CQ is promising because it increases the pH of intracellular vacuoles, binds to the cellular receptors, changes the glycosylation and because of its selective and reversible immunomodulator effect on human CD4+ T cells.
HCQ exerts similar mechanism of action: a) increases the pH; b) modulation of activated immune cells; c) reduces the number of proinflammatory cytokine and other mediators to control inflammation.
243 It has also been suggested by Roldan et al.
244 a likely involvement of HCQ in iron homeostasis during SARS-CoV-2 infection, which is a similar mechanism to other viral infections in humans.
[250] [251] [252] The little difference between the therapeutic and the toxic dose of CQ is also known, and poisoning is related to cardiovascular complications that can be fatal.
Using either CQ or HCQ, then, requires strict prescription and self-medication is not advised.
249 Based on the RECOVERY data, it was concluded that HCQ is not effective in patients admitted with COVID-19.
This highlights the importance of randomized clinical trials and the collection of clear data on the efficacy and safety of medications.
Ivermectin (39) (FIG.
7) is an FDA-approved broad-spectrum antiparasitic agent used in the treatment of tropical diseases, such as onchocerciasis, lymphatic filariasis, strongyloidiasis and lice.
There is also evidence of its effectiveness in the management of myiasis, trichinosis, malaria, leishmaniasis, trypanosomiasis, Chagas disease and schistosomiasis as well as bed bugs, inflammatory skin lesions, epilepsy, neurological diseases, tuberculosis and some cancers.
266 It is known that ivermectin is capable of inhibiting the bond between a virus and the nuclear transport mediated by the superfamily of importin proteins (IMP α/β1) 267 Based on the fact that SARS-CoV-2 is a RNA virus deeply related to SARS-CoV, studies on SARS-CoV proteins have revealed a potential role for IMP α/β1 during infection in signal-dependent nucleocytoplasmic shutting of the SARS-CoV nucleocapsid protein.
273 Furthermore, the SARS-CoV accessory protein ORF6 has been shown to antagonize the antiviral activity of the STAT1 transcription factor by sequestering IMP α/β1 on the rough ER/Golgi membrane.
274 Considering the ivermectin nuclear transport inhibitory activity, such drug is widely believed as a promising therapeutic approach against SARS-CoV-2.
Recently, Caly et al.
275 A different mechanism of action that consolidates the use of ivermectin against COVID-19 is its immunomodulatory property.
The inflammatory response (proinflammatory cytokine) is exaggerated in patients with the extreme case of the disease, which is likely explained by the hypoxiainducible factor (HIF-1α) that is activated by the virus when no inhibitory medication is administered.
This understanding can be explained by the study conducted by Kosyna et al.
276 , whose lab tests with and without ivermectin aimed to examine whether the properties of the bond between HIF-1α and IMP α/β1 and between HIF-1 and nuclear localization signals were affected by the hypoxia mechanism on the cellular level.
The authors concluded that ivermectin inhibited both IMP α/β1 and HIF-1α.
Regarding This hinders its indication and clinical decisions.
Thus far, data indicate ivermectin is useful at the early stages of the disease, even the epidemiologic profile of COVID-19 shows significant differences regarding the age of patients for the affected countries and patients with comorbidity.
279 Therefore, large scale randomized clinical trials are necessary to standardize clinical, laboratory and image evaluations, as well as combined drug therapy with vitamins and zinc, for example.
280 Financially, ivermectin is inexpensive and its doses and protocols are well established for different purposes.
In addition, this drug has little side effects.
281 Indole, also named benzo [b] pyrrole, is a planar bicyclic heteroaromatic, whose ten π electrons move across its structure making this chemical group behave as a weak base.
282, The indole ring is the most abundant heterocyclic in nature and is commonly found in biologically active natural products, such as vegetables and seafood.
It is also present in the structure of the essential amino acid tryptophan, which interferes with protein synthesis and with the regulation of physiological mechanisms such as precursors for serotonin and vitamin B3.
283 288 and adding the indole ring to spirothiazolidinones conducted to better influenza A/H3N2 inhibition.
289 Now, the antiviral activity of indole and its derivatives for COVID-19 therapy is supposed.
Arbidol (40) is also a promising drug to fight COVID-19 (FIG.
7) .
This drug is classified as antiviral and has been used for 25 there is high expression of ECA2, identified as human receptor for virus entry.
290 Notwithstanding, a different clinic trial concluded that Arbidol monotherapy is best for the patient than LPV/RTV.
291 Despite the results, both investigations recognize their own limitations due to small cohort size and lack of placebo-control group.
According to the authors, these limitations are inherent to pandemic times when placebo-control groups are difficult to conduct due to life-threatening conditions.
Most studies with Arbidol use 200 mg/3*day 164, 290, 291 following the Chinese guidelines.
Previous investigations on the pharmacokinetics of Arbidol in healthy Chinese patients showed that a single 800 mg oral dose is sufficient for Cmax de ~4,1μM.
293 This value was obtained through invitro assays that pointed the drug as effective and promising against SARS-CoV-2.
Hence, clinical trials are still necessary to confirm the efficacy of Arbidol at elevated doses to treat COVID-19.
293 Rizatriptan (RZT) (41) (FIG.
7) is used to treat migraine and is a selective receptor of serotonin (5-HT) type 1B and 1D, structurally and pharmacologically related to other selective antagonists at these receptors.
294 Its structure is based on an indole ring replaced with methyltriazole at 5-position and unsubstituted ethanamine replaced with methyl at 3-position, the substitution sites are the same of melatonin (MLT).
After virtual triage through molecular dock at spike-ACE2 interface, ligations π-cation, interactions π-π and hydrogen bonds were identified between RZT and the SARS-CoV-2 protein complex.
As one of the outstanding compounds in the analysis, in-vitro tests are still necessary.
187 It is noteworthy that overdosing of the drug can trigger dizziness, fainting, cardiac issues, hypertension, bradycardia and vomiting.
Despite the safety of the drug in regular doses, there are no reports on either in-vitro or in-vivo tests to support the theoretical data and the antiviral action thus far.
One last indole derivative that could be repurposed to treat COVID-19 is melatonin (MLT) (42) (FIG.
7) .
MLT is classified as a hormone and nutraceutical, as it is naturally produced by the pineal gland and released into the bloodstream.
It regulates the sleep-wake cycle as well as our mood, learning and memory, fertility, reproduction and the immune system.
From a chemical perspective, it is an indole derivative with a methoxy group at 5-position and one ethylacetamide at 3-position.
295 Regarding its antiviral potential, MLT acts indirectly through anti-inflammatory, antioxidant and immune modulating activities.
296 An investigation with murine infected with Semliki Forest Virus (SFV) and West Nile Virus (WNV) showed the efficiency of MLT in reducing mortality rates for these viruses as well as in reducing the levels of pro-inflammatory cytokines.
297 The anti-inflammatory and antioxidant properties of MLT point to its antiviral effects in humans.
298 Based on computational data, Zhou et al.
299 suggested using combined medication to fight SARS-CoV-2.
One such combination involves MLT plus mercaptopurine in synergic action against the following targets: PL pro , ACE2, c-Jun signal and anti-inflammatory vias.
Therefore, experimental studies on modifications of ACE2 pathways caused by MLT are useful to understand this drug.
299 On the other hand, it has been suggested that using this neuro-hormone can mitigate the extreme form of the disease, the acute respiratory syndrome that has caused most deaths by SARS-CoV-2 cases.
Despite its safety for humans, the lack of data on the relevance of its use for COVID-19 patients Emetine (43) is an approved anti-protozoal drug used against amebae with reported inhibitory activity for enterovirus infections, 301 Zika virus and Ebola by interfering with the process of viral replication and entry in host cells.
302 Emetine is an isoquinoline alkaloid that presents 4 methoxy groups in its structure (FIG.
7) .
Studies also confirm emetine has in-vitro activity against coronaviruses, including SARS-CoV and MERS-CoV.
99 assays to clarify the activity of these drugs and the mechanism involved in the antiviral action of emetine both in isolation and combined to other drugs.
Another alkaloid candidate to repurpose against COVID-19 is homoharringtonine (HHT) (44) (FIG.
8) .
HHT is an FDA-approved drug in semi-synthetic form known as omacetaxine.
This drug displays antitumoral activity in the treatment of myeloid chronic leukaemia.
The mechanism of action implicates the ribosomal bond to prevent protein translation.
In addition to antitumor activity, there are data in the literature that describe antiviral activity of HHT against several types of viruses including CoVs.
304, 305 A recent in-vitro evaluation of anti-SARS-CoV-2 activity displayed EC 50 of 2.10 µM.
However, the mechanism of action is not yet clear, which demands further investigation on ideal doses of HHT to achieve the clinical results expected of a COVID-19 therapeutic drug.
110 The first reports on tetraethylthiuram disulfide, disulfiram (DSF) (45) (FIG.
8) , date back to 1881.
However, only in the 1940s that DSF would become popular when it was discovered that it could form copper chelates which favoured the death of micro-organisms and enabled treatment of intestinal parasites.
[306] [307] [308] In 1945, DSF alcohol sensitivity was discovered accidentally and it was soon used in the clinical treatment of alcohol dependence.
309, 310 DSF is used to treat alcohol dependence because it irreversibly inhibits the acetaldehyde dehydrogenase enzyme and modifies cysteine residues in its active site.
This change prompts the formation of a disulphide bond between two cysteine residues in the active site.
311 DSF effectiveness is based on its similarly to several proteins yielding a range of biological activities, such as antitumoral, 312, 313 antimicrobial, 310 and anti-SARS and MERS-CoV.
314 Adding to the list of drugs to be repurposed against SARS-CoV-2, recent studies indicate that DSF is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues that suppress the natural cycle of the enzymes, suggesting broad-spectrum characteristics.
312, 314 CoVs have two viral enzymes, M pro and PL pro , that are cysteine protease involved in the formation of structural and non-structural proteins that constitute the viruses and favour control of host cells.
315 Different assays, such as proteolytic and binding synergy assays, were also conducted and described.
314 Although outcomes indicate DSF for anti-MERS-CoV and anti-SARS-CoV therapy, to the present date (15 th June 2020), no other article was published claiming the availability of the compound as promising anti-SARS-CoV-2.
Recently, an announcement was published on the Oxford University website on the results of one Randomized Evaluation of COVID-19 therapy.
317 More specifically, the study focused on dexamethasone (46), a corticosteroid with fluorine at 9-position (FIG.
8) .
The drug is mostly used as anti-inflammatory, which works by inhibiting vasodilation, reducing leukocyte migration to the inflammation site and increasing vascular permeability.
318 Immunotherapy is an effective intervention in viral infections.
Most attempts at immunotherapy were successful in fighting viruses similar to SARS-CoV-2.
The principal methods include vaccine, neutralizing antibodies (nAbs) candidates and convalescent plasma.
35, 61, 67, 68, 156, 319, 320 In addition, according to the evidences from viral infections (Ebola, Influenza, SARS and MERS), immunotherapeutic interventions can reduce viral load and mortality rate of patients.
321, 322 Development of either monoclonal (mAbs) or polyclonal (pAbs) neutralizing antibodies is a commonly adopted immunotherapeutic alternative due to its specificity, purity, low contamination by blood-transmitted pathogens and relative safety.
However, there are limitations to the use of nAbs once its development and large-scale production for clinical use are a complex, expensive and slow process.
321 Promising scientific investigations have suggested using mAbs or pAbs as prophylactic and therapeutic measures against influenza 323 and HCoVs, such as MERS-CoV 324 and SARS-CoV.
325 Targets reported as promising for HCoVs immunotherapy were cytokine, 326 S1-receptor-binding domain (S1-RBD), S1 N-terminal domain (S1-NTD) and some other region of subunit S2 in order to block the RBDs bonds to their respective receptors and to interfere either with S2-mediated membrane fusion or with the entry in the host cells, thus inhibiting infection.
327 These researchers have encouraged the development of nAbs with cross reactivity potential and/or cross neutralization effect on SARS-CoV-2 infections, as shown by Tian et al.
328 Data suggest that mAb CR3022 can be developed as therapeutic candidate, either isolated or combined with neutralizing antibodies to prevent and treat COVID-19, given that it could potently form bonds with SARS-CoV-2 RBD (KD of 6.3 nM).
A different study by Wang et al.
329 reported the discovery of a human mAb (47D11) that promoted cross neutralization of SARS-CoV and SARS-CoV-2 in a culture of cells through an independent receptor-binding inhibition mechanism that targets a conserved epitope on the spike HCoVs RBD mentioned above.
It is also reported the on-going investigation of convalescent plasma or immunoglobulin as last resource to improve the survival rate of patients with several viral infections such as H 5 N 1 avian influenza, 330 334 A possible explanation for the efficacy of convalescent plasma is that immunoglobulin antibodies in the plasma of recovered patients can suppress viremia.
156 Shen et al.
335 reported that five patients with extreme symptoms of COVID-19 received blood transfusion containing convalescent plasma with specific SARS-CoV-2 antibodies.
After a series of blood transfusions, the improvement in clinical status of patients was observed.
Viral loads also decreased and became negative within 12 days after the transfusion, and SARS-CoV-2-specific ELISA and nAbs titers increased following the transfusion.
In spite of the limited sample, the authors concluded that convalescent plasma transfusion benefited patients infected with SARS-CoV-2.
Therefore, testing safety and efficacy of transfusing convalescent plasma in patients infected with SARS-CoV-2 can be of value.
319, 336 Among the modalities of immunotherapy, vaccines are expected to be more promising, hence the global engagement in their production.
Over the last decade, the scientific community and the vaccine industry had to answer urgently to the epidemics of H1N1, Ebola, Zika and, more recently, SARS-CoV-2.
Vaccine development is an expensive and slow process with high risks of failure, which often motivate developers to follow a linear sequence of steps with several breaks for data analysis and fabrication processes.
Therefore, it is fundamental that vaccines be developed through faithful methods even if it takes longer to move them onto clinical trials or to make a large number of doses available, a challenge during a pandemic.
337 Developing efficient vaccines for SARS-CoV-2 will be essential to reduce the severity of the disease, viral shedding and transmission to control future outbreaks.
Prior to the COVID-19 pandemic, multiple strategies were used to generate vaccines for the first HCoVs (SARS-CoV and MERS-CoV).
338 Several studies related to SARS-CoV vaccine production targeting the protein S, due to its function in the receptor binding and fusion to the host membrane, were successful in animal tests against that coronavirus.
339-341 These vaccines employed live-attenuated virus vaccines, killed virus, DNA vaccines and viral vector vaccines.
Theoretically, these techniques could be applied to develop SARS-CoV-2 vaccines given their similarities from both the genomic perspective and the mechanisms employed in the invasion and infection of host cells.
326 Gao et al.
342 promoted the pilotscale production of a purified inactivated SARS-CoV-2 virus vaccine candidate (PiCoVacc), which induced SARS-CoV-2-specific nAbs in mice, rats and non-human primates.
In addition, three immunizations using two different doses (3 μg or 6 μg per dose) in macaques provided partial or complete protection against the SARS-CoV-2 challenge, respectively, without observable antibodydependent enhancement of infection.
These data reinforce the use of PiCoVacc in the next steps of clinical trials targeting SARS-CoV-2 still for the present year.
Given the magnitude of the COVID-19 pandemic, it has become indispensable to work as fast as possible to develop vaccines for global distribution.
However, protocols are necessary to safekeep the population's health.
Hence, before allowing human testing of COVID-19 vaccines, regulatory organizations must evaluate their safety against a series of virus strains and more than one animal model.
They also must demand preclinical evidences that experimental vaccines prevent infectioneven if it means waiting weeks or months for models to become available.
This is time well-spent, once testing vaccines without investing the due amount of time to completely understand the risks can lead to setbacks for current and future pandemics.
343 repurposing potential relies on the mechanism for other viruses, such as Hepatitis C, by inducing conformational changes that compromise RdRp activity.
It was also possible to identify benzene derivatives used to treat cancer (Dasatinib and Imatinib) and one antiviral (Darunavir), but predominant in-silico and some in-vitro outcomes demand more conclusive studies.
Representative of benzoic derivatives, NTZ displayed significant inhibitory activity against pro-inflammatory cytokines, which can benefit control of ARDS, in spite of an unclear mechanism against SARS-CoV-2.
Quinoline derivatives, HCQ and CQ, were some of the first drugs investigated.
After several in-silico, in-vitro and in-vivo assays, based on results presented by RECOVERY to the present date, unfortunately, these drugs were proven ineffective in hospitalized COVID-19 patients.
Ivermectin represents macrolide derivatives and is suggested as promising due to both immunostimulatory activity and inhibitory activity on nuclear transport when administered at the early stages of the disease.
Among indole derivatives, Arbidol was pointed out as useful for reducing viral binding and releasing of intracellular vacuoles that contain the virus.
Nonetheless, clinical trials are still necessary after dose adjustment for better outcomes.
Both indole derivatives (emetine and HHT), in spite of promising results, were only submitted to in-silico and in-vitro tests, thus demanding further investigation on their toxicity and mechanism of control.
Hence, the currently available data on the classes of drugs investigated here revealed that drugs were considered promising mainly after in-silico tests only.
In fact, the inefficacy of some of these drugs became evident after in-vitro or in-vitro tests, as CQ and HCQ, whose clinical trials failed to confirm the so-expected anti-SARS-CoV-2 activity.
It is necessary to highlight the importance of clinical trials with drugs considered promising in theoretical studies, with due calm and openness to question and refute hypotheses, in the absence of scientific evidence to support their use to treat COVID-19.
Similarly, the careful analysis of practices involving patients with the extreme form of the disease is also important to identify new alternatives, such as the results recently published by RECOVERY on dexamethasone.
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All authors researched data for the article, contributed substantially to discussion of the content, wrote the article and reviewed and edited the manuscript before submission.
key: cord-304056-2bo0s0hz authors: Lezotre, Pierre-Louis title: Part I State of Play and Review of Major Cooperation Initiatives date: 2014-12-31 journal: International Cooperation, Convergence and Harmonization of Pharmaceutical Regulations DOI: 10.1016/b978-0-12-800053-3.00002-1 sha: doc_id: 304056 cord_uid: 2bo0s0hz Abstract The basic principle of international cooperation is to establish bilateral and multilateral efforts to leverage the human, scientific and financial resources and the knowledge and experience of other key regulatory authorities to avoid duplication of efforts, to make activities more efficient and to allow the focussing of limited resources on higher-risk areas of concern.
This increased cooperation between worldwide regulators has necessitated proactive deliberate efforts towards convergence/harmonisation of regulation, practices and requirements to eliminate or reduce differences.
Cooperation and harmonisation of standards in the pharmaceutical domain are already a reality and have become increasingly important during recent decades, with a high level of commitment to these activities by all stakeholders.
The worldwide Drug Regulatory Authorities (DRAs) have been working to end an isolationist attitude that cannot resolve current worldwide issues and challenges caused by an ever increasing globalisation.
As a result, many cooperation and harmonisation initiatives have been established at the bilateral, regional and global levels as a response to the changing geo-economic-political situation.
The spectrum of collaboration varies from simple informal technical cooperation to full integration of systems and regulations.
Indeed, all these initiatives can be very different in scope (some are part of a broader harmonisation initiative), level of harmonisation (depending on the political support/commitment), organisation (well-structured versus simple discussion) or advancement (established process vs. pilot projects), but they all work towards convergence of requirements and/or practices.
All these multiple worldwide cooperation and harmonisation programmes have evolved rapidly over the past decades.
This book section provides the current status of this complex and broad phenomenon of cooperation, convergence and harmonisation in the pharmaceutical sector.
It reviews all major global, regional and bilateral cooperation initiatives.
Many aspects of increased globalization also have profound implications on pharmaceutical regulation worldwide.
In general, globalization of the economy (with increased travel of people and exchange of goods, finance, and information), and also globalization of the pharmaceutical market (including development, manufacture, and distribution activities), requires increased cooperation and harmonization of pharmaceutical standards and regulation.
Pharmaceutical industries have asked for better harmonization of requirements for the development and manufacture of pharmaceutical products to avoid duplication of work that ultimately creates delays in drug availability [23] .
In this context, harmonization of pharmaceutical regulations has naturally become an important topic of discussion among worldwide Drug Regulatory Authorities (DRAs).
Over the past several decades, they have been working to end an isolationist attitude that cannot resolve current worldwide issues and challenges.
As a result, many cooperative initiatives (bilateral, regional, and global) were established, and harmonization efforts have been enhanced.
All these initiatives can be very different in scope (some are part of a broader harmonization initiative), level of harmonization (depending on the political support/commitment), organization (well structured versus simple discussion), or advancement (established process versus pilot projects), but they all work towards harmonization of requirements and/or practices.
Increased exchange of information on a regular basis (e.g., more than 26 countries and international organizations from Australia to Vietnam now have agreements to share information with the United States Food and Drug Administration [US FDA]) [24] also contributes to the natural convergence of requirements and practices.
Harmonization models can be distinguished by their scope and objectives.
Indeed, the spectrum of collaborations varies from simple technical cooperation to full integration of systems and regulations: ▸ Integration model: In this type of agreement, most of the time driven by political decision, deeper harmonization of regulation is achieved with the creation of supranational central authorities in order to support integration and/or creation of a single market (e.g., EU, the Association of Southeast Asian Nations [ASEAN] ).
In this case, harmonization of standards and regulations is critical in reducing trade barriers.
In this model, countries give up some of their autonomy on certain matters by transferring the power to make decisions to the common supranational authority or by automatically recognizing decisions from the other party (via mutual agreement mechanisms).
The African Medicines Registration Harmonization (AMRH) initiative has defined five identifiable levels of harmonization ( Figure 1 ).
To facilitate cooperation, a Mutual Recognition Agreement (or Arrangement) (MRA) can be signed by one or more parties to mutually recognize or accept some or all aspects of one another's requirements.
They can be concluded at the technical level (e.g., the Status and Future Plans," November 2009. confidentiality arrangements between the US FDA and European Medicines Agency [EMA] , or the MRA between EU and Australia) or at the government level (e.g., European Treaty).
These multilateral initiatives are major projects as they involve multiple organizations and countries and represent the highest degree of harmonization.
The objective of this technical and scientific intergovernmental cooperation is to globally discuss scientific issues that support the decisions made by individual governments and international regulatory bodies in order to achieve global scientific consensus.
The goal is to facilitate the development of new medicines and to make them available to the maximum number of people worldwide.
There is no intent of full integration of systems and regulations.
The main difficulty faced by these initiatives is the complexity and management of the structure due to the important number of participants (e.g., the World Health Organization [WHO] has 194 Member States) and the diversity of needs, challenges, and level of development of its members.
The World Health Organization (WHO) was established in 1948 as a specialized agency of the United Nations (UN) [25] .
It is accountable to its Member States and works closely with other entities of the UN system.
This agency has a very broad scope of responsibilities as it is the directing and coordinating authority for international health matters and public health within the UN system.
WHO is well known for some of its work (e.g., the coordination of influenza surveillance and monitoring activities, emergency assistance to people affected by disasters, mass immunization campaigns or actions against Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome [HIV/AIDS], tuberculosis, and malaria).
However, WHO undertakes many more activities because it is responsible for providing leadership on global health matters, shaping the health research agenda, setting norms and standards, articulating evidence-based policy options, providing technical support to countries, and monitoring and assessing health trends.
Most of these core functions, as further defined in its "11th General Programme of Work," [26] rely on cooperation and harmonization of standards.
This focus on regional and global collaboration, and especially aid from developed countries to developing countries, is aligned with the UN Millennium Development Goals (MDGs).
a a The United Nations Millennium Development Goals (MDGs) are eight international goals that UN Member States (and international organizations) have agreed to achieve by the year 2015.
They are derived from the United Nations Millennium Declaration, signed in September 2000, which endorsed a framework for development and commits world leaders to combat poverty, hunger, disease, illiteracy, environmental degradation, and discrimination against women.
These MDGs are interdependent and several relate either directly or indirectly to health.
WHO is therefore very involved in this process and works with countries to achieve the health-related MDGs.
Indeed, the objective of these MDGs is that countries and development partners work together to improve the global situation and resolve major issues.
A number of specific targets and indicators have been identified to monitor progress towards the goals.
Goal 8 ("Develop a global partnership for development") specifically recognizes the role of developed nations and addresses global cooperation and partnerships.
WHO has worked in the area of pharmaceuticals since its creation approximately 60 years ago.
During this time, many products and services have been created that are widely recognized as core functions of WHO.
The role of WHO in pharmaceutical regulations is based on its constitutional mandate and various World Health Assembly (WHA) resolutions.
This support is twofold.
One aspect relates to the development of internationally recognized norms, standards, and guidelines.
The second relates to providing guidance, technical assistance, and training in order to enable countries to implement global guidelines to meet their specific medicines regulatory environment and needs [27] .
All countries that are members of the UN may become members of WHO by accepting its constitution.
Other countries may be admitted as members when their application has been approved by a simple majority vote of the World Health Assembly (WHA).
Territories that are not responsible for the conduct of their international relations may be admitted as associate members upon application made on their behalf by the member or other authority responsible for their international relations.
Members of WHO are grouped according to regional distribution.
WHO's strength lies in its neutral status and nearly universal membership.
Today, it represents 194 countries and two associate members (Puerto Rico and Tokelau).
One country is an observer (Vatican) [28, 29] .
The organization is headed by the Director-General, b but the WHA is the supreme decisionmaking body for WHO.
It generally meets in Geneva, Switzerland in May of each year, and is attended by delegations from all Member States.
Its main function is to determine the policies of the organization.
The Health Assembly also appoints the Director-General (on the nomination of the Executive Board), supervises the financial policies of the organization, and reviews and approves the proposed budget.
The work of the Assembly is supported by the Executive Board, which it elects.
This executive arm of the Assembly is composed of 34 members technically qualified in the health field.
Members are elected for three-year terms.
The main board meeting, at which the agenda for the forthcoming Health Assembly is agreed upon and resolutions for forwarding to the Health Assembly are adopted, is held in January.
A second shorter meeting in May, immediately after the Health Assembly, is held to address more administrative matters.
The primary functions of the Board are to give effect to the decisions and policies of the Health Assembly, to advise it, and generally to facilitate its work.
Under the leadership of the Director-General, c more than 8,000 people from more than 150 countries work for WHO.
This WHO staff includes health professionals (including medical doctors, public health specialists, epidemiologists, and scientists) as well as managers, economists, administrators, and other professionals.
They are located in country offices, six regional offices, and at the headquarters in Geneva, Switzerland [30] .
One of the unique aspects of WHO is its decentralized structure.
WHO's work is a great combination of actions at the country, regional, and global levels.
These efforts to decentralize its structure are aimed at getting closer to the ground (field) where decisions made can be more responsive to actual needs.
Indeed, this decentralized and regionalized structure provides WHO with multiple opportunities for engaging with countries.
WHO's global headquarters is located in Geneva, Switzerland.
The team based at the global headquarters supports and builds on all of the regional and local efforts.
It sets global policies and standards, facilitates technical support to regions and countries, monitors and publicizes progress, and helps mobilize political and financial support.
At the WHO headquarters, medicine activities are conducted within the cluster of Health Systems and Services (HSS) and are coordinated by the Department of Essential Medicines and Health Products (EMP).
This department (which employs about 100 staff members [31] ) is involved in the harmonization of pharmaceutical regulations because it coordinates various activities in the areas of quality assurance (e.g., the International Pharmacopoeia, International Nonproprietary Names [INN] , prequalification of medicines, counterfeit medicines), regulation and legislation (e.g., International Conference of Drug Regulatory Authorities [ICDRAs]), and safety and efficacy (e.g., drug alerts).
These activities comprise guideline development, workshops, and training courses, coordination and promotion of pharmacovigilance for global medicine safety, regulatory and other information exchange, and review of narcotic and psychotropic substances.
WHO Member States are grouped into six regions, each of them having a regional office: ▸ WHO Regional Office for Africa in Brazzaville, Republic of Congo.
▸ WHO Regional Office for Europe in Copenhagen, Denmark.
▸ WHO Regional Office for Southeast Asia in New Delhi, India.
▸ WHO Regional Office for the Americas/Pan American Health Organization (PAHO) in Washington DC, United States.
▸ WHO Regional Office for the Eastern Mediterranean in Cairo, Egypt.
▸ WHO Regional Office for the Western Pacific in Manila, The Philippines.
Each of WHO's regional offices are the first point of contact for country offices that need extra technical or financial help.
These regional offices also give special attention to adapting global policies to fit specific needs in their regions.
Indeed, the regional level is important in the WHO organization as it links the global strategy and plan with the country's reality and needs.
They play a key role in the implementation of WHO norms and standards by ensuring that: ▸ Country and regional needs are taken into consideration when WHO norms and standards are developed ▸ Global guidelines and internationally recognized norms and standards are appropriately implemented in their regions (in the context of their own specific regulatory environment and challenges) by providing guidance, technical assistance, and training In addition to global activities coordinated from WHO headquarters, WHO regional and country offices can also carry out a variety of medicine-related activities specific to their regions.
In addition to the regional and headquarters offices, WHO has 145 country offices that cover 159 Member States.
d There are also two field offices (the WHO Humanitarian Assistance Office in Pristina, Kosovo and the West Bank and Gaza Office) and offices covering two different areas, the US-Mexican border field office in El Paso, Texas (US), and the Office of Caribbean Program Coordination in Barbados.
WHO has also established "WHO liaison offices" in key locations (e.g., at the European Union in Brussels, Belgium, at the African Union and the Economic Commission for Africa in Addis Ababa, Ethiopia, in Washington DC, US, and at the UN in New York City) and more than 10 "technical offices" (e.g., the European Observatory on Health Systems and Policies in Berlin, Germany) [32] .
d Some countries that do not have a physical WHO country office are served by the WHO Representative of another country (for instance, the WHO Representative to Malaysia covers not just Malaysia, but also Brunei, Darussalam, and Singapore) .
Approximately 40% of WHO country offices are either owned or supported by the government and ministries of health.
Some of these WHO country offices are located in independent premises either rented or owned by WHO, while others are located within ministries of health or UN common premises.
These country offices are led by the Head of WHO Office (HWO), who are designated by the Director-General and by the respective regional Directors.
The HWO manages WHO core functions at the country level and provides leadership in the following key functional areas: ▸ Advocacy, partnership, and representation ▸ Support for policy development and technical cooperation ▸ Administration and management It is important to note that WHO is focused on needs of countries and emphasizes in particular the decentralization process that is aimed at increasing WHO's impact on health and development at the country level.
This country focus tailors WHO's technical collaboration to the needs and capacities of each Member State, with a special emphasis on the poorest countries and most fragile contexts.
The key principles guiding WHO cooperation in countries are [33] : ▸ Ownership of the development process and projects by the country ▸ Alignment with national priorities and strengthening national systems ▸ Harmonization with the work of sister UN Agencies and other partners in the country towards better aid effectiveness ▸ Collaboration as a two-way process that fosters Member States' contributions to the global health agenda WHO's country presence is the platform for effective cooperation with countries for advancing the global agenda, contributing to national health strategies and planning, and bringing country realities and perspectives into global policies and priorities.
According to the above principles and its structure, WHO is indeed able to focus on countries' needs and better define its priorities to actively support the development, implementation, monitoring, and assessment of national health policies, strategies, and plans.
But it also allows for better monitoring implementation of global agreements such as the Millennium Development Goals (MDGs) and the International Health Regulations (IHR [2005] ).
These activities in countries are governed by the Country Cooperation Strategy (CCS), which is WHO's key instrument to guide its work in countries.
It is a medium-term vision (generally covering four to six years) for its technical cooperation with a given Member State, in support of the country's national health policy, strategy, or plan.
It is an organization-wide reference that guides partnership, planning, budgeting, and resource allocation.
WHO also established the Department of Country Focus (CCO) to support and advocate for WHO country offices, develop the capacity of WHO country teams for effective engagement in partnership platforms, and facilitate and monitor WHO's engagement in the aid effectiveness agenda at the country level.
For example, CCO provides support for the development, dissemination, and use of the Country Cooperation Strategy.
▸ Expert Committees: Expert committees have an important role in WHO activities.
They are defined in the WHO constitution.
e In addition to the constitution, regulations for expert advisory panels and committees are also included in the WHO document entitled "Regulations for Expert Advisory Panels and Committees."
f An expert committee is the highest official advisory body to the Director-General of WHO as well as to all the organization's Member States.
It is established by the WHA or by an Executive Board decision.
There are various types of WHO expert committees.
For example, the WHO Expert Committee on Specifications for Pharmaceutical Preparations (ECSPP) has been providing, for more than 60 years, recommendations and tools to assure the quality of medicines from their development phase to their final distribution to patients.
There is also the Expert Committee on Biological Standardization (ECBS), which is as old as the ECSPP.
In addition to its structured organization, the WHO has been supported since its creation by its "collaborating centers."
These are institutions such as research institutes and parts of universities or academies that are designated by the Director-General to carry out activities in support of WHO programs.
Currently there are over 800 WHO collaborating centers in over 80 Member States working with WHO in several areas (one of them being "Pharmaceuticals").
Several collaborating centers may exist for the same topic (e.g., international classifications or traditional medicines) and form a specific network to help WHO regarding this specific topic.
of Causes of Death.
WHO also started to publish its Bulletin, which is today an international peer-reviewed monthly journal of public health with a special focus on developing countries.
j In its early years, WHO's priority was the prevention and control of specific diseases (e.g., malaria, tuberculosis, smallpox, yaws, onchocerciasis, and venereal disease), some of which are still a problem today.
They also focused on women's and children's health and nutrition, and environmental sanitation.
WHO's work has since grown to cover other (sometimes new) health problems (including polio, HIV/AIDS, and Severe Acute Respiratory Syndrome [SARS] ), but it also works to control tobacco and alcohol use and to promote diet and physical activity to prevent the four main noncommunicable diseases (cardiovascular disease, cancer, chronic lung diseases, and diabetes) [36] .
WHO has also been increasingly involved in the global regulation and control of medicines.
In 1977, the first essential medicines list was released two years after the WHA introduced the concepts of "essential drugs" and "national drug policy."
One hundred and fifty-six countries today have a national list of essential medicines.
WHO has also funded many projects over the years to facilitate global cooperation and harmonization of standards.
The purpose of all these activities in the pharmaceutical domain is aimed at increasing global and equitable access to safe, effective medicines of assured quality.
This specific goal is derived from the overall objective of WHO to improve and maintain global public health.
This objective has been regularly reiterated in several WHA resolutions and during other events such as the ICDRAs.
In 1978, the International Conference on Primary Health Care (Alma-Ata, Kazakhstan) set the historic goal of "Health for All," to which WHO continues to aspire.
More recently, the UN MDGs have further clarified the objectives and priorities of global cooperation derived from the UN Millennium Declaration signed in September 2000.
One of WHO's mandates is "to develop, establish and promote international standards with respect to food, biological, pharmaceutical and similar products" [37] .
WHO Member States (especially developing countries) rely on WHO for expertise and guidance in regulation, safety, and quality assurance of medicines through development and promotion of international norms, standards, guidelines, and nomenclature.
To achieve this goal, WHO relies on cooperation and uses its decentralized organization to facilitate implementation of projects and agreed-upon standards.
The harmonization activities are initiated according to the WHO's Medicines Strategy.
Trigger actions to initiate a new project or development of a standard are given at different levels and bodies (i.e., the WHA, Executive Board Resolutions, ICDRAs, or WHO programs and j Since it was first published in 1948, the Bulletin has become one of the world's leading public health journals.
As the flagship periodical of WHO, the Bulletin draws on both WHO experts (as editorial advisors, reviewers, and authors) and external collaborators.
clusters).
These projects and standards are then developed through a vast global consultation process involving WHO Member States, national and regional authorities, international agencies, and with specialists from industry, national institutions, nongovernmental organizations, etc.
Project updates and approved standards become publically available through the extensive list of WHO publications to support national, regional, and global health strategies.
k Because the global dissemination and exchange of information is important, WHO secures the broad international distribution of its publications and encourages their translation.
l This ensures the widest possible availability of authoritative information and guidance on health matters.
The Department of EMP, based at the WHO global headquarters in Geneva, works closely with expert committees, other regulators, and relevant WHO collaborating centers to develop and implement these harmonization activities.
This department coordinates these activities globally with the support of WHO's regional advisors and country project staff in each of the regional offices and many country offices.
Each of the regional offices has two to five professionals coordinating the Medicines Strategy, and 40 WHO country offices have full-time pharmaceutical policy experts [38] .
It is worth mentioning that in addition to its normative activities and harmonization projects, WHO also assists countries in capacity building by assessing regulatory systems.
It does this by facilitating cooperation and information exchange between countries and by providing technical support.
It is very important to involve all countries (whatever their development level), and to facilitate the implementation of norms and standards.
Finally, WHO has developed relationships with a lot of nongovernmental and civil society organizations on a global basis via the Civil Society Initiative (CSI) , and also at regional and national levels.
The objectives of WHO's relations with nongovernmental organizations (NGOs) are to promote the policies, strategies, and activities of WHO to facilitate their implementation.
WHO has a large repertoire of global normative work relevant for all levels of development.
In the area of medicines, a lot of standards, norms, and classifications have been developed, and forums/networks have been created to enhance global cooperation.
Important initiatives are presented below.
k WHO publishes practical manuals, handbooks, and training material; internationally applicable guidelines and standards; reviews and analyses of health policies, programs, and research; and state-of-the-art consensus reports that offer technical advice and recommendations for decision makers.
Also, the WHO Technical Report Series makes available the findings of various international groups of experts that provide WHO with the latest scientific and technical advice on a broad range of medical and public health subjects.
l In 1978, the World Health Assembly turned multilingualism into a WHO policy by establishing six official languages (Arabic, Chinese, English, French, Russian, and Spanish) .
Since the adoption of a 1998 resolution, all governing bodies' documents and corporate materials have been made available online in all official languages.
The International Conference of Drug Regulatory Authorities (ICDRAs) provides drug regulatory authorities of WHO Member States with a forum to meet and discuss ways to strengthen collaboration and harmonization of pharmaceutical regulations.
This is a key accomplishment of WHO that has been instrumental in guiding DRAs, WHO, and interested stakeholders to develop national, regional, and international medicines regulation, and it continues to be a cornerstone of international harmonization of medicines regulation.
These conferences have been held since 1980, and they have involved both developed and developing countries.
The 14th ICDRAs, held in Singapore from November 30 to December 3, 2010, involved 345 participants from over 90 agencies.
The 15th ICDRAs, which took place in Tallinn, Estonia from October 23 to 26, 2012, was attended by over 300 participants from 100 countries.
The aim of these conferences is to promote the exchange of information and collaborative approaches to issues of common concern.
Topics discussed include quality issues, herbal medicines, homeopathy, regulatory reform, medicine safety, counterfeiting, regulation of clinical trials, harmonization, new technologies, and e-commerce.
Recommendations are proposed for actions to take among agencies, WHO, and related institutions.
It is worth mentioning that the idea to create ICH began to formulate after background discussions between the US, the European Union (EU), and Japan during the 5th ICDRAs conference in Paris, France in 1989 [39] .
As a platform was established to develop international consensus, the ICDRAs continues to be an important tool for WHO and DRAs in their efforts to harmonize regulation and improve the safety, efficacy, and quality of medicines on a worldwide basis.
The WHO constitution mandates the production of international classifications on health.
These internationally endorsed classifications, developed through the WHO network m are very important as they facilitate the storage, retrieval, analysis, interpretation, and comparison of data.
They support global cooperation and harmonization by providing a consensual framework that governments, healthcare providers, and consumers can use as a common language.
They also permit the comparison of data not only within populations over time, but also between populations.
WHO reference classifications are the International Classification of Diseases (ICD), the International Classification of Functioning, Disability and Health (ICF), and the International Classification of Health Interventions (ICHI).
In addition, related and derived classifications (based on the reference classifications) have also been developed (e.g., the Anatomical Therapeutic Chemical Classification with Defined Daily Doses (ATC/DDD) that classifies m WHO has designated a number of collaborating centers to work with it in the development, dissemination, maintenance, and use of the WHO International Classifications.
therapeutic drugs according to the organ/system on which they act, and their chemical, pharmacological, and therapeutic properties).
The WHO International Clinical Trials Registry Platform (ICTRP) is a global initiative that aims to make information about all worldwide clinical trials involving humans publicly available.
This activity was launched during the 58th WHA in 2005 n following discussions and recommendations from a Ministerial Summit on Health Research in Mexico City, Mexico in November 2004.
The ICTRP is not itself a clinical trials registry, but a central repository that can be searched using the WHO search portal (http://apps.who.int/trialsearch/).
All items in the trials registration data set are copied from individual registries onto the WHO central repository, and data is updated regularly.
Indeed, details on clinical trials come directly from one of the primary registries o in the WHO Registry Network (e.g., the European Clinical Trials Register that became a member of the Network in September 2011 p ).
By consolidating clinical trials information from several worldwide sources using standardized data set format/criteria, and by implementing unambiguous identification (i.e., a Universal Trial Number [UTN] ), the ICTRP not only facilitates the exchange of information, but also promotes harmonization of this information.
Harmonization is also further achieved because WHO proactively supports countries/regions in establishing WHO-compliant clinical trials registries or policies on trial registration.
Quality assurance is a wide-ranging concept covering all matters that individually or collectively influence the quality of a product.
This is a major public health challenge, particularly in light of growing cross-border health issues and the growing international dimensions of trade.
The quality of pharmaceuticals has been a concern of WHO since its inception.
The development of norms, standards, and guidelines to promote quality assurance is an integral part of WHO's constitution, and has been endorsed and supported through numerous WHA resolutions.
More recently, the WHO Medium-Term Strategic Plan for 2008-2013 requested that the organization develop international standards, recommendations, and instruments to assure the quality of medicines, whether produced and traded nationally or internationally.
n Resolution WHA 58.34 called on the global scientific community, international partners, the private sector, civil society, and other relevant stakeholders to "establish a voluntary platform to link clinical trials registers in order to ensure a single point of access and the unambiguous identification of trials with a view to enhancing access to information by patients, families, patient groups and others."
o A Primary Registry in the WHO Registry Network is a clinical trial registry with at least a national remit that meets WHO Registry criteria for content, quality and validity, accessibility, unique identification, technical capacity, and governance and administration.
p The European Clinical Trials Register provides public access to information extracted from the EU clinical trial database ("EudraCT").
The WHO Medicines Quality Assurance Program, which is part of the EMP Department, produces norms, standards, and guidelines on the quality assurance of pharmaceuticals.
These regulatory tools are prepared through a vast global consultative process, and are ultimately approved by the WHO ECSPP, q which meets annually.
The report of each meeting (Technical Report Series) includes newly adopted guidelines in its annexes.
When adopted, the norms, standards, and guidelines become international harmonized standards intended for use by national DRAs, manufacturers, and other interested parties.
Many important international standards and projects have been developed in this area: ▸ Good manufacturing practice (GMP) ▸ Guidelines for regulatory approval (e.g., the guidelines on stability testing or on registration requirements to establish the interchangeability of multisource generic pharmaceutical products and the proposal to waive in vivo bioequivalence requirements) ▸ Prequalification of medicines, laboratories, and supply agencies ▸ Model certificates for quality assurance-related activities ▸ Quality control testing ▸ New specifications for inclusion in the Basic Tests Series and the International Pharmacopoeia ▸ International Chemical Reference Substances (ICRS) r ▸ The INN program Some of these international guidelines and projects are further developed below.
▸ Good Manufacturing Practice: Good Manufacturing Practice (GMP) is the part of quality assurance that ensures products are consistently produced and controlled to the quality standards appropriate to their intended use and as required by the marketing authorization.
GMP is aimed primarily at diminishing the risks involved in any pharmaceutical production that cannot be eliminated through testing of the final product.
s GMP covers all aspects of production: from the starting materials, premises, and equipment, to the training and personal hygiene of staff.
Detailed, written procedures are essential for each process that could affect the quality of the finished product.
Panel on the International Pharmacopoeia and Pharmaceutical Preparations.
r ICRS are used by laboratories to test pharmaceuticals for the purpose of quality control.
These substances are mainly used for validating the results from specific tests, and as primary standards for calibrating secondary standards.
WHO's collection of ICRS is now maintained by the Council of Europe's European Directorate for Quality of Medicines and HealthCare (EDQM) , which also distributes the substances worldwide.
EDQM is responsible for obtaining candidate material, testing it to ensure its purity and suitability, and reporting results with recommendations to WHO.
s The main risks are the following: unexpected contamination of products causing damage to health or even death; incorrect labels on containers, which could mean that patients receive the wrong medicine; and insufficient or too much active ingredient resulting in ineffective treatment or adverse effects.
Recognizing the importance of GMP in international commerce of pharmaceutical products, WHO developed requirements early on.
The first WHO draft text on GMP was prepared in 1967 by a group of consultants at the request of the 20th WHA [40] .
It was subsequently submitted to the 21st WHA under the title "Draft Requirements for Good Manufacturing Practice in the Manufacture and Quality Control of Medicines and Pharmaceutical Specialties" and was accepted.
In 1968, the revised text was discussed by the WHO ECSPP and published as an annex to its 22nd report.
The text was then reproduced, with some revisions, in 1971 in the Supplement to the 2nd edition of the International Pharmacopoeia (Ph.
Int.).
Since then, WHO has further defined its general principles and requirements regarding GMP [41] , and it has also established several detailed guidelines covering specific needs for GMP of active pharmaceutical ingredients [42] , pharmaceutical excipients [43] , sterile pharmaceutical products [44] , biological products [45] , blood establishments [46] , pharmaceutical products containing hazardous substances [47] , investigational pharmaceutical products for clinical trials in humans [48] , herbal medicinal products [49] , radiopharmaceutical products [50] , and water for pharmaceutical use [51] .
Finally, it also developed guidelines of a more general scope such as validation [52] , risk analysis [53] , technology transfer [54] , and inspection [55] , and has created appropriate training materials for countries.
Many countries have formulated their own requirements for GMP based on the WHO GMP.
The International Pharmacopoeia (Ph.
Int.)
comprises a collection of quality specifications for pharmaceutical substances (i.e., active ingredients and excipients) and dosage forms together with supporting general methods of analysis.
It is intended to serve as source material for reference or adaptation by any WHO Member State.
Clearly defined steps are followed in the development of new monographs.
The Ph.
Int.
is published by WHO with the goal of achieving a wide global harmonization of quality specifications for selected pharmaceutical products, excipients, and dosage forms.
The Ph.
Int., or any part of it, has legal status whenever a national or regional authority expressly introduces it into appropriate legislation.
The history of the Ph.
Int.
dates back to 1874 when the need to standardize terminology and to specify dosages and composition of drugs led to attempts to produce an international pharmacopoeia compendium.
The first conference, called by the Belgian Government and held in Brussels in 1902, resulted in an agreement for the unification of the formulae of potent drugs, which was ratified in 1906 by 19 countries.
The outcome considerably influenced the subsequent publication of national pharmacopoeias.
In 1947, the Interim Commission of the WHO took over the work on pharmacopoeias previously undertaken by the Health Organization of the League of Nations.
The 3rd WHA, held in May 1950, formally approved the publication of the "Pharmacopoea Internationalis" and recommended, in accordance with Article 23 of the WHO Constitution, "the eventual inclusion of its provisions by the authorities responsible for the pharmacopoeias."
It was thus recommended that the "Pharmacopoea Internationalis" not be used as a legal pharmacopoeia in any country unless adopted by the pharmacopoeial authority of that country.
This first edition, published with the aim of creating a worldwide, unified pharmacopoeia, relied on collaboration with national pharmacopoeia commissions for its preparation.
In 1975, the purpose of the Ph.
Int.
was reconsidered.
It was decided that the publication should focus more on the needs of developing countries (because developed countries had established their own pharmacopoeias), and recommended only simple, classical chemical techniques that had been shown to be sound.
Since 1979, the drugs appearing in the Ph.
Int.
have therefore been selected from the list of essential drugs based on the first report of the WHO Expert Committee on the Selection of Essential Drugs.
Also, whenever possible, classical procedures are used in the analytical methods so that the use of expensive equipment is minimized in the application of the Ph.
Int.
to facilitate its implementation by developing countries.
The work on the Ph.
Int.
is carried out by the WHO ECSPP in collaboration with members of the WHO Expert Advisory Panel on the International Pharmacopoeia and Pharmaceutical Preparations and other specialists [56] .
The process involves consultation with, and input from, WHO Member States and DRAs, WHO collaborating centers and national drug quality control laboratories in all six WHO regions, standard-setting organizations and parties including regional and national pharmacopoeias, and manufacturers around the world.
In 1950, The WHA adopted a resolution [57] to create the International Nonproprietary Names (INN) Program in order to identify pharmaceutical substances unambiguously on a worldwide basis, and to provide a universal, unique, nonproprietary name to be used in Pharmacopoeia monographs.
It began operating in 1953 when the first list of INNs for pharmaceutical substances was published.
Today, this program is coordinated by the WHO EMP department.
The selection of a new INN relies on a strict procedure [58, 59] .
This process is supported by the Expert Advisory Panel on the International Pharmacopoeia and Pharmaceutical Preparations, which provides advice on proposed names following an application made by the manufacturer or inventor.
The procedure also involves the WHO Secretariat, which examines the suggested names for conformity with the general rules, similarities with published INNs, and potential conflicts with existing names.
After a time period for objections has lapsed, the name will obtain the status of a recommended INN and will be published as such in "WHO Drug Information" if no objection has been raised.
To make INNs universally available, they are formally placed by WHO in the public domain, hence their designation as "nonproprietary" names (also known as "generic names").
The existence of this international nomenclature for pharmaceutical substances is important for the clear identification, safe prescription, and dispensing of medicines to patients, but also for communication and exchange of information among health professionals and scientists and regulators worldwide.
It provides them with a unique and universally available designated name to identify each pharmaceutical substance.
Today, INN names are widely used and globally recognized.
At present, more than 8,000 INNs have been published, and this number is growing every year.
The majority of pharmaceutical substances used in medical practice are designated by an INN, and their use is already common in research and clinical documentation.
Nonproprietary names are intended for use in pharmacopoeias, labeling, product information, advertising and other promotional material, drug regulation and scientific literature, and as a basis for product names (e.g., for generics).
Also INN collaborates closely with numerous national drug nomenclature bodies.
The use of INN names is normally required by national authorities and also by the European Community.
As a result of ongoing collaboration, national names such as British Approved Names (BAN), Dénominations Communes Françaises (DCF), Japanese Adopted Names (JAN), and United States Adopted Names (USAN) are nowadays, with rare exceptions, identical to the INN.
In addition to the quality standards, WHO also developed norms and standards for pharmacovigilance, and promotes information exchange on medicine safety.
The aim is to assure the safety of medicines by ensuring reliable and timely exchange of information on drug safety issues, promoting pharmacovigilance activities on an international basis, and encouraging participation in the WHO Program for International Drug Monitoring [60].
In 1968, WHO established its Program for International Drug Monitoring in response to the thalidomide disaster in 1961.
At the end of 2010, 134 countries were part of the WHO Pharmacovigilance Program.
An international system for monitoring adverse drug reactions (ADRs) using information derived from Member States was established in 1971.
This allows WHO to issue a rapid Drug Alert whenever a serious problem in the safety of any medicinal product arises.
WHO headquarters in Geneva is responsible for policy issues, while the operational responsibility for the program rests with the WHO Collaborating Centre for International Drug Monitoring, Uppsala Monitoring Centre in Sweden.
A common reporting form was developed, agreedupon guidelines for entering information were formulated, common terminologies and classifications were prepared, and compatible systems for transmitting, storing and retrieving, and disseminating data were created.
The ADRs database in Uppsala currently contains over three million reports of suspected ADRs.
In 2003, a WHO Advisory Committee on Safety of Medicinal Products (ACSoMP) was established to guide WHO on general and specific issues related to pharmacovigilance.
Additionally, a network of "information officers" was established in 1980 to allow a direct relationship between WHO and all national DRAs in Member States.
Each national information officer is charged with providing information to WHO on the safety and efficacy of pharmaceutical preparations, and with securing prompt transmission to national health authorities regarding new information on serious adverse effects.
This certification scheme was initially adopted by the 22nd WHA in 1969 [61], but since then it has been amended.
It is an administrative instrument that requires each participating Member State, upon application by a commercially interested party, to attest to the competent authority of another participating Member State whereby: ▸ A specific product is authorized for placement on the market within its jurisdiction, or if it is not authorized, the reason why that authorization has not been accorded.
▸ The manufacturing plant in which it is produced is subject to inspections at suitable intervals to establish that the manufacturer conforms to GMP as recommended by WHO.
▸ All submitted product information, including labeling, is currently authorized in the certifying country.
The primary document delivered under this scheme is the Certificate of Pharmaceutical Product (CPP), but two other documents can be requested within the scope of the scheme.
The first is a statement of licensing status of pharmaceutical product(s), and the second is a batch certificate of a pharmaceutical product (this document is rarely applied other than to vaccines, sera, and biologicals).
These documents are used by DRAs of importing countries in their decision to approve, renew, extend, or vary a license.
WHO created models for these confidential documents and listed the information that such certificates need to include.
Obligations that certifying authorities need to fulfill in order to be able to deliver a certificate have also been defined [62]: ▸ Possess an effective national licensing system, not only for pharmaceutical products, but also for responsible manufacturers and distributors.
▸ Have GMP requirements, in agreement with those recommended by WHO, to which all manufacturers of finished pharmaceutical products are required to conform.
▸ Effective controls must be in place to monitor the quality of pharmaceutical products registered or manufactured within its country, including access to an independent quality control laboratory.
▸ Have a national pharmaceuticals inspectorate, operating as an arm of the national DRA, and having the technical competence, experience, and resources to assess whether GMP and other controls are being effectively implemented, and the legal power to conduct appropriate investigations to ensure that manufacturers conform to these requirements by, for example, examining premises and records and taking samples.
▸ Support administrative capacity to issue the required certificates, to institute inquiries in the case of complaint, and to notify expeditiously both WHO and the competent authority in any Member State known to have imported a specific product that is subsequently associated with a potentially serious quality defect or other hazard.
GMP standards provide the basis for the WHO Certification Scheme that relies on the capacity, experience, and expertise of the certifying authority of the exporting country.
This scheme is a great example of cooperation between countries and is an important tool to support a regulatory system in developing countries that do not have enough capacity, resources, or expertise.
Biological medicinal products, such as vaccines, blood and blood products, diagnostics, gene therapy, biotechnology products, cytokines and growth factors, and cell and tissue products, rely heavily on international standardization to ensure their quality and their equivalence across manufacturers.
This is especially true due to the increasing globalization in the production and distribution of these biological medicines.
Over the past 50 years, WHO has worked to standardize these biological materials by establishing international biological reference materials t as well as developing international guidelines and recommendations on the production and control of biological products and technologies.
Guidelines provide more general information on a range of topics of interest to national DRAs and manufacturers (e.g., "Guidelines on Evaluation of Similar Biotherapeutic Products, SBPs"), whereas recommendations establish the technical specifications for manufacturing and quality control of specific products (e.g., "Recommendations to Assure the Quality, Safety and Efficacy of BCG Vaccines").
WHO has also released many other documents on general topics (such as "Regulation and Licensing of Biological Products in Countries with Newly Developing Regulatory Authorities" [63] and "Good Manufacturing Practices for Biological Products" [64]) or on a specific type of product (e.g., blood products and related biologicals, cells and tissues, cytokines, or vaccines) to facilitate control of these biological products on a worldwide basis.
These norms and standards have been developed to assist WHO Member States in ensuring the quality and safety of biological medicines and related in vitro biological diagnostic tests worldwide.
By adopting these guidance documents in their pharmacopoeias or equivalent legislation, each country ensures that the products produced and used in their country conform to current international standards.
By advising national DRAs and manufacturers on the control of biological products, regulatory guidance documents also establish a harmonized regulatory framework for products in international markets.
WHO accomplishes its biological program through the WHO collaborating centers and the WHO ECBS.
Members of the ECBS are scientists from national control agencies, academia, research institutes, public health bodies, and the pharmaceutical industry acting as individual experts and not as representatives of their respective organizations or employers.
Its work is based on scientific consensus achieved through this international consultation and collaboration.
This committee, which directly reports to the Executive Board, has met on an annual Additionally, WHO has been particularly active in the specific field of blood products and related biologicals.
It has provided technical guidance and quality assurance tools to DRAs, National Control Laboratories, and manufacturers to support implementation of quality and safety systems for the production and control of blood products and related in vitro diagnostic devices worldwide.
Indeed, many countries have significant difficulties in fulfilling their responsibilities in this field because processing blood (with inherent variability due to the nature of the source materials) is a highly specialized process that requires a high degree of expertise.
This development of WHO International Reference Materials and Guidelines supports the technical capacity of national DRAs and assures the compliance of manufacturers to quality and safety measures globally in order to prevent transmission of diseases via blood products.
It also contributes to technology transfer, global cooperation, and harmonization of regulations via the Blood Regulators Network (BRN).
Finally, the WHO has been very involved in the development of standards and guidelines regarding vaccines due to the importance of these products in public health.
v Moreover, WHO established the "prequalification of vaccines" (regarding the acceptability, in principle, of vaccines from different sources for supply) to help the United Nations Children's Fund (UNICEF) and other UN agencies that purchase vaccines.
Finally, through its regulatory pathways initiative it also helps to address the challenges faced by developing countries that are targets for clinical trials or introduction of new vaccines not registered in the country of manufacture.
The objective is to support the establishment of regulatory mechanisms for the licensing of products in those countries that have not yet fully developed the expertise for the review of technical applications.
This is achieved via workshops and technical assistance in collaboration with the European Medicines Agency (EMA) through its Article 58 Scientific Opinion procedure, w the US FDA, and other national DRAs in developed countries.
A Developing Countries' Vaccine Regulators Network (DCVRN) was created in September 2004, and regional initiatives were also established.
In many countries (developed and undeveloped), there is recognition of the significant need for research and development of medicines specifically for pediatric use (or data from pediatric studies using medicines that have been developed for adults).
This lack of pediatric data became an important problem despite many initiatives from different regions or countries.
The lack of suitable pediatric medicines, paired with inconsistent regulatory frameworks, poses significant risks to a particularly vulnerable patient population.
The overall aim of the PmRN x is to promote availability of quality medicines (including biological medicines and vaccines) for children by facilitating communication, collaboration, and regulatory harmonization across manufacturing, licensing, and research [66] .
More specifically, among several objectives, this network tries to: ▸ Provide a forum for discussion between worldwide DRAs to build awareness of pediatric medicines regulatory considerations ▸ Facilitate the collaboration, discussion, and work towards consensus on regulatory standards for pediatric medicines (i.e., the development of international recommendations and common standards for clinical trials and registration of medicines for children based on the existing ICH, EMA, and US FDA guidelines) ▸ Strengthen licensing (approval) systems for pediatric medicines by increasing regulatory cooperation, information sharing, and training Traditional medicines y have been used in many countries throughout the world over many centuries.
Today, these medicines still represent an important part of healthcare in some countries.
z For example, more than 100 countries have regulations for herbal medicines, but practices of traditional medicine vary greatly from country to country and from region to region, as they are influenced by factors such as culture, history, personal attitudes, and philosophy.
However, while it is often necessary to tailor legislation and delivery to reflect the needs and traditions of the individual countries, a number of themes and issues are common, such as the importance of practitioner training, the issues related to safety, the need to enhance research into both products and practices, and the importance of labeling.
Also, the use of traditional medicines has expanded globally and has gained popularity in the last few decades.
Specifically, these practices have not only continued to be used for primary healthcare of the poor in developing countries, but have also been used in other countries where conventional medicines are predominant in the national healthcare system.
aa With this tremendous expansion in the use of traditional medicines worldwide, safety and efficacy as well as quality control of herbal medicines and traditional procedure-based therapies have become important concerns for many of these countries.
For this reason, WHO has been increasingly involved in developing international standards and technical guidelines for these types of medicines, and also in increasing communication and cooperation between countries [67] .
The challenge now is to ensure that traditional medicines are used properly, and to determine how research and the evaluation of traditional medicines should be carried out.
Supported by several WHA and Executive Board resolutions, WHO has developed and issued a series of technical guidelines (e.g., guidelines for the assessment of herbal medicines, research guidelines for evaluating the safety and efficacy of herbal medicines, and guidelines for clinical acupuncture research).
In 1997, WHO developed draft guidelines for "methodology on research and evaluation of traditional medicine" that was finally approved in April 2000 [68] .
The purpose of this document is to promote the proper development, registration, and use of traditional medicines and to harmonize the use of certain terms in traditional medicine.
Moreover, in 2006, WHO established a global network (called the International Regulatory Cooperation for Herbal Medicines [IRCH]) to allow communication and exchange between worldwide regulatory authorities responsible for the regulation of herbal medicines.
The mission of this program is "to make quality priority medicines available for the benefit of those in need."
This is achieved through evaluation and inspection activities, and in cooperation with national DRAs and partner organizations.
The list of prequalified medicinal products (updated regularly) is used principally by UN agencies (including UNICEF and the Joint United Nations Programme on HIV/AIDS [UNAIDS]) to guide their procurement decisions.
But, the list has also become a vital tool for any agency or organization involved in bulk purchasing of medicines, as demonstrated by the Global Fund to Fight AIDS, Tuberculosis and Malaria.
The strategy is to apply unified standards of acceptable quality, safety, and efficacy and to build the capacity of staff from national DRAs, quality control laboratories, and from manufacturers or other private companies, to ensure quality medicines.
Technical assistance, training, and capacity building are an important part of the program [70] .
When a product is included on the WHO list, the relevant product dossier has been evaluated and the manufacturing sites inspected by WHO-appointed assessors and inspectors and found to comply with WHO standards.
WHO also recognizes the evaluation of products by some major DRAs that apply stringent standards for quality, including, but not limited to, the US FDA, EMA, and Health Canada.
bb However, it is important to note that the inclusion of a product (or a laboratory) on this list does not imply any approval by WHO because it is the sole prerogative of national authorities.
WHO inspections are done by a team of inspectors, including: ▸ An inspector/expert from one of the Pharmaceutical Inspection Co-operation Scheme (PIC/S) countries ▸ A WHO representative (inspector/expert) ▸ An inspector (or inspectors) as an observer from the national DRA of the country in which the laboratory is located At the end of 2011, the WHO list of prequalified medicines included 269 products (manufactured in 25 countries); a total of 23 quality control laboratories had been prequalified (covering all WHO 6 regions).
The program had also prequalified its first active pharmaceutical ingredients (APIs) [71].
The above projects are specifically related to the harmonization of pharmaceutical regulations and regulatory standards related to medicinal products.
However, it is important to note that several other WHO projects not directly related to the harmonization of pharmaceutical bb When a product is listed with a reference to US FDA or EMA, the alternative listing procedure was used, and the products have been added to the list relying on the assessment and inspections conducted by the US FDA or EMA.
regulations cc have been or are also very important because they facilitate implementation of common systems, agreements on terminology, and the establishment of a forum for exchange of not only information, but also expertise and experience.
These other WHO projects ultimately facilitate overall dialogue, cooperation, convergence, and harmonization between countries and regions.
Moreover, other more general projects can also promote regional and subregional collaboration and harmonization of the regulation.
For example, one of the principles of the general EC-ACP-WHO Partnership established in 2004 dd was to "strengthen existing collaborative arrangements (e.g.
pooled procurement in the Caribbean) and catalyse the creation of new ones, which can work together to achieve pooled procurement, common policies and harmonization of legislation."
In addition, WHO publishes many documents regarding pharmaceuticals and regulations (i.e., newsletters, periodicals, reports status, or special publications such as the WHO Blue Book [72] ) that allow the diffusion and exchange of information and data everywhere in the world.
For example, "WHO Drug Information" is a quarterly journal, launched in 1987, which provides an overview of topics relating to medicine development and regulation that is targeted to a wide audience of health professionals and policymakers.
It communicates the latest international news and trends.
Finally, some other specific WHO projects are also very important in facilitating the implementation of the international standards.
These following projects need to be reviewed even though they are not directly related to the harmonization of pharmaceutical regulations because they demonstrate the key role of WHO in the global regulatory system, and therefore show how this organization has the legitimacy to further coordinate global harmonization.
▸ WHO Review of Drug Regulatory Systems: To ensure that public health is appropriately supported, national regulatory capacity needs to be regularly assessed, areas of weakness need to be identified, and necessary measures need to be taken.
The objectives of this review are to strengthen national regulatory and control capacity through the identification of specific needs and the provision of appropriate technical support and training.
This is done via the evaluation of existing legal framework, regulations, and control activities in order to assess the national regulatory capacity against a set of predefined parameters.
WHO can then provide technical input if gaps are identified.
This activity is very important, especially in developing countries, to ensure that international standards can be appropriately implemented at the national level.
It is also an important tool to have a clear status of national regulatory systems to evaluate appropriate needs from developing countries and therefore necessary support from regional and international organizations.
The WHO multicountry study (involving only 10 countries) also showed that such assessments represent significant opportunities to learn more about the strengths and weaknesses of DRAs and the different strategies used to improve drug regulation performance [75] .
The International Health Regulations (IHR), first adopted by the Health Assembly in 1969 and then significantly revised in 2005 in consideration of the growth in international travel and trade and the emergence or reemergence of international disease threats and other public health risks [76] , were finally adopted by the 58th WHA on May 23, 2005 and entered into force on June 15, 2007.
The IHR is an international legal instrument that is binding on all the WHO Member States.
These global rules were developed and implemented to enhance national, regional, and global public health security.
Its aim is to help the international community prevent and respond to acute public health risks that have the potential to cross borders and threaten people worldwide.
The stated purpose and scope of the IHR are "to prevent, protect against, control and provide a public health response to the international spread of disease in ways that are commensurate with and restricted to public health risks, and which avoid unnecessary interference with international traffic and trade."
The IHR has been used for the H1N1 pandemic crisis [77] .
The revised IHR requires countries to strengthen their core surveillance and response capacities so that they can report certain disease outbreaks and public health events to WHO.
Building on the unique experience of WHO in global disease surveillance, alert, and response, the IHR defines the rights and obligations of countries to report public health events, and establishes a number of procedures that WHO must follow in its work to uphold global public health security.
As mentioned above, this document was not specifically developed for pharmaceutical products, but is an important global tool that enhances cooperation between all countries in the world.
Indeed, even if this agreement does not specifically relate to the harmonization of pharmaceutical regulations, it is very interesting for many reasons.
First, this project helps strengthen worldwide capacities for public health and global cooperation in general, which is important for the implementation of harmonized global standards.
More importantly, this is one of the first agreements that manages public health as a truly global issue and proposes further action using an integrated international approach and network.
It shows that further integrated global cooperation in the area of health (with WHO being at the center of this cooperation to coordinate this effort) is possible and beneficial [78].
The mission of WHO's program on medicines and pharmaceutical policies is to support the achievement of the health-related MDGs by assisting governments and organizations to ensure global and equitable access to safe, effective medicines of assured quality.
Goal 8 ee and Target 8E ff are particularly applicable to WHO harmonization activities in the pharmaceutical domain.
Many of WHO's activities in the pharmaceutical domain support the achievement of these MDGs because they globalize the resolution of major public health issues (that cannot be resolved at the national/local level), they promote collaboration between countries and regions, and they provide tools and standards to allow such international collaboration.
Since its creation, WHO has indeed played a significant role in the global harmonization of pharmaceutical regulations.
As per its mandate and the responsibilities defined in its constitution, it has developed and maintained numerous international standards, norms, guidelines, classifications, and nomenclatures through a rigorous, international, and independent scientific consultative process.
In addition to this normative role, WHO has also established important networks to facilitate global cooperation.
For example, ICDRAs has been an important player in global regulatory harmonization.
It launched many projects that have facilitated and promoted harmonization and cooperation between countries [79].
Cooperation projects have also been pioneered over the years with a specific interest in essential medicines.
gg The WHO Prequalification Program has been an important step since it demonstrated that cooperation in the domain of medicine evaluation is possible and beneficial.
Indeed, this program has been very positive and its scope has continually been extended since its creation in 2001.
It has clearly accelerated the access of essential medicines worldwide (especially in low and middle income countries) [80] .
This model should be used to further develop regional and global collaboration for medicine evaluation.
The example of the 2010/2011 pilot WHO/East African Community (EAC) collaborative procedure initiated to facilitate registration of prequalified medicines in the EAC [81] was positive.
The overall aim was to identify a framework for WHO/EAC, for joint evaluation and approval of dossiers and inspections of medicine manufacturing sites, and to ensure that these assessments are integrated into national regulatory decision making.
Two assessors each from three EAC countries (Kenya, Tanzania, and Uganda) and six WHO assessors jointly assessed two product dossiers submitted by a single manufacturer.
The dossiers were submitted in parallel, and with identical content, to each participating EAC country and to WHO.
The products were both prequalified.
The principal benefit of this joint assessment was that once the products had been jointly assessed and approved by WHO/EAC, they were granted immediate access to the markets of each of the countries that had participated in the joint assessment.
Also, such joint assessment contributes to harmonization of regulatory requirements at the regional level.
This pilot WHO/EAC project also exemplifies the role of WHO in providing technical assistance to countries and supporting local capacity building.
Indeed, by acknowledging the important role of adequate systems to implement sound and effective pharmaceutical regulation, WHO has supported developing countries in addressing their deficiencies or capacity problems through various types of training, assessment of regulatory capacity, and the recommendation of institutional development plans.
These activities have been very beneficial in the past, but work needs to continue and grow in this domain, as problems still exist.
Indeed, the extent of implementation of standards varies from one region to another.
There are a number of factors that explain observed weaknesses of medicine regulation, and these differ from country to country and depend also on the individual health systems.
Countries may vary regarding their registration system, and not all of them can implement a comprehensive medicine evaluation and registration system.
Also, WHO encourages regional and international collaboration among national DRAs in order to promote the harmonization of requirements and practices, and to strengthen professional competence [82] .
However, as recognized in its Medicine Strategy Plan, cooperation with regional harmonization initiatives and organizations should be further enhanced [83] .
Closer cooperation and coordination should also be sought with other global initiatives such as ICH.
Further assistance to countries and cooperation with other regional and global initiatives are indeed possible and can be facilitated by WHO's regionalized structure.
This specific threelevel organization provides multiple opportunities for engaging with countries.
The headquarters focus on initiation, development, and global coordination of harmonization projects, while regional offices focus on technical support and building national capacities to support implementation.
WHO's presence in countries also allows a close relationship with ministries of health and its partners inside and outside of government.
This work at the regional and country levels is critical in ensuring that local and regional needs and challenges are taken into consideration when international standards and projects are developed.
To conclude, although some improvements may address current challenges, WHO has been very successful and beneficial for all Member States (developing and also developed countries).
It has promoted evidence-based debate, analysis, and recommendations for health through its own work and that of the numerous formal and informal networks and collaborating centers around the world.
These networks facilitate lively cooperation between scientists across nations and allow governments to jointly tackle global health problems.
Development and promotion of global norms and standards in medicine is one of WHO's efforts that is widely perceived as being in an area in which WHO has a comparative advantage.
This advantage is due to the recognition of WHO as the global leader and coordinating authority on global public health.
The achievement of the MDGs and the renewal of primary healthcare are indeed unthinkable without WHO's norms and standards, policy guidelines, and technical cooperation.
This is why the development and promotion of global norms and standards are an area of continued focus for WHO [84] .
It is indeed critical that WHO continue its work towards better harmonization and cooperation in the pharmaceutical domain.
Acknowledging the unique neutral and independent role of WHO, its numerous successes in the past, and its nearly universal membership, it would be appropriate to further extend the leadership of WHO in this domain.
This increased responsibility in the coordination of medicines would also further fulfill its mandate "to act as the directing and co-ordinating authority on international health work."
[85] The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) is a 20-year-old program.
This unique initiative was established with the objective to bring together the DRAs of Europe, Japan, and the United States and experts from the pharmaceutical industry in these three regions to discuss scientific and technical aspects of pharmaceutical product registration.
The drug regulatory systems in all three regions share the same fundamental concerns for the safety, efficacy, and quality of medicines.
However, many time-consuming and expensive experiments have been repeated in all three regions to meet specific regional requirements.
The goal of ICH has been to increase harmonization of technical requirements to ensure that safe, effective, and high-quality medicines are developed and registered in the most efficient and cost-effective manner in order to be delivered to the maximum number of patients in the world without delay.
These activities have been undertaken to promote public health, prevent unnecessary duplication of clinical trials in humans, and minimize the use of animal testing without compromising safety and effectiveness.
By making recommendations on ways to achieve greater harmonization of technical requirements for product registration, the objective is indeed to reduce or obviate the need to duplicate the testing carried out during the research and development of a new product.
Since its inception in 1990, ICH has evolved, through its Global Cooperation Group (GCG), to respond to the increasingly global face of drug development, so that the benefits of international harmonization for better global health can be realized worldwide.
This ICH mission is embodied in its current Terms of Reference: ▸ To maintain a forum for a constructive dialogue between regulatory authorities and the pharmaceutical industry on the real and perceived differences in the technical requirements for product registration in the EU, US, and Japan in order to ensure a more timely introduction of new medicinal products, and their availability to patients; ▸ To contribute to the protection of public health from an international perspective (added upon revision in 2000); ▸ To monitor and update harmonized technical requirements leading to a greater mutual acceptance of research and development data; ▸ To avoid divergent future requirements through harmonization of selected topics needed as a result of therapeutic advances and the development of new technologies for the production of medicinal products; ▸ To facilitate the adoption of new or improved technical research and development approaches which update or replace current practices, where these permit a more economical use of human, animal, and material resources, without compromising safety; ▸ To facilitate the dissemination and communication of information on harmonized guidelines and their use such as to encourage the implementation and integration of common standards.
ICH is comprised of representatives from six parties (the founding members of ICH) that represent the regulatory bodies and research-based industry in the EU, Japan, and the US: Since 1990, when ICH was initiated, members have been added: ▸ The International Federation of Pharmaceutical Manufacturers & Associations (IFPMA), the global nonprofit, nongovernmental organization, founded in 1968 to represent the research-based pharmaceutical, biotech, and vaccine sectors.
Its members are comprised of over 20 leading international companies and over 40 national and regional industry associations covering both developed and developing countries.
IFPMA is very involved in all subjects related to the improvement of global health.
It has been closely associated with ICH since its inception to ensure contact with the research-based industry (especially outside the ICH regions).
IFPMA provides the ICH Secretariat.
This important group of nonvoting members was established as a link between ICH and non-ICH countries and regions.
The ICH organization consists of the ICH Steering Committee, ICH Coordinators, ICH Secretariat, and ICH Working Groups.
The ICH Global Cooperation Group (GCG) and the ICH Medical Dictionary for Regulatory Activities (MedDRA) Management Board are subcommittees of the ICH Steering Committee.
The Steering Committee is the body that governs the ICH, determines the policies and procedures, selects topics for harmonization, and monitors the progress of harmonization initiatives.
This committee, established at the first ICH meeting in April 1990, has met at least twice a year since, with the location rotating between three regions (EU, Japan, and US).
During these committee meetings, new topics are considered for adoption, reports are received on the progress of existing topics, and maintenance and implementation of the guidelines are discussed.
Each of the six ICH parties has two seats on the ICH Steering Committee.
Each of the observers nominates nonvoting participants to attend the ICH Steering Committee Meetings.
IFPMA also participates as a nonvoting member.
Meetings of the ICH MedDRA Management Board, ICH GCG, and the Regulators Forum also occur during the same week as the Steering Committee meeting.
ICH Working Groups are the key players of the ICH harmonization process.
They are responsible for the development, implementation, or maintenance of ICH guidelines.
Each of the six ICH parties is represented in every working group.
The official membership of an Expert Working Group/Implementation Working Group (EWG/IWG) is usually limited to two officials per party (one Topic Leader and one Deputy Topic Leader).
One of these topic leaders is nominated Rapporteur (and sometimes a second is nominated Co-Rapporteur) by the Steering Committee.
ICH Observers and Interested Parties hh can also nominate one representative.
The pharmacopoeial authorities and representatives from the self-medication industry and the generic industry were invited to participate in the various Working Groups.
Finally, the three regulatory parties of the Steering Committee officially designate a Regulatory Chair when a new ICH topic is formally adopted.
The Regulatory Chair, designated among the three regulatory parties, regularly presents reports to the Steering Committee and ensures, in close collaboration with the Rapporteur, timely execution of the ICH process and adherence to the concept paper and business plan, including scope and timelines.
Depending on the type of harmonization activity required, the Steering Committee will endorse the establishment of one of three types of Working Groups: ▸ Expert Working Group (EWG): These Working Groups are appointed by the Steering Committee when new topics are accepted for harmonization.
The objective of each EWG is to review the differences in requirements between the three regions and develop scientific consensus required to reconcile those differences.
It is charged with developing a harmonized guideline that meets the objectives defined in the concept paper and business plan.
▸ Implementation Working Group (IWG): An IWG's task is to develop questions and answers (Q&A) to facilitate implementation of existing guidelines.
▸ Informal EWG/IWG: These Working Groups are formed prior to any official ICH harmonization activity.
Their objective is to develop a concept paper and business plan.
Working Groups meet in the same week as the Steering Committee and report on their progress to the Committee.
These one-week meetings are key for the ICH organization as they allow for a regular review of efforts and achievements and adjust them if necessary.
ICH Discussion Groups are established to discuss specific scientific considerations or views (e.g., Gene Therapy Discussion Group) to facilitate the exchange of information on a specific topic, and ultimately the harmonization of the requirements.
The Coordinators are fundamental to the smooth running of the ICH and are nominated by each of the six parties.
An ICH coordinator acts as the main contact with the ICH Secretariat and ensures that ICH documents are distributed to the appropriate persons within the area of their responsibility.
Each party has also established a contact network of experts within their own organization or region in order to ensure that, in the discussions, they reflect the views and policies of the cosponsor they represent.
The way this network operates differs according to the administrative structure of the party concerned.
Due to structural differences within the EU and MHLW, ICH technical coordinators are also designated from the EMA and PMDA, respectively.
They support the ICH coordinator and facilitate every action of the Steering Committee members in the region, mainly by applying their scientific knowledge.
Their roles include acting as a contact point between the experts within the EMA and PMDA and the ICH coordinator at the main regulatory body, and as a contact point with the ICH Secretariat.
The ICH Secretariat operates from the IFPMA offices in Geneva (Switzerland), and provides support to the ICH Steering Committee.
The Secretariat is primarily concerned with preparations for, and documentation of, meetings of the Steering Committee, as well as coordination of preparations for Working Group (EWG, IWG, and Informal WG) and Discussion Group meetings.
The Secretariat also provides administrative support for the GCG and the MedDRA Management Board, and maintains the ICH website.
The MedDRA Management Board, appointed by the ICH Steering Committee, has overall responsibility for direction of MedDRA, an ICH standardized dictionary of medical terminology.
The Board oversees the activities of the MedDRA Maintenance and Support Services Organization (MSSO), which serves as the repository, maintainer, developer, and distributor of Med-DRA.
The Management Board is composed of the six ICH Parties, the Medicines and Healthcare Products Regulatory Agency (MHRA) of the UK, Health Canada, and WHO (as Observer).
The IFPMA acts as a nonvoting observer on the Management Board and also chairs the Board.
As stated in its mission statement adopted by all parties in May 2005, this group "promotes a mutual understanding of regional harmonization initiatives in order to facilitate the harmonization process related to ICH guidelines regionally and globally, and to facilitate the capacity of drug regulatory authorities and industry to utilize them."
This group ensures that the benefits of ICH harmonization extend beyond the three ICH regions (Japan, EU, and US).
The role of the GCG has changed over time as the focus on collaboration with the non-ICH regions increased.
From its creation to today, three phases can be differentiated: ▸ First Phase (1999 to 2003 : Information Sharing Outside ICH: During these first three years, the GCG mandate was to share information outside ICH (via preparation of brochures, presentations at international meetings, etc.).
The objectives were to make available to any country or pharmaceutical company that requested it information on ICH, ICH activities, and ICH guidelines.
To this end, the group created a series of brochures intended to guide its activities as it answers requests for information and responds to non-ICH regulators and industry: • ▸ Second Phase (2003 Phase ( to 2007 : Integration and Collaboration with RHIs: On November 9, 2003, the ICH GCG released their terms of reference in which they extended their action to act as the primary representative of the ICH Steering Committee outside the ICH regions, and equally as such as a conduit for non-ICH parties to the ICH Steering Committee.
To do so, the group developed a privileged relationship with other non-ICH harmonization initiatives.
This key activity of the GCG had three advantages: • To share ICH discussions and actions with the non-ICH regions (allowing, when possible, harmonization and implementation of ICH activities on a worldwide basis) • To promote and organize the involvement of the non-ICH regions experts in ICH discussions (via expert meetings, comments on Step 2 Guidelines, and training on Guidelines) • To facilitate interregional collaboration in order to promote transparency, better understanding of challenges and potential solutions to harmonization issues, leverage collective experience and knowledge (allowing easier standardization and development of Good Harmonization Practice) When, in 2003, the GCG decided to include representatives from the non-ICH regions, the relationship with the non-ICH regions became more collaborative and proactive, and the focus shifted from information sharing to a two-way dialogue to set up training and work on implementation.
The results of these collaborations allowed the organization of workshops in the regions (e.g., APEC Workshops on Clinical Research Inspections in 2008 and 2009 in Thailand, the SADC Quality Guideline Workshop in 2008 in Zambia, and the APEC Quality Guideline Workshop in 2008 in China).
As an example, the GCG also endorsed the APEC Life Sciences Innovation Forum (LSIF) sponsored workshop on ICH Quality Guidelines (Q8, Q9 and Q10), held in September 2007 in Seoul, South Korea.
This workshop was a great success for the spread of ICH concepts and recommendations in this region as it was attended by more than 400 participants (i.e., regulators, policymakers, academia, and industry) from 17 countries.
This type of workshop allows for practical explanation of ICH guidelines, but also opens up discussion and exchange on the anticipated challenges and opportunities associated with the implementation of ICH guidelines in order to better prepare implementation.
The participation of these individual countries is distinct and complementary to the participation of official RHI representatives.
In June 2008, the inaugural meeting of the expanded GCG occurred.
Today, the key focus of the GCG continues to be the implementation of ICH guidelines via the organization of training that began in 2007.
This training is indeed an important means for the promotion of better understanding of ICH and ICH guidelines beyond the ICH regions.
It developed a framework and mechanism for policy [91-2], a procedure for selection and prioritization, a template for training requests, definitions of roles and responsibilities for the organization and coordination of training activities, and a clearinghouse of training events for public access.
These training activities (most of the time coordinated with the respective RHIs) involve ICH experts.
During the meeting in October 2007 in Yokohama, Japan, the ICH Steering Committee also decided to complement the GCG with the Regulators Forum.
The ICH Regulators Forum is the latest idea implemented by ICH to increase communications and sow relationships between worldwide DRAs in order to ensure adoption and implementation of ICH guidelines.
Following a proposal from the US FDA in 2007, the first meeting occurred in Portland, Oregon, US in June 2008.
This is a good complement to the GCG activities and includes authorities from the three ICH regions, the Observers, the RHIs, and individual DRAs such as Australia, Brazil, China, Chinese Taipei, India, Korea, Russia, and Singapore.
This ICH Regulators Forum allows frank discussion and the sharing of expertise among DRAs regarding best practices and challenges related to the implementation of ICH guidelines and their impact on regulatory systems.
This discussion assists in identifying training and capacity needs for action by the GCG.
More importantly, it also builds mutual understanding, relationships, and trust.
In the 1980s, many varied efforts of harmonization of pharmaceutical regulatory requirements were conducted.
First, the European Community, who was developing a single market for pharmaceuticals, had shown that harmonization among different countries (with different medical cultures/practices and regulatory systems) was possible.
At the same time, bilateral discussions between Europe, Japan, and the US on the possibility for harmonization were ongoing.
The concretization of these ad hoc discussions happened during the World Health Organization (WHO) International Conference of Drug Regulatory Authorities (ICDRAs) in Paris in 1989, where specific plans were agreed to.
Following this meeting, the three authorities approached IFPMA to discuss a joint regulatory-industry initiative on international harmonization.
The spirit and concept of ICH was then agreed on between the different parties.
In April 1990, ICH was officially created at its inaugural Steering Committee meeting, hosted by the EFPIA in Brussels, Belgium.
Representatives of the regulatory agencies and industry associations of Europe, Japan, and the US met primarily to plan an international conference, but at the meeting the wider implications and terms of reference of ICH were also discussed.
During this first meeting, the structure (including a Steering Committee and Expert Working Groups) and the focus of ICH activities (harmonization of safety, efficacy, and quality guidelines for human drugs and biological products) were agreed on.
Eleven topics were selected for discussion at the first conference.
Finally, it was agreed to expand the membership of the Steering Committee to include representatives from WHO, EFTA, and Canada as observers because the harmonized guidelines could be useful to other non-ICH regions.
Additionally, agreement was reached on the full name of ICH.
This name was chosen because one of the objectives of this group was to organize international conferences on harmonization.
Today, this name is associated with the overall initiative.
The ICH members officially confirmed their commitment to ICH in a statement following the 2nd Steering Committee Meeting: The Parties cosponsoring this Conference, represented at the 2nd Steering Committee Meeting in Tokyo, 23-24 October 1990 re-affirmed their commitment to increased international harmonization, aimed at ensuring that good quality, safe, and effective medicines are developed and registered in the most efficient and cost-effective manner.
These activities are pursued in the interest of the consumer and public health, to prevent unnecessary duplication of clinical trials in humans and to minimise the use of animal testing without compromising the regulatory obligations of safety and effectiveness.
This Conference will provide a unique opportunity for regulators and industry to reach consensus on the steps needed to achieve this objective through greater harmonization of technical requirements and to set out practical and realistic targets for harmonising requirements where significant obstacles to drug development and the regulatory process have been identified.
Recognising the substantial progress which has already been made in achieving harmonization within Europe and through bilateral contacts between Europe, Japan, US, and other regions, the Conference will seek to make further progress through a trilateral approach, with clearly defined priorities, methods of work and recommendations to both industry and regulatory authorities.
Whilst the Conference will be an important step forward, it is not seen as an end in itself, but as a stage in a developing process, at a high level, between regulators and industry.
The Conference, its preparations, and follow-up activities will be conducted in an open and transparent manner and the presence of observers from other regulatory authorities and WHO is welcomed as a means of ensuring that the benefits of progress towards harmonization can be utilized world-wide.
The Conference will not only look at existing issues but will, based on past experience, seek to minimise future divergence of new registration requirements, as a consequence of technical progress.
This initial ICH statement is important because it provides the spirit of ICH that has been followed and implemented in all subsequent ICH activities since.
From its creation in 1990 to 2000, the initial focus of ICH was to promote technical and scientific exchanges and discussions in order to find consensus on divergent technical requirements for registration of medicinal products in the ICH regions.
The goal was indeed to remove redundancy and duplication in the development and review process, such that a single data set could be generated to demonstrate the quality, safety, and efficacy of new products.
During this first phase of its activities, the ICH structure and process were defined, a lot of harmonization activities started, and several guidelines/standards developed.
These first harmonization discussions were directed to both technical scientific content (related to quality, safety, or efficacy topics) and to format and communication tools (development of E3 and the start of MedDRA, Electronic Standards for Transmission of Regulatory Information (ESTRI) and Common Technical Document (CTD) projects).
During these first 10 years, there was a growing interest in ICH products beyond ICH countries, and ICH recognized early that harmonization within the ICH regions would not suffice.
However, during these first years, discussions and activities focused mainly on harmonization among ICH parties (even though ICH agreed to include observers as a link to the non-ICH regions) because it was important to start the process with a limited number of committed parties.
In November 2000, the 5th International Conference on Harmonization (ICH5) in San Diego, California, US marked the end of the first 10 years of ICH activity.
This conference provided an opportunity to evaluate results and to identify future needs in the area of international harmonization.
At the conference, results were presented of a survey on utilization of ICH guidelines confirming the positive contribution of ICH in improving the international drug regulatory approval process, thus speeding the availability of new medicines to the public.
In its statement titled "The Future of ICH" released at ICH5, the Steering Committee emphasized its intentions to focus the second phase of ICH on implementing and maintaining existing guidelines, preventing disharmony, encouraging scientific dialogue and harmonization in new areas, and undertaking efforts towards global cooperation with non-ICH regions and countries.
During its second phase, ICH continues to develop and implement tripartite guidance on specific technical requirements, and also increase its effort on the implementation of harmonized regulatory communication tools (i.e.
MedDRA, CTD, ESTRI, etc.)
between authorities and industry.
Indeed, one of the areas of focus of this second phase is to ensure adequate implementation and maintenance of all the guidelines developed since 1990.
Today, new guidelines continue to be developed, but less frequently.
These new guidelines cover important technical subjects related to pharmacovigilance (i.e., guidelines E2D, E2E, and E2F) or improvement of quality systems (i.e., guidelines Q8, Q9, and Q10).
New emerging topics (such as gene therapy) have also been discussed.
However, the main challenge of ICH is now to maintain and update the collection of guidelines already developed (i.e., follow the evolution of science, the experience gained, etc.).
The second focus and priority of this ICH phase has been, and continues to be, the extension of relationships with non-ICH regions.
It began with the creation of the GCG as a subcommittee of the ICH Steering Committee in 1999.
Since this time, ICH has developed its relationship with non-ICH regions and tried to facilitate the implementation of its standards and guidelines on a broader territory via collaboration and training.
Even if some relationships existed before, the GCG has been key for this geographical extension, and its role increased over time by moving from information sharing (via preparation of brochures, presentations at international meetings, etc.)
to a collaborative and proactive dialogue (via the incorporation of non-ICH regions and countries in the group).
Further evolution of the ICH structure and the GCG's Terms of Reference are expected to continue to promote greater involvement of global regulators [92-1,92-2].
The first activity of ICH was to organize the ICH1 Conference in 1991, one year after its creation, in order to exchange points of view and discuss divergences among different parties involved.
Since ICH1, five additional conferences have been organized (Table 1) .
These regular, well-attended conferences helped communicate the results of the harmonization activities to the largest possible audience.
They were designed as an open forum (in breakout sessions) to gather additional public comments and provide updates on ICH's scientific activities.
These six conferences were well attended (e.g., 2,400 participants representing industry and authorities of over 40 countries for ICH3 and 1,300 participants representing industry and authorities of over 140 countries for ICH5).
The early ICH conferences were very important in increasing visibility on the process of harmonization and for ensuring that the process was carried out in a transparent manner.
ICH5 focused primarily on the finalization and completion of the CTD guideline.
The last ICH conference organized, ICH6, focused on areas such as new technologies and global cooperation with regulatory harmonization initiatives outside the ICH regions.
The three satellite sessions (related to "Partnerships in Harmonization," "Gene Therapy," and "MedDRA Users' Group") also confirmed the priorities of the meeting.
During this conference, opportunities and new challenges for regulatory harmonization were discussed.
The practical implementation of the CTD was also reviewed.
After ICH6, no additional international conferences were scheduled.
ICH7 was planned to have taken place in Europe in 2007, but it was then canceled.
Instead, in May 2007, the ICH Steering Committee decided to replace these large international ICH conferences with smaller and more frequent regional public meetings at the time of the ICH Steering Committee meetings in the region (in order to benefit from the presence of Steering Committee Members and ICH Experts).
Now that the ICH process is well recognized, these smaller regional meetings allow for a better focus on regional issues and challenges.
It also provides everyone the opportunity to meet with regulators and industry experts involved in ICH activities, to be regularly informed on recent developments, and to exchange information on different hot topics.
Following this decision, regional meetings have been organized: ▸ In Europe, the first EU regional public meeting took place in Brussels, Belgium in November 2008.
▸ In North America, the first regional public meeting took place in Washington, DC, US in October 2008.
▸ In Asia, the first regional public meeting took place in Tokyo, Japan in November 2007.
The ICH Process was first drawn up at the Steering Committee meeting in Washington, DC in March 1992, and amended in Tokyo, Japan in September 1992.
Since then, the ICH procedures have been revised several times .
Moreover, the new principles of governance, agreed to at the ICH Steering Committee meeting in June 2012, have revised the role of regulator and Suggestions for new harmonization initiatives may arise in a number of forums (ICH Regional Guideline Workshops; regional and international conferences, workshops, and symposia dealing with research and development (R&D) and regulatory affairs; recognized associations; testing and registration of medicines, etc.).
From the suggestion of a new harmonization action to the development of a new guideline (or modification of an existing guideline), there are three sequential steps: • Submission of a Concept Paper to the ICH Steering Committee by an ICH party or an Observer • Endorsement by the Steering Committee • Establishment of a Working Group The Concept Paper is the start of all ICH harmonization activities.
This document provides a short summary of the proposal (maximum two pages) and provides the information indicated below: • Type of Harmonization Action Proposed: For example, a new harmonized tripartite guideline and recommendation, or a revision of an existing guideline (indicating the category of procedure).
• Statement of the Perceived Problem: Brief description with an indication of the magnitude of the problem currently caused by a lack of harmonization, or anticipated if harmonization action is not taken.
• Issues to be Resolved: A summary of the main technical and scientific issues that require harmonization.
• Background to the Proposal: Further relevant information (e.g., the origin of the proposal, references to publications, and discussions in other forums).
• Type of Expert Working Group: Recommendation on whether the EWG should be a six-party group (for topics related to the R&D of a new drug substance and product) or an extended EWG (e.g., GMP).
If necessary, further documentation and reports may be added to the Concept Paper.
Depending on the category of harmonization activity, a Business Plan may also be required.
The Business Plan outlines the costs and benefits of harmonizing the topic proposed by the Concept Paper.
Only when the ICH Steering Committee endorses a Concept Paper, and where appropriate a Business Plan, can the harmonization activity be initiated.
A preliminary determination will be made on whether the topic is of sufficient interest to all parties and can be accommodated within the ICH work schedule.
The Steering Committee takes the following points into account when discussing a Concept Paper: • Objectives and Expected Outcome of the harmonization action • Categories of the ICH process • Composition of the EWG or IWG appointed to discuss the technical issues • Setting a Timetable and Action Plan for the EWG/IWG The Concept Paper may need to be revised and updated to reflect the Steering Committee discussions and conclusions.
If the Steering Committee agrees that a topic may warrant further consideration and a Business Plan needs to be developed, an informal EWG/IWG will be formed and the group will work through e-mail, teleconference, and rarely, face-to-face meetings.
The first tasks of the informal EWG/IWG will be to finalize a Concept Paper and develop a Business Plan.
The revised Concept Paper and Business Plan will be sent prior to, and presented at, the next Steering Committee meeting.
At its meeting in Yokohama, Japan (in June 2006), the ICH Steering Committee agreed to have the final versions of the Concept Papers and Business Plans available on the ICH website, for public information.
Depending on the type of harmonization activity proposed, the ICH Steering Committee will endorse the establishment of either an EWG or an IWG.
ICH harmonization activities fall into four categories.
As presented in Table 2 , these four categories cover the creation and development (stepwise progression), implementation, revision, and maintenance of guidelines.
No procedure is in place for the withdrawal of existing ICH guidelines because it happens very rarely.
When Guideline Q1F (Stability Data Package for Registration Applications in Climatic Zones III and IV) was withdrawn, an explanatory note was released following the endorsement of the withdrawal by the ICH Steering Committee at its meeting in Yokohama, Japan in June 2006.
Withdrawal notifications were also released by the EMA, MHLW, and US FDA.
▸ The Formal ICH Procedure: The formal ICH procedure follows a stepwise approach consisting of five steps with "decision points" at Step 2 and Step 4 that enable the Steering Committee to monitor the progress of the harmonization topics.
This procedure is followed for the harmonization of all new ICH topics.
A streamlined procedure is also available when necessary.
The procedure is initiated with the endorsement, by the Steering Committee, of a Concept Paper and Business Plan.
An EWG with membership as specified by the Concept Paper is subsequently established.
The EWG works to develop a draft guideline and bring it through the various steps of the procedure that culminate in Step 5 and the implementation in the ICH regions of a harmonized tripartite guideline.
• Step 1: Consensus Building When the Steering Committee adopts a Concept Paper as a new topic, then the process of consensus building begins.
The EWG prepares an initial consensus technical document, based on the objectives set out in the Concept Paper and in consultation with experts designated to the EWG.
The initial draft and successive revisions are circulated for comments within the EWG, providing fixed deadlines for receipt of those comments.
Work is conducted via e-mail, teleconferences, and web conferences.
If endorsed by the Steering Committee, the EWG will also meet face-to-face at the biannual Steering Committee meetings.
Interim reports on the progress of the draft are made to the Committee on a regular basis.
When consensus is reached among all EWG members, the EWG signs the Step 1 Experts Signoff sheet.
The Experts Document with EWG signatures is submitted to the Steering Committee to request adoption under Step 2a of the ICH process.
Step 2a is reached when the Steering Committee agrees, based on the report of the EWG, that there is sufficient scientific consensus on the technical issues for the technical document or recommendation to proceed to the next stage of regulatory consultation.This technical document is made public on the ICH website.
On the basis of the technical document, the three ICH regulatory parties take the actions they deem necessary to develop the "Draft Guideline."
The consensus text approved by the three regulatory ICH parties is signed off by the three regulatory ICH parties as the Step 2b Draft Guideline.
• Step 3: Regulatory Consultation and Discussion Regional Regulatory Consultation: At this stage, the guideline embodying the scientific consensus leaves the ICH process and becomes the subject of normal wide-ranging regulatory consultation in the three regions.
In the EU it is published as a draft CHMP Guideline, in Japan it is translated and issued by the MHLW for internal and external consultation, and in the US it is published as draft guidance in the Federal Register.
Step 2 guidelines released for consultation are also available on the ICH website.
DRAs and industry associations in non-ICH regions may also comment on the draft consultation documents by providing their comments to the ICH Secretariat.
After obtaining all regulatory consultation results, the EWG that organized the discussion for consensus building will be resumed.
The same procedure described in Step 1 is used to address the consultation results into the Step 2 final document.
The draft document to be generated as a result of Step 3 is called the Step 4 Draft Guideline.
The Step 4 document with regulatory EWG signatures is submitted to the Steering Committee to request adoption as Step 4 of the ICH process.
Step 4 is reached when the Steering Committee agrees, on the basis of the report from the regulatory chair and the regulatory rapporteur of the EWG, that there is sufficient scientific consensus on the draft guideline.
This endorsement is based on the signatures from the three regulatory parties to ICH affirming that the guideline is recommended for adoption by the regulatory bodies of the three regions.
In the event that one or more parties representing industry have strong objections to the adoption of the guideline on the grounds that the revised draft departs substantially from the original consensus, or introduces new issues, the regulatory parties may agree that a revised document should be submitted for further consultation.
In this case, the EWG discussion may be resumed.
The Step 4 final document is signed off on by the Steering Committee signatories for the regulatory parties of ICH as an ICH harmonized tripartite guideline at Step 4 of the ICH process.
• Step 5: Implementation Having reached Step 4, the harmonized tripartite guideline moves immediately to regulatory implementation, the final step of the process.
This step is carried out according to the same national or regional procedures that apply to other regional regulatory guidelines and requirements in the EU, Japan, and the US.
Information on the regulatory action taken and implementation dates are reported back to the Steering Committee and published by the ICH Secretariat on the ICH website.
In the EU, ICH guidelines are submitted to the CHMP for endorsement and the timeframe for implementation is established (usually six months).
ICH Guidelines are available on the EMA website.
In Japan, ICH texts are translated into Japanese and subsequent Pharmaceutical and Medical Safety Bureau notification for the promulgation of guidelines written in Japanese is issued with an implementation date.
The notifications on guidelines in Japanese and also English attachments (ICH Texts) are available on the PMDA website.
In the US, the US FDA publishes a notice with the full text of the guidance in the Federal Register.
Step 4 guidance is available for use on the date it is published in the Federal Register.
They are available on the US FDA website.
▸ The Q&A Procedure: The Q&A procedure is followed when additional guidance is considered necessary to aid in the interpretation of certain ICH harmonized tripartite guidelines and ensure a smooth and consistent implementation in the ICH regions and beyond.
The Q&A Procedure is initiated with the endorsement of the Steering Committee of a Concept Paper.
In the case of major implementation activities, the Steering Committee may also consider the need for a Business Plan.
An IWG with membership as specified by the Concept Paper is subsequently established.
The development and adoption of the Q&A follow an established process.
Questions received from stakeholders are collected, analyzed, reformulated, and ultimately used as model questions for which standard answers are developed and posted on the ICH website.
The incoming questions are not answered individually, rather they serve to highlight areas that need additional clarification and are then used to develop a model question that will be answered in the Q&A document.
Based on the level of guidance given by the answers, the IWG will assess whether the Q&A document should be a Step 2b document and published for comments or a Step 4 document and published as final.
The document should be Step 2b if, based on the answers provided, it sets forth substantial new interpretations of the guideline(s).
The document should be a Step 4 if, based on the answers provided, it sets forth existing practices or minor changes in the interpretation or policy of the guideline(s).
The document then follows the normal path of a Step 2b/Step 4 document as per the formal ICH procedure.
The revision procedure applies when an existing adopted guideline needs to be revised or modified.
It is almost identical to the formal ICH procedure (i.e., five ICH steps).
The only difference is that the final outcome is a revised version of an existing guideline rather than a new guideline.
The revision of a guideline is designated by the letter R1 after the usual denomination of the guideline.
When a guideline is revised more than once, the document will be named R2, R3, R4, and so on with each new revision.
The maintenance procedure is used to add standards to exist ing guidelines and/or recommendations or to provide an update based on new information.
This procedure has been used to amend the addendum of Guideline S5(R2), "Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility," and Guideline M3(R1), "Maintenance of the ICH Guideline on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals," on November 9, 2000.
It is currently applicable for changes to the Q3C Guideline on Residual Solvents, the Q4B Annexes, and M2 Recommendations.
In each case, the procedure is used when there is new information to be added or when the scientific/technical content is out-of-date or no longer valid.
For the Q3C guideline, this maintenance procedure is used to revise the permitted daily exposure (PDE) as new toxicological data for solvents become available.
Since its creation, and pursuant to its main goal, ICH has released a number of guidances, each harmonizing technical requirements for registration of medicinal products.
For each technical topic, the relevant EWG discussed the important question of whether there is scientific justification for the different regional requirements, and whether it would be possible to develop a mutually acceptable guidance.
The objective of this scientific discussion is to reach a scientific consensus whatever the time and effort it requires [93] (and not a "compromise" that would be an unacceptable decrease of certain regional requirements without scientific justification/ basis).
ICH has also worked on broader projects (e.g., MedDRA and CTD), which have been critical for the international exchange of information.
The ultimate goal of ICH activities is to remove redundancy and duplication in the development and review process such that a single set of data could be generated to demonstrate the quality, safety, and efficacy of a new medicinal product.
The Steering Committee has given priority to harmonizing the technical content of the sections of the reporting data.
The first ICH Guideline to deal with harmonizing the format of reporting data was E3, "Content and Format of Clinical Study Reports."
This Guideline describes a single format for reporting the core clinical studies that make up the clinical section of a registration dossier.
The goal of developing a harmonized format has led to the creation of the ICH Guideline M4, "The Common Technical Document" (CTD), further described below.
At the first ICH Steering Committee meeting it was decided that the topics selected for harmonization would be divided into Safety, Quality, and Efficacy in order to reflect the three criteria that are the basis for approving and authorizing new medicinal products.
Since then, ICH has created a fourth category called Multidisciplinary, which covers crosscutting topics that do not fit uniquely into one category or another.
Therefore, today ICH topics are divided into four categories (Quality, Safety, Efficacy, and Multidisciplinary) and ICH topic codes are assigned according to these categories.
A summary of harmonized topics is provided below.
An updated list of these guidances (including their status) can also be downloaded from the ICH website (and also from the US FDA, PMDA, and EMA websites).
The guidelines under this category provide harmonization of information related to the development, manufacturing, and testing of medicines.
They specifically cover stability testing (Q1), validation of analytical procedures (Q2), impurities testing (Q3), pharmacopoeial text harmonization and interchangeability (Q4), quality information on biotechnological products (Q5), specifications (test procedures and acceptance criteria) (Q6), GMP (Q7), pharmaceutical development (Q8), quality risk management (Q9), and pharmaceutical quality systems (Q10).
In addition, the ICH Steering Committee endorsed on April 11, 2008 the development of a new Q11 guidance related to the development and manufacture of drug substances (chemical entities and biotechnological/biological entities).
The guidelines under this category provide harmonization of information related to in vitro and in vivo preclinical studies.
They cover all preclinical studies performed during the development of new pharmaceutical products, such as carcinogenicity studies (S1), genotoxicity studies (S2), toxicokinetics and pharmacokinetics studies (S3), toxicity studies (S4), reproductive toxicology studies (S5), pharmacology studies (S7), and immunotoxicology studies (S8).
Guideline S6 specifically addresses preclinical safety evaluation for the biotechnological products.
The ICH Steering Committee also endorsed on May 10, 2007 the development of a new S9 guideline that provides preclinical guidelines on oncology therapeutic development.
Finally, the photosafety evaluation of pharmaceuticals was endorsed as a new topic (S10) by the ICH Steering Committee in June 2010.
The guidelines under this category provide harmonization of information pertaining to the clinical evaluation of pharmaceutical products.
Most of these guidelines relate to the assessment and management of safety data (E1 and E2 Guidelines).
These guidelines cover: • All the above Efficacy guidelines can be applied to all therapeutic classes of drugs.
Until now, ICH has focused the discussion on general (i.e., nontherapeutic class-specific) guidances.
However, there are, in some therapeutic classes, individual drug evaluation guidelines among the three regions.
Differences between guidelines can result in obstacles to the mutual use and acceptance of clinical data.
At the Steering Committee meeting in September 1998, it was agreed that this should be adopted as a new area of work for ICH, with the first such guideline being undertaken as a "pilot study" to assess the feasibility of extending work in this area.
It was agreed to develop the first therapeutic class-specific guideline for antihypertensive drugs.
No other guideline for clinical evaluation of a specific therapeutic category has been developed since this guideline (E12).
This category was created to include guidelines covering topics that do not fit uniquely into one of the above three categories.
In addition to the technical guidelines described in previous sections, ICH set up EWGs to harmonize Medical Terminology (M1: MedDRA), Drug Dictionaries (M5), and the format and organization of data in regulatory applications (M4: CTD) in order to ease the exchange of information.
The creation of electronic standards (M2: ESTRI) was also critical for the quick exchange of common, agreed-upon data.
In November 2010, the ICH Steering Committee endorsed the establishment of an EWG for the Electronic Common Technical Document (eCTD) and assigned the topic code "M8" (even though work in relation to the eCTD had previously been undertaken by the M2 EWG).
All these harmonization initiatives are critical achievements that required a lot of effort from their respective working groups.
They are important activities that greatly contributed to the international harmonization of pharmaceutical regulations because they harmonized and facilitated the exchange of information between regulators and pharmaceutical companies.
Due to the importance of these initiatives, each of them is detailed in the specific subsections below.
Guideline M3 covers a specific topic relating to both safety and efficacy issues.
For this reason, it has been classified as a "Multidisciplinary Topic."
This joint safety and efficacy guideline provides principles for nonclinical strategies (i.e., scope, timing, and duration of nonclinical safety studies) in relation to the conduct of clinical trials.
It helps to reduce the differences between the ICH regions and it also provides recommendations to reduce animal use during research and development (e.g., inclusion of any in vivo evaluations as additions to general toxicity studies instead of performing separate studies).
This guideline is definitively aligned with the overall ICH objectives, as its purpose is to facilitate the timely conduct of clinical trials, reduce the use of animals in accordance with the 3Rs (reduce/refine/replace) principles, and reduce the use of other drug development resources.
It clearly promotes the safe and ethical development and availability of a new pharmaceutical as quickly as possible.
Finally, the ICH Steering Committee endorsed (in June 2010) the "Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk" as a new topic (M7).
MedDRA was developed by an ICH EWG in the early 1990s.
It was designed to support the classification, retrieval, presentation, and communication of medical information internationally and throughout the product regulatory cycle.
Prior to MedDRA, different medical dictionaries (and also different versions of these dictionaries) were used, such as the World Health Organization Adverse Reaction Terminology (WHO-ART), the Coding Symbols for a Thesaurus of Adverse Reaction Terms (COSTART) from the US FDA, and the Japanese Adverse Reaction Terminology (J-ART) from the MHLW.
At that time, several worldwide authorities were looking for a more cost-and time-efficient way of processing suspected adverse reaction reports (e.g., the United Kingdom Medicines Control Agency [UK MCA] were developing a new system of coding called ADROIT).
It became obvious that this activity should fall under the auspices of ICH.
The goal of ICH in developing MedDRA was to have an internationally recognized standard, and medically rigorous and well-maintained terminology to facilitate communication.
It is indeed one of the most important ICH projects for ensuring the global exchange of clinical information.
This international medical terminology is particularly important in the electronic transmission of adverse event reporting (both in the pre-and post-marketing areas), as well as in the coding of clinical trials data.
The MedDRA dictionary is a multi-axial terminology that provides a set of terms that consistently categorizes medical information.
It includes terminology for symptoms, signs, diseases and diagnoses, and therapeutic indications.
It is organized by System Organ Class (SOC), divided into High-Level Group Terms (HLGT), High-Level Terms (HLT), Preferred Terms (PT), and finally into Lower-Level Terms (LLT).
The MedDRA dictionary has been translated into many languages.
As the terminology itself does not contain specific guidelines for its use, an ICH working group has been charged to develop two guides: ▸ "MedDRA Term Selection: Points to Consider": This document was created to achieve consistency in the way users assign particular terms to particular symptoms, signs, diseases, etc.
▸ "MedDRA Data Retrieval and Presentation: Points to Consider": This document provides guidance on retrieval and on sorting and presenting data in the most understandable and reproducible way for the benefit of drug development, pharmacovigilance, and risk management.
These two documents provide a best practice approach for the use of MedDRA.
They are revised for each new MedDRA version release.
In addition, the MedDRA dictionary includes Standardized MedDRA Queries (SMQs) that were developed (in collaboration with CIOMS) to facilitate the retrieval of MedDRA-coded data as a first step in investigating drug safety issues in pharmacovigilance and clinical development.
SMQs are groupings of terms from one or more MedDRA System Organ Classes (SOCs) that relate to a defined medical condition or area of interest.
They are intended to aid in case identification.
Because the terminology requires constant updating and maintenance, it was agreed that a Maintenance and Support Services Organization (MSSO) would be needed to carry out this task and to distribute the terminology, on license, to users in industry and regulatory agencies.
The MSSO, contracted by ICH with technical and financial oversight by the MedDRA Management Board, is tasked to maintain, develop, and distribute Med-DRA.
Since the release of version 1.0 in 1994, MedDRA has become the accepted international standard for all worldwide regulatory activities (MedDRA is not yet mandatory in the US).
As a single global, standardized medical terminology, MedDRA speeds the exchange of clinical information, facilitating research and safety monitoring, and making the regulatory approval process more efficient and responsive.
Different translations of MedDRA have been released.
In March/April 2008, MedDRA was also implemented in the WHO VigiBase, providing a global repository of MedDRA-coded safety data that can be used as a substantial tool for pharmacovigilance.
During a meeting on October 27-28, 2007 in Yokohama, Japan, the MedDRA Management Board announced fee reductions for lower revenue subscribers.
These reductions are in keeping with the MedDRA Management Board's goal of facilitating the use of Med-DRA for all users.
Since January 2007, access to MedDRA has been free for academic organizations, hospitals, healthcare providers, and other users involved in noncommercial activities.
The objective of the Electronic Standards for Transmission of Regulatory Information (ESTRI) project was to facilitate international electronic communication.
To this end, an ICH Multidisciplinary Expert Working Group (called M2 EWG) was established during the ICH meeting of 1994 in Brussels, Belgium.
The M2 EWG was to evaluate and recommend ESTRI that meet the requirements of the pharmaceutical companies and DRAs from the three ICH regions.
Since 1994, the M2 EWG has developed the technological framework and recommended solutions for international information exchange.
This was obtained by gathering requirements, assigning specific tasks, evaluating international standards and products, and recommending a functional architecture.
This project included the verification of procedures for consistent, accurate information transfer, and the evaluation of encryption technologies and key certification procedures for the transfer of regulatory information.
The working group has undertaken test projects to define logical electronic communication standards in order to ensure the integrity of information and data exchange between pharmaceutical companies and authorities.
To allow flexible change, some of the activities of the EWG result in recommendations that do not follow the formal ICH step process.
They are agreed upon in the EWG, signed by all parties of the EWG, and are endorsed by the ICH Steering Committee at its different meetings.
These recommendations, which have been modified and improved over time, provide various open international standards that allow for the international transmission of information regardless of the technical infrastructure.
To facilitate the use of these recommendations, the M2 EWG has also developed a glossary for the technical terms.
Today, six M2 Recommendations are available.
They cover and standardize general aspects, but also the choice of file format and information transfer as described in Table 3 .
Recommendations were also prepared for the choice of physical media (i.e., floppy disks, CD-R, and DVD-RAM).
Because these physical media are not relevant anymore, these recommendations were retired in June 2008.
In addition to the Recommendations, the M2 EWG also developed several specifications with regard to the electronic exchange of information: ▸ The first specification developed by the M2 EWG was related to the electronic transfer of the Individual Case Safety Report (ICSR) presented in ICH Guideline E2B (Data Elements for Transmission of Individual Case Safety Reports).
Following the development of the E2B guideline, it became necessary to work on an electronic specification to guide the pharmaceutical companies on how to provide the information required by the E2B guideline.
Indeed, successful electronic transmission of ICSR relies on the definition of common data elements (provided in the E2B guideline), but also a standard electronic transmission procedure.
The first version of this specification was approved by the Steering Committee under Step 2 in 1997.
Since then this specification has been modified because its implementation and use had to be aligned with the evolution of the ICH E2B and M1 (MedDRA) guidelines.
As a result of this activity, adverse event (AE) data can be extracted, populated, and electronically transmitted in the manner specified by the ICH ICSR message from safety and surveillance databases.
Even if it has required a lot of work, the implementation of electronic reporting of ICSRs based on the ICH E2B, M1, and M2 standards progressed very rapidly across the ICH regions.
Thanks to these standards, pharmaceutical companies can now exchange case reports electronically via gateway with some DRAs (such as the US FDA or EMA).
▸ The second specification developed by the M2 EWG was the Electronic Common Technical Document (eCTD) created as the electronic message for the Common Technical Document (CTD) detailed in ICH Guideline M4.
This specification has since been maintained by the eCTD IWG.
The eCTD specification, based on XML (Extensible Markup Language) technology, allows for the electronic submission of the CTD from applicant to regulator, taking into consideration the facilitation of the creation, review, lifecycle management, and archiving of electronic submissions.
While the table of contents is consistent with the harmonized CTD, the eCTD also provides a harmonized technical solution to implementing the CTD electronically.
This eCTD specification is applicable to all modules of initial registration applications and for other submissions of information throughout the lifecycle of the product, such as variations and amendments.
The backbone has been developed to handle both the regional and common parts of submissions.
Implementation of eCTD has begun across the ICH partner and observer regions.
For example, since January 1, 2008, all electronic submissions to the US FDA are required to be in eCTD format.
▸ In 2003, the M2 EWG published the first version of the Study Tagging File (STF) specification, which is supplemental to the eCTD.
This specification has since been modified several times.
For each study included in Modules 4 and 5 of an eCTD submission, the STF includes information allowing for the identification of all the files associated with this specific study.
This is additional information to the eCTD backbone files that already include many items, but do not contain enough information on the subject matter of several documents (e.g., study report documents) to support efficient processing and review of applications.
The Common Technical Document (CTD) is one of the major and most well-known achievements of ICH, and like all other big harmonization projects of ICH, required much effort.
It provides a harmonized structure and format for regulatory applications.
The objective is to reduce the time and resources needed to compile applications for registration of medicines in the different ICH regions.
Additionally, this new common format allows DRAs to have more consistent reviews, helping them to perform analysis across applications and to exchange information among them.
Before the development of the CTD, each region had its own requirements for the organization of technical reports in the submission and for the preparation of the summaries and tables.
In Japan, applicants had to prepare the GAIYO, which organized and presented a summary of the technical information.
In Europe, expert reports and tabulated summaries were required, and written summaries were recommended.
The US FDA had specific guidelines regarding the format and content of the New Drug Application (NDA).
In 1996, the ICH industry representatives proposed assembling the information generated during the development of a product in the same order.
This proposal followed an industry survey in May 1996 that assessed the time and resources needed to convert an EU Marketing Authorization Application (MAA) into a US NDA (and the reverse).
This survey showed that an average of three to four months and 20 to 30 people were required for the conversion from one format to the other.
With the acceptance in all three regions, the CTD now avoids the need to generate and compile different regional versions of most of the registration dossier sections.
The CTD was adopted as an ICH topic at the Steering Committee Meeting that took place just before the ICH4 meeting (July 1997).
The CTD specifications reached Step 2 of the ICH process at the Steering Committee meeting in July 2000.
After public consultation, Step 4 was achieved at the ICH5 Conference in San Diego, California in November 2000.
On September 12, 2002 (at the Washington, DC meeting), numbering and section headers were then edited for consistency and use in the eCTD.
The CTD consists of five modules (Module 1 is region specific, and Modules 2, 3, 4, and 5 are intended to be common for all regions): ▸ Module 1 includes administrative information (i.e., application form) and proposed prescribing information.
▸ Module 2 summarizes data included in Modules 3, 4, and 5 and is organized in seven subsections: • CTD The CTD is defined by a general ICH Guideline (M4) and three specific technical guidelines (M4Q, M4S, and M4E, which cover the quality, safety, and efficacy parts of the CTD, respectively).
A Q&A document is associated with each of these four guidelines to facilitate implementation of the CTD.
The ICH parties agreed to implement this harmonized format in the three regions by July 2003.
It is indeed used today in the three ICH regions: it is mandatory in the EU and Japan, and "highly recommended" in the US (the current legislation does not allow the US FDA to make it mandatory).
Moreover, this format is also used in other countries (e.g., Australia, Canada, Turkey, etc.
), and derivatives of the CTD have been developed in other regions (e.g., the ACTD developed by the ASEAN countries).
This harmonized format is indeed one of the great successes of the ICH process.
While the realization of the CTD took many years, there is now a common format for the regulatory submissions across the three ICH regions (Europe, Japan, and the US) and beyond.
This facilitates pharmaceutical companies in making simultaneous filings in the ICH regions as it eliminates the extensive work previously required to convert from one format to another.
However, the CTD is not a "Global Dossier."
It remains only a harmonization of format instead of a harmonization of content.
This initial misunderstanding, certainly created by the desire of many people to accelerate the harmonization of technical requirements, led to a lot of criticism against this new format.
However, the CTD cannot be a truly global identical dossier (including the same information/data/level of detail) if all technical requirements are not fully harmonized.
Moreover, the submission's content may also be different for several reasons, such as different individual regulations, legal status, or requirements, and different manufacturing situations for the three regions.
Indeed, although the CTD provides a common format for regulatory applications, the actual content must still meet local regulations, laws, and statutes.
As a result, despite being presented in the same order, the required content of Modules 2 to 5 may vary by region.
For example, the Integrated Summary of Efficacy/Integrated Summary of Safety (ISE/ ISS) that were requested by the US FDA before the implementation of the CTD are still needed.
Because these integrated summaries are unique to the US, the Table of Contents of the CTD does not specifically include them.
A specific US FDA guidance was released in June 2007 to help pharmaceutical companies decide where to place these US-specific ISE/ISS documents within the structure of the CTD.
To conclude, even if the CTD is "only" an agreed-upon common format for the modular presentation of summaries, reports, and data, it provides obvious advantages.
The CTD allows companies and DRAs to harmonize the terms and way of communication [94] .
Having the same "language" will certainly help the harmonization of content, and ultimately the harmonization of technical requirements.
Indeed, regulatory reviews and communication with the applicant will be facilitated by a standard document of common elements.
In addition, exchange of regulatory information between DRAs will be simplified.
This increase of communication between authorities and between authorities and pharmaceutical companies will obviously facilitate expertise and opinion sharing (related to the safety, efficacy, and quality of the development product) in a timely manner that will ultimately provide benefits to patients by providing quality medicines more quickly on the market.
Like MedDRA, the objective of this project was indeed to support all aspects of pre-and post-approval pharmacovigilance activities as well as communication of regulatory information.
For example, MedDRA and the harmonization of drug dictionaries are critical in the transmission of the ICSR presented in ICH guideline E2B (Data Elements for Transmission of Individual Case Safety Reports).
The transmission of structured data (especially electronically) does imply the use of controlled vocabularies.
Before the ICH initiative, there was no harmonized standard to document information and data on medicinal products.
Regulators in the different regions had established their own standards, which differed in data format, content, language, and applied standard terminology (e.g., terminology used for substances, routes of administration, pharmaceutical forms, etc.).
The WHO Drug Dictionary, or a modified version of this product, was sometimes used.
This lack of internationally harmonized standards related to core sets of medicinal product information and medicinal product terminology made the scientific evaluation, comparison, and exchange of drug data (especially in the area of pharmacovigilance) very difficult.
The activity on the M5 Guideline only began in 2003.
Following the example and success of MedDRA, the ICH Steering Committee at its meeting in November 2003 agreed to launch this new harmonization initiative and to develop a new tripartite guideline that defines the Data Elements and Standards for Drug Dictionaries.
During the ICH meeting in Tokyo, Japan in February 2003, WHO presented a white paper regarding the concepts of a global drug-coding dictionary.
During this meeting, the Steering Committee agreed to convene an informal discussion group in Brussels, Belgium during the ICH meeting in July 2003 to allow for a discussion of this proposal.
An Informal Working Group was then established to develop a Concept Paper and prepare a Business Plan.
The M5 Guideline was released for consultation at Step 2 of the ICH process on May 10, 2005, along with controlled vocabulary lists for routes of administration and units of measurement.
This Guideline was subsequently submitted to the ISO for development under this process.
Step 2 Guideline was updated based upon feedback received during consultation in 2005, as well as additional considerations following its submission to ISO for development as an international standard.
Key parts of this updated guideline will be incorporated into the ICH "Implementation Guide for Identification of Medicinal Products Message Specification," which is currently undergoing development as an ISO standard.
▸ Achievements So Far: For two decades, the ICH process has achieved much success and benefited both DRAs [95-1] and pharmaceutical industries .
More importantly, this harmonization has been pursued in the interest of patients and public health to prevent unnecessary duplication of clinical trials in humans and to minimize the use of animal testing without compromising the regulatory obligations of safety and effectiveness.
To achieve this objective, the goal of ICH has been to promote international harmonization by bringing together representatives from the EU, Japan, and US to discuss and establish common guidelines and standards.
Through the ICH process, considerable harmonization has been achieved in the technical requirements for the registration of pharmaceuticals for human use.
This is now a mature harmonization initiative.
Since its creation, over 50 harmonized guidelines have been developed in the areas of quality, safety, and efficacy in order to eliminate duplication in the development and registration process.
Moreover, common harmonized tools for regulatory communication (MedDRA, CTD, ESTRI) have also been made available.
This represents an extraordinary contribution to the global harmonization of pharmaceutical regulations.
These guidelines already form a solid basis for harmonized application of technical requirements during the registration process.
While the technical output of the ICH process has been very positive, the importance of the unique way in which ICH operates should also be noted.
Indeed, in addition to the practical harmonization of specific technical items, one of the major outcomes of ICH has been to create a forum that allows experts from different countries and with different backgrounds to communicate, exchange, discuss, and share their experience and information in a structured manner.
This is of course an essential first step to any harmonization.
Finally, another important achievement of ICH is to be well recognized on a worldwide basis.
ICH guidelines have been adopted and are now followed outside the ICH regions (e.g., Switzerland, Canada, and Australia, and also many RHIs).
Although ICH's initial focus was the development of guidelines for use in the ICH regions, increased globalization drastically modified the international cooperation environment.
In response to a growing interest from beyond the ICH regions in the use of ICH guidelines, the ICH Steering Committee took the first step in March 1999 of establishing the ICH GCG.
In November 2003, new terms of reference and rules were endorsed for the GCG with the aim of establishing partnerships beyond the ICH regions to promote a better understanding of ICH guidelines globally.
Since then, RHIs from across the globe, but also representatives from DRAs and Departments of Health (DoH) that are either a major source of API or clinical trials data have been invited to participate in the GCG meetings and listen to technical topics at the level of the Steering Committee (at the biannual ICH meetings).
In addition, as per a decision of the ICH Steering Committee in November 2010, invited RHIs and DRAs/DoH may now also nominate technical experts as active members of ICH EWGs.
The implementation of ICH recommendations and standards outside the three ICH regions is indeed very important as it allows industry to better develop medicinal products for the global market.
As a consequence of this expansion to non-ICH regions, training and capacity building have become a key focus of the ICH GCG.
In 2006, the GCG implemented a strategy for addressing training and capacity needs to help ensure the most effective use of resources, opportunities, and the realization of desired outcomes.
Over the past few years, the GCG has responded to numerous requests for training, providing ICH expertise both for the development of training programs and for the delivery of the training itself.
Today, the GCG and the ICH Steering Committee continue to implement new tools to promote a better understanding and use of ICH guidelines and recommendations 96] .
One of the drivers of this success is in the fact that this harmonization process is based on scientific consensus developed between industry and DRA experts.
Before ICH, the industry and regulators never sat at the same table in an international forum to discuss the science of drug development in order to develop best practices across different regions.
This joint effort allows not only for the involvement of the best experts (from both the authorities and pharmaceutical industries) in specific technical discussions, but also for ensuring that discussions take into account both the regional legislations and the practical impact on the development of pharmaceutical products.
This inclusion of both industry and regulators increases commitments to the common goal (i.e., implementation of the ICH tripartite, harmonized guidelines, and recommendations) that has obviously been a key factor in the success of ICH.
The results of a survey on the impact of ICH, presented during the ICH6 Conference in Osaka, Japan, showed a high degree of satisfaction by both DRAs and industry with the completed ICH guidelines, and continuous support from both sides for ICH activities.
The second driver of ICH's success is linked to its well-defined structure and process.
In the beginning years of ICH, the Steering Committee organized its structure around the working groups, which included world-recognized experts.
This decision was critical because it allowed ICH to have very robust scientific and technical recommendations, most of the time accepted and implemented without fundamental criticism.
The Steering Committee has of course also been key as the governing body that gives direction, selects the topics for harmonization, and ensures completion of projects in a timely manner (not always easy when one's goal is consensus).
In addition to the structure, the Steering Committee has also been able to define a process that supported this incredible harmonization task in a structured and organized way, supported by different players such as the ICH Secretariat and coordinators.
Indeed, the stepwise approach that has been put in place for the development of guidelines (the defined five-step process with decision points at Step 2 and Step 4) has been very important.
This approach allowed for the creation of comprehensive drafts by a small number of experts (the best environment for facilitating focused discussion and development of consensus) and public review before implementation (which promotes transparency, and avoids surprises and post-approval issues).
The creation of Concept Papers and Business Plans that the Steering Committee put in place at a later stage are also fundamental to (1) define clear goals, and (2) help to monitor progress towards the predefined goals.
Finally, the review of progress during regular meetings also ensures commitment, follow-up, and therefore the seriousness of this initiative.
Finally, the extension of ICH beyond the ICH regions was possible because the Steering Committee understood early on that its activity could not be restricted to the ICH regions with the increasing globalization of drug development and manufacture.
Indeed, research and manufacture of new products is not confined to the three ICH regions any longer.
Clinical trials are carried out throughout the world and many non-ICH countries are involved in the development and manufacture of pharmaceutical products.
To increase transparency and promote collaboration outside ICH regions, the Steering Committee accepted observers (e.g., Canada), worked with other international organizations (EFTA and WHO), and involved other regions/countries in this process via the ICH GCG, which evolved over time.
All these actions allowed the ICH work to be expanded to most of the regions/countries in the world, and its harmonization benefits to be available worldwide.
The collaboration with non-ICH regions is today one of the priorities of ICH in order to increase commitment of these regions and facilitate worldwide implementation of ICH recommendations.
▸ Limitations and Challenges for the Future: As mentioned above, ICH has been an incredible contributor to the international harmonization of pharmaceutical regulations.
ICH has been successful in achieving harmonization (initially of technical guidelines and then on the format and content of registration applications), and has positively impacted the global development of new drugs.
All parties agree that there is a need to maintain this harmonization in the interest of the patient and public health.
Now that the process and networks are in place, it seems indeed obvious that ICH needs to continue its activities as one of the major players in the international harmonization of pharmaceutical regulations.
Further harmonization activities should be continued in a focused manner.
However, in an evolving international environment, some aspects of this initiative need to be reviewed as new approaches may be needed.
Indeed, some aspects of this initiative may be optimized in order to better handle new and future challenges.
The first challenge of ICH, which the Steering Committee has already acknowledged, is the implementation and maintenance of already developed guidelines.
The current magnitude of successful harmonization actions and the need for these to remain current in a rapidly changing environment calls for focusing more effort on the implementation and monitoring of ICH commitments.
Development of IWGs or task forces to manage this challenge will be key to its success.
This focus on implementation and maintenance should not, however, impact the work on new harmonization topics that still need to be discussed.
These new topics for harmonization need to be rigorously assessed for need (i.e., scientific merit/emerging science) and feasibility (i.e., expected outcome, timeline, and resource requirements).
Another major challenge for ICH is to confirm its worldwide expansion and to continue to develop and strengthen its collaboration and partnership outside the ICH regions in order to better integrate these regions into the ICH process.
At the time of ICH establishment, it was agreed that its scope would be confined to registration of new drugs and medicines in Western Europe, Japan, and the US because the vast majority of the new drugs were developed and manufactured in these three regions.
However, since then, there has been strong involvement of other parts of the world.
Canada and Australia are key markets for pharmaceuticals, and are often involved in global clinical studies.
More recently, the emergence of other countries has been recognized in all areas, including the pharmaceutical industry.
As already recognized by the ICH Steering Committee, the success of ICH in the ICH regions only will not be relevant any longer.
The modification of the landscape obliges ICH to review and broaden its objectives.
The current organization (with the GCG) that initially responded to this increased globalization may not be the most appropriate solution for future stages of development.
The ICH organization and systems need to be reviewed and revised to better serve these broader objectives.
In 2000 (during ICH5), the ICH Steering Committee reviewed its structure and concluded that this structure continues to be appropriate.
However, in order to increase transparency, they welcomed appropriate participation of other interested parties in a flexible and ad hoc manner on topics that also affected them.
A decade later, the new evolving environment requires a bigger revision of its structure and process.
The ICH Steering Committee understands this urgent need and has declared that a new ICH organizational structure will be adopted.
The Steering Committee will set the framework for new rules on governance, decision making, and membership [92-1].
Finally, ICH has to become more proactive in new emerging topics to prevent future disharmony.
The gene therapy topic is an interesting example that demonstrates the previous lack of commitment of ICH to "proactive harmonization."
In September 2002, the ICH Steering Committee established a Gene Therapy Discussion Group (GTDG) in recognition of the rapidly evolving area of gene therapy medicines.
The GTDG developed several ICH consideration documents in this area.
Despite this first positive step/outcome, the development of these consideration papers and the activities towards the development of a new multidisciplinary guideline (Guideline M6) was discontinued in September 2011 because "currently the ICH regions do not have the resources to support the development of further ICH consideration documents" in this domain .
Recently, the ICH Steering Committee started to define a new proactive approach to identify and creatively pursue advancements in science .
If ICH succeeds in these challenges, it will certainly become a real international organization/forum (vs. a multiregional initiative) where proactive discussion on all past and new technical requirements for registration of pharmaceuticals for human use will be discussed.
However, some of these challenges are not new.
ICH acknowledged these challenges years ago and has already tried to resolve them without succeeding (e.g., proactivity), confirming the difficulties of this task.
To face these challenges, ICH needs to revise its structure and engage a new phase in order to address the evolution of regulations and the globalization of drug development and manufacturing, and to promote better proactivity in harmonization.
The ongoing ICH reform is obviously an important milestone toward resolution of current limitations.
Europe was the first major regional bloc established after World War II.
Following this, there have been many regional harmonization activities throughout the world, especially over the past 30 years.
Countries in different regions of the globe have organized themselves into closer economic and political entities.
These movements have transformed the world, both economically and politically, as they create new opportunities and also new challenges (e.g., the management of regulations and standards disharmony).
These regional harmonization initiatives include members with closer interests and needs, compared to global initiatives, allowing further harmonization and cooperation.
This level of harmonization is also essential for developing countries that may not have access to all global harmonization discussions due to sparse resources or lack of expertise.
Regional cooperation can represent their interests and challenges and allow them to be heard at the global level.
ii This level of cooperation is also essential for establishing region-wide pooled procurement systems.
Very diverse initiatives (each with a different scope, objective, structure, and working model) were established due to different cultural, historical, and political contexts.
They range from a simple technical and scientific intergovernmental cooperation model to an advanced integration model.
ii Although all countries are part of WHO, many countries are not represented at ICH where global standards are developed.
However, most of the major regional harmonization initiatives are today represented via the ICH GCG group.
The political and economic development of each region, and sometimes subregions, has indeed shaped the level of harmonization in the pharmaceutical area: ▸ Scenario 1 -Pharmaceutical harmonization in the context of an economical and political integration: In certain regions, economic integration among countries implies integration of pharmaceutical regulations and the harmonization of technical standards.
This degree of integration varies from one region to another (and sometimes from one subregion/country to another), but the harmonization of regulations and policies and standards are very important to create a consistent regional legislative framework and a common certification system for products across regions.
Europe is the best example in terms of advanced harmonization and integration with the development of a centralized system, institutions, and procedures for the registration of medicines to be marketed in the single market.
jj ▸ Scenario 2 -Pharmaceutical harmonization in the context of a general political agreement: Other initiatives follow a general political agreement, mostly signed to avoid conflicts or wars in certain areas in the world or to facilitate economic growth and trade within a region (e.g., Asia-Pacific Economic Cooperation [APEC]), without an integration goal.
The output of this harmonization initiative is variable, but most of the time does not produce a deep harmonization of pharmaceutical regulations because it is not the primary objective of the agreement and therefore the resources and efforts from the countries for this pharmaceutical regulation harmonization are variable.
▸ Scenario 3 -Pharmaceutical harmonization based on a specific intergovernmental agreement: In other regions, a simple technical and scientific intergovernmental cooperation has been established, focusing solely on the harmonization of pharmaceutical regulations.
This is the case of the PANDHR initiative in the Americas where regional integration has not been the objective because countries continue to present very different systems and degrees of development, and there are no political commitments to create a single market.
Countries only cooperate to promote harmonization without creating common legislation and procedure.
This is a scenario that produces good harmonization of pharmaceutical regulations because this is the focus of the initiative, compared to Scenario 2 above, which is a derivative of a broader political agreement.
However, the risk and difficulty of this scenario is its implementation.
Because there is not an ultimate economic and political goal (e.g., developing a single market as in Scenario 1), implementation of the agreed-upon recommendations in the national law is somewhat difficult.
Its success clearly depends on the commitment of each country.
It is important to understand that the scenarios discussed above can also be considered as steps.
Harmonization is a moving process and harmonization initiatives evolve over time.
For example: jj This central system is supported by the national DRAs that also continue to operate their own registration systems for products limited to national markets.
• The European model was initiated to stop war between its countries (Scenario 2), but has in the time since evolved to an integration model to create further economic and political bonds ( Scenario 1).
• ASEAN is another evolving initiative that may follow the European model.
Today, it is between Scenario 1 and 2.
This evolution to a more integrated model is obviously easier when the members are somewhat limited in number and share common geographical, historical, and cultural roots.
It is indeed very difficult to imagine that APEC or PANDRH will evolve towards integration models such as Europe or ASEAN.
The European Community was created after World War II in order to develop a more peaceful Europe by promoting cooperative projects.
Since then, it has rapidly evolved to become a unique partnership between 28 European countries.
The main goal of the community is the progressive integration of Member States' economic and political systems, and the establishment of a single European market based on the free movement of goods, people, money, and services.
The European Union (EU) is not a federation like the United States of America (US), nor is it simply an organization for cooperation between governments like the United Nations.
It is, in fact, unique in that the countries that make up the EU (its "Member States") remain independent sovereign nations, but pool their sovereignty in order to gain a strength and world influence that none could have on their own.
kk With approximately 500 million people (representing 7% of the world's population), the EU is today the world's third largest population after China and India, representing a huge single market.
The EU's Gross Domestic Product (GDP) is now bigger than that of the US, and it is the world's biggest exporter and importer [98] .
Diversity is an important characteristic of the EU as symbolized by its motto, "United in Diversity," with many differences existing among its Member States.
This diversity is a positive attribute of the Union.
However, considering the 24 official languages and the major historic, social, cultural, and economic differences between Member States, its development has not been easy.
Its diversity has also influenced its organization and the way the harmonization process has been structured.
It is therefore very important to understand the history and organization of the EU in order to understand how the European pharmaceutical regulation has been structured over time.
effectively alone and where cooperative action at the community level is indispensable.
These include major health threats and issues with a cross border or international impact, such as pandemics and bioterrorism, as well as issues relating to free movement of goods, services, and people.
Acknowledging that all countries share common values (i.e., ensure high standards of public health and equity in access to quality healthcare), it is therefore logical that the EU has developed common standards for medicines.
Moreover, the implementation of a single market requires harmonization of the pharmaceutical market.
The ability to travel freely, or to live and work anywhere in the EU, only makes sense if EU citizens can be sure to obtain the same level of healthcare wherever they go.
Therefore, a number of European Community rules have been adopted to ensure the highest possible degree of protection of public health while promoting the free movement of medicines in an internal market without barriers.
The European Commission (EC)'s role is not to mirror or duplicate national activities, but to coordinate them.
Work on healthcare at the community level adds value to Member States' actions, particularly in the area of illness prevention, including activity on the safety and efficacy of medicines [99] .
Today, the European pharmaceutical system is well developed and the vast majority of requirements have been harmonized.
This successful European cooperation in pharmaceuticals is also recognized on a worldwide basis due to its major contribution to the global harmonization of pharmaceutical regulations (via its active involvement in international initiatives such as ICH and WHO).
Today the EU is composed of 28 Member States, but the size of the EU has changed over time as it has continually expanded since European integration first began in 1951 with only six countries ( Table 4 ).
The final three enlargements (in 2004, 2007, and 2013 ) expanded the EU Member States from 15 to 28, and were rooted in the collapse of communism.
It was a historic advancement that offered an unexpected and unprecedented opportunity to extend the Union into Central and Eastern Europe.
Today, the landmass of the EU covers 4 million km 2ll and can rightly claim to represent a continent (Plate 1).
Stretching from the Atlantic Ocean to the Black Sea, it reunites Western and Eastern Europe for the first time since they were separated by the Cold War.
In the future, the EU will continue to grow as an increasing number of countries express interest in membership.
The Treaty on European Union sets out the conditions for such accession (Articles 6 and 49): Any European country which respects the principles of liberty, democracy, respect for human rights and fundamental freedoms, and the rule of law may apply to become a member of the Union.
The applicant country must meet a core of criteria (e.g., having stable institutions and a functioning market economy) in order to ensure that EU principles will be respected and that EU rules and procedures will be effectively implemented.
This is a long and rigorous process that starts when the country submits an application to the Council.
Today, Iceland, the former Yugoslav Republic of Macedonia, Montenegro, Turkey, Albania, Bosnia and Herzegovina, Kosovo, and Serbia are candidates to join the EU, some of these countries being in more advanced stages of negotiation with the EU than others.
Membership is only granted when the necessary requirements are met and when candidate countries have demonstrated that they will be able to fulfill their part as members.
in the EU regulatory network.
For example, the IPA program supported the participation of nominated representatives of the concerned countries in selected meetings and training courses as observers.
The program also supported the organization of conferences to prepare the countries for integration into the European regulatory network for medicines.
These activities helped identify areas where additional action might be needed to ensure the smooth transposition of the EU "acquis communautaire" mm into the national legislation of these future EU Member States.
▸ The Specific Case of Iceland, Liechtenstein, and Norway: In July 2009, Iceland submitted its application for EU membership and the accession negotiations have now been opened.
Norway, despite two failed attempts by referendum to enter the European Community in 1972 and the EU in 1994, remains undecided whether or not it will apply once again for EU membership.
Presently, however, neither Norway nor Liechtenstein are candidates for EU membership.
However, even if these three countries are currently not part of the EU, it is important to note that they have a specific strong relationship with the Union through the European Economic Area (EEA) Agreement that entered into force on January 1, 1994.
This agreement allows these three EEA European Free Trade Association (EFTA) States nn to participate in the EU internal market on the basis of their application of internal market relevant acquis.
oo All new relevant Community legislation is dynamically incorporated into the Agreement and thus applies throughout the EEA, ensuring the homogeneity of the EU internal market.
Also, the EEA Agreement allows for EEA-EFTA States to participate in the internal market's relevant Community programs and agencies, albeit with no right to vote.
In the pharmaceutical sector, Norway, Iceland, and Liechtenstein have adopted the complete Community acquis on medicines, and are consequently parties to the European procedures.
In the case of the Centralized Procedure, the representatives from these three countries do not vote, but their position is stated separately in the opinion, where relevant, in the minutes of the Committee and in the case of divergent opinions appended to the Committee's opinion.
Their position is not counted in reaching the Committee's opinion [103] .
According to Decision No.
74/1999 of the EEA Joint Committee (which entered into force on January 1, 2000), when decisions on approval of medicinal products are accepted by the Community, these three countries will accept corresponding decisions on the basis of the relevant acts.
The Liechtenstein authorities have transposed into their national legislation a provision that makes Commission Decisions automatically applicable on their territory.
However, legally mm "Acquis communautaire" is a French term referring to the cumulative body of EU laws, comprising the EC's objectives, substantive rules, policies, and in particular, the primary and secondary legislation and case law -all of which form part of the legal order of the EU.
nn The European Free Trade Association (EFTA) is an intergovernmental organization set up for the promotion of free trade and economic integration to the benefit of its four Member States: Iceland, Liechtenstein, Norway, and Switzerland.
Although Switzerland has many agreements with the EU, it is today not part of the EEA Agreement due to the rejection of accession by the Swiss people.
oo The EEA Agreement is concerned principally with the four fundamental pillars of the internal market, "the four freedoms" (i.e., freedom of movement of goods, persons, services, and capital).
binding acts from the Community (e.g., Commission Decisions) do not directly confer rights and obligations in Norway and Iceland, but first have to be transposed into legally binding acts in these states [104] .
Since the end of World War II, the EU has steadily become more established and organized.
The unique European model (not a federation but a more integrated than simple cooperation between governments) requires a complex organization that not only protects the independent sovereignty of the Member States, but also allows for the delegation of some of decision-making powers to shared supranational institutions.
Today, the structure in place was specifically designed to represent the interests of the Community, the Member States, and the European citizens.
Within this overall European structure and context, many special domains have been harmonized and organized to support the functioning of the single market.
A number of institutions, committees, and technical bodies ( Table 5 ) play a significant role in the European pharmaceutical system.
The roles and characteristics of these are briefly described in the following sections.
▸ The European Parliament is the directly elected EU institution that represents the interests of the EU's citizens.
Its 766 members are elected once every five years.
Its origins go back to the 1950s and the founding treaties, but the Lisbon Treaty significantly increased its role in the decision-making process and budget approval.
Its legislative powers were reinforced by the extension of the co-decision procedure.
Today the European Parliament is firmly established as a co-legislator, has budgetary powers, and exercises democratic control over all the European institutions.
Its work is organized through a system of specialized committees that review and prepare legislative proposals and reports to be presented at the plenary assembly.
The Committee on the Environment, Public Health and Food Safety is responsible for the legislation covering pharmaceutical products and the EMA.
The European Parliament has three working locations: Brussels (Belgium), Luxembourg, and Strasbourg (France).
Luxembourg is home to the administrative offices of the General Secretariat.
Meetings of the entire Parliament, known as "plenary sessions," take place in Strasbourg and sometimes in Brussels.
Committee meetings are also held in Brussels.
▸ The Council of the European Union represents the individual Member States.
It meets in different configurations and is attended by one minister from each of the EU's national governments (depending on the agenda).
Health-related discussions are handled by the Employment, Social Policy, Health and Consumer Affairs Council (EPSCO).
As with the European Parliament, the Council was set up by the founding treaties in the 1950s.
It is a key decision-making body that, among other responsibilities (e.g., coordination of the EU's economic policies and foreign and security policy) shares lawmaking and budgetary powers with the European Parliament.
Its work is facilitated by the Committee of Permanent Representatives (COREPER), which is responsible for preparing the work of the Council of the European Union (all issues must pass through COREPER before they can be included in the agenda for an EU Council meeting).
This committee consists of the Member States' ambassadors to the EU.
These permanent national representatives and their team are located in Brussels, Belgium, and protect national interests at the EU level.
▸ The European Commission (EC) is independent of national governments as it represents and upholds the interests of the EU as a whole.
It acts as the "guardian of the Treaties" but remains politically accountable to the Parliament.
Like the Parliament and Council, the EC was set up in the 1950s under the EU's founding treaties.
A new Commission, which is formed by a President (designated by the Member States and approved by the Parliament) and the "commissioners" (each of them responsible for a specific policy area), is appointed every five years.
Its role is to draft proposals for new European laws (which are presented to the European Parliament and the Council for adoption).
It is also the EU's executive arm because it is responsible for implementing the decisions of the Parliament and the Council.
This means managing the day-to-day business of the EU: implementing its policies, running its programs, allocating its funds, and representing the EU in international negotiations.
The day-to-day running of the Commission is done by its administrative officials, technical experts (via its various committees and groups), translators, interpreters, and secretarial staff (which represent more than 20,000 people).
This staff is organized in departments, known as Directorates-General (DG), and "services" (such as the Legal Service).
The overall coordination is provided by the Secretariat-General.
Each DG is responsible for a particular policy area and is headed by a Director-General who is answerable to one of the commissioners.
The regulation of medicinal products was previously under the DG Enterprise and Industry, but this policy area has been transferred to the DG Health and Consumers (SANCO) as of March 1, 2010.
The Commission is based in Brussels (Belgium), but it also has offices in Luxembourg, representation in all EU countries, and delegations in many capital cities around the world.
This "institutional triangle" produces the policies and laws (such as European pharmaceutical legislation) that apply throughout the EU.
The Court of Justice upholds the rule of these European laws and makes sure that this EU legislation is interpreted and applied in the same way in all EU countries.
The other institutions of the EU (the European Council and the Court of Auditors) are critical for the functioning of the EU, but are not directly involved with the development and harmonization of pharmaceutical legislation.
The EU institutions are supported by a number of other bodies (e.g., the European Central Bank, the European Ombudsman, etc.).
Specialized agencies (e.g., the EMA, the European Centre for Disease Prevention and Control, and the Executive Agency for Health and Consumers) have also been established to handle certain technical, scientific, or management tasks.
This Agency is headed by an Executive Director (who is its legal representative responsible for all operational and staffing matters) and has a staff of about 900 full-time members [107] .
The Management Board is the supervisory body responsible for setting the Agency's budget, approving the annual work program, and ensuring that the Agency works effectively and cooperates successfully with partner organizations across the EU and beyond.
In addition to its staff, the EMA is composed of seven committees that conduct the main scientific work of the Agency.
These committees and their characteristics are reviewed below: • human use.
The CHMP plays a vital role in the EU marketing procedures as it is responsible for: -Conducting the initial scientific assessment and issuing opinions on an MAA for medicines registered via the Centralized Procedure (these opinions are used by the EC as a basis for its legally binding decisions) -Coordinating post-marketing activities for medicines registered via the Centralized Procedure -Arbitrating disagreements between Member States during Mutual Recognition and Decentralized Procedures (Arbitration Procedure) -Acting in referral cases, initiated when there are concerns relating to the protection of public health or where other community interests are at stake (Community Referral Procedure) This Committee (and its working parties) also provides assistance to companies during development, prepares scientific and regulatory guidelines, and cooperates with international partners on the harmonization of regulatory requirements for medicines.
• The Committee for Orphan Medicinal Products (COMP), established by Regulation (EC) No 141/2000, is charged with reviewing applications from companies seeking "orphan medicinal product designation" for products they intend to develop for the diagnosis, prevention, or treatment of rare diseases (so-called "orphan drugs").
This committee is also responsible for advising the European Commission on the establishment and development of a policy on orphan medicinal products in the EU, and assists the Commission in drawing up detailed guidelines and liaising internationally on matters relating to orphan medicinal products.
• submitted by pharmaceutical companies, and to adopt opinions on these plans.
This includes assessing applications for full or partial waivers and assessing applications for deferrals of pediatric studies.
This Committee also assesses data generated in accordance with the agreed-upon PIPs, provides opinions on the quality, safety, or efficacy of a medicine for use in the pediatric population (at the request of the CHMP or a Member State), and supports the development of the European Network of Pediatric Research at the European Medicines Agency (Enpr-EMA).
ss • The Committee for Advanced Therapies (CAT) is a multidisciplinary committee established in accordance with Regulation (EC) No 1394/2007.
It is responsible for providing scientific opinions on advanced-therapy medicinal products (ATMPs) and any scientific questions related to this field.
For example, it prepares a draft opinion on each ATMP application before the CHMP adopts a final opinion on the granting, variation, suspension, or revocation of a marketing authorization for the medicine concerned.
• The Committee for Medicinal Products for Veterinary Use (CVMP) is responsible for preparing the Agency's opinions on all questions concerning veterinary medicinal products.
• The Pharmacovigilance Risk Assessment Committee (PRAC) is the last committee established by the EMA to implement the new EU pharmacovigilance legislation.
It is responsible for assessing and monitoring safety issues for human medicines.
This includes the detection, assessment, minimization, and communication relating to the risk of adverse reactions, while taking the therapeutic effect of the medicine into account.
It also has responsibility for the design and evaluation of post-authorization safety studies and pharmacovigilance audits.
Its recommendations are considered by the CHMP when it adopts opinions for centrally authorized medicines and referral procedures, and by the CMDh when it provides a recommendation on the use of a medicine in Member States.
These EMA scientific committees are comprised of members of all EU and EEA-EFTA states (Iceland, Liechtenstein, and Norway); some committees include patients' and doctors' representatives.
They are supported by a number of working parties and related groups that have expertise in a particular scientific field.
The Committees consult with them on scientific issues relating to their particular field of expertise and delegate to them certain tasks associated with the scientific evaluation of an MAA or drafting and revision of scientific guidance documents.
In particular, the CHMP is supported by an important number of groups (i.e., the Biologics Working Party, the Scientific Advice Working Party, or the numerous scientific advisory groups specialized by therapeutic area); some are standing parties and some temporary groups.
All these groups are made up of members selected from the European expert list maintained by the EMA.
Indeed it is worth noting that the EMA evaluation system works through a network of European experts made available to the Agency by the national DRAs of all EU Member States and of the three EEA-EFTA States (Iceland, Liechtenstein, and Norway).
This system brings together the scientific resources and expertise of all these countries in a network of over 4,500 European experts who serve as members of the Agency's scientific committees, working parties, or scientific assessment teams.
The EMA is today considered as the model of fruitful cooperation between national DRAs, working together within a Community body to serve Community purposes.
Also, to ensure that the European system is accessible to everyone, in 2005 the EMA launched a dedicated office to provide special assistance to small-and medium-sized enterprises ( -For the collection, preparation, storage, distribution, and appropriate use of blood components in blood transfusions -For the transplantation of organs, tissues, and cells The role of the EDQM is essential in Europe in facilitating mutual recognition of quality control tests carried out on medicines and ensuring that patients receive the same quality of pharmaceutical products throughout Europe.
There is a substantial amount of interaction between the EMA and the EDQM.
For example, the EDQM representatives participate as observers of the EMA's Quality Working Party (QWP) and Biologics Working Party (BWP) meetings, the GMP Inspection Services Group meetings, as well as HMPC meetings at the EMA.
It is important to note that the European Member State plays a significant role in the European pharmaceutical system.
The EMA works closely with the 28 EU Member States as well as the EEA-EFTA countries (Norway, Iceland, and Liechtenstein).
Member State representatives are members of the Agency's management board while the Agency's scientific committees and its network of 4,500 scientific experts are nominated by the Member States.
Without their support and expertise, the EMA would be unable to deliver on its responsibilities and mandate as laid down in European legislation.
It is also important to realize that many medicines available in Europe are not authorized by the EC on the recommendation of the EMA.
Many products are still approved and supervised by the national DRAs via the Mutual Recognition Procedure, the Decentralized Procedure, or National Procedure.
To coordinate their efforts, the Member States established the Heads of Medicines Agencies (HMA) group, which is a network of the heads of the national DRAs.
This HMA is comprised of more than 40 national agencies, some also having responsibility for veterinary products, medical devices, and cosmetics, and also pricing and reimbursement of products.
The EMA is also a member of the HMA.
The first meeting of the HMA took place in Amsterdam (The Netherlands) at Schiphol Airport, on February 20, 1996.
The HMA is focused on EU coordination and harmonization, decision making, and consensus on strategic issues of the European Medicines Regulatory Network.
Its aim is to foster an effective and efficient European Medicines Regulatory system.
More specifically, it works towards the following key objectives [111]: ▸ Addressing key strategic issues for the European Medicines Regulatory Network, such as the exchange of information and sharing of best practices ▸ Collectively being responsible for all areas of medicines regulation, including the Mutual Recognition and Decentralized Procedures ▸ Focusing on the development, coordination, and consistency of the Network ▸ Supporting the Network by providing high-quality professional and scientific resources ▸ Providing a focus for making the most effective use of scarce resources across the Network, such as developing and overseeing arrangements for work sharing To fulfill these objectives, the HMA has been working on both general issues (i.e., strategy for telematics, and regulatory and scientific training) and technical and scientific topics (i.e., harmonization of clinical trials, coordination of products testing, and European Risk Management Strategy) is support of the European Medicines Regulatory Network.
The HMA's website contains the MRI Product Index database, which includes all medicines approved in the Member States according to the Mutual Recognition Procedure.
One interesting program that has been developed is the Benchmarking of European Medicines Agencies (BEMA).
The BEMA program assesses the systems and processes in individual agencies against a set of agreed-upon indicators.
This is a good opportunity to exchange best practices and ensure harmonization of practices (i.e., assessment, inspection, etc.)
between regulators within the Network.
Coordination among the national competent authorities is not a simple task due to the heterogeneity of these national organizations.
Indeed, these authorities differ in size, historic origins, roles, resources, expertise, and funding.
Acknowledging these differences and also the legal, scientific, social, political, and financial challenges facing the Network, the HMA adopted a strategic paper that provides a plan of action for 2011-2015 [112] .
This second plan (the first one covered 2006-2010), highlights a number of key themes and areas of focus (i.e., pharmacovigilance, clinical trials, and communication) and also the need for international cooperation.
The HMA is supported by the Heads of Medicines Agencies Management Group, the Permanent Secretariat, and working groups covering specific areas of responsibility.
Iceland, and Liechtenstein) appointed for a renewal period of three years.
Observers from the European Commission and accession countries also participate in the meetings.
It also has many interactions with the EMA to facilitate harmonization in several areas (i.e., pediatric regulation, variation regulation, and pharmacovigilance).
It holds monthly meetings at the EMA (which also provides the Secretariat of the CMDh).
In practice, approximately half of the time of the CMDh meeting is dedicated to discussions on procedural and regulatory issues, development of guidance documents, and oversight of the activities of the various CMDh subgroups and working groups, while the other half is devoted to trying to reach agreement for applications referred to the CMDh in the case of disagreement between Member States.
The gradual harmonization of pharmaceutical regulation in the EU has been dictated by the development and expansion of the community.
It represents a good example of successful harmonization and also demonstrates the influence of the political and economical decisions on the harmonization process and its outcomes.
▸ The Birth of the European Union: The historical roots of the EU lie in World War II.
Following this bloody, horrific war, several leaders in Europe wanted to ensure that war could never happen again.
Their goal was to develop a peaceful Europe and to stop the frequent wars via the promotion of cooperative projects.
This initiative has been critical but not easily accomplished due to the post-war geopolitical situation and the beginning of the 40-year-long Cold War that split Europe into East and West.
On September 19, 1946, Winston Churchill called for a "kind of United States of Europe" in a speech given at the Zurich University.
Many attempts at cooperation were made in the following years (e.g., the Customs Convention between Belgium, Luxembourg, and the Netherlands, and the Organization for European Economic Cooperation).
In 1949, West European nations created the Council of Europe.
uu It was a first step towards cooperation between them, but some countries wanted to go ever further.
On May 9, 1950, France's Foreign Minister Robert Schuman presented a plan for deeper cooperation and for the creation of an organized Europe, which would prove indispensable to the maintenance of long-term peaceful relations.
This proposal (known as the "Schuman Declaration") is considered to be the beginning of the creation of what is now the EU.
May 9 has since been designated as "Europe Day" to celebrate this event.
The idea of this plan (inspired by Jean Monnet, top advisor of the French government) was to promote European peace by (1) eliminating the age-old opposition of France and Germany, and (2) creating a framework and organization open to the participation of the other countries in Europe.
It proposed that the Franco-German production of coal and steel be placed under a common high authority and that this new productive unit be open to all European countries willing to participate.
The double objectives of this proposal were (1) to set up common foundations for economic development as a first step in the federation of Europe, and (2) to make war materially impossible [113] .
Based on the Schuman plan, six countries (Germany, France, Italy, The Netherlands, Belgium, and Luxembourg) signed the Treaty of Paris on April 18, 1951 to establish the European Coal and Steel Community (ECSC) in order to run their coal and steel industries under a common management.
It is important to note that the independence and the powers of the high authority have been critical, and differentiated the EU from other traditional intergovernmental organizations.
Indeed, the establishment of the ECSC was the first step towards a supranational Europe.
For the first time the six Member States of this organization relinquished part of their sovereignty, albeit in a limited domain, in favor of the European Community.
Building on the success of their first treaty, the six countries decided to expand cooperation to other economic sectors.
On March 25, 1957, under Belgian Minister for Foreign Affairs, Paul-Henry Spaak, they signed the Treaty of Rome, establishing the European Economic Community (EEC) (or "common market") allowing persons, goods, services, and capital to move freely across borders.
The same day, they also signed a second treaty to create the European Atomic Energy Community (EURATOM).
Despite the construction of the Berlin Wall in August 1961, which increased the division between the East and the West, the cooperation between European countries continued to increase in different areas (e.g., food and agriculture, aerial navigation, the environment, etc.).
On July 1, 1968, the six countries created the world's largest trading group by removing customs duties on goods imported from each of the six countries to the others, allowing free cross-border trade for the first time.
They also applied the same duties on their imports from outside countries.
This EU Internal Market was reinforced in 1986 with the adoption of the "Single European Act" (which entered into force on July 1, 1987) to remove the final obstacles.
In 1993, the single market and its four freedoms (movement of goods, services, people, and money) had finally been fully established.
Additional agreements, such as the Schengen Agreement in 1995, have since been signed to further facilitate movement within Europe.
Today, this single market represents the core of the EU.
In 1991, following the collapse of communism across Central and Eastern Europe and the dissolution of the Pacte de Varsovie, a decade began that would be critical for the future of Europe.
On December 13, 1997, EU leaders agreed to start the process of membership negotiations with 10 countries of Central and Eastern Europe (Bulgaria, the Czech Republic, Estonia, Hungary, Latvia, Lithuania, Poland, Romania, Slovakia, and Slovenia).
The Mediterranean islands of Cyprus and Malta were also included.
In December 2000, treaty changes agreed to in Nice (France) and finally signed on February 26, 2001 were entered into force on February 1, 2003 and opened the way for enlargement of the EU by reforming its institutions and voting rules.
This enlargement to the Eastern European countries became effective on May 1, 2004 and January 1, 2007.
Six years later, on July 1, 2013, the accession of Croatia brought the number of Member States to 28 countries.
A single currency (Euro [€]) was introduced on January 1, 1999 in 11 countries (joined by Greece in 2001) for commercial and financial transactions only.
Notes and coins were introduced in January 2002.
This introduction of the single currency followed a long stepwise process that started in the 1970s with the creation of the "Exchange Rate Mechanism" to maintain monetary stability.
The next important step of integration (i.e., development of a political union with fully functioning institutions) took time and faced many challenges.
The debate on the "constitutionalization" of Europe started in 1984 when the European Parliament adopted Altiero Spinelli's report proposing, in a "draft treaty on European Union," a fundamental reform of the European Community.
In the 1990s, two important treaties transformed the community: ▸ The Treaty on European Union (signed in Maastricht [The Netherlands] on February 7, 1992, entered into force on November 1, 1993) represented a new stage in European integration as it opened the way to political integration.
It was a major EU milestone, introducing the concept of European citizenship and setting clear rules for the future single currency and for foreign and security policy.
Under the treaty, the name "European Union" officially replaced "European Community."
▸ The Treaty of Amsterdam (signed on October 2, 1997, entered into force on May 1, 1999), built on the achievements of the treaty from Maastricht, laid down plans to reform EU institutions, gave Europe a stronger voice in the world, and concentrated more resources on employment and the rights of EU citizens.
Building on this transformation of the Community, the adoption of a European constitution and major institutional reform became an important topic of discussion for two reasons: ▸ Succeeding treaties have spurred progress in the building and reforming of Europe and its institutions.
This long process marked by ever-closer integration progressively transformed Europe from an economic community to a political union.
▸ The combination of the various treaties and protocols signed over 50 years has made the European structure and legislation more and more complex.
Although the EU will certainly continue to grow, it is difficult to predict the next steps of integration due to the current geopolitical situation and the instability caused by the financial crisis.
The evolution of pharmaceutical regulation harmonization and cooperation in Europe represents an excellent example and model that needs to be analyzed in detail as it shows the different important steps necessary for harmonization success.
A large body of legislation has been developed, with progressive harmonization requirements since the 1960s.
The first European Directive related to pharmaceutical products (Directive 65/65/EEC [114]) was signed on January 26, 1965.
This text provides the European definitions of a "Medicinal Product" and a "Substance" and set up some fundamental principles for the creation of the European pharmaceutical system such as: ▸ No medicine may be placed on the market of a Member State unless a marketing authorization has been issued by the competent authorities following the review of an application submitted by the person responsible for placing that product on the market.
▸ Quality, safety, and efficacy are the basis for the evaluation of an application by the competent authorities.
▸ The information included in the application should be updated on a regular basis.
Following this first Directive, many texts followed over the years to further detail the European principles and requirements led by Directive 65/65/EEC, to organize and structure the European system, and to add new requirements related to specific types of products or emerging problems.
Major texts and important steps in the development of the European pharmaceutical system are discussed below.
However, it is important to note that many other legislative texts, guidelines, and other recommendations (including harmonized quality, and nonclinical and clinical requirements) have been prepared and released over the years to support the major legislatives texts listed in this section.
Directive 65/65/EEC was complemented by two additional Directives (Directives 75/318/EEC and 75/319/EEC) in May 1975 to provide further details on the analytical, nonclinical, and clinical standards and protocols to be applied during the development of medicines, and how the results of such studies should be presented in the MAA.
Directive 75/319/EEC also established the idea of Expert Reports (that would later influence the structure of the CTD), the CPMP (that would later be part of the EMA), and the first Multi-State Licensing Procedure, which would then evolve progressively to become the current Mutual Recognition Procedure (MRP).
Further clarification of requirements was provided by Directive 83/570/EEC (which also modified the Multi-State Licensing Procedure to facilitate its use), and Directive 87/21/ EEC (which established the notion of combination products and created a route for abridged applications in case of generics and literature-based applications).
In 1987, Directive 87/22/EEC established the Concertation Procedure, which provided a simple community-wide licensing opinion (via a mandatory referral to the CPMP) for all new biotechnology products and optionally for high technology medicinal products [115] .
It was an important new step in building the European pharmaceutical system as this new procedure (the forerunner of the current Centralized Procedure) required further cooperation between National DRAs compared to the Multi-State Licensing Procedure previously established.
However, both procedures were still based on voluntary cooperation between the relevant national authorities, and each Member State remained solely responsible for granting the Marketing Authorization.
In 1989, legislators extended the scope of the previous Directives to specific types of products: vaccines, toxins or serums, and allergens (Directive 89/342/EEC); radiopharmaceuticals (Directive 89/343/EEC); and products derived from human blood or human plasma (Directive 89/381/EEC).
Additionally, on April 23, 1990, Directive 90/219/EEC laid down the first common measures related to genetically modified organisms (GMOs); several additional texts have since then been released on this topic over the years.
Finally, extension of the scope of the harmonization of homeopathic products was only made in 1992 via the adoption of Directive 92/73/EEC.
In 1991, Directive 91/356/EEC, which laid down the principles and guidelines of GMP, was adopted.
In 1992, four new Directives covering the distribution of medicines were adopted to further establish the EU Internal Market and facilitate the free movement of products.
They especially harmonized wholesale distribution (Directive 92/25/EEC), the classification of products as subject to medical prescription or not (Directive 92/26/EEC), the labeling of products (Directive 92/27/EEC), and advertising principles (Directive 92/28/EEC).
Despite all these texts adopted since 1965, the resulting progress of completing the single market in pharmaceuticals was not satisfactory.
It was therefore decided to fundamentally improve the authorization procedures.
A new European pharmaceutical system was then created in 1993 (but only implemented in January 1995).
This new system, still in place today, is based on two major texts that established, for the first time, "European decisions" binding to the Member States: ▸ Following the adoption of these European procedures, it was necessary to harmonize the system to vary the terms of marketing authorization.
This was done via the adoption of two Regulations in 1995: Regulation 541/95 (for the MRP) and Regulation 542/95 (for the Centralized Procedure).
Additionally, acknowledging the increased complexity of the European pharmaceutical legislation, it was agreed to assemble all previous Directives in one single text.
This codifying Directive, Directive 2001/83/EC adopted on November 6, 2001, was necessary because all the Directives adopted since 1965 had been frequently and substantially amended.
Therefore, this Directive regroups all legal requirements agreed-upon since 1965 (except requirements and legal provisions provided by Regulation 2309/93).
This new Directive has already been amended several times since its adoption, some of these amendments being the result of a major general review of the legislation and system discussed below.
In 2000, as directed by Regulation 2309/93 (Article 71), the Commission conducted a major review of the operation of the new system implemented in 1995.
The goal of this audit, contracted out to independent auditors, was to review the extent to which the results achieved over the first five years have met the objectives (namely to enhance the creation of a single market in medicinal products, while ensuring the protection of public health and the development of the pharmaceutical industry).
The audit report [116] , known as the "Cameron McKenna Andersen Report," includes the results of the extensive consultation carried out involving individual companies, all DRAs responsible for the authorization of medicines and the EMEA, patient and professional associations, trade associations, and relevant ministries.
This audit highlighted the overall satisfaction with this new system, as both procedures had been perceived as contributors in both a qualitative and quantitative way to create a harmonized European Community pharmaceutical market.
Ninety-two percent (92%) of companies and 97% of DRAs in the EU were satisfied or very satisfied with the Centralized Procedure.
There was also general recognition of the very considerable contribution made by the EMEA and the EU telematics strategy to the successful operation of the system.
However, this report also identified several issues and listed several possible improvements to the system.
These criticisms were primarily directed towards the MPR for which it was agreed that the lack of real supervisory, management support, and liaison between Member States had altered the application of the central principle of mutual recognition.
Concerned Member States were continuing to assess applications.
Regarding the Centralized Procedure, it was felt that it should be opened up to a broader range of products and that the "decision-making process" of the Commission (post-CPMP opinion) should be reduced and improved.
Finally, it was also interesting to note that the European procedures had not yet produced any real dividends in terms of cost efficiencies through economy of scale.
There was also a need to reduce the administrative burden where there were no public health implications (e.g., in relation to minor variations to existing approvals).
This evaluation of the regulatory processes was not only very timely with the emerging technical challenges (e.g., gene therapies, etc.
), but also with the political challenges in preparation for EU expansion [117] .
Indeed, there was little doubt that the upcoming major enlargement of the EU (in 2004, and involving 10 additional countries) would accentuate the weaknesses of the system if both the structural and process issues were not resolved by then.
Based on this review of the EU pharmaceutical legislation and various public hearings, the EC concluded that on the whole the system had proven appropriate and suitable for its purpose and therefore it was recommended that it keep its main principles and structures.
However, the EC also proposed several adaptations of the system and legislation in order to better achieve four major objectives [118]: ▸ Assure a high level of public health protection, notably by increased supervision of the market through the strengthening of inspection procedures and of pharmacovigilance.
▸ Complete the single market for pharmaceutical products, taking into account the stakes of globalization, and establish a regulatory and legislative framework that favors the competitiveness of European industry.
▸ Respond to the challenges of the future enlargement of the EU.
▸ Rationalize and simplify the system and improve its overall coherence and visibility and the transparency of its procedures.
These proposals, such as opening up the Centralized Procedure to a broader range of products, establishment of a fast track procedure and conditional authorization, improvement of the transparency of the system, strengthening pharmacovigilance and supervision requirements, abolition of the renewal, control of the effective use of marketing authorization with the "Sunset Clause," improvement of the decision-making process after CPMP opinion, re-organization and increase of the role of the EMEA and its committees, major modifications to the MRP and creation of the Decentralized Procedure, and harmonization of data protection periods [119,120], have been further debated with the Parliament and the Council over subsequent years.
Most of them have finally been implemented via the adoption of new or revised legislation and/or guidelines.
One of the major legislative impacts has been the adoption of Regulation ( Finally, in addition to these critical texts that created the European system and general requirements, it is worth mentioning the following additional legislative texts adopted over the past 10 years on important specific subjects (see Part I-2.
The current European pharmaceutical system has progressively developed over the years via the adoption of agreed-upon policies.
Since 1985 many texts have been adopted with the aim of achieving a single market for pharmaceutical products.
As noted above, several European institutions and technical bodies, together with the EU Member States, are involved in the harmonization of European pharmaceutical regulation.
The European harmonization process lies in the adoption of EU laws [121] that can be categorized as follows: ▸ The "primary" legislation: The Treaties are binding agreements between EU member countries.
They state EU objectives, rules for EU institutions, how decisions are made, and the relationship between the EU and its Member States.
They also form the basis or ground rules for all EU actions.
This means that every action taken by the EU is founded on treaties that have been approved voluntarily and democratically by all EU member countries.
For example, if a policy area is not cited in a Treaty, a law cannot be proposed in that area.
▸ The "secondary" legislation: This is derived from the principles and objectives set out in the Treaties.
It includes the following texts: • Regulations are the most direct form of EU law.
As soon as they are passed, they have binding legal force throughout every Member State and must be applied in its entirety across the EU.
National governments do not have to take action themselves to implement EU regulations (i.e., regulations do not require any transposition by the national authorities).
• Directives are legislative acts that set out a goal that all EU countries must achieve.
National authorities have to adapt their laws to meet these goals, but are free to decide how to do so.
vv Directives are used to bring different national laws in line with each other, and are particularly common in matters affecting the operation of the single market (e.g., product safety standards).
They may concern one or more Member States, or all of them.
• Decisions are individual acts relating to specific cases and are addressed to specific parties.
They are binding only on those to whom they are addressed (e.g., an EU country or an individual company), and are directly applicable (no need for implementation into national law).
Decisions can come from the EU Council (sometimes jointly with the European Parliament) or the EC.
vv Each directive specifies the date by which the national laws must be adapted (giving national authorities room to maneuver within the deadlines necessary to take account of differing national situations).
• Recommendations are not binding, but allow the institutions to make their views known and to suggest a line of action (without imposing any legal obligation on those to whom it is addressed).
• Opinions are not binding.
They are an instrument that allows the institutions to make a statement in a nonbinding fashion; in other words, without imposing any legal obligation on those to whom it is addressed.
They can be issued by the main EU institutions (Commission, Council, Parliament), the Committee of the Regions, and the European Economic and Social Committee.
The European Parliament and the Council of the EU share legislative power, which means they are empowered to adopt European laws (directives and regulations).
In principle, it is the Commission that proposes new "legislative texts," ww but it is the Parliament and Council that adopt them.
The Commission and the Member States then implement them, and the Commission ensures that the laws are correctly applied.
The vast majority of European laws are adopted jointly by the European Parliament and the Council using a procedure known as "co-decision."
xx This means that the directly elected European Parliament has to approve EU legislation together with the Council (the governments of the 28 EU countries).
In addition to this "ordinary legislative procedure," there are also other special legislative procedures (which apply only in specific cases) where the Parliament has only a consultative role.
The requirements and procedures for the marketing authorization of medicinal products, as well as the rules for variations to the terms of marketing authorizations and for the constant supervision of products after they have been authorized, are primarily laid down in Directive 2001/83/EC and Regulation (EC) No 726/2004 (and their subsequent amendments).
These texts additionally lay down harmonized provisions in related areas such as the manufacturing, wholesaling, or advertising of medicinal products for human use.
In addition, various laws have been adopted to address the particularities of certain types of medicinal products and promote research in specific areas.
In addition to the legal texts, many additional Community or international documents and recommendations have been developed and support the harmonization and cooperation in the EU.
The "Introduction and General Principles" of Annex 1 of Directive 2001/83/EC, as ww The European Commission is the only institution empowered to initiate legislation.
Before proposing a new text, it assesses the potential economic, social, and environmental consequences that they may have by preparing "impact assessments" (which set out the advantages and disadvantages of possible policy options) and by consulting interested parties.
The Commission will propose action at the EU level only if it considers that a problem cannot be solved more efficiently by national, regional, or local action.
This principle of dealing with things at the lowest possible level is called the "subsidiarity principle," and has been reaffirmed in the Lisbon Treaty.
xx The co-decision procedure was introduced by the Maastricht Treaty on European Union (1992) , and strengthened and made more effective by the Amsterdam Treaty (1999) .
With the Lisbon Treaty that took effect on December 1, 2009, this procedure has been renamed "ordinary legislative procedure" and has become the main legislative procedure of the EU's decision-making system.
amended, acknowledged these scientific and technical recommendations (i.e., "The rules governing medicinal products in the European Community," ICH guidelines, and monographs of the European Pharmacopoeia).
All Community rules in the area of medicinal products for human (and veterinary) use are compiled in "The Rules Governing Medicinal Products in the European Union" (EudraLex), published by the EC.
Volume 1 of this publication contains the body of the EU pharmaceutical legislation (i.e., Regulations, Directives, Decisions, etc.).
The subsequent volumes include guidelines yy developed to support this basic legislation: zz ▸ Volume 2 (also known as "Notice to Applicants"), first published in 1986, contains all regulatory guidelines related to procedural and regulatory requirements (i.e., the presentation and content of the dossiers), and also the application forms.
It was prepared and is regularly updated by the European Commission in consultation with competent authorities of the Member States and the EMA.
This Notice has no legal power.
In case of doubt, therefore, reference should be made to the appropriate Community Directives and Regulations.
Also, in July 2012, the information contained in Chapter 7 of Volume 2A (concerning general information on procedures for marketing authorization) was transferred to EMA and CMDh websites.
▸ Volume 3 consists of all the scientific guidelines for medicinal products for human use prepared by the Committee for Medicinal Products for Human Use (CHMP) in consultation with the competent authorities of the EU Member States.
The guidelines are intended to provide a basis for practical harmonization in the manner in which the EU Member States and the EMA interpret and apply the detailed requirements for the demonstration of quality, safety, and efficacy contained in the Community Directives.
An updated list of scientific guidelines is accessible on the EMA website.
▸ Volume 4 contains guidance for the interpretation of the principles of GMPs for medicinal products for human and veterinary use.
▸ Volume 9 contained pharmacovigilance guidelines for medicinal products for both human use (Volume 9A) and veterinary use (Volume 9B).
Volume 9A was replaced by the EMA "Guidelines on Good Pharmacovigilance Practice (GVP)" in 2012 [122] .
▸ Volume 10 contains guidance documents applying to clinical trials.
Finally, in addition to the published rules listed above, a lot of other documents that do not have the status of a law or guideline (i.e., questions and answers [Q&A], recommendations, public statements, position papers, reflection papers, etc.)
are released by the EMA to provide additional guidance.
Moreover, templates (e.g., assessment templates and guidance), internal standard operating procedures (SOPs), work instructions (WINs), and policy covering both general and specific topics (e.g., pharmacovigilance, inspection, etc.)
have been developed by the EMA to improve consistency in activities and evaluations and to help ease the exchange of information.
Many technical requirements have been harmonized and published in Europe to ensure that medicinal products throughout Europe are of equal quality, safe, and efficacious.
These are the three basic criteria that are always evaluated and taken into consideration when establishing the risk and benefit ratio.
These criteria are evaluated through the quality, nonclinical, and clinical information included in all applications.
Of course, the level of quality/nonclinical/clinical documentation varies depending upon the type of products and the level of development, but they are always the basis of approval for the registration of a clinical trial or a new product.
Legal provisions related to these technical requirements are included in Annex 1 of Directive 2001/83/EC and other relevant Regulations or Directives.
In addition, scientific and technical guidelines are also prepared by the EMA's Committees (i.e.
CHMP, COMP, PDCO, etc.)
and its Working Parties (in consultation with the competent authorities of the EU Member States).
Guidelines developed by other technical bodies (e.g., the European Pharmacopoeia) or international bodies are also used in Europe.
For example, Europe is a founder and member of ICH, and therefore all ICH guidelines are also applicable in Europe.
▸ Quality: Many European requirements are in place regarding the quality of the products (active substance, excipients, and finished products).
Detailed scientific guidelines have been developed to adequately cover pharmaceutical development, manufacture, packaging, control (i.e., specifications, analytical procedures and validation, and impurities), stability evaluation, and post-approval changes.
Moreover, guidelines for certain types of products (i.e., biologics, radiopharmaceuticals, medicinal gases, or herbal medicinal products) have been specifically released to take into account their specific challenges.
These technical and scientific guidelines, together with the Q&A document, provide a common interpretation of the European legislation and ensure harmonization of quality requirements.
Also, in addition to these guidelines, it is worth mentioning two other publications that have been critical in the harmonization of the quality aspect of medicinal products available on the European market: • Good Manufacturing Practice (GMP) is one of the most important harmonized requirements that have been issued.
As per Directive 2001/83/EC and Directive 2001/20/EC, all products (including investigational medicinal products) have to comply with the principles and guidelines of GMP.
These GMP principles are laid down in Directive 2003/94/EC.
In addition, the EC has published detailed GMP guidelines in line with those principles in EudraLex (Volume 4).
This volume covers both the basic requirements for Medicinal Products (Part I) and for Active Substances used as Starting Materials (Part II).
Particular considerations and conditions for specific products (biological products, radiopharmaceuticals, medicinal gases, products derived from human blood or plasma, herbal medicinal products, excipients, etc.)
are also in place or under discussion.
Under this EU system, manufacturers and importers of medicines located in the EEA are subject to a manufacturing authorization and come under the supervision of the competent authorities of the Member States (the Supervisory Authorities), who are responsible for issuing the authorizations for those activities taking place in their territories.
• The European Pharmacopoeia (EP), established on July 22, 1964 by eight countries, aaa is a collection of standardized specifications, so-called monographs, which define the quality reference standard for medicines.
Today, the Convention has been ratified by more than 35 European countries and the EU.
European Directive 2001/83/EC refers to the mandatory character of EP monographs in the preparation of dossiers for MAA in the EU.
The EP is also applicable in all the signatory states of the Convention for the elaboration of an EP, and is used as a reference by many other countries (there are more than 20 Observers).
The EP is published by the EDQM and covers active substances, excipients, substances or preparations for pharmaceutical use of chemical, animal, human or herbal origin, homoeopathic preparations and stocks, antibiotics, as well as dosage forms and containers.
The texts of the European Pharmacopoeia also apply to biologicals, blood and plasma derivatives, vaccines, and radiopharmaceutical preparations.
▸ Nonclinical: All aspects of nonclinical testing and programs are covered under general guidelines (e.g., GLP) bbb or discussions on nonclinical strategies to identify and mitigate risks for first-in-human clinical trials or guidelines specific to a type of testing (i.e., pharmacology, aaa Belgium, France, Germany, Italy, Luxembourg, the Netherlands, Switzerland, and the United Kingdom.
bbb The principles of Good Laboratory Practice define a set of rules and criteria for a quality system concerned with the organizational process and the conditions under which nonclinical health and environmental safety studies are planned, performed, monitored, recorded, reported, and archived.
pharmacokinetics, single and repeat dose toxicity, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and local tolerance).
Most of these guidelines have in fact been developed under the auspices of ICH.
As for the quality requirements, specific nonclinical guidelines have also been developed for certain types of products.
Numerous clinical guidelines are available, which cover all phases of clinical development, from early on (i.e., clinical pharmacology and pharmacokinetics studies) to the design of Phase 3 studies (disease and patient characteristics, advice on selection of endpoint, duration, control groups, and choice of comparator, etc.).
Due to the specificities of each group of products, guidelines have been organized by therapeutic area, and some focus on certain types of products (herbal medicinal products or radiopharmaceuticals and diagnostic agents).
Additionally, general guidelines have also been released to provide advice on general considerations and topics during drug development that are not disease-specific (e.g., "Guideline on Missing Data in Confirmatory Clinical Trials," "Extrapolation of Results from Clinical Studies Conducted Outside Europe to the EU Population," "Clinical Trials in Small Populations," "Data Monitoring Committee," "Choice of a Non-Inferiority Margin," and "Excipients in the Label and Package Leaflet of Medicinal Products for Human Use").
In addition to these numerous scientific guidelines, it is worth mentioning the development and implementation of GCP in Europe for investigational medicinal products.
This harmonization of GCP has been critical for the recognition of data between European countries, and therefore cooperation on clinical aspects of drug development.
Directive 2001/20/EC is the framework legislation that provides for additional directives, accompanying guidelines, and detailed guidance documents.
These guidelines and guidance documents are published in EudraLex (Volume 10).
Finally, it is important to note that there has been a lot of effort put forth in past years regarding harmonization of the European pharmacovigilance system.
This system is coordinated by the EMA, but also involves national competent authorities ccc and the European Commission.
It includes a broad range of activities such as the review of Risk Management Plans (RMPs) and PSURs, the development and maintenance of the EU reporting and data warehouse system for case reports (EudraVigilance), signal-identification activities in the EU, and the coordination of EU rapid alert and incident management systems for timely and adequate responses to new safety data.
The EU legal framework of pharmacovigilance was provided in Regulation (EC) 726/2004 and Directive 2001/83/EC.
Additionally, relevant ICH guidelines have been implemented, and Volume 9 of EudraLex has been dedicated to this key public health function.
It included a number of detailed guidelines, definitions, standards, and information regarding the precise execution of pharmacovigilance-related procedures.
ccc In some Member States, regional centers are in place under the coordination of the national competent authority.
In December 2008, following a public consultation, the EC decided to further harmonize the system (to ensure it is optimally effective, robust, and transparent) via the adoption of two additional texts [123, 124] .
The final new legislation [125] was finally published on December 31, in the Official Journal of the European Union.
On June 19, 2012, the Commission Implementing Regulation (EU) 520/2012 was adopted, complementing the 2010 pharmacovigilance legislation that started to apply in July 2012.
Finally, some pharmacovigilance incidents in the Union have shown the need for further improvements of the 2010 legislation.
These issues have been addressed by Directive 2012/26/EU and Regulation No 2012/1027/EU, which started to apply in 2013.
Due to the number and importance of improvements that need to be implemented [126, 127] , many observers consider this new pharmacovigilance legislation as the biggest change to the EU legal framework since the creation of the EMA in 1995.
The implementation of this new pharmacovigilance legislation required a lot of effort from the EMA [128].
This was a major activity because several processes needed to be established or amended (e.g., the establishment of a new Pharmacovigilance Risk Assessment Committee [PRAC] replacing the CHMP Pharmacovigilance Working Party).
Also, an important change of the new legislation is the increased direct involvement of the EMA in the pharmacovigilance of nationally authorized products, in addition to the centrally authorized products.
For example, the EMA has released the "Guidelines on Good Pharmacovigilance Practice (GVP)", which replace Volume 9 of EudraLex [129] .
This new set of guidelines applies to all medicines authorized in the EU, whether centrally or nationally authorized.
The EMA is also working with other groups to continuously improve the safety monitoring of medicines.
This includes its central coordinating role in PROTECT, ddd its support of the European Network of Centres for Pharmacoepidemiology and Pharmacovigilance (ENCePP), eee its work with the US FDA on AE signal detection activities, and its notifications to the WHO of any measures taken in the EU on medicines that may have a bearing on public health protection in third-world countries.
Finally, the Heads of Medicines Agencies have also put in place a multi-annual program (called the European Risk Management Strategy [ERMS] ) which aims to strengthen European pharmacovigilance systems by putting in place efficient measures allowing for the early detection, assessment, minimization, and communication of a medicine's risk throughout its lifecycle.
These guidelines apply to more than one specific area and have been prepared through the collaboration of several working parties.
They provide advice and guidance on specific ddd PROTECT is a project of the Innovative Medicines Initiative (IMI), which is aimed at strengthening the monitoring of the benefits and risks of medicines in Europe by developing innovative tools and methods that will enhance the early detection and assessment of adverse reactions.
eee ENCePP is a network that supports independent, post-authorization studies on the safety and benefit/risk aspects of specific medicines.
important topics (i.e., pediatrics, cell therapy and tissue engineering, vaccines, biosimilars, gene therapy, and pharmacogenomics).
The EU harmonization activities related to certain of these topics are further discussed in the following sections.
It is also important to note that cooperation in the areas of inspection (e.g., GMP, GLP, GCP, or PhV) is critical.
Although the responsibility for carrying out inspections rests with the national competent authorities of Member States, the coordination of these inspections by the EMA (and the agreement of common standards) has been an important step that allows for: • Increased cooperation between Member States • Reduced duplication of work (due to the recognition of inspections performed by other Member States) • Ensuring the same level of quality of medicinal products, and the data generated during their development, wherever the location of the manufacturing site or studies A European system for the authorization of medicinal products has been created with the objective of ensuring that safe, effective, and high-quality medicines can quickly be made available to all citizens across the EU.
Today, the European system offers several routes for the authorization of medicinal products: ▸ The Centralized Procedure (laid down in Regulation (EC) No 726/2004) is compulsory for certain types of products: products derived from biotechnology processes, advanced therapy medicines, orphan medicines, or products intended for the treatment of certain specific diseases.
For medicines that do not fall within these categories (the "mandatory scope"), companies can also submit an application if the medicinal product constitutes a significant therapeutic, scientific, or technical innovation, or if it is in any other respect in the interest of public health.
Applications for the Centralized Procedure are made directly to the EMA and lead to European marketing authorization.
This authorization, binding in all Member States, is granted by the EC (based on the opinion of the relevant EMA committee).
It is valid for the entire Community market, which means the medicines may be put on the market in all Member States.
This is the ultimate integration model in this domain because there is a single application, a single evaluation, and a single authorization allowing direct access to the single market of the Community.
▸ The Mutual Recognition Procedure (MRP) (laid down in Directive 2001/83/EC), applicable to the majority of conventional medicinal products, is based on the principle of recognition of an already existing national marketing authorization by one or more Member States.
Should any Member State refuse to recognize the original national authorization on the grounds of potential serious risk to public health, the issue is referred to the CMDh to find a consensus.
In that case, the CMDh uses its best efforts to reach an agreement on the action to be taken (within the 60-day time period foreseen in the legislation).
When this fails, the matter is then referred to the EMA/CHMP for arbitration (see below for details).
At the end of the MRP and Decentralized Procedure, national marketing authorizations are granted in the Member States involved, whereas the Centralized Procedure results in a single marketing authorization (called a "Community marketing authorization") that is valid across the EU, as well as in the EEA-EFTA states (Iceland, Liechtenstein, and Norway).
Purely national authorizations are still available, but are limited to medicinal products to be marketed in one Member State only.
In addition to the above registration procedures, another European procedure called "referral" has been established.
This Community Referral Procedure is used to resolve disagreements (e.g.
between Member States during an MRP or a Decentralized Procedure), address specific concerns relating to the safety or efficacy of a medicine or a class of medicines, or when there is a need to harmonize national decisions across the EU.
In a Referral Procedure, the EMA is requested to conduct, on behalf of the European Community, a scientific assessment of a particular medicine or class of medicines.
The problem is "referred" to the CHMP so that the Committee can make a recommendation for a harmonized position across the EU.
Referral Procedures can be started by the EC, any Member State, or by the pharmaceutical company.
At the end of the referral, the Committee makes a recommendation, and the European Commission issues a decision to all Member States reflecting the measures to take to implement the CHMP recommendation.
Finally, it is important to note that, in addition to the harmonization of procedures for the authorization of medicines, the system also ensures harmonization and coordination of the pre-and post-authorization activities: ▸ Pre-authorization activities: Companies can request scientific advice (or protocol assistance in the case of medicines for "orphan" or rare diseases) from the EMA at any stage of medicine development, whether the medicine is eligible for the Centralized Procedure or not.
This European procedure helps the company to make sure that it performs the appropriate tests and studies so that no major objections regarding the design of the tests are likely to be raised during evaluation of the marketing authorization application.
▸ Post-authorization regulatory activities (i.e., variations or extensions and transfers of marketing authorizations, renewals, PSURs, and notifications) have also been harmonized and are coordinated via the Centralized, MRP, or Decentralized Procedures.
This ensures that the same quality, safety, and efficacy of products are maintained during the entire lifecycle management of the products throughout Europe (e.g., availability of new formulations, extension of indications, etc.).
After years of extensive discussions involving ethical aspects [130] , the European Commission adopted a proposal on September 29, 2004 [131] .
This proposal led to new legislation (Regulation (EC) No 1901/2006) that entered into force in the EU on January 26, 2007.
Today, this amended text (and its several associated guidelines and other published information) [132] sets up a system of requirements, rewards, and incentives together with lateral measures to ensure that medicines are researched, developed, and authorized to meet the therapeutic needs of children (representing over 20% of the total European population [133]).
In practice, this new regulation established an expert Pediatric Committee (PDCO) within the EMA, which is responsible for providing opinions on the development of medicines for pediatric use.
The key objectives of the regulation are: • To ensure high-quality research in the development of medicines for children aged 0 to 17 years of age • To ensure, over time, that the majority of medicines used by children are specifically authorized for such use • To ensure the availability of high-quality information about medicines used by children In 2008, a communication from the EC (Communication 2008/C 243/01) provided guidelines on the format and content of applications for agreement or modification of a pediatric investigational plan.
Many additional procedural and scientific guidance documents have also been released by the EMA to facilitate the implementation of this new regulation.
The EU introduced a new Orphan Medicinal Product legislation in 2000 in order to provide incentives for the development of medicinal products for rare disorders.
Harmonization of requirements for these types of products is critical to allow for multinational clinical studies and to limit the development challenges due to the small number of patients.
Prior to this European legislation, a number of Member States had adopted specific measures to increase knowledge on rare diseases and improve their detection, diagnosis, prevention, and treatment.
However, these initiatives were few and did not lead to any significant progress in research on rare diseases.
Procedure for the designation of orphan medicines with the technical Committee for Orphan Medicinal Products (COMP), which is responsible for the scientific examination of applications.
Designated orphan medicines are assessed centrally on a European level by the CHMP, rather than in each Member State separately.
This regulation also put in place incentives for the research, marketing, and development of such products (e.g., fee waivers, a 10-year market exclusivity period postauthorization, and scientific assistance for marketing authorizations).
Following its entry into force and its associated rules and guidelines, the number of orphan medicines authorized has increased significantly [134] .
This Directive's aim is to protect public health while securing the free movement of herbal medicines within the Community.
While most individual herbal medicines will continue to be licensed nationally by Member States, the process for licensing and information on herbal substances and preparations will be increasingly harmonized across the EU.
For example, in order to further integrate these special medicines in the European regulatory framework, a Committee for Herbal Medicinal Products (HMPC) was established at the EMA in September 2004 (replacing the CPMP Working Party on Herbal Medicinal Products).
The major tasks of this scientific Committee are to establish Community monographs for traditional herbal medicines, and to prepare and maintain a list of herbal substances that have been in medicinal use for a sufficient period of time, and so are not considered to be harmful under normal conditions of use [135] .
The procedures for clinical trials in Europe used to vary from one country to another.
There were different national approaches regarding the approval and notification systems, documentation requirements, and timelines [136] .
In October 2004, in order to coordinate the implementation of the new harmonized requirements across the Member States, the HMA established the Clinical Trials Facilitation Group (CTFG).
The CTFG (attended by representatives from the National DRAs, EC, and the EMA) acts as a forum for discussion on the agreement of common principles and processes to be applied throughout Europe.
It also promotes harmonization of clinical trial assessment decisions and administrative processes across the national DRAs.
This group established a Voluntary Harmonization Procedure (VHP) for the assessment of multinational CTAs [138] .
During this three-phase procedure, DRAs from all Member States involved assess the application, though each Member State remains ultimately responsible for the approval of the CTA in its own country.
However, there is a coordinated validation phase (phase 1) and voluntary cooperation of the Member State during the assessment phase (phase 2) before the usual formal national process (phase 3).
Phases 1 and 2 of the procedure are coordinated by a VHP coordinator.
The "acceptability statement" obtained through this VHP procedure is then included in the subsequent national CTA applications.
From March 2009 to April 2010, 30 applications were evaluated through the pilot VHP procedure; 23 of these applications received a positive opinion [139] .
The average procedural time was 52 days (which is significantly less than the average time of standard national procedures).
The overall feedback from sponsors was positive, except that: Directive 2001/20/EC and its associated texts and guidelines are a very important step in the harmonization of procedure for the registration and conduct of clinical trials in Europe.
Implementation of this Clinical Trials Directive into national legislation of all 27 EU Member States was completed in 2006.
Principles like Clinical Trial Authorization by the national DRAs within defined maximum timelines led to significant harmonization of the clinical trial approval process.
However, it has been agreed that this new system needs further harmonization in order to achieve the ultimate objective [142] .
Indeed, the actual assessment of a request for authorization of a clinical trial is done independently by the Member States concerned.
The legislation does not provide for a mechanism whereby the Member States are obliged to reach a common conclusion regarding a clinical trial involving different Member States.
This lack of obligation and detailed direction implied different interpretation from Member States and therefore created implementation issues.
As a consequence, sponsors have to respond to the various required changes and adapt their protocol in view of diverging assessments by the DRAs.
This situation requires additional time and effort by the pharmaceutical industry (without added value for the patients).
In 2010, following a public consultation and a long and thorough impact assessment ( The proposal has been submitted to the European Parliament and the Council who engage in ordinary legislative procedure.
This proposal, once adopted by the EU-legislator, is going to replace the 2001 Clinical Trials Directive.
It is expected to come into effect in 2016 and to provide major revisions to the current system (e.g., single assessment outcome, simplified reporting procedures, etc.).
Finally, it must be noted that other important topics related to the regulation of medicines are also coordinated at the community level (by the EC and the EMA) in order to have harmonized regulatory actions and enforcements, and to complete the single pharmaceutical market.
These harmonization initiatives are at different stages of development: • To support cooperation and harmonization activities, the EU needed systems and knowledge management support.
The implementation of this telematics (the integrated use of telecommunications and informatics) strategy, coordinated by the EMA, is critical to increase efficiency and transparency across the European Medicines Regulatory Network.
In addition to the standards for Electronic Submissions (eSubmissions) that were developed and published, a central set of Pan European systems and databases was created.
These systems and databases exchange information with systems of external stakeholders and DRAs, while staying separate from them.
They also help provide high-quality information on medicinal products to the general public and support the monitoring of the post-authorization risk and benefit balance of medicines in the EU.
The following critical projects and tools have been developed under this program (some of them are still under development): ▸ EudraCT: The Community's electronic database for clinical trials containing information submitted by sponsors.
It informs DRAs of ongoing clinical trials in all Member States and EEA countries, enabling an overview of multi-state trials.
The system also alerts DRAs in the case of early interruption or termination.
▸ EudraGMP: Community database on manufacturing and import authorizations and GMP certificates.
The EMA launched the first release in April 2007.
This system is used by EU GMP inspectors to share information (i.e., GMP authorization, noncompliance with GMP information resulting from inspection activity, planned inspection activity, and "rapid alerts" arising out of faulty manufacture).
▸ EudraNet: Private electronic network linking the members of the European Medicines Regulatory Network and EMA.
It ensures that both electronic mail between members of the Network and their access to the EU telematics systems is secure.
▸ EudraLink: The European Medicines Regulatory Network's secure file transfer system used for exchanging information for regulatory purposes.
It operates independently of EudraNet, so that it can be used by applicants and marketing authorization holders, as well as the regulatory organizations within the network to transfer files.
▸ EudraPharm: The Community's database of authorized medicinal products.
Some functionalities of this database are still under development.
▸ EudraVigilance: System monitoring the post-authorization safety of medicines through safety reports (i.e., suspected adverse reaction reports).
It is designed to receive, process, store, and make available information.
One of the objectives of this system is the early detection of possible safety signals to facilitate the regulatory decision-making process (based on a broader knowledge of the adverse reaction profile of medicines).
the EMA to receive, validate, store, and make available information for review marketing authorization applications.
The system's key benefit is its ability to take advantage of the lifecycle management functionality built into the eCTD by easily allowing the full extent of the current valid documentation as well as its submission history.
▸ EU Telematics Controlled Terms (EUTCT): Central repository and publication system for a controlled term list used in the European Medicines Regulatory Network.
The establishment of the EU has not been easy, but it has represented the desire to end conflicts in Europe.
Since its creation, the EU has been successful in delivering peace between Member States and has reunited a fractured continent via the promotion of cooperative projects (i.e., economic and social).
This cooperative initiative went beyond the initial objectives of its founders.
Ever deeper integration has been pursued while embracing new members.
The membership of the EU has grown from 6 to 28 nations, bringing the EU's population to half a billion people.
It has created stable institutions, a single market, and a single currency.
Despite numerous challenges, ggg the EU has survived, and is today a major economic and commercial power.
Although improvements are still needed in certain areas, the EU represents a unique model of successful cooperation, harmonization, and integration between countries of different languages, cultures, history, and levels of development.
In the pharmaceutical sector, much has been achieved towards the consolidation of the European system of evaluation and supervision of medicines.
Several challenges have already been overcome, but outstanding issues still need to be resolved to further support and improve public health in Europe, free movement and access to medicines in the community, and the competitiveness of the Union.
Taking into consideration its successes and challenges, this section provides a balanced evaluation of the current situation.
It demonstrates that harmonization of pharmaceutical regulation in Europe can be considered a real and quick success in general (considering the major changes it required), but acknowledges some specific areas where work is still needed.
For all these reasons, the development of the EU and its European pharmaceutical "regulation/ system" is a great example that needs to be further evaluated and discussed.
Although this model of harmonization and integration may not be fully applicable to other cases, this experience can certainly help other regional or global harmonization initiatives.
Since the adoption of the first pharmaceutical directive in 1965, many topics have been harmonized.
The past 50 years have seen a gradual convergence of pharmaceutical legislation in Europe.
Today, a considerable package of harmonized legislation (in the form of the pharmaceutical "acquis communautaire") is in place to support two objectives: the protection of public health and the free movement of products.
These provisions/texts applicable to medicinal products are included in EudraLex.
They include binding legislation (i.e., Regulations and Directives), but also numerous technical guidelines and recommendations to facilitate the implementation of these common principles.
A well-structured European pharmaceutical system has also been established.
In addition to the European institutions necessary to harmonize and create the European pharmaceutical legislation, technical European bodies have also been established.
Today, the evaluation and supervision of medicines in Europe is shared between European and national bodies that form a complex but well-organized network of approximately 5,000 technical and regulatory experts.
Words like "networking," "work sharing," and "harmonization" became common and remain crucial for the future.
The establishment of the EMA as a key coordinator of this system was an important decision for the integration and harmonization of practices and standards to support and promote the single European pharmaceutical market.
The primary aim of this centralized system was to create conditions in which a single scientific evaluation of the highest possible standard would lead to rapid access to an integrated market of innovative and good cost-effective treatments.
This objective, in large measure, has been achieved.
The EMA, which is comprised of experts provided by national DRAs, has today established itself as a leading world agency for the evaluation of medicines.
Its contribution to the effectiveness and efficiency of the EU system, and therefore to the protection of public health and to the achievement of an operational internal market, is well recognized by all stakeholders.
The effectiveness of the system has been maintained despite its growing complexity.
Indeed, the increase in the number of centralized applications hhh and other procedures, EU enlargement, and new regulations have led to an increased workload and an enlarged scope of responsibility for the EMA over the past 10 years.
These changes have led to the creation of new committees (COMP, PDCO, CAT, HMPC, PRAC) that require the implementation of additional procedures and new tools.
These structural changes and increased responsibilities should be monitored closely in the future to avoid risks of inconsistencies, overlapping, bureaucracy, and rigidity.
Also, it is critical to continue to monitor financial compensation of national DRAs and to regularly assess the involvement of each Member State in the EU pharmaceutical system to ensure availability of appropriate resources and expertise [150, 151] .
Within this legal framework and European pharmaceutical system, community authorization procedures (Centralized, MRP, or Decentralized) have been in place since the mid-1990s.
The centrally coordinated tasks include assessments led by rapporteurs and co-rapporteurs, inspections, and pharmacovigilance through the medicine's lifecycle.
Although the national DRAs have prime responsibility for the efficient operation of MRPs and Decentralized Procedures, national marketing authorizations, and clinical trial authorizations for human medicines, the EMA has an important role in supporting these noncentralized functions.
For example, the EMA maintains the Eudravigilance database and the EudraCT database, and supports a range of scientific committees and the coordination group for MRPs and Decentralized Procedures [152] .
The criteria for the approval of medicines and other technical topics have been extensively harmonized within the EU.
Many technical and regulatory guidelines have been released in all areas (quality, nonclinical, and clinical).
There has been a specific focus in recent years to improve the European pharmacovigilance system, to simplify the variation system, to harmonize the requirements for clinical trials, and to implement an advanced therapies regulation.
The establishment of the European Pharmacopoeia has also been very important to ensure standardization of specifications and quality of medicines in the EU.
All these measures and actions described above have led to improved marketing authorization procedures, the harmonization of data protection in the EU, better access to medicines for children, orphan drug development, clinical trials, and a new regulatory framework for advanced therapies.
Lifecycle management of products has also been improved (i.e., the revised legislation on variations to reduce the administrative burden by streamlining the circumstances obliging industry to file applications).
The next review of the European system will be noteworthy because it will evaluate if new measures (developed following the last review in 2000) improved the system and produced real dividends in terms of cost efficiencies through economy of scale (via the reduction of the administrative burden where this did not have public health implications).
It is also worth mentioning that this European system is solid enough to stand the challenges of new therapeutics.
The current structure, forum, and processes allow "proactive" harmonization.
Indeed, most of the harmonization initiatives are created to discuss existing disharmonies on specific topics.
At the beginning, the European harmonization effort, related to pharmaceutical regulation, was focused on disharmonies between countries.
Today, even if disharmonies do still exist on some specific subjects, many topics have been successfully harmonized.
The processes and structures that have formed over the years now allow the system to cover new subjects for which no national regulations and requirements have been developed yet.
Developing this new regulation at the EU level automatically creates harmonized requirements (this can be called "proactive harmonization").
.
This group, which included EMA staff and members of the CHMP and its working parties, generated recommendations on how the EMA should tackle these new emerging topics not covered by the existing national, regional, or global regulations and standards.
▸ EMA Innovation Task Force (ITF): In order to provide support for medicine innovation in the EU, the EMA established an internal horizontal cross-sectorial group to focus on emerging therapies and technologies.
The ITF brings together competences from the areas of quality, safety, efficacy, pharmacovigilance, scientific advice, orphan drugs, and good practices compliance, as well as legal and regulatory affairs.
One of the objectives of the ITF is to address the impact of emerging therapies and technologies on current scientific and regulatory requirements.
Its scope also encompasses areas for which there are no established scientific, legal, and regulatory experience.
One of their tasks is to identify areas for legal, regulatory, and technical guidance preparation and proposals for consideration by the EMA Committees and working parties, and to contribute to relevant EC initiatives and legislation [154] .
The EU today is recognized as a major player in the international harmonization of pharmaceutical regulations.
It has developed privileged relationships and initiated cooperation projects with other countries outside the European Community (major developed countries and emerging markets).
For example, the EMA cooperates with many of the world's largest regulatory bodies outside the EU iii in areas such as inspections, safety of medicines, and exchange of information on issues of mutual concern.
The establishment of the International and European Cooperation Sector, formed in February 2012 and responsible for the development, coordination, and implementation of the Agency's international strategy and activities (including confidentiality arrangements with countries outside the EU), demonstrates the EMA commitment to international cooperation [155] .
Also, collaboration has been initiated with China, India, and Russia on pharmaceuticals, and it is partnering with international organizations (i.e., ICH, WHO, and PIC/S).
This work should continue and also be extended.
It is indeed important to support the development of globally harmonized standards and requirements in order to ensure fair competition with other parts of the world for the development of medicines and to avoid delay in the availability of essential medicines for European patients.
Ensuring against falsified medicines, resolution of pandemic issues, product development in emerging markets, and reliability of clinical data produced outside Europe are good examples where international cooperation is necessary to ensure adequate protection of public health in Europe.
In spite of all the above-mentioned major progress and regular improvement of legislation by the European Commission, there is still room to improve the EU pharmaceutical system.
On the regulatory side, issues dealing with the implementation and interpretation of Community legislation by Member States continue to create obstacles to the free movement of medicines.
Stakeholders continue to raise concerns regarding market fragmentation linked to disparities in national pricing and reimbursement schemes (despite the adoption of Directive 89/105/EEC in the early days of the European pharmaceutical system), unnecessary regulatory burdens caused by divergences in the implementation of Community legislation (e.g., clinical trials requirements), and a lack of commercial interest in national markets that are economically less attractive.
European patients still suffer from inequalities in the availability and affordability of medicines.
This situation could worsen and create significant inequalities between patients in accessing medicines if it is not resolved.
Additionally, Europe has been losing ground when it comes to innovation and competitiveness in the pharmaceutical market.
In its communication of December 10, 2008 [156] , the EC recognized that further harmonization is necessary to resolve shortcomings in the EU pharmaceutical market in furthering increased globalization of this sector.
To improve this issue, the EC confirmed its objective to continue to progress towards a single and sustainable pharmaceuticals market [157] .
To further support and improve the public health in Europe and free movement of medicines within the community, and to maintain its competitiveness, the EU needs further harmonization in several areas, such as: novel medicines by patients, mainly due to increased pressure to cut healthcare budgets.
In certain countries, medicines are not made available due to administrative requirements and poor economic rewards.
A lack of transparency and harmonization with regard to pricing, reimbursement, and relative effectiveness remains a challenge [158] .
In contrast to the benefit-risk assessment carried out by regulators, national HTA bodies compare the "relative effectiveness" of medicines and take their financial cost into account.
This post-marketing national HTA evaluation can lead to national differences due to different country needs.
The addition of different requests (i.e., different type of studies) from regulators and HTA bodies can also delay availability of new products.
To resolve this major issue, the European Network for Health Technology Assessment (EUnetHTA) was established to support effective collaboration between national HTAs.
Also, the EC gave the political mandate to the EMA to begin interacting with HTA bodies when it published the conclusions of the Pharmaceutical Forum in October 2008. kkk Since then, the EMA has begun to collaborate with national HTA bodies and with EUnetHTA [159] .
This interaction focuses on centralized approved products and aims to facilitate communication between EMA and HTA bodies early in a medicine's development and throughout the medicine's lifecycle.
As mentioned above, the harmonization of price and reimbursement evaluation is critical in supporting a European pharmaceutical market.
However, it will be a very difficult and long process to implement due to political and budgetary aspects and differences in pharmaceutical markets and healthcare budgets existing between Member States.
The European Clinical Trials Directive (Directive 2001/20/EC) has been an important and necessary step in the harmonization of European pharmaceutical regulation.
The principles defined in the Declaration of Helsinki (in 1964) and the ICH GCP E6 guideline (in 1996) allowed some harmonization of clinical practices and protection of clinical patients.
But, before this Directive came into force, the rules for performing clinical trials (i.e., regulatory procedures and requirements) varied significantly in the European Community as they were based on differing regulatory approaches in the Member States.
This new legislation promoted harmonization of clinical trial practices allowing important improvements related to the protection of patients (i.e., safety and ethical concerns) and reliability of data, and facilitated the exchange of information between DRAs.
However, despite this progress, important negative effects of this new legislation have been reported (e.g., the increase in bureaucracy and administrative costs).
The number of clinical trials carried out in the EU has fallen by 15% in recent years, while administrative kkk The Pharmaceutical Forum was set up in 2005 by the European Commission as a three-year process in order to find relevant solutions to public health considerations regarding pharmaceuticals, while ensuring the competitiveness of the industry and the sustainability of national health care systems.
More specifically, this forum analyzed three key themes: information to patients on pharmaceuticals, pricing and reimbursement policy, and relative effectiveness.
costs and delays have doubled [160] .
It is still labor intensive and costly to duplicate largely identical administrative procedures for multinational clinical trials.
Additionally, sponsors spend a great deal of time retrieving the relevant national information and requirements and preparing customized applications without added value for the patient and the regulators (the core scientific information is the same, but the format and administrative information and forms differ).
It is indeed a problem for a large pharmaceutical company, as it usually requires additional dedicated departments with the necessary resources to track differences in national requirements and follow the many parallel procedures.
But it is even more problematic for SMEs or academic sponsors for whom these costs can reach prohibitive levels.
This multiplication of parallel procedures also has an important impact on the DRAs.
Indeed, available resources are used in multiple assessments of the same core information in different Member States, which clearly delays the start of clinical studies.
It is important to note that this duplication of assessments does not necessarily increase the quality of the assessment, as the necessary specific expertise might not always be readily available in all the Member States concerned.
This is a nonefficient use of national resources without added value for the patients or science.
This implementation problem is partly due to the legal framework that has been chosen for harmonization in this area.
As with all Directives, the Clinical Trials Directive had to be transposed in national laws.
Unfortunately, in this case, the objectives of the directive were transposed into divergent national legislations, somewhat missing the harmonization goal and making multinational trials difficult to perform.
In its 2009 consultation paper [161], the EC proposed options to improve the situation.
One of the best options is to continue with the harmonization process.
This would mean creating a real European system of authorization for clinical trials to avoid duplication of assessment.
It would avoid the inconsistent assessment conclusions and requests, encourage appropriate use of resources and expertise (for both the sponsors and DRAs), and ensure common implementation of the principles laid down in the Clinical Trials Directive.
The VHP initiative seems to be a good first step.
It allows for a better implementation of the EU Clinical Trials Directive principles and further harmonizes the conduct of clinical trials in Europe.
However, this procedure cannot be considered as the ultimate solution because it does not resolve all issues [162] .
More specifically: • There are still parallel CTA assessments by multiple DRAs.
• There are still major differences between countries regarding the time it takes to issue approval.
• This is a voluntary cooperation and there are differences in the level of interest and responsiveness between countries.
• The current procedure does not remove specific national requirements or differences between national assessments (this is a cooperation effort, not a harmonization of requirements).
• This process does not accelerate the First Patient Enrolled (FPE) in Europe.
To resolve these outstanding issues, the current VHP procedure should be revised to become a real MRP where the assessment will be conducted by only one reference Member State.
The content of the dossier should also be fully harmonized between countries.
The establishment of a Centralized Procedure through a new Regulation (which will deliver a Pan-EU approval) would also be very helpful for certain types of products that require specific expertise not available in all EU countries (e.g., advanced therapies), for orphan drugs, and/or for pediatric medicines.
This centralized process for CTA would be a good bridge between the EMA Scientific Advice process and the Centralized registration procedure.
The system for registration of clinical trials would then mimic the system already in place for the registration of medicinal products with a combination of three types of procedures: • Centralized Procedure for specific products such as biotechnology and advanced therapies • Mutual Recognition Procedure for other multinational clinical trials • National Procedure for a clinical trial involving only one Member State This reorganization of the system and procedures, supported by the EC [163] and most of the shareholders involved in clinical trials [164] , would utilize the current structures and expertise in Europe, would build on the experience acquired with the registration process, and would facilitate patient access to clinical trials and to new technology within the community.
It would allow the necessary flexibility and different levels of review for interventional trials (e.g., a small national study with a well-known entity does not need the same type of evaluation, organization, and bureaucracy as a Phase 1 study with a new fusion protein or a large multinational Phase 3 study).
Measures should be put in place to ensure that such reorganization would allow this flexibility and avoid any further increase of delay and administrative costs and burdens.
For example, "recognition" of other assessments should be the focus, and "nonrecognition" should be limited to major issues (that should be clearly defined).
These "nonrecognitions" of assessment by another country should be rare to avoid regular arbitration or appeals that would further delay the start of the clinical studies.
Selection of Reference Member States (RMS) should also be defined because many parameters are involved in such selection (i.e., expertise, resources, balanced workload between countries, etc.).
Finally, this new cooperative system should not result in the simple addition of national requirements, but a harmonized scientific assessment that would be implemented equally in all Member States.
This next step in the harmonization of a clinical trial in Europe would certainly be beneficial for patients, sponsors of clinical trials (pharmaceutical companies, but also small entities or academic centers), and DRAs.
Some of the above proposals have already been recommended by the European Commission [165] .
The recent adoption of a "Proposal for a Regulation of the European Parliament and of the Council on clinical trials on medicinal products for human use, and repealing Directive 2001/20/EC" [166] by the Commission represents an important step in the improvement of the current system.
However, this process will take time to implement, and national interests will need to be overcome.
Finally, the assessments of Ethics Committees also need to be reviewed and improved.
The Clinical Trials Directive is based on the concept of one Ethics Committee opinion per Member State concerned.
However, several Member States maintain a decentralized system where the single Ethics Committee opinion is based on the opinion of several local committees.
As a consequence, in the EU there are approximately 1,900 Ethics Committees involved in the assessment of clinical trials [167] .
Also, better harmonization of responsibilities between DRAs and Ethics Committees must happen across Europe [168] .
It is agreed that ethical issues fall within the responsibility of Member States.
However, current practices need to be reviewed in order to smoothly integrate an improved harmonized system and to protect European clinical trials subjects.
These programs are important to make new therapies available to patients as soon as possible.
They should be handled on a European basis in order to ensure that every European person, wherever their location, has the equal right to access these new medicines at the same time.
Today, this difference in access within Europe is clearly contrary to the overall European objective to ensure that all patients within the Community have the same access to the same quality products throughout Europe.
Of course, the harmonization of these requirements and procedures should be carefully implemented to avoid the creation of delays compared to the current situation.
▸ Pharmacovigilance: The EU pharmacovigilance system demonstrates that cooperation and harmonization of regulations and practices in Europe is beneficial to patients.
Indeed, merging the EU national pharmacovigilance systems into one network increases the quantity of data/reports/ information, which facilitates the early detection of possible safety signals, and therefore the monitoring of product safety.
Unfortunately, the Mediator issue in France has shown that the EU pharmacovigilance system needs to be improved to be fully functional.
This topic has been one priority of the European network.
The ongoing implementation of the new legislation by the EMA and the Member States will be critical.
Although the Mutual Recognition and Decentralized Procedures have improved over time, challenges still exist, and the principle of these procedures (i.e., recognition of another country's assessment) is not always respected.
In both these procedures, Member States can only refuse to recognize other countries' assessments if they feel that this recognition could have a "potential serious risk to public health."
Unfortunately, this reason for disagreement is vague enough to allow flexibility for Member States.
In 2006, a guideline was released [171] to further clarify how this risk should be defined.
However, some national DRAs continue to have a broad interpretation of "potential serious risk to public health," and trigger EMA arbitrations for grounds that do not fall under this specific category [172, 173] .
In addition to the specific issues discussed above, more general challenges can also impact the harmonization of European pharmaceutical regulation.
Although these general considerations are not specific to the pharmaceutical sector, they can influence the establishment and implementation of pharmaceutical regulation.
Therefore, they need to be understood and integrated when developing implementation plans and timelines: • .
This major difference in workload between countries demonstrates a big gap in work sharing and certainly highlights differences in national DRAs' expertise and resources and pharmaceutical companies' interests for each national market.
• One of the complexities and difficulties of the EU system is the division of activities undertaken at the national level (e.g., clinical trial responsibility, scientific advice handling, etc.)
and at the EU level (e.g., equal scientific advice handling, assessment of pediatric investigational plans, etc.).
This requires many communications and infrastructures between the EU and national players.
• External economic or political factors could also influence the harmonization of European pharmaceutical regulation.
For example, the modification of European borders via new enlargement of the EU (even if the EU leaders have agreed to mark a pause for now, discussion on the accession of countries such as Turkey, Iceland, and Serbia are still ongoing).
Additionally, the possible creation of a "Mediterranean Union" desired by past French President Sarkozy could also impact the scope and timelines of the next steps of harmonization and integration.
Finally, it will be important to see if and how the two new functions created by the Treaty of Lisbon (President of the EU Council and High Representative of the Union for Foreign Affairs and Security Policy) will benefit the EU.
The first important dossiers after the creation of these two functions (global financial crisis, global security, and support to Greece) have indeed still been handled by the political leaders of major Member States (i.e., France and Germany).
It is clear that the European system is integrally linked to its own history.
This model cannot fully fit every harmonization initiative in the world because every situation and need is different.
However, it is worth reviewing the lessons learned from this 50 plus years old initiative.
This first Regional Harmonization Initiative (RHI) overcame a lot of challenges, and has since developed into a strong regional harmonized pharmaceutical regulation and system.
This success demonstrates that an organized cooperation and harmonization can facilitate the development of high standards and practices.
More specifically, the European initiative clearly demonstrates that a structured stepwise approach is necessary: ▸ First, it is necessary to set up major principles (Directive 65/65/EEC).
▸ Second, it is critical to provide specific detailed requirements and to further detail the agreed principles (Directives 75/318/EEC, 75/319/EEC, etc.).
▸ Third, a structured and organized system is needed to implement the principles and requirements.
Technical bodies need to be established to control medicines and manage the establishment of common procedures (especially centralized types).
In Europe, it was key that the national DRAs provide expertise and resources to European bodies not only to ensure appropriate availability of resources, but also to ensure full adhesion of the countries into the system and adequate communication between all players of the system (national and European).
▸ When all the basic principles and a system are in place, additional more specific requirements can be discussed so that the system can take into account particular needs (i.e., specific requirements for specific products, population, etc.)
in order to have a more coherent system.
▸ Finally, it very important to monitor the system and regularly review the extent to which this system and measures support the harmonization goals and meet the predefined objectives.
Evolution of the environmental impact (i.e., globalization, change of membership, change of political commitment, and need for new requirements due to emerging problems, etc.)
also has to be taken into consideration, and the regulation and system needs to be carefully adjusted to ensure its longevity.
Another lesson learned from Europe is the importance of cooperation.
To be successful and ensure effective functioning of this system, cooperation between the different entities of the system (EMA, HMA, National DRAs, EC) has been, and remains, critical.
Even if the European pharmaceutical system is complex, it is well organized.
The provision by the Member States of high-quality scientific resources for the evaluation and supervision of medicines is a critical factor for the success of the EU system.
Indeed, scientific excellence (as a result of EU-wide pooling of expertise and data) has been a key strength.
In this respect, it should be stressed once again that such excellent progress has been highly dependent on close collaboration between the EMA and the national DRAs within the context of the EU regulatory network, and in particular on the valuable input of high-quality specialist expertise provided by the Member States.
This provision of national resources, coordinated by the EMA, is one of the features of the EU regulatory network.
This success also relies on political support for this European harmonization initiative in order to support the creation of the single market.
Without this political commitment (and therefore associated funds and resources), it would have certainly been much more difficult and taken more time to create this system.
It is recognized that other harmonization initiatives in the world are certainly suffering from the lack of such political commitment, especially when such harmonization is not driven by the willingness to create a single market (i.e., integration model).
Finally, the EU has also clearly demonstrated that better organization at the regional level is extremely critical to ensuring the success of global harmonization and cooperation.
Even if all regions are not working towards integration like Europe, this example of better coordination and representation should be followed and discussed in other regions of the world.
Indeed, this example demonstrates that a well-organized and coordinated regional structure is beneficial to all stakeholders [176]: ▸ Individual countries via better representation and better access to international activities/agreements/decisions through regional structure (this is especially true for small countries with less expertise and resources).
Individual countries also benefit from the infrastructure (i.e., databases or training programs) and good practices developed at the regional level.
▸ Regions because they allow better representation of interests (Europe has more power than a combination of small countries' voices, and has an impressive network of experts).
▸ International cooperation and harmonization initiatives because they facilitate communication by reducing the number of contacts and seats at the international level (but provide a structure for dissemination of information).
For example, having all EU countries represented at ICH would not be possible.
This regional coordination is very important for the future of global initiatives (such as ICH or WHO projects), but it is even more important in the management of a worldwide health crisis (e.g., pandemic influenza).
This European coordination system should be implemented in other regions of the world because the coordination of rapid and efficient communication of information and actions during such a crisis helps the overall coordination of the situation.
For example, in the recent case of pandemic influenza, it was critical to have central coordination (not only global, but regional).
The EMA (using its "crisis management plan") allowed Europe to respond rapidly and efficiently to the challenges of an outbreak of pandemic influenza by: ▸ The fast-track review of vaccines (using its best experts) ▸ Monitoring the safety of centrally authorized pandemic-influenza vaccines and antiviral medicines ▸ Liaising and coordinating activities with critical partners, including the EC, EU Member States, other European Agencies (such as the European Centre for Disease Prevention and Control), and international partners (such as WHO and regulatory bodies of non-EU countries) to ensure timely exchange of information and coordination of activities relating to the pandemic ▸ Coordinating the communication of relevant information to the public, healthcare professionals, and the media All of these activities would be less efficient if performed by each individual country.
Political and economic development in the Pan-American region has resulted in interest in regional economic integration.
Several subregional integration groups have emerged in this area since the 1970s.
Harmonization of pharmaceutical regulations and technical standards is a component of this economic integration, but the degree of progress in this area varies a lot from one subregion to another (and even from one country to another).
In light of these various economic integration initiatives, the need became evident for an entity in which the different countries of the region could share experiences and expertise.
The Pan-American Network for Drug Regulatory Harmonization (PANDRH) was created in November 1999.
This is a regional initiative established to promote drug regulatory harmonization throughout the Pan-American region within the framework of national and subregional health policies.
This continental forum is not a supranational entity, and its decisions represent recommendations to be assimilated into the subregional integration initiatives.
The mission of this network is "to promote the harmonization of pharmaceutical regulation covering aspects of quality, safety, efficacy and rational use of pharmaceutical products, the strengthening of National Regulatory Authorities (NRA) capacity within the Region of the Americas based on the right of the population to access quality medicines, recognizing advances in science and technology and within the context of national and sub-regional realities" [177] .
The objective of this initiative is to facilitate regional harmonization of medicinal drug requirements and guidelines for specific regulatory issues.
This objective is achieved by adopting recommendations for implementation at national and regional levels, and also by supporting the development of training on specific important topics.
However, this initiative also has broader objectives such as: ▸ Promoting and maintaining a constructive dialogue among DRAs, the pharmaceutical industry, and other sectors ▸ Strengthening the DRAs of the region ▸ Encouraging convergence of drug regulatory systems in the Pan-American region ▸ Facilitating technical cooperation among countries in collaboration with subregional integration groups.
Since 2003, PANDRH has been a member of the ICH Global Cooperation Group (GCG).
This membership broadens PANDRH's role because this regional harmonization initiative is now also involved in global harmonization.
PANDRH provides a way to disseminate recommendations on drug regulatory harmonization of global initiatives.
It also ensures that regional specificities and challenges will be considered when new global recommendations are discussed.
▸ DRAs of all Pan American Health Organization (PAHO) Member States ▸ Regional pharmaceutical industry associations: Latin American Association of Pharmaceutical Industry (ALIFAR) and Latin American Federation of the Pharmaceutical Industry (FIFARMA).
▸ Academia ▸ Consumer groups and professional associations It also includes representatives from the five subregional trade integration groups within the Americas (Plate 2) that are themselves multinational cooperation initiatives but are working on a broader integration with emphasis on political and/or financial interest: ▸ The Andean Community is a Community established in 1969 (by the Cartagena Agreement) that currently regroups four countries (Bolivia, Colombia, Ecuador, and Peru).
Chile and Venezuela have also been part of this initiative in the past and some others countries are observers.
These countries decided voluntarily to join together for the purpose of achieving more rapid, better-balanced, and more autonomous development through Andean, South American, and Latin American integration.
They also created a free trade area (including the four current members plus Venezuela).
This integration initiative is broad and regroups several areas, one of them being health.
The integration of health is governed by the Andean Health Body, which coordinates the actions aimed at improving the healthcare of member countries.
It gives priority to cooperative mechanisms that promote the development of subregional supranational systems and methodologies.
These actions are also coordinated with the other subregional, regional, and international organizations.
Discussions include many topics such as the development of a pharmaceutical policy model, the evaluation of medicinal products, and a surveillance network.
▸ SICA (The Central American Integration System) is the institutional framework of subregional integration in Central America.
This is the latest step of a long integration process in the region.
It was created in December 1991 (by the signing of the Tegucigalpa Protocol) by the States of Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua, and Panama.
This initiative also involves the Dominican Republic as an associated State and some regional and extra-regional observers (Mexico, Chile, Brazil, China, Spain, and Germany).
The headquarters of the General Secretariat is located in El Salvador.
The first objective of this integration process in Central America was to transform the area into a region of Peace, Liberty, Democracy, and Development, based firmly on the respect, tutelage, and promotion of human rights (following a history of political crisis, conflict, and dictatorial rule in the region).
Health topics are covered by the Executive Secretariat of the Council of Ministers of Health in Central America (SE-COMISCA).
Several projects are under discussion in this subregion, such as the basis for quality assurance of drugs and a pharmacovigilance system.
▸ MERCOSUR (the "Common Market of the South") was created in 1991 (by the signature of the Treaty of Asuncion) and encompasses five Latin American countries (Argentina, Brazil, Paraguay [which is currently suspended], Uruguay, and Venezuela).
The purpose of this agreement was to set up a common market and eliminate trade barriers among the signatory parties.
MERCOSUR has been involved in several health projects (such as implementation of GMPs with training and joint inspections and development of programs on vaccine regulation and control) to promote cooperation between its members and harmonization of specific pharmaceutical regulations in this subregion.
To date, there is no mutual recognition system.
▸ NAFTA (North American Free Trade Agreement) was implemented in January 1994 to remove most barriers to trade and investment among the US, Canada, and Mexico.
The objective of this agreement was to establish procedures to facilitate trade and investment on the North American continent.
This trade liberalization had some positive impact and created one of the largest trade blocs in the world, but some downsides have also been reported by economists (who have shown that NAFTA has not been able to produce an economic convergence).
NAFTA has had a minor impact on the harmonization of pharmaceutical regulations in the region and has not been able to resolve the problem of parallel import of pharmaceutical products between Canada and the US.
One of the major components of this initiative is the Pan-American Conferences on Drug Regulatory Harmonization held every two to three years.
These conferences are the highest instance of the PANDRH Network.
They serve to define priority areas for harmonization and to endorse standards, guidelines, and other recommendations, including norms and procedures and Steering Committee membership.
They also provide a forum for discussing issues of common interest in drug regulation.
Participants include all interested parties such as the DRAs of all PAHO Member States, representatives of the regional pharmaceutical industry associations, academia, consumer groups, professional associations, and representatives from the five subregional trade integration groups within the Americas.
The 1st PANDRH Conference took place in November 1997 (in Washington, DC, US).
PAN-DRH was then officially created during the 2nd PANDRH Conference in November 1999 (also in Washington, DC).
Following these first two conferences, subsequent conferences took place to review ongoing activities of the Working Groups.
PANDRH mimics the ICH structure.
It is organized around three major bodies: ▸ The Steering Committee (SC), which ensures operational management of this initiative between conferences, is composed of: • Seven members from five national DRAs (one from each of the subregional economic groups) and two industry representatives (FIFARMA and ALIFAR) • Seven alternate members from five different national DRAs (one from each of the subregional economic groups) and two industry representatives (FIFARMA and LIFAR) • Regulators from other countries (not represented on the SC), representatives from nongovernmental organizations (NGOs) recognized by PAHO/WHO, and other stakeholders invited by the SC who may also participate in SC meetings as observers Members of the committee serve for a period of four years, with staggered rotation to maintain continuity.
The SC meets at least twice every year.
Its primary role is (1) to establish the agenda for the biennial Pan-American conferences, and (2) to follow up on conference recommendations by establishing and monitoring the progress of Working Groups.
The responsibility of this group is to promote progress between conferences through the coordination, promotion, facilitation, and monitoring of the harmonization activities.
▸ The Technical Working Groups are specifically formed to work on topics and areas identified for harmonization.
The members are experts in their specific subject matter.
A Working Group may include the following categories of members: • Main Members that represent the national DRA of a country in each of the five subregional blocs, the regional Industry Associations ALIFAR and FIFARMA, and those designated by the Secretariat • Alternate Members designated to attend the meetings instead of the principal members • Observers from any country generally nominated by a participating national DRA (the observers do not retain voting rights) • Expert resources (as needed) to support a specific activity of the group (expert resources do not have voting rights) The national DRAs of countries not represented in the Working Group can designate focal points to follow the activity of the group.
Each Working Group has a coordinator (and an alternate) who chairs and coordinates the meetings, leads the development of documents, and reports periodically to the SC on the progress of the group.
In general, the first task of a new Working Group is to conduct a survey to identify the differences in regulatory requirements among countries in order to prepare a work plan.
Then, the group reviews international and regional and/or national recommendations and guidelines and prepares a harmonized proposal.
When the harmonized standard is developed, the Working Group is in charge of designing training and helping in implementation of this standard by assisting countries in the dissemination and education concerning this new rule.
Technical Working Groups meet in conjunction with SC meetings or separately (determined by a work plan and resources).
▸ A Secretariat, provided by PAHO, supports the initiative technically and administratively.
It monitors the PANDRH website, serves as a focal point for the coordination and dissemination of information, coordinates activities arising from recommendations of the conferences and SC, and acts as liaison and a representative of the Network in global and interregional harmonization organizations (ICDRAs, ICH, etc.)
As in other regions of the world, there is a need to promote harmonization of pharmaceutical regulations to facilitate the availability of safe, effective, and good-quality products and thereby protect public health.
PAHO initiated communication among the different members of the pharmaceutical sector in the Americas in order to facilitate communication among the different subregional blocs (and also the countries not already covered by these blocs) and organize regional harmonization.
The first Pan-American conference took place in November 1997 (in Washington, DC, US).
This conference was considered the first step towards the establishment of PANDRH.
During this first conference, the scope and the term "harmonization" were defined (as the search for common ground within the framework of recognized standards, taking into account the existence of different political, health, and legislative realities among the countries of the Region).
The structure and financial support of PANDRH were also discussed at this first conference.
However, PANDRH was officially created during the 2nd Conference (November 1999 in Washington, DC) following a consultation in Caracas, Venezuela in January 1999, and also several ad hoc discussions and meetings (Meeting of Americas' Regulators in Washington, DC in November 1997, Regional Working Group on Bioequivalence in Caracas in January 1999, and Regional Working Group on GCP in Buenos Aires in May 1999).
During this second conference, the mission statement and objectives of the SC were agreed upon.
This initiative was then officially recognized by the 42nd Directing Council of the PAHO in September 2000.
Resolution CD42.R11, which was approved during this Council, provided strong support from Ministers of Health of the Member States in the region to PANDRH and to the process of drug regulatory harmonization.
During PANDRH Conference V (in Buenos Aires in November 2008), the regulations governing PANDRH (mission, structures, and procedures) that were originally created during the 2nd Conference were slightly modified to incorporate lessons learned during its first few years of establishment [178].
Harmonization proposals are developed by the Technical Working Groups.
These groups primarily use WHO documents as the basis for developing regional guidelines.
Other international guidelines including ICH and selected regional (e.g., EU, American subregional) or national technical documents are also used as the basis for harmonization proposals and as reference materials.
After a Working Group has agreed on a draft harmonized document, it is posted on the website for external comment.
Comments are reviewed by the Working Group to prepare the final version of the document that will be presented for adoption by the Conferences through the SC.
Conclusions and recommendations of the Conferences are to be adopted by consensus (if consensus cannot be reached, the different points of view have to be recorded).
During its seventh meeting (in June 2006 in Washington, DC, US), the SC established a system of phases and stages for its harmonization process.
This system, which mimics the ICH process, is composed of five phases, with each having substages: Final technical documents are intended for use at the national level (through the subregional integration groups), but this implementation is at the discretion of each country.
Members of the SC are responsible for monitoring implementation in their subregion.
PANDRH is also discussing strategies to follow up the implementation of its recommendations at the national and subregional levels.
In addition to the biennial Pan American Conferences on Drug Regulatory Harmonization that allow for communication and exchange, PANDRH is also committed to training all interested parties (including regulators and industry).
Such training covers major topics such as GMP inspection, GCP, GLP, bioequivalence, etc.
The initial priorities that the PANDRH defined during the first conference were GMP (to facilitate the implementation of GMP in the region and ultimately to develop mechanisms for mutual recognition of inspection), bioequivalence, and GCP.
Additional topics were then added, each of these considered critical in the development of the network and in the protection of public health in all concerned countries.
Currently, there are 13 areas of priority that have been selected by PANDRH (for which Working Groups have been established): Several recommendations developed so far are based on WHO recommendations.
For example, WHO Report 32 was the basis for the discussion on GMPs, and the WHO and ICH guidelines were used to build consensus on GCPs.
Most of the selected topics are technical and have been chosen in order to ensure the quality, safety, and efficacy of the products approved, and that these products are adequately promoted and maintained.
The work on drug classification is also key to ensuring a common language and facilitating subsequent harmonization discussions.
Combat against drug counterfeiting has also been selected, as this is a major issue in this region directly affecting public health in all countries and requiring a multidisciplinary, multi-sectorial, and crossborder perspective.
Finally, the activity on drug registration is a broader project, and is very important for ensuring implementation of PANDRH recommendations and for reaching full harmonization of pharmaceutical regulations.
This is critical in ultimately developing a collaborative regional or subregional registration process and system and sharing of expertise and resources between countries.
This group drafted a proposed list of harmonized requirements for drug registration in the Americas [179] .
The current list of selected topics above will certainly be amended in the future if new emerging topics (creating potential health public issues in several countries of the region) need to be discussed and resolved at a regional level.
For example, the Working Group on Biotechnological Products has been established following a roundtable session of the 5th PANDRH Conference.
This roundtable session was organized to discuss biotechnological products (and also the specific issue of biosimilars).
Biosimilars present a clear risk for the patient (if they are not well controlled), but also a major opportunity for increased access to cheaper essential medicines (if they are well regulated).
These biotechnological/biologic products have unique technical challenges that require technical and specific expertise.
PANDRH will have to work on this topic collaboratively with WHO, which has already released recommendations on this topic.
PANDRH's scope of harmonization and cooperation includes technical guidelines, regulatory processes, and the strengthening of national DRAs through harmonization of processes and standards to improve and assure drug quality.
By adopting its recommendations and standards, countries in this region can clearly improve the quality of their regulatory system and provide access to quality, safe, and effective drugs.
Moreover, PANDRH plays an important role in the global harmonization of pharmaceutical regulations.
It is an important link between global organizations/forums and the regions.
Through its involvement in the ICH GCG, it increases: ▸ The integration of the regional challenges/priorities/vision in the development of international standards ▸ The implementation of such international standards in the region This regional initiative is one of the most difficult to operate because it includes very different regulatory systems and structures (from the most developed system such as the US FDA to the most undeveloped countries in the world).
This initiative also has to take into account the existence of very different political, health, and legislative realities among the countries that correspond to very different priorities, interests, and resources.
This reality creates difficulties in the management of projects and the establishment of consensus [180] .
However, this disadvantage also provides opportunities and benefits as the most developed DRAs can help to mentor the less developed ones.
Recognizing preexisting asymmetries in the region, PANDRH has become a forum to discuss common issues on drug regulation and share knowledge and expertise.
Not all the countries are involved in actually developing the proposals, but all of them participate in the decision of adopting them via the conferences.
By promoting the collaboration of experts from different countries/subregions, and also from both the public sector (authorities and academia) and private sector (industry), PANDRH has developed quality recommendations (frequently based on WHO or other international reports and recommendations).
It must be noted that PANDRH is clearly dependent on PAHO/WHO.
Without this support and investment, PANDRH would certainly not be viable.
Indeed, this financial, technical, and administrative support from PAHO/WHO, which represents an important recognition (both in and outside the region), is critical for the following reasons: ▸ As for all such multinational initiatives, one of the challenges of PANDRH is funding.
PANDRH's budget is primarily supported by PAHO, but additional funds also come from governments, the pharmaceutical industry, international organizations, and registration fees from training courses.
▸ Resources from involved countries are limited.
PAHO, by providing a Secretariat, has structured this initiative and allows the practical development of the harmonization projects.
▸ WHO provides critical technical help for the preparation of PANDRH recommendations.
Most PANDRH guidelines and documents are indeed based on WHO reports.
The 6th Conference of PANDRH, held in July 2011 (which included over 300 participants from 26 countries), focused its discussions on the theme "Strengthening National Health Regulatory Authorities."
Several working groups presented the conclusions of their work and their recommendations and actions.
The topics also addressed during this Conference included the role of PANDRH as coordinator of international cooperation, PAHO's recognition of national regulatory reference authorities (ANMAT-Argentina, ANVISA-Brazil, INVIMA-Colombia, and CECMED-Cuba), implementation of the PANDRH guidelines in the subregions, and innovative activities of the national DRAs in surveillance or in treatment compliance.
This conference concluded with the approval of a strategic orientations document.
The main recommendations were aimed at developing more effective cooperation among countries to guarantee, inter alia, the adoption and implementation of the different technical documents produced.
The major challenges for the future (what PANDRH will be assessed on) is the implementation of both its own and ICH's recommendations.
This will determine if this initiative delivers on its promises and if the countries that form this initiative are committed to this harmonization.
Because DRAs of all countries in the region participate in the conferences, it is expected that recommendations and guidelines will be adopted and implemented by the individual countries and incorporated in the discussion at subregional economic groups.
However, it may not always be so straightforward/automatic, and the implementation of its recommendations may become one of the major challenges of this regional initiative because its members have no obligation to implement harmonized standards.
The decision to develop a 2013-2020 PANDRH strategic plan to guide future development of the network, and ensure flexibility, scientific rigor, and representation of all stakeholders in the network [181], will certainly strengthen this initiative.
The Gulf Cooperation Council (GCC), also known as the Cooperation Council for the Arab States of the Gulf (CCASG) is a political and economic union.
Established in 1981, this trade bloc comprises six Arab states of the Arab Gulf.
It represents one of the wealthiest country groupings in the world due to its extensive oil and gas reserves.
Its population is approximately 42 million and its gross domestic product (GDP) is estimated at approximately US $917 billion [182] .
The GCC has been active in political affairs outside its territory.
Due to the instability of the Middle East region, the GCC has been heavily involved in diplomatic discussions to solve the different conflicts and problems of the region (i.e., Iraq/Iran war, Iraqi invasion of Kuwait, Iraqi situation after the breakdown of the former regime, Israeli/Palestinian war, etc.).
The objectives are to avoid the expansion of war and eliminate violence and terrorism in the region in order to support regional development and modernization.
In order to achieve unity, the GCC promotes the coordination, integration, and interconnection between its Member States in various fields.
One of the first objectives of the GCC is to formulate similar regulations in different areas, including health.
Cooperation and coordination in health are under the responsibility of the Council of the GCC Health Ministers (CHM).
Under its oversight, the Gulf Central Committee for Drug Registration (GCC-DR) was established to provide Gulf States with safe and effective medicines at a reasonable cost.
This committee works towards this objective by promoting cooperation and harmonization among Member States.
This initiative covers prescription, nonprescription, generics, and biologics.
On the international side, the GCC represents the region at the ICH Global Cooperation Group (GCG).
The current GCC members are six Arab states of the Arab Gulf (Plate 3): Bahrain, Kuwait, Oman, Qatar, Saudi Arabia, and the United Arab Emirates (UAE).
Iran and Iraq are currently excluded although both nations have a coastline on the Persian Gulf.
Yemen is currently not part of the union.
This country is, however, involved in some GCC initiatives (i.e., activities related to the health sector) in view of a future accession.
For example, Yemen is a member of the Council of the GCC Health Ministers (CHM).
The Supreme Council is the highest authority of the GCC and is formed by the heads of the Member States.
Presidency of the GCC Supreme Council rotates, and it convenes annually in a regular session, though additional extraordinary sessions may also be scheduled.
This Supreme Council is supported by the Ministerial Council, composed of the Ministers of Foreign Affairs of Member States or other ministers acting on their behalf.
The Ministerial Council proposes policies, lays out recommendations, and coordinates existing activities in all fields.
Resolutions adopted by other ministerial committees are referred to the Ministerial Council, which in turn refers relevant matters to the Supreme Council for approval.
The CHM is the highest regional level of authority in the area of health.
It consists of Health Ministers from each of the GCC Member States (plus Yemen, though presently not a member).
It meets for two to three days twice a year, and these meetings are open to all regulators from the GCC Member States and Yemen.
WHO (via its Regional Office for the Eastern Mediterranean, EMRO) also attends as an observer.
The CHM is supported by an Executive Board to whom an Executive Office General Director reports.
The Executive Office is located in Riyadh, Saudi Arabia.
At the working level, a GCC-DR was established to oversee the different activities in the pharmaceutical sector.
The Steering Committee of the GCC-DR is composed of two members from each of the Member States (including Yemen), and meets at least four times per year.
The membership is limited to government agencies or DRAs.
The Executive Office also appoints two of its affiliates as advisors (nonvoting members) to the Steering Committee.
This committee is responsible for the registration of the pharmaceutical companies and their products as well as for the preparation of technical regulations and guidelines.
To develop a new guideline, the GCC-DR Steering Committee uses the resources of the Member States by assigning the drafting of the specific guideline to either a single Member State or several Member States.
Technical working groups can also be set up to help in developing the guideline.
Within the Executive Office, a permanent GCC-DR Secretariat was also created to support the organization.
The role of this Secretariat is to facilitate the harmonization activities through administration, coordination, and communication.
It is also responsible for receiving and reviewing registration files for completeness and for preparing Steering Committee meeting agendas.
The GCC was created on May 25, 1981, and its unified economic agreement was signed by its Member States on November 11, 1981 in Riyadh, Saudi Arabia.
The primary objective was to achieve "coordination, integration and interconnection between Member States in all fields in order to achieve unity between them" [183] .
This integration plan was developed in detail during the first 20 years following the establishment of the GCC.
On December 31, 2001, the GCC Supreme Council adopted, during its 22nd Session in Muscat, Oman, a revised economic agreement that accelerated this integration.
This revised agreement enhanced and strengthened economic ties and increased harmonization among Member States.
In Chapter II, the agreement defined specific areas that needed to be harmonized in order to support the GCC common market, health being one of these areas.
Article 12 also promotes joint projects and adoption of integrated policies between Member States.
Having finally completed all requirements, the GCC common market was declared in December 2007 and came into force as of January 2008.
This launch of the common market removed barriers to cross-country investment and service trade.
GCC cooperation in the health sector began in the mid-1970s when the GCC health ministers held informal meetings such as the one held in Geneva (May 16, 1975) during the general assembly of WHO.
Such cooperation was then formalized with the establishment of the Conference of the Health Ministers of the Arab Countries in the Gulf, which held its first meeting in February 1976.
Since 1991, it has been called CHM.
As mentioned previously, under the CHM, the GCC-DR was established in 1999 to provide the Gulf States with safe and effective medicines.
The scope of the GCC-DR's harmonization and cooperation efforts in the pharmaceutical sector covers technical guidelines and regulatory processes.
This includes the registration of pharmaceutical companies and products as well as good manufacturing practice (GMP) inspection.
Under the oversight of the CHM, the GCC-DR Steering Committee is responsible for the selection and prioritization of topics, the assignment of the development of guidelines and policies, and the subsequent review and approval of the resulting recommendations.
When a new topic is selected for harmonization, the GCC-DR Steering Committee assigns the development of the guideline/policy to either a single Member State or several Member States, and a technical working group is then established.
The membership of this working group is at the discretion of the assigned Member State(s).
It may include regulatory, industry, and academic experts.
Technical working groups meet regularly (independently of the Steering Committee meetings).
An annual meeting is also held with both the Steering Committee and relevant invited experts to discuss policy and regulations.
ICH guidelines are often used as reference material when developing GCC-DR guidelines.
Other international guidelines (including WHO recommendations), available national technical documents, and guidelines from other regions (e.g., EU) are also used.
Once developed by a working group, the draft guideline is posted on the GCC and the Saudi Food & Drug Authority (SFDA) websites (http://www.sgh.org.sa and http://www.sfda.gov.sa/ en/Pages/default.aspx).
They are also circulated to all Member States for comment.
At the end of the consultation period, the working group reviews all comments received, finalizes the document, and proposes its adoption by the GCC-DR Steering Committee.
Following its adoption, the General Director of the Executive Office submits the guideline to the CHM for final approval.
GCC-DR Steering Committee members are responsible for monitoring the implementation of the adopted guidelines in their countries.
Each country reports whether it encounters any problems in implementing the guidelines during an annual meeting where the GCC-DR activities are evaluated.
Standard practices and operating procedures have been developed to govern all steps of the harmonization process (i.e., selection and prioritization of topics, solicitation of comments, approval/ implementation of guidelines and responsibilities of the different bodies, as well as funding).
Additional procedures also cover the process in place for the registration of products and companies.
The GCC-DR is financed by Member States (using established quotas of contributions) and by registration fees.
The status of its activities is communicated through its website, and also through presentations at national and international meetings, workshops, and conferences.
Although the Executive Office organizes GMP training, there is currently no official structured training program within this initiative.
Each Member State is responsible for providing training to their regulators.
The GCC-DR has initiated work on several general topics related to the development and registration of all medicinal products (GMP and GMP inspection, bioequivalence studies, stability, good laboratory practice [GLP] , and clinical trials).
The group also decided to harmonize practices on post-marketing activities via the development of guidelines on post-marketing surveillance (covering the counterfeiting problem) and pharmacovigilance.
Finally, recommendations on specific types of products (biosimilars, sera and anti-venom, vaccines, and blood products) are also under discussion.
The guidelines listed above are at different stages of development (under discussion, drafting in progress, approved, or implemented).
They are all based on ICH, WHO, US FDA, and/or EMA recommendations.
In addition to these guidelines, the GCC-DR also established a common central procedure for the registration of both the pharmaceutical companies and the pharmaceutical products.
The establishment of a common system of registration and control of medicines was discussed at the first meeting of the CHM in 1976.
This subject was a recurrent topic of discussion until actual implementation of this procedure in 2001.
Since its implementation, the registration of both medicines and pharmaceutical companies has slowly transitioned from the national to the GCC registration procedure as shown in the Table 6 .
Under this procedure, dossiers (including fees) are submitted to the GCC-DR Secretariat.
Each country reviews the dossiers and forwards its recommendations to the GCC-DR Steering Committee.
The committee's resolutions are adopted by the majority of the attendant members' votes (four countries is the minimum that must be represented).
GMP inspection and analysis of samples by the accredited laboratories are also part of this central procedure.
After the central approval, each country must adopt this central approval nationally.
As mentioned above, the GCC-DR is responsible for GMP inspections, but also for the approval of quality control laboratories and for the review of technical and post-marketing surveillance reports.
All these central activities increase the harmonization and integration of the pharmaceutical sector.
Since its creation and the signing of its initial unified economic agreement in 1981, the GCC has cooperated in many different fields (i.e., political, military, security, legal, economic, environment, and health) and developed common policies in support of achieving full integration.
This integration goal was reemphasized in 2001 when the GCC Supreme Council adopted a revised Economic Agreement.
In January 2008, the launch of the GCC common market marked an important step in the GCC's integration.
In the health sector, cooperation began earlier.
Before the signature of the unified economic agreement in 1981, the Health Ministers had decided to cooperate in the area of health.
Since the initial discussions by the Health Ministers, many objectives have been fulfilled.
The development of common guidelines, cooperation in the domain of GMPs, and the establishment of a central registration procedure for companies and products are certainly the major achievements from this group.
The unified purchase of drugs (i.e., common tenders concept) is also one of the most important achievements of the CHM.
It has ensured the purchasing of high-quality registered products from registered companies (national or international) for a more affordable price as it increased the amount of products purchased.
But it has also ensured the use of the same products by all Member States, which is indeed an important step in the integration process and the creation of the common market.
This cooperation allows the member countries to implement common drug policies and adopt an efficient drug quality surveillance reporting system to monitor the efficacy and safety of the registered drugs [184] .
Recognizing all the above achievements, and despite clear increases in cooperation, the GCC has, however, not yet fully achieved its goal of unity in the pharmaceutical sector.
Indeed, this group has selected an integration model that will require stronger ties between countries.
For example, the central registration procedure still involves national reviews and is longer than the national registration [185] .
Moreover, approvals delivered via this central procedure still have to be adopted by each member country.
This integration process is not as advanced as in Europe, where there is a rapporteur that conducts the review of the application on behalf of the group and where the EC approves drugs on behalf of all European countries.
Harmonization of the regulation (via both regional integration and international cooperation) is critical for this region for the following two reasons: ▸ First, this region is highly dependent on medicines developed and manufactured in other countries and regions.
Even if pharmaceutical companies (both international and regional) are increasing their investment in the Middle East region, this region is still primarily an import-oriented market.
All GCC countries share the same characteristic of being high importers of pharmaceutical products.
More than 70% and 80% of pharmaceuticals consumed in Oman and Saudi Arabia, respectively, are imported [186] .
It is critical for the region to ensure that products from other countries have been developed and manufactured following acceptable standards and requirements.
▸ Second, we have seen that most of the GCC-DR recommendations and guidelines are based on other international work (i.e., ICH, WHO, etc.).
The GCC is therefore dependent on the resources and expertise of these international organizations to develop its own state-of-the-art requirements and standards.
The next step in the integration process of the GCC region will certainly be a better and bigger sharing of resources and expertise.
The challenges of this next step will be the development of an organization and infrastructure to support such evolution.
Today, the regulatory expertise in the different countries is varied, with Saudi Arabia being the leader in the region.
This country represents the biggest pharmaceutical market of the region, with approximately 65% of the pharmaceutical sales of the GCC [187], and its regulatory system is recognized as the most developed of the region.
In 2010, the regulatory agency in Saudi Arabia, the SFDA, employed 150 people in its drug sector with approximately 50 reviewers, compared to less than 10 in most of the other GCC countries.
The ongoing development of a common and central system needs to ensure that the less developed countries of the regions will benefit from this cooperation without impacting the more developed countries in this sector.
Another challenge for this group, like for all other harmonization initiatives, is the implementation of the agreed-upon standards.
The GCC needs to work on measures, including the development of a structured training program, to facilitate the implementation and follow-up of recommendations.
Today, the Southern African Development Community (SADC) is comprised of 15 Southern Africa States, and its headquarters are located in Gaborone, Botswana.
Each of the SADC Member States is at varied stages of socio-economic development, but are predominantly underdeveloped.
Its aggregated gross domestic product (GDP) is approximately US $457 billion, with South Africa representing a significant portion of this amount.
Its estimated total population is approximately 250 million [188] , with an average population growth rate of 2.2% and an average fertility rate of 4.9 births per woman of childbearing age.
Approximately 50% of this population lacks sustainable access to affordable and quality essential medicines.
The average life expectancy is 39.7 years (the lowest in the world) [189] .
The SADC objectives (listed in Article 5 of the SADC Treaty) support regional integration and increased economic, social, and political cooperation in order to promote peace and security, economic growth, well being of the population, and protection of the environment and natural resources of the region.
To achieve this major and broad objective, the SADC has launched projects and defined specific actions (e.g., harmonization of policies and creation of appropriate institutions and mechanisms).
Additionally, the SADC has had major milestones, such as the formation of the SADC Free Trade Area (FTA) in 2008, and set future goals, including the establishment of the common market by 2015 and the creation of a single currency by 2018.
The first achievement related to the formation of the SADC FTA took place on August 17, 2008 at Sandton, South Africa during the 28th Summit of SADC heads of state and government.
Acknowledging that regional cooperation was critical to addressing the health problems of the region, the SADC decided to include health in its program of action.
The need for harmonization of registration and control of medicines was further justified in 1999 when the disparities of legal systems and levels of development affected the implementation of a regional bulk purchasing initiative (involving five medicines used to treat tuberculosis) [190] .
The SADC health program was developed taking into account global and regional health declarations and targets.
To enhance this regional health integration within a legally enforceable framework, a protocol on health matters was developed.
SADC also has access to the international network because it is part of the ICH Global Cooperation Group (GCG).
The The Summit, comprising all the heads and/or governments of SADC Member States, is the highest regional authority and therefore the supreme policymaking institution of SADC.
It is responsible for the overall direction and control of the community.
Its structure and functions are enumerated in Article 10 of the SADC treaty.
The Summit usually meets in the Member State holding the deputy chairpersonship of SADC at the time (additional meetings can also be held if necessary).
The main objective of the Organ on Politics, Defense and Security, under the oversight of the Summit, is to promote peace and security in the region.
The structure, operations, and functions of the Organ are regulated by the protocol on politics, defense, and security cooperation, which was approved and signed by the Summit at its meeting in August 2001 in Blantyre, Malawi.
Since 1999, the SADC leadership has been based on the Troika system, which includes the chair, incoming chair, and the outgoing chair of SADC (other Member States may be co-opted into the Troika if necessary).
The Troika represents the Summit between annual meetings and makes quick decisions on behalf of SADC that are ordinarily made during the Summit meetings.
This system allows the organization to execute tasks and implement decisions expeditiously.
It also allows the provision of policy direction to SADC programs and operations between regular SADC meetings.
This Troika system is applied at the Summit level, but is also applicable for the Organ on Politics, Defense and Security, the Council, the Integrated Committee of Ministers, and the Standing Committee of Officials.
To support the SADC activities, a central Secretariat was formed.
This body is defined as the principal executive institution of SADC responsible for the coordination of the harmonization of policies and strategies to accelerate regional integration.
It is responsible for the management of SADC meetings, and financial and general administration.
It is also involved in strategic planning, management of SADC programs, and the implementation of decisions of SADC policy organs and institutions.
One of the characteristics of the SADC is its emphasis on a decentralized institutional arrangement ( Figure 2) .
Following previous negative experiences and failures in regional discussions, the founder states agreed that Member States should be the principal players in the formulation and implementation of policy decisions.
Therefore in addition to the central SADC institutions, SADC National Committees were established by the SADC treaty.
These SADC institutions at the national level are present in each Member State and include key stakeholders from government, the private sector, and civil society.
Their functions are (1) to provide national feedback and input in regional strategy and planning, and (2) to ensure the proper implementation of these agreed-upon regional strategies, protocols, and programs at the national level.
This Southern African Union was created in 1980 by nine founding Member States (Angola, Botswana, Lesotho, Malawi, Mozambique, Swaziland, United Republic of Tanzania, Zambia, and Zimbabwe) with the adoption of the Lusaka Declaration on April 1, 1980 in Lusaka, Zambia.
At that time, this alliance was called the Southern African Development Coordination Conference, and its main objective was to coordinate development projects in order to lessen economic dependence on South Africa, then under apartheid.
The formation of this alliance was the culmination of a long process of consultations begun in the 1970s when it became clear to the leaders of the founder countries that the improvement of living standards would require regional cooperation.
This cooperation was directed initially towards the political liberation of the region.
Following the decolonization and the political independence of Southern African countries, and acknowledging the poverty and economic problems of the region, the leaders of these countries saw the promotion of economic and social development through cooperation/integration as the next logical step.
On August 17, 1992 (in Windhoek, Namibia) , a new declaration and treaty was signed during the Summit of Heads of State and Government.
Article 2 of the treaty gave a legal basis to the organization and promoted it from a coordinating conference into a development community.
The SADC was then established to spearhead economic integration of Southern Africa.
This strengthening of the integration process in Southern Africa was aligned with the overall African continental efforts to promote closer economic relations (as defined in the treaty signed in 1991 to establish the African Economic Community).
In March 2001, SADC country Heads of State and Governments met in Windhoek, Namibia.
During this extraordinary Summit, many important decisions were made that triggered an amendment to the SADC treaty.
First, the Summit decided to restructure SADC institutions and to establish SADC national committees in order to facilitate the implementation of a more coherent and better-coordinated strategy.
The extraordinary Summit also approved the preparation of the RISDP by the secretariat.
The purpose of this 15-year plan (which was adopted in August 2003 and launched in March 2004) was clearly to deepen regional integration by providing SADC Member States with a consistent and comprehensive program of long-term economic and social policies.
This plan reemphasizes the major objectives of the organization, reviews the socio-economic indicators and challenges of the region, and analyzes all the important domains for the integration process (including health).
It also provides objectives and specific targets for priority intervention areas, and specifies plans and timeframes for implementation and monitoring of its important measures.
For example, in the health domain, the plan proposes to coordinate, harmonize, and monitor the implementation of regional policies and to standardize the qualification and accreditation systems.
The cooperation in the health domain started in 1997 with the development of the SADC health program.
Three key policy documents were important in the implementation of this SADC health program: As defined in Article 22 of the SADC Treaty, protocols were established in each area of cooperation.
These protocols spell out the objectives and scope of, and institutional mechanisms for, cooperation and integration.
Each protocol (which is approved by the Summit and is registered with the secretariat of the United Nations Organization and the Commission of the African Union) is binding for the Member States that are party to the protocol.
More than 20 protocols have been developed in all domains of integration.
The protocol on health [192] covers all aspects related to health (from the control of major communicable and noncommunicable diseases to the health laboratory service and institutional mechanisms).
Article 29 states that Member States should cooperate in the harmonization of procedures for pharmaceuticals, quality assurance, and registration, and also in the production, procurement, and distribution of affordable essential drugs.
The implementation plan of this protocol (which further defines and prioritizes the actions to facilitate implementation of the protocol) fixes the integration of regional regulatory processes and the establishment of a mutual recognition as a 2011-2015 past, present, and future milestone [193] .
In line with the SADC Health Protocol, a pharmaceutical program was developed to address issues related to the access to quality medicines in all Member States.
This program was approved in June 2004.
This SADC pharmaceutical harmonization initiative and cooperative activities include the development of technical guidelines and policies relating to the registration and control of medicines across the SADC Member States.
The initiative aims to improve the quality, safety, and efficacy of medicines circulating within the region, and to establish and maintain a regional shared network system for DRAs.
The ICH and WHO guidelines, as well as other guidelines, form the basis as reference materials for the development of regional guidelines, with agreement on the adoption of international guidelines whenever possible.
Potential topics for harmonization are identified at the level of the subcommittee of Ministers of Health, often with the input of senior ministerial health officials and MRA forum experts.
The process of harmonization is initiated through the SADC Secretariat, which prepares and submits for decision an agenda to the Ministers of Health.
Within this context, the SADC Pharmaceutical Business Plan was released in June 2007.
This 2007-2013 plan identified priority areas, objectives, and major activities that needed to be implemented both at regional and national levels to improve access to quality and affordable essential medicines (including African traditional medicines).
For example, strengthening regulatory capacity (and ensuring that fully functional DRAs are in place with an adequate enforcement infrastructure) and facilitation of the trade in pharmaceuticals within the regions were key strategies developed in the plan.
The monitoring and ongoing evaluation of this plan (its implementation was estimated at US $16 million) was also described (see Figure 3 , which explains the relationship between the different players of the plan).
Under the oversight of the Ministers of Health, a group of designated senior officials monitored the implementation of the plan via the establishment of technical subcommittees or task teams.
This group of senior officials (from the health departments of each Member State) was also supported by the Secretariat.
The sector of the Secretariat responsible for supporting the operations of the pharmaceutical harmonization initiative takes place under the directorate of the SHD&SP.
National Health Ministries also play a significant role (by coordinating and leading the implementation of programs at the national level), and report on progress through their SADC National Committees.
Finally, other stakeholders (e.g., professional associations, research institutions, DRAs, etc.)
are also involved and requested to provide expertise and feedback on specific actions of the plan.
In 2004, the Medicines Regulatory Forum was created as a technical subcommittee to promote the harmonization and enhancement of the pharmaceutical regulations in the region.
This standalone committee is made of the heads of the national regulatory bodies.
The SADC has released guidelines on several topics.
These guidelines regulate the following general areas: ▸ The conduct of clinical trials: these guidelines provide a framework (information to be submitted, review process, etc.)
and refer to the entire ICH GCP (this is not a replacement or subimplementation of the ICH GCP).
▸ Registration of medicines: "Guidelines for Submitting Applications for Registration of a Medicine" were released in 2007.
An application form is also available.
▸ Good manufacturing practices.
▸ Pharmacovigilance (only basic rules are provided).
▸ Advertising.
▸ Recalls.
▸ Registration of nutritional supplements, vaccines, and traditional medicines.
▸ Bioavailability and bioequivalence.
▸ Stability studies.
▸ Import/export (with an emphasize on GMP).
Most of the above guidelines are based on, or cross-reference, ICH and WHO guidelines and recommendations.
These international bodies provide much of the technical assistance to SADC initiatives.
When they exist, national rules and requirements are also used (e.g., the GCP requirements from South Africa).
Guidelines have also been developed to cover the following topics that are of specific interest for the region: ▸ Pharmaceutical wholesale ▸ HIV vaccine clinical trials ▸ Donations of pharmaceutical products It should be noted that the SADC efforts in the pharmaceutical area include African traditional medicines.
These products are an important part of the healthcare environment of these countries.
One of the cooperation projects is to establish a regional databank of traditional medicines and medicinal plants, and to develop regional policies and legal frameworks for the practice of these traditional medicines.
Finally, SADC is trying to establish a joint procurement system and to harmonize standard treatment guidelines/lists among countries.
These two actions will facilitate the use of the same medicines within the region and therefore allow further harmonization of the pharmaceutical environment.
Since its inception in April 1980, SADC has demonstrated that regional cooperation and integration is possible and useful for Southern Africa.
One of the foremost achievements of SADC has been to put in place a regional program (the SADC Programme of Action) with numerous projects covering cooperation in various economic sectors.
The formation of the SADC FTA on August 17, 2008 was an important first step in this ongoing integration process.
The overall and ultimate goal of SADC is integration by 2020; this is a very ambitious plan.
Presently, the level of cooperation varies for each area.
In some areas, this cooperation only aims to coordinate national activities and policies.
In others, the cooperation goes towards real integration.
For example, on foreign policy, the main objective is coordination and cooperation, but in terms of trade and economic policy, a tighter coordination is in progress with a view to one day establishing a common market with common regulatory institutions.
In the health and pharmaceutical domain, many harmonization projects have been established despite challenges.
Indeed, as recognized in the SADC pharmaceutical business plan, the region has many weaknesses, such as weak regulatory systems (leading to many unregistered products), lack of adequate capacity and trained personnel, outdated medicine and intellectual property laws, and noncompliance to GMP (leading to inadequate availability of medicines and poor and inconsistent quality of these medicines in some Member States).
Even if there is a political will, it is very difficult for the authorities of this region to resolve this situation as they are confronted by two major problems: ▸ The management of major diseases (such as HIV/AIDS, tuberculosis, malaria, etc.)
▸ The lack of adequate resources and finances to support all health initiatives The combination of the two above problems, common to all developing countries, slows down the development of other health activities.
All the efforts and resources in the domain of health are rightfully dedicated to the prevention and treatment of the major public health concerns.
Activities such as the development of adequate regulatory function and framework or the development and harmonization of pharmaceutical requirements are therefore negatively impacted.
Even if all SADC Member States have national medicine policies, legislation, and regulation in place, some of these policies have been draft documents for many years (up to 15 years).
A number of the laws date back from the 1960s (some even to the 1930s).
It is clear that such policies and legislation need revisions to include recent developments and meet current standards in public health and medicines.
Such revisions and updates would help the implementation of the SADC harmonized recommendations and guidelines.
However, despite the numerous weaknesses and problems that the region faces, the SADC was able to promote cooperation between Member States in order to improve access to quality medicines.
There have been several major accomplishments in the development and harmonization of pharmaceutical requirements, such as the development of pharmaceutical guidelines for the registration and control of medicines, the establishment of the pharmaceutical business plan, and the establishment of the "Medicines Regulatory Forum."
Moreover, the SADC has now analyzed (with its pharmaceutical business plan) the weaknesses, opportunities, and overall priorities in the pharmaceutical domain (i.e., regulation and control of medicines).
The road map includes the assessment and strengthening of DRAs (work performed in collaboration with the WHO), combat against the spread of counterfeit medicines, the development of regional training programs, and the establishment of accredited quality control (QC) laboratories.
To support this road map and other areas of harmonization, the structure of the SADC institution will certainly have to be modified (as done in the past).
In order to be successful, SADC will also need to continue to work with external organizations.
Support and technical assistance from ICH and WHO will continue to be critical.
But, communication and cooperation with other groups and regions (e.g., the New Partnership for Africa's Development [NEPAD]) will also be necessary to coordinate the efforts on the entire continent and share the available resources, financial support, and expertise.
This is especially important because some SADC members are also part of other African subregional initiatives.
Finally, the next important phase for SADC is the implementation of the agreed-upon standards, recommendations, and plans (e.g., How will the proposed actions to "strengthen national DRA capacity to implement harmonized SADC guidelines" be managed?).
Implementation is a challenge for all harmonization initiatives.
This is especially true for this region due to all the weaknesses carried by these countries and the lack of resources and finances.
However, the lack of appropriate regulations in some countries may paradoxically become an opportunity; the coordination of the development of the regulation (based on the WHO and ICH recommendations) can be viewed as an a priori harmonization.
Moreover, it is interesting to note that the SADC structure presents a specificity not found in other harmonization initiatives.
In addition to the standard centralized bodies (i.e., Summit, Council of Ministers, Committee of Senior Officials, Central Secretariat, etc.
), the SADC has established National Committees.
These national SADC contact points could become critical for this implementation phase.
This unusual model may also be useful for other worldwide initiatives.
The Association of Southeast Asian Nations (ASEAN), established in 1967, has very broad objectives.
The aims and purposes of the Association, stated in its Declaration, include: ▸ The acceleration of economic growth, social progress, and cultural development in the region through joint endeavors in the spirit of equality and partnership in order to strengthen the foundation for a prosperous and peaceful community of Southeast Asian Nations ▸ To promote regional peace and stability through abiding respect for justice and the rule of law in the relationship among countries in the region ▸ To promote active collaboration and mutual assistance on matters of common interest in the economic, social, cultural, technical, scientific, and administrative fields ▸ To provide assistance to each other in the form of training and research facilities in the educational, professional, technical, and administrative spheres ▸ To maintain close and beneficial cooperation with existing international and regional organizations with similar aims and purposes, and explore all avenues for even closer cooperation among them The ASEAN region has a population of approximately 590 million, a total area of 4.5 million square kilometers, a combined gross domestic product (GDP) of US $1,500 billion, and a total trade of about US $1,500 billion [194] .
Its estimated annual pharmaceutical imports and exports is US $9.5 billion [195] .
Among the three pillars of the ASEAN Community (Political-Security, Economic, and Socio-Cultural) agreed upon by the ASEAN Leaders in the Declaration of ASEAN Concord II (signed on October 7, 2003 in Bali, Indonesia), the establishment of a single market by 2020 is an important goal.
Its objective is to allow the creation of a stable and prosperous ASEAN economic region in which there is a free flow of goods, services, and investments in order to reduce poverty and socio-economic disparities.
At the 12th ASEAN Summit in January 2007, the Leaders affirmed their strong commitment to accelerate the establishment of an ASEAN Economic Community (AEC) by 2015 and signed the Cebu Declaration on the Acceleration of the Establishment of an ASEAN Community by 2015.
In 1992, in moving towards this ultimate goal, ASEAN launched the ASEAN Free Trade Area (AFTA) and defined priorities (e.g., healthcare) where regional integration should be accelerated.
One of the basic criteria to support AFTA, and ultimately a single market, is the harmonization of standards and regulations.
Therefore, recognizing the importance of the harmonization of standards to facilitate and liberalize trade and investment in the region, ASEAN has established the ASEAN Consultative Committee on Standards and Quality (ACCSQ) to harmonize national standards with international standards and implement mutual recognition arrangements on conformity assessment to achieve its end goal of "One Standard, One Test, Accepted Everywhere."
The ACCSQ monitors the harmonization of standards and regulations in many different areas (i.e., pharmaceutical products, but also cosmetics, medical devices, food, electrical and electronic equipment, automotive products, wood-based products, etc.).
Harmonization in the pharmaceutical area is coordinated by the Pharmaceutical Product Working Group (PPWG).
The objective of this group is to harmonize the technical procedures and requirements applicable to the ASEAN pharmaceutical industry in the region, taking into account other regional and international developments on pharmaceuticals.
Since 2003, ASEAN has been a member of the ICH Global Cooperation Group (GCG).
This membership helps ASEAN to become an important component in the global harmonization process, as it constitutes a way to disseminate the ICH recommendations on drug regulatory harmonization.
It also ensures that ASEAN specificities and challenges will be considered when new global recommendations are discussed.
The The highest decision-making body of ASEAN is the meeting of the ASEAN Heads of State and Government (the ASEAN Summit) that is convened annually.
Additional ministerial meetings are also held regularly.
Committees of senior officials, technical working groups, and task forces have been created to support the ASEAN Summit and Ministerial meetings and conduct the agreed ASEAN activities.
The ACCSQ was established to coordinate the harmonization of national standards with international standards.
This committee reports to the ASEAN Senior Economic Official Meeting (SEOM) that is under the supervision of the ASEAN Economic Ministers (AEM).
The PPWG, under the supervision of the ACCSQ, was created to coordinate the harmonization activities related to the pharmaceutical area.
The scope of activities of the PPWG includes the following: ▸ Exchange information on the existing pharmaceutical requirements and regulations implemented by each ASEAN member country.
▸ Review and prepare comparative studies of the requirements and regulations.
▸ Review the harmonized procedures and regulatory systems currently implemented in others regions in order to develop harmonized standards, regulations, and procedures for the region.
For each specific topic selected for harmonization, the PPWG sets up ad hoc committees and assigns one of the Member States as the project leader.
Membership of the Ad Hoc Committee is on a voluntary basis.
The core members of the PPWG are the chair and co-chair, representatives from the DRAs from each ASEAN Member State, a representative from the ASEAN Secretariat, as well as representatives from pharmaceutical industry associations.
Delegates from additional Member States can also participate in PPWG meetings as observers.
In addition, ACCSQ members and invited experts may attend the annual PPWG meeting.
The Ad Hoc Committee meets prior to the PPWG meetings.
Additionally, the PPWG operates through self-sponsorship (i.e., each Member State is responsible for its own funding for traveling or hosting meetings).
WHO has also contributed to the process in the past.
PPWG activities are supported by the ASEAN Secretariat, which was established on Feburary 24, 1976 to coordinate the ASEAN branches and to implement ASEAN projects and activities.
In 1992, the mandate of the ASEAN Secretary-General was enlarged to initiate, advise, coordinate, and implement the agreed-upon ASEAN activities.
Finally, it should be noted that another working group, the ASEAN Working Group on Pharmaceuticals Development (AWGPD) (under the supervision of the ASEAN Health Ministers Meetings), also participates in the regional harmonization of pharmaceutical regulations through its activities on traditional medicines, good manufacturing practices (GMPs), good clinical practices (GCPs), counterfeiting drugs, and pharmacovigilance [196] .
ASEAN was officially established with the signature of its declaration (the Bangkok Declaration) on August 8, 1967 in Bangkok, Thailand by the five original Member Countries (i.e., Indonesia, Malaysia, Philippines, Singapore, and Thailand).
Brunei Darussalam joined on January 7, 1984, Vietnam on July 28, 1995, Laos and Myanmar on July 23, 1997, and Cambodia on April 30, 1999.
The ACCSQ was formed in 1992 to facilitate and complement the AFTA.
Efforts towards specific harmonization of pharmaceutical regulations have been initiated by the ACCSQ since 1992.
The Pharmaceutical Product Working Group was then established in September 1999 in Kuala Lumpur, Malaysia following a decision by the ACCSQ during its 13th meeting (March 1999 in Manila) .
During its inaugural meeting during September 6-7, 1999, the PPWG formulated its terms of reference and set up a work plan (i.e., goals, strategy, activities, expected output, and status).
Subsequent meetings focused on the status review of ongoing harmonization activities, and discussion and adoptions of final recommendations.
The ASEAN also decided to develop relationships with other countries.
They developed "bilateral agreements" with a number of countries (Canada, India, the US, the Russian Federation, Pakistan, etc.
), other regions (Europe, GCC, SADC, Andean Group, Mercosur), and international organizations (United Nations, UNESCO).
But one of the most important developments was the creation of the "ASEAN Plus Three" cooperation to promote the East Asia region.
This cooperation began in December 1997 with the convening of an informal summit among ASEAN leaders and their counterparts from East Asia, namely China, Japan, and the Republic of Korea.
It was then formalized in 1999 with the issuance of a joint statement on East Asia cooperation at the 3rd ASEAN Plus Three summit in Manila, Philippines.
The ASEAN Plus Three leaders expressed confidence in further strengthening and deepening East Asia cooperation at various levels and areas, particularly in economic, social, political, and other fields.
Public health and harmonization of standards are topics under discussion among others.
Several bilateral economic arrangements have already been signed, and may be the basis for the possible establishment of an East Asia free trade area in the future [197] .
In November 2007, two important documents were ratified: ▸ First, the ASEAN charter which spells out the principles to which all 10 Member States adhere to was signed.
This legal framework, which entered into force on December 15, 2008, serves as a firm foundation in formulating the ASEAN community by providing legal status and an institutional framework for ASEAN.
It also codifies ASEAN norms, rules, and values, sets clear targets for ASEAN, and presents accountability and compliance.
▸ Second, the ASEAN leaders also signed the Declaration on the ASEAN Economic Community (AEC) blueprint that provides the elimination of forms of nontariff measures and market access limitations in order to transform ASEAN into a single market.
The draft guidelines developed by the Ad Hoc Committees are reviewed, discussed, and then adopted, by consensus, during the PPWG meeting.
These standards are then endorsed by the ACCSQ.
The PPWG harmonization process includes the following steps: ▸ Exchange and review of information on existing pharmaceutical requirements and regulations of the Member States.
▸ Compare the requirements and regulations to identify key areas for harmonization.
▸ Create an Ad Hoc Committee (and assignment of a lead country) to prepare the draft "harmonized product," which most of the time is based on guidelines or recommendations already available (in one of the ASEAN countries, internationally, or in another regions).
▸ Circulate the draft to all Member States for comments.
▸ Consolidate comments into the revised draft.
▸ Discuss and adopt (by consensus agreement) the draft by the PPWG.
▸ Endorsement of the document and recommendation by the ACCSQ.
▸ Dissemination of the adopted documents (via the ASEAN website or seminars/ meetings).
▸ Compulsory implementation of the recommendation by the Member States.
In order to organize, coordinate, and monitor the implementation of the agreed-upon recommendations and guidelines, the PPWG set up a specific Task Force and working group to focus on a Mutual Recognition Arrangement (MRA) and implementation.
They developed a standard operating procedure (SOP) and plan of action.
They also assessed the status of the implementation of requirements (i.e., adoption into the national systems) in order to develop appropriate training (to government and industry) to increase understanding of the ASEAN guidelines and fill the gaps among the Member States.
The first project of the ASEAN PPWG was to compare the existing product registration requirements for pharmaceuticals of ASEAN member countries in order to help define key areas for harmonization.
This report was finalized in 1999.
Following this assessment, the group developed the ASEAN Common Technical Requirements (ACTRs) for pharmaceutical product registration in the ASEAN region.
These requirements are sometimes based on the existing national requirements, WHO guidelines and recommendations from other regions (e.g.
the ASEAN guidelines for "The Conduct of Bioavailability and Bioequivalence Studies" were created from the EMA/CPMP Note for Guidance).
But most of the ASEAN ACTRs have been developed via the adoption or modification of the ICH guidelines.
They cover all the quality, nonclinical, and clinical aspects already developed by ICH.
Labeling requirements, administrative data (i.e., Certificate of Pharmaceutical Product (CPP), Letter of Authorization, Application Forms, etc.
), and the glossary have also been discussed.
The final ACTRs were endorsed by the ACCSQ at its 21st meeting (in March 2003) .
Guidelines to ACTR (e.g., Process Validation and Stability) have also been developed.
The group also developed an ASEAN Common Technical Dossier (ACTD) for pharmaceutical product registration.
Like the ICH CTD, this initiative reduces the time and resources needed to compile applications for registration in different countries.
Regulatory reviews and communication with the applicant is also facilitated by a standard document of common elements.
This ACTD is based on the ICH CTD, but is organized into four parts only (the overview and summaries are included at the beginning of the relevant Parts I, II, and III instead of being grouped under a separate section as in Module 2 of the ICH CTD): ▸ Part I: Activities have also been conducted in the area of GMPs.
On April 10, 2009, the ASEAN Economic Ministers (at the 14th ASEAN Summit and related summits in Pattaya, Thailand) signed the ASEAN MRA for GMP inspection of manufacturers of medicines.
This arrangement establishes the mutual recognition of GMP certifications and/or inspection reports (issued by inspection bodies) that will be used as the basis for regulatory actions such as granting of licenses and supporting post-marketing assessment of conformity of these products.
The PPWG also worked on a bioavailability/bioequivalence study reporting format and a post-market alert system.
The objective of the ASEAN post-marketing alert system is to share information relating to defective or unsafe medicines, and also cosmetics, health supplements, and traditional medicines.
This pilot project was launched in April 2005 and then adopted by the PPWG in February 2006.
The two major accomplishments of the PPWG are the ACTD and the ATCRs.
The ACTD is the common format for marketing authorization application dossiers, while the ATCRs are the set of written materials intended to guide applicants to prepare application dossiers in a manner that is consistent with the expectations of all ASEAN DRAs.
A series of guidelines for the implementation of the ATCRs is being finalized.
Most of the ASEAN recommendations strictly follow the ICH guidelines and recommendations.
Indeed, ASEAN is a good example of the influence of the ICH outside the ICH regions and of the integration and implementation of ICH standards outside ICH frontiers.
Beyond these harmonized technical aspects of the pharmaceutical product registration that need to continue, the ultimate goal of the ASEAN PPWG is clearly to implement a system where countries fully cooperate in enhancing mutual regulatory capacities and resources.
With the ongoing challenges posed by the globalized economy, and in particular the huge economic growth of China and India, which may have specific impacts on the region, this association of countries is clearly committed to full integration (with the goal to establish an ASEAN economic community by 2015) and moving towards the European community model.
The ultimate steps in the pharmaceutical harmonization process will certainly be the development of ASEAN pharmaceutical directives, the development of a Pan-ASEAN registration process (with a centralized procedure), and the establishment of an ASEAN regulatory agency.
But the full implementation of this supranational system will take time.
It will only be possible when the ASEAN has developed common legislation and structure (i.e., commission, parliament, etc.
), as in Europe.
The harmonization of pharmaceutical regulations can, however, continue before such an organization is in place.
The next logical step is the creation of an MRA procedure.
Indeed, this type of procedure is not binding for the countries (and therefore does not require common legislation) and requests only a "facilitator body" and not a supranational evaluation agency.
This procedure would be similar to the old "multi-state" procedure that Europe established in 1975 as a first step towards the creation of the system that we know today.
ASEAN is also committed to increased relations with external partners.
The creation of the ASEAN Plus Three cooperation may indeed promote the harmonization of pharmaceutical regulations in the much broader Asia region.
Outside the region, ASEAN and its PPWG clearly want to increase relationships and cooperation with other regional organizations, and also international bodies (i.e., UN, WHO, ICH).
This development, which is outside its current framework, could indeed strengthen this initiative by increasing its exposure on an international basis, therefore allowing this organization to play a pivotal role in the international community.
The Asia-Pacific Economic Cooperation (APEC) is a forum, established in 1989, to facilitate economic growth, cooperation, trade, and investment in the Asia-Pacific region.
This region accounts for approximately 40% of the world's population, approximately 54% of world gross domestic product (GDP), and about 44% of world trade [198] .
Since its creation, this intergovernmental grouping has worked to reduce tariffs and other trade barriers across the Asia-Pacific region in order to liberalize trade and investment and facilitate business within the region.
APEC also works to create an environment for the safe and efficient movement of goods, services, and people across borders in the region through policy alignment and economic and technical cooperation.
To support its "Three Pillars" (i.e., Trade and Investment Liberalization, Business Facilitation, and Economic and Technical Cooperation), APEC has been active in a broad range of more than 50 topics (from fisheries, agriculture, and tourism to terrorism, finance, and intellectual property).
This broad range of topics, under which hundreds of specific projects have been developed, reflects the complex factors and issues related to economic development, growth, and the pursuit of open trade and investment for a region.
Several of these topics can influence the health and pharmaceutical sector (such as Intellectual Property or Science and Technology), but two specifically focus on this area: ▸ The Health topic, managed by the "Health Working Group," focuses mainly on the prevention and management of infectious diseases (naturally occurring or due to bioterrorism) in the region.
This working group is not involved in the discussion related to pharmaceutical regulation.
▸ The Life Sciences topic, managed by the Life Sciences Innovation Forum (LSIF), addresses key challenges in the health and pharmaceutical sector in order to create the right policy environment for life sciences innovation.
The harmonization of standards and the regional and international cooperation are two of the tools used to achieve the objectives.
As a member of the ICH Global Cooperation Group (GCG) since 2003, APEC LSIF promotes the implementation of the ICH guidelines through its workshops.
It also keeps ICH informed on the status of the different ongoing initiatives in the region.
APEC has 21 member economies from the broad Asia-Pacific region, which spans four continents (Plate The 21 members of APEC recognize that strong economies and harmonization initiatives are not built by governments alone, but by partnerships between government and its key stakeholders, including industry, academia, research institutions, and interest groups within the community.
Therefore, APEC actively involves these key stakeholders in the work of the forum.
At the working level, representatives from the private sector are invited to join many APEC working and expert groups.
This process provides an important opportunity for industry to provide direct input into APEC's ongoing work.
APEC has official observers, the Association of Southeast Asian Nations (ASEAN) Secretariat being one of them.
These observers participate in APEC meetings and have full access to documents and information.
APEC operates as a cooperative, multilateral economic, and trade forum.
APEC policy direction is provided by APEC Leaders from the member economies.
The Life Sciences Innovation Forum (LSIF), under the Committee on Trade and Investment, is a tripartite forum involving representatives from government and academia, and also from industry.
It brings together scientific, health, trade, economic, and financial considerations to create the right policy environment for life sciences innovation.
All the APEC activities are supported by the APEC Secretariat, which is based in Singapore and operates as the core support mechanism for the APEC process.
It provides coordination, technical, and advisory support, as well as information management, communication, and public outreach services.
The idea of APEC as a cooperative to enhance economic growth and prosperity, and to strengthen the Asia-Pacific community, was first publicly mentioned by the former Prime Minister of Australia (Bob Hawke) during a speech in Seoul, South Korea in January 1989.
Later that year, 12 Asia-Pacific economies met in Canberra, Australia to establish APEC.
In November 1994, APEC's vision was reiterated by APEC Economic Leaders during their meeting in Bogor, Indonesia.
During this meeting, the Economic Leaders adopted what are referred to as the "Bogor Goals."
These goals of "free and open trade and investment in Asia-Pacific no later than 2020" were based on a recognition of the growing interdependence of the economically diverse region, which comprises developed, newly industrializing, and developing economies.
Due to the heterogeneity of the region, it was agreed that the pace of implementation would take into account differing levels of economic development among APEC economies.
In 1995, a framework for meeting the Bogor Goals (referred to as "the Osaka Action Agenda") was adopted.
This action plan focused on three key areas: ▸ Trade and Investment Liberalization ▸ Business Facilitation ▸ Economic and Technical Cooperation Following this first action plan, several other plans have been adopted over the years to support the implementation of the Bogor Goals.
Specific topics (such as climate change and severe acute respiratory syndrome [SARS]) were also discussed.
Recognizing the global financial crisis as one of the most serious economic challenges ever faced, the leaders highlighted the importance of reducing the gap between developed and developing members.
This meeting included discussions related to regional economic challenges (implementing a structural reform and food supply and price), the social dimension of globalization, the enhancement of human security in the region, and the problem of climate change.
The LSIF and the Health Working Group held their first joint meeting in March 2011 in Washington, DC, US to explore possible areas of cooperation.
This meeting followed the 2010 recommendations of the APEC Senior Official endorsing a new Terms of Reference for the Steering Committee on Economic and Technical Cooperation.
It was then agreed that the role and operations of the Health Working Group would be reviewed with a view to merge, disband, or reorient this body.
The LSIF leads the activities related to the regulatory convergence in the pharmaceutical area within the Asia-Pacific region.
Both APEC and the LSIF have recognized the benefits of convergence related to the pharmaceutical standards within the region.
To achieve this goal, these two groups rely on other regional and global harmonization initiatives.
Indeed, the LSIF is working towards the adoption and implementation of existing harmonized international guidance and regulatory best practices.
It also provides the ability to access funds to advance projects.
Unlike ASEAN, the objective is not to proactively develop specific regional harmonized guidance.
This practice is in line with the overall APEC goals to facilitate cooperation and trade in the region, and to operate on the basis of nonbinding commitments and open dialogue.
As already mentioned, APEC has no treaty obligations required of its participants, and there is no plan for integration (unlike ASEAN, which follows an integration model like Europe).
Recognizing this specific context, the objective of LSIF is "regulatory convergence" with gradual alignment over time between member economies.
The distinction with "regulatory harmonization" is that "regulatory convergence" does not typically involve or require active harmonization of regulations that would be unrealistic within the APEC environment.
The objectives and priorities of the LSIF, listed in its strategic plan approved by the APEC ministers in 2004, are very broad.
This plan includes recommendations on four different sectors: Research, Development, Manufacturing and Marketing, and Health Services.
The goal was to develop recommendations that would contribute to a more efficient, effective, and coordinated policy approach to support innovation and health in the region.
These recommendations have applications in many different areas (legal, finance, scientific, regulatory, infrastructures, etc.).
One of the recommendations from this strategic plan follows: "harmonization of standards for life sciences products and services and mechanisms for collaboration and exchange of information among economies were recognized as critical elements" [199] .
The principle was to review policies, standards, and regulatory mechanisms against international best practices in order to move towards regional convergence.
The objective was also to achieve close collaboration and to facilitate the use of international standards and global best practices through collaboration with outside bodies such as the ICH GCG.
The LSIF has been very active in sponsoring a series of workshops on anti-counterfeiting, ICH quality guidance, clinical trials, and good clinical practice (GCP) inspection.
However, it has been recognized that the LSIF has not been used to its full potential to promote regulatory convergence and cooperation compared to some other RHIs [200] .
What was missing was the engagement of regulators and the appropriate industry people in this equation, together with the lack of a more focused strategic framework and multiyear plan for medical products.
In 2008/2009, acknowledging the lack of strategic and effective approaches, the LSIF decided to react and strengthen its organization: ▸ In June 2009, the LSIF took an important step towards harmonization by establishing, in Seoul, South Korea, the APEC Harmonization Center (AHC).
This followed a proposal from South Korea in August 2008 (at the APEC LSIF VI in Lima, Peru) that was endorsed by the APEC leaders in November 2008 in a Joint Ministerial Statement.
As an LSIF organization, this center has its own structure (including a Director, a Secretariat, and an Advisory Board of LSIF Experts), and also its own website (www.apec-ahc.org).
This organization includes representatives from government, industry, and academia.
Its mandate is to provide a platform to address and solve priority concerns of APEC members on regulatory convergence.
Following the establishment of the AHC, several workshops took place.
In general, they focused on the regional regulatory convergence, but also discussed specific problems such as multiregional clinical trials and the biosimilar concept.
The purpose of these workshops is to allow government, regulators, academics, and the pharmaceutical companies to discuss and exchange information and views on the harmonization of standards.
Funding and support from the AHC has allowed for the delivery of more than a dozen workshops since June 2009.
▸ In addition to the AHC, APEC also decided to establish a Regulatory Harmonization Steering Committee (RHSC) within the LSIF structure to strategically coordinate regulatory convergence in the region.
The RHSC brought together senior officials from regulatory authorities and representatives from industry coalitions.
This committee provides leadership and direction on regulatory priorities.
During its inaugural meeting in Seoul, South Korea in June 2009, the RHSC discussed and finalized its Terms of Reference and started to identify priority projects.
Since then, the RHSC has initiated several projects and developed a Strategic Framework on Regulatory Convergence of Medical Products by 2020 to coordinate activities [201] .
Since the creation of the APEC AHC and RHSC, considerable progress has been made with the design, development, and implementation of a more strategic, coordinated, and sustainable approach.
This includes the Strategic Framework and the creation of priority work areas (PWAs), each of which is associated with a roadmap that defines an overall strategy to achieve the ultimate goal of greater regulatory convergence by 2020 in the area of medical products.
Each project or activity undertaken must now support the roadmap and in turn move APEC closer to the 2020 goal.
This is a better-structured organization that moves away from individual, uncoordinated activities and workshops to a more directed, coordinated approach with parties and individuals that are in a position to effect change and commit resources.
The workshops, organized and funded by the AHC and led by the RHSC membership, are now tied to a directed roadmap and strategic framework representing the collective efforts and commitment of many economies.
These workshops served as a diagnostic of issues, challenges, and opportunities associated with a particular area of focus, with recommendations coming back to the RHSC for consideration.
All workshops are championed by the regulators of various APEC economies (for example, the US for medical product quality and supply chain integrity, Korea for biotechnological products and pharmacovigilance, Singapore for cellular-and tissue-based therapies, Chinese Taipei for good review practices and combination products, and Thailand for GCP inspections).
Finally, this organization is partnering with other regional and international players in an effort to promote synergy and more effective use of resources.
A good example here is the supply chain roadmap.
This is a global issue and requires a global, coordinated approach.
The RHSC roadmap is being implemented through the direction of an oversight committee that includes the WHO, EMA, EDQM, and the DRA of Nigeria.
In doing so, APEC takes account of and complements like initiatives, and can serve as a catalyst to global action.
Up to now, the APEC did not proactively develop guidance or harmonized standards and requirements.
The objective is to promote convergence via the dissemination of international harmonized information and recommendations (i.e., ICH guidelines).
To achieve these goals, the group has developed and funded several projects.
In 2008, the LSIF released an "Enablers of Investment Checklist," a voluntary guidance tool for member economies to assess and improve their innovative life sciences sector investment opportunities.
One of the six principles covered by the checklist is "Efficient and Internationally Harmonized Regulatory Systems."
Under this principle, the LSIF promoted the development and implementation of focused efforts on harmonization towards international standards through recognized international organizations (i.e., ICH).
Moreover, to support this objective, the LSIF also proposed development of the following: ▸ A regulatory framework (transparent, predictable, and science-based) that allows for the quick introduction of new innovative products ▸ An efficient clinical trial regulatory system focused on safety, efficacy, and ethical standards ▸ An adequate number and level of training programs for regulatory personnel ▸ The publication of proposed regulations for stakeholder comments (which should be taken into account) ▸ Laws providing for stakeholder consultation throughout the regulatory drafting and review process ▸ Participation in international joint clinical trials Performance metrics have also been defined to assess the implementation of the recommendations.
Finally, some of the other principles on this checklist also support cooperation and convergence as they assess the resources, exchange programs, intellectual property rights, and interagency coordination of life science policy and regulation.
In addition to the "Enablers of Investment Checklist," LSIF has also developed projects focusing on specific topics of interest, such as: ▸ Clinical Trials: The area of clinical trials was selected as one of the LSIF priorities in its strategic plan.
Assessment and improvement of the clinical trial system and regulation in each country has also been recommended in the LSIF "Enablers of Investment Checklist."
The goal was to put in place an effective regulation infrastructure (by harmonizing regulatory practices and policies according to international best practices and standards).
This activity includes work on regulatory process and framework (incorporating interagency review of new policies, guidances, and regulations), implementation and promotion of good clinical practices (GCPs)/good manufacturing practices (GMPs), protection and enforcement of intellectual property, establishment of clinical trials registries, and implementation of ICH recommendations.
To implement this goal and strengthen the DRAs' capacity to harmonize practices, a first workshop on "Review of Drug Development in Clinical Trials" was held in March 2008.
Several additional workshops concerning clinical trials and GCP (including clinical research inspection) have since been set up on this subject.
The first workshop organized by the AHC in 2009 focused on the opportunities and challenges of multiregional clinical trials.
Each of the workshops serves to refine recommendations and showcase the China-Japan-Korea Tripartite Research Initiative that is exploring possible ethnic differences between the three countries.
As a result of workshops, two roadmaps have been developed: one for GCP inspection (under the leadership of Thailand), and one for multiregional clinical trials (under the leadership of Japan) [202] .
The focus will address gaps and needs not addressed by any other institution or regulatory authority to date.
▸ Counterfeit Medicines: Another area of interest for LSIF has been the increase in counterfeit medicines in the region.
A series of seminars and workshops have been organized since January 2008 to examine ways to combat this problem.
The LSIF has also developed an Anti-counterfeit Medical Product Action Plan.
The objective of this plan is to share best practices in the detection and prevention of counterfeits to both DRAs and industry professionals, and organize systems to reduce the threat and occurrence of counterfeit medicines.
Finally, it is important to note that APEC also promotes capacity building for its members.
This objective is met through the organization of workshops, training courses, and seminars that enable people, businesses, and government departments to improve their skills and knowledge [204] .
The primary focus of APEC is clearly the economy, and its objectives center on the facilitation of trade and business between member economies (with no integration plan).
The Asia-Pacific region has consistently been one of the most economically dynamic regions in the world.
Since the establishment of APEC in 1989, the total amount of trade has grown significantly [205] .
APEC's work under its three main pillars of activity has helped drive this economic growth.
In 2010, APEC conducted an assessment to determine what progress has been made against the Bogor Goals of free and open trade and investment.
The results were positive, showing that member economies have taken concerted action and progressed in a wide array of economic, trade, investment, and social areas.
Average tariffs in the region have been reduced from about 17% in 1989 to approximately 5.8% in 2010.
Nontariff barriers have also been reduced thanks to APEC's work on trade facilitation.
This progress by APEC towards the Bogor Goals contributed to a more than five-fold increase in members' total trade (goods and services) between 1989 and 2010 (from US $3.1 trillion to US $16.8 trillion).
Finally, these activities contributed to real benefits for people across the entire Asia-Pacific region.
Over the span of 10 years, from 1999 to 2009, poverty was reduced by 35% (poverty levels are measured by calculating the population living on less than US $2 a day) [206] .
APEC represents a large region and approximately 40% of the world's population.
This is obviously an advantage in facing the challenge of globalization.
However, this size and magnitude can also be a disadvantage in terms of management.
Indeed, this region is very heterogeneous with countries at the two extremes of the development spectrum (i.e., very developed and very undeveloped countries).
Due to this disadvantage and the heterogeneity of this large region, it is difficult to adopt a treaty and to impose obligations on these members.
For this reason, APEC operates on the basis of nonbinding commitments where each country has the choice to implement the decisions.
The implementation of economic measures (i.e., reduction of taxes and trade barriers to increase trade between members) is possible since it can quickly benefit all members.
However, the lack of a treaty or obligations on members can sometimes be more challenging for more drastic long-term reforms (i.e., the harmonization of standards), as member economies have different priorities.
The diversity of the APEC region means that member economies will gradually move closer together in requirements and approaches, but not everyone will implement the measures at the same time.
Capacity and local realities must be taken into account.
Though technical cooperation is part of APEC's objectives (i.e., APEC is very involved on specific topics such as climate change), it is the second priority behind economic development.
The health topic, managed by the Health Working Group and the Life Science Innovation Forum, has clearly been funded because this topic has an impact on the economy.
As stated on the APEC website, "Life sciences innovation is critical to growth and socio-economic development as healthy people produce healthy economies.
Efficient and effective delivery of patient focused products and services can improve a population's longevity, wellness, productivity and economic potential" [207] .
However, even if the above challenges are important, very positive outcomes have to be noted in terms of regulatory convergence in the pharmaceutical area.
Indeed, this organization supports convergence via the funding of projects and workshops.
LSIF was able to focus its effort on projects that impact all member economies (developed or developing), such as the coordination of multicountry clinical trials, the implementation of GCPs, the quality of medicines, the counterfeit medicines problem, and the emergence of biosimilars.
LSIF also creates a forum that allows exchange of information between very different countries and between all the players (regulators, industry, and academia).
This communication and dissemination of harmonized standards is very important, and is as essential as the development of the standards itself.
In 2008/2009, acknowledging a lack of strategic coordination, APEC and LSIF decided to better organize the activities.
First, they established the AHC to facilitate the exchange of information and the creation of a network.
Second, they created the RHSC to strategically coordinate regional convergence.
Since this revision of LSIF's structure and the creation of these two supporting bodies, significant progress has been made and APEC has since declared that further harmonization to "achieve convergence on regulatory approval procedures" is targeted for 2020 [208] .
To support this goal, many important projects have been initiated on critical topics, such as product quality and supply chain integrity [209] , good review practices [210], GCP inspection [211], pharmacovigilance [212] , biotechnology products [213], etc.
All these changes and projects today represent great promise for this region, and the tools to be developed could also support global cooperation and convergence.
The challenge is now to implement the plan and to continue to coordinate the projects in order to achieve the desired objectives.
The recent establishment of the RHSC Regulatory Network (including DRAs not currently part of the RHSC) will certainly support the implementation of agreed-upon measures.
Many different types of bilateral cooperation have been established over the years.
lll It would be difficult to list and discuss them all as several dozen exist.
However, all these types of bilateral cooperation and agreements can be grouped into three categories based on their scope and objectives: ▸ Cooperation between Two Developed Countries: The objective of such cooperation is to exchange good practices and harmonize standards to avoid duplication of efforts (e.g., for orphan drugs).
For example, the EU and the US developed a privileged relationship and the exchange of officials and staff between US FDA and EU Authorities allow for a closer collaboration, exchange, and therefore better understanding of each other.
▸ Cooperation between One Developed Country and One Developing Country: This type of cooperation focuses on training, mentoring, and support from the developed country to the developing country.
The objective is indeed to build expertise and capacity in the developing country based on the experience of the developed country.
For example, the US FDA has established several agreements with developing countries (e.g., Brazil, Mexico, South Africa, Taiwan, etc.)
▸ Cooperation between Two Developing Countries: By pooling experience and resources, two countries can better tackle issues of common interest.
This type of cooperation allows for better allocation of sparse resources, and also increases interest for pharmaceutical companies (two small markets with different requirements would be less attractive to industry).
For example, Brazil has cooperation projects with Cuba, Dominican Republic, Mozambique, and several other countries [214, 215] .
One of the most advanced bilateral collaborations is between Australia and New Zealand.
It represents a good example of a bilateral cooperation and harmonization model working towards a full integration of systems.
Indeed, after several years of convergence and harmonization, Australia and New Zealand agreed to establish a joint Australia New Zealand lll Bilateral cooperation can involve two countries, but it can also mean the collaboration of a regional entity with another party.
For example, the European Union has been collaborating with Australia, Canada, the US, and Japan, but also with the GCC group.
Therapeutic Products Agency (ANZTPA).
This new Agency will ultimately replace Australia's Therapeutic Goods Administration (TGA) and the New Zealand Medicines and Medical Devices Safety Authority (Medsafe).
During the first meeting of the ANZTPA Implementation Ministerial Council (Melbourne, January 28, 2012), Ministers from both countries agreed on key elements to establish the joint Trans-Tasman Agency, and also how the joint regulatory scheme will be organized over a five-year period [216-1].
Since then, the framework of the ANZTPA is under discussion .
This cooperation/harmonization initiative was begun with the objective of sharing expertise and resources in order to provide health benefits for consumers by creating a world-class scheme.
It is also expected that this single approval process for both countries will increase efficiency, improve the standards of medicines produced in the two countries, reduce regulatory costs for industry, and facilitate further economic integration [217] .
This initiative is a great example of successful bilateral harmonization and cooperation, and emphasizes the importance of a staged approach for this type of project.
It also shows that such ultimate integration of systems is challenging.
Indeed, the agreement for a joint regulatory scheme was first reached in 2003, but this project was not able to proceed because New Zealand was unable to pass enabling legislation.
Negotiations between the countries were also suspended in July 2007 [218] .
The increased collaboration between Europe and the US in the pharmaceutical domain is another interesting example of bilateral cooperation.
Though this cooperation does not follow an integration model, it is a well-developed bilateral initiative.
It is a stepwise and structured program that is interesting as it provides a clear example of what such bilateral collaboration can achieve in a nonintegration process, and also outlines its limitations.
It also provides examples of the measures and organization necessary to support such bilateral work.
The European Union (EU) and the United States of America (US), in addition to their collaboration within the scope of multilateral frameworks such as the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), have also established strong regulatory and scientific bilateral cooperation in the pharmaceutical sector.
This bilateral cooperation promotes public health, safer trade of products, and harmonization of regulations.
Over the years, the scope of this transatlantic collaboration has increased, and today represents a good example of what bilateral cooperation can achieve.
This collaboration mainly involves the European Commission (EC), the European Medicines Agency (EMA) and the United States Food and Drug Administration (US FDA).
However, it is important to note that the US FDA also maintains an active relationship with national DRAs throughout Europe.
Confidentiality arrangements with the US have been signed at the European level (EC and EMA) and also at the national level with Austria, Belgium, Denmark, France, Germany, Ireland, Italy, the Netherlands, Sweden, and the United Kingdom.
This is particularly important for collaboration in the area of inspections.
It also allows the US FDA to exchange information on products not approved via the Centralized Procedure (this exchange is done through the relevant Reference Member States [RMSs]).
The leaders of the EU and the US agreed on a framework for advancing transatlantic economic integration and established the Transatlantic Economic Council (TEC) to oversee the efforts outlined in the framework, with the goal of accelerating progress and guiding work between EU-US Summits.
Moreover, confidentiality agreements have been established to create a framework allowing the exchange of confidential information between the EU and the US FDA as part of their regulatory and scientific processes.
They include information on advanced drafts of legislation and regulatory guidance documents, as well as nonpublic information related to ensuring the quality, safety, and efficacy of medicinal products for human (and veterinary) use.
An implementation plan has also been agreed upon between all parties to allow for a successful exchange of information and documents between the EU and the US FDA in accordance with the terms of the confidentiality agreements.
The objective of this implementation plan was to describe the processes by which each party will undertake information and document exchange as envisioned by the confidentiality agreements.
Also, to facilitate this transatlantic pharmaceutical cooperation, the US FDA and the EMA have established "liaison officials."
These liaison officials remain employed by their home organizations, but their physical location in the partner agency is designed to facilitate collaboration.
Their role is to facilitate regulatory and scientific cooperation between the US FDA and the EMA, and to coordinate information exchange.
They also increase awareness of interaction opportunities with the EMA and the US FDA, and potential new areas of common interest [219,220].
In 2003, the scope of this bilateral cooperation intensified with the establishment of confidentiality arrangements between the parties.
These agreements signed on September 12, 2003 were then extended on September 15, 2005.
In September 2010, these confidentiality agreements were extended again, and are now in effect for an indefinite period without the need for further renewal.
These two 2010 official statements of authority and confidentiality commitment [223, 224] restate the agreement to pursue in-depth collaboration and exchange of confidential nonpublic information between the US FDA and the EMA.
It is interesting to note that these statements reiterate that the shared information includes confidential commercial or trade secret information (the US FDA is required by current legislation to ask pharmaceutical companies before sharing trade secret information with counterpart DRAs).
At the EU-US Summit on April 30, 2007, further momentum was given to regulatory collaboration with the signature of the Framework for Advancing Transatlantic Economic Integration between the European Union and the United States of America by EC President José Manuel Barroso, German Chancellor Angela Merkel, and US President George W. Bush.
This document called for more effective, systematic, and transparent regulatory cooperation, and the removal of unnecessary differences between regulations.
It also specifically requested the promotion of "administrative simplification in the application of regulation of medicinal products."
The objective of this bilateral process is more towards cooperation than harmonization per se.
Exchange of information between the parties allows for a better understanding of each other's systems and requirements, and therefore builds confidence and recognition facilitating convergence.
This EU-US cooperation also tries to avoid future disharmony by upstream regulatory cooperation on new medicines legislation [225] .
The exchange of information and practices are well structured and occur on a regular basis, but the exchange can also be done on an ad hoc basis if necessary.
▸ Regular Exchange: The EMA and US FDA exchange a list of specific information on applications (both pre-authorization of new molecules and post-authorization of marketing products), including decisions made for such applications on a quarterly basis.
They also exchange other information such as a list of Good Clinical Practice (GCP) inspections or pharmacovigilance topics (either product-or nonproduct-related issues).
▸ Ad Hoc Exchange: In addition to the exchange of new drafts of final legislation or guidelines (prior to publication), the EU and US FDA also exchange information relating to scientific advice, difficulties in relation to the evaluation of applications, and urgent drug safety issues and other issues impacting public health.
These types of information are exchanged prior to their release into the public domain.
Meetings or workshops on regulatory issues of mutual concern are also organized on an ad hoc basis.
Finally, the EMA and the US FDA publish an annual report summarizing their interactions under the confidentiality arrangements.
These arrangements also provide for annual meetings between the US FDA, the EMA, and the EC to monitor the operation of activities within the scope of the agreed-upon implementation plans.
However, it should be noted that the sharing of product-related information is limited to medicinal products evaluated or authorized in accordance with the EU Centralized Procedure, as well as medicinal products authorized at the national level by the EU Member States, which are subject to arbitration or referral in accordance with European Community procedures [226].
Initiatives related to general topics are reported below.
In addition to these initiatives, cooperation has also been established in certain specific scientific areas or for a specific type of product (i.e., oncology, pharmacogenomics, nanotechnology, Advanced Therapy Medicinal Products [ATMP], blood products, and vaccines).
Under the auspices of the Transatlantic Economic Council, on November 28, 2007 the EC hosted the "Transatlantic Administrative Simplification Workshop" in Brussels, Belgium, which was co-chaired by the EC and the US FDA and organized in collaboration with the EMA and the Heads of the EU National Medicines Agencies (HMA).
The key objective was to identify opportunities for administrative simplification through transatlantic cooperation in the removal of unnecessary burdens of administrative practices and guidelines.
This would allow more human and fiscal resources to be focused on greater innovation and efficiency in the development of quality products.
It was agreed that this project should not require change to legislation, and of course, the simplifications should maintain or increase current levels of public health protection.
As a follow up to the Transatlantic Administrative Simplification Workshop, a "Medicines Regulation Transatlantic Administrative Simplification Action Plan" was published in June 2008, outlining administrative simplification projects to be taken forward.
This document promoted further cooperation and pilot collaboration programs in major areas such as inspections, biomarkers, counterfeit medicines, risk management (content and format), scientific advices, biosimilars, pediatrics, and advanced therapies.
During the annual EC/EMA-US FDA bilateral meeting in September 2010, it was agreed that the majority of projects in the original plan had been successfully completed and that most of the pilot projects had been extended and became "standard" cooperation [227] .
Ongoing developments and new initiatives in transatlantic administrative simplification are now included in the annual reports on interactions between the US FDA and the EMA.
Several projects have been initiated to increase collaboration on GMP and GCP inspections.
Ad hoc exchanges on specific products, quality defects, product shortages, and on draft guidelines also took place.
▸ GMP Inspections: Several pilot projects were first initiated in the context of the Transatlantic Administrative Simplification Workshop deliverables.
An initial project (established in cooperation with the European Directorate for the Quality of Medicine and the Australian Therapeutic Goods Agency) was conducted between December 2008 and December 2010 and related to GMP inspections of Active Pharmaceutical Ingredients (API) manufacturers [228] .
The project's objective was to determine whether greater international collaboration and information sharing could help to better distribute inspection capacity, thus allowing more sites to be monitored and reducing unnecessary duplication.
The second project, related to finished products, allowed EU-US FDA joint inspections and was aimed at developing ways of working together on joint inspections of routinely scheduled sites in the territory of the US or EU, to reduce duplicate inspections and the resulting burden on both the pharmaceutical industry and the DRAs.
This pilot phase, conducted under confidentiality agreements, allowed the development of new tools for work sharing and the exchange of information in order to share inspection reports and to organize joint inspections.
Increased transparency and visibility of inspections performed by participating authorities allowed a successful collaboration between authorities on manufacturing sites of common interest.
It also increased the number of inspections performed that were of value to more than one authority.
This pilot phase confirmed that such collaboration in the area of GMP inspections led to a reduction in duplicate inspections, more efficient use of combined inspectional resources, and wider global inspectional coverage.
Following the successful conclusion of the pilot, it was agreed to maintain the cooperation established [229] .
In December 2011, the US FDA and the EMA decided to further enhance their GMP inspection cooperation by moving from confidence building to reliance upon [230] .
This initiative, launched in January 2012, allows the EMA and the US FDA to share inspections of manufacturing sites in each other's territories.
This important step follows the positive experience acquired through the pilot joint inspection programs and other information sharing projects that have occurred over several years.
This strategy allows some inspections on each other's territories to be deferred or waived completely based on a number of considerations and on a risk-based approach [231] .
This strategy is applicable to GMP inspections related to manufacturing sites located in the US and the European Economic Area (EEA), mainly focusing on routine post-authorization and surveillance inspections as a first step [232] .
The result of this arrangement could free up inspection resources that would then become available for inspections to other regions.
Ongoing EMA-US FDA joint inspection pilot projects will continue according to the agreedupon procedures [233, 234] as it remains important to maintain mutual confidence and build further mutual understanding of GMP inspection approaches.
Some successful pilot programs will also be expanded to new partners such as the ongoing collaboration on GMP inspections of active substance manufacturers [235] .
Due to the increased globalization of pharmaceutical product clinical development, and based on previous positive experiences in the GMP field, the EMA and US FDA agreed to launch a pilot EMA-US FDA GCP initiative.
The objective of this GCP initiative, conducted between September 2009 and March 2011, was to reinforce and systematize periodic information exchanges on GCP-related activities between the US FDA and EMA.
These included the exchange of GCP inspection plans to improve inspection coverage, the exchange of information on applications to help identify candidates for collaborative inspections, and the exchange of inspection outcomes and reports (both negative and positive) and their potential impact.
Conduct of collaborative GCP inspections and the sharing of information on interpretation of GCP (such as draft guidelines or policies) were also part of this project.
The pilot initiative has been very productive.
A considerable amount of information has been exchanged on many products [236] , and this communication (which included 23 teleconferences and four face-to-face meetings) has facilitated improvements in the inspection coverage and decision-making processes of the agencies.
The 13 collaborative inspections conducted under the initiative have contributed greatly to each agency's understanding of the other's inspection procedures.
They have also led to the identification of potential improvements to these procedures.
Both agencies have learned several general lessons during the process [237].
In addition, exchanges of views on interpretation of GCP documents have also been organized.
During the pilot initiative, the EMA and the US FDA have shared different pieces of GCP-related guidance documents, position papers, and policies in order to harmonize the agencies' understanding of GCP and to standardize the requirements for industry wherever convergence would be beneficial for the clinical research process.
At the end of the program, both parties considered this pilot initiative very successful and agreed to continue this collaboration, incorporating lessons learned with the broader aim of moving from "confidence building" to the mutual acceptance of inspectional findings.
The agencies will also expand the scope of the initiative to sites outside the US and EU [238].
Although not defined as a cluster, interactions in the area of safety continue to play an important part in the ongoing collaboration between the US FDA and the EMA.
▸ Videoconferences take place on a bimonthly basis and include product-related issues and issues related to risk management.
Usually five to six products are discussed at these teleconferences.
▸ Regular informal teleconferences in order to exchange information on emerging safety and strategic issues.
▸ EMA shares the Early Notification System on a monthly basis and the US FDA sends advance notice of publication of its quarterly update reports on potential safety signals.
▸ Joint projects have also been established, such as the collaborative project on the Progressive Multifocal Leukoencephalopathy Research Agenda to stimulate research into this important safety issue that affects some biological agents.
The objective of this program is to allow interaction between the EMA and the US FDA assessors and sponsors during product development.
This dialogue between the two agencies' assessors and sponsors on scientific issues [240] aims to optimize product development and avoid both unnecessary testing replication and unnecessary diverse testing methodologies.
Such a procedure can be valuable for products developed for indications for which development guidelines do not exist, or if guidelines do exist, the EMA's and the US FDA's recommendations differ significantly.
Experts from the EMA and the US FDA exchange views and discuss draft responses to questions from the applicants on their clinical development programs or on new biomarkers.
General principles for this voluntary parallel scientific advice were published in 2009 by the EMA and the US FDA [241] .
It is important to understand that this is a parallel procedure, and unfortunately, not joint advice.
The goals of the EMA and US FDA are primarily to share information and perspectives, rather than specific harmonization of study or regulatory requirements (although they recognize that harmonization is a beneficial outcome).
After this procedure, the two agencies conduct their individual regulatory decision-making process regarding drug development issues and marketing applications.
Each agency provides independent advice to the sponsor regarding questions posed according to their own usual procedures and timelines.
The advice of each agency may therefore still differ after the joint discussion.
However, in many cases, these discussions between regulators achieved a high degree of alignment and helped industry move closer to a global development plan [242] .
In 2010, following a rather slow acceptance in previous years (due to hesitation from industries to use this procedure that does not commit the two agencies to issue common advice), the EMA and the US FDA discussed seven new parallel scientific advice procedures.
WHO experts were involved in two of these procedures, due to the therapeutic area covered by the request.
In addition to the formal parallel scientific advice exchanges between the US FDA and the EMA, ad hoc informal scientific advice teleconferences between the agencies took place for five products in 2010 [243].
"Clusters" or specific areas of mutual interest have been identified, and a more structured working relationship has been established.
These clusters (i.e., oncology, pediatrics, orphan medicines, pharmacogenomics, blood products, biosimilars, and vaccines) facilitate the exchange of information through teleconferences relating to applications for marketing authorization and extensions of indications, including risk management plans [244] .
The latest cluster established, with a focus on biosimilars [245], significantly increased cooperation between the agencies.
The recent announcement from the EMA stating that the agency will now accept data from reference product batches sourced outside the EU for biosimilar product applications [246] will certainly boost the EU-US cooperation in this domain and the global development of biosimilar products.
This decision follows the US FDA proposal to also accept comparative data referencing a product that is not approved in the US [247].
The EU-US FDA collaboration on orphan drug development has been important.
Discussions between the EMA and the US FDA usually include sharing of information on applications submitted in order to approach and discuss criteria for designation.
A common application form has been designed and agreed to so that sponsors can apply for orphan designation (of the same medicinal product for the same use) in both jurisdictions using this common form, facilitating the exchange of information.
Since 2010, discussions have also included analysis of different opinions.
On February 26, 2010, the US FDA and the EMA announced that they had agreed to accept the submission of a single annual report mmm from sponsors of orphan products designated for both the US and the EU [248] .
Each regulatory body continues to conduct their own review of the annual report to assure the information meets their own requirements.
The use of one single report benefits both the sponsor and the two regulatory agencies.
The sponsors benefit from the elimination of duplication of efforts to develop two separate reports, and the regulators can better identify and share information throughout the development process of an orphan product.
Collaboration in pediatrics is governed by the principles agreed to in 2007 [249] .
This framework includes information exchange (product-specific and general issues) and invitation of the other party to relevant pediatrics meetings.
The two main objectives are (1) to avoid exposing mmm These reports provide information on the status of the development of orphan medical products, including a review and status of ongoing clinical studies, a description of the investigation plan for the coming year, any anticipated or current problems in the process, difficulties in testing, and any potential changes that may impact the product's designation as an orphan product.
children to unnecessary trials, and (2) to facilitate the development of global pediatric development plans that are based on scientific grounds and that are compatible for both agencies.
In practice, the cluster on pediatrics organizes monthly teleconferences between the EMA's pediatric team and the US FDA during which Pediatric Investigational Plans (PIPs) are discussed in detail and information between the two agencies is exchanged.
In addition, more general questions have also been addressed, such as extrapolation, choice of endpoints, and patient/parent reported outcomes.
From September 2009 until September 2010, 42 products and four general topics were discussed [250] .
Since the end of 2009, US FDA representatives have been able to participate in certain EMA PDCO discussions and vice versa.
The EMA has also provided the US FDA access to its internal database that includes scientific details on all PIPs.
Several guidelines have been developed at the ICH level (ICH Q8, Q9, Q10) in order to facilitate the implementation of "Quality by Design."
Taking into account the global perspective of pharmaceutical manufacturing, the EMA and US FDA agreed that it would be beneficial if at this early stage of implementation assessors from the US and EU could exchange their views on the implementation of ICH concepts and relevant regulatory requirements using actual applications.
A three-year pilot program, operating under the US-EU Confidentiality Arrangements, started in April 2011.
This program allowed parallel evaluation of "Quality by Design" aspects of applications submitted to the EMA and the US FDA at the same time [251-1].
On August 20, 2013, the EMA and US FDA published the lessons learned and Q&A resulting from the first parallel assessment.
Both agencies found the pilot program extremely useful to share knowledge, facilitate a consistent implementation of the ICH guidelines, and harmonize regulatory decisions to the greatest extent possible [251-2].
The bilateral collaboration between the EU and the US has been extremely productive, and today it is recognized as a very successful initiative.
Its scope has increased over the years, from the basic exchange of information and harmonization of format to close collaboration and discussion of divergent positions.
The liaison placement in each organization has also been an important decision to facilitate such cooperation.
This increase in interaction, in a relatively short period of time, has been driven in part by reaction to crises and in part by proactive measures to enhance EMA-US FDA communication and collaboration [252] .
The establishment of the Transatlantic Administrative Simplification project in 2007 has also been beneficial as it initiated several pilot projects that further demonstrated the need for, and benefits of, such collaboration.
In general, activities in all the clusters have increased over time, and there has been an overall increase in the number of ad hoc requests for teleconferences on specific products and topics.
Following a significant increase between 2008 and 2009, the total number of monthly US FDA and EMA interactions (i.e., teleconferences, document exchanges, etc.)
now averages about 55 per month, excluding document exchanges relating to cluster and pilot activities.
Significant achievements have also been made in several critical areas for public health such as orphan medicinal products (with the agreement on a single annual report), drug development (with the establishment of the parallel scientific advice procedure and collaboration on pediatric development), GCP and GMP inspections (with several successful pilot projects that increased collaboration), and safety of products (with close collaboration and regular exchange of safety information, risk management, and safety alerts).
Exchange of draft regulation (before release in the public domain) has also facilitated harmonization of practices and exchange of opinions.
Finally, tools for more effective tracking have also been developed.
All these achievements confirm that collaborations between countries have a positive impact on public health.
It is particularly evident in certain areas such as orphan drug development (for diseases affecting a small population) or the exchange of information relating to urgent drug safety issues (to better assess and understand risks).
It is also important to note that this successful collaboration allows not only for the convergence of practices, but more importantly, this exchange of information and communication builds confidence in each other's systems, practices, and evaluations, allowing for a sharing of activities in certain areas.
This is already the case in the area of inspection.
In December 2011, EMA spokesperson Monika Benstetter stated that "each agency is now relying on its partner for drug manufacturing facility inspection data."
[253] The success of this transatlantic cooperation is partly due to the fact that it has been well structured and organized over the years.
The establishment of clusters and then the creation of the liaison officials' positions nnn strengthen regulatory cooperation between the agencies.
These decisions have been extremely beneficial from the perspective of education and timely communication.
A large number of staff visits and exchanges also took place, and there is now more routine involvement in the scientific work of both agencies.
The US FDA representatives take part as observers in Committee for Medicinal Products for Human Use (CHMP) discussions, and the EMA representatives are provided with access to webcasts of US FDA Advisory Committees.
However, other parameters such as those listed below have also been critical for this success, and clearly demonstrate their importance of this type of cooperation and harmonization initiative: ▸ First, it is clear that the political commitment to increased cooperation has been important.
Indeed, closer collaboration was evident after the signing of the "Framework for Advancing Transatlantic Economic Integration between the European Union and the United States of America" in 2007 by EC President José Manuel Barroso, German Chancellor Angela Merkel, and US President George W. Bush.
▸ Second, the establishment of confidentiality agreements, which since 2010 are effective for an indefinite period, allow both parties to exchange inspection reports or other nonpublic product-related information.
This was critical in the establishment of collaboration as this communication on specific practical cases allowed the parties to nnn Since 2009, the FDA has seconded a permanent representative to the EMA's office in London.
Since early 2010, the EMA has seconded a representative to the FDA's offices.
discuss the similarities and differences of opinion when assessing product applications and documentation.
Although necessary, sharing only public information (i.e., new regulations and guidelines) does not provide this opportunity.
▸ Third, this bilateral collaboration benefited from the fact that both parties had the same level of maturity and development of their systems and regulations, and similar public health needs and challenges (even if they were not always identical).
▸ Lastly, the step-by-step approach established has been helpful because it provided clear priorities (with the clusters), allowed progressive exchange of information (from ad hoc requests to regular teleconference and nonpublic product information exchange), and time for each party to evaluate the partner agency's system and practices (with several specific pilot projects and visits/exchange of staff).
Although it took some time and a lot of effort, these different steps were beneficial as they facilitated transparency and confidence building.
This clear understanding of similarities and differences of practices is a prerequisite to foster a culture of convergence of each agency's assessments and evaluations.
To conclude, this bilateral collaboration is now very developed and has moved from confidence building and exchange of information, to recognition of each other's information and data for decision making.
Its success so far supports the continuation of this collaboration and even its extension, as confidence in each other's system continues to increase.
Although it is recognized that each party will remain ultimately responsible for public health in their territories, closer cooperation and convergence are obviously possible in many domains.
Finally, it would be beneficial to continue to expand successful projects to additional partners (as has been the case for GMP inspections of active substance manufacturers [254] ) in order to foster greater international collaboration and information sharing.
In addition to the bilateral, regional, and global regulatory initiatives described in previous sections, other technical and scientific harmonization projects have also been initiated.
Although these projects do not enter in the scope of this research (as they do not specifically relate to regulatory harmonization), it is important to mention them, as the standards they develop are often used by the regulatory harmonization initiatives.
The following organizations and projects ooo have indeed supported the harmonization of standards in the pharmaceutical domain: ▸ The Pharmacopoeial Discussion Group (PDG) involves (since 1989) the European Pharmacopoeia (EP), the Japanese Pharmacopoeia (JP), and the US Pharmacopeia (USP) to harmonize pharmacopoeial standards (i.e., excipient monographs and selected general chapters).
It works in collaboration with ICH, and WHO became an observer in May 2001. ooo This list of organizations/projects below is provided as an example and does not represent an exhaustive list.
▸ The International Organization for Standardization (ISO) is the world's largest developer and publisher of international standards (with a network of the national standards institutes of 163 countries and a central secretariat in Geneva, Switzerland).
This is a nongovernmental organization that today has more than 19,000 international standards and other types of normative documents covering many technical areas.
▸ The Pharmaceutical Inspection Co-operation Scheme (PIC/S) facilitates (since 1995) ppp cooperation and networking in the field of good manufacturing practice (GMP) in order to lead the international development, implementation, and maintenance of harmonized GMP standards and quality systems of inspectorates in the field of medicinal products.
The PIC/S activities include the development and promotion of harmonized GMP standards and guidance documents, the training of inspectors, and the assessment of inspectorates.
This initiative currently includes more than 40 worldwide pharmaceutical inspection authorities.
▸ The Council for International Organizations of Medical Sciences (CIOMS) is an international, nongovernmental, nonprofit organization that was established jointly by WHO and the United Nations Educational, Scientific and Cultural Organization (UNESCO) in 1949.
It includes over 55 international, national, and associate member organizations representing many of the biomedical disciplines, national academies of sciences, and medical research councils.
One of the objectives of CIOMS is to facilitate and promote international activities in the field of biomedical sciences, and its activities include programs on drug development and international nomenclature of diseases.
▸ The World Medical Association (WMA) is an international organization founded in 1947 to represent physicians.
Today, it includes 100 National Medical Associations, and its goal is to achieve consensus on the highest international standards of medical ethics and professional competence.
The Declaration of Helsinki (developed in 1964) is the WMA's best-known policy statement.
Finally, other groups of experts have also worked and released recommendations on specific topics related to the harmonization of pharmaceutical regulations (e.g., the PhRMA's [Pharmaceutical Research and Manufacturers of America] Simultaneous Global Development project [255] or the nonprofit TransCelerate BioPharma project [256] ).
All these projects contribute to the global convergence and harmonization of pharmaceutical regulations.
Many harmonization initiatives have been established over the past several decades because regulators understand that cooperation can help in resolving the new challenges brought on by globalization.
Understanding the importance and advantages of cooperation and ppp The Pharmaceutical Inspection Convention (PIC) had been operating since 1970. harmonization in supporting their mandate to promote and protect public health, many countries and regions have strongly enhanced their collaboration with other countries bilaterally and multilaterally at the regional and global levels.
The globalization of the pharmaceutical market has highlighted several problems that have been associated with data generated from foreign countries and with imported products.
For example, in 2008, deaths associated with heparin imported from China into the US was due to contamination of its pharmaceutical ingredients at a Chinese plant, and in Panama, the diethylene glycol found in cold and fever medicine killed many people [257] [258] [259] .
These problems have been a wake-up call, and they further increased the recognition of benefits to be derived from leveraging the activities and resources of foreign counterpart DRAs [260] .
For example, the US has strongly increased their international collaboration in the pharmaceutical domain.
US legislators decided that such international cooperation and harmonization activities are an integral part of the US FDA's mission.
Indeed, the Food and Drug Administration Modernization Act of 1997 stated that one of the missions of the FDA is to "participate through appropriate processes with representatives of other countries to reduce the burden of regulation, harmonize regulatory requirements, and achieve appropriate reciprocal arrangements" [261] .
Since then, the US FDA's international work has grown exponentially, especially over the past decade, to respond and adapt to the new global society [262] .
It has increased communication qqq and developed regulatory cooperation with other countries (bilaterally and multilaterally).
The US FDA's role in harmonization and multilateral relations is to coordinate and collaborate on activities with various international organizations (i.e., WHO, ICH, PANDRH, and APEC) and individual countries on international standards and harmonization of regulatory requirements.
In pursuit of appropriate international collaboration, the US FDA utilizes a wide variety of International Arrangements, including "Confidentiality Commitments" rrr and "Memoranda of Understanding and other Cooperative Arrangements."
sss The EMA is one of the US FDA's closest regulatory partners.
with China, uuu the US FDA must increase its capacity for inspecting and analyzing Chinese products before they are shipped to the US.
In order to accomplish this, the US FDA established an office in Beijing, China in November 2008 and employed 14 people (with additional employee hiring planned in the following years [263] ).
It has allowed for solid relationships with Chinese regulators and exporters, and has trained more than 1,600 manufacturers and regulators on US safety standards in two years [264] .
Finally, there has been increasing recognition within the US FDA of the need to strengthen regulatory capacity and provide technical and scientific expertise to developing countries to ensure that products exported to the US meet US FDA standards and adequate levels of patient protection.
Many cooperative initiatives have been established to meet this goal [265] .
other countries and regions, including the US, EU, Australia, Canada, Singapore, and China.
These bilateral collaborations are based on confidential agreements vvv and include information sharing.
Proactive exchange of staff has also been agreed upon with some DRAs ( ).
Japan's PMDA has also developed privileged relationships with China and South Korea following the pandemic influenza crisis [271, 272] .
Since 2007, this tripartite initiative has specifically cooperated on clinical research and promoted regional clinical trials [273, 274] .
In February 2009, the Advisory Council approved the PMDA International Strategic Plan as a framework for its international activities [275] .
This plan outlined the strategies for bilateral, regional, and global cooperation, and established an internal office in charge of international affairs.
In line with this International Strategic Plan, further goals (to be attained by 2020) were published in November 2011 .
Finally, a Roadmap for the PMDA International Vision was released in April 2013.
In this roadmap, the PMDA defines more specific actions to support its international vision .
The primary objective of this increase in international collaboration was to urgently resolve the "drug lag" www that has impacted the Japanese pharmaceutical market in the past (2.4 years in 2006).
Many measures have been taken to improve the clinical testing environment (including the promotion of global clinical trials) and expedite drug approval decisions (via, among other measures, the increase of collaboration with the other worldwide DRAs).
A global, simultaneous drug development approach has also been strongly recommended.
Many actions, including release of guidelines, have been taken to facilitate such global development [277] .
In addition to the US and Japan, other major DRAs of developed countries (such as Health Canada and the Australian TGA) also recognized the important added value of global cooperation and therefore increased their involvement in international activities.
The EU, based on its prior experience of harmonization and cooperation from the establishment of its own system, has also developed external bilateral and multilateral collaborations and is today an important international partner.
Although these diverse, coexisting, bilateral, regional, and global initiatives create complexity, it is important to note that they are complementary.
Global harmonization does not preclude having regional harmonization and regional harmonization does not preclude bilateral agreements.
In fact these three levels of harmonization and cooperation bring about different added value: ▸ Bilateral agreements allow for a bigger exchange of information, including productspecific data, through confidential agreements and the development of privileged relationships (and trust) between regulators as they allow for assessment of one another's vvv In the case of China, a cooperative agreement has been established.
www Drug lag is defined as the difference of availability of new medicines between the US and Japan.
systems and practices.
xxx These assessments are indeed critical for confidence building and can ultimately support the signing of agreements, allowing for recognition of inspection or the exchange of nonpublic information (e.g., EU/US collaboration and confidentiality agreements).
Bilateral collaboration also helps strengthen relationships, which would be more difficult in the context of a multilateral initiative, and facilitates training and mentoring activities between developed and developing countries.
▸ Regional harmonization allows for the harmonization of policies between countries that are usually closer in term of systems, cultures, and levels of development.
It is indeed easier to harmonize closed systems and policies between countries of similar culture and environment (for example, it is more difficult to harmonize systems and policies between Asia and North America because they have very different medical practices and cultures).
This level is essential for global harmonization because it provides a structure.
Achieving global harmonization without a supporting regional organization structure is impossible.
This regional level allows for inclusion of regional realities and difficulties in global discussions, and eases the diffusion and implementation of the global recommendations.
▸ Global harmonization is the highest level of harmonization.
Compared to regional harmonization, the global harmonization initiative is not driven by economic objectives; the goal is not to create a free trade area or a single market, but to develop global consensus and standards in order to allow the world's population to have access to medicine and innovative therapies.
To conclude, these bilateral, regional, and global cooperative activities have been beneficial as they supported the harmonization of requirements globally and therefore facilitated the availability of safe and efficacious medicines, critical in promoting global public health, on a worldwide basis.
Many topics and standards have already been partly or fully harmonized at a bilateral, regional, or global level.
For example, most of the requirements regarding the conduct of nonclinical studies, and also the GMP and good clinical practice (GCP) principles, have been agreed on, allowing for joint inspection projects.
A common format of application has been developed, and many technical aspects have been harmonized through the ICH's work.
Collaboration has also been increasing in resolving major topics requiring global interaction, such as orphan drug evaluation yyy and development of medicines for the pediatric population.
zzz Confidence and trust have been built between developed countries through pilot projects, but xxx For example, bilateral collaboration allows two countries to assess their respective inspection systems or systems to control critical information (such as trade secrets).
Such assessments of each other systems could be possible in the case of multilateral collaboration, but would be more complex.
yyy Because only a small number of the population is affected by these life-threatening diseases or serious conditions, it is critical to have global requirements in order to facilitate global clinical studies.
Moreover, the pharmaceutical industry has been reluctant to invest in the research and development of medicinal products to treat these conditions.
The development of global requirements allows quick access to the global market and therefore allows a better return on investment.
zzz It is critical that countries cooperate in this area to avoid exposing children to unnecessary trials.
also through the location of official liaisons in other DRAs to facilitate collaboration.
This has been positive, and this new type of interaction is very promising as it increases relationships and allows for the better exchange of experiences and information.
aaaa The establishment of liaisons in other countries also allows more proactive measures and risk analysis in the area of quality systems and inspections [278] .
Exchanges of information between DRAs have also dramatically increased.
This regular communication between regulators facilitates evaluation of risk (e.g., via exchange of safety alerts) and assessment of new medicines.
Finally, systems have been put in place to help developing countries (e.g., CPP scheme, prequalification of medicines, Article 58 of European Regulation (EC) No 726/2004, etc.).
However, without underestimating all these important positive outcomes, it is clear that differences still exist and that further efforts will be required to support this ongoing harmonization process.
There are still differences between countries in terms of standards and strategies to assess compliance against standards.
The conduct of global clinical studies continues to present many challenges (i.e., related to registration, conduct of the studies, and also the use of data), and there are still several clinical trial registries and databases in use.
The safety of medicines has been one of the main focuses of DRAs in the past due to major problems and events, but there has not been a real effort toward worldwide harmonization regarding risk-mitigation strategies.
Additionally, new standards continue to be developed by different bodies (i.e., ICH vs. regional organizations) in parallel that not only duplicate efforts, but also create disharmony (e.g., biosimilars requirements had first been developed by individual countries and also by WHO).
There is also a significant difference in the level of implementation of harmonized standards (i.e., the ICH recommendations/guidelines) between countries, and the CTD format has still not been implemented in all countries.
It has also been reported that differences between developed and developing countries has in fact continued to increase in the past several years due to the increased complexity of technologies associated with the development of new therapies.
Even between two close partners like the US and EU, which have developed a privileged partnership and strong cooperation, there are still important differences in standards.
For example, the US is still requiring two placebo-controlled studies to determine efficacy of a new medicine, while the EU is more interested in comparative studies using an active comparator.
This difference is due to different legal requirements and scientific opinions regarding the value of such comparative data [279] .
This situation may change in the future with the growing interest in the US for "comparative effectiveness" promoted by the Obama administration.
Finally, this complex worldwide harmonization context (with increased communication and exchange of experience, information, and good practices) requires good communication and coordination between all these ongoing initiatives.
Even if such communication was initiated by WHO and ICH (with the GCG group), further improvement would still be needed.
This enhanced coordination of international cooperation would indeed be beneficial, as it would provide the necessary transparency regarding the focus and responsibility of each initiative (i.e., development of standards, coordination of implementation of recommendations, etc.).
aaaa Exchange of information and best practices has been one of the most important outcomes of the EU/US bilateral collaboration.
It would also facilitate the appropriate use of resources and expertise, and therefore avoid duplication of efforts or conflicting recommendations and actions.
Overlapping membership between the initiatives bbbb may not be fully efficient, and can create confusion and duplication of work.
Although the increased coordination of these diverse initiatives would be beneficial, it will certainly be challenging.
It will need to be thoroughly structured and implemented, and it will also be critical that the coordinated body is a recognized and experienced entity, with appropriate mandate and power.
Under this Directorate, the US FDA's Office of International Programs serves as the agency's focal point for all international matters and is responsible for maximizing the impact of the US FDA's global interactions.
Additional US FDA reorganizations were also announced in 2012 to further respond to drug industry globalization [268].
Also, in addition to China, the US FDA now has staff stationed permanently in India the total number of shipments of US FDA-regulated products from China increased from approximately 1.3 million to 2.1 million.
Of the 2.1 million entry lines arriving in 2011, 30% were drugs and devices, and 12% were human food products
key: cord-014687-0am4l5ms authors: nan title: SPR 2012 date: 2012-03-29 journal: Pediatr Radiol DOI: 10.1007/s00247-012-2356-8 sha: doc_id: 14687 cord_uid: 0am4l5ms nan Dear Colleagues, I confess I haven't read many "Welcome Letters" at the beginning of the SPR program book over the years.
Perhaps the only defensible benefit of this is that there is no preconception about the content of this message…or the length.
I will be brief.
This meeting is about building bridges…bridges from our past to the future and bridges between all of us who believe fundamentally in maintaining or improving the health of our children.
The content, which is detailed on subsequent pages, speaks for itself.
This material will be presented during the sessions with an appreciative look back at past accomplishments-the legacy of our subspecialty-with a vision to the future of pediatric imaging.
We can only measure how broad and deep our successes have been by connecting with these beginnings.
Looking beyond the titles (and the speakers), I think you will see that the material is not only about techniques and tactics but about ideas, insights, energy, all conspiring in the creative process … an aggregate for excellence in pediatric imaging.
The content is also punctuated by a strong presence of our clinical colleagues.
Again, this builds bridges.
How can we maintain and expand these relationships?
Moreover, the connections between science and clinical practice are evident in the structured blending of scientific papers and topical presentations by both imaging and clinical experts.
This blending is also "fraternal" in that there will sometimes be disagreement and critical commentary, but this is essential in the advancement of medicine.
Support and criticism make a stronger mortar.
In the end, this gathering is about fostering a connected community, including technologists, nurses, physicists and other allied health experts including industry experts.
Finally, the emblem of pediatric radiology has always been embossed by cooperation, passion, commitment, and humanistic care.
I believe the program content, the presenters and you, the participants, all embrace this.
I hope that you will feel the spirit and the passion of the meeting and all of us will in many ways be better able to care for children because of this-even if you never read this message!
Donald P. Frush The Gold Medal of The Society for Pediatric Radiology is our most distinguished honor.
The SPR Medal is awarded to pediatric radiologists who have contributed greatly to the SPR and our subspecialty of pediatric radiology as a scientist, teacher, personal mentor and leader.
Marilyn Goske has always wanted to make a differenceand what a difference she has made!
Her role as an educator, and her lifelong commitment to improving training for residents, fellows, faculty, medical staff and radiologic technologists has resulted in many wonderful initiatives that have benefited all in pediatric radiology.
The work she is most proud of-the Cleveland Clinic Web Based Curriculum, working with the leadership of SPR's Philanthropic Campaign for Children, launching the Image Gently Campaign and the pediatric research component within the American College of Radiology's Dose Index Registry share a common theme: educating others in providing the best care possible for children.
Born in Berea, Ohio, Marilyn's father, George, was a chemical engineer.
Her mother, Cornelia aka "Corky", loved writing as one of the first women journalists for the Associated Press and later teaching, passions she passed on to her daughter.
While Marilyn was blessed with a strong female role model in her mother, it was her brother, James, who was her cheerleader, always pushing her to dream big.
He encouraged her to follow in his footsteps first at Ohio University, then on to the Ohio State College of Medicine to pursue an MD degree during an era when nursing would have been a more conventional goal.
Marilyn met her husband Rick on a double date in college-unfortunately, they were with different dates!
Luckily, they were able to get together for an actual date with each other 18 months later.
They quickly became engaged and married within a year of that first true date.
When Rick started his residency in internal medicine, Marilyn transferred to the University of Connecticut School of Medicine in Farmington.
It was here that she met her first pediatric radiologist-and what a giant-Mike Ozonoff!
When Rick moved on to a neurology residency in Rochester, New York, Marilyn followed and met another pediatric radiology giant: Beverly Wood, at Strong Memorial Hospital.
Beverly proved to be a wonderful teacher, mentor, co-researcher and lifelong friend.
Marilyn describes Beverly as inspirational and "fearless" in trying new technologies.
It was during her time in Rochester that Marilyn went to her first SPR meeting and, not surprisingly, won the 1984 Caffey Award for her work on "Experimental Neonatal Intraventricular Hemorrhage: Clinical, Radiographic and Pathologic Features."
By then Marilyn had two young children and moved on to the private sector, practicing part-time for several years first in Rochester, then in Cleveland, Ohio.
Her years in private practice were particularly helpful in learning the importance of patient oriented service-and paved the way for her intuitive public relations strategies when designing the Image Gently campaign in later years.
Dr. Goske was asked to join the Cleveland Clinic in 1990, as the first full-time section head of pediatric radiology.
It was here that she built a new section and spearheaded the web based education program for pediatric radiology residents with co-founder Janet Reid.
This important free web site with 65 modules is used widely by over 200 radiology residencies nationally and internationally.
Her passion for education continued, inspiring her to complete a medical education fellowship focused on professionalism within the Cleveland Clinic Lerner College of Medicine.
Her work towards this fellowship has led to many creative educational initiatives including yearly educational summits at the SPR.
She was named chair of the Professionalism committee of the RSNA where she along with her committee have sponsored interactive workshops on this topic dear to her heart.
Dr. Goske's energy and effective leadership skills brought her to become involved in the Society for Pediatric Radiology, first as the coordinator for SPR's first video-taped course in 1994.
Mentors Diane Babcock and Carol Rumack proposed her for the Nominating Committee.
This was followed by Chair of the Membership Committee, where she organized the first formal survey of the Society, then as a Board member, then as Secretary, and finally as President and Chair of the Board of Directors, completing 12 years on the SPR Board.
Working together with Stuart Royal, she successfully energized the Campaign for Children raising funds for the Research and Education Foundation of the SPR and expanded the work of prior presidents in further organizing the Corporate Support committee.
Marilyn's years as President and Chairman of the Board of the SPR were highly successful with many unique strategic goals.
She was instrumental in the founding of the junior SPR.
She led the wonderful 2007 SPR national meeting in Miami which included the first educational summit to enhance knowledge in adult learning and resident competencies.
Most people would rest after completing their arduous year as President but as Chairman of the Board, Marilyn was just beginning!
She moved to Cincinnati Children's Hospital, joining the radiology department and was named the Dr. Corning Benton Endowed Chair for Radiology Education where she got to work with Dr. Janet Strife, another influential mentor and friend.
Acknowledging SPR's long focus on reducing radiation doses in the imaging of children but concerned about the lack of change in practice by a majority of radiologists despite increasing reports of possible side effects, Marilyn developed a public relations and awareness campaign.
Her goal was to inspire all to work towards decreasing radiation exposure to children when possible.
With the help of many, she founded the Alliance for Radiation Safety in Pediatric Imaging and the Image Gently campaign, initially focusing on CT.
Her ability to encourage numerous experts and societies to work together and get involved in "child sizing the amount of radiation used" has resulted in a groundswell of research and activity in this area.
Currently 69 organizations with over 800,000 members have joined the Alliance including 24 international societies.
The web site, www.imagegently.org, has been immensely successful filled with free information pamphlets in over 12 languages, PQI projects, and modules for parents, physicians, and technologists.
The Image Gently Campaign has received several awards including the Associations Advance America Honor Roll, RT Image magazine group with the "most influence in radiology" and the Most Effective Philanthropy Program from Aunt Minnie.
Image Gently has spawned the creation of the adult-focused Image Wisely campaign.
The Alliance has been named by the Joint Commission, U.S. Food and Drug Administration, and the American Medical Association in their influential statements on radiation dose as providing much needed guidance and information.
Marilyn's exceptional talent is inspiring and coordinating experts in multiple fields to work together towards common goals.
She continues to work hard on the Image Gently campaign with more safety and quality messages planned for the coming years.
She is also proud of her work with the ACR Dose Index Registry and Quality Improvement Registry in CT Scans in children in working toward developing diagnostic reference levels with a talented consortium of pediatric radiologists, medical physicists and technologists.
She has acted as a national and international expert in her work with the International Atomic Energy Agency, the World Health Organization and the National Council on Radiation Protection in Medicine and the FDA.
Dr. Goske's multiple committee appointments are taken seriously, and her work is always meticulous, well thought out, and brought to successful completion.
She has been an active member of numerous national and international societies including the John Caffey Honorary Society, ACR, RSNA, ESPR, AAWR, and SCORCH.
It is important to remember that Dr. Goske is also a successful researcher with numerous grants obtained through the SPR REF including the Thorne Griscom Education award and the RSNA Scholar grant.
She has published over 80 peer-reviewed articles, 19 electronic publications, 7 chapters, and presented 26 scientific exhibits as well as given numerous scientific presentations.
An articulate and engaging speaker, she has been invited to give over 130 lectures locally, nationally and internationally.
While Marilyn has been very focused on her work with the SPR, she believes that it is her amazing family and their love that really fuels her life.
Her husband Rick is an internationally known neurologist and researcher in multiple sclerosis.
Her adult children Jamie and Brian, both in Manhattan, remain close, and spending quality time together as a family remains the joy of her life.
Whether it is relaxing together in Florida cooking or fishing, or taking an exotic vacation to India, being with Rick, Jamie and Brian makes her the happiest.
Marilyn's genius is partly refusing to take "No" as an answer.
Along the way, at every turn there were those who believed that what she wanted to do couldn't be done.
Her approach was to draft the nay-sayers to the team and charge ahead with their willing and enthusiastic help.
Daniel Burnham might have been talking about Marilyn and not about his plan for the city of Chicago when he said: Make no little plans; they have no magic to stir men's blood and probably themselves will not be realized.
Make big plans; aim high in hope and work, remembering that a noble, logical diagram once recorded will not die, but long after we are gone be a living thing, asserting itself with evergrowing insistence.
As an amazing change agent, inspirational leader, and wonderful role model, the SPR is proud to honor Marilyn Goske with the 2012 Gold Medal.
She made big plans!
Dorothy I. Bulas, MD Pioneer Honorees were first acknowledged in 1990 as a means to honor certain physicians who made special contributions to the early development of our specialty.
It is time to reevaluate the meaning of the Pioneer Honoree.
The subspecialty of Pediatric Radiology has been in existence now for more than 50 years.
We are beyond "the early development"; we must recognize other pioneering paths and should consider contributions to the subspecialty beyond the bounds of a modality, a technique, an observation or a change in practice.
Whatever this advancement is, it must be forged with vision, innovative ideas, and the ability to enable and sustain science and application.
George S. Bisset, III, M.D.
Why George Bisset?
Has he been part of the Pediatric Radiology landscape these last ten years?
Been part of the dialogue that has been increasingly influential across all of Radiology, a conversation steeped in a deep tradition of excellence in diagnosis and treatment, and the safety and welfare of our children?
Been a leader in science and application?
Part of the landscape?
No.
But he has been beyond that and has worked tirelessly within the horizon, surveying…a step before, but guiding us on towards our destiny.
A conversant?
Part of the dialogue?
Maybe.
But he has been defining thought and concept upon which such conversation is born and nurtured.
Part of the science and application?
Yes, as much as anyone who promotes, who facilitates and sustains discovery, then here we are.
Horizons, innovation, and the gift of en-abling…What else is needed to define a true pioneer?
How was this done?
Simply stated, George Bisset has devoted at least the last decade to the advancement of our specialty in truly novel ways through his leadership, especially in RSNA and the ABR.
In the RSNA, as the Scientific Program Committee Chair several years ago he was instrumental in the conception, development and implementation of the integration of scientific papers and refresher course topics.
This has been a resounding success, is currently used in other categories during RSNA and is a model for other meetings, including the Annual Meeting for the Society for Pediatric Radiology over the past few years.
Pediatric Radiology was first in this effort.
George continued to endorse topics that were marquees for Pediatric Radiology over the year in his education role on the RSNA Board of Directors.
He endorsed and implemented the Pediatric Campus concept at the 2011 RSNA.
Early returns are that this was an extremely successful model to consolidate experts in pediatric radiology (and those interested in this subspecialty), pertinent science, education and administration.
George is now the President of RSNA, perhaps the most widely respected scientific and educational organization for our profession across the globe…and I would argue, with more promise for our future success in Pediatric Radiology than has ever existed.
And George Bisset, who through two terms as the Pediatric Trustee for the Board of Trustees for the ABR, again, was on the horizon of a critical, sometimes perilous, and complete transformation of our certification examination process, always mindful of his constituency and colleagues, his duty as a physician, and the public and patients.
This required delicate diplomacy, forward thinking, professionalism, and enlistment of a cadre of experts from within our subspecialty to assure excellence in pediatric radiology through ABR certification.
He was also a leader in the development, validation and implementation of the imagerich computer based examination model (the pediatric CAQ) now the standard for the new ABR examinations.
With these successes in mind, who better to embody the concept of bridging horizons that is the theme for this entire meeting?
If you were looking for more numbers and accolades, I apologize.
Here are some: more than 225 contributions to medical and scientific literature, advancing care through pediatric body CT and MR imaging research, a litany of presentations and invited lectures, Vice Chairs, Chairs, Chiefs, Boards of Directors, committee member and committee leader, clinical excellence including as a pediatric cardiologist and interventional radiologist, a superb speaker and author.… all are on his CV but I believe serve really as signposts for his gifts, some of those mentioned above, that a CV simply cannot convey.
He could have played it safe with all of these successes on his CV.
But pioneers don't play it safe.
They are on the horizon, too busy defining thought and enabling (our) advancement-building bridges.
I believe it is time to reevaluate the meaning of the Pioneer Honoree and I have the greatest honor and pleasure of introducing George S. Bisset, III for the Pioneer Award for 2012.
Linked with past awardees, he continues an exceptional legacy and I don't believe his explorations and discoveries are finished… Donald P. Frush, MD The Society bestows Presidential Recognition Awards on members or other individuals whose energy and creativity have made a significant impact on the work of the Society and its service to its members.
In 1999, David Kushner was recognized by the SPR with its first Presidential Recognition Award for his vision and foresight in working with both the American College of Radiology (ACR) and the Society for Pediatric Radiology (SPR) in developing an important new relationship and for his service to the SPR.
In summarizing his considerable efforts for that award, I noted that he "contributed substantively to the increased visibility of the SPR within the ACR.
His tenure as our Treasurer placed our organization on a firm financial foundation."
With the current award, the Society recognizes his indefatigable continuing efforts on our behalf including: His work with the ACR: 1.
Establishing a pediatric radiology caucus at the annual ACR meeting, 2.
Convincing the ACR of the value of managing specialty societies by making the SPR its first successful new model for imaging society management, 3.
Advocating tirelessly for pediatrics and children's health within the ACR by serving on the Council Steering Committee and then as ACR Council Vice Speaker and Speaker, 4.
Helping establish the first pediatric commission, assuring that pediatric issues will receive support of the College and its resources while serving on the Board of Chancellors of the ACR for the past five years.
The SPR's "Image Gently" campaign was a beneficiary of this pediatric commission of the Board of Chancellors, 5.
Continuing to shepherd and contribute to the pediatric component of the ACR practice guideline process.
His work with the SPR: Since his earlier award, David has served as: Foundation from 2000 to 2003, including the launch of the formal fundraising effort, "the Campaign for Children," 2.
SPR President 2003 -2004 , organizing and running a very successful meeting in Savannah, 3.
Chair of the Board of Directors of the SPR from 2004 to 2005, including leading a strategic planning process that resulted in a new, more focused division of labor amongst Board members and defined Board responsibilities.
David was born in Fargo, North Dakota, received a BA from the University of Minnesota, and received his medical education at the University of Pennsylvania.
This was followed by two years of training in pediatrics at Children's Hospital, Boston.
He then did a two-year fellowship at the National Institute of Health in Bethesda, performing research in embryology and teratology.
He returned to Massachusetts General Hospital for training in diagnostic radiology.
This was followed by a year of residency in pediatric radiology at Children's Hospital Boston, followed by a one-year fellowship.
He then became director of the pediatric radiology section at Massachusetts General Hospital, a position he held from 1979 to 1988.
From 1988 to 2005, David was chief of the Division of Diagnostic Imaging and Radiology at Children's National Medical Center in Washington, DC attracting a strong faculty, training many fellows and promoting research.
During that time, he served as a volunteer radiologist and pediatrician to inner city healthcare systems aiding the indigent and homeless, and developing telemedicine capabilities linking free clinics with radiology experts.
In 2005, our man inside the beltway moved a bit outside by accepting the medical directorship of radiology at the Children's Hospital of the King's Daughters in Norfolk, Virginia, and Professor of Radiology and Pediatrics at the Eastern Virginia Medical School.
He assures me that life there is good, being a bit more "laid back" with fishing and sailing just outside the door.
He also finds time for Italian cooking and practicing jazz on his several guitars.
Fortunately for all of us in the SPR, David is close enough to our central office and the ACR that he will be able to continue work on our behalf for many years to come.
The Society bestows Presidential Recognition Awards on members or other individuals whose energy and creativity have made a significant impact on the work of the Society and its service to its members.
The 2012 SPR Presidential award is given in recognition of Stuart's numerous significant and outstanding contributions to the SPR over many years of service.
The awardee is selected by the Honors Committee, a committee comprised of the three most recent past presidents of the Society.
Dr. Royal is a proud native of Birmingham, Alabama.
He is a second generation physician who came naturally to his desire to care for children as the son of a pediatrician, Arnold Royal, who took care of children in the Birmingham community until he was 79 years old.
Dr. Royal attended Rice University in Houston, Texas followed by MD and MS degrees from the University of Alabama at Birmingham.
He subsequently moved to San Francisco, where he completed a pediatric internship followed by a diagnostic radiology residency at the University of California, San Francisco.
Dr. Royal credits Dr. Charles Gooding at UCSF for influencing his decision to pursue a career in pediatric radiology.
During his internship Stuart observed Dr. Gooding make a plain film diagnosis of TAPVR, type 3 on a severely ill and perplexing newborn, and he was immediately hooked into radiology.
While at UCSF Dr. Royal was also appointed as a National Institute of Health research fellow in the Department of Radiology.
Following residency, Stuart completed a fellowship in pediatric radiology at The Children's Hospital Medical Center in Boston.
From Boston, Stuart returned to his roots in Birmingham, Alabama in 1980, where he was appointed as a pediatric radiologist at the University of Alabama and subsequently The Children's Hospital in Birmingham.
In recognition of his outstanding leadership skills and accomplishments at The Children's Hospital, Dr. Royal was appointed as the Radiologist-in-Chief in 1987 , and subsequently the Harry M. Burns endowed Chair of Pediatric Radiology.
He also holds appointments as Clinical Professor of Radiology and Pediatrics at the University of Alabama at Birmingham and serves on The Children's Hospital Board of Trustees.
At Alabama Dr. Royal has earned the high esteem of his colleagues, referring physicians, and staff for his outstanding clinical acumen as a diagnostic radiologist and for his undaunting commitment to excellent care of children.
Colleagues describe Stuart as one who fosters a strong work ethic, high commitment to teaching, and sincere compassion for children.
In 2006, Stuart was the recipient of the Children's Advocate award by Childcare Resources for improving the quality of care and access to radiological services for underserved children in Birmingham.
Stuart has been married to the love of his life, Barbara Royal, for the past 40 years.
Stuart and Barbara are the proud parents of two very accomplished children, Jeremy a budding radiologist in training at the University of Alabama, and Rachael, who has an MBA and works as a Vice President for Moody's in New York.
Stuart and Barbara are also the proud grandparents of three grandchildren.
In conversation, Stuart is quick to pull out his iPhone and share the latest pictures of family members while recounting their latest activities and milestones.
Throughout his professional career, Dr. Royal has worked tirelessly to advance the mission of the Society for Pediatric Radiology.
He is past president and chairman of the board of the SPR and has served on numerous SPR committees.
He ran a highly successful SPR meeting in New Orleans in 2005.
Those in attendance will recall the jubilant parade Stuart led through the streets of New Orleans to culminate the meeting.
As President and then Chair of the SPR board, Stuart played a critical and instrumental role in bringing the SPR management contract under the umbrella of the ACR.
The synergy achieved by the SPR-ACR relationship has yielded results well beyond a simple management contract.
Pediatric radiology and SPR now have a voice at the "radiology table."
Stuart has also been a strong advocate for supporting translational research to advance the care of children via imaging.
To help achieve this goal, he has worked aggressively to secure increased funding for the Society of Pediatric Radiology Research and Education Foundation.
Following the launch of the REF's Campaign for Children in 2000, Stuart made it his personal mission to work with the leadership of the Society, both past and present, to discuss major gifts to the Foundation.
Through Stuart's personal effort, the Foundation received pledges for many significant leadership gifts, including from SPR Pioneers Drs.
Hooshang Taybi and Ed Singleton and from himself and Barbara.
The SPR is highly fortunate to have benefited from Stuart's numerous contributions and dedication to the care of children.
The Society is very proud to bestow the 2012 President's Award on Dr. Stuart A. Royal.
The Society extends Honorary membership to individuals outside of pediatric radiology who have made outstanding contributions to the care of children.
This evening, Dr. Harvey L. Neiman, whose leadership of the American College of Radiology is resoundingly praised, is the recipient of the 2012 Honorary Member Award.
For 2012, as in 2007 when his contributions were similarly recognized, Dr. Neiman's selection by the Society for Pediatric Radiology Honors Committee was made in appreciation for the strength of his efforts to further the SPR's philosophy, goals, and programs for responsible diagnosis and treatment of the young patient as embodied in the ACR and SPR's "Image Gently" campaign.
Image Gently has succeeded not only in raising awareness of the great diagnostic benefits we can offer to pediatric patients but also directs us to acknowledge the downside of overzealous diagnostic efforts where excessive radiation becomes a risk.
Importantly, the "Image Gently" campaign, an upbeat, positive program rather than a punitive one, a smile rather than a frown, makes pediatric and all radiologists aware that their best practice reflects balanced, educated, up-to-date utilization of state-of-the art technology with exercise of responsible leadership in protecting the pediatric patient.
For adults, awareness of the need for patient protection is communicated in Image Wisely.
Dr. Neiman's vision and successful achievements are evident on every page of his curriculum vitae.
A consummate strategist in assembling teams to make forward-looking goals a reality, Harvey now stands at the top of our specialty as the first physician Executive Director of the American College of Radiology.
At this time in big-business medicine, as we see the physician, leader of the patient care team, being diminished to one of many "providers," it is so important for our patients' well-being for us to recognize the obligations commanded by our training, clinical experience and commitment.
Dr. Neiman's recognition of the need for physicians' leadership in improving the quality of patient services and his development of programs in all areas of the college's activities have been just short of miraculous-Image Wisely for adults, Quality and Safety including the Performance Guidelines and Accreditations, Education, Government Relations, Economics, Imaging Metrix, ACRIN, and the new Radiology Leadership Institute-to name only a few.
All have contributed significantly to the care of our patients and the stature of our specialty.
Dr. Neiman was born in Detroit and attended Mumford High School.
From Wayne State University, he received his B.S.
in 1964 and his MD in 1968.
Harvey's postgraduate training was at the University of Michigan, where he was a resident in Radiology (1969 -1972 ), Chief Resident (1971 , and a 1972-73 fellow in Angiography (Cardiovascular Radiology), receiving ABR certification in 1973 and a CAQ in Vascular and Interventional Radiology in February 1995.
Dr. Neiman often expresses his gratitude to and profound respect for his mentor and beloved chief at the University of Michigan, Dr. William Martel.
Dr. Neiman was Chief of Cardiovascular Radiology at Walter Reed Army Hospital and a lecturer in Cardiovascular Radiology at the AFIP from 1973 -1975 .
In 1975 , he joined the Northwestern Radiology faculty, rising to Professor in 1981, and for ten years he headed up the section of Angiography and Sectional Imaging, advancing its technology and honing the skills of Northwestern's radiology residents.
Harvey also offered a highly sought-after fellowship in Interventional Radiology, US, and CT.
In 1985, Dr. Neiman left Northwestern to assume the Chair in Medical Imaging at the Western Pennsylvania in Pittsburgh.
I was the first woman to have completed his fellowship in US, CT, and Interventional Radiology at Northwestern and accompanied him to Pittsburgh.
His tenure at West Penn attests to his talent in making his visions a reality: the department became a highly respected, successful academic private practice notable in many areas including ultrasound, breast and women's imaging, and interventional radiology.
Harvey instituted an excellent radiology residency program in 1988 as well as fellowship programs in 1986 in the areas of excellence noted above.
During the 40 years since Harvey received his MD, he has been awarded honors from many national, international, and specialty societies, has been an invited lecturer over 181 times on ultrasound, interventional radiologic, radiologic educational, management, turf issues, disruptive and new technology topics to name just a few.
Dr. Neiman, who was a founder of the SRU (Society of Radiologists in Ultrasound), has to his credit 122 peer-reviewed articles, 69 scientific presentations and 20 exhibits, a text co-authored with Dr. James Yao, Angiography of Vascular Disease (1984) , and 26 book chapters.
He has received many honors including fellowship in the American College of Radiology, American Institute of Ultrasound in Medicine, Society of Radiologists in Ultrasound and the Society of Cardiovascular and Interventional Radiology (now SIR).
As part of his strong commitment to the future leaders of radiology, for Diagnostic Radiology he has served as a member of the Residency Review Committee of the Accreditation Council for Graduate Medical Education.
He has been a member of the American College of Radiology and its committees and commissions for many years including the Commissions on Education, Ultrasound, and Economics.
He also served as chair of the Commissions on Ultrasound and Economics.
From 1994 to 2002 , he was a member on the ACR Board of Chancellors, serving as its chairman 2000-2002.
He was President of the Radiology Advocacy Alliance from 1998 to 2000.
In 2003, nine years ago, Dr. Neiman became the ACR's Executive Director.
He currently serves in this position, where his excellent business skills, knowledge of health policy and economic issues, and strong administrative background have furthered our specialty.
His goal, to ensure that the ACR's resources benefit all radiologists and patients across all economic strata, is evident in his actions at the College.
Harvey has a devoted, wonderful family that often included me and my youngest daughter on many Pittsburgh occasions.
His beautiful, elegant wife of many years, Ellie Neiman, is here tonight to celebrate with him the SPR's recognition of his many achievements.
Dr. Neiman has two accomplished, lovely daughters, Jennifer, extremely successful in her marketing career, and Hilary, an attorney.
Jennie's husband, Dr. Seth Kligerman, one of many young radiologists whom Harvey has mentored, is on the radiology staff at the University of Maryland.
How Harvey has had time between, through, and among all of these achievements to have become mentor, colleague, and friend to me and to so many others who have been inspired by his ability to see into the future and to shape it in a positive way is remarkable.
Now that Dr. Neiman has taken all of radiology under his wing, not just its component parts, the future of our specialty, one of the best, can be assured but also recognized for its centrality to all of medicine.
It is my honor and privilege to introduce to you Harvey L. Neiman MD, FACR as this year's Society for Pediatric Radiology Honorary Member.
The Singleton-Taybi Award is given in honor of Edward Singleton and Hooshang Taybi, in recognition of their personal commitment to the educational goals of the SPR.
Initiated in 2006, the Award is presented annually to a senior member of the SPR whose professional lifetime dedication to the education of medical students, residents, fellows, and colleagues has brought honor to him/her and to the discipline of pediatric radiology.
It comes as no surprise to those who know him that Dr. Daneman, "Dr. D" as some of us call him, has been named the 2012 recipient of the Singleton-Taybi Award in recognition of his many years of dedication to the education of residents, fellows, and colleagues.
Born in South Africa in 1947, he received his Medical Degree at the University of the Witwatersrand, Johannesburg, receiving the Harwood-Nash award for the most successful student in Surgery.
Initially, Dr. D thought he would become a pediatric surgeon; but after passing the Part I examination offered by the Royal Australasian College of Surgeons, he changed his mind and began his training in diagnostic radiology.
He chose a radiology residency at the Royal Prince Alfred Hospital, in Sydney, Australia.
This included a year in pediatric radiology at the Royal Alexandra Hospital for Children in Sydney where his interest and love of pediatric radiology began.
Dr. D then had the foresight to pursue pediatric radiology fellowship training at the Hospital for Sick Children in Toronto, Canada.
After completing the fellowship, he was immediately offered a position as staff radiologist at "Sick kids."
He became Director of Body Imaging in 1984 and Radiologist-in-Chief in 1988 serving in that capacity for 7 years.
His management style was simple but effective.
He chose staff that were young, but smart and innovative.
He nurtured them and provided them with all the tools they needed to become successful professionals, like him.
But contributing to his own department was not enough for him.
He also found the time and strength to contribute, teach, train, and help pediatric radiologists in the most remote portions of the globe in every continent, which resulted in recognition from prestigious organizations in places such as South America, Israel, Europe, Taiwan and Australia: he is an Honorary member of the European Society for Pediatric Radiology and the Sociedad Latinoamericana de Radiologia Pediatrica as well as other national societies.
Dr. D is an "institution" inside the great institution that is Sick Kids.
His teaching is unique and praiseworthy in being enthusiastic, provocative, and fun at the same time.
His lectures have been regarded as both instructive and practical by his students and trainees due to his special gift of making the most complicated things look as simple as possible.
In sharing his diagnostic knowledge and know-how, he passes his own, innate teaching spirit on to his apprentices.
He has earned several awards for this, including the outstanding teacher award granted by the University of Toronto fellows at Sick Kids for the past 5 consecutive years.
Dr. D receives numerous invitations to present at national and international meetings and symposia and has been invited as a visiting professor to more than a hundred institutions across the globe.
He does not only teach us the ins and outs of Pediatric Radiology, but he makes sure that we learn to love it and understand the importance not only of good practice but also the imperative to pass knowledge on by teaching and publishing.
Dr. D is someone who inspires us to reach beyond our limits, someone we want to emulate.
He shares his knowledge, his wisdom, and his advice freely.
He shares with us the most incredible secrets of his own career, so we understand from his personal experience.
Dr. D never tells you what to do, he suggests to you, in an incredible articulate fashion, what you want to do yourself.
Dr. D has been and is for many of us, more than an educator, more than a mentor, he is our "coach."
Well before this concept was introduced into medicine by A. Gawande 1 , Dr. D intuitively had the vision to "coach" his trainees, trying to get the best out of them, without pressure, but with love and passion, and especially emphasizing the importance of achieving a worklife balance in order to prevent the now so common "stress and burnout" affecting the radiology community 2 .
He warned us that many high achievers reach their goals only at the expense of their personal lives, but Dr. D has been as successful personally as he is professionally.
His wonderful wife of 40 years, Louise, his two daughters and his recently newborn granddaughter serve as sources of strength and pride.
He is a truthful and generous friend to many, both in and out of radiology.
It is not uncommon for many of us, who came through Sick Kids, to come back and visit and be invited to his house to share a wonderful dinner with other invitees, who may be radiologists from North America or from other parts of the globe visiting Sick Kids to learn from him.
Dr. Daneman's research has widely influenced the field of Pediatric Radiology.
Examples include the work of Dr. Daneman and his colleagues on intussusception, which has promulgated the use of ultrasound for diagnosis, and the use of air enema for reduction.
This approach has been adopted as standard practice at many institutions in North America and across the globe.
To share his research with others in the field, Dr. Daneman has authored or co-authored more than 200 publications, including peer reviewed articles and book chapters on a wide range of topics related to the imaging of children.
Dr. D is one of those rare people who are irreplaceable.
He is a superb teacher, a gifted academician, a capable administrator, and a person called "friend" by so many of us.
We are thrilled and proud to present our Society's Singleton-Taybi award to Dr Alan Daneman in recognition of his lifelong accomplishments and personal commitment to the educational goals of the SPR.
We cannot imagine anyone more deserving of this award than Dr. D. Thank you "coach"!
Monica Epelman, MD and Oscar Navarro, MD John Caffey, MD 1895 -1978 Dr. Caffey was regarded throughout the world as the father of pediatric radiology.
His classic textbook, Pediatric X-Ray Diagnosis, which was first published in 1945, has become the recognized bible and authority in its field.
The seventh edition of this book was completed several months before his death in 1978.
It has been among the most successful books of its kind in the medical field.
Dr. Caffey was born in Castle Gate, Utah on March 30, 1895 .
It is interesting that he was born in the same year that Roentgen discovered the x-ray.
Dr. Caffey was graduated from University of Michigan Medical School in 1919, following which he served an internship in internal medicine at Barnes Hospital in St. Louis.
He spent three years in Eastern Europe with the American Red Cross and the American Relief Administration, and returned to the United States for additional training in medicine and in pediatrics at the Universities of Michigan and Columbia, respectively.
While in the private practice of pediatrics in New York City at the old Babies Hospital of Columbia University College of Physicians and Surgeons, he become interested in radiology and was charged with developing a department of pediatric radiology in 1929.
He frequently expressed appreciation and admiration for the late Ross Golden, Chairman of Radiology at Columbia Presbyterian Hospital, who allowed him to develop a separate department of diagnostic radiology without undue interference, and who was always available to help and advise him.
Dr. Caffey's keen intelligence and inquiring mind quickly established him as the leader in the fields of pediatric x-ray diagnosis, which recognition became worldwide almost instantaneously with the publication of his book in 1945.
Dr. Caffey received many awards in recognition of his achievements.
Outstanding among these were the Mackenzie Davidson Medical of the British Institute of Radiology in 1956, the Distinguished Service Award of the Columbia Presbyterian Medical Center in 1962, the Outstanding Achievement Award of the University of Michigan in 1965, the Howland Award of the American Pediatric Society in 1967, the Jacobi Award of the American Medical Association in 1972, and the Gold Medal Award of the American College of Radiology in 1975.
He had been a member of the American Journal of Roentgenology.
He was a counselor of The Society for Pediatric Radiology and was an honorary member of the European Society of Pediatric Radiology.
Dr. Caffey's contributions to the pediatric radiologic literature were many.
He was instrumental in directing attention to the fact that a prominent thymic shadow was a sign of good health and not of disease, an observation that literally spelled the end to the practice of thymic irradiation in infancy.
Infantile cortical hyperostosis was described by him and is called "Caffey's Disease."
Dr. Caffey in 1946 first recognized the telltale radiographic changes that characterize the battered child, and his students helped disseminate his teachings about these findings.
It was Dr. Caffey who first recognized and descried the characteristic bony changes in vitamin A poisoning.
He recognized and described the findings associated with prenatal bowing of the skeleton.
In 1963, three years after his retirement from Babies Hospital, he joined the staff of the Children's Hospital of Pittsburgh as associate radiologist and as Visiting Professor of Radiology and Pediatrics at the University of Pittsburgh School of Medicine.
Although Dr. Caffey came to Children's Hospital and the University of Pittsburgh in an emeritus position, he worked daily and on weekends throughout the years he was there.
In Pittsburgh, he made four major new contributions to the medical literature.
He described the entity, "idiopathic familial hyperphosphatasemia."
He recognized and described the earliest radiological changes in Perthes' Disease.
He called attention to the potentially serious effects of shaking children, and used this as a subject of his Jacobi Award lecture.
He described, with the late Dr. Kenny, a hitherto unrecognized form of dwarfism that is now known as the Caffey-Kenny dwarf.
The John Caffey Society, which includes as its members pediatric radiologists who have been intimately associated with Dr. Caffey, or who have been trained by his students, was established in 1961.
This society is now among the most prestigious in the field of radiology.
His book and the society named in his honor will live on as important memorials to this great man.
His greatness was obvious to all who worked with him.
He was warm, kind, stimulating, argumentative, and above all, honest in his approach to medicine and to x-ray diagnoses.
His dedication to the truth was expressed in his abiding interest in the limitations of x-ray signs in pediatric diagnosis and in his interest in normal variation in the growing skeleton.
He was concerned with the written and spoken word and was a skilled semanticist.
His book and his articles are masterpieces of language and construction.
He stimulated and was stimulated and loved by all who had the privilege of working with him.
Radiology and Pediatrics have lost a great man, but they shall ever have been enriched by his presence.
Interstitial lung disease, which is more common in infants than older children, is defined as a rare heterogeneous group of parenchymal lung conditions primarily due to underlying developmental or genetic disorders.
Affected infants typically present with clinical syndromes characterized by dyspnea, tachypnea, crackles, and hypoxemia.
Mainly due to a lack of evidence based information regarding underlying pathogenesis, natural history, imaging findings, and histopathologic features of interstitial lung disease, the understanding of interstitial lung disease in infants has been limited in the past.
However, in recent years, the understanding of interstitial lung disease in infants has been substantially improved primarily due to: 1) advances in imaging technology for better detection; 2) improvement of thoracoscopic techniques for lung biopsy; 3) established pathologic criteria for consistent diagnosis; and 4) development of new classification system based on underlying etiology of the interstitial lung disease.
In fact, several forms of interstitial lung disease in infants that exhibit distinct clinical, radiological, and pathological patterns are currently emerging.
The overarching goal of this article is to review a new classification system, imaging findings, and pathological correlation of interstitial lung disease in infants.
Improved understanding of this often challenging disorder can aid in early and accurate diagnosis, which in turn, will result in improved patient care.
Large Airway Disease in Pediatric Patients: Impact of Advanced Post-processing Techniques Catherine M. Owens, BSc MBBS MRCP FRCR The introduction of multidetector row computed tomography (MDCT) scanners has altered the approach to imaging the pediatric thorax.
In an environment where the rapid acquisition of CT data allows general hospitals to image children instead of referring them to specialist pediatric centers, it is vital that general radiologists have access to protocols appropriate for pediatric applications.
This lecture will focus on the main principles of volumetric CT imaging that apply generically to all MDCT scanners and in particular we describe the reconstruction techniques for imaging the pediatric thorax and the low-dose protocols used in our institution on a 64-slice dual source CT scanner.
Examples of important clinical applications with the impact and added value of post processing are also given.
Neoplasms, by definition, comprise an abnormal uncoordinated proliferation of cells that persists even after the inciting stimulus as ceased.
The resulting mass may be benign or malignant and arise from any tissue that is normally found in the location where the mass develops.
Thus, tumors of the chest may arise from bone, lung, pleura, lymphatics, muscle, etc.
Whether benign or malignant, chest masses may be incidental findings on imaging obtained for other reasons.
This presentation will focus on malignant tumors of the chest, address the imaging characteristics and staging of the most common chest malignancies and discuss characteristics that may aid in distinguishing these lesions from their corresponding benign or infectious counterparts.
Included in this presentation will be the most common chest wall malignancies (Ewing family of tumors and rhabdomyosarcoma), mediastinal malignancies (lymphoma, germ cell tumors, and neurogenic malignancies) and pulmonary primary malignancies (pleuropulmonary blastoma and carcinoid).
The changing appearance of selected tumors in patients treated with new targeted therapies will be introduced.
Lung disease is the most common chronic disease of childhood, but young children cannot perform the breathing maneuvers required for the most commonly used method for assessing lung function, spirometry.
CT provides exquisite structure information about the lung but concerns regarding the long-term consequences of the relatively high radiation dose limit its use particularly in the pediatric population.
Magnetic resonance imaging (MRI) has the potential to provide regional information about the lung without the use of ionizing radiation.
While conventional proton MRI has found widespread clinical application in most organs of the body, MRI of the lung lags behind because the lung is intrinsically difficult to image with MRI.
The strength of the MR signal depends on the physical density of protons in the tissue being imaged and the local environment of the protons.
The lung has a low physical density and thus a low proton density so little MR signal is generated by the lung.
Furthermore, the magnetic susceptibility effects from its many air-tissue interfaces cause what little signal is generated to rapidly decay so that the lung typically appears dark on conventional proton MR images.
A variety of strategies have been developed to overcome the inherent difficulties of MRI of the lung, resulting in recent substantial improvements in image quality.
Additionally by administering an inhaled gaseous contrast agent, such as the hyperpolarized noble gases helium-3 or xenon-129, direct visualization of lung airspaces in an MR image is possible.
A number of unique strategies for evaluating the structure and function of the human lung using hyperpolarized gas MRI have been developed.
Although the level of structure detail possible with lung MRI may never equal that of CT, MRI may nonetheless has the potential to provide clinically useful information and be a sensitive, effort independent test of pediatric lung disease.
For a matter of time, we will focus in this presentation only on the following: Intestinal malrotation A normal visceral situs can be inferred sonographically in relation to the right-sidedness of the superior mesenteric vein, to the retromesenteric location of D3 and to the right iliac position of the ileocecal valve.
Conversely, intestinal malrotation is likely when the 3 aforementioned features are reversed.
In addition, CDU can display the whirlpool pattern in case of midgut volvulus or internal hernia, alleviating the need for preoperative opacification.
The reliability of US in diagnosing intussusception is well documented since the early 1990s.
The value of US in predicting the success or failure of pneumatic reduction and/or bowel necrosis is more debatable, based upon a coexisting bowel occlusion, the presence of interloop fluid, bowel wall changes (intramural air, dilated mural vascular channels), absent blood flow at CDU.
The continuous down-grading of US in comparison to CT, and the opposite conclusions of various series regarding imaging of pediatric appendicitis are based upon different prerequisites and definitions.
Historically and in most USA institutions, sonography reports are either negative (entire normal appendix), positive (abnormal inflamed appendix), or equivocal (non-visualization or partial visualization of appendix).
The equivocal group is then logically investigated by a subsequent abdominal CT.
In Europe, some USA centers, and in our practice, US reporting include 4 groups and take into account ancillary findings: 1. normal appendix (blind-ended, lamellated, compressible, <6 mm in diameter, without peristalsis; 2. appendix not depicted, no secondary signs; 3. appendix not depicted, with one of the following: hyperchoic mesenteric fat, fluid collection, local dilated small bowel loop; 4. appendix inflamed.
Group 3 represents most cases of perforated appendicitis, groups 1 and 2 the negative sonogram.
CT is then indicated only in obese patients and to assess the feasibility of percutaneous interventions.
Inflammatory bowel disease In the recent literature, MR enterography is often preferred to CT enterography.
Small bowel series look prehistoric and US is rarely mentioned.
Sonography however is very valuable both for screening children presenting with abdominal pain, diarrhea, weight loss, or GI bleeding and for following the course of the disease and searching for complication.
Hypervascularization has been proved to parallel the disease activity.
Initially mentioned by Dr. Rita Teele, the interest of US for differentiating high-intermediate/low varieties of imperforated anus has been re-emphasized more recently.
A perineal rectal cul de sac distance of 15 mm is quoted as the significant cut-off value.
US can also display rectourinary fistulae outlined by air.
Update on MDCT and MRI of Hepatobiliary Disease in Children: What's New Lisa H. Lowe, MD A variety of disorders may affect the pediatric liver.
Recent advances in histopathological knowledge and imaging techniques have led to important changes that radiologists must be aware of in order to allow for an accurate limited differential, and in some cases, specific, diagnosis.
This presentation will focus on recent developments that have lead to a better understanding of the embryopathogenesis for fibropolycystic liver diseases (including choledochal cysts and Caroli disease), histopathological findings that have led to new classification systems for of pediatric vascular anomalies, technological advances and contrast agents in magnetic resonance imaging that are useful to characterize and limit the differential diagnosis of hepatic masses.
Diagnostic Errors in Pediatric Abdominal Imaging: Diagnostic Pearls and Pitfalls George A. Taylor, MD This presentation reviews the types of diagnostic errors in abdominal imaging occurring over a 13-year period in an academic pediatric radiology practice.
Radiologists engage in two interrelated processes when interpreting imaging studies: perception and analysis.
Failures in perception (failure to identify an important finding) are a common source of diagnostic error in pediatric imaging, while failures in the analytic portion of the process (over-or faulty interpretation of a finding) are not as common.
Under-interpretation of findings can be related to a number of perceptual and visual phenomena including visual isolation where attention is selectively focused on a main area of the image while less or no attention is given to secondary areas, and satisfaction of search which occurs when additional lesions remain undetected after detection of an initial lesion.
Many analytic errors are the result of commonly used heuristics or shortcuts in reasoning.
These include the availability heuristic in which likelihoods are based on memory of a similar case, the framing effect in which a different diagnosis is reached based on how the information is presented, and the anchoring heuristic in which the initial impression is difficult to change, despite conflicting new information.
Another recognized pitfall is blind obedience, in which a diagnostician stops thinking when confronted by authority.
This authority can be human or technical (reliance on a laboratory value).
Finally, diagnostic errors can result from an attitude of overconfidence.
Examples of these heuristics and strategies to minimize cognitive errors will be discussed.
Marta Hernanz-Schulman, MD, FAAP, FACR This session will consider abdominal masses that present in the neonatal period, spanning developmental, inflammatory and neoplastic conditions.
Time constraints do not allow an exhaustive list or description, but the more important or frequent lesions are discussed.
The presentation is subdivided by systems.
The renal section discusses various conditions presenting with hydronephrosis, such as ureteropelvic junction obstruction and duplication anomalies, followed by autosomal recessive polycystic kidney disease and multicystic dysplastic kidney, cystic entities commonly presenting in the perinatal period.
Neoplastic renal entities include lesions with benign behavior, such as ossifying renal tumor of infancy, with the discussion extending to entities with very poor prognosis such as clear cell sarcoma and rhabdoid tumor, while discussing the congenital mesoblastic nephroma, its histologic subtypes and the differences in their presentation, imaging findings and clinical behavior.
Suprarenal lesions include the adrenal hemorrhage, congenital neuroblastoma and subdiaphragmatic sequestration.
Hepatic lesions include developmental anomalies that present as mass lesions, such as choledochal cysts, vascular lesions such as congenital and infantile hemangiomas, and neoplastic lesions such as the mesenchymal hamartoma and hepatoblastoma.
Differences in clinical presentation, imaging characteristics and behavior of the lesions are discussed.
The section on pancreatic lesions discusses pancreatic cysts and pancreaticoblastoma.
GI tract and mesenteric lesions include duplication cysts, lymphangioma, and meconium pseudocyst, and their relationship to bowel obstruction and persistent perforation.
Ovarian cysts can present as large masses in neonatal girls, and should be high in the differential diagnosis of large masses encountered in female infants; the imaging characteristics of simple and complicated cysts are described, as well as their course and potential complications.
Pediatric Procedures: From Imaging to Intervention The Spectrum of Vascular Anomalies in Pediatric Patients: Multimodality Imaging Evaluation and Current Treatment Patricia E. Burrows, MD Vascular anomalies are categorized into two main groups, vascular tumors and vascular malformations.
Genetic and molecular regulation of vascular genesis of angiogenesis, and mutations responsible for some of the vascular malformations, have been delineated.
In order to implement future targeted treatment of vascular lesions, accurate diagnosis is important.
Imaging modalities that are effective in distinguishing the various types of vascular anomalies and demonstrating the extent include ultrasonography with Doppler interrogation, MRI and various forms of MR vascular flow imaging, conventional angiography and venography.
Techniques used to image lymphatic channel anomalies, conventional lymphangiography, lymphoscintigraphy and infrared fluorescent lymphangiography.
In this presentation, common forms of vascular anomalies will be described and rare or recently recognized anomalies will be mentioned.
Current treatment of the different forms of vascular anomalies will also be discussed, including pharmacotherapy using beta blockers, angiogenesis inhibitors and mTOR inhibitors.
Endovascular techniques used in treating vascular malformations, including embolization and sclerotherapy will be presented.
Pediatric vascular disease is extremely varied, with a wide range of conditions requiring diagnostic or therapeutic intervention.
Technological improvements in non-invasive imaging modalities such as MRI and CT have reduced the need for diagnostic angiography; however, with advances in interventional techniques, arteriography in the pediatric patient is now often performed for therapeutic reasons.
Pediatric arteriography presents unique issues and challenges.
Tremendous variability in patient size and physical maturity limits the ability to standardize technical aspects of performing arteriography.
In addition, radiation protection, sedation/anesthetic support, monitoring of fluid balance, and maintaining patient warmth must be considered.
A regimented protocol for assessment of the pediatric patient must be followed, with review of the indications for the study requested, and review of patient-specific issues such as coagulation profile, concurrent medical disease, patient weight, and anesthetic concerns.
Appropriate patient monitoring is imperative to ensure patient safety.
Vascular access can be quite challenging.
Ultrasound and micropuncture access techniques have tremendously improved successful access while reducing associated complications.
The smallest catheter that can accomplish procedure objectives should be used.
For most diagnostic cases, 4 French systems can be used for children>10 kilograms, while 3 French catheters are preferred in those <10 kg.
Intraprocedural heparinization (75-100 IU/kg) is also more often used, especially in children weighing less than 10-15 kg.
Rates and volumes of contrast injected for pediatric arteriography are not standardized, as in adult patients.
In general, contrast dose should be limited to 6-8 mL/kg, and 4-5 mL/kg in premature infants and neonates.
All these new technique are less invasive, improve patients' outcomes and reduce morbidity.
They are also cost-effective as patients are discharged home earlier and recover faster from the intervention.
The future holds promising new technologies such as High-Intensity Focused Ultrasound (non-invasive method of thermal ablation) and nanoparticles for drug delivery.
Pediatric interventional radiology will continue to be an essential part of these minimally invasive therapies.
Musculoskeletal Imaging: From Planning to Performance Kirsten Ecklund, MD The purpose of this talk is to review advanced MR imaging techniques currently being used in the evaluation of pediatric musculoskeletal tumors.
The goals of these techniques include improved image resolution and quality, lesion tissue characterization, and increased acquisition speed.
Diffusionweighted (DW) and perfusion imaging will be emphasized; however, whole body, metallic artifact mitigation, and volumetric sequences will also be discussed.
DW MRI is based upon the Brownian motion of water within extra and intra-cellular spaces which depends upon tissue cellularity.
DWI can aid in the differentiation of benign from malignant lesions, which generally have restricted diffusion.
There is even greater potential for DWI in the assessment of tumor response to therapy.
The apparent diffusion coefficient (ADC) maps are critical to accurate interpretation of diffusion sequences.
ADC maps distinguish between restricted diffusion and T2 effect, both of which appear bright on DWI.
Both qualitative and quantitative tissue assessments can be made with DWI.
Challenges for DWI in the pediatric musculoskeleton include susceptibility artifacts from bone, motion vulnerability, and geometric distortion at larger fields of view.
Our current protocols and parameters for DWI will be presented.
contrast-enhanced (DCE) MR using one of a variety of vendor specific sequences.
Qualitative and quantitative assessments of inflow and distribution of contrast have been shown to help differentiate between benign and malignant lesions and to evaluate drug efficacy during therapy.
This technique is especially promising in those patients undergoing antivascular and antiangiogenic therapy.
Tal Laor, MD Congenital abnormalities of the musculoskeletal system can result in alterations of limb size, configuration, and/or segmentation.
These disorders often affect both the osteocartilaginous skeleton as well as the surrounding soft tissues and can be localized or diffuse.
In this session, we will focus on the imaging features of several congenital abnormalities that result in a small or short limb, in altered configuration of a limb, or in abnormal segmentation.
Deformities of both upper and lower limbs will be examined.
Like congenital abnormalities, developmental disorders of the pediatric musculoskeletal system can be limited to a single area or can affect numerous sites within the body.
For example, neonatal brachial plexopathy is a localized disorder that produces characteristic musculoskeletal alterations about the shoulder girdle and elbow of affected children.
The alterations of morphology and function of the shoulder develop over time with growth of the child and change in response to a variety of therapies.
We will review the features of developmental anomalies of the pediatric musculoskeletal system and evaluate the role that imaging plays in the initial evaluation and in the subsequent assessment of these children during treatment.
Multimodality Imaging of Skeletal Trauma in Children: Using All of the Tools Peter J. Strouse, MD Skeletal trauma is a common indication for imaging throughout the pediatric age range.
Newborns may suffer birth trauma.
Infants and toddlers may be subject to abusive injury.
Children of all ages may suffer accidental injury.
Older children and adolescents are increasing hurt in sporting activity and vehicular accidents.
Fracture patterns vary with maturation of the child.
Interference with normal growth is a potential complication.
Imaging of skeletal trauma begins with radiography.
Proper anatomic and age specific radiographic technique assures optimal diagnostic yield.
Radiography suffices in most cases to diagnose fracture or confirm normalcy.
"Clinical correlation" aids in diagnosis.
Ultrasound, CT, MRI and nuclear medicine may play a role in specific instances where plain radiographs are non-diagnostic or to better delineate certain fractures.
Arthrography and conventional tomography have occasionally been used in the past and tomosynthesis may prove useful.
Follow-up radiographs may be useful for diagnosis or confirmation of some fractures.
This presentation will focus on the imaging of acute skeletal injury.
Technique and approach for plain radiography will be emphasized.
Specific indications and roles for ancillary imaging techniques will be defined and illustrated with representative cases.
Although classically thought of as a disease of adulthood, stroke is much more common in the pediatric population than was once appreciated.
This may be due to many factors, not the least of which is increased awareness due to the presence of subspecialty stroke teams now fairly commonplace in many children's hospitals, and the fairly recent advent of more advanced imaging technique such as diffusion-weighted imaging (DWI) and its routine use in imaging the central nervous system (CNS) in the child and adolescent.
Causes of stroke in children can be protean, and range from idiopathic on one end of the spectrum, to traumatic on the other, with many causes in between, many of which may not be intuitive to the clinician without further research.
Moyamoya disease and its many causes, such as sickle cell disease (SCD), trisomy 21 and neurofibromatosis type I (NF I) can all lead to stroke in children, as can congenital clotting deficiencies such as Factor V Leiden deficiency and congenital cardiac lesions with their resultant shunting of blood between the left and right cardiac circulations.
Although usually arterial in nature, strokes may arise from the venous system in clinical scenario of venous thrombosis with resultant venous infarctions.
Factors contributing to venous thrombosis in children and adolescents can be due to dehydration (especially in the very young), severe iron deficiency anemia, inflammatory bowel disease and exogenous hormone ingestion such as is seen with oral contraceptives (OCP) in young women.
Advanced Imaging Techniques for Neuroimaging in Pediatric Patients: Where Are We Now?
Blaise V. Jones, MD The past decade has seen a large number of advanced imaging techniques introduced to the clinical armamentarium of the pediatric radiologist.
From the development of multidetector CT scanners that can obtain whole head diagnostic studies in less than 2 s to the routine use of 3 T MR imaging, technical advances have dramatically changed our ability to diagnose and manage neurological disorders in children.
However, all of these advances are not of equal clinical utility, and it is imperative that the pediatric radiologist be well versed in their judicious and appropriate application.
This presentation will discuss the effective use of volume CT scanning, CTA, SWI, ASL, fMR, pMR, and other advanced imaging techniques in the diagnosis of neurological disorders presenting in childhood.
At the conclusion of the presentation the attendee will have a better understanding of how to ideally apply these technologies in practice.
A Spectrum of Abnormality in Pediatric Neck: Practical Imaging Choices and Interpretation Caroline D. Robson, MBChB Learning Objectives: 1.
Become familiar with an optimized imaging approach for head and neck infections 2.
Recognize the complications of head and neck infections 3.
Recognize the utility and interpretation of imaging for neck masses This talk will cover the imaging approach and interpretation of findings in head and neck infection and neck masses.
Infection includes acute complicated sinusitis, coalescent mastoiditis, neck infection and local and intracranial complications.
Optimized imaging protocols and image interpretation for neck masses will also be discussed and illustrated.
Acute complicated sinusitis is diagnosed when acute sinusitis is accompanied by orbital symptoms (e.g.
proptosis) and/or mental status changes, seizures or other neurological findings.
Coalescent mastoiditis is diagnosed when otomastoiditis is accompanied by tenderness and/or swelling over the mastoid process.
CT and MR provide complementary information.
CT is obtained with contrast.
MR sequences include fatsuppressed T2, T1, diffusion, and fat-suppressed contrastenhanced T1 weighted images with MR venography.
Intracranial complications include epidural abscess, subdural empyema, meningitis, cerebritis, brain abscess, venous thrombosis and venous infarction.
The limitations and usefulness of CT in the diagnosis of neck abscess will be illustrated.
The imaging approach to masses depends on patient age, and the size and location of the mass.
US, CT, MR, and nuclear medicine studies provide complementary information.
As for infection, optimized imaging approaches and key imaging features for various masses will be discussed.
Embryology and Diagnostic Approach in Spinal Dysraphism L. Santiago Medina, MD, MPH and Esperanza Pacheco-Jacome, MD Congenital anomalies of the spine are malformations that can be confusing due to the complexity of their embryology, and to the sometimes unclear classifications and terminology.
The purpose of this review is to give a clear and basic understanding of the different stages of the embryological development of the spinal cord, starting with the bilaminar disc in the first week of gestation.
During the second week, the formation of a trilaminar disc (gastrulation), the notochord, and the formation of the neural tube or neurulation.
Also, a review of the development of the distal cord: conus medullaris, filum terminale, ventriculus terminalis, by a different mechanism, canalization and retrogressive differentiation.
Beside the embryological review, a case correlation will be presented using MR imaging to demonstrate these malformations.
Open spina bifida entities include meningocele and myelomeningicele.
Closed or occult spinal dysraphism (OSD) is characterized by a spinal anomaly covered with skin and hence with no exposed neural tissue.
OSD spectrum includes dorsal dermal sinus, thickened filum terminale, diastematomyelia, caudal regression syndrome, intradural lipoma, lipomyelocele, lipomyelomeningocele, anterior spinal meningocele and other forms of myelodysplasia.
Several studies have shown that MRI and ultrasound have better overall diagnostic performances (i.e., sensitivity and specificity) than plain radiographs for detection of occult spinal dysraphism.
For H1N1, most patients had mild illness but a small percentage required mechanical ventilation and ICU admission.
The high risk groups include children <5 years old and those with chronic medical conditions in particular neurodevelopmental impairment.
Pediatric mortality was 7.5% of all deaths associated with the pandemic reported in the U.S.
In both conditions, the most prominent radiographic and CT features were airspace disease including ground glass opacities (GGO) and consolidation, commonly with multi-focal and bilateral involvement.
Pleural effusion, adenopathy and cavities were absent.
In some patients with viral infection, respiratory symptoms may be mild but are complicated by neurological manifestations.
A brief review of MRI features in H1N1 related encephalopathy including acute necrotizing encephalopathy (ANE) will be given.
Bernard F. Laya, DO Tuberculosis (TB) is a worldwide major public health problem with one-third of the world's population being infected.
It is a leading cause of death and disability from infection worldwide.
Children are amongst the most vulnerable group because of their immature immune status.
A child usually gets TB infection after being exposed to a sputum-positive adult.
Depending on many factors, the infection can lead to latency or TB disease.
It can affect virtually any organ in the body and can be devastating if left untreated.
TB in children remains a diagnostic challenge.
In addition to history of TB exposure, signs and symptoms, laboratory and microbiologic tests, medical imaging remains a valuable tool in its diagnosis.
Although findings are nonspecific, the radiograph is the most commonly ordered initial imaging tool for screening and diagnosis of pulmonary and musculoskeletal involvement.
Computed tomography and magnetic resonance imaging offer more detailed assessment especially in cranial and abdominal involvement.
Medical imaging is also utilized to follow up patients during or after anti-TB treatment.
Knowledge of the common imaging patterns, pitfalls and dilemma are very important in establishing the diagnosis of TB in children.
The pathophysiology of pediatric TB will be discussed as it correlates with imaging findings.
The wide spectrum of imaging manifestations in various modalities will be presented.
Imaging updates along with pitfalls and dilemma in the interpretation will also be discussed.
TB can affect almost every organ system but the author will present cases that are more commonly encountered.
and concurrent CT/ PET-CT (k00.33) panels.
There were more indeterminate nodule predictions by PET-CT (n038 of 75; 51%) and concurrent CT/PET-CT (n 023; 31%) than by CT alone (n012; 16%).
The overall accuracy of CT alone was 71%, PET-CT alone 45% and concurrent review 60%.
Worst case sensitivity and specificity were 85% and 44% for CT alone, 60% and 19% for PET-CT alone, and 67% and 48% for concurrent CT/PET-CT.
Conclusions: PET-CT assessment of pulmonary nodules in children is feasible but limited by non-diagnostic quality CT images and atelectasis caused by sedation.
Subjective assessment of nodules by PET-CT does not appear to improve the ability to distinguish benign from malignant histology in children with solid malignancies.
Semi-quantitative nodule assessment using the standardized uptake value may improve the performance of PET-CT in this setting and will be investigated in the future.
Purpose or Case Report: NEC is the most common lifethreatening medical/surgical emergency of the gastrointestinal (GI) system in neonates, with an incidence up to 10% in infants weighing <1500 g. With advances in treatment of NEC, increased survival rates result in rise in post-NEC GI complications such as feeding intolerance.
Development of post-NEC bowel strictures results from healing of involved bowel and can result in bowel obstruction.
It has been routine to study the bowel of infants after medical treatment for NEC by contrast enema and small bowel follow-through prior to initiating feeding.
However, in order to "Image Gently" we are attempting to decrease the radiation exposure to these patients.
We postulate that in patients with no abnormal bowel dilation prior to initiation of feeds, the incidence of colonic stricture would be so low that routine enemas would be unnecessary and could be eliminated from the workup.
recorded as present or absent in 6 anatomic abdominal regions defined as: 1 and 2-from the dome of the diaphragm to top of L2 to the right and left of midline, respectively; 3 and 4-from top of L2 to the iliac crest to the right and left of midline, respectively; 5-from the iliac crest to the top of the sacrosciatic notch; 6-below the top of the sacrosciatic notch.
We assessed the frequency of findings in each region and how often findings in regions 5 and 6 were associated with findings in regions 1-4.
95% confidence intervals were calculated.
Results: The fewest pertinent findings were present in region 6 in 10.2% (51/501) (95% CI: 7.7-13.1%) of radiographs.
Findings included: abnormal bowel 6% (n031), bowel gas paucity 1.4% (n07), pneumatosis 0.4% (n02), inguinal hernia 0.8% (n04) and osseous abnormalities 1.2% (n06).
Pertinent findings were present in region 5 in 67.7% (339/501) (95% CI: 63.4-71.8%).
Findings included: abnormal bowel 43.7% (n0 219), bowel gas paucity gas 19.6% (n098), pneumatosis 1.6% (n08), free air 0.2% (n01), and abnormal bowel with pneumatosis 2.6% (n013).
Among 51 patients with an abnormality in region 6, 49 (96.1%) also had an abnormality within at least one of regions 1 through 4.
Among the 342 patients with an abnormality in region 5 or 6, 338 (98.3%) also had an abnormality within at least one of regions 1 through 4.
Catheter/tube tips were located in region 5 in 6.8% (n034) and region 6 in 1.4% (n07) of radiographs, respectively.
Pneumatosis was present most frequently in regions 3 (5.8%), 4 (4.0%), and 5 (4.2%).
Free air was present most frequently in regions 1 (1.6%), regions 2 and 3 (0.6% each).
Conclusions: Our preliminary data suggest that pertinent findings on neonatal portable abdominal radiographs are rarely isolated to the pelvis, implying that gonadal shielding of regions 5 and 6 should not compromise diagnostic accuracy.
Purpose or Case Report: The purpose of this study was to determine the sensitivity, specificity, and positive and negative predictive value of ultrasound in diagnosing appendicitis when the appendix is visualized, using three diagnostic categories: positive, negative, and equivocal.
The 3-category diagnostic accuracies for appendiceal diameter and radiologist impression were compared.
Methods & Materials: A retrospective study was performed evaluating all right lower quadrant ultrasound reports dictated over a 5-month period.
Included studies were interpreted as positive, negative, or equivocal for appendicitis.
Report impressions that did not specify one of these categories and studies where the appendix was not seen were excluded.
The pathologic diagnosis of appendicitis was considered the gold standard for a positive diagnosis.
Because virtually all pediatric surgical cases in the region are referred to our hospital, it was assumed that the patient did not have appendicitis if surgery was not performed.
Logistic modeling using appendiceal diameter as the independent variable established cutoff diameters of ≤6 mm0negative, >8 mm0positive, and 6<x≤ 8 mm0equivocal.
Sensitivity, specificity, and positive and negative predictive values were calculated for both methods.
Results: Of the 571 patients imaged during the study period, 255 (45%) met the inclusion criteria; 34 (6%) reports did not categorize the impression and in 282 (49%) studies the appendix was not seen.
Surgery was performed on 122 (48%) subjects, of which 101 (83%) had a pathologic diagnosis of acute appendicitis.
Using the radiologist impression, there were 68 true positives (TP), 2 false positives (FP), 8 false negatives (FN), and 150 true negatives (TN), for a sensitivity of 97% (95% CI090-100%), specificity of 95% (CI090-98%), PPV of 89% (CI080-95%), and NPV of 99% (CI095-100%).
There were 27 (11%) cases reported as equivocal; of these, 7 (26%) had a pathologic diagnosis of appendicitis.
Using appendiceal diameter, there were 46 TP, 6 FP, 2 FN, and 161 TN, for a sensitivity of 88% (CI077-96%), specificity of 99% (CI096-100%), PPV of 96% (CI0 86-99%), and NPV of 96% (CI092-99%).
There were 41 (16%) cases categorized as equivocal; of these, 25 (61%) had a pathologic diagnosis of appendicitis.
Conclusions: The use of diagnostic categories enables high PPV and NPV with a relatively low number of equivocal cases.
Using appendiceal diameter alone results in higher PPV than that of the radiologist impression, but also yields a higher number of equivocal cases.
Purpose or Case Report: Intussusception is an obstructive condition that a segment of the bowel invaginates into adjacent bowel and is an urgency that needs to be reduced as soon as possible to prevent bowel ischemia or necrosis.
When it occurs in the infants and young child, it usually develops idiopathically and reduced by the technique such as air enema under the fluoroscopic guidance or saline enema under the ultrasound guidance.
The success rate of the non-surgical reduction has reported over 95%.
The pneumatic reduction is to introduce air into the patients' rectum via a ballooned Foley catheter by hand pump.
A mercury manometer connecting with the Foley catheter calibrates the pressure of insufflation, and the recommended maximum pressure is 120 mmHg.
The hydrostatic reduction is to drip saline from a bag at the height of 3 ft that may be different according to the institute's guidelines.
Until recently, in both techniques, the precise measurement of the intraluminal pressure was not possible.
With the purpose of intraluminal pressure monitoring, we introduced a specific digital pressure gauge that can measure both pneumatic and hydrostatic pressure in various units and can give the collected data to our workstation.
With the data, we purpose to make the curve of intraluminal pressure during the procedure and to compare the pattern and difference in both techniques of reduction, which permits suggesting the better technique for reducing the intussusception.
From February 2011, we performed the intraluminal pressure monitoring in 8 children; 4 cases of pneumatic reduction and 4 cases hydrostatic reduction.
The results indicated that the curves were similar in both techniques.
However, the hydrostatic reduction tended to need longer time and higher pressure difference than the pneumatic reduction.
We assumed that a higher flow rate and more fluctuating introduction of pressure in the pneumatic reduction made the difference between both methods.
Purpose or Case Report: Anecdotal evidence suggests that some pediatric brain tumors, particularly pilocytic astrocytomas, demonstrate greater enhancement on delayed post-contrast T1 weighted MR imaging compared to early post-contrast imaging.
Our goal was to compare immediate post-contrast imaging with delayed post-contrast imaging in pediatric brain tumors.
Methods & Materials: The research protocol was granted exempt status by the institutional review board (COMIRB# 10-1354).
Between November 2010 and March 2011, all patients for whom an MRI of the brain was ordered to follow-up known brain tumor or to evaluate new brain tumor underwent a modified protocol that included both immediate and 10 min delay post-gadolinium images.
The modified protocol sequence order was DWI, T1 MPRAGE, contrast injection, immediate post-contrast T1 MPRAGE, T2, FLAIR, 10 min delayed post-contrast T1 MPRAGE.
This sequence allowed both immediate and delayed post-contrast imaging to be performed without scanner downtime.
A total of 67 unique patients met inclusion criteria of an enhancing primary brain tumor with the delayed imaging performed within a window of 6-19 min.
These studies were evaluated independently by 2 pediatric neuroradiologists (NS and LF) and a radiology resident (JM) to compare the degree of lesion conspicuity and edge discrimination, and to determine an overall preference.
In cases of discrepancy between reviewers, final grading was determined by group consensus.
Results: Delayed post-contrast images were preferred in 55/67 tumors (82%), immediate post-contrast images were not preferred in any case, and 12/67 cases were graded as equivalent (18%).
Notably, delayed imaging was preferred for 19/20 pilocytic astrocytomas (95%).
In 9/67 cases (13%), the improvement in conspicuity and edge discrimination on delayed imaging was felt to have a potential impact on clinical management, e.g.
by extending resectable or treatable tumor margin.
Conclusions: Delayed post-contrast imaging can be performed easily by reordering existing MRI protocols, and no scanner downtime is required for this protocol change.
Our results indicate that pediatric brain tumors (particularly pilocytic astrocytomas) often enhance more conspicuously on delayed imaging, that no diagnostic confidence is lost on delayed imaging, and that clinical management may be more accurately guided.
3-directions) were retrospectively reviewed, after IRB approval.
Both sporadic (n07) and NF-1 associated optic gliomas (n06) were included.
Tumors confined to intraconal optic nerve were excluded due to technical limitation of EPI DWI within the bony orbit.
Apparent diffusion coefficient (ADC) of the solid portions of the tumor was measured using the region of interest method both at baseline and on follow-up MRI.
Results: For the 13 patients, 7 required treatment (surgery and/or chemotherapy) for symptomatic tumor progression (5 sporadic OPG and 2 cases of NF-1).
Median follow-up interval was 3.1 years (range: 7 months to 10.5 years, SD 2.7 years).
There was no difference in ADC values between sporadic and NF-1 associated OPG.
OPG that required therapy had significantly higher baseline mean ADC (1589 x 10-6 mm2/s, SD 0.22), whereas OPG with low mean ADC (1255 x 10-6 mm2/s, SD 0.18) remained clinically asymptomatic or stable (Stratified Mann-Whitney test, p00.0002).
An increase in mean ADC of 100 x 10-6 mm2/s doubled the odds the patient will require treatment (OR02.16, 95% CI: 1.51-3.10, p<0.0001).
Enhancement patterns did not predict tumor behavior or NF-1 status (Fisher's exact test, p00.192).
Conclusions: Significantly higher mean ADC was seen in OPG that required therapy for tumor progression.
MRI may be a useful prognostic tool in further defining a subset clinically significant sporadic or NF-1 associated OPG.
Purpose or Case Report: Fast brain MRI imaging has recently been introduced for assessing brain ventricles in patients with ventriculo-peritoneal shunts (VPS).
However, there is minimal literature on how this method could be beneficial for other indications.
Compared to CT, this method reduces lifetime cumulative radiation dose and has multiplanar capabilities.
It is much faster than full brain MRI, reducing exam costs and potentially reducing the need for sedation.
However, fast brain MRI has lower resolution than a standard brain MRI and cannot detect hemorrhage.
The goal of our study is to retrospectively assess additional clinical applications for fast brain MRI.
Methods & Materials: Patients who had undergone fast brain MRI were found in XenoBase, a database which references deidentified electronic medical records at our institution.
XenoBase was then used to identify subgroups by diagnosis.
One of the most common diagnoses was arachnoid cyst.
After IRB approval, patients with fast brain MRI and arachnoid cyst were identified so that their fast brain MRI could be compared to any full brain MRI and to clinical outcomes.
Fast brain MRI findings were excluded if there was not a full brain MRI within 1 year.
Agreement with full brain MRI, and documented effect on clinical management were coded for statistical review.
Results: Between 2008 and 2011, there were 14 arachnoid cyst patients with 16 fast brain MRI exams.
In 12 members of this group, fast brain MRI was used alone or in comparison with a full brain MRI in the decision to follow or discharge the patient.
2 exams failed to report a pituitary cyst and mass effect which were reported on prior exams.
6 patients had no complications in the year following their fast brain MRI.
The most common complication was headache (4 cases).
Conclusions: Fast brain MRI reduces radiation exposure, time, cost, and the need for sedation in VPS patients.
These benefits may cautiously be extended to other patient groups also requiring routine imaging, such as those with arachnoid cysts.
Disclosure: The authors disclose that they will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Susceptibility-weighted imaging (SWI) is a new technique that exploits susceptibility differences in different tissues, to provide a different type of tissue contrast.
SWI is a gradient echo sequence utilizing both phase and magnitude data to achieve exquisite sensitivity to tissue magnetic susceptibility effects.
It is extremely sensitive to even minute amounts of paramagnetic substances and therefore for detection of blood products (hemosiderin, ferritin), deoxygenated blood, calcium, iron, and small vein depiction.
It is particularly suited for vascular imaging, especially in cerebral ischemia.
SWI has been shown to be more sensitive in detecting cerebral microbleeds than is conventional T2*-weighted gradient-recalled echo imaging (GRE) partly because of its inherent sensitivity, increased spatial resolution and the thinner slices acquired.
Recent studies have shown that SWI detected more microbleeds in more patients, irrespective of their location and that SWI may be more sensitive in detecting traumatic lesions than CT or MRI.
Methods & Materials: Between January 2011 and October 2011 we have performed SWI on 75 pediatric patients (from 1 day to 15 yrs old) affected with different pathologies.
MRI was performed using either a 3-or 1.5-T magnet (Achieva ; Philips Healthcare, Best, The Netherlands).
Among these patients 62 also received a classic GRE sequence.
Results: Our experience confirms a greater sensibility of SWI than GRE in detecting microbleedings and larger bleedings.
SWI is much more helpful in patients younger than 1 year since it can distinguish hemorrhagic and non hemorrhagic punctuate white matter lesions detected as hyperintensities on T1-W images.
It is more accurate in distinguishing petechial hemorrhages and ischemic foci in patients with germinal matrix hemorrhages.
SWI can provide important diagnostic information not only in vascular pathologies but also in tumors showing the altered tumor microvascularity, the degree of intratumoral necrosis and the presence of subtle defects of the BBB within the surrounding parenchymal tissue.
The technique also allows the detection of possible bleedings in infectious lesions.
Conclusions: SWI is a valuable technique in the evaluation of a wide variety of intracranial pathologies in the pediatric population and its use is advisable on a routine basis.
Purpose or Case Report: A common problem with pediatric MR imaging is motion.
Diffusion-weighted imaging (DWI) is highly sensitive to the effects of both rigid-body motion and brain pulsation.
The resulting motion artifacts can manifest in several ways, and include blurring (motion between volumes), slice 'drop-outs' (motion during the diffusion encoding), and aliasing (motion during the parallel imaging/ghost calibration scan).
In order to improve image quality in pediatric DWI and avoid the potential for repeat scans, we have implemented our GRAPPA-accelerated diffusion tensor (DT)-EPI sequence on over 1,600 pediatric patients at our institution, and have developed tailored reconstruction software to correct for large motion and reduce the need for general anesthesia (GA).
Methods & Materials: GRAPPA-accelerated EPI DTI data was collected on 100 patients at 1.5 T and 1,500 patients at 3 T between the ages of 1 month and 18 years old.
With these data, we investigate the use of a method to select the best GRAPPA and ghost calibration weights; 3D rigid-body realignment with importance weighting; a new volume rejection criterion for large motion; together with other procedures to lower image noise and reduce phase artifacts.
All image reconstruction was performed using compiled threaded MATLAB code on the vendor's (GE) multiprocessor reconstruction hardware.
Results: Using the maximum motion detected from the 3D realignment procedure on the 1,600 DTI exams, 51% of patients did not move (either through co-operation or GA, denoted here by <1 mm of motion); 28% of patients moved between 1-3 mm; 13% between 3-5 mm; 6% between 5-10 mm; and 2% between 10-18 mm.
Except for 3 patients in total, the motion correction steps employed here were extremely robust in correcting for motion.
In addition, the motion correction steps employed here do not blur or negatively affect the data on non-moving patients.
Conclusions: Our proposed integrated reconstruction scheme suggests that correcting for different types of motion at different stages of the DTI acquisition is critical to provide scans of diagnostic quality.
Apart from the very rare instance (0.2%) where the patient moved continuously an extensively throughout the scan, our motion correction procedures employed here are extremely robust.
Using this scheme, we show that highly diagnostic motion-corrected DTI data can routinely be read on our patient database within 10 min.
.
Participating sites followed mutually agreed upon vendor-specific DTI protocols used for neonates in clinical practice: SS-EPI; b 0700;~2 mm3 voxel; 30-32 directions; TE~84 msec; 1 acquisition; parallel imaging; multi-channel (8-32) head coil.
Tensor data was analyzed at one site using internally developed software in IDL.
B0 and eddy current distortion were assessed by analyzing grid line distortion on ACR slice 5 ; deviations from linearity were measured in the phase encoding direction.
ACR slice 7 was used to assess SNR of the center and periphery of the b00 image, and fractional anisotropy (FA) of the phantom, which should approach 0.
Deviations from specified sequence parameters were recorded.
Results: Both Siemens scanners had some image degradation due to vibration susequently corrected by the vendor.
Nonlinear gridline distortion was highest on the GE followed by Philips but was corrected with the use of 2nd order shimming.
Eddy current distortion was consistently smaller than acquisition voxel size after image registration.
TE varied from 74-92 msec; acquisition bandwidth range was 1563-2928 Hz/pixel.
The 2 mm slice thickness was problematic on clinical GE scanners resulting in non-isotropic voxels.
Across vendors, SNR was consistently higher in the image periphery than center by a factor of 1.
30-0.45 ).
The greatest difference was showed in patients with FHC.
No association was found between the value of LDL-C and IMT.
The 62.5% received drug treatment at the time of evaluation.
In DBT-1 a no association was found between the IMT and the levels of HbA1c and lipids.
And in the OB group found no association between IMT and BMI Z score.
FHC 31%, 8% of DBT-1 and 6% of OB showed atherosclerotic plaques and intima irregularities.
Conclusions: The IMT ultrasonography was able to demonstrate that patients with chronic diseases, with increased cardiovascular risk in adulthood, showed early alterations of carotid intima media in children.
This allows clinical applications of preventive and therapeutic guidance.
A benchmark for precision of flow assessment was determined from differences between aortic and pulmonary arterial flows, which should agree.
The precision of flow when utilizing a slow flow structure (vein) and a fast flow structure (artery) was then determined, serving as an indicator of the precision of slow flow measurements.
This was done in pairs for the upper body (SVC flow vs difference between ascending and descending aortic flow), lower body (IVC flow vs descending aortic flow) & total body (SVC flow+IVC flow vs ascending aortic flow).
Paired t-tests were used to identify significant differences in the respective arterial and venous flow rates.
With an f-test, the relative precision of the arterial flow (determined from aortic and pulmonary arterial flow) was compared against the precision of agreement between upper, lower, and total body flows, each of which has a slow flow venous component, thus assessing precision of venous flow measurement.
Results: Arterial & venous flow measurements were strongly correlated for the upper body (r00.89), lower body (r00.96) and total body flow (r00.97); net aortic and net pulmonary trunk flow rates were also tightly correlated (r00.97).
Moreover, there was no significant difference in the precision of arterial & venous flow measurements (t-test, p00.31).
Lower venc (200 cm/s or lower) improves the accuracy and precision associated with low velocity venous flow, a finding that tended to but it did not reach statistical significance.
Conclusions: With 4-D PC MRI, the accuracy and precision of venous flow quantification are comparable to arterial flow quantification, though venc settings may improve venous flow assessment.
Disclosure: Dr. Tariq has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
The spatial resolution requirements for imaging tiny target vessels in neonates have so far precluded its use of CEMRA in this patient population.
The purpose of our study, therefore, was to explore the potential of high resolution CEMRA at 3.0 T in neonates with suspected congenital vascular anomalies Methods & Materials: 16 neonates (mean age 9.4 days, range 1-28 days; mean weight 2.7 kg, range 1.5 kg-4.2 kg) underwent CEMRA at 3.0 T. CEMRA was performed under controlled apnea on a 32-channel 3.0 T system using either a 12-channel head-neck coil (9 pts) or a 15-channel adult knee coil (7 pts).
Gd contrast delivery was calculated based on a total dose of 0.2 mmol /kg.
Parallel imaging factors of 4-6 were used, generating 0.7 x 0.5 x 0.6 mm voxels in an acquisition time of 17-22 s. Images were scored for quality and the presence of artifacts independently by two observers.
The CEMRA series were evaluated to identify pulmonary arterial and venous anatomy.
Extra-pulmonary vascular anatomy was divided into 12 sections for image quality assessment on MPR.
Results: The MRA data sets had high mean image quality and low mean artifact grading scores.
No significant difference was evident between the two observers for scoring image quality and artifact (p00.9), and there was good interobserver agreement (k00.66, 95% CI: 0.26-0.81).
The pulmonary arteries were confidently visualized to at least the 2nd order branches in all 16 patients, at least the 3 rd order branches in 15 (93%).
Out of 176 evaluated segments, 164 (93%) were graded as the vessel "very well visualized with excellent definition of vessel" PA abnormalities were diagnosed in 10 patients and PV anomalies in 6 patients.
Correlation was made with open surgical findings in 12 cases, confirming the CEMRA diagnoses with no discrepancy between the operative and imaging conclusions.
The results of our study suggest that high resolution CEMRA at 3.0 T is extremely promising in neonatal CCHD and, in combination with echocardiography, may often provide comprehensive information for treatment planning.
With appropriate use of physiological motion compensation, parallel imaging and coil configuration, voxel volumes less than 0.5 mm3 are routinely achievable.
Purpose or Case Report: It is not well understood whether transfusion-dependent children (mainly those who have hereditary anemias subject to excessive iron levels from regular blood transfusions) with no or mild cardiac iron overload and without signs of heart failure exhibit early changes in cardiac dysfunction.
This study aims to evaluate intramyocardial deformation and subsequent cardiac dysfunction in such patients using the cardiovascular magnetic resonance technique of myocardial tagging as well as assess iron overload by another cardiovascular magnetic resonance technique of T2 star (T2*).
We hypothesize that transfusion dependent children undergoing chelation therapy will be at high-risk for cardiomyopathy despite normal or slightly elevated iron levels and no signs of heart failure due to progressive myocardial deformation as assessed by myocardial tagging; that is, we expect abnormal mechanical parameters to exist despite T2* values which correspond to normal or slightly elevated myocardial iron levels.
Methods & Materials: 8 patients (mean age 15 yrs) requiring regular blood transfusions for various reasons (e.g.
Thalassemia, Sickle Cell disease, other anemias) were investigated using Harmonic Phase magnetic resonance tagging and T2* imaging.
Various myocardial mechanical parameters were quantified and correlated to cardiac iron levels as measured by T2*.
Results: 2 children with no overt signs of heart failure (as measured by ejection fraction and volume indices) exhibited normal T2* iron levels in combination with global circumferential abnormal myocardial strain and 4 children exhibited regional strain.
Conclusions: This preliminary data suggests that transfusiondependent children without overt signs of heart failure exhibit early signs of cardiac dysfunction (an effect of strain as assessed by myocardial tagging) in direct relation to normal or slightly elevated myocardial iron levels (as measured by T2*).
A larger sample size of children could support the idea of abnormal myocardial mechanics being a diagnostic imaging marker for the development of myocardial iron overload and eventual iron-mediated cardiomyopathy.
The choice of coil and field of view varied according to the age and size of the patient.
Imaging was performed on a 1.5 T magnet (Philips MRI system).
After the cine gradient echo time of flight sequences were acquired, the dynamic keyhole MR angiographic sequence was applied in the coronal orientation.
This sequence commenced 2-5 s after initiation of the first bolus administration of 2-10 mL of undiluted gadopentetate dimeglumine (0.5 mmol/mL, Magnevist) at a rate of 2-3 mL/sec followed by 10-20 mL of saline at a rate of 2 mL/sec.
Image acquisition was repeated multiple times without delay for 60 s during shallow breathing.
Contrast material was administered through a peripheral or central venous line, and sometimes via simultaneous bilateral extremity injections.
Rarely manual hand injection was performed.
The parameters were as follows: CENTRA k-space filling, acquired voxel size 1-1.2 x 1-1.2 x 2-3, reconstructed voxel size 0.4-1 x 0.4-1 x 1-1.5, number of dynamics 8-20, keyhole percentage 25-38%, SENSE (parallel imaging) factor 2x1-2x2, dynamic times 1-3.6 s, and injection rate 1-3 cc/second.
Image analysis was based on original contrast-enhanced data sets, MIP and volume rendering.
The quality of the MRV was graded by a subjective scale from 1-5 (10excellent, no limitations; 50nondiagnostic).
Comparison with unenhanced MR venographic sequences and Doppler ultrasound were available in all cases.
Results: 4D MRV provided diagnostic information on patency of venous segments in all cases, including the presence of venous occlusion or stenosis, the segments involved, and collateral venous pathways.
Conclusions: 4D MR venography using the method outlined is a reliable means of estimating central venous thrombosis in children, using short dynamic scan times with high spatial and temporal resolution, creating an effect similar to conventional venography.
Paper #: PA-033 Analysis of Optimal Phase Interval for Prospective EKG-Triggered Cardiac CT in Children Prakash Masand, MD, Texas Children's Hospital, Savivek Bogale, Lorna Browne, Rajesh Krishnamurthy Purpose or Case Report: Evaluate high temporal resolution cine MRI of the heart in children to determine relationship of heart rate to the optimal motion free interval during the cardiac cycle, and to derive a chart depicting optimal reconstruction intervals across a range of heart rates which could be applied for prospective EKG triggered CT imaging of the heart.
Methods & Materials: Pediatric patients who had high temporal resolution cine Steady State Free Precession sequence (50 frames acquired across a single cardiac cycle) performed as part of a MRI/MRA of the heart from 2005-2011 were included.
A single reader analyzed the high temporal resolution cine data to determine the location and duration of the motion-free phase interval in systole and diastole for heart rates ranging from 60-140 bpm.
The following information was collected: age, heart rate, duration of systole and diastole, EKG phase and phase percentage of motion-free interval, and duration of motion-free interval.
Heart rates were grouped in ranges of 10 (61-70, 71-80, 81-90 etc.
), with at least 25 patients in each group.
Wilcoxon sum rank test was performed to compare the static durations of systolic and diastolic phases for each heart rate group.
Two-tailed P-values<0.05 were considered to indicate statistical significance.
A chart matching heart rate range to the optimal phase of image acquisition and reconstruction was created.
Results: Optimal reconstruction intervals for motion-free imaging of the heart in systole and diastole, and single best phase for a given heart rate are as follows: (Please see Table 1 ) Mean duration of the motionless phase in systole and diastole for a given heart rate is as follows: (Please see Table 2 ) Conclusions: This is the first time that variability in location of the motion-free interval within the cardiac cycle with respect to heart rate in children has been studied in rigorous fashion.
This chart will have immediate application in the so-called 'target scan', which uses prospective EKG triggered acquisition of data in combination with half-scan reconstruction to minimize radiation dose with respect to retrospective EKG gated imaging.
Results: A total of 10 patients were found to have aberrant PDA by CCTA.
Nine patients had a PDA emanating from the brachiocephalic artery to the pulmonary arteries and one patient had PDA emanating from the carotid or the subclavian artery according to the operative report.
All ten patients had echocardiography in addition to CCTA.
Nine of the ten patients underwent surgery.
One patient was lost to follow up.
Comparison between CCTA and echocardiography to operative report showed the following: 3 out of the 10 echocardiograms agreed completely with CCTA and operative report.
7 out of the 10 echocardiograms did not report aberrant PDAs which were seen on CCTA and confirmed during surgery.
In one patient, the echocardiogram reported a double aortic arch which was found to be an aberrant PDA from the LEFT brachiocephalic artery; a finding that changed the surgical management and pre-surgical planning.
The results were statistically significant with a p value of <0.05.
Conclusions: Diagnostic accuracy of ductus arteriosus anatomy is important in pre-surgical planning and management of patients with ductal dependent pulmonary circulation.
Cardiac CT angiography is more accurate as compared to echocardiography in diagnosis of aberrant patent ductus arteriosus. )
in patients younger than 1 year of age.
The prospective ECG-gated protocol scanned 50% of the cardiac cycle with 10 msec padding in patients with heart rates greater than 100 bpm.
Two experienced radiologists evaluated the image quality and course of the coronary arteries.
Image quality was assessed based on the degree of homogeneity of vascular enhancement, presence of stair-step artifacts, image signal and noise as well as the overall subjective image quality based on a 4 point scale.
The course of the left and right coronary arteries was also evaluated based on a 4 point scale, focusing on how well the evaluator was able to visually follow the coronary artery from its origin to its termination.
Radiation dose was recorded and compared.
Results: The mean score of the homogeneity of vascular enhancement, stair step artifacts, image noise and signal, overall image quality, and score given to visualization of the course of the coronary arteries showed no significant difference (p>0.35) in the retrospective and prospective ECG gated groups.
The mean estimated effective dose was significantly lower for the prospective ECG gated group compared to the retrospective group, (0.83 mSV vs 2.39 mSV) respectively (p<0.0005).
Conclusions: Prospective ECG-gated cardiac MDCT provides comparable assessment of coronary anatomy, image quality with significantly less radiation dose when compared to the retrospective ECG-gated MDCT.
Prospective ECG gated cardiac MDCT is a powerful adjunct to the treatment and surgical planning of pediatric patients with congenital heart disease less than 1 yr of age with lower radiation dose.
Methods & Materials: 3 pediatric neuroradiology lectures were recorded and made available to 29 radiology residents at a university program through on-line streaming video viewed through an internet link.
Topics included brain tumors, phakomatosis, and congenital brain malformations.
One lecture per week was recommended prior to case conferences that reviewed the same topic.
Pre-and post-tests and a feedback survey were administered.
Nonparametric paired sign test and analysis of covariance were used to evaluate changes in test scores overall and according to feedback responses.
Spearman's partial rank correlation coefficient was used to evaluate the relationship between the number of viewed videos and test scores.
Results: Twenty-nine residents completed the pre-test and 28 the post-test.
The means (SD) scores were 59.3% (12.0%) and 64.8% (14.2%) respectively.
There was a significant improvement in test scores (p 00.019).
Residents that agreed/strongly agreed that the streaming technology lectures were convenient had greater improvement than those who did not (14.0 vs. -3.2%, p00.001).
Similarly, those who agreed/strongly agreed that being able to replay a lecture was helpful had greater improvement than those who did not (10.9 vs. -6.7%, p 00.001).
Finally, those who agreed/ strongly agreed that the streaming technology lecture format was a better teaching tool had greater improvement than those who did not (10.8 vs. -4.7%, p00.003).
Significant positive correlation between number of videos watched postconference and improvement was present (Spearman's rho0 0.48, p00.013).
Conclusions: On-line streaming video with live case conferences enhances radiology resident learning of pediatric neuroradiology.
Step (MPPS) software provided an accurate measurement of scan time.
Visual charting made gaps in utilization apparent.
Technologists and nursing notes correlated to gaps identified barriers and opportunities for improvement.
Nursing and Anesthesiology reduced redundancies.
Standardized protocols lead to more consistent scan times.
Appointment access for sedated MRI was measured by the third available appointment.
Results: Manually entered data points were time consuming, inconsistent and unreliable.
The process improvement was most effective when fewer more reliable data points were used to evaluate the effect of change.
The program resulted in a reduction of appointment access for sedated MRI from 30 days to 2 days with no change in the hours of operation.
Magnet utilization was increased from 58% to 73%.
Induction outside the scan room provided the most efficient process tested.
We ranked first in utilization in a Children's Healthcare Cooperation of America (CHCA) survey as measured by exams per scanner.
Patient preference for a.m. scheduling was shown by survey and corroborated by scheduling data.
Consistent scan times were achieved by protocol standardization augmented by indication driven decision support.
Conclusions: A consise definition of MR utilization established a metric that was used in the process cycle of Analyze-Optimize-Measure.
Anesthesia induction outside the magnet was the most efficient practice but required collaboration between nursing, MR technologists and Anesthesiology.
Protocol standardization were valuable aspects of process improvement essential to optimizing parallel sedation.
These adjustments reduced appointment access from 30 days to 2 days, increased utilization from 58% to 73% and produced a number one rank in utilization by CHCA survey.
We exploited the synonym option in Epic order entry to translate indications into procedures mapped to specific protocols for MRI neuroimaging.
The order screen allows the provider to enter an indication.
That indication is linked through a synonym option to a specific exam and protocol.
The recommendation is based upon institutionally created clinical care guidelines, and can be accepted with a single click to complete the order.
The requesting provider retains the option to override the recommendation.
An step in process development utilized an order queue established within the Epic inbasket.
A Pediatric Radiologist monitored the queue and communicated with referring providers to obtain additional history and educate toward best imaging practice.
These interactions facilitated development of a robust index of clinical indications used to create the synonym pool.
Results: MRI neuroimaging indications were expanded into a robust data set linked to specific MRI exams aligned with specific protocols.
The synonym option within Epic created the opportunity for the requesting provider to simply enter an indication which drives the procedure and recommended protocol.
Provider satisfaction has been high and concurrence with recommendations nearly universal.
Conclusions: Indication driven order entry was achieved through the synonym option in order entry within the Epic EMR.
Imaging recommendations are based upon institutional clinical care guidelines developed through consensus.
A robust compilation of pediatric MRI neuroimaging indications has been created and linked to specific exams and protocols.
Compliance with the indication driven recommendation has been high.
Modifications of the current system are currently under development for all cross sectional modalities and organ systems.
Paper #: PA-039 Cost-Effectiveness of Routine Neonatal Renal Ultrasound in Non-Syndromic Complex Congenital Heart Disease Elfrides Traipe, TCH, extraipe@texaschildrens.org; Jill V. Hunter, Marthe Munden Purpose or Case Report: To assess the prevalence of abnormal renal ultrasound in non-syndromic complex congenital heart disease (CCHD) and assess the cost-effectiveness of routine renal ultrasound in this population.
We restrospectively reviewed the initial neonatal renal ultrasound and any subsequent renal imaging in 97 patients with non-syndromic CCHD.
Etiologies included hypoplastic left heart syndrome (HLHS), transposition of the great vessels (TGA), coarctation of the aorta (CoA), truncus arteriosos (TA), double outlet right ventricle (DORV) with or without patent ductus arteriosus.
Patients were recruited consecutively as part of a prospective trial for pre-and post-operative magnetic resonance imaging of the brain.
Results: The neonatal pre-operative renal ultrasounds were analyzed in 41 female and 56 male patients.
Only 1 of the 97 patients showed any congenital renal anomaly.
This patient was born with hypospadias, that would have routinely stimulated neonatal follow up.
Conclusions: Knowledge of embryology would not lead us to anticipate a high co-incidence of congenital renal and cardiac pathology.
Based on this statement and our findings, our recommendation to improve cost-effectiveness is not to perform routine neonatal renal ultrasound in non-syndromic CCHD, but only if otherwise clinically indicated.
Purpose or Case Report: To conduct a meta-analysis of the diagnostic performance of contrast enhanced voiding urosonography (ceVUS) in comparison to voiding cystourethrography (VCUG) or direct radionuclide cystography (DRNC).
A literature search was conducted for studies published on ceVUS in the pediatric age group.
Studies were included if the ultrasound contrast agents (USCA) Levovist® (Bayer-Schering Pharma, Germany) or SonoVue® (Bracco, Italy) were used and enough data was available to extract 2x2 tables.
If the ceVUS was compared to both VCUG and DRNC in the same patients the results for each were analyzed separately.
A bivariate hierarchical model that takes into account the heterogeneity in ceVUS sensitivity and specificity in different studies was used for the assessment of the summary diagnostic metrics.
The summary ROC curve was derived and presented graphically from the parameters of the model.
Additionally, the 95% confidence intervals (CI) and the positive (LR+) and negative (LR-) likelihood ratios were calculated.
Results: Out of 127 publications only 30 comparative studies fulfilled the inclusion criteria.
These encompassed 26 ceVUS studies in comparison with VCUG and 4 with DRNC with regards to detection of vesicoureteric reflux.
In 26 studies the USCA Levovist® and in 4 SonoVue® were used.
A total of 2549 children with 5078 pelvi-ureteral-units (PUUs) were included in the meta-analysis.
ceVUS compared to VCUG and DRNC had a sensitivity of 90% (CI: 85-93) and a specificity of 92% (CI: 89-94) with LR+and LR-of 11.7 and 0.11, respectively.
The performance of ceVUS was better when compared to DRNC than to VCUG (sensitivity 94%, specificity 95% versus 90% and 92%, respectively.
The meta-analysis of the diagnostic performance of ceVUS regarding the urethra included 880 patients (682 boys).
Excellent imaging of urethral anatomy was reported in over 97% of the patients.
However, currently there is only one comparative study with 146 patients available.
In this study 100% sensitivity and 100% specificity were reported.
Conclusions: Sufficient evidence is available clearly demonstrating the high diagnostic performance of ceVUS compared to VCUG or DRNC regarding the detection or exclusion of VUR in children.
These findings combined with the absence of radiation should be convincing reasons for promoting the widespread use of ceVUS in children.
Disclosure: Dr. Darge has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Prenatal ultrasound (US) has increased identification of infants with asymptomatic renal pelvic dilatation.
Society for Fetal Urology (SFU) grading is used in the sonographic evaluation of pediatric hydronephrosis.
Based on US findings, a nuclear medicine diuretic renogram may assess renal function, which could result in operative intervention.
Standardized protocols for diuresis renography, the "welltempered renogram," already exist; however, no current study has assessed effect of intravenous hydration (IV) status with US in the evaluation of childhood hydronephrosis.
Our study assesses the effect of hydration on SFU grading.
In this prospective IRB approved study, pediatric patients diagnosed with pelvicaliectasis requiring a diuretic renogram were recruited to undergo pre and post hydration renal US.
A urinary catheter was placed followed by renal US pre and post IV hydration (10 ml/kg normal saline bolus).
Imaging was performed by the same sonographer on the same US machine.
A well-tempered renogram was then performed.
All images were reviewed by two blinded radiologists, one pediatric radiologist who assigned SFU grades to each kidney.
Results: Data were collected from 34 studies, with ages ranging from 6 weeks-16 years, with an average age of 22 months.
There were 28 unique patients.
Of these, 23 underwent a single renogram, 4 underwent two renograms, and 1 underwent 3 renograms.
One patient had a solitary kidney due to MCDK.
Thus, there were 33 usable paired sonograms (67 kidneys) for analysis.
SFU grades were compared in the pre-and post-hydration US for each kidney.
Two-sided statistical tests were done to assess whether SFU grades changed significantly after hydration (sign test).
52 of 67 (78%) kidneys remained the same grade post hydration.
When there was a difference, most demonstrated an increase (13 of 15 kidneys and p<0.01).
No change in SFU grade pre-and post-hydration differed by more than 1.
Only 1 kidney went from grade 2 to grade 3.
SFU above grade 2 is considered clinically significant.
No kidney that was grade 3-4 pre-hydration became grade 0-2 post-hydration.
When SFU is dichotomized grade 0-2 vs. 3-4, there was no significant change in grade from pre to post hydration (p01).
Conclusions: Hydration does not appear to have a clinically significant effect on SFU grade.
Therefore, performance of a "well-tempered" US is unnecessary.
Disclosure: Dr. Lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Study type was significantly associated with both total and weighted score (both p < 0.0001).
RUS was better and MAG3 was worse than VCUG, RNC, and DMSA, which did not differ from each other.
Other factors associated with worse total scores included patient age 1-3 years (p<0.001) and non-white race (p00.04).
Gender, prior testing history, wait time, and parent education were not associated with total scores.
In the multivariate model, RUS remained the best, MAG3 the worst, and DMSA, VCUG, and RNC in the middle (p<0.0001).
Compared directly, DMSA and VCUG total score did not differ (p00.59).
Conclusions: This study documents significant differences among GUI studies with respect to the patient and family experience, but there was no overall difference between DMSA and VCUG.
These findings may be useful to aid decision-making when considering GUI for pediatric patients.
We retrospectively reviewed the imaging findings of 29 consecutive patients with histologic diagnosis of CD (17 males, 12 females; mean age 14.6 years; age range 5-24 years) who underwent MRE between 1/21/ 2011 and 10/10/2011.
The MRE was performed in a Siemens Avanto 1.5 Tesla scanner.
Standard departmental volume of polyethylene glycol and fluid were administered for bowel distention.
The imaging protocol included DWI with eight b values ranging between 0 sec/mm2 and 800 sec/ mm2 and gadolinium enhanced dynamic 3D VIBE (volume interpolated breath hold exam) in the coronal plane.
The studies were qualitatively evaluated in a blinded fashion by two board certified radiologists.
Disease activity was defined as bowel wall thickening and enhancement in the gadolinium enhanced images.
DWI abnormality was defined as bowel thickening, increase signal on DWI images and decrease signal on ADC maps of the ileum.
Intra voxel incoherent motion (IVIM) DWI parameters were used as quantitative biomarkers for the analysis of slow diffusion (D) and fast diffusion fraction (f).
Results: Gadolinium enhanced VIBE images identified abnormal thickening and enhancement of the ileum in 11/29 (38%) patients.
DWI identified abnormal signal in 11/29 (38%) patients.
The sensitivity and specificity of the qualitative DWI for identifying ileitis, as shown by gadolinium enhanced imaging, were 82% and 89%, respectively.
Quantitative analysis showed statistically significant difference in IVIM maximal values for f (fast diffusion fraction) between abnormal (mean00.67, std00.17) and normal (mean00.8, std00.14) ileum segments (p00.012).
There was statistically significant difference in IVIM maximal values for D (slow diffusion) between abnormal (mean 02.2 μm2/ms, std 0 0.7 μm2/ms) and normal (mean 02.7 μm2/ms, std 0 0.6 μm2/ms) ileum segments (p00.0084).
Abnormal loops of bowel had decreased slow and fast diffusion parameters.
Conclusions: Diffusion weighted imaging has excellent sensitivity and specificity for the detection of active ileitis in pediatric CD.
Furthermore, quantitative IVIM model parameters provide effective biomarkers for this condition.
IVIM DWI has the potential to assess bowel inflammation without intravenous contrast enhancement and further increase our understanding of CD.
Methods & Materials: Forty pediatric patients (median age 13.8 years, range 10.0-17.7) with suspected (n035) or confirmed IBD (n05) were included and underwent gastroileocolonoscopy with biopsies followed by MRE (median interval 20 days, range 6-55).
The MRE results were compared with macroscopic and microscopic assessment of the ileum.
The clinical importance of the MRE results was registered.
Results: Crohn disease (CD) was diagnosed in 25 cases, ulcerative colitis (UC) in 12, and IBD unclassified (IBDU) in three.
Macroscopic ileitis was detected in 15/25 (60%) of CD cases and in 2/12 (17%) of UC (backwash ileitis).
Microscopic inflammation was found in another four CD cases and one IBDU patient.
In total, discrepancy between macroscopic and microscopic inflammation was found in 9 CD, 2 UC and one IBDU patients.
The sensitivity of MRI was 64% (against macroscopy and/or microscopy) to 71% (against macroscopy alone), while the specificity was 100% and 92%, respectivley.
MRE findings was decisive for diagnosis in 4/40 (10%) and led to treatment adjustments in 11/40 (28%) in the following six months.
Conclusions: MRE is a reliable method for imaging of intestinal inflammation in pediatric IBD, and can be supportive or essential for clinical treatment decisions.
Results: Of 15 children with EC, 7 had CT imaging of the abdomen and pelvis.
These 7 children ranged in age from 15 months to 16 years (mean 10.2 years +/− 6.3) with a male predominance (n04, 57%).
The most common presenting symptoms were abdominal pain (n06), bloody diarrhea (n03), and rectal bleeding (n03).
EC was characterized as a dense and predominant eosinophilic inflammatory infiltrate in the lamina propria and/or epithelium without granulomas.
CT scans were abnormal in 6 (86%).
No colonic luminal contrast was present in 2 patients, and in one of these, the colon appeared normal.
Abnormal CT findings included cecal wall thickening (n05, 71%), mucosal enhancement without colonic wall thickening, (n01, 14%), mesenteric lymph node enlargement (n02, 29%), terminal ileal thickening (n02, 29%), jejunal and ileal thickening (n01, 14%), and pneumatosis (n01, 14%).
Of the 5 patients with cecal involvement, 4 primarily involved the cecum with less severe or no ileal or downstream colonic involvement.
Pneumatosis extended along the length of the colon with rectal predominance.
Conclusions: The predominant CT finding in our EC series was wall thickening, most severe in the cecum with variable extent downstream with mild or no involvement of the terminal ileum.
Although there is overlap, these findings are different from the most common patterns encountered with ulcerative colitis or Crohn disease and should raise the possibility of EC in children presenting with abdominal pain and bloody diarrhea.
Purpose or Case Report: Timely identification of childhood arterial ischemic stroke (AIS) is critical to development of acute treatment strategies.
We present our experience prior to and following development of a pediatric stroke alert system (SAS).
Through multi-disciplinary collaboration in a tertiary care setting, a pediatric SAS was established in 2008.
We describe the system, imaging protocol evolution, and impact upon the time between admission and MRI initiation (time-to-MRI) in patients with childhood AIS.
Of 74 patients in our stroke database (COMIRB #05-0339), 27% met inclusion criteria for stroke alert initiation (acute focal neurological deficit within 12 h).
Eleven pre-2008 and nine post-2008 patients met criteria.
We compared the time-to-MRI between these two groups, utilizing a two-tailed t-test.
Results: The pediatric SAS has two phases: I-neurological evaluation and II-imaging and treatment consideration.
Phase I stroke alert is initiated when a child presents with an acute focal neurologic deficit.
If neurology confirms stroke symptoms and CT head is negative for an alternative etiology, a stroke alert is called prompting an emergent brain MRI.
If MRI confirms an acute stroke, hyperacute therapies are considered.
Initial MRI protocol included DWI, T2, FLAIR, 3D TOF COW MRA, 2D TOF neck MRA and fat saturated T1 neck imaging.
After internal quality review, T1 MPRAGE brain and contrast enhanced 3D neck MRA were added.
The sequence order was also altered so diagnostic sequences were scanned first (DWI and COW MRA).
There was a trend towards decreased time-to-MRI in the post-2008 group (mean0152 min, SD +/− 120) as compared to the pre-2008 group (mean 340 min, SD0+/−304; p00.10).
Conclusions: Institution of a pediatric SAS improved urgent neurologic evaluation and demonstrated a trend towards shorter time-to-MRI.
Ongoing quality review has enhanced imaging quality and decreased time-to-MRI.
Continued refinement of Pediatric SAS's will be critical to the success of recently funded phase I clinical trials in the evaluation of hyperacute therapies.
Results: There were 6 female and 3 male infants.
The mean post-gestational age at presentation was 20 days (range 0-90 days), while the corrected age was less than 30 days for all patients.
7 patients presented with seizures and signs of infection; 1 presented with lethargy and later proved to have protein C deficiency.
MRI was performed 0-12 days from presentation in these 8 patients.
Another patient with known protein C deficiency underwent MRI at 3D for followup of screening US abnormalities.
There were a total of 27 deep cerebral white matter lesions: 21 frontal, 4 parietal, 2 temporal lobe.
Lesions were fluid signal cavities with restricted diffusion.
Larger lesions had dependent debris.
All lesions had associated hemorrhage and most lesions had evidence of adjacent small vessel venous thrombosis.
Lesions imaged after Gad showed peripheral enhancement.
Three lesions were seen to increase in size on follow-up imaging.
Three patients, 2 with meningitis confirmed via microbiology and 1 with presumed meningitis by CSF counts, underwent surgical aspiration of a total of 6 lesions.
All specimens were sent for pathology and culture and were negative for microorganisms.
Conclusions: Recognizing the MR appearance of necrosis and liquefaction after deep white matter cerebral venous infarction in neonates can distinguish this entity from cerebral abscess and potentially avoid an unnecessary neurosurgical aspiration procedure.
All four children were initially treated with aspirin but experienced recurrent events on therapy.
All four were subsequently anticoagulated.
Two children have remained on warfarin for 6-7 years without recurrent events, while the other two had recurrent events despite adequate anticoagulation.
These two children underwent uncomplicated coil embolization of the affected vertebral artery segment, and they have remained symptom-free for five and 20 months since then.
Conclusions: DAVA was diagnosed by CA in 3/4 patients.
All four children with DAVA in our series suffered recurrent strokes despite aspirin therapy.
Two of the four experienced further strokes on anticoagulation, necessitating endovascular therapy.
These findings suggest that DAVA in children may require CA to diagnose, and that it may be refractory to standard adult therapies.
Ongoing multicenter efforts in childhood AIS should further evaluate the diagnostic approach and recurrence risk of childhood DAVA. )
and cerebral gray matter abnormalities were present in 6 (1%).
Posterior fossa lesions were seen on US in 1.6% , but mastoid views were included in only 50% of the centrally read US.
Conclusions: In the largest extreme preterm cohort to date with near-term MRI and serial US, 19% had mod-severe WMA on brain MRI, similar to previous reports.
Cerebellar abnormalities were detected more frequently by MRI than by US.
Neurodevelopmental outcomes at 18-22 months and school age will assess the relative and combined values of MRI and US as outcome predictors.
, an analysis technique based on task-free resting state fMRI recording, can be useful in assessing disruption of connectivity in certain disease states, including epilepsy.
In healthy control subjects, functional connectivity reveals strong bilateral interhemispheric connectivity in such system as sensory-motor, visual, auditory as well as dorsal attention and default mode networks.
In patients with epilepsy associated with unilateral diffuse hemispheric disease such data is limited.
Differences in the pattern of activation would suggest alteration in connectivity in these entities.
This finding would impact the typical interpretation of this data that is becoming routinely collected for epilepsy pre-surgical evaluation.
Methods & Materials: Siemens (Erlangen, Germany) system, 3-Tesla (Trio) scanner was used for imaging (EPIBOLD sequence, TE030 ms, flip angle090°).
Resting state fMRI scan were performed in both awake and anesthetized patient.
Awake patients were instructed to relax and rest while keeping their eyes open.
Analysis was performed using 1000 Functional Connectomes Project scripts based on AFNI and FSL software packages.
Resting state data were analyzed for connectivity with the following seeds: Somatomotor, Visual, Auditory, and Default Mode (posterior cingulated cortex (PCC)).
Results: We applied this technique to evaluate 12 patients with hemispheric seizure disorders, including Rasmussen's, neonatal infarct and migration disorders.
All the subjects demonstrated some deviation from typical interhemispheric connectivity with a spectrum of findings.
The figure below shows connectivity patterns in a patient with cortical dysplasia.
While some interhemispheric connectivity remained in somatomotor (SM) and auditory (A1) systems, it was disrupted in Visual (V1) and default mode (PCC) networks.
Variable patterns were found across the cases that corresponded to lesion side, supportive of disruption in interhemispheric connectivity as measured by fMRI.
Conclusions: Resting state functional connectivity patterns are well documented in healthy subjects.
These results suggest that interhemispheric connectivity disruption is a typical feature of unilateral diffuse hemispheric disease though variable in presentation, either being limited to select systems or demonstrating broad disconnect between the two hemispheres.
These results should be carefully considered when evaluating data for pre-surgical epilepsy evaluation.
Purpose or Case Report: Premature birth is associated with white matter injury leading to a wide ventricular system.
However,normative standards for ventricular size are lacking for this particular group.Aims: We aimed to, in a controlled, population based Norwegian cohort of ex-prematures without major handicaps, and for men and women separately,to 1) create standards for radiological indices of ventricular dilatation, 2)investigate associations of these measurements with subjectively assessed ventricular size,3) examine differences in ventricular size between ex-prematures and healthy controls Methods & Materials: The initial birth cohort included 217 neonates, birth weight below 2000 g (low birth weight)born within Hordaland county, Norway, between April 1st 1986 and August 8th 1988.
113 of 174 eligible survivors (without major handicaps)underwent MR examination during the period January 2006 to May 2007.
103 of these were expremature (born before gestational age 37 weeks) and were included in this sub-study.
Based on T2 weighted images, the ventricular size was subjectively judged as being normal, mildly, moderately or severely dilated by an experienced paediatric neuroradiologist, while objective measurements were performed in a blinded fashion, by a second observer (SMA) using an imaging software program (Nordic Ice®).
Results: The normative standards for the ventricular system in ex-premature young adults showed wide variations, in particular for the occipital horns.
The agreement between subjective and objective assessment of ventricular size was good.
Ex-prematures had smaller heads than those born term (control group).
There was no difference in ventricular size between the two groups, even after adjusting for head size.
Ex-premature males had larger ventricles than females; however, the difference disappeared after adjusting for head size.
Conclusions: Young adults born prematurely with a birth weight below 2000 g do not have larger lateral ventricles than healthy controls born term, even after correcting for a smaller head size.
Paper #: PA-057 Best Practice for Reproducibility When Measuring T2*: Implications for Liver and Cardiac Iron Assessment Mark Ferguson, MD, Radiology, Seattle Children's Hospital, markferg@uw.edu; Randolph Otto, Seth D. Friedman Purpose or Case Report: Patients with red blood cell transfusion-dependent conditions receive high amounts of iron that can lead to abnormal iron accumulation in tissues resulting in organ damage.
While the liver is the dominant excess iron storage organ, iron related cardiotoxicity is a leading cause of morbidity and mortality in patients with transfusion-dependent thalassemia.
Therefore, accurate determination and tracking of tissue iron levels in both the liver and the myocardium is important for patient prognostication as well as monitoring treatment changes.
While multi-echo gradient echo MRI (T2*) is widely used and validated method employed for iron assessment, less attention has been given to derived metrics.
Specifically, the literature almost exclusively reports and uses the mean value for T2* from a pixel-wise (PW) map.
Infrequently used is the median.
The median is a potentially superior metric than the mean because it is insensitive to outliers.
Outliers will always occur in data because of either noise or imperfect vessel exclusion.
To compare mean versus median on reproducibility of T2* measurement, 23 subjects who had paired heart/liver measurements were examined.
The entire liver (excluding vessels) and the interventricular septum myocardium were traced on representative images from each series.
Mean and median T2* values were generated from the pixel maps.
R2* (1000/ T2*) and coefficient of variation (CV) were computed on a patient-by-patient basis.
These measures were then summarized for the group.
Results: Markedly higher R2* values were observed in both heart and liver using median summary measures (liver: t0−2.79, p0.01, heart: t0−2.8, p0.01).
These findings were accompanied by lower CV's (better reproducibility) for the median approach (liver: t01.89, p0.07; heart: t01.91, p0.07).
Conclusions: The consistent difference in derived T2* values between the methods (median>mean) should be considered when comparing derived R2* values to established normal ranges.
CV data support that using the median as the final summary metric will always outperform mean metrics for measuring change in R2*.
This finding has immediate implications for the scientific literature and for guiding therapeutic management over time.
Results: Twelve children (age 5mo-13yo; M:F 6:6) with GSDs (7 ABCA mutations, 4 SP-C mutations, 1 undefined mutation) and 16 children (age 2wk-18yo; M:F 10:6) with other DLDs (including pulmonary interstitial glycogenosis, neuroendocrine cell hyperplasia of infancy, lymphocytic interstitial pneumonia, lipoid pneumonia, diffuse alveolar damage, granulomatous infection, capillaritis and other pulmonary hemorrhage syndromes) were identified.
CT findings with highest sensitivity for GSDs were ground glass attenuation (83%), parenchymal cysts (67%), and interstitial thickening (58%).
Parenchymal cysts, honeycombing, and pectus excavatum were more specific for GSDs compared to other DLDs (p<0.05).
The combination of either parenchymal cysts and honeycombing or ground glass attenuation and pectus excavatum provided the highest specificity (100%) but low sensitivity (25%).
The combination of parenchymal cysts and ground glass attenuation provided good specificity (81%) and modest sensitivity (50%).
No combination of findings provided both high sensitivity and specificity.
Conclusions: Ground glass attenuation is the most sensitive finding for GSDs, while parenchymal cysts, honeycombing, and pectus excavatum are more specific findings for GSDs than other chronic DLDs of childhood.
However, no single finding or combination of findings on chest CT is both highly sensitive and specific for GSDs, and chest CT cannot substitute for genetic testing or lung biopsy for the differentiation of GSDs from other DLDs.
PEs were observed in 33 scans of patients without a history of congenital heart disease (CHD), and 26 PEs in 12 scans of patients with a history of CHD.
Z-axis scan lengths for the chest CT exams ranged from 10.1-33.3 cm.
A z-axis scan length of 14 cm centered 3.5 cm below the carina captured all PEs in all patients, and a length of 12 cm centered 3.5-4 cm below the carina in patients with CHD.
A z-axis scan length of 8 cm centered 5 cm below the carina was sufficient to capture at least one PE in all patients, and a length of 8 cm centered 4-5 cm below the carina in patients with CHD.
The radiation effective dose of the chest CTPA exams ranged from 3-10 mSv.
Limiting the z-axis scan length on CTPA exams to 14 cm or 8 cm would have resulted in a 20% or 40% decrease in z-axis coverage, respectively, and estimated radiation effective dose reduction of 21-42% due to less radiation exposure to the intrathoracic structures, thyroid gland and upper abdominal viscera.
Conclusions: Limiting the z-axis scan length coverage for CTPA exams based on a model of the typical anatomic distribution of PEs relative to the reference level of the carina permits a substantial reduction of radiation dose in children without reducing the sensitivity for detection of pulmonary emboli.
Purpose or Case Report: To determine whether the addition of multiplanar reformation MDCT images affects reader performance parameters and provides added diagnostic value compared to the use of axial CT MDCT images alone for diagnosing PE in children.
This was an institutional review board-approved retrospective study of 60 consecutive pediatric patients who underwent CTPA for clinically suspected PE.
Two faculty pediatric radiologists and two radiology residents independently reviewed each study initially using only axial MDCT images and later using MPR MDCT images in any x-, y-, or z-axis for detecting PE.
Diagnostic accuracy, confidence level, and interpretation time of MPR MDCT images were compared to axial MDCT images using McNemar's test and paired t-tests.
The kappa coefficient was calculated to assess interobserver agreement.
Diagnostic accuracy was compared between faculty pediatric radiologists and radiology residents by logistic regression whereas confidence level, interpretation time, and added diagnostic value were evaluated with analysis of variance (ANOVA).
Results: The final study cohort consisted of 60 CTPA studies from 60 children (28 M/32 F; mean age 14.7 years).
Nine (15%) of 60 CTPA studies were found to have PE.
Diagnostic accuracy in correctly detecting PE ranged from 91.7 to 100% (mean096.7%), with no significant differences between the use of axial and MPR MDCT images.
Logistic regression indicated no significant difference in diagnostic accuracy of detecting PE between faculty pediatric radiologists and radiology residents for axial MDCT images (P0.48) or MPR MDCT images (P0.24).
Confidence level and interobserver agreement were significantly higher and average interpretation time was longer in evaluating PE with MPR MDCT images compared to axial MDCT images for all reviewers (P<.001).
Compared to faculty pediatric radiologists, significantly greater increases in confidence level, interobserver agreement, interpretation time, and added diagnostic value using MPR MDCT images compared to axial MDCT images to diagnose PE were found for radiology residents (P<0.001).
Conclusions: Use of MPR MDCT images in diagnosing PE on CTPA in children significantly increases confidence, interobserver agreement, and interpretation time among faculty pediatric radiologists and radiology residents.
Because MPR MDCT images provide significantly greater improvements in reading parameters for residents than for faculty members, their routine use should be encouraged for trainees.
Paper #: PA-062 Chest CT in Children, Anesthesia and Atelectasis Beverley Newman, MD, Radiology, Stanford University, bev.newman@stanford.edu; Elliot Krane, Terry E. Robinson Purpose or Case Report: In spite of advances in CT equipment and speed, sedation/ anesthesia is required in many young children for optimal quality CT for detailed parenchymal evaluation; resultant atelectasis is a common and important quality issue.
Our purpose was to evaluate the safety and effectiveness of a standardized lung recruitment technique.
Methods & Materials: With IRB approval and parental informed consent, 49 controlled ventilation, low dose, chest CT's (cooperative effort between anesthesia, pulmonology and radiology) were performed in 38 children (7 had 2-4 CTs) (21 F, 16 M; ages .02-5.13 yrs, mean 2.5 yrs).
Indications included cystic fibrosis 8; ciliary dyskinesia 4; chronic or interstitial lung disease 16; evaluate pulmonary metastases 10.
CT parameters were 80-100kVp, 25-80mAs, IV contrast 11.
Various prior methods employed by the pediatric anesthesiologists to maintain lung inflation had unpredictable results (a brief survey showed 5/9 nonintubated anesthetized cases had problematic atelectasis).
A standardized intubation technique was therefore adopted: 1.Use of a tight fitting face mask during induction and IV placement, inspiratory pressures of 20-25 and PEEP of 5.
2.
Introduction as early as possible using an appropriately sized cuffed endotracheal tube.
3.
Alveolar recruitment maneuvers-10-12 3 s breaths to 40 cm H2O/5 (32-35 in 1st 6 cases).
4.
Three breaths at 25/5, inspiratory breathold followed by 25-30 cm on 4th breath for scout and inspiratory scan, and complete ventilator disconnection for expiratory scan.
Recruitment breaths repeated before each scan.
Two experienced readers reviewed and scored the images on a 5 point scale for overall quality and atelectasis.
Results: All studies were completed safely with no procedural complications.
One child had propofol-related postoperative emergence delirium.
All CT scans were diagnostically good to excellent with small subsegmental atelectasis in 8 (6/8 were the initial cases with lower recruitment pressures) and segmental atelectasis in 2.
13 cases had prior CTs, without this technique, that were suboptimal due to moderate procedural atelectasis, in spite of tracheal intubation in the majority of cases.
Conclusions: An intubation lung recruitment technique can be performed safely and consistently by different individuals using a standardized protocol.
Procedural atelectasis that affects quality is reliably absent and repeat sequences are not needed.
Obtaining a high-quality dynamic airway imaging study is critical for accurate interpretation and subsequent medical decision-making.
The ideal MRI sleep study is one that allows successful completion while maintaining spontaneous breathing without artificial airway, which can be an anesthesia challenge.
Dexmedetomidine has been shown to have sedative properties paralleling natural sleep with minimal respiratory depression.
We hypothesized that dexmedetomidine compared to propofol would have less effect on upper airway tone and airway collapsibility and provide favorable conditions with less airway interventions required during dynamic MRI airway imaging in children with OSA.
In this prospective study, we examined the requirement for airway intervention for propofol (100-200 mcg/kg/m) and dexmedetomidine(1-3 mcg/kg/h) in children and adolescents with OSA.
Severity of OSA was analyzed by overnight polysomnography.
For children with history of mild OSA there was no intervention unless oxygen saturation decreases below 90%; while for children with history of moderate/severe OSA, an artificial airway was placed when oxygen saturation decreased below 85%.
Results: Demographics and OSA severity by polysomnography were comparable.
Requirement for artificial airway by severity of OSA as documented by polysomnography will be shown.
MRI sleep studies required airway intervention in 3/26(12%) children in the dexmedetomidine group versus 7/ 29 (24%) children in the propofol group.
MRI sleep studies were successfully completed without the use of artificial airways in 23 children (88%) in the dexmedetomidine group versus 22 children (76%) in the propofol group.
Conclusions: Safe and effective anesthetic management is a key factor in obtaining good quality MR images of the airway.
Although there was no statistical significant difference in the need for airway intervention between drugs, dexmedetomidine provided acceptable sedation for MRI sleep studies with less airway intervention in children with OSA.
Dexmedetomidine may be the preferred agent for sedation during MRI sleep studies in children, and may offer benefits to children with sleep disordered breathing requiring anesthesia or sedation for other diagnostic imaging studies.
An open mouth and administration of CPAP resulted in smaller AP diameter of the retroglossal airway compared to images without CPAP due to CPAP pressure pushing the tongue posteriorly.
In patient 1 volume of oral cavity anterior to the tongue increased from 7.41 mL to 11.74 mL.
Meanwhile, the AP diameter of the retroglossal airway decreased from 4.8 to 1.4 mm (71% decrease).
In patient 2 the mouth was initially closed but parted when the pressure of CPAP was added with the oral volume increasing from 3.69 ml to 15.80 ml.
The AP measure of the retroglossal airway decreased from 8.3 mm to 2.8 mm (66% decrease).
In patient 2 the mouth was then closed and CPAP reapplied resulting in an AP measurement of 11.0 mm (33% increase).
The AP diameter difference between CPAP and no CPAP were tested with paired t-test, but were not statistically significant (p00.1475).
Conclusions: Positive airway pressure on a patient by full facemask and an open mouth can have an adverse effect on the retroglossal airway.
This adverse effect is an important consideration in the use of positive airway pressure to support airways for OSA, or during emergency resuscitation when a full facemask is used.
Paper #: PA-065 The Purpose or Case Report: A nanoparticle blood pool iodinated contrast agent (NCTX) has been designed and tested in preclinical animal models.
We report data in animal models exemplifying its advantages over conventional contrast in the setting of CT pulmonary angiography Methods & Materials: NCTX blood pool nanoparticles of 125 nm diameter with an encapsulated total iodine concentration of~125mgI/ml were administered by intravenous injection to mice, rabbits, dogs, pigs and sheep.
(These studies were actually conducted for other purposes and a review of the data revealed the similarities that motivated this paper.)
Total injected volumes were~5 ml/kg in large animals, and as high as 10 ml/kg in small animals to provide satisfactory vessel enhancement.
Iohexol or iopamidol was administered for comparative studies with conventional contrast.
In a subset of pigs, iatrogenic pulmonary arterial emboli were introduced prior to contrast administration.
Toxicity studies were conducted in mice and monkeys.
Results: The visualization of pulmonary vessels using NCTX blood pool nanoparticles was generally at least equivalent to using conventional contrast, and superior in several cases, particularly in small veins and when bolus timing of the conventional contrast was suboptimal.
In all cases, satisfactory vessel enhancement was achieved for a duration of several hours following a single infusion of NCTX blood pool nanoparticles.
There was no evidence of renal toxicity, and only transient elevation of hepatic enzymes at relevant dose levels.
Conclusions: NCTX nanoparticle blood pool agents demonstrate several advantages over conventional glomerularfiltered iodinated contrast agents for CT pulmonary angiography in animal models, including no nephrotoxicity, no dependence on bolus injection technique, superior depiction of small veins, and capability of re-imaging for follow-up studies without needing contrast re-injection.
Potential applications in human pediatric subjects include the diagnostic and post-therapeutic evaluation of cardiopulmonary anomalies and pulmonary embolism, especially in patients with renal insufficiency or tenuous vascular access.
Disclosure: Dr. Annapragada has indicated that he is a stock holder and consultant for Marval Biosciences Inc. Paper #: PA-067 Cardiovascular Image Quality Using a Nanoparticle CT Contrast Agent: Preliminary Studies in a Pig Model Rajesh Krishnamurthy, Radiology, Texas Children's Hospital, rxkrishn@texaschildrens.org; Ketan Ghaghada, Prakash Masand, Abhay Divekar, Eric Hoffman, Ananth Annapragada Purpose or Case Report: Image quality in a separate study using a long circulating, liposomal-based nanoscale blood pool iodinated contrast agent (NCTX) suggests clinical utility in pediatrics, potentially reducing difficulties in contrast-CT of children with congenital heart disease (CHD) including the size of intravenous cannula, need for accurate timing, inability to simultaneously opacify multiple targets of interest (requiring repeated contrast administration and/or repeated imaging).
Methods & Materials: Six pigs (average weight 30 kg) were imaged after slow intravenous infusion of NCTX (105 mg I/mL) at an iodine dose of approximately 900 mg I/ kg (8.5 mL/kg).
Retrospective EKG gated CT imaging was performed 3 h later using a 128-slice dual-source CT scanner at 80 and 120 kVp.
Two radiologists analyzed and graded (on a 5-point scale with 1: unreadable, 5: Excellent) images aimed at anatomic structures relevant to CHD.
Quality of images obtained at 80 and 120 kVp were compared.
Uniformity of contrast opacification was measured using a ROI-based CTnumber method at various intracardiac and extracardiac sites and mean non-uniformity was calculated.
Results: There was excellent agreement between the two readers on all counts at 120 kVp.
80 kVp images received lower scores for coronary morphology (4/5), and aortic valve visualization (3.5/5), but were comparable in other aspects.
Pulmonary artery and pulmonary vein branch visualization extended up to the 5th generation in all cases.
Visualization of coronary artery branches was possible up to the second generation, with good arteriovenous separation.
Subtle morphologic features including crista terminalis, Thebesian valve, foramen ovale, membranous septum, and chordae of the mitral valve were demonstrated in all cases.
Automated functional analysis and myocardial mass quantitation was feasible in all cases.
There was no significant difference in blood pool attenuation between the atria, ventricles, and extracardiac vasculature on quantitative assessment.
No image artifacts were visible on the reconstructed images.
Conclusions: These findings suggest that NCTX promises to be superior to conventional contrast agents for CT imaging of complex congenital heart disease, due to the absence of nephrotoxicity, avoidance of repeated contrast administration, and reduced number of scans performed.
Avoiding the need for accurately timed scans precludes the need for large bore intravenous access.
These attributes make it a promising agent that warrants further studies.
Disclosure: Dr. Annapragada has indicated that he is a stock holder and consultant for Marval Biosciences Inc. Paper #: PA-068 Theoretical Cost and X-Ray Dose Reduction in Pediatric Congenital Heart Disease Imaging by the Use of a Nanoparticle Contrast Agent Robert Bell, The University of Texas-Houston; Rajesh Krishnamurthy, Gabriela Espinosa, Christopher Petit, Ananth Annapragada Purpose or Case Report: The purpose of this study is to determine the effective, population averaged reduction in costs and radiation dose that can be achieved in the diagnosis of congenital heart disease by use of a nanoparticle long circulating blood pool contrast agent.
Methods & Materials: A Markov model of the decision tree followed at the Texas Children's Hospital in the image based diagnosis of congenital heart disease was constructed in TreeAge software.
The model included CT Angiography, MR Angiography, cardiac catheterization, and echocardiography diagnostic modalities.
Patient records, accumulated between 2003 and 2011 were examined to inform the model.
The radiation dose and cost for each step were encoded as penalty functions.
Markov simulations were run for two decision trees: (1) utilizing CT angiography and (2) replacing conventional CT angiography with blood-pool agent based CT angiography.
The overall population X-ray dose and accrued cost was calculated for each pass through the model.
Results: X-ray dose distributions for the example populations showed substantial reductions per CT study, as much as 50%.
Averaged over the population, since a sizeable fraction of patients are diagnosed without ever being exposed to any X-ray based modality, reductions were more modest, but still substantial.
Costs per CT study were slightly higher when the blood pool contrast agent was used.
When the diagnostic probability using the blood pool agent increased, it led to an automatic overall cost reduction.
Conversely when the diagnostic probability remained unchanged, costs rose, commensurate with the increased cost of the contrast agent.
Conclusions: The use of a blood pool contrast agent for CT angiography leads to substantial reduction in radiation dose in the setting of congenital heart disease.
Cost reductions are more modest, and are driven almost completely by the reduction in the number of MR and invasive angiography procedures resulting from increased diagnostic success using blood pool based CT angiography.
The model as constructed does not account for potential workflow changes that might result from the use of a new contrast agent.
Actual reductions realized may therefore be higher.
Disclosure: Dr. Annapragada has indicated that he is a stock holder and consultant for Marval Biosciences Inc. Paper #: PA-069 Frequencies and Patterns of Situs Discordance in Chest and Abdomen Justin Boe, Stanford, justinj.boe@gmail.com; Beverley Newman, Shreyas Vasanawala, Frandics Chan Purpose or Case Report: Incidence of situs anomalies, including heterotaxy and situs inversus, is estimated at 0.02% of population.
As the first step in the segmental analysis of structural heart disease, the determination situs position is of fundamental importance.
Abdominal situs, as defined by splenic position and morphology, and cardiac situs, as defined by atrial morphology, are usually but not always in agreement.
Echocardiographers also employ the relative position of the great arteries and vein at the hiatus to determine cardiac situs.
We evaluate the frequencies of discordances among abdominal, hiatal and cardiac situses.
Methods & Materials: With retrospective IRB approval, imaging records from 2001 to 2011 were reviewed for the diagnosis of cardiac situs inversus and heterotaxy.
Patients who had cardiac CT or MRI were included.
Images were evaluated on a 3D-processing station by a cardiac radiologist.
Cardiac situs was determined by the morphology of the atrial appendages.
When an atrial appendage was not adequately visualized, cardiac situs was assessed by the relative position of the main pulmonary artery and bronchi.
Hiatal situs was determined by the relative position of the aorta and the systemic venous return, and abdominal situs by the position and morphology of the spleen.
Results: Thirty-five cases were identified, with 23 cardiac CT and 12 MRI.
Patients' age ranged from 1 day to 35 years old.
In the abdomen, the numbers of situs inversus, asplenia, and polysplenia were 11 (32%), 12 (34%), and 12 (34%).
For the heart, the numbers of situs solitus, inversus, rightisomerism, and left-isomerism were 2 (6%), 13 (37%), 11 (31%) and 9 (26%).
The abdominal and cardiac situses were discordant in 5 (14%) cases.
Polysplenia had the highest number of discordance with the heart.
Hiatal situs was discordant with the abdomen in 5 cases (16%) and with the heart in 8 (25%) cases.
Conclusions: Situs disagreement between the abdomen and the heart is not uncommon and they should be documented separately in radiology reports.
Hiatal situs, as used by echocardiographer, disagrees with the cardiac situs in a quarter of the cases.
It should be used with caution in the segmental analysis.
Paper #: PA-070 Diminished ASL Intracranial Perfusion in Children with Neurofibromatosis Type 1 Kristen Yeom, MD, Stanford University, kyeom@stanford.
edu; Cynthia Campen, Patrick Barnes Purpose or Case Report: Neurofibromatosis type 1 (NF1), a neuro-cutaneous syndrome affecting 1/3500 children is associated with moyamoya syndrome (MMS).
However, no comparisons of cerebral perfusion in patients with NF1 and NF1-associated MMS to healthy controls exist.
We hypothesize cerebral blood flow (CBF), as measured by magnetic resonance imaging (MRI) arterial-spin-labeled (ASL), is diminished in children with NF1 compared to healthy controls, with the lowest levels seen in patients with NF1-associated MMS.
Methods & Materials: Twenty children aged 2-18 years with NF1, four with MMS, and 26 age-matched controls underwent ASL CBF on a 3 T magnet.
Pseudocontinuousspin-echo-ASL technique was used.
Measurements were taken bilaterally in cerebral cortical-subcortical regions, and the deep gray nuclei.
Trends in measurements as a function of disease severity were tested with the Jonckheere-Terpstra test for ordered alternatives.
A Bonferroni-adjusted p-value less than 0.0013 was considered significant.
Results: We identified 6/12 areas with significantly diminished ASL CBF (ml/100 g/min) in patients with NF1 (midrange), and NF1-associated MMS (lowest) compared to healthy controls (highest).
These included the: thalami (left: p00.0002, right: p00.0004); superior/middle temporal lobes (left: p 00.0012, right: p 00.0009); temporooccipital lobes (left: p00.0006, right: p00.0003); occipital poles (left: p 00.0008, right: p 00.0001); centrum semiovale (left: p00.0022, right: p00.0005); and left parietal lobe (p00.0012).
Conclusions: Cerebral perfusion diminishes in a graded fashion in children with NF1 and NF1-associated MMS, particularly in the posterior circulation and the MCA-PCA posterior watershed zones.
Future studies may demonstrate an important role for ASL in the presymptomatic diagnosis of cerebral vasculopathy, and the definition of NF1-related vasculopathy patterns.
Paper #: PA-071 Cingulate Gyrus MRI Sign in Pediatric NF1 Patients: A Novel Imaging Marker Nadja Kadom, MD, Radiology, Children's National Medical Center, nkadom@childrensnational.org; Nabila Hai, Rhea Udyavar , Amir Noor, Gilbert L. Vezina, Maria T. Acosta Purpose or Case Report: We observed a magnetic resonance imaging (MRI) signal abnormality in the anterior cingulate gyrus of pediatric patients with neurofibromatosis type 1 (NF1).
The cingulate gyrus could play a role in cognitive deficits of NF1 patients.
The first objective here is to document inter-rater reliability scores for visual detection of this sign.
The second objective is comparing ADC values of the cingulate gyrus in areas of visually abnormal MRI signal in NF1 patients to matched normal MRIs to confirm a pathophysiological basis of the visual MRI sign.
Methods & Materials: Retrospective analysis, IRB approved, 61 NF1 patients and 38 matched controls.
In the visual assessment part, two blinded neuroradiologists rated presence or absence of MRI signal abnormality in the cingulate gyrus in three different age groups of NF1 patients mixed with normal controls.
Cohen's Kappa inter-rater reliability coefficients were calculated.
The same blinded neuroradiologists evaluated the cohort one year later, this time by agreement at the workstation.
In the ADC measurements part, two researchers, one blinded, manually placed ROI's in the anterior and posterior cingulate regions of 26 NF1 patients and their matched controls, and student t-test was used to assess for significance of differences in measured values.
Results: Cohen's Kappa for the three age groups showed very good agreement (Kappa coefficients were either 0.9 or 1.0).
Rater agreement at the workstation was 100%.
All subjects with a positive finding also had NF1 and the sign was not seen in any of the normal controls.
The prevalence of the sign was 43%.
ADC measurements showed significantly higher ADC values in the anterior cingulate gyrus of NF1 patients when compared to normal controls and also when compared to the posterior cingulate gyrus in NF1 patients.
Conclusions: Our results show that visual T2/FLAIR MRI abnormalities in the anterior cingulate gyrus are present in 43% of patients with NF1 from ages 2 to 19 years.
ADC measurements confirm a pathophysiological basis for this finding.
Future correlation with clinical manifestations, such as learning and behavioral manifestations in patients with NF1, are under way to further evaluate the clinical importance of this finding.
Tract-Based Spatial Statistical Analysis of Diffusion Tensor Imaging in Pediatric Patients with Mitochondrial Disease Seth Friedman, PhD, Seattle Children's, seth.friedman@ seattlechildrens.org; Andrew V. Poliakov, Sandra L. Poliachik, Dennis W. Shaw Purpose or Case Report: Often diagnosed at birth or in early childhood, mitochondrial disease presents with a variety of clinical symptoms, particularly in organs and tissues that require high energetic demand such as brain, heart, liver, and skeletal muscles.
In a group of pediatric patients identified to have complex I or I/III deficits, but with white matter tissue appearing qualitatively normal for age, we hypothesized that quantitative DTI analyses might unmask deficits in microstructural integrity.
Methods & Materials: DTI and structural MR brain imaging data were collected in 10 pediatric patients with confirmed mitochondrial disease and 10 clinical control subjects matched for age, gender, scanning parameters, and date of exam.
Paired Tract-Based Spatial Statistics (TBSS) were performed to evaluate differences in fractional anisotropy (FA) and mean diffusivity (MD).
Results: In patients with mitochondrial disease, significant widespread reductions in FA values were shown in white matter tracts.
MD values were significantly increased in patients, having a sparser distribution of affected regions compared to FA.
Results of TBSS statistical analysis will be shown.
To be shown in green is the mean FA skeleton which represents the centers of main white matter tracts.
All results p<.05.
Red and yellow represent a significant increase, blue and light blue represent a significant decrease.
Conclusions: Despite qualitatively normal appearing white matter tissues, patients with confirmed mitochondrial disease have widespread microstructural changes measurable with quantitative DTI.
This supports the evaluation of such metrics in other populations where gross imaging features may be normal.
To extend our studies to patients with other PLP1 mutations, we analyzed the brains of 52 male PMD patients (ranging in age from 2 to 45) and 9 female carriers for whom the PLP1 genotype had been determined and analyzed by MRI.
For each patient we measured, white matter volume (WMV) and the intercaudate distance (ICD).
The MRI data were correlated with functional disability scores (FDS) using a system we developed for clinical evaluation of PMD patients and which was validated by assessments of 22 PMD patients.
Brain volume and segmentation were measured using NIH image 1.62.
The average number of coronal slices analyzed from each patients MRI was 60 slices.
When graywhite contrast was not adequate, then the Intercaudate distance (ICD) and intercaudate ratio were measured as described in Caon et.
al., (2003) .
Results: Comparison of the MR measurements and the FDS demonstrated that white matter volume inversely correlates with functional disability, suggesting that the initial disability does correlate with the extent of myelination.
The intercaudate distance also correlated with the FDS, and may usefully substitute when gray-white matter segmentation is not possible.
Conclusions: PMD is a clinically and genetically heterogeneous disease caused by mutations in the gene encoding the major CNS myelin protein, proteolipid protein (PLP).
Myelin is a major target of disease pathogenesis in most cases of PMD, but how the various mutations cause clinical disability is not fully understood.
Our data demonstrate that the extent of brain white matter atrophy, measured directly by volumetric fractionation, or indirectly by analyzing the intercaudate ratio, is significantly correlated with the patient's functional disability.
White matter atrophy is thus the main cause of clinical disability in patients with PMD of all ages and mutation type.
Paper #: PA-074 Maturational Effects on Language Localization in Children Demonstrated by fMRI Susan Palasis, MD, Children's Healthcare of Atlanta at Scottish Rite, spalasis@yahoo.com; Binjian Sun, Laura L. Hayes, Richard A. Jones Purpose or Case Report: Language localization is of paramount importance when contemplating surgery in children with intractable epilepsy or brain tumors.
The potential risk of injury to language centers in the developing pediatric brain needs to be weighed against the potential benefits of surgery.
In the past, language localization was crudely and invasively determined using the WADA test.
Most institutions are now transitioning to non invasive localization using functional MRI (fMRI).
The purpose of our study was to analyze language localization relative to age in children using age appropriate language paradigms and fMRI.
Methods & Materials: Forty three healthy, English speaking, right handed children underwent fMRI evaluation for language localization.
The studies were performed on a 3 T system.
Three novel age appropriate language block paradigms were utilized, targeted both to expressive and receptive language processing.
These paradigms were the auditory category decision task (AUDCAT), the auditory description decision task (ADDT), and the Listening task.
The spatial statistical maps generated by the fMRI data were fused to the 3D anatomical MRI dataset.
Language areas were localized and statistical analysis was performed with age as the variable in a general linear model.
Results: Our results demonstrate a distinct trend in language localization and lateralization with brain maturation.
In the young age groups (less than 12 years) the localization tended to be less focused and bilateral in the frontal and temporal regions of the brain.
In the older age groups (greater than 12 years), language became more localized and lateralized to the expected left sided pattern.
The findings were more robustly demonstrated with the ADDT task and were statistically significant (p<0.05).
Conclusions: Our study clearly demonstrates the plasticity of language centers in the maturing pediatric brain.
This observation is significant for neurosurgical planning and rehabilitation in the pediatric population.
(3) no SLC26A4 mutations were found in 16, 12 and 47 subjects, respectively.
Significantly higher association with SLC26A4 mutations was found in bilateral EVA+V/C dysplasia (16/18).
Double mutations of SLC26A4 is more often associated with combined EVA+ V/C dysplasia, while a single mutation with EVA only.
Cochlear aplasia without EVA (0/2) and SNHL with normal imaging (3/21) are less likely associated with SLC26A4 mutation.
Conclusions: SLC26A4 mutation is highly associated with EVA and V/C dysplasia.
Once EVA with or without V/C dysplasia are found at imaging, genetic investigation is recommended for SLC26A4 mutation because of possible thyroid involvement.
moderate-severe HIE who were randomized to cooling (33.5°C for 72 h).
There were 73 in the hypothermia group and 63 in the control group.
All MRIs were reviewed by a cental reader masked to the clinical findings, groupings, and outcomes.
The MRI findings were scored according to pattern and extent of injury, including involvement of the cerebral hemispheres, basal ganglia, thalami, internal capsules, and other structures.
Brain injury scores were correlated with death or disability at 18 months postnatal age.
Results: No MRI abnormalities were observed in 38 of 73 infants (52%) in the hypothermia group and in 22 of 63 infants (35%) in the control group (P00.08).
Infants in the hypothermia group had fewer areas of injury (12%) as compared with the control group (22%, P0 0.02).
There were 51 of the 136 infants with death or disability at 18 months.
The brain injury score correlated with outcome of death or disability (P00.001) and disability among survivors (P00.0001).
Conclusions: Fewer areas of brain injury on MRI were observed following whole-body hypothermia.
The MRI brain injury score is a marker of death or disability at 18 months following hypothermia for term HIE.
.
Presence or absence of the "red dot" on FA color maps was correlated to clinical (ataxia, oculomotor abnormalities etc.)
and morphological data, and to FA and MD measurements.
Results: The "red dot" was absent in JS and HGPPS (genetic cross wiring impairment diseases) and present in COMA and WVS (no reported gene abnormalities so far) as in normal controls.
JS and COMA presented on MRI molar tooth appearance.
HGPPS presented "split pons" appearance.
JS and COMA patients presented oculomotor apraxia, WVS and HGPPS palsy of the horizontal gaze.
Mirror movements were found in 2 JS and in WVS.
Ipsilateral responses are present in HGPPS.
WVS presented multiple cranial nerves impairment.
In JS, FA and MD values of SCP, PT and PC were significantly lower than in normal controls (p >0.01).
In HGPPS high FA and low MD were found in PC and PT (p >0.01) and normal in SCP.
Conclusions: The "red dot" absence is unrelated to morphological or clinical abnormalities.
Absence of the "red dot" is associated to abnormal measurements of FA and MD in PC and PT( low in JS and high in HGPPS).
These findings indicate a pivotal role for the PC in the physiopathology of these diseases.
The "red dot" absence seems to be a marker of genetic cross wiring diseases.
In this view, COMA and WVS should not be considered as part of these diseases.
Sonographic Predictors of Intermittant Testicular Torsion in the Pediatric Patient Jennifer Williams, MD, Pediatric Radiology, Texas Children's Hospital, jlwilli1@texaschildrens.org; Marthe Munden Purpose or Case Report: Intermittent testicular torsion (ITT), defined as sudden onset unilateral scrotal pain with spontaneous resolution, is difficult to confirm both clinically and sonographically.
The purpose of this study was to determine if sonographic predictors exist for diagnosing ITT in the pediatric patient.
Methods & Materials: A search of the PACS data system for patients presenting with suspected intermittent testicular torsion was performed.
Fifteen patients with a total of 20 episodes presenting over a 2 year period were found.
A retrospective review of the medical records for clinical presentation, surgical outcome, and comorbidities was performed.
Scrotal ultrasound images and reports were reviewed for testicular size and echotexture, testicular flow, epididymal appearance, vascular bundle appearance, and presence of hydrocele.
Results: An abnormal appearance of the vascular bundle was found in 85% of episodes (17/20).
Initial absence of testicular flow followed by reperfusion during the scan was seen in 30% of episodes (6/20); 45% had increased flow (9/ 20), 10% had decreased flow (2/20), and 15% had normal flow (3/20) .
Nine of the 15 patients had surgery; of these 8 were found to have evidence of ITT and 1 was found to have acute testicular torsion.
Of patients with ITT, 88% (7/8) had an abnormal vascular bundle.
Testicular flow was not initially visualized but returned during the exam in 50% of patients (4/8), was increased in 38% of patients (3/7) and was decreased in 13% patients (1/8).
Conclusions: ITT is a difficult diagnosis.
The most reliable sonographic indicator is an abnormal spermatic cord, found in 85% of episodes and 88% of surgically proven ITT.
Dedicated views of the spermatic cord must be obtained in order to differentiate an abnormal epididymis from an engorged vascular bundle (the so-called pseudomass).
Attention to testicular flow is of particular importance.
While visualization of a transition from no or decreased testicular flow to normal flow during the sonogram is certainly diagnostic of ITT, increased testicular flow should not lead to false reassurance.
Purpose or Case Report: Testicular torsion is a common acute condition in boys requiring prompt and accurate diagnosis.
The objective was to evaluate ultrasound accuracy and findings, and clinical predictors in testicular torsion in boys presenting to the Stollery pediatric emergency department (ED) with acute scrotal pain.
Methods & Materials: Retrospective review of US, surgical and ED records for boys aged 1 month to 17 years, presenting with acute scrotum from 2008 to 2011, was performed.
Age, demographics, clinical symptoms, physical findings, US and surgical techniques, findings and diagnoses were recorded.
Surgical results and follow-up were used as the gold standard as all pediatric urology in our region is performed at our centre.
Results: 343 patients presented to ED with acute scrotum with the following diagnoses: 35 testicular torsion, 11 possible torsion-detorsion, 3 torsion of appendix testes, 135 epididymo-orchitis, and 159 other.
Of 266 US performed, 29 boys had torsion confirmed by surgery.
There were 8 inconclusive US reports, none of which had torsion at surgery or follow-up.
The false positive rate of US was 1.5% (4 patients), and there were no false negatives.
Six torsion patients had no US.
Median time from ED to US and surgery for torsion patients was 159 and 303 min.
Six patients had non-salvageable testes.
Diagnostic accuracy of US compared to surgery was 96% for torsion and 67% for other.
Sonographic heterogeneity was seen in 80% of patients with testes that the surgeon felt were non-viable at surgery and 72% of patients with viable testes (p00.35).
Sudden-onset scrotal pain (92%), abnormal position (86%) and absent cremasteric reflex (91%,) were most prevalent in torsion patients.
Conclusions: Color Doppler US is accurate and sensitive for diagnosis of torsion in the setting of acute scrotum.
Despite heterogeneity on pre-operative US, many testes were felt to be salvageable at surgery.
Rate of salvage of torsion was high.
Common symptoms and findings of torsion were sudden onset of pain, abnormal testicular position and absent cremasteric reflex.
Paper #: PA-080 Diagnostic Twists of Tubal Torsion Srikala Narayanan, MD, Children's National Medical Center, snarayan@childrensnational.org; Anjum N. Bandarkar, Dorothy Bulas Purpose or Case Report: Fallopian tube torsion is a rare cause of acute pelvic pain in a young female and requires prompt diagnosis for immediate surgical intervention.
Our purpose is to review varied imaging findings of surgically proven cases of tubal torsion.
Methods & Materials: Retrospective review of our data base from 2007 to 2011 revealed 7 cases of surgically proven fallopian tube torsion.
Ages ranged from 9 to 15 years of age.
All had pelvic ultrasound performed, 3 cases had additional CT performed for acute pelvic pain.
Results: US findings included thickened dilated tubular hypoechoic structure (5), cystic mass (4); adnexal (3), midline (1).
Five cases had normal ovaries bilaterally (2 with paratubal cysts).
CT imaging findings include dilated, fluid filled, thickwalled tube with internal hyperdensity (40HU) likely debris/ hemorrhage in 1 case.
Additional findings included cystic adnexal mass (3 cases), beak sign (1 case) and increased vascularity (1 case).
Secondary signs included free fluid (5), peritubular fat stranding (1), vascular congestion and thickening of the broad ligament (1) and enlarged draining vein (1).
Laparoscopic salphingectomy was performed in 3 cases (including 2 cases with isolated tubal torsion).
Laparoscopic detorsion was performed in a total of 4 cases.
In addition, laparoscopic cyst drainage was performed in 2 out of these 4 cases.
Detorsion with paratubal cystectomy and hemorrhagic ovarian cystectomy was performed in 1 of the 4 cases.
Conclusions: Imaging diagnosis of tubal torsion can be difficult.
It can occur in isolation with a dilated thickened tubular structure adjacent to a normal ovary or potentially mimic appendicitis, pyosalpinx, complex adnexal cyst or cystic adnexal neoplasm.
Presence of normal ovaries, beaked tapered tubular structure with intratubal fluid level and hemorrhage may help in making the diagnosis.
It is important to recognize this entity in a patient with acute pelvic pain to facilitate prompt tubal sparing surgery.
Paper #: PA-081 Adjusted Renal Length in Pediatric Bone Marrow Transplant Recipients Nicholas Bodmer, MD, University of Washington, nbodmer@gmail.com; Teresa Chapman, Sangeeta Hingorani, Marguerite Parisi Purpose or Case Report: Bilateral nephromegaly has been observed in the bone marrow transplant (BMT) patients at our institution.
This study aims to quantify this observation, thereby providing radiologists with an adjusted baseline agedetermined renal growth curve for BMT patients.
Methods & Materials: A retrospective clinical chart and imaging review was performed on 185 patients who underwent BMT between 2006 and 2010 and who had abdominal imaging including the kidneys.
Ultrasound, CT, and MRI exams were used for renal length measurement.
Renal lengths were assessed for each age group, first as an average length of all the patients within that age group overall, and subsequently as an average renal length by age group divided into the following time frames after transplantation: 0-30 days, 31-90 days, 91-180 days, and 181+ days.
Clinic chart information collected included BUN, creatinine, weight, and medication use.
Results: Renal length was measured using 278 imaging cases, distributed across each age group as follows: 6-12 months, N 011; 13 months-2.5 years, N 030; 2.6-4.5 years, N033; 4.6-7.5 years, N038; 7.6-11.5 years, N0 51; 11.6 years and higher, N0115.
Renal lengths were greater, on average, within every age group, compared with previously established normative age-related renal lengths (Rosenbaum et al.)
.
The augmented renal lengths universally were observed in the 0-30 day post-transplantation timeframe.
Return to normal renal lengths typically occurred by 6 months post transplant.
Clinic chart review revealed that the majority (87%) of patients received nephrotoxic medication within two weeks of imaging.
Conclusions: Pediatric BMT patients have larger kidneys in the absence of known renal disease than age-matched peers.
A revised, age-based renal length chart for post-BMT patients has been generated which should help prevent the misdiagnosis of nephromegaly in this population, eliminating unnecessary diagnostic evaluations.
Multiple etiologies to explain renal enlargement in these patients are possible, including fluid overload, nephrotoxic medication, or direct effect of the transplant.
Purpose or Case Report: MR urography can be a comprehensive exam for anatomical and functional pediatric renal evaluation.
Quantification of renal function may benefit when dynamic contrast enhanced images can be obtained at high spatiotemporal resolution and with minimal respiratory motion artifacts.
Though respiratory triggering may decrease motion artifacts, it results in loss of temporal resolution by a factor of about three.
A two-echo gradient echo sequence with segmented outer k-space sampling and view-sharing/Dixon image reconstruction (DISCO, DIfferential Subsampling with Cartesian Ordering) was chosen as a starting point due to its high temporal resolution.
It was then modified to enable respiratory triggering while maintaining temporal resolution of one temporal frame every one to two respirations, with segments of k-space only acquired in the expiratory phase of respiration.
Imaging parameters were: 12°flip angle, ± 167 kHz bandwidth, TR~3.56, matrix 256x200, FOV 28-34 cm, slice thickness 4 mm, and 2x2 spatial acceleration.
With IRB approval and informed patient consent 9 consecutive patients referred for MRI renal function evaluation were recruited (age range; 0.5 to 9.6 years, mean±SD: 3.99±3.6 years; males 78% females 22%), and scanned on a GE 3 T MR using a 32-channel torso array with the respiratory-triggered high spatiotemporal resolution technique to extract regional GFR maps.
Two readers by consensus assessed image qualitative SNR, motion artifacts and volumetric fat-water suppression performance.
Results: Data acquisition was obtained to completion in all subjects without triggering failure.
Temporal resolution was approximately 12 s for two respiratory cycles.
No case had major fat suppression failure, whereas minor fat suppression failure was seen in 11% (95% C.I.
0 to 37%).
All cases had diagnostically acceptable SNR.
No motion artifacts were noted in 7/9 cases, while some artifacts with ghosting in 2/9 cases.
Regional GFR maps could be successfully extracted for each patient without the need for image registration.
Attached figure shows image quality.
Conclusions: View-sharing offsets loss of temporal resolution from respiratory triggering.
Thus, high spatiotemporal resolution renal dynamic contrast enhanced respiratory triggered images can be obtained with minimal motion artifacts in a pediatric clinical setting to evaluate renal function.
Disclosure: Dr. Chowdhury has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Methods & Materials: The study cohort was selected from the IRB approved Children's Oncology Group AREN03B2 study.
Cases are evaluated for pre-operative WT rupture based on central review of surgical/pathology findings.
70 WT cases with rupture were matched to 70 WT controls by age and tumor weight (within 6 months and 50 g).
CT scans were independently reviewed by 2 blinded radiologists, for presence/absence of rupture and the following CT signs: poorly circumscribed mass, perinephric fat stranding, peritumoral fat planes obscured, retroperitoneal fluid, ascites beyond cul-de-sac, peritoneal implants, ipsilateral pleural effusion, intratumor hemorrhage.
Sensitivity, specificity of CT for assessing pre-operative WT rupture was determined.
The relationship between CT signs and rupture was assessed by McNemar's test, and the most predictive CT signs determined by backward selection multivariate logistic regression.
Results: Sensitivity, specificity for detecting WT rupture were: reviewer 1-53.7%, 88.4%, reviewer 2-70.2%, 88.4%.
Kappa coefficient for interobserver agreement was substantial: 0.76 (p<0.0001).
All CT signs tested, except peritoneal implants and intratumoral hemorrhage, had significant association with tumor rupture (p<0.01).
For reviewer 1, ascites and fat stranding around tumor were most predictive (Odds ratio 18.359 and 10.554, P<0.01).
For reviewer 2, ascites and retroperitoneal fluid were most predictive (OR 8.345 and 4.916, P<0.01).
Conclusions: CT has high specificity but relatively low sensitivity for detecting preoperative WT rupture.
The presence of ascites beyond cul-de-sac is the best indicator of preoperative rupture, followed by fat stranding and retroperitoneal fluid.
Paper #: PA-084 The Failed Pyeloplasty: Evaluation with MR Urography Damien Grattan-Smith, Children's Healthcare of Atlanta, damien.grattansmith@mac.com; Ricahrd Jones, Stephen Little, Wolfgang Cerwinka, Hal Scherz, Andrew Kirsch Purpose or Case Report: To identify imaging characteristics associated with failed pyeloplasty seen with MR urography.
We have performed MR Urography in 142 children following pyeloplasty.
From this group, 16 children had follow-up surgical intervention with repeat pyeloplasty or balloon dilatation of the UPJ.
Imaging features reviewed included degree of hydronephrosis, calyceal transit times, renal transit times, signal intensity versus time curves, as well as functional analysis based on volumetric and Patlak differential function and change in the asymmetry index.
Results: All children who underwent a second surgical procedure had delayed calyceal transit times.
The degree of hydronephrosis and renal transit times were either stable or worse when compared to pre-operative evaluation.
Functional derangement could show stability, slight improvement or deterioration.
The asymmetry index estimated the severity of the obstruction.
Conclusions: MR urography is valuable in the evaluation of children who have undergone pyeloplasty.
The calyceal transit time appears to be the most reliable discriminator when comparing successful and failed pyeloplasty.
Calyceal transit times may be prolonged before the hydronephrosis becomes progressive.
Disclosure: Dr. Grattan-Smith has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Initial attempts at interpreting functional MR Urography (fMRU) can be challenging.
A time intensive navigation through a multitude of both subjective and objective functional results is necessary to render a useful interpretation.
This is a guided review of fMRU, noting the important functional findings in high-grade unilateral pelvicalyceal dilatation (PCD), in the absence of ureterectasis, with a contralateral normal kidney allowing for an optimal functional comparison.
Methods & Materials: A retrospective functional evaluation of 16 cases with unilateral pelvicalyceal dilatation (PCD), without prior pyeloplasty, was conducted.
The fMRU studies were carried out according to a standard protocol and post-processing using the CHOP-fMRU software.
This included IV hydration, bladder catheterization and IV Furosemide administration.
Fifteen minutes after diuretic administration, a dynamic coronal 3D fat saturated T1 sequence was performed in a supine position over 15 min.
A sagittal 3D T1 and delayed single coronal T1, both fat saturated, followed in a supine and/or prone position.
The following functional features were evaluated: visualization of the ureter, the presence of a contrast-urine level and swirling of contrast in the dilated renal pelvis.
The functional results included in the analysis were calyceal transit time (CTT), renal transit time (RTT), time-to-peak (TTP), parenchymal volume (PV), differential renal functions (volumetric-vDRF, Patlak-pDRF and volumetric Patlak-vpDRF) and the difference between vDRF and pDRF.
Results: 16 patients were comprised of 8 males and 8 females with an age range of 0.1-17.0 years (median 0.8 yrs).
Of the kidneys with PCD, the ureter was visualized in 10, 3 during the dynamic sequence, 4/9 during supine delay and 3/7 only in prone position.
A contrast-urine level was present in 14 of the dilated systems, and swirling in 6.
The ureter was visualized during dynamic sequence in all contralateral normal kidneys and at no time was swirling or a contrast-urine level identified.
The average functional parameters are seen in Table 1 .
A statistically significant (p<0.05) difference between the normal and dilated pelvicalyceal systems was achieved in TTP, pDRF and vpDRF for this small sample size.
Conclusions: Awareness of multiple functional features and the range of calculated results may aid in subsequent combined interpretation of the fMRU with the morphologic analysis.
Disclosure: Dr. LeCompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
2007 and 2011.
Demographics, clinical presentation , diagnostic studies and treatment outcomes were evaluated.
Post-procedure imaging was evaluated for clot burden reduction (patency) and residual venous stenosis by two-reader consensus.
Results: Ten patients (5 male; 5 female, mean age 16 years, range 15-18) presenting with acute upper extremity swelling and pre-procedure imaging revealing 100% occlusion of the axillary and subclavian veins received successful endovascular therapy.
All 10 patients underwent infusion catheter placement for thrombolysis with tissue plasminogen activator or urokinase.
6 patients received additional pharmacomechanical treatment.
Angioplasty was also performed in all patients.
The mean treatment duration was 33 h (range 16-62).
Post-procedural imaging revealed that 9 of 10 patients achieved 75-100% patency (clot burden reduction) and 1 patient achieved 50-75% patency.
The residual venous stenosis was graded: 5 patients had 0-25% stenosis, 2 patients had 25-50% stenosis and 3 patients had 75-100% stenosis.
All patients were discharged on full anticoagulation therapy with low molecular weight heparin.
9 patients had surgical rib resection postthrombolysis with an average length of time from thrombolytic therapy to surgery being 32 days (range 14-73).
3 patients had re-thrombosis events during the follow-up period (mean 10 months; range 1-34), with one re-thrombosis event occurring within one week of thrombolytic therapy, prior to surgery and the other two occurring 3-5 weeks post-rib resection.
There were no procedure related complications.
One patient was lost to follow-up after initial successful catheter directed therapy.
Conclusions: Percutaneous endovascular techniques such as pharmacomechanical thrombolysis and angioplasty appear to be feasible and safe options for Paget Schroetter Syndrome in otherwise healthy adolescent patients.
In attempt to prevent rethrombosis and chronic symptoms, we refer all patients for adjunctive surgical decompression.
Future larger studies are needed to address optimal strategies for these patients.
Combined 3D Fluoroscopy Image Guided Percutaneous Intervention with Real-Time Optical Sensing at the Tip of a Needle for Tissue Characterization Rami Nachabe, Philips, rami.nachabe@philips.com; John M. Racadio, Drazenko Babic, Ross Schierling, Jasmine Hales, Benno Hendriks Purpose or Case Report: To investigate the feasibility and potential of real-time tissue characterization at the tip of a needle with diffuse optical spectroscopy (DOS) sensing capabilities during 3D fluoroscopy guidance using cone beam CT and dedicated needle path planning software.
Methods & Materials: A C-arm X-ray system that combines fluoroscopy and 3D imaging from a cone beam CT was used to image a woodchuck with hepatocellular carcinoma (HCC).
The imaging system enabled needle path planning, which was used to perform insertion and navigation of a needle toward the liver tumor.
The needle was integrated with optical fibers for real-time tissue spectral sensing at its tip.
Optical spectra measurements were obtained continuously as the needle passed through healthy liver tissue and then into the tumor.
From the diffuse optical spectra measurements, the following clinical parameters were extracted for tissue characterization: blood volume fraction, blood oxygenation, lipid volume fraction and tissue light scattering (related to tissue density).
The tissue parameters were compared for healthy liver and tumor using the Kruskal-Wallis test.
Results: The tissue density of the healthy liver was lower than that of the tumor.
Higher blood and lipid volume fractions as well as oxygenation levels were observed in the healthy liver as compared to the tumor.
All differences were statistically significant (P<0.01).
Additionally, a much wider heterogeneity in tissue density was observed in the tumor as opposed to the healthy liver.
Conclusions: Differences in tissue properties between tumor and healthy liver enable discrimination between these two types of tissues.
Adding real-time optical sensing at the tip of a needle to 3D fluoroscopy image guidance is a feasible technique that complements the imaging information with relevant physiological parameters; it facilitates more precise definition of tumor boundaries despite any target motion during needle insertion.
Disclosure: Dr. Racadio has disclosed that he is a consultant for Philips Healthcare and receives travel reimbursement.
Rami Nachabe, Drazenko Babic and Benno Hendriks are employees of Philips Healthcare.
Methods & Materials: Two children aged 6 months and 3 months were treated at this institution for liver failure resulting from urea cycle disorders, with a hepatocyte transplant procedure.
The recipient liver was irradiated prior to transplant to facilitate engraftment.
The procedure involves the injection of prepared hepatocytes from a suitably screened, compatible donor, via a main portal vein branch into the recipient liver.
In both procedures access to the umbilical vein was achieved by the surgery service and a 4 French arterial sheath was placed.
A 4 French angled catheter was used for diagnostic runs and to access the right and left main portal vein.
A 3 French Fogarty catheter (Edwards Lifesciences, Irvine, Ca) was placed to isolate each portal vein branch in turn and hepatocytes injected using hand injections.
Pressures in the main, right and left portal veins were measured and hand injections of contrast made at regular intervals.
Careful attention must be paid for evidence of pruning of portal branches, indicating occlusion of small portal branches, or portal to hepatic vein shunting.
If shunting is seen, infusion must be stopped as embolism of hepatocytes into pulmonary arteries may result with serious clinical sequelae Results: In both patients, the desired number of hepatocytes were successfully delivered into the recipient liver.
In both cases, mild pruning of the portal vein branches was evident at the end of the procedure.
Portal vein presssures remained steady.
There was no venographic or clinical evidence of pulmonary arterial embolization.
Conclusions: The interventional radiologist plays a central role in the hepatocyte transplant procedure.
Familiarity with catheterizing portal branches from an umbilical vein approach, measuring venous pressures, using small occlusion catheters and recognizing venographic end points such as portal vein pruning and portal to hepatic vein shunting are necessary to the safe and successful completion of this new technique.
Purpose or Case Report: The aim of the study was to evaluate the trends in term of type of tube placed, number of procedures per year, number and age of the patients as well as the number of procedures per patient and the interval of time between two placements, and finally the irradiation burden borne by the patients.
Methods & Materials: After REB approval the radiologic files of the patients who underwent naso-duodenal-jejunal (NDJ) or gastro-jejunal (GJ) or jejunal (J) tube placement under fluoroscopy over the past five years (2006 to 2010) were extracted from the RIS and reviewed.
The results were tabulated as a single batch and stratified by year.
Results: Eighty-nine patients representing 234 procedures (155 NDJ, 77, GJ, 2 J) were included.
Only 38 patients underwent a single procedure.
The average number of procedures per patient was 2.6 with a maximum of 12 during the study period.
The average patient's age was 55.3 months (SD074.88, median0 11.43).
The average fluoro time per procedure was 7.2 min (SD08.3, median05.0).
The average interval between two procedures was 58 days (SD0108,44, median018).
The average fluoroscopy time per patient combining those having a single procedure and those having multiple ones, was 19.57 min (range 0.3 to 151.7, SD024.36, median012.45).
Conclusions: Fluoroscopic placement of enteric tubes delivers a significant amount of irradiation.
Our data led to two interventions with respect to insertion and management of the tubes.
On one hand, when the attempt pursued by a radiologist is not successful after 10 min of fluoroscopy other strategies should be considered including another operator or an alternative technique for tube positioning.
On the other hand, information will be distributed toward the clinicians and nurses in order to improve the management of these tubes and avoid fortuitous displacement which was responsible of a significant amount of repeated procedures leading to undue irradiation.
Purpose or Case Report: To evaluate drug elution pharmacodynamics of doxycycline in an albumin-based solution, as used in percutaneous imaging-directed therapy of aneurysmal bone cyst (ABC) and microcystic lymphatic malformation (LM) Methods & Materials: Doxycycline mixed with 25% human serum albumin (HSA), and doxycycline mixed with saline solution (both 20 mg/ml) were evaluated using a fluid diffusion chamber system over 8 h, recording pH and doxycycline concentration.
Static pH and doxycycline concentrations were recorded every 5 min for the first 180 min, then every 30 min for a total of 8 h, averaged over 3 trials in each of the HSA and saline systems.
Statistical analysis evaluated standard deviation and rate of change for the 3 trials in each system.
Drug elution dynamics data were correlated with clinical experience in the doxycycline/albumin treatment of 49 patients (233 treatments) with aneurysmal bone cyst (ABC) and 63 patients with 1263 lymphatic malformation microcysts.
Results: Drug elution was linear in both the HSA and saline systems, with statistically significant (p<.001) slower elution drug release from the albumin system as compared with the doxycycline and saline solution, both over 3 and 8 h. Purpose or Case Report: To describe a successful Interventional Radiologic approach to the management of Paget Schroetter Syndrome presenting as acute arm swelling in adolecent athletes.
Methods & Materials: Institutional Review board approval was obtained for this retrospective study.
Five patients aged 14 to 18 years (mean 16.5 years) were treated at this Institution over a 2 year period all presenting with acute arm swelling (July 2009 -July 2011).
Ultrasound confirmed subclavian vein thrombosis in all cases.
All were treated with placement of an infusion catheter (EV3, Plymouth, MN), infusion of Tissue plasminogen Activator (TPA) at a rate of 1 mg/hour overnight and aspiration of remaining clot with a "Trellis" (Bacchus Vascular, Santa Clara, CA, USA) thrombectomy device.
Results: Clot was successfully removed in all five patients.
Complete clearance of clot was confirmed by contrarst venography in all cases.
In four patients balloon angioplasty of a narrrowing at the junction of the subclavian and brachiocephalic veins was carried out.
In one, the thrombus recurred within 6 h. The patient was retreated the next day with aspiration of clot using the "Trellis" device and an infusion catheter placed with low dose (0.5mgs/hour) TPA commenced until surgical review; this patient was operated on within 48 h of final thrombolysis.
All patients were seen by a Vascular Surgeon with an interest in this condition.
All underwent surgical decompression; at end of the study period all patients were asymptomatic.
Conclusions: Interventional Radiologic management of acute axillo-subclavian thrombosis due to Paget Schroetter syndrome is safe and highly successful in the adolescent population.
Early recurrence of thrombus is not uncommon and prompt surgical consultation with a view to early surgical decompression is recommended.
Purpose or Case Report: Diagnostic reference levels (DRL) or target radiation dose ranges for pediatric CT scans are needed in the U.S.
The first U.S. pediatric CT dose index registry (QuIRCC) within the American College of Radiology recorded estimates of patient radiation dose using a new method (SSDE) based on body width(BW) for the purpose of developing Diagnostic Reference Levels (DRL).
In addition to developing DRL at the 75th percentile, the purpose of this study was to determine the SSDEs associated with the lower range of acceptable image quality through subjective image quality evaluation.
Methods & Materials: Six children's hospitals participated in a retrospective review of abdominal CT with IV contrast on patients <18 yrs of age.
From 939 exams, each site submitted de-identified images for selected cases based on SSDE and patient width.
A total of 106 cases were selected from the lowest, first quartile and median SSDE.
Six investigators reviewed 3 images from each case under identical viewing conditions and rated them for subjective quality according to a score sheet and reference scale of images with known quantum mottle.
Cases were considered non-diagnostic if at least 3 of 6 reviewers ranked them as such.
Results: First, second, and third quartile SSDE and CTDI-vol32 values from 6 sites for each BW will be shown.
6/ 106 cases were ranked non-diagnostic by the reviewers.
4/6 non-diagnostic cases were below the 10th percentile based on SSDE.
5/6 of "non-diagnostic" cases had SSDE less than the 25th percentile.
The unacceptable case with SSDE above the 25th percentile (16 cm, SSDE 8.2 mGy) was due to subcutaneous metal implant with artifact.
The QuIRCC 75th percentile using CTDIvol 32 for a 5 yr old is 7.1 mGy which is 30% lower than the ACR CT accreditation data's published 75th percentile.
Conclusions: This consortium developed target dose ranges (DRLs) for CT of the abdomen with IV contrast for routine exam indications based on evaluation of image quality that establish lower and upper ranges (25-75 percentile) of patient dose(using SSDE) associated with clinically acceptable images.
This study demonstrates that pediatric radiologists in this consortium are comfortable interpreting images at or above the 25 percentile SSDE and judged all but one image within this target range as diagnostically acceptable.
Table 1 ).
With the exception of neonate chest, most used age-based techniques; only two centers reported using thickness.
No survey used grids for wrist images, while 2/3 of the surveys used grids for chest and abdomen exams in 5-year-olds.
At the most common SID there was up to a 60 kVp variation (5year-old chest AP) and up to 8-fold variation in mAs (13 year old Scoli Lat).
Only two surveys used equipment that displayed the new IEC exposure index.
Conclusions: Participants report variability in the techniques and methods used to acquire common radiographic studies, reflecting differences between detector types and users.
Radiologists, technologists, medical physicists, manufacturers, and the FDA have an opportunity to work together to standardize the techniques based on detector type to optimize radiation exposure for pediatric radiographic exams.
Disclosure: Dr. Don has indicated that he performs contract research for Carestream and that he is on the speaker's bureau for Siemens and receives an honoraria.
Purpose or Case Report: This study assesses community adoption of CT radiation dose guidelines after a 10-year international initiative to reduce medical radiation exposure in children.
Size-specific dose estimates (SSDE) from community pediatric body CT scans are compared to SSDE from matched scans obtained at a children's hospital that adheres to Image Gently Campaign principles.
We reviewed 112 pediatric CT scans (14 chest (C), 80 abdomen/pelvis (AP),18 chest/abdomen/ pelvis (CAP)) transferred from 32 community imaging centers to our university children's hospital between July 2010 and February 2011.
Community scans were acquired with variable parameters and reconstructed with traditional filtered back projection (FBP).
Comparison was made to 432 children's hospital CT scans, performed in accordance with principles of the Image Gently Campaign.
Because iterative reconstruction (IR) software was added to our scanner during the study, enabling us to reduce CTDIvol by 60%, children's hospital scans were divided into two groups: A) 213 scans obtained with standard weightbased pediatric protocols and FBP (October 2009-October 2010; 58 C, 110 AP, 45 CAP) and B) 219 scans obtained with reduced-dose weight-based pediatric protocols and blended IR/ FBP (October 2010-April 2011; 85 C, 104 AP, 30 CAP).
CTDIvol and greatest lateral dimension were recorded from each scan and were used to calculate SSDE.
Mean SSDE from community scans was compared to mean SSDE from children's hospital groups A and B.
Statistical analysis was performed with Student's t-test.
Results: Patient age range was 0-17 years in both community and children's hospital groups.
Mean SSDE for community C, AP, and CAP scans was 1.7, 1.3, and 1.6 times higher than mean SSDE for matched scans in control group A (p<0.001) and 5.0, 2.8, and 3.7 times higher than mean SSDE for matched scans in control group B (p<0.0001).
Conclusions: SSDE was significantly higher for community pediatric body CT scans than for matched scans performed at a children's hospital that adheres to Image Gently Campaign principles.
Results suggest that more community outreach and education are required in implementation of low-dose CT protocols outside of children's hospitals.
Concurrent use of IR provides a means of achieving even greater SSDE reduction than is possible with FBP alone and should be encouraged.
Paper #: PA-095 Optimization of Tube Voltage and Current in Size-based Pediatric CT Imaging: A Phantom Study Boaz Karmazyn, MD, Radiology, Riley Hospital for Children, bkarmazy@iupui.edu; Yun Liang, Keith Kaser, Peter Johnson, Mervyn Cohen Purpose or Case Report: Determine the change in CT dose index (CTDIvol) required to maintain the same quantum mottle noise when using lower tube voltages (80 and 100 kVp) relative to 120 kVp in different sized cylinder water phantoms (CWP) representing a wide range of pediatric body sizes.
We performed 256 MDCT scans of 10, 20, 25, and 35 cm CWP.
Thirty scans were performed for each phantom.
The tube currents ranged from 50 to 500 mAs with increments of 50 mAs, and the tube voltage levels were 80, 100, and 120 kVp.
The noise (standard deviation in HU) was measured using center region of interest (ROI) that was 80% of phantom's area.
Two other ROIs (each 2% of the area) were placed at the center and periphery of the phantom images to measure noise gradient.
Results: In the smallest (10 cm) CWP, approximately the same noise level was maintained with all three tube voltages without a significant change in CTDIvol.
For the 20, 25, and 35 cm phantoms, the average CTDIvol needed to be increased by 2%, 4%, and 19%, respectively, to maintain same noise level when the voltage was decreased from 120 to 100 kVp.
The average CTDIvol needed to be increased by 15%, 22% and 52% to maintain the same noise level in the 20, 25, and 35 cm CWP when the tube voltage was decreased from 120 to 80 kVp.
The difference between central and peripheral noise increased on average by 11.1%, 19.6%, 23.7%, and 28.0% in the CWP of 10, 20, 25, and 35 cm, respectively.
In each CWP, the central to peripheral noise difference was more pronounced (up to 3.7% more) with decrease in kVp from 120 to 100 or 80.
Conclusions: Noise measurements in the water phantom model indicate that tube voltage could be decreased from 120 to 80 in CWP of 10 cm without significant change in CTDIvol.
It is also possible to decrease the voltage from 120 to 100 kVp with a minimal (< average 5%) increase in dose in CWPs of 10, 20, and 25 cm.
The noise gradient increases with larger CWP and smaller kVp.
Paper #: PA-096 Comparison of Radiation Dose Estimates, Image Noise, and Scan Duration in Pediatric Body Imaging Using 320-Row and 64-Row CT Jennifer Johnston, MD, Radiology, Cincinnati Children's Hospital Medical Center, jhtai@yahoo.com; Daniel J. Podberesky, Erin Angels, Terry T. Yoshizumi, Greta Toncheva, Donald P. Frush Purpose or Case Report: To compare effective dose (ED) estimates, image noise, and scan duration for pediatric chest, abdomen and pelvis protocols using 320-row and 64-row CT scanners in various acquisition modes.
Methods & Materials: Organ doses were measured using 20 MOSFET dosimeters.
Dose, scan duration, and noise measurements were made in a 5-year-old anthropomorphic phantom for conventional helical, 160-detector helical and volume acquisition modes for chest, abdomen and pelvis protocols on a 320-row CT, and for helical mode on a 64row CT (Aquilion ONE and Aquilion 64, Toshiba Medical Systems, Otawara, Japan) using similar scan parameters representing currently used clinical protocols.
Mean organ doses from three runs for each protocol, in combination with ICRP 103 tissue weighting factors, were used to obtain ED for each protocol.
Noise was measured as the standard deviation of Hounsfield units in 3 equivalent locations at 4 levels for each protocol with an ROI tool.
ED and noise were compared with a paired T-test or sign test.
Results: Compared to helical acquisitions on the 64-row CT, ED of all tested acquisition modes on the 320-row volume CT were significantly lower for chest, abdomen/pelvis (AP) and chest/abdomen/pelvis (CAP) protocols (Table) .
Scan durations were lower across the board on the 320-row volume CT.
Compared to acquisitions on the 64-row CT, noise was in general similar to those on 320-row CT protocols, but some acquisition protocols on the 320-row CT produced greater noise (Table) , specifically volume acquisition for chest CT and 160-detector helical and volume modes for AP and CAP protocols.
Conclusions: Dose savings can be achieved for chest, AP and CAP CT examinations on a 320-row CT scanner compared to helical acquisition on a 64-row CT, with shorter scan durations.
Image noise was in general comparable between protocols.
Although noise differences between some modes did reach statistical significance, the impact on overall image quality will need to be studied further.
Paper #: PA-097 The Observed to Expected Total Fetal Lung Volume as a Predictor of Short-and Long-Term Morbidity in Surviving Infants with Congenital Diaphragmatic Hernia Emily Stenhouse, The Royal Hospital for Sick Children, emilysten@doctors.org.uk; Neil Patel, Judith Simpson, Watt Andrew, Gregor Walker, Carl Davis Purpose or Case Report: Observed-to-expected total fetal lung volume (O:E TFLV) is a validated MR measure which we have previously demonstrated to be significantly reduced in non-surviving infants with Congenital Diaphragmatic Hernia (CDH).
Our aim was to investigate the relationship between O:E TFLV and short-and long-term morbidity outcomes in surviving infants with CDH.
Methods & Materials: A retrospective analysis of cases of isolated left-side CDH referred to our institution for fetal MR evaluation between 24-35 weeks.
MR imaging studies were performed on a 1.5 T Philips system using a phased array body coil.
The observed TFLV was calculated by multiplying the summed area of the region of interest by the section thickness.
The expected TFLV was calculated with a formula previously described in the literature using the gestational age of the fetus.
The observed TFLV was expressed as a percentage of the expected TFLV at a given gestation.
Morbidity outcome data was obtained from the case records of all surviving infants.
Specific measures of illness severity relating to short-term intensive care management and long-term outpatient management were recorded.
Differences in O:E TFLV between outcome groups were assessed by t-test.
Results: 18 liveborn infants with isolated left-side CDH and antenatal MR scans were identified.
Scans were performed at 24-35 weeks gestation.
12 infants survived to discharge; gestation 38.5 (36.0 -39) weeks, birth weight 3.17 (2.03-3.66) kg.
Median length of admission was 38 (23-103) days, median duration of follow-up was 3.1 (0.7-5.4) years.
O:E TFLV was significantly lower in non-surviving infants; 23 vs. 37%, P0 0.005.
O:E TFLV was significantly lower in infants who received High Frequency Oscillation Ventilation (HFOV) versus those who were conventionally ventilated (29% vs 41%, P00.05).
O:E TFLV was also significantly lower in those infants who had a length of admission greater than the median of 38 days (29% vs. 43%, P00.02).
O:E TFLV trended lower with other measures of increased morbidity; inhaled nitric oxide use, patch repair of diaphragm, rehospitalisation within 1 year, supplemental feeding at discharge, gastro-oesophageal reflux, and developmental delay.
Conclusions: As well as predicting survival, lung volume measurement by O:E TFLV is a promising predictor of outcome and morbidity in surviving infants with CDH.
Further studies in larger populations are required to provide quantitative predictive risk data.
Characterization of the Inherent Acoustic Noise of a Dedicated NICU MRI System Jean Tkach, PhD, Cincinnati Children's Hospital Medical Center, jean.tkach@cchmc.org; Yu Li, Ron G. Pratt, Christopher Villa, Beth M. Kline-Fath, Charles Dumoulin Purpose or Case Report: We have developed a small foot print 1.5 T MRI scanner specifically for neonatal imaging that can be easily installed in a Neonatal Intensive Care Unit (NICU).
The scanner has a maximum patient bore diameter of 21.8 cm (without RF coil), and roughly twice the gradient performance of the best conventional adult whole-body 1.5 T MR systems.
It is known that sensory stimulation such as acoustic noise can elicit autonomic instability in both term and preterm neonates.
The inherent noise properties of the NICU MRI system were measured as part of the initial safety evaluation of the system and compared against that of a conventional 1.5 T MRI system.
To evaluate the inherent acoustic noise characteristics of the NICU MRI scanner, Sound Pressure Level (SPL) measurements were performed on it and on a conventional adult sized whole body 1.5 T HDx GE MRI system (GE Healthcare, Waukesha, WI).
A Brüel & Kjaer model 2250 sound level meter (Brüel & Kjaer Sound & Vibration Measurement A/S, Denmark) was used to perform the SPL measurements for 6 several different MR acquisitions (spin echo, gradient echo, fast RF spoiled gradient echo, fully balanced steady state free precession, gradient echo echo planar, and diffusion weighted) using acquisition parameters consistent with clinical protocols.
The MR sequences, acquisition parameters, noise measurement equipment and methodology were identical for the two MR systems.
The maximum SPL in units of A weighted decibels (dBA) was recorded for each of the MR acquisition/MR system combinations evaluated.
Results: The maximum SPL values measured during each of the 6 MR acquisitions were lower for all sequences (average 11.33dBA (range05-18dBA)) for the NICU MRI unit as compared to the conventional MRI scanner ( Table 1 ).
The average measured maximum SPL value, reported in dBA, across all 6 acquisitions was 86.2±2.6 for the NICU scanner, and 97.5±2.9 for the conventional MRI scanner.
The highest SPL values were measured for the diffusion-weighted sequence: 85 and 103dbA, for the NICU and conventional MRI scanner respectively.
Conclusions: Because of the smaller dimensions of the gradient coils in the NICU MRI system, acoustic noise is less than that of conventional MRI scanners despite the superior gradient performance of the smaller coils.
The lower inherent acoustic noise level of the NICU system provides improved safety for the neonate, and facilitates siting of the unit in the NICU.
Disclosure: Dr. Tkach has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Paper #: PA-099 Late Neurologic Events in Extremely Premature Infants Carlos Guevara, MD, Radiology, Duke University, cjg7@duke.edu; Brett Bartz, Caroline L. Hollingsworth, Caroline W. Carrico, Michael C. Cotten, Charles M. Maxfield Purpose or Case Report: Germinal matrix hemorrhage (GMH) is a major complication of prematurity.
Persistence of germinal matrix and immature neurovascular autonomic regulation in the premature neonate is thought to predispose to GMH.
Most GMH in premature population occurs during the first 4 days of life, and yet the persistence of the germinal matrix to 32 weeks gestation may allow for post-natal GMH outside of the immediate perinatal period.
To our knowledge, this is the first systematic review of late GMH (after the first week of life) in a large population of extremely preterm neonates (less than 28 weeks of gestation).
This IRB approved retrospective review included patients weighing less than 750 g or born at less than 28 weeks of gestation from 2008 through 2010.
The study population included 150 infants who had a head ultrasound (HUS) within the first week of life and at least one follow HUS after the first week of life.
All HUS were reviewed by three experienced pediatric radiologists for the presence and grade of ICH or late developing hemorrhagelike lesions (HLL).
Infants with and without HLL were evaluated for several clinical variables, including neurodevelopmental outcomes (Bayley scales).
Results: Average gestational age of study population was 25.1 weeks.
The incidence of GMH in the first week of life was 34% Grade 1, 38.6% Grade 2, 4.9% grade 3/ 4, and 2.2% posterior fossa.
New echogenic foci (HLL) at the caudothalamic groove were seen in 13.3% after the first week of life.
70% of these lesions were bilateral.
A four-fold increase in incidence of HLL was seen in infants <750 g compared to those> 750 g. Higher grade hemorrhages were not seen in this patient population, although 6% of infants had late posterior fossa hemorrhages.
The clinical course of infants with HLL trended towards a higher incidence of stressors, but this was not statistically significant.
The Psychomotor Development Index scores were lower than those infants without hemorrhage.
Conclusions: Small HLL at the caudothalamic groove are common in extremely preterm infants after the first week of life.
Higher grade (2-4) hemorrhages were not seen.
There were no cases of intraventricular extension and no direct complications.
If isolated, this finding necessitates no follow-up imaging, but may be associated with poor neurodevelopmental outcome.
Disclosure: Dr. Guevara has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: TOF/APV is a rare congenital heart lesion in which pulmonary arteries may become aneurysmally dilated and compress adjacent airways.
Pulmonary arterioplasty is often required to relieve tracheobronchial compression in addition to intracardiac repair.
The purpose of this study was to review pre and postnatal imaging findings and their impact on patient management and clinical course.
Methods & Materials: A retrospective database search identified 9 infants with TOF/APV between 2005-2011 (4 fetal diagnosed cases and 5 diagnosed postnatally).
For FDC, prenatal ultrasound (US) and fetal MRI were correlated with postnatal CT for the size of the central pulmonary arteries, airway compression, and presence / distribution of air trapping/atelectasis.
For all cases postnatal CT findings (between 3-9 days of age) were correlated with clinical management and outcome.
Results: Prenatal diagnosis of TOF/APV was suggested sonographically, based on dilated central PAs, between 21-28 weeks gestational age (GA).
Fetal MRI, performed between 32-37 weeks GA confirmed the diagnosis and aneurysmal central PAs and demonstrated air trapping &/or atelectasis in 3/4 with normal appearing lungs in 1 fetus.
Size of the PAs (4/4) and presence and distribution of lung abnormality (3/4) correlated closely between fetal MRI and postnatal CT, although detailed visualization of the central airway/ vascular relationships were better defined on CT. Fetal MRI identified an unexpected diaphragmatic hernia (DH) not seen on US.
For the PND cases, CT showed aneurysmal PAs and airway compression with air trapping &/or atelectasis in 4/5 infants.
Seven infants with airway obstruction on CT required pulmonary arterioplasty; 1 infant with no air trapping did not have arterioplasty.
7/8 operative patients survived, one with concomitant DH died at age 22 days due to hemorrhagic shock.
One FDC was inoperable due to poor cardiac function and died at age 7 days.
Conclusions: Prenatal MRI correlates well with postnatal CT for assessing pulmonary artery size and location and severity of lung abnormality in patients with TOF/APV, this allows for appropriate management planning and may negate the need for an immediate postnatal CT. CT accurately depicts the location and extent of airway compression and resultant air trapping or atelectasis, serving to guide the need for and extent of the arterioplasty procedure.
Paper #: PA-101 Craniosynostosis Syndromes: Prenatal Findings by US and MRI Eva Rubio, MD, CNMC, rubioeva@yahoo.com; Anna Blask, Alexia Egloff, Dorothy Bulas Purpose or Case Report: Craniosynostosis with associated malformations is a feature of several related syndromes resulting from a FGFR or Twist genetic mutation.
Syndromes include Apert, Crouzon, Pfeiffer, and Carpenter syndromes.
Our purpose was to review imaging findings which aid in suggesting the diagnosis prenatally.
We retrospectively reviewed prenatal US and MRI findings in 6 cases with prenatal (5 with postnatal/molecular) diagnosis of a craniosynostosis syndrome: 3 cases of Apert, 1 case of Carpenter, and 2 cases of Pfeiffer syndrome.
Results: 5/6 cases were correctly diagnosed prenatally.
In the second trimester findings may be subtle, with mild calvarial changes; digit abnormalities, in particular, may elude the imager in unsuspected cases.
Although the diagnosis could be made with either modality, the full spectrum of abnormalities was best appreciated using a combined imaging approach of MRI and US.
By US many salient features were depicted: Turribrachycephaly/trigonocephaly/cloverleaf (6/6); Syndactyly (4/4); Polydactyly (1/1).
Agenesis of the corpus callosum was identified by US in (2/2) cases.
Conversely, MRI, performed in all cases, contributed additional observations not well seen by US: the fetal airway was well delineated in all cases (6/6); a low lying spinal cord was noted (1/1), midface hypoplasia (6/6) and migrational/sulcation abnormality (1/1).
Additional findings of absent ductus venosus with biliary atresia (1/1), abdominal wall defect (1/1) and renal anomalies (1/1) were seen with both modalities.
Reimaging in later pregnancy depicted important changes (2/2), including worsening hydrocephalus and resolution of suspected airway occlusion.
Conclusions: US and MRI are complementary modalities in evaluating fetuses with craniosynostosis.
Airway patency, midface hypoplasia, spinal cord abnormalities and intracranial abnormalities are often better seen with MRI.
Fetal activity, digits, bone detail, and cardiac anomalies are better appreciated by US.
Findings may be subtle in the second trimester.
Repeat imaging in later pregnancy may reveal specific information affecting delivery planning.
Paper #: PA-102 PCPRA Best Paper 2011 Hyperpolarized Carbon-13 MRSI for Pediatric Disease John MacKenzie, MD, Department of Radiology and Biomedical Imaging, UCSF, john.mackenzie@ucsf.edu; Yi-Fen Yen, Linda Nguyen, Jeffrey Gu, John Kurhanewicz Purpose or Case Report: To study the potential of carbon-13 MR spectroscopic imaging (13 C-MRSI)-a radiation free molecular imaging strategy-for the detection and treatment monitoring of pediatric disease.
Methods & Materials: The potential of 13 C-MRSI to detect pediatric disease was tested in rodent models of pediatric arthritis.
Animals were induced with arthritis and subsequently given intravenous hyperpolarized 13 C-pyruvate, and imaged.
The amount of 13 C-lactate produced from pyruvate in normal and arthritic joints was measured both at single points in time and dynamically at either 3 or 14 Tesla.
The 13 C-MRSI data were compared with clinical measures of arthritis, cell stimulation studies, and joint changes on conventional anatomic MRI and histology.
Results: Alterations in lactate production as measured by 13 C-MRSI appear to depict sites of arthritis and correlate with other more established but potentially less reliable or more invasive measures of disease status.
Imaging robust mouse models of pediatric disease may be feasible at 14 Telsa.
This method may also be translated from high-field to clinical equipment with reasonable hardware and software modifications that allow detection of hyperpolarized 13 C compounds.
13 C-MRSI depicts increased lactate production at specific regions of inflammation within arthritic joints and is confirmed by histological inspection and anatomic MRI.
On average, lactate production is increased by 60% in areas affected by inflammation.
Conclusions: The intravenous injection of hyperpolarized carbon-13 compounds and subsequent imaging with 13 C-MRSI provides a unique molecular imaging strategy to noninvasively monitor pediatric disease.
This non-invasive imaging strategy may eventually provide clinical utility for several pediatric diseases involving inflammation, infection and tumor.
Disclosure: Dr. MacKenzie has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Methods & Materials: Using the Hemangioma-Vascular Malformation Clinic registry at Cincinnati Children's Hospital, we searched for patients diagnosed with KHE whose evaluation included MRI.
Twenty such patients were found, although three of the patients had no pre-therapy MRIs.
The imaging studies were reviewed by the authors with assessment of the following characteristics: location, margin definition, soft tissue involvement, and pre and post contrast signal intensity.
Results: Location: Lesion location was as follows: trunk (9), extremity (3), extremity plus trunk (3), and head/neck (2).
Signal: All lesions were dark on T1 weighted sequences with diffuse enhancement after contrast administration.
The majority of the lesions were bright on T2 weighted sequences, but there were 3 cases that had heterogenous to low T2 signal (with all involving the retroperitoneum).
Of the 17 cases, only one had both high arterial and venous flow by MRI.
Margin definition: Four of the lesions had well defined borders (greater than 50% well circumscribed) with minimal to no adjacent infiltration/edema.
Two of those four cases were exophytic masses.
The remaining 13 cases were poorly defined lesions with adjacent infiltrative fluid signal intensity and enhancement.
Tissue/Organ involvement: Tissue/organ involvement was counted if abnormal fluid-signal intensity or enhancement was identified at that site.
Review of these cases showed fifteen patients with muscular involvement.
Dermal and subcutaneous involvement was observed in all but 4 cases, with the uninvolved lesions being isolated and deep.
Additional sites of suspected involvement included bone (3), pleura (1), penis (1), and pancreas (2).
Conclusions: KHE is a rare neoplasm of infancy with a spectrum of features by MRI.
Poorly defined lesions are much more frequent than well-circumscribed masses.
However, pathologic correlation of such infiltrative margins is usually not available as treatments after biopsy are primarily medical rather than surgical.
Common additional MRI features include predominant involvement of muscle, subcutaneous fat, and skin over viscera and bone with lesions generally showing increased T2 signal and enhancement.
Is Dedicated Chest CT needed in Addition to PET CT for Evaluation of Pediatric Oncology Patients?
Ibrahim Tuna, Montefiore Medical Center, dristuna@yahoo.
com; Jeffrey Levsky, Jeremy Rosenblum, Rosanna Ricafort, Benjamin Taragin Purpose or Case Report: To evaluate the diagnostic accuracy of low dose CT performed during PET-CT as compared to dedicated chest CT in the assessment of pulmonary findings in children with malignancy.
The institutional review board approved this HIPAA compliant research.
Pediatric oncology patients, ages between 0-21, with known solid malignant tumors who were referred to PET-CT and standard chest CT within 30 days for staging or assessment of treatment response between 01-2008 and 01-2011 were eligible for this retrospective study.
Radiology reports were reviewed for potential discrepancies.
Two radiologists re-evaluated the standard chest CT and low dose chest CT portion of the PET CT of the discordant cases, while comparing with the most recent prior studies.
Studies were scored for pulmonary nodules, bony metastasis, adenopathy, and pleural effusions.
True discrepancies were assessed by a panel of pediatric oncologists to judge whether the differences in reports might lead to a significant change in management.
Results: 120 (57 female, 63 male) patients were identified.
31 radiologic reports of 16 different patients (8 female, 8 male) had potential discrepancies based on review of the reports.
The primary tumors were rhabdomyosarcoma (n0 6), Hodgkin's lymphoma (n 03) and others (n 07).
Reevaluation of the original images showed true discrepancies in 3.3% (4/ total 120).
In 2 studies, the discrepancy had no clinical significance.
In 2 studies, a pulmonary nodule was identified on standard chest CT which was not described on the PET-CT.
Both of these patients had rhabdomyosarcoma.
One of these patients had findings that pediatric oncologists considered significant enough to alter patient management.
Conclusions: We found a low false negative rate for clinically significant findings on the low dose portion of PET-CT as compared to standard chest CT.
In the future, improvements in acquisition technique and post processing of the CT portion of the PET-CT may further improve its diagnostic utility, obviating the need for a routine separate diagnostic CT, thereby minimizing radiation exposure in these young patients.
Methods & Materials: 98 low-dose CTA examinations were performed in pediatric patients over a three year period to evaluate suspected vascular traumatic injury with some patients receiving scans of more than one area of the body.
Areas scanned in this include the head and/or neck (N054), chest (N017), abdomen and/or pelvis (N013), upper extremity (N08) and lower extremity (N017).
In 80 of these patients, suspected vascular injury was due to a history of either blunt (N 041) or penetrating (N 039) trauma.
64 patients were referred directly from the emergency department, while 27 were inpatients and the remaining 7 were referred from an outpatient setting.
Patients (32 F:66 M) ranged in age from 0 to 23 years old (mean age 11).
Studies were performed on a 64-channel MDCT scanner with 80 or 100 kV, 40 to 200mAs, 1.0 to 1.5 mm section thickness, reconstructed with 50% overlap, and 0.8 to 1.5 pitch.
Contrast medium was power-injected using weight-based protocols to optimize iodine delivery.
Exams were interpreted on a workstation using advanced imaging techniques.
Patient radiation dose was calculated in all cases.
Clinical outcome was assessed through a 6 month follow-up when possible.
Results: All studies were technically adequate.
76.5% (N078) of studies revealed no vascular injury, while 23.5% (N023) revealed acute vascular pathology.
Vascular injuries included vascular occlusion (N012), vasospasm (N03), narrowing/dissection (N04), pseudoaneurysm (N02), and transection (N0 1).
Extravascular traumatic findings were demonstrated in 51.0% (N050), including fractures, lung injury, soft tissue hematomas, and a ruptured Baker's cyst.
Of the patients with acute vascular findings, 43.4% (N010) underwent surgical management (including 6 for vascular injury), while 52.1% (N012) were managed conservatively.
One patient with active extravasation was managed with angiographically-guided embolization.
In no case was catheter angiography required to confirm CTA findings.
Conclusions: Low dose CTA is a reliable means to screen pediatric patients emergently for acute vascular injury.
Vascular and non-vascular pathology can be diagnosed noninvasively for efficient patient management.
Paper #: PA-106 Elasticity Measurement by Acoustic Radiation Force Impulse (ARFI) Technique of Normal Liver, Kidney and Spleen in Healthy Children Mi-Jung Lee, Radiology, Severance Children's Hospital, mjl1213@yumc.yonsei.ac.kr; Myung-Joon Kim Purpose or Case Report: There are many previous studies about using acoustic radiation foce impulse (ARFI) value to measure the elasticity of tissue, mainly the liver in adult patients.
However, there was limited study about ARFI measurement in the children.
The purpose of this study is to evaluate the ARFI value in the normal liver, kidney and spleen in healthy children and to evaluate the effect of sex, age, and body mass index (BMI).
The study prospectively enrolled healthy pediatric volunteers who are under 18 years old, and underwent abdominal ultrasonography and ARFI between July 2011 and August 2011.
ARFI velocity measuring was performed by 4-9 MHz linear probe for children under 5 years old and 1-4 MHz convex probe for older children.
ARFI velocity was measured three times at each organ.
However this measurement was stopped if the child cannot tolerate.
Results: Two hundred two children (M:F092:110; mean age, 8±4.7 years) were enrolled.
And ARFI measurement was performed only two time for some organs in three children.
The mean ARFI value was 1.12±0.20 m/s in liver, 2.20±0.49 m/s in right kidney, 2.33±0.53 m/s in left kidney, and 2.25 ± 0.41 m/s in spleen.
ARFI velocity was not different between boys and girls.
However, ARFI velocity was different between right and left kidneys (p00.001).
The ARFI value of right kidney, left kidney and spleen was correlated with age, height, weight and BMI (p<0.001).
However, the ARFI value of liver was not correlated with these parameters.
Conclusions: ARFI measurement is feasible in children with only three times acquisition for each abdominal organ.
The mean ARFI velocity was increased according to the age, height, weight and BMI in kidney and spleen, but it was constant in liver.
Disclosure: Dr. Lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Diagnostic image quality can be achieved over a wide range of radiation exposure in digital radiography.
"Exposure Factor Creep" or "Dose Creep'" in which technologists tend to increase dose to avoid the appearance of noise has been well described.
Using the ALARA principle, acceptable images can be achieved while minimizing dose.
At our institution "dose creep" has been observed in bedside pediatric chest radiography.
To address this we coupled a data mining tool with a continuous quality improvement (CQI) initiative which educates individual technologists on appropriate technique.
Methods & Materials: Radiation dose in digital radiography is estimated from an exposure index, a proprietary format that varies among manufacturers.
Our institution uses a Fuji Computed Radiography system which calculates an S, or Sensitivity value, that provides an approximation of the radiation dose to the imaging plate, using an inverse scale.
Overexposed bedside chest radiographs were defined by a S value less than 150.
A data-mining program was developed to extract from the DICOM header the S value and other relevant information, on a monthly basis.
These data were used to provide training and feedback on a one-on-one-basis.
Results: With ad hoc feedback and group training initiatives prior to implementation of this new system, approximately 16.7% (344/2057) of bedside chest radiographs were overexposed over a four month period.
After one-on-one intervention with the technologists, preliminary findings reveal a trend towards fewer overexposed radiographs with approximately 9.2% (40/435) with S<150.
Conclusions: Our tool provides a simple method for systematically identifying overexposed radiographs and the corresponding responsible technologists.
We anticipate that this personalized educational program will continue to reduce the proportion of overexposed radiographs and thus the radiation dose to our pediatric patients.
Purpose or Case Report: Ensuring radiation protection for children undergoing CT scans is challenging due to rapidly changing technology, differences in CT equipment and potential lack of understanding of unique aspects of scanning children.
The Joint Commission has named technologists' training as an "action" item.
We developed 8 online training modules to fill potential gaps in CT technologists' education.
Methods & Materials: Four modules were created by pediatric radiologists, radiologic technologists and medical physicists; 4 were developed by education/training experts from major CT vendors (GE, Philips, Toshiba, Siemens) through the Medical Imaging Technology Alliance.
4 modules were created as Microsoft Word documents containing de-identified images and edited by education specialists at the American Society of Radiologic Technologists and the Alliance for Radiation Safety in Pediatric Imaging.
They were converted to audio/video format using question/answer narration.4 vendor modules were created in Microsoft Powerpoint format and edited.
All 8 modules were converted into Adobe Captivate learning program to achieve uniformity of appearance.
Modules are hosted on the ASRT server and linked to the Image Gently website.
A certificate may be printed as documentation of completion.
Results: All 8 modules are available at www.imagegently.org.
Two introductory modules discuss basics of CT equipment and medical physics related to radiation dose in children.
The third and fourth modules discuss dose-saving strategies for neu-roCT and body CT. Four vendor-produced modules address unique aspects of equipment design such as automatic exposure control and dose saving strategies for children.
Conclusions: Through collaborative efforts with medical imaging professionals and vendors, we have developed 8 free online modules addressing radiation protection for children.
CT technologist training in specific dose saving strategies for children is variable and limited.
These modules have the potential to improve CT technologists' understanding of equipment.
End Confusion which focused attention on improving communication with patients and families.
There is little research regarding health literacy (HL) in radiology.
The purpose of our study was to determine if an educational intervention (brochure) improves HL for parents whose child will undergo a fluoroscopic study.
Methods & Materials: An education exemption was obtained from the IRB.
A multidisciplinary team developed brochures for 5 fluoroscopic procedures.
Participants were randomly selected and asked to complete a survey to assess their knowledge of the procedure and use of radiation both before and after reading a brochure.
A final survey to rate and gain feedback about the brochure was completed.
Results: Median age of children whose parents participated (n0120) was 4 years.
VCUG was most commonly performed (46%).
Prior to the brochure, 92% of participants knew the name of the test their child was having.
After the brochure, 99% knew the name (p < .0001).
Prior to the brochure, 81% felt informed about the test, whereas 99% felt informed after (p<.0001).
Test scores showed an improvement in parent knowledge about the procedure with a median increase of 20 points after the brochure (scale of 1-70; p<.0001).
Even after reading the brochure, 23% of parents wanted more information.
Prior to the brochure, 68% of parents knew the test involved radiation compared to 100% afterwards (p<.0001).
Parents improved their understanding of the relative amount of radiation compared to background from 25% before to 79% after the brochure (p<.0001).
Overall, 99% rated the brochure >2 on a 3-point scale with 92% rating the brochure 3 (p<.0001).
Written feedback was uniformly excellent.
Conclusions: Improving HL for parents is part of the mission of radiology medical professionals.
Our study demonstrates that there is room for improvement in communicating with parents about fluoroscopy.
Straightforward information for parents provided as a brochure improves their understanding of radiologic fluoroscopic procedures.
Paper #: PA-110 Compendium of Resources for Radiation Safety in Medical Imaging Anum Minhas, Duke University, anum.minhas@duke.edu; Donald Frush Purpose or Case Report: Diagnostic imaging, including ionizing radiation modalities, maintains a foremost role in evaluation of medical disorders.
There is increasing awareness and need for information across varied sectors about low level radiation and potential risks.
Many medical/scientific organizations have resources discussing radiation risk and management.
However, there is no one resource compiling the same available information.
Methods & Materials: Websites, including those of national and international medical organizations (e.g., ACR, "Image Gently" Alliance, IAEA) were reviewed for information on radiation dose, risk, justification, optimization, guidelines (which included general information about improvement in quality and dose reduction without specific mention of optimization techniques), appropriateness criteria, and general principles of radiation safety for radiography, fluoroscopy/angiography, and CT.
This information was divided by modalities and separated into adult and pediatric populations.
Information from organizations that were not arbitrarily considered to be national (e.g., subspecialty society, regional organization, individual institution/practice) was not reviewed.
The resources were then organized into 8 tables, organized by modality.
Websites with training modules were noted as well.
Results: 29 websites were explored.
Overall, less information is available about medical radiation safety in children compared to adults.
Across both, most information is available on CT, then fluoroscopy, and finally radiography.
Across all groups and modalities, there is no information available for patients/parents on optimization, appropriateness, or guidelines, with the exception of adult radiography where there were some guidelines.
Conclusions: This compendium on medical imaging radiation serves as a collective resource for communities including the public and regulatory organizations.
Additionally, the compendium can be used to determine redundant or deficient areas, providing opportunities for more comprehensive and efficient efforts in medical radiation protection for patients.
Inappropriate and Cloned Histories in Children: How Big a Problem is It?
Leann Linam, MD, Radiology, UAMS/ACH, llinam@uams.
edu; Chetan C. Shah, S Bruce Greenberg Purpose or Case Report: ACR standards require appropriate clinical history for obtaining imaging examinations.
Cloning clinical histories is a federal violation.
Our purpose is to determine the frequency of inappropriate histories (IH) and/or cloning histories (CH) at a tertiary children's hospital.
Methods & Materials: Three pediatric MOC radiologists reviewed clinical histories for radiographs obtained at a tertiary children's hospital on 3 randomly selected dates (2 weekdays and 1weekend day) for appropriateness and cloning.
Appropriate histories have associated ICD-9 codes.
Cloning is defined by identical clinical histories occurring on 3 consecutive days and could be clinically appropriate or inappropriate.
Only the first patient radiograph on a day was included.
χ2 testing was performed to determine significant differences.
Results: 14% (54/388) of exams had IH.
IH were significantly more common in inpatients than outpatients (p< 0.0001).
NICU examinations accounted for 52% of all IH and were significantly more frequent than other inpatient locations (p0.006).
The CVICU examinations accounted for 11% of all IH and was the second most common patient location for IH, but not significantly different from other inpatient locations (p00.09).
The increased frequency in IH on the weekend reflects a change in patient mix with fewer outpatient examinations performed than on weekdays and was not significant (p00.07).
The most common IH included: evaluate ETT or evaluate lungs (15 each).
Cloning only occurs in inpatients and was combined with IH in 48% of patients with CH.
The NICU accounted 63% of CH which was significantly greater than other inpatient locations (p00.026).
Conclusions: 1 in 7 radiographs had IH which can lead to misdiagnoses or nonpayment by insurance companies.
Inpatients, especially the NICU were the most common patient locations.
Cloning was also a common problem and was frequently combined with IH.
Identifying the extent of IH allows for corrective educational measures to be instituted which should improve compliance with existing medical and legal standards for ordering radiographs.
Paper #: In Vivo Validation of Size-Specific Dose Estimates (SSDE) Through Breast Entrance Skin Dosimetry (ESD) During Pediatric Chest CT Angiography Sjirk Westra, MD, Radiology, Massachusetts General Hospital, swestra@partners.org; Xinhua Li, Mannudeep Kalra, Bob Liu, Suhny Abbara Purpose or Case Report: SSDE is a new CT dose measure that corrects scanner console CT Dose Index (CTDI) for cross-sectional body diameter, being a better estimate of absorbed dose in individual patients of varying body size.
SSDE has been developed through phantom studies and computer simulations of CT dose, but has not yet been validated in vivo.
The purpose of our study was to determine correlation between SSDE and measured breast entrance skin dose (ESD) for pediatric chest CTA across a variety of scanning techniques, scanner models and patient sizes.
Methods & Materials: Our study was IRB-approved, with waiver of written informed consent.
During 42 consecutive chest CTA exams done on 4 different scanners over a period of 7 years, we measured mid-sternal ESD as an approximation of breast dose with skin dosimeters, which was also expressed as mammogram equivalents.
For each scan, we recorded patient age, weight, effective mA, kVp, console CTDIvol-32 cm and DLP-32 cm (from which we calculated age-adjusted effective dose (ED)).
We measured effective chest diameter Ø to convert CTDI to SSDE, and we correlated SSDE with measured breast ESD, using linear regression.
We evaluated image quality with regard to answering the clinical question.
(Table) , due to systematic introduction of automatic exposure control, low kV and high pitch scanning techniques.
All studies were of diagnostic image quality to address the clinical question.
Conclusions: SSDE is a valid measure of CT dose in pediatric patients undergoing chest CTA over a wide range of scanner platforms, techniques, and patient sizes, and may be used to model breast and other organ dose, and to document results of dose reduction strategies over time.
Purpose or Case Report: The purpose of this project was to create an automated system capable of quantifying slice-byslice CT image quality and radiation dose data based on patient size.
The information generated from this system should enable size-specific optimization of CT scan parameters in order to obtain images of diagnostic quality at the lowest possible radiation doses.
Methods & Materials: A mathematical model was developed to predict CT image noise based on kVp, effective mAs, and water-equivalent diameter of the patient.
A conical water phantom was used to calibrate the model on multiple scanners and accounting for different operational modes and scan parameters, including tube voltage (kVp), tube current (effective mAs), bowtie filter, and focal spot size.
A software application was created to process image data from the scout topogram and incorporate DICOM metadata from the axial images.
A database and data viewing application were developed to display individual and aggregate study data.
All of these systems were integrated and automated to enable real-time monitoring of image quality and radiation dose as a function of patient size.
Results: Since the completion of the automated system, 565 CT exams have been processed.
A search application allows the user to find an individual study or a collection of studies based on parameters such as body part imaged or study protocol.
The viewing application displays slice-by-slice patient diameter, radiation dose, and image quality for each study.
Radiation dose estimates are adjusted for patient size, yielding size-specific dose estimates.
The application also graphs individual study data compared to those of comparative studies that are included in the search.
Conclusions: We have successfully developed an automated system that monitors CT image quality and radiation dose data based on patient size.
The system enables simultaneous real-time monitoring of all studies performed on all CT scanners at our institution.
Specifically, the system enables size-specific radiation dose estimates at every scan level.
This system will be used to guide protocol adjustments in order to optimize CT image quality and thus optimize radiation dose.
Disclosure: Dr. Larson has disclosed that he has a patent application in process through CCHMC for CT radiation dose reduction.
Purpose or Case Report: At many institutions, CT scan parameters for children are determined by patient age or weight.
AAPM Task Group 204 recommends cross sectional body dimension, such as patient width to determine Size Specific Dose Estimates.
The purpose of our study was to develop prediction models of body width based on patient age and weight and compare these models with actual measured body widths for children undergoing body CT. Methods & Materials: 6 children's hospitals participated in a 3-month retrospective review of abdominal CT scans on patients <18 years of age after local IRB approval.
Recorded values included patient width(cm) from an axial image at the level of the splenic vein, patient age (yrs) and patient weight (lbs).
A regression model for predicting patient width as a function of age and weight was determined.
Results: 939 exams, 472 had all 3 measurements.
Both age and weight were significant predictors of patient width (p<.0001).
There was also a significant interaction between weight and age (p<.0001), indicating that the relationship between patient width and weight depended on the age of the patient.
The R2 for the regression model for predicting patient width from age and weight individually were 0.65 and 0.83 respectively.
The R2 for the model including both age and weight and their interaction was 0.86 leaving 14% of the variation unexplained.
The regression equation for this model is: Patient width 014.1 + 0.34 x Age(yrs)+ 0.12 x Weight(lbs)-0.003 x Age x Weight.
Despite the R2 of 0.86 for the model using both age and weight, the average error (RMSE) for predicting patient width compared to a direct measurement of width was 1.9 cm.
The plot of observed minus predicted values (residuals) versus predicted values indicates that the best model (combination of weight and age) results in measurable errors of predicted patient width relative to direct measurement.
Conclusions: A combination of both patient age and weight results in a more accurate patient width prediction than using age or weight alone.
While age and weight can be used to predict body width, this is not sufficiently accurate for generating CT protocols.
Therefore, direct measurement of body width form either physical measurement on the patient or from the scout view or an axial image is preferred to select appropriate scan parameters for pediatric abdominal CT. Paper #: PA-115 Automated Size-Adjusted Dose Monitoring for Pediatric CT Dosimetry Olav Christianson, Clinical Imaging Physics Group, olav.
christianson@duke.edu; Ehsan Samei, Donald Frush Purpose or Case Report: The potential health risks associated with low levels of ionizing radiation have created a movement in the radiology community to minimize radiation dose during CT imaging; this is especially important for pediatric patients due to their increased sensitivity to radiation.
It is thus essential to accurately assess the risks to pediatric patients undergoing CT imaging.
Current efforts to monitor radiation dose, however, are limited because they do not account for differences in risk from ionizing radiation due to variability in patient size, age, and gender.
In this context, we developed an automated size-adjusted dose monitoring program capable of performing patient-specific risk estimation to facilitate protocol optimization.
Methods & Materials: DICOM routing software was used to send dose reports and scout images to an image repository on a dosimetry server.
Optical character recognition was used to extract dose-relevant data from dose reports; patient size was determined from corresponding scout images.
Based on anatomical location, risk estimation conversion coefficients (qfactors) were determined for each series in the dose reports.
The q-factors were adjusted according to patient size, age, and gender and then multiplied by the DLP to estimate the risk to each patient.
This process was applied to the cohort of pediatric patients undergoing CT examination at our institution.
To evaluate the impact of including patient size, age, and gender, risk estimates were obtained excluding and including the dependencies on size, age, and gender.
The results were computed in units of cancer incidence per 1000 cases exposed (cpt).
Results: The average patient-generic risk estimate for a pilot group of patients undergoing body CT was 0.15±0.14 cpt.
By including patient size, the risk estimate was increased to 0.26 cpt±0.27 cpt.
By including patient age and gender, the average risk estimate was further increased to 1.0 cpt±0.72 cpt.
Conclusions: We developed a new size-adjusted dose monitoring program for pediatric CT dosimetry.
Comparisons between patient-generic and our new patient-specific risk estimates show that failure to consider patient size, age, and gender resulted in risk estimates that were too low by a factor of seven.
Additionally, the increase in standard deviation we observed demonstrates that our method of including patient size, age, and gender is sensitive to the inherent variability in the patient population.
Disclosure: Dr. Christianson has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Treatment of prenatally diagnosed lung masses is controversial, with many specialists recommending elective surgical removal in the first year of life because of a reported or perceived increased risk of infection and malignancy, while other centers recommend a conservative approach to management.
The natural history of unresected lung masses is not clear.
In our center, our standard recommendation is prophylactic resection of asymptomatic lesions, although not all families choose this option.
We asked whether respiratory morbidity increased during the time prior to elective resection of prenatally diagnosed lung masses.
Methods & Materials: Ninety-eight pregnant women carrying fetuses with chest masses were imaged by ultrasound (US) and magnetic resonance imaging (MRI).
Medical records of the liveborn infants were retrospectively reviewed.
Results: Fetal diagnosis of a lung mass was made at a mean of 27 weeks gestation (range 17-32 wks).
Intrauterine fetal demise was documented in 4 pregnancies.
There was one elective termination of pregnancy.
Three infants were lost to follow up.
Thus, outcomes were available for 90 children (59% M, 41% F) with prenatally diagnosed lung masses.
Significant respiratory morbidity (RM) was defined as the occurrence of pneumonia, asthma, chronic coughing or wheezing, or respiratory symptoms severe enough to require an Emergency Room visit or hospitalization.
Of the 76 children who had surgical removal of their lung mass, 34 (45%) had RM prior to surgery.
Fifteen out of 90 children did not have surgery but have been followed expectantly, and 3 of 14 (21%) developed some form of RM.
Fifteen of 76 (20%) infants had immediate and significant RM (tachypnea, grunting, increased work of breathing, increased oxygen requirements or need for intubation) in the newborn period leading to urgent surgery (range of age at surgery: 1-10 d; mean 2.5 d).
Of the 61 initially asymptomatic infants, 17 (28%) developed RM prior to elective removal of the mass (range 6-96 weeks, mean 17 weeks).
Of the lesions removed, histology revealed: cystic adenomatoid malformation (CCAM) 59%, CCAM + sequestration 21%, Sequestration 8%, congenital lobar emphysema (CLE) 7%, CCAM+CLE 1%, other 3%.
Conclusions: The risk of respiratory morbidity appears to be increased during the time prior to elective resection of prenatally diagnosed lung masses, which may be important for parents and pediatric specialists to consider when deciding whether to remove an initially asymptomatic lung mass.
Purpose or Case Report: It is now accepted that fetal MRI with its superior tissue resolution can be very helpful in clarifying anomalies detected during obstetrical ultrasound.
This is particularly the case with intracranial abnormalities, although indications are expanding.
The current English medical literature, though, appears to be focused on evolving MRI techniques and how MRI compares to ultrasound with regards to image quality and detection of additional findings which may alter the diagnosis.
However, we found no study specifically evaluating the clinical relevance and impact of the information obtained by fetal MRI to the specialists who counsel and treat these patients.
A "Satisfaction and Clinical Impact" survey was created and sent to all the members of our Fetal Diagnosis and Treatment Group, asking specifically how the clinicians rated their satisfaction with this type of imaging, its influence on their counseling and on various clinical decisions, both prenatal and postnatal.
Results: We received responses from 37 specialists in 10 different clinical disciplines.
The greatest number of respondents came from our obstetricians (28%), many of whom perform their own ultrasounds, and from members of our medical geneticists/genetic counselors (27%), although 46% of respondents were from various other clinical disciplines, both medical and surgical.
There was a surprisingly high degree of satisfaction overall with the quality of the images and with the type and amount of information provided.
Most respondents indicated they felt fetal MRI was "moderately" or "extremely" useful for their particular clinical decisions, and most respondents agreed that fetal MRI impacted "moderately" or "significantly" on counseling and management of these pregnancies.
Impact appeared greatest on the counseling of the parents and their decision to terminate/pursue the pregnancy, and the least impact was on issues around delivery.
Conclusions: Fetal MRI, in addition to providing images of better quality, particularly in certain conditions, has clinical value in that it directly impacts on the counseling of parents and on clinical decisions.
2006-2011.
Ultrasound reports were reviewed to determine sonographic diagnoses.
Selected patients from this cohort underwent MRI using GE 1.5 Tesla magnet without contrast (Sequences included SSFSE, FIESTA, FGRE or dual echo in 3 planes).
The images were reviewed and multiple characteristics were assessed for specifiying the area of obstruction.
The features included: presence of normal fluid-filled bowel, small rectum for gestational age, signal of meconium in the rectum, and meconium filled dilated bowel.
Results: 46 cases of sonographically suspected bowel obstruction were identified during the study period; 27 of these underwent fetal MRI.
Of these 27 cases, 4 had normal MRI and postnatal outcomes, 2 cases did not have postnatal findings available, and 2 had postnatal meconium peritonitis but no obstruction.
One case of congenital chloride diarrhea was diagnosed by fetal MRI.
A variety of bowel abnormalities were observed amongst the remaining 18 cases.
Proximal obstruction was diagnosed in 8 cases: jejunal atresia (n07) and multiple atresia (n01).
Distal obstruction was diagnosed in 10 cases: ileal atresia (n03), meconium plugging (n04), closed gastroschisis (n01), enteral duplication cyst (n01), and imperforate anus (n01).
Characteristic patterns of features were identified amongst these 18 cases that specified the location of obstruction.
These patterns of findings allowed accurate localization of the level of obstruction in all cases when compared to postnatal findings.
Distal obstruction was characterized by normal fluid-filled small bowel and high T1 signal in distended loops.
Jejunal atresia was characterized by multiple loops of dilated bowel with high T2 signal primarily in the left upper quadrant.
Small rectum for gestational age was not consistently associated with proximal or distal atresia.
Conclusions: Evaluation of fetal MRI with attention to specific features allows localization of bowel obstruction.
This may aid in counseling and postnatal management, including the need and type of postnatal imaging study.
Early diagnosis and treatment of PH may prevent clinical deterioration.
PVT may produce a spectrum of imaging appearances, which has not been fully recorded in the literature.
The goal of this paper is to review the spectrum of imaging appearances of neonates and survivors of neonatal PVT with special emphasis on the role of US and to correlate these findings with the clinical findings including outcome.
Methods & Materials: A retrospective review of 133 consecutive neonates admitted between 1999-2003 and diagnosed with PVT was conducted.
Diagnosis was established by US at a mean age of 9 days (range: 1-40).
Health records, initial and follow-up (f/u) imaging were reviewed.
Findings were classified as non occlusive, single branch, PVT (grade 1); occlusive PVT (grade 2) and PVT with extensive parenchymal ischemia (grade 3).
Results: PVT was diagnosed in 133 patients, 70 of whom were followed up to for 2 years or longer.
Twelve patients were excluded due to liver disease, 22 expired and 29 were lost to f/u.
Of the 70 in whom f/u was available, at the time of initial diagnosis, grade 1 PVT was present in 27, all were on the left.
Grade 2 PVT was diagnosed in 28 and grade 3 PVT in 15.
On f/u physical exam, findings were unremarkable in 68/70 patients.
Liver function tests (LFT) and thrombophilia assessment were available in 25 children, mild LFT abnormalities were noted in 9 and 6 children had evidence of thrombophilia.
US exams were available in 37/70 children.
Among the 37 survivors of neonatal PVT, US was regarded as normal in only 14 children; 16 showed left lobar atrophy (LLA), 5 had slowly progressive splenomegaly without other signs of PH, and 2 developed clinically significant PH requiring shunting.
Conclusions: PVT has a wide spectrum of imaging appearances, it is possibly underdiagnosed and clinically unsuspected.
Varying degrees of LLA are likely a sequela of clinically silent left PVT.
US is a sensitive method for the detection of disease and assessment of progression.
Paper #: PA-120 Fetal MRI in Arthrogryposis Hedieh Eslamy, MD, Radiology, Lucile Packard Children's Hospital, hkeslamy@gmail.com; Erika Rubesova, Louanne Hudgins, Britton Rink, Richard A. Barth Purpose or Case Report: To present the fetal MRI findings in fetuses with a prenatal diagnosis of arthrogryposis and correlate with postnatal outcome or autopsy results.
Arthrogryposis refers to contractures involving more than one joint which often represent deformational changes secondary to decreased or absent fetal movement.
Prognosis varies widely dependent on diagnosis, ranging from isolated contractures in amyoplasia to lethality in some cases.
We hypothesized that fetal MRI may demonstrate central nervous system (CNS) pathology and muscle abnormalities which are important for predicting postnatal outcome.
Methods & Materials: We identified 6 fetuses with a diagnosis of arthrogryposis between January 2010 and October 2011.
All had fetal MRI which was performed on a GE 1.5 Tesla magnet, with SSFSE, FIESTA and FGRE sequences in 3 planes.
The fetal MRI's were evaluated for CNS and muscle abnormalities.
The extremities were evaluated for: muscle mass, increase in subcutaneous fat (indicative of muscle atrophy), and extremity joint positioning.
These findings were subsequently correlated with the clinical exam of the neonates, pathology in the abortus and karyotype when available.
Results: Results of fetal US, amniocentesis, fetal MRI and post-natal or post-termination outcomes will be summarized.
Five fetuses had ≥2 limb joint contractures.
A sixth case had neck hyperextension and lateral flexion associated with akinesia and hydrops.
On MRI, no structural brain or spine abnormalities were identified.
The abnormalities detected in the extremities were: severe decrease in muscle mass associated with increased subcutaneous fat (3 cases); normal muscle mass (2 cases); moderate decreased muscle mass associated with increased subcutaneous fat (1 case).
In the 3 cases that delivered, the diagnoses were amyoplasia (2) and distal arthrogryposis (1).
In a fourth case that underwent elective termination, autopsy was consistent with amyoplasia.
Two cases are pending delivery.
Conclusions: While fetal MRI can be useful to rule out CNS anomalies, it may also provide important information on decreased muscle mass as an important prognostic sign in a fetus with arthrogryposis.
In our series, severely decreased muscle mass was predictive of amyoplasia, and joint contractures limited to hands and feet with preserved proximal muscle mass was predictive of distal arthrogryposis.
Both diagnoses are associated with relatively good prognosis and usually normal intelligence.
Purpose or Case Report: The purpose of this study is to assess the effects of iterative reconstruction technique (IRT) on image quality metrics measured in child-sized anthropomorphic phantoms as kVp is changed.
Methods & Materials: CT scans were performed on anthropomorphic phantoms with sizes of 1, 5 & 10 years (ATOM Phantoms, CIRS, Norfolk Virginia) using low dose pediatric chest protocols (1.6, 3 & 6 mSv) to determine baseline noise and dose levels.
Subsequently three voltage levels (120, 100 & 80 kVp) were used while adjusting mAs to maintain baseline CTDIvol and without mAs adjustment which allowed varied CTDIvol.
Images were reconstructed using 100% filtered back projection (FBP) and blends FBP: IR (80:20, 60:40, & 40:60) .
Parameters including CTDIvol, dose length product, scan length, kVp, and mAs, were recorded for each scan.
Image noise, contrast:noise (CNR), and signal:noise (SNR) data were recorded from ROIs in phantoms and dilute iodine contrast filled syringes (5, 3, 1.5%).
Results: As kV is lowered from 120 to 80, image noise is doubled if mAs is not increased to maintain CTDIvol, and CNR is increased but SNR is decreased due to the increased image noise.
As kVp is lowered from 120 to 80, image noise is increased nominally (8-21%) if mAs is increased to maintain CTDIvol; therefore the increase in CNR and decrease in SNR is negligible.
CTDIvol is reduced >300% in all phantom scans as kV is reduced from 120 to 80.
IRT reduces image noise by up to 36% [range 10-41%] in all phantom sizes and in clinical images.
As CTDIvol is maintained in patient scans, image noise, CNR, and SNR are reduced in patients (p<0.05), resulting in improved image quality.
Conclusions: When lowering kVp, compensation with increases in mAs is necessary to maintain CTDIvol.
However, lower target CTDIvol can be achieved when adding IRT as image noise can be decreased.
For these phantoms, CNR and SNR improved using all [selected] levels of IR, even when kV was reduced, resulting in lower CTDIvol in phantoms.
At all kVp settings when IRT is applied, image noise is reduced, resulting in improved CNR and SNR for all phantoms.
Disclosure: Dr. Bardo has indicated she is in the speaker's bureau and receives an honorarium from Koninklijke Philips.
Paper #: PA-122 Adaptive Iterative Dose Reduction in Evaluation of the Pediatric Abdomen with Ultra-Helical 320-Channel MDCT Jeffrey Hellinger, MD, Stony Brook University, jeffrey.
hellinger@yahoo.com; Bernice Hoppel, Richard Mather, Monica Epelman Purpose or Case Report: Radiation reduction is paramount for pediatric patients.
Ultra-helical 320-channel MDCT allows for rapid acquisitions at low dose.
We evaluated the ability of a new adaptive iterative dose reduction algorithm (AIDR) to reduce noise in low-dose ultra-helical pediatric abdominal CT scans.
AIDR is an iterative algorithm that adaptively reduces noise in the raw and image domains while preserving image structure.
The raw data from 14 consecutive low-dose pediatric abdomen exams was gathered.
A dose simulation tool which adds noise to raw projection data was employed to simulate tube current at 1/4 of baseline mA.
Data were reconstructed with both standard filtered back projection and with AIDR.
Regions of interest were drawn in the liver and lumbar musculature to determine the signalto-noise (SNR), contrast-to-noise (CNR) and overall diagnostic quality of each data set.
Statistical significance was determined using a Student's t-test.
Subjective image quality was evaluated by two reader blind review using a five point scale (50excellent, 10unacceptable).
Results: The SNR and CNR were significantly lower for the 75% dose reduction datasets compared to the original filtered back projection reconstructions (SNR: 3.59 vs 2.20, p<0.001; CNR: 1.24 vs 0.75, p00.01).
When AIDR was applied to the 75% dose reduction data, the SNR and CNR improved to be superior to the native case (SNR: 3.59 vs 5.48, p<0.001; CNR: 1.24 vs 1.95, p00.02).
The average image quality score for the low dose datasets with AIDR was 4.2 compared to 3.4 with standard filtered back projection at the baseline mA Conclusions: AIDR significantly improves the image quality of pediatric abdominal CT images.
With a simulated 75% reduction in dose, AIDR produces images with significantly greater SNR and CNR.
The subjective image quality scores for AIDR showed dramatic improvement over standard filtered back projection.
AIDR processing algorithms with ultra-helical 320 MDCT will allow 75% reduction in radiation exposure while achieving the same diagnostic quality as compared to routine pediatric abdomen MDCT radiation protocols with filtered back projection processing algorithms.
Purpose or Case Report: To explore incorporating ASIR into pediatric head CT protocols, to reduce patient radiation dose while maintaining image quality.
Methods & Materials: An Alderson RANDO head phantom was estimated to approximate the size of a 7-year-old child's head, and was scanned at decreasing 10% mA intervals (100 to 50%, 150 to 75 mA) relative to this institution's age-based head CT protocols.
Each of these studies was then was reconstructed at 10% ASIR intervals (0% to 100%), and a 100 mm2 ROI was obtained in a consistent location behind the frontal bone to estimate noise (SD).
Using this phantom data, our ventriculoperitoneal (VP) shunt follow-up CT protocol was modified, and patients were scanned at 20% ASIR with approximately 20% mA reductions relative to our normal age-based mAs.
These ASIR studies were then anonymously compared to older non-ASIR head CT studies from the same patients (with identical kVp/slice thickness) by two blinded attending pediatric neuroradiologists.
All studies were evaluated subjectively for diagnostic utility (1-4), sharpness (1-5), noise (1-4), and artifacts (1-4).
50-100 mm2 ROIs were drawn in consistent locations to estimate noise in air, bone, CSF, and white matter (WM).
Results: The phantom study suggested similar same noise levels at 100% mA/0% ASIR (3.9) and 80% mA/20% ASIR (3.7).
12 patients (average09, range01 to 17 years) were then scanned at approximately 20% mA reductions, with an average of 349 days (range027 to 871 days) between the ASIR study and prior non-ASIR study.
The average CTDIvol and DLP values of the 20% ASIR studies were 22.4 mGy and 338.4 mGy-cm, and for the non-ASIR studies were 28.8 mGy and 444.5 mGy-cm, representing statistically significant decreases in the CTDIvol (22.1%, p00.00007) and DLP (23.9%, p00.0005) values.
There were no significant differences between the ASIR studies and non-ASIR studies in respect to diagnostic acceptability (p00.33), sharpness (p0 0.45), or noise (p00.84).
There was a non-significant trend that the ASIR studies had a lower artifact score (1.8 vs 2.1, p0 0.06).
There was good to perfect (kappa00.5 to 1.0) agreement.
The ASIR studies had statistically significant decreased CSF noise (3.0 vs 4.4, p00.0000008), but no noise differences were seen in air (p00.46), bone (p00.26), or WM (p00.22).
Conclusions: Our findings suggest that ASIR can provide dose reductions in pediatric head CT without affecting image quality.
Purpose or Case Report: To validate the T2 map as a noninvasive quantitative biomarker of fatty infiltration of muscles and to determine whether the T2 map can differentiate between boys with DMD and healthy boys.
Methods & Materials: Two groups of boys with similar ages (range 5-15 years) were evaluated: 42 boys with DMD (mean age 10.4 years) and 29 healthy boys (mean age 11.7 years).
MR images were performed at 3 T. Fatty infiltration of the pelvic and thigh muscles on T1-weighted images (WI) was graded from 0 to 4.
On T2 maps with and without fat suppression, the muscle with the greatest fatty infiltration on T1-WI was selected, and a region of interest was placed to obtain T2 values.
T2 values from T2 maps with fat suppression were subtracted from values of T2 maps without fat suppression and designated as the "T2 fat value."
T2 fat values were obtained from the same muscles in all boys.
Comparison was made between the T2 fat values of the two groups.
The upper reference limit of the reference interval (RI) of T2 fat values was obtained from the control group to establish the normal range and applied to both groups to determine the accuracy of the T2 map.
Results: The gluteus maximus muscle had the greatest fatty infiltration on T1-WI.
Median T2 fat value was 73.0 msec for DMD (95% RI 193.8, range 29.2-175.6) and 7.5 msec for the control group (95% RI 19.2, range 1.4-21.6).
When applied to the two groups, the upper reference limit of the RI for control patients yielded 100% sensitivity, 93% specificity, 95% positive predictive value, and 100% negative predictive value.
Conclusions: Utilization of T2 maps for the quantitative measurement of fatty infiltration of muscles can clearly differentiate between DMD and normal control boys with a high degree of accuracy and precision.
This advanced noninvasive technique may potentially replace invasive muscle biopsies currently used for diagnosis.
Purpose or Case Report: Prior work has shown that the gluteus maximus muscle has the greatest T2 relaxation time on MR imaging using T2 mapping in boys with Duchenne muscular dystrophy (DMD).
However, an increased T2 value on T2 relaxation time mapping may reflect both fatty infiltration and inflammation of the muscle.
Fatty infiltration characteristically follows inflammation in this disease process.
Therefore, the purpose of this study was to determine the contribution of each component (fat and inflammation) within gluteus maximus muscles and to correlate each component to clinical assessments.
Methods & Materials: Forty-six boys with DMD (ages: 5-15 years) were recruited.
MR imaging of the pelvis using T2 maps with and without fat suppression were performed.
The T2 map "fat values" (T2 value calculated from the T2 map without fat suppression [FS] minus T2 map with FS) and the T2 map "inflammation value" (T2 value from the T2 map with FS) were obtained.
Clinical assessments typically used to evaluate DMD patients (including clinical functional score, 30 ft run, Gower score, and 4 step-up time) were also performed.
Spearman correlation coefficients between fat and inflammation values and the clinical assessments were calculated.
Results: There was a statistically significant correlation between the fat value of the gluteus maximus muscle and each clinical assessment test (p<0.05).
However, the inflammation value of the gluteus maximus muscle did not correlate with any clinical assessment.
Conclusions: In DMD, the amount of fatty infiltration of the gluteus maximus muscle has excellent correlation with clinical assessment.
The amount of inflammation of the gluteus maximus muscle, however, does not correlate with clinical function.
Therefore, further study is needed to determine whether components (fatty infiltration or muscle inflammation) of the single most involved muscle reflect the components of all the muscles of the pelvis and thighs and whether the cumulative muscle involvement of each component represents clinical disease severity.
Utility of Contrast-Enhanced MR Imaging in Children with Osteonecrosis: Does Gadolinium Help?
Lamya Atweh, MD, Radiology, Texas Children's Hospital, laatweh@texaschildrens.org; Robert C. Orth, Wei Zhang, R. Paul Guillerman, Herman Kan Purpose or Case Report: At our institution, gadolinium contrast-enhanced MR sequences are often obtained to assess epiphyseal and non-epiphyseal osteonecrosis in children.
Several studies have shown that dynamic contrast-enhanced sequences may provide prognostic information about long-term complications and healing of osteonecrosis.
To our knowledge, no studies have determined the added value of routine post-contrast MR imaging in assessing acute complications related to chronic osteonecrosis.
The purpose of this study was to evaluate the utility of intravenous gadolinium contrast in the MRI identification of complications in children with an established diagnosis of osteonecrosis.
Methods & Materials: 64 patients were restrospectively identified (age range: 1.75 years to 25.75 year; M:F 0 59:80) with an imaging diagnosis of chronic osteonecrosis who underwent 139 contrast-enhanced MR studies between 1/2000 and 9/2011.
The pre-and post-contrast MR images were consensus reviewed by two CAQ pediatric radiologists.
Pre-and post-contrast images were reviewed at separate times.
The pre-contrast images were available during the review of post contrast images.
Studies were assessed for: osteonecrosis location (epiphyseal, non-epiphyseal osteonecrosis, or both), joint effusion, marrow edema, and epiphyseal collapse.
95% confidence interval (CI) and Cohen's kappa coefficient(κ) was calculated to assess observed agreement.
Results: The diagnosis of osteonecrosis without complicating features was made in 49.6% (CI: 41.3-58.0%) (69/139) of pre-contrast studies and 53.2% (CI: 45.0%-61.5%) (74/ 139) of post-contrast studies.
When chronic osteonecrosis with complicating features was identified,pre-and postcontrast images idenfied joint effusion in 44.9% (57/127) and 51.2% (65/127) (κ00.686, p<0.001); marrow edema in 50.4% (70/139) and 46.8% (65/139) (κ00.727, p<0.001); and epiphyseal collapse in 51.2% (65/127) and 42.5% (54/ 127) (κ00.796, p<0.001), respectively.
Myositis or muscle strain was incidentally diagnosed in 12.2% (17/139) pre-contrast and 10.1% (14/139) post-contrast (κ 0 0.674, P <0.001) studies.
Conclusions: The high observed agreement between the pre-and post-contrast MR images shows that the addition of intravenous gadolinium may not be necessary in the majority of children with chronic osteonecrosis.
Paper #: PA-127 Systematic Protocol for Assessment of the Validity of BOLD MRI in a Rabbit Model of Inflammatory Arthritis at 1.5 Tesla Michael Chan, BHSc, University of Toronto, mw.chan@ utoronto.ca; Afsaneh Amirabadi, Anguo Zhong, Antonella Kis, Rahim Moineddin, Andrea S. Doria Purpose or Case Report: Blood oxygen level-dependent (BOLD) MRI has the potential to identify regions of early hypoxic and vascular joint changes in inflammatory arthritis.
At this point, there is no standard protocol for data analysis of BOLD MRI measurements in musculoskeletal disorders.
Standardization of the technique is paramount to compare results between studies and assess the validity of this technique in tissues outside the blood-brain barrier.
Our objective is to optimize BOLD MRI reading parameters in a rabbit model of inflammatory arthritis by determining the diagnostic accuracy of (1) statistical threshold values (r>0.01 vs r>0.2), (2) summary measures of BOLD MRI contrast [ the mean of the % BOLD signal differences within the region of interest (ROI) (diff_on_off) and the percentage of suprathreshold voxels within the ROI (PT%)], and (3) voxel activation algorithm (positive, negative, and positive_negative).
Methods & Materials: Using BOLD MRI protocols with a carbogen stimulus on a 1.5 T magnet, we imaged injected and contralateral knee joints of 21 juvenile rabbits at baseline, and days 1, 14 and 28 after a unilateral intra-articular injection of carrageenin.
Nine non-injected rabbits served as controls.
Receiver operating characteristic (ROC) curves were plotted to determine the diagnostic accuracy of the reading parameters.
The BOLD measures from [(injected knee-control knees)/control knees] were counted as positive cases, while the BOLD measures from [(contralateral knees-control knees)/control knees] were regarded as negative cases.
Areas under the curve (AUCs) were calculated to determine the most accurate parameters.
Results: Using diff_on_off and positive_negative activations as constants, r>0.01 was found to be more accurate than r>0.2 (p00.03 at day 28).
Comparison of diff_on_off and PT% yielded no statistically significant difference (p> 0.05).
Finally, positive_negative activations for diff_on_off and negative activations for PT% using r>0.01 were the most diagnostically accurate (AUC00.78, p<0.01 at day 28, and AUC00.90, p<0.01).
Conclusions: From the results of this study, the most diagnostically accurate and clinically relevant reading parameters included the use of a more lenient threshold of r>0.01, a diff_on_off measure of BOLD contrast, and a positive_negative voxel activation algorithm.
PT% may used as an ancillary measure of BOLD contrast.
Quantitative versus Semi-Quantitative MR Imaging of Cartilage in Blood-Induced Arthritic Ankles Andrea Doria, MD, PhD, Diagnostic Imaging, The Hospital for Sick Children, andrea.doria@sickkids.ca; Ningning Zhang, Carina Man, Pamela Hilliard, Ann Marie Stain, Victor Blanchette Purpose or Case Report: To cross-sectionally compare the ability of a scoring system (semi-quantitative method) with a manual segmentation technique (quantitative method) to evaluate the status of the articular cartilage of growing ankles of children with blood-induced arthritis.
Methods & Materials: 12 boys, 11 with hemophilia (A, n09; B, n02) and 1 with von Willebrand disease, median age 13 (range, 6-17) underwent a high resolution MRI protocol at 1.5 Tesla, x-rays, and physical examination using the Hemophilia Joint Health Score (HJHS) system.
Two blinded radiologists scored the MRI examinations for cartilage items (horizontal component: surface erosions, scores 0-2 and vertical component: cartilage degradation, scores 0-4) according to the semiquantitative method (International Prophylaxis Study Group MRI scale).
An experienced operator applied a validated quantitative 3D-MRI method (horizontal components: AC, VC, VCtAB, ThCtAB; vertical component: ThCcAB) to corresponding high resolution MR images of ankles.
Results: Internal correlation of the semi-quantitative method components was substantial (r00.72, P<0.0001, tibia) to high (r00.91, P<0.0001, talus) in any site of investigation, but it was site-specific with the quantitative method, being significant only in the talar trochlea (r00.86, P<0.0001).
External correlation of corresponding components of the semiquantitative and quantitative methods was moderate (r00.55, P00.005) to poor (r00.39, P00.05) for horizontal components, and non-existent for vertical components.
Components of the semi-quantitative method highly correlated with lifetime number of previous ankle bleeds (r00.74-0.84, P<0.0001), Pettersson x-ray (r00.87-0.94, P<0.0001), and HJHS scores (r00.91, P<0.0001).
This correlation was poor (r00.42, P0 0.04) to moderate (r0−0.56, P00.004) for horizontal components of the quantitative method.
Conclusions: The biologic concepts of the semi-quantitative and quantitative MRI methods are distinct for assessment of ankles.
The semi-quantitative method is valid for assessing cartilage changes in cross-sectional studies of blood-induced arthropathy, however the quantitative method is suboptimal or less powerful for this purpose.
Paper #: PA-129 Shoulder MR Arthrography In Skeletally Immature Patients Nancy Chauvin, MD, Department of Radiology, The Children's Hospital of Philadelphia, chauvinn@email.
chop.edu; Camilo Jaimes, Victor Ho-Fung, Diego Jaramillo Purpose or Case Report: There has been a well documented increase in sports participation in children which has lead to an increase in sports-related injuries.
To date, there are no studies describing the value of shoulder MR arthrography compared with the gold standard, arthroscopy.
We retrospectively reviewed 80 MR shoulder arthrograms obtained in pediatric patients between 2004 and 2010 who underwent subsequent shoulder arthroscopy.
Interpretation of the images was performed by three pediatric radiologists who were blinded to the arthroscopy findings.
Images were evaluated in consensus and independently.
Assessment included evaluation of the osseous structures, labral-ligamentous complex, joint space and the rotator cuff interval.
The MR results were compared with reported surgical findings.
Sensitivity and specificity were calculated.
Results: Nine patients were excluded due to technical reasons.
Of the remaining 71 patients, 48 were boys (9.7-18.5 years, mean 15.7 years) and 23 were girls (12.7-19.3 years, mean 15.6 years).
At arthroscopy, 53 patients (74%) had injury to the anterior inferior glenoid labrum.
MR sensitivity was 92% for depiction of Bankart-type injuries with a specificity of 94%.
37 patients (52%) had Hill Sach lesions and MR had sensitivity of 86% with specificity of 88%.
24 Superior Labrum Anterior Posterior (SLAP) tears (33%) were identified at arthroscopy with MR sensitivity of 67% and specificity of 89%.
Overall, MR arthrography had a positive predictive value of 96% for identification of a surgical lesion.
Agreement between the observers was high.
Interobserver reliability was calculated with an intraclass correlation coefficient (ICC)of 0.638 with a Cronbach's Alpha of 0.841.
Conclusions: MR shoulder arthrography can accurately depict labral and osseous injury and provides pertinent preoperative information.
A Novel Multi-Channel MR Coil for Improved Pediatric Elbow Coil Imaging Suraj Serai, PhD, CCHMC, suraj.serai@cchmc.org; Randy Giaquinto, Kathleen Emery, Charles Dumoulin Purpose or Case Report: Single flex coils or adult size coils are currently used for imaging the pediatric elbow.
This frequently results in uncomfortable patient positioning, motion, poor fat suppression, low SNR and there is currently lack of a dedicated pediatric elbow coil in the commercial market.
Our goal was to explore the usefulness of a new coil array dedicated for pediatric elbow imaging and to compare quantitative & qualitative imaging findings to commercially available coils.
Methods & Materials: An eight channel elbow coil was designed.
The coil frame was designed to be rigid and lightweight.
Seven identical loop coils were built into a polycarbonate frame and an eighth coil built into a paddle that fits into the top frame.
The coil elements were constructed with heavy copper to provide a high Q-factor and increased SNR.
The complete coil including electronics & covering, weighs only 1.4 kg.
MR Imaging under IRB approval was performed on a GE 1.5 T scanner using a routine clinical elbow protocol including T1W, PDW, T2W, Fat-Sat, Non-fat-sat, 2D & 3D sequences.
Subjects were positioned feet-first with the elbow on the side & were subjectively assessed for comfort level.
Images obtained from the new coil & from the current commercial coils were compared for SNR.
Results: Scan positioning was reported to be comfortable.
SNR was between 20-25% higher as compared to the routine coils.
Fat saturation was uniform, indicating that the magnetic susceptibility of the coil is well-matched to human anatomy.
Anatomical detail depiction was subjectively better for anatomic features such as trochlea.
Detection & diagnostic confidence of elbow disorders were improved with the new coil & greatly decreased motion artifacts were observed.
Conclusions: The new pediatric elbow coil provided excellent image quality, patient acceptance and clinical performance improvements over existing coils.
The open coil design also allows for imaging of the elbow in a partially flexed position or in a cast.
The advantages provided by the new coil are expected to include shortened image acquisition times (via parallel imaging) & increased SNR.
Disclosure: Dr. Serai has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Incremental Value of Knee Radiography in the Interpretation of Pediatric Knee MRI Yen-Ying Wu, Texas Children's Hospital, yxwu@ texaschildrens.org; Robert C. Orth, Wei Zhang, R. P. Guillerman, Herman Kan Purpose or Case Report: The ACR Appropriateness Criteria recommendation for the imaging work-up of knee pain is radiography followed by MRI.
In many cases, MRI is performed prior to review of radiographs or the referring subspecialist does not feel radiographs add value, particularly when ligamentous injury is suspected.
The purpose of this study is to determine if radiography adds incremental value in the interpretation of knee MR studies electively referred by pediatric sports medicine and orthopedic subspecialists.
Knee MRI studies referred from pediatric sports medicine physicians or pediatric orthopedic surgeons between 9/2008 and 9/2011 (N0194, ages 4-18 years, M:F087:107) with accompanying radiographs were identified.
Patients were separated into 3 groups based on MRI findings: normal, ligamentous injury, or osteochondral injury (osteochondral lesions, bone contusions/fracture, and avulsion injury).
Knee radiographs were consensus reviewed by two CAQ pediatric radiologists blinded to MRI findings and categorized into the same groups.
Radiograph and MRI findings were compared and categorized into 3 groups: neutral if radiograph and MRI findings were the same, misleading if findings were discordant, or helpful if radiographs improved MR interpretation.
The latter group was analyzed for impact on MR diagnosis.
Results: For 194 knee radiographs, 166 were normal, 2 showed ligamentous injuries, and 26 showed osteochondral injuries.
When radiographs were interpreted as normal (N0 166), by MR 44% were normal, 33% had ligamentous injury, 10% had osteochondral injury, and 13% had both ligamentous and osteochondral injury.
When radiographs were interpreted as ligamentous injury (N02), by MR 50% were normal and 50% had ligamentous injury.
When radiographs were interpreted as osteochondral injuries, by MR 8% had ligamentous injury, 38% had osteochondral injury, and 54% had both ligamentous and osteochondral injury.
Subset analysis of true positive radiographs (N025) found 56% to be helpful and 44% to be neutral in MR diagnosis.
For radiographs considered helpful, 0% resulted in a change in MR diagnosis.
In regards to the influence of radiographs on MR interpretation, 37% (72/194) were misleading, 56% (108/194) were neutral, and 7% (14/194) were helpful.
Conclusions: A minority of pediatric knee radiographs aided MR diagnosis, and none resulted in a change in diagnosis.
Pediatric knee MRI and interpretation should not be predicated on radiologist review of knee radiographs in this subset of patients.
Paper #: PA-132 Sonographic Evaluation of Pediatric Skeletal Lesions: Is it worthwhile?
Henrietta Rosenberg, MD, Radiology, The Mt.
Sinai Medical School, Henrietta.Rosenberg@mountsinai.org; Amish Patel, Neil Lester Purpose or Case Report: The purpose of this paper is to demonstrate how ultrasound(US) may serve as a readily available, cost-effective, non-invasive, non-ionizing, practical tool for the evaluation of a variety of skeletal abnormalities in the pediatric age range.
We reviewed the clinical and imaging findings in 31 patients seen during the past 2 years in whom US demonstrated abnormalities related to the skeletal system, excluding patients with hip joint effusions or DDH.
Results: US proved useful in the following situations: evaluation hard superficial immobile mass (osteoma shin) (1), absent medial end clavicle on X-ray in region of neck mass (US showed ABC medial end clavicle)(1), to determine if soft tissue mass involves adjacent bone nodular fasciitis surrounding clavicular head (1), for diagnosis and followup fracture (displaced/non-displaced) in infants (4), diagnosis osteomyelitis in patients with cellulitis (4), question of fracture underlying cephalohematoma or subgaleal hematoma (4), rib mass (osteochondroma) (1) or mass costochondral junctions (contour deformities costochondral cartilage) (6), firm posterior knee mass (Baker's cyst) (1), firm anterior knee mass (septated cystic mass suprapatella region due to rheumatoid disease) (1), immobile hard scalp mass due to epidermoid cranial vault (1), painful mass occipital bone with soft tissue components extending through the skull externally and internally due to Langerhan's histiocytosis (1), indeterminate mass clavicle clinically thought to be post-traumatic sequellae, resolved on follow-up (1), assessment craniosynostosis (3), for differentiation of pathological entity from normal anatomic structure (lump on back of slender baby proved to be normal posterior spinous process) (1).
Conclusions: US is worthwhile for evaluation of wide range of pediatric skeletal abnormalities and helps to determine if the a lesion is one that is "touch" or "don't touch".
To maximize diagnostic accuracy, the imager should have thorough knowledge of the clinical history, physical findings, laboratory and other imaging findings.
In equivocal cases or in those patients in whom the field of view (FOV) is insufficient for complete visualization of an obvious lesion or if malignancy is suspected, US serves to triage those patients in whom further imaging is necessary.
High Incidence of Vertebral Fractures in Children with Acute Lymphoblastic Leukemia 12 Months After the Initiation of Therapy Mary Ann Matzinger, MD FRCP(C), University of Ottawa, matzinger@cheo.on.ca; Nazih Shenouda , Brian Lentle, Josée Dubois, Helen R. Nadel Purpose or Case Report: Vertebral fractures due to osteoporosis are a potential complication of childhood acute lymphoblastic leukemia (ALL).
To date, the incidence of vertebral fractures during ALL treatment has not been reported Methods & Materials: We prospectively evaluated 155 children with ALL during the first 12 months of leukemia therapy.
Lateral thoracolumbar spine radiographs were obtained at diagnosis and 12 months.
Vertebral bodies were assessed for incident vertebral fractures using the Genant semi-quantitative method, and relevant clinical indices such as spine bone mineral density (BMD), back pain and the presence of vertebral fractures at diagnosis were analyzed for association with incident vertebral fractures.
Results: Of the 155 children, 25 (16%, 95% Confidence Interval [CI] 11% to 23%) had a total of 61 incident vertebral fractures, of which 32 (52%) were moderate or severe.
Thirteen of the 25 children with incident vertebral fractures (52%) also had fractures at the time of diagnosis.
Vertebral fractures at diagnosis increased the odds of an incident fracture at 12 months by an odds ratio of 7.3 (95% CI 2.3 to 23.1, p00.001).
In addition, for every 1.0 standard deviation reduction in spine BMD Z-score at diagnosis, there was 1.8-fold increased odds for incident vertebral fracture at 12 months (95% CI 1.2 to 2.7%, p00.006).
Conclusions: Children with ALL have a high incidence of vertebral fractures 12 months after diagnosis, and the presence of vertebral fractures and reductions in spine BMD Zscores at diagnosis are highly associated clinical features.
Purpose or Case Report: To provide objective measures of acetabular morphology utilizing volume-rendered CT and to better characterize normal acetabular development in adolescents.
Implications for the diagnosis of femoroacetabular impingement (FAI) will be discussed.
Methods & Materials: 146 hips in 73 consecutive patients (36 female, 37 male; ages 13-20 years) who underwent abdominal and pelvic CT for non-hip related complaints were retrospectively examined.
Examinations were performed for a variety of complaints, including abdominal pain, nephrolithiasis, vomiting etc.
Patients with obvious hip pathology were excluded.
Pelvic rotation was eliminated, and pelvic inclination was measured and corrected to 60°u tilizing a volume rendered CT model.
Measurements of femoral head diameter (FHD), anterior femoral head coverage (FHCA), and posterior femoral head coverage (FHCP) were obtained.
Femoral head area (FHA) was defined as π(FHD/2)2.
Percent anterior femoral head coverage (%FHCA) was defined as (FHCA/FHA)*100.
Percent posterior femoral head coverage (%FHCP) was defined as (FHCP/FHA)*100.
Acetabular version by volume-rendered CT (AVVR) was defined as (FHCP/FHCA).
Results: Average pelvic inclination angle (sd) was 70.9 (5.6) for females and 64.8 (6.3) for males.
Average (sd) %FHCA was 22.7 (4.9) for males and 18.6 (5.6) for females.
Average (sd) AVVR was 2.39 (0.57) for males and 3.42 (1.19) for females.
Among males, average AVVR decreased with subject age.
On the other hand, there was little change in average AVVR with age among females.
Conclusions: Average AVVR is greater for females than males, and this difference becomes more striking with increasing subject age.
This represents an unexpected finding given the reported increased incidence of "pincer" type FAI among females.
Characterization of acetabular morphology among adolescents with clinical FAI should consider subject age and gender.
In this regard, volumerendered CT is capable of providing an objective measure of acetabular morphology.
Mistakes in Musculoskeletal Plain Film Interpretation James Crowe, Pediatric Radiology, Texas Children's Hospital, jecrowe@texaschildrens.org; George S. Bisset Purpose or Case Report: To evaluate the mistakes made by trained pediatric radiologists when interpreting radiographs of the extremities obtained for the evaluation of outpatient acute pain (mostly post-traumatic).
We retrospectively evaluated all radiographs and associated interpretations obtained during a 6 month period from April 15, 2011, to October 15, 2011, of the elbows, wrists, knees and ankles in pediatric outpatients who presented with acute pain in the affected area.
All radiographs were previously interpreted by a CAQ-certified pediatric radiologist varying in experience from 1 year to 57 years.
745 abnormals were identified, including 305 elbows, 168 wrists, 175 knees and 97 ankles.
All radiographs were determined to be "as dictated", missed significant finding, or overcall.
Attention was focused on the missed findings and overcalls.
Results: Findings were as follows: elbow radiographs-14 missed findings and 10 overcalls, wrist radiographs-12 missed findings and 5 overcalls, knee radiographs-9 missed findings and 0 overcalls, ankle radiographs-14 missed findings and 10 overcalls.
This resulted in a total of 49 missed findings (6.6% of abnormals) and 25 overcalls (3.4% of abnormals).
Of the 49 misses, 49% were fractures.
The highest mistake percentage occured in the ankles where the combined misses and overcalls approached 25%.
This was also the location where we found the highest percentage of missed fractures (9.0%) Conclusions: When just abnormal cases were considered, fully trained pediatric radiologists have a mistake rate of approximately 9.8%, if misses and overcalls are included.
From a quality improvement perspective, we will review all of the types of misses and overcalls to expose common themes.
Longitudinal Assessment of Osteoporosis in a Blood-Induced Hemophilia Rabbit Model Using Quantitative Ultrasound Kuan-Chieh Wang, University of Toronto, kc.wang@ utoronto.ca; Afsaneh Amirabadi, Anguo Zhong, Christopher Tomlinson, Andrea S. Doria Purpose or Case Report: The reduction of physical activities in hemophilic patients may lead to bone demineralization and consequent osteoporosis.
Quantitative Ultrasound (QUS) is free of ionizing-radiation, relatively inexpensive, and easy to use that making this technique suitable for follow-up of hemophilic children with clinical suspicion of osteoporosis.
To our knowledge, no previous study has investigated the value of QUS for longitudinal assessment of growing bones in an animal model which is paramount for clinical translation of the technique once change in measurements could relate to either the baseline pathology or physiologic bone growth variability.
The objective of this study is to investigate the intra-and inter-operator reliability of QUS over time, and its ability to discriminate bone loss in pathologic vs control knees of a rabbit model of blood induced arthritis.
Methods & Materials: Sixteen juvenile white New Zealand rabbits distributed into two groups: 8 received 8 intraarticular blood injections over 17 weeks (n 0 8 pathologic and 8 contralateral knees), and 8 noninjected rabbits were used as controls (n 016 knees).
Midshaft tibia speed-of-sound (SOS) was measured at baseline, and weeks 8 and 17 of the experiment.
Two operators scanned each site twice at each time point.
QUS measurements were compared to microCT (reference standard) on week 17 to validate the study results.
Results: The SOS measured in the control group increased significantly (P<0.001) over the 17 week period.
There was not such an increase in the arthritis SOS value (P>0.05).
In both groups the overall intra-operator coefficient of variation of SOS measurements was 6% at baseline and decreased to 2% at week 17 likely due to increased tibia size.
The inter-operator reliability was 6% at baseline and 3% at week 17.
With regard to the effect of bone growth on QUS measurements for the control group (n 016), SOS values increased by 419.13 m/s, whereas for the pathologic group (n08), they only increased by 195 m/s.
Statistically significant differences in ratios of SOS between final/baseline results were noted (P 00.016) between the pathologic and control groups.
Conclusions: The longitudinal use of QUS has an acceptable intra-and inter-operator reliability.
Even accounting for the significant impact that bone growth has on QUS measurements over time, QUS can differentiate pathologic from control knees in the proposed animal model and holds potential for clinical use in the assessment of osteoporosis in hemophilic children.
Methods & Materials: The study was approved by the institutional review board.
68 pediatric patients with 73 abdominal tumors (34 malignant and 39 benign lesions) underwent diffusion-weighted MR imaging (DWI) on clinical 1.5 T (n039) and 3 T (n029) MRI scanners.
ADC maps were generated from b0500 DWI and ADC values were retrospectively and independently measured by two radiologists.
ADC values of benign and malignant tumors were compared with the Welch two sample t-test.
A p value of 0.05 was considered to indicate statistical significant differences.
In addition, a receiver operating curve analysis (ROC) was performed to determine the optimal cut-off ADC value for differentiating benign and malignant tumors.
Results: The mean ADC value (mm2/sec) of benign tumors was 1.681 x 10-3 for the first reader and 1.679 x 10-3 for the second reader.
The mean ADC value (mm2/sec) of malignant abdominal tumors was 1.018 x 10-3 for the first reader and 1.113 x 10-3 for the second reader.
The differences between benign and malignant tumors were statistically significant (p<0.001 for both readers).
ROC analysis revealed an optimal cut-off ADC value for differentiating malignant and solid tumors as 1.1 x 10-3 mm2/sec.
Conclusions: Diffusion-weighted imaging with ADC maps can be used to differentiate between benign and malignant pediatric abdominal tumors.
Creation of a Database to Evaluate Imaging Findings in Long-Term Survivors of Pediatric Malignancy Alexander Towbin, MD, Radiology, Cincinnati Children's Hospital Medical Center, alexander.towbin@cchmc.org; Seth Hall Purpose or Case Report: Over the past 20 years, there have been significant improvements in the treatment of pediatric malignancies.
Improved therapy has led to an increase in the number of long-term survivors.
Many of these survivors are now experiencing late effects as a result of the original disease process or its treatment.
These late effects are frequently identified on imaging.
The purpose of this study is to create a database of the imaging findings of long-term survivors of pediatric malignancy in an attempt to begin to classify the findings and identify associations.
Methods & Materials: After IRB approval, the institutional cancer registry was searched to identify all patients younger than 20 years of age who were diagnosed with a solid tumor between 1980 and 2005.
Patients were included in the database if they survived for more than 2 years from the date of their initial diagnosis.
The electronic medical record system was then used to obtain demographic and treatment information for each included patient.
The dictated reports from all cross-sectional imaging studies evaluating the chest, abdomen, or pelvis performed more than two years from the date of diagnosis were then reviewed.
Each positive imaging finding was classified by the involved organ.
Results: After querying the institutional cancer registry, 909 patients were identified who met the inclusion criteria for this database.
The most common neoplasms were neuroblastoma, Wilms tumor, and astrocytoma.
Of the included subjects, 420 had imaging of the chest, abdomen, or pelvis.
Overall, 2851 reports were evaluated and classified.
Findings were most commonly identified in the lungs, musculoskeletal system, kidneys, liver, and lymph nodes.
Conclusions: A database examining the late effects in longterm survivors of pediatric malignancies was created.
This database has the potential to help identify the radiologic manifestations of the complications of cancer therapy and thus help guide rationally determined long-term risk-benefit ratios in the treatment of pediatric malignancies.
Imaging Followup of Lymphoma in Pediatric Patients: Is Pelvic CT Necessary?
Javier Lopez Bueno, MD, Children's Hospital of Eastern Ontario, jlopezbueno@cheo.on.ca; Nishard Abdeen Purpose or Case Report: Pelvic CT is often included in the imaging followup of patient with lymphoma before, during and after treatment to assess response to treatment and monitoring for relapses.
While such followup is expected to improve detection of relapse, there is little objective evidence of its effectiveness in lymphoma.
Anecdotally, there are few pelvic relapses in pediatric patients with lymphoma regardless of primary site.
We hypothesize that pelvic CT could be avoided as part of the followup without adverse impact on survival or in the detection rate of relapses, and with subsequent significant reduction in the radiation dose, particularly to the gonads.
Methods & Materials: Research ethics board approval was obtained.
Patients diagnosed with lymphoma and with at least one year of followup at our tertiary care pediatric hospital were included.
Sex, age, type of lymphoma, stage, primary site, site of relapse if any as well as the number of CT scans of the head, neck, chest, abdomen and pelvis were recorded.
Results: A total of 29 patients met study criteria.
There were 21 males and 8 females, with an average age of 11.9 years (range 3-17 years).
Eighteen patients had Hodgkin disease (62%) and eleven had non-Hodgkin lymphoma (38%).
Mean length of followup was 3.8 years (range 1-12 years).
An average of 4.5 pelvic scans per patient were performed for surveillance (range 0-12).
Three relapses were detected.
Of these only one was in the pelvis, in a patient whose initial T cell non-Hodgkin lymphoma was extensive and involved the neck, chest, abdomen and pelvis.
Conclusions: This study suggests a low incidence of pelvic relapse in pediatric patients with lymphoma.
The routine use of pelvic CT in surveillance protocols may therefore be of little benefit while imposing a significant radiation burden.
Our study is limited by small sample size and short length of followup.
Further large scale studies are required.
(ESFT) is performed by measuring the size of the tumors before and after chemotherapy.
The proposed method of measuring tumor size, however, differs amongst RECIST 1.1 (Response Evaluation Criteria in Solid Tumors), WHO (World Health Organization) and COG (Children's Oncology Group) response criteria.
In our project, we assessed whether response classification differs between the three different methods.
Methods & Materials: After IRB approval, we retrospectively analyzed MRI studies of 55 patients with Ewing Sarcoma who were treated at Stanford and UCSF Medical Centers.
Tumor size was assessed before and after therapy.
Tumor measurements were obtained using RECIST 1.1 (longest single diameter), WHO (longest diameter and perpendicular diameter), and COG criteria (three measurements to calculate tumor volume).
Tumor response was assessed by the differences in sizes of the tumors before and after treatment using four response categories: progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR).
Concordance between the three response classification systems was assessed using Cohen's kappa (k) coefficient and percentage of disagreement per response category.
Results: The k statistic for concordance in COG/WHO, COG/ RECIST and RECIST/WHO were 0.663, 0.210 and 0.166 respectively.
Disagreement rates for RECIST/WHO, COG/ WHO, and COG/RECIST were 12.73, 34.55, and 47.27% respectively.
Using tumor volume, twenty-six patients were reclassified: twenty-four cases of Stable Disease coded by RECIST were reclassified as Progressive Disease by COG and two cases of Partial Response coded by RECIST were reclassified as Complete Response by COG.
Conclusions: This study demonstrates poor agreement between the RECIST 1.1 and COG response criteria in ESFT.
Given the degree of discordance between response criteria in ESFT, evaluation of the prognostic impact of each of these classification systems may guide selection of the optimal system for future use in this disease.
Imaging Recognition of Chylous Ascites Following Surgery for Abdominal Neuroblastoma Zeyad Metwalli, MD, Baylor College of Medicine, metwalli@bcm.edu; R. P. Guillerman, Heidi V. Russell, Eugene S. Kim Purpose or Case Report: Surgical resection is a standard part of multimodality treatment of neuroblastoma, the most common abdominal malignancy of infancy and early childhood.
Chylous ascites is a rarely reported complication of surgery for abdominal neuroblastoma, and is likely underrecognized, posing the risk of nutritional deterioration and sepsis.
To facilitate early diagnosis and institution of appropriate therapy, we present the salient imaging findings of the largest known series of chylous ascites following surgery for abdominal neuroblastoma.
Methods & Materials: All patients with abdominal neuroblastoma complicated by post-operative chylous ascites over a five-year period at a large children's hospital were identified by a database search.
A retrospective review of the imaging studies and clinical charts was conducted.
Results: Chylous ascites developed following surgical resection of abdominal neuroblastoma in 5 of 36 patients, with the diagnosis made between postoperative days 20 and 33.
Four cases were high-risk neuroblastoma and one was intermediaterisk neuroblastoma.
All 5 cases involved resection of an adrenal mass and dissection around the abdominal great vessels.
All 5 cases manifested with abdominal distention on physical exam, and ascites was suspected clinically in 3 cases.
Computed tomography (CT) in all 5 cases revealed a large volume of ascites of near-water attenuation (range of −3 to 16.5 Hounsfield units).
The 3 cases imaged with ultrasound (US) showed hypoechoic or anechoic ascites without septations.
The chylous ascites resolved after 1-4 months of treatment with dietary fat restriction, medium chain triglycerides, intravenous octreotide, or peritoneal catheter drainage.
Conclusions: Chylous ascites is an under-recognized complication of surgical resection for abdominal neuroblastoma, occurring in 14% of patients in this series.
The diagnosis is supported by the demonstration on CT or US of a large volume of ascites causing abdominal distention 3-5 weeks post-operatively.
The ascites is typically near-water in attenuation rather than fatty in attenuation and should not be misattributed to peritonitis, hemorrhage, bowel leak, or early tumor recurrence.
Cervical Spine Injuries In Patients With Suspected Physical Abuse Nadja Kadom, MD, Radiology, Children's National Medical Center, nkadom@childrensnational.org; Zarir P. Khademian, Tanya Hinds, Katherine Deye, Allison M. Jackson, Eglal Shalaby-Rana Purpose or Case Report: To evaluate the incidence and nature of cervical spine injuries and relationship to posterior fossa abnormalities in children who underwent brain and cervical spine MRI as part of the clinical workup for suspected physical abuse.
Methods & Materials: Authors retrospectively analyzed records of eighty-five children less than three years of age who were documented by the Child Protective Services at a level one pediatric trauma center over a period of four years (2006) (2007) (2008) (2009) (2010) .
Only patients who underwent both MRI imaging of the cervical spines (c-spine) in addition to brain imaging as part of the clinical workup were included.
Cspine and posterior fossa of brain MRIs were independently reviewed by two pediatric neuroradiologists, both blinded to clinical details.
C-spine abnormalities (bone marrow edema, cord edema, intrathecal blood, disc pathology, soft tissue/ ligamentous injury, vascular injury) were documented and correlated with abnormalities seen in the posterior fossa (blood, brainstem edema, cerebellar edema).
Results: At this time, 40/82 patients have been reviewed.
Twenty patients (50%) had both cervical spine injuries and posterior fossa abnormalities.
There were no patients with isolated cervical spine injuries without posterior fossa abnormalities, but there were five patients (12.5%) that had posterior fossa abnormalities in the absence of c-spine injuries.
Fifteen patients (37.5%) did not have any spinal or posterior fossa imaging abnormality.
None of the patients had bone marrow edema, disc pathology, or intrathecal blood.
One patient had vascular neck injury and cord edema.
Conclusions: Our results show that the incidence of cervical spine injury in children under investigation for abusive head trauma is as high as 50%.
Our data show further that cervical spine injury predicted posterior fossa injury in all patients, while presence of posterior fossa injury predicted concomitant c-spine injury in only 75%.
The incidence of c-spine trauma we found in these patients is higher than reported elsewhere in the literature and may impact whether or not routine c-spine MRI will be included in national imaging guidelines for children under investigation of abusive head trauma.
Pediatric Skull Fracture Andre Loyd, PhD, Biomedical Engineering, Duke University, aml6@duke.edu Purpose or Case Report: Skull fractures are often seen in the setting of Non accidental trauma (abuse) abuse, and are usually attributed to falls from heights above 1 m. Part of the difficulty in assessing height is due to uncertainties in actual distance.
Objective: To determine what types of skull fractures can occur in pediatric and adult post-mortem human specimens during controlled impacts on hard surfaces from various heights.
Methods & Materials: Skull fracture patterns in postmortem human specimens from a unique bank of pediatric specimens (30-week gestation to 16-years-old, n013) were subjected to controlled drops from both arbitrarily low heights (15 and 30 cm) and high heights (2 m) onto an aluminum platen.
The specimens were dissected from the neck at the occipital condyles and intracranial were sealed inside the head using PMMA.
The heads were dropped on to five different impact locations.
Fractures were identified using palpation and high resolution MDCT.
Results: No specimens between 33-weeks-gestation and 24days-old sustained fractures from the 15-30 cm drops.
Three out of four (75%) specimens ages between 5-and 22-months old fractured due to the 15 or 30 cm drops.
The 9-and 16-year-old specimens and all adult specimens survived the 15-30 cm drops.
All specimens subjected to the 2 m drop fractured.
The specimen between 11-months and 22-months sustained either a linear fractures or diastatic fractures from the 15 cm and 30 cm drops.
The results indicate that some aged infants and young children can sustain skull fractures by being dropped or falling from relatively low heights.
Drops, as low as 15 cm, can cause linear and diastatic fractures in pediatric skulls.
The presences of compliant sutures and fontanelles in neonatal heads allow the head to deform during impact.
These data add very important information to mechanisms of skull fractures across ages, including ages in which child abuse is a consideration.
Evaluation of a New Classification System for Temporal Bone Fractures in Children Aimed at Increasing Prognostic Value Badriya Al-Qassabi, MD, McGill University, albahlania1@ yahoo.com; Lucia Carpineta, Rania Ywakim, Bahar Torabi, Andrew M. Zakhari, Lily H P. Nguyen Purpose or Case Report: To compare a new classification of temporal bone fractures which specifically evaluates involvement of the otic capsule against the traditional classification system (transverse versus oblique versus longitudinal), to evaluate whether this new classification is able to better identify patients at risk of adverse otologic outcome and neurologic complications in the pediatric population.
Methods & Materials: A retrospective hospital chart review was performed by ENT colleagues searching for all patients with temporal bone fractures seen at our center over the past 10 years.
This was followed by a blinded review of the CT heads by a resident and a trained pediatric radiologist with neuro expertise.
These CTs were evaluated for petrous involvement, otic capsule involvement and any associated intracranial lesions.
This information was then correlated with clinical outcome measures including post-traumatic hearing deficit, facial nerve palsy, persisting CSF leak and global neurologic sequelae.
The new classification was compared to the traditional one, and specifically analysed for the ability to better predict the clinical outcomes.
Results: Expectedly, pediatric temporal bone fractures were infrequent and otic involvement even more rare.
Fractures with involvement of the otic capsule (versus otic sparing) were found more frequently in boys.
They were also more likely to be associated with immediate otologic signs and neurologic findings on presentation.
These fractures also had the highest association with conductive hearing deficit (>60%) and were twice as likely as otic sparing fractures to be associated with immediate facial nerve palsy and with more important concomitant intracranial injuries such as midline shift.
Conclusions: While our numbers are small, our results suggest a trend that when temporal bone fractures show involvement of the otic capsule, there is higher risk of adverse otologic outcome and neurologic complications even in the pediatric population.
Absence of a Causal Relationship Between MR Detected Subdural Hematomas (SDH) in Neonates with Hypoxic-Ischemic Encephalopathy (HIE) Deniz Altinok, Children's Hospital of Michigan; Jay Shah, Harut Haroyan, Gulcin Altinok, Nitin Chouthai Purpose or Case Report: The existing controversy regarding subdural hemorrhages noted in patients with HIE is an important discussion in the medical, legal and child-welfare realms.
It is our goal to provide additional information to this critical debate through MR findings on patients with clinically diagnosed HIE.
Methods & Materials: All patients born with clinically diagnosed HIE, and treated at Children's Hospital of Michigan in the past 8 years were examined; those with Head MRI taken within 19 days of life were selected.
In total, 41 patients fit the criteria, this included: 22 Males and 19 Females, and an age range of 2-19 days at scan (average age of 10 days at scan).
All traumatic births, coagulopathies, and other pertinent clinical findings were noted.
MR Imaging was reviewed and reported by a blinded pediatric neuroradiologist, these reports were then compared to the "original read".
Results: All 41 patients were confirmed radiologically to have HIE.
The causes of HIE in all cases examined were either intrauterine/delivery asphyxia, aspiration, or congenital disease.
Of these 41 cases, the findings were: 6 SDH, 11 parenchymal hemorrhages, 4 intraventricular hemorrhages, 4 cephalohematomas, 3 subarachnoids, 1 large subcutaneous hemorrhage and 1 instance of MCA stroke.
All 6 patients with MR Detectable SDH had 1 or more confounding factors (1 Meningitis, 3 Coagulopathies, 4 Chest Compressions, 1 Cardiac Malformation, 1 PPH, 1 Severe Pulmonary Hemorrhage requiring Transfusion of Plasma and pRBC).
Conclusions: It has been hypothesized that SDH is often found incidentally in children diagnosed with HIE, this is however a dubious conclusion considering our results.
In fact, the presence of SDH and HIE concomitantly is low even when including a population with traumatic births such as ours.
Sports-Related Concussion in Children: An MRI and MRS Study Kim Cecil, PhD, Cincinnati Children's Hospital Medical Center, kim.cecil@cchmc.org; Todd A. Maugans, James L. Leach, Mekibib Altaye Purpose or Case Report: The pathophysiology of sportsrelated concussion (SRC) is poorly understood, especially for children.
Following SRC and mild traumatic brain injury in adults, a few MRI and proton MRS studies have identified axonal injury with declines in the neurometabolite N-acetyl aspartate (NAA).
We wanted to examine a SRC adolescent population with proton MRS, diffusion tensor imaging (DTI) and other MRI methods within 72 h of concussion and with short term followup to determine if there were differences in imaging metrics with age and sex matched healthy control participants.
Methods & Materials: Twelve children, ages 11-15 years, who experienced SRC were evaluated with ImPACT neurocognitive testing, T1-weighted MRI, susceptibility weighted imaging (SWI), DTI, proton MRS, and phase contrast angiography (PCA) at less than 72 h, 14 days and 30 days or greater post-concussion.
Healthy, age and sex matched controls for each SRC participant were recruited and evaluated at a single time point.
Quantitative imaging metrics included fractional anisotropy, metabolite concentrations, and global cerebral blood flow (CBF).
Group comparisons were examined by paired t-test or Wilcoxon signed rank test.
Correlational data employed Spearman rank correlation.
Results: ImPACT results revealed significant differences in initial total symptom score (TSS), and reaction time (RT) for the SRC group compared with the control group, with TSS resolving by a mean of 14 days and RT at 30 days.
No evidence of structural injury was observed qualitatively for either group.
Analyses between groups or over time within the SRC group found no decreases in NAA or elevation of lactic acid upon MRS, and no changes in fractional anisotropy upon DTI.
Within the SRC group, significant changes in the global CBF were observed.
Improvement towards control values occurred by 14 days for 27% and by 30 days for 64% of SRC group participants.
Conclusions: Pediatric SRC affects global CBF without evidence of structural or metabolic injury.
Predictive Value of High Resolution MR Imaging of Brain and Sella in Children with Clinical Optic Nerve Hypoplasia for Hypopituitarism Charles Glasier, Radiology, Arkansas Childrens Hospital, glasiercharlesm@uams.edu; Raghu H. Ramakrishnaiah, Julie Shelton, Chetan C. Shah, Paul H. Philips Purpose or Case Report: To review the spectrum of CNS abnormalities and their incidence in children with optic nerve hypoplasia and to calculate the sensitivity and specificity of magnetic resonance imaging in predicting endocrine abnormalities.
Methods & Materials: This is an IRB approved retrospective study of 44 children with clinical optic nerve hypoplasia who underwent MRI of the brain and orbits as part of the clinical workup in a tertiary care pediatric hospital.
High resolution MRI studies were performed on 1.5 Tesla scanners.
MRI studies were reviewed for optic nerve hypoplasia, absent or ectopic posterior pituitary, absent pituitary infundibulum, absent septum pellucidum, migration anomalies and hemispheric injury.Radiologists were blinded to patients endocrinologic status.All patients had clinical evaluation by a pediatric Neuro-ophtalmologist and endocrinologist.
A standardized panel of serologic testing that included serum cortisol, ACTH, TSH, and free T4 levels were performed on all patients.
Statistical analysis was performed to determine the sensitivity and specificity of MR findings in predicting endocrinologic deficiency.
Results: Study included 44 children(26 males and 18 Females) who had clinical optic nerve hypoplasia.
The mean age of the study population was 3 yr (SD:4.7 Yr).15 children had unilateral and 29 children had bilateral optic nerve hypoplasia by MRI.6 children had absent posterior pituitary bright spot and 9 had ectopic posterior pituitary,7 had absent infundibulum,1 had complete callosal agenesis,3 partial callosal agenesis and 16 had callosal thinning.
9 had absent septum pellucidum.
2 had hypopituitarism.
Of the 12 patients with Hypopituitarism 8 had abnormal abnormal pituitary on MRI, 3 had absent septum pellucidum, and 1 child had migration abnormality.
None had corpus callosal abnormality.
The sensitivity and specificity of MRI in predicting Hypopituitarism by demonstration of abnormal pituitary is 75% and 81% respectively.
The positive predictive value and the negative predictive value is 60% and 90% respectively.
Among the 32 patients with normal endocrinologic function, none had pituitary abnormalities on MRI.
Conclusions: Pituitary abnormalities are the most common intracranial abnormality in patients with optic nerve hypoplasia followed by absent septum pellucidum.
Detection of pituitary abnormalities by the MRI has high specificity and high negative predictive value for endocrine abnormality.
Paper #: PA-148 CT Imaging Pearls for Shunted Pediatric Brains Srikala Narayanan, MD, Children's National Medical Center, snarayan@childrensnational.org; Nadja Kadom Purpose or Case Report: Shunted pediatric patients frequently present emergently with symptoms that could indicate shunt malfunction, such as headache and vomiting.
Here, we present imaging pearls on non-contrast head CT in shunted children.
Methods & Materials: Illustration of each of the following: 1.
Shunt tip and volume averaging-Consider location of side holes and use of multiplanar reformatted images.
2.
Shunt at burr hole-Consider radiolucent shunt parts.
3.
Shunt rupture in the neck-Remember to investigate the lower extracranial shunt parts.
4.
Shunt in cyst/subdural shunt (vs dislocation)-Consider primary shunt location in a cyst rather than shunt dislocation.
5.
Enlarged temporal horns-Look for it.
In infants occipital horns may dilate first.
6.
Enlarged 3 rd ventricle-Look for bulging of lateral walls.
7.
Sulcal effacement-Use the "three shades of gray" rule.
8.
Small cisterns-Detecting shape distortion can help.
9.
Periventricular edema-Easily overlooked because of similar low density compared to ventricular fluid.
10.
Slit-ventricle -Requires cautious reporting.
Conclusions: Careful evaluation of CT images in shunted pediatric patients can reveal important clues for making an accurate diagnosis, even when prior images are not available.
Successful Treatment of Mice with Creatine Transporter Deficiency Kim Cecil, PhD, Cincinnati Children's Hospital Medical Center, kim.cecil@cchmc.org; Diana M. Lindquist, Matthew R. Skelton, Gail J. Pyne-Geithman, Joseph F. Clark Purpose or Case Report: Creatine transporter deficiency (CTD) is an untreatable X-linked mental retardation syndrome with severe cognitive and speech impairment.
Patients are identified by an absence of creatine in the brain on MR spectroscopy (MRS) and distinguished from two creatine synthesis deficiency syndromes with genetic testing.
For CTD, the absence of the transporter (SLC6A8) prevents creatine from crossing the blood brain barrier and entering brain cells.
A brain specific CTD knockout mouse was developed replicating key features of the human disease and establishing an animal model for treatment of CTD.
We report the successful treatment of the CTD knockout mouse and present confirmation by MRS. Methods & Materials: Brain specific knockout and littermate control mice were randomly assigned and treated with on one of three supplements: AgentX (confidential), creatine or maltodextrine as placebo.
1H and 31P MRS data were collected on a 7 T MR system (Bruker).
Mice (N016) were studied with MRS after 9 weeks of supplementation.
Single voxel 1H data were acquired on a 144 uL voxel covering the cerebrum using a double spin echo sequence.
31P data were acquired with an ISIS sequence from the same voxel.
Metabolite quantification was performed with jMRUI and compared between groups and over time with statistical tests for significance (t-tests, ANOVA).
Results: Creatine and phosphocreatine levels in the brain were all significantly higher after 9 weeks supplementation of AgentX in knockout mice, compared to creatine and placebo fed knockout mice (Phosphorus MRS [144 uL brain voxel] with phosphocreatine (PCr) (0 ppm) observed only in AgentX treated knockout mice.
Adenosine triphospate (ATP) gamma (−2.5 ppm), alpha (−7.5 ppm) and beta (−17 ppm) peaks are noted in all three knockouts.
Conclusions: Successful treatment was achieved in a SLC6A8 brain specific knockout mouse for the second largest known cause of X-linked mental retardation in humans, CTD.
Disclosure: Dr. Cecil has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Prevalence of Abusive Injuries in Siblings and Contacts of Abused Children Kenneth Feldman, MD, General Pediatrics/Children's Protection Program, University of Washington/Seattle Children's, kfeldman@u.washington.edu Purpose or Case Report: Siblings and children who share a home with a physically abused child are thought to be at high risk for abuse.
However, rates of injury in these children are unknown.
Disagreements between medical and CPS professionals are common and screening is highly variable.
Our objective was to measure the rates of occult abusive injuries detected in contacts of abused children using a common screening protocol.
This was a multi-center, observational cohort study of 20 child abuse teams who shared a common screening protocol.
Data were collected for all children <10 years undergoing evaluation for physical abuse and their contacts.
For contacts of abused children, the protocol recommended physical examination for all children <5 years, skeletal survey and physical exam for children <24 months, and physical exam, skeletal survey and neuroimaging for children <6 months old.
Results: Among 2,825 children evaluated for abuse, 618 met criteria as "physically abused" and these had 477 contacts.
For each screening modality, screening was completed as recommended by the protocol in approximately 75% of cases.
Of 133 contacts who met criteria for skeletal survey, new injuries were identified in 16 (12.0%).
None of these fractures had associated findings on physical examination.
Physical examination identified new injuries in 6.2% of 257 eligible, examined contacts.
Neuroimaging failed to identify new injuries among 19 imaged, eligible contacts less than 6 months old.
Twins were at significantly increased risk of fracture relative to other non-twin contacts (56.3% vs 11.9%, OR 19.9).
Conclusions: These results support physical examinations and skeletal survey, regardless of physical examination results, for contacts of abused children <24 months of age.
Too few children had cranial imaging to change recommendations to image contact children less than 6 months old.
Even for children where no injuries are identified, these results demonstrate that abuse is common among children who share a home with an abused child.
They support including contacts in evaluations and interventions (foster care, safety planning, social support) designed to protect physically abused children.
The project was supported by the Health Resources and Services Administration/Maternal shown that pediatric rib fractures may be a marker for significant intrathoracic injury.
This information has been used to suggest that children with rib fractures and no underlying intrathoracic injury may have sustained them due to insufficient bony mineralization and minor trauma rather than inflicted injury.
Methods & Materials: IRB approval was obtained for a retrospective review of all children under 3 years of age with imaging diagnosis of rib fracture over a 6-year period at two University hospitals.
Children with prior thoracotomy, previously recognized metabolic bone disease, and prematurity <36 weeks were excluded.
Medical records were reviewed and children with documented abuse or accidental trauma were evaluated.
Children with indeterminate injury mechanisms were excluded.
Sixty-six patients with rib fractures were included in analysis, 47 due to abusive injury and 19 due to accidental trauma.
Children were analyzed for associated intrathoracic, abdominal or intracranial injury, additional fractures and retinal hemorrhage.
Results: Abused children were younger (4.7+/−6.1 months) than accidentally injured children (18.9+/−11.1 months, p< 0.001).
Children with rib fractures due to accidental trauma had a higher incidence of intrathoracic injury compared to those due to abusive injury (53% vs 13%,p<0.001).
There was no difference in the incidence of abdominal or intracranial injury between groups.
Mortality and ICU admission rates were similar.
Abused children had a higher total number of rib fractures (mean 5.5 vs 3.0, P<0.009) and were more likely to sustain additional fractures outside of the thoracic cavity (77% vs 63%, P<0.001).
Conclusions: Abuse is a more common cause of rib fractures in young children than accidents.
Children with rib fractures due to abusive trauma are less likely to have intrathoracic injury compared to those sustaining rib fractures due to accidental trauma.
This suggests differences in mechanism of injury between groups.
Pediatric Elbow Fractures: A Different Angle on an Old Topic Shannon Zingula, MD, Pediatric Radiology, Cincinnati Children's Hospital Medical Center; Kathleen Emery, Christopher G. Anton Purpose or Case Report: The 3 most common elbow fractures classically reported in pediatric orthopedic texts are supracondylar (SC) (50-70%), lateral condylar (LC) (20%), and medial epicondylar (ME) fractures (10%) with fractures of the proximal radius (including but not limited to fractures of the radial neck) being relatively uncommon (5-10%).
Our experience at a large children's hospital suggests a different distribution.
Purpose: 1) to describe the frequency of different elbow fracture types in a large pediatric population, and 2) to determine the fracture types that were occult on initial radiographs but detected on follow-up.
Methods & Materials: Review of medical records identified 468 children, median age 6 years and interquartile range for age of 4-8 years (range, 0.8-18 years) diagnosed with elbow fractures at our institution from October 2010 through July 2011.
Initial and follow-up radiographs were reviewed in blinded fashion independently by two experienced pediatric musculoskeletal radiologists to identify fracture type(s) on initial and follow up radiographs.
Note was made of fractures identified on follow up only.
Results: The most common fractures included SC (n0254, 54%), radial neck (RN) (n080, 17%), and LC fractures (n066, 14%).
As compared to classically referenced incidences, RN fractures were seen significantly more (p<0.0001) and ME fractures (n025, 5%) significantly less (p0.0008) than would be predicted.
In 26 patients without fracture seen on initial films, occult fractures were seen on follow up; SC (n012, 46%) and RN fractures (n08, 31%) were most common.
The frequency of RN fractures compared to the overall group (31% vs. 17%) approached but did not reach statistical significance (p00.06).
34 patients with one fracture had additional fractures seen on follow-up not seen initially with olecranon fractures most frequent (n018, 53%.)
This was significantly more common than the number identified on initial radiographs (n033, 7%) (p<0.0001).
Conclusions: SC fractures are the most frequent elbow fracture seen initially and in follow up followed by RN and LC fractures in a distribution different than classically described.
The relatively high frequency of RN and olecranon fractures detected on follow up speaks to their potentially occult nature.
Careful attention to these areas is warranted in patients with initially normal radiographs.
Purpose or Case Report: Previous studies have found that fractures involving the spine, hands and feet are rare on skeletal surveys for suspected child abuse, leading some authors to suggest eliminating views of these regions from the initial skeletal survey protocol.
The purpose of this study was to assess this recommendation by performing a historical review of these injuries in a population undergoing screen-film based skeletal surveys for suspected abuse.
This cross-sectional, retrospective IRB approved study reviewed the reports of the initial skeletal surveys of all children <2 years of age with suspected abuse imaged between April, 1988 and December, 2001 .
Infants underwent skeletal surveys according to ACR standards acquired on a mammographic type screen-film imaging system with at least 13 line pairs per millimeter resolution.
Studies in toddlers were performed using a par speed screenfilm system.
Results: 62% (225/365) of all skeletal surveys demonstrated positive findings, and 44% (98/225) had >1 fracture.
5.5% (20/365) of all studies had fractures involving the spine, hands or feet.
Of all positive skeletal surveys, 8.9% (20/ 225) had fractures involving the spine, hands or feet, and 20.4% (20/98) of all patients with >1 fracture on skeletal survey had fractures involving these regions.
Conclusions: These data, acquired in the screen-film era, suggest that fractures of the spine, hands and feet may not be rare in infants and toddlers in cases of suspected child abuse.
The benefits of eliminating views of these regions from the initial skeletal survey should be carefully weighed against the cost of missing these potentially important injuries in at-risk pediatric populations.
Purpose or Case Report: Dating fractures is critical in cases of suspected infant abuse, but there are little scientific data to guide radiologists, and dating is generally based on personal experience and conventional wisdom.
We previously reported a scientific scheme for dating fractures in infants based on an analysis of subperiosteal new bone and callus formation in birth-related clavicular fractures.
We hypothesize that when used as a guide this system can significantly improve the ability of radiologists to accurately date fractures in young infants.
Methods & Materials: 103 radiographs of presumed birthrelated clavicular fractures in infants 0-3 months were reviewed by 2 pediatric radiologists with 2 (reader A) and 15 (reader B) years experience in two reading sessions separated by one year.
For the first read, no guidelines were provided.
Training was carried out prior to the second session, and readers were given the dating scheme as a guide during fracture analysis.
Readers were asked to provide an estimate of the minimum and maximum fracture age in both sessions.
The primary outcome was whether or not the reader's estimated range for fracture age included the actual fracture age.
A secondary outcome was the width of the estimate of fracture age.
These outcomes were compared across the two reading sessions.
Results: The rate of correct response significantly increased after training for each reader (reader A: 66% to 89%, p<.0001; reader B: 76% to 86%, p0.041).
The width of estimated fracture age after training was significantly smaller for each reader (reader A: mean width 17 days to 13 days, p<.0001; reader B: 25 days to 15 days, p0.001).
Conclusions: Our results suggest that the ability of a radiologist to accurately date fractures can improve significantly when provided with a scientifically based system outlining patterns of fracture healing.
This scheme can be applied in radiologic practice and may prove particularly useful in cases of suspected abuse, where fracture dating often has forensic implications.
Purpose or Case Report: To demonstrate the acute and subacute features of proximal femoral physeal fractures in the abused child.
Also to demonstrate how to recognize this injury in patients with unossified femoral heads.
The database of patients with suspected non-accidental trauma, accumulated over 12 years, was reviewed.
254 out of a total of 599 patients (43%) were proven to be cases of non-accidental trauma, as determined by the child abuse pediatrician.
From these 254 patients, the cases of proximal femur growth plate fractures were identified.
Results: 7 patients with proximal femur growth plate fractures were identified for a prevalence of 2.8%.
One patient had bilateral proximal femoral fractures, for a total of 8 fractures in 7 patients.
5 were boys, 2 were girls with ages ranging from 2.5 mos to 2 yrs 2mos.
In 4 patients, the fracture was revealed on imaging performed because of refusal to bear weight; in the other 3 patients, the fracture was found during imaging for the skeletal survey.
The fracture was on the left side in 7 cases and on the right side in 1 (the patient with bilateral fractures).
In all of the fractures, there was lateral displacement of the femoral shaft.
In 3 fractures, the femoral head was not yet ossified simulating the appearance of a dislocation.
Location of the femoral head in the hip joint was verified by ultrasound or CT (CT abdomen had already been done in 1 patient) thus delineating the presence of a physeal fracture.
6/8 fractures were Salter-Harris I and the other 2 were Salter-Harris II fractures.
The fracture was acute in 2 cases and subacute in 6 cases.
In these 6 subacute cases, periosteal reaction and/or calcifying subperiosteal hemorrhage was present in 3, and irregularity and scalloping of the metaphysis was present in the other 3.
Conclusions: Proximal femoral growth plate fractures are quite uncommon in non-accidental trauma.
The injuries are typically Salter-Harris I or II fractures, seen more often in the healing phase.
In the presence of an unossified femoral head, the laterally displaced femoral shaft can simulate hip dislocation; this can be clarified with hip sonogram.
Purpose or Case Report: In recent years, metal stents have been used to overcome airway obstruction in children for whom no better surgical option is available.
These devices are not designed for use in the airway, however, and may cause significant complications.
Bioabsorbable airway stents may avoid some of the problems associated with metal stents.
Methods & Materials: This is a retrospective review of all endoluminal insertions of bioabsorbable airway stents at a single institution from April 2010 to September 2011.
Custom-made polydioxanone stents of various sizes (Ella DV, Ella, Czech Republic) were used.
Results: Twelve stents were inserted in the airways of seven children.
Indications were: recurrent obstruction after slide tracheoplasty (2), persistent airway compression after correction of a congenital cardiac lesion (2), collapse of stem cell supported tracheal homograft, tracheomegaly following fetal balloon insertion, and syndromic tracheobronchomalacia (TBM).
Eleven stents (diameters 6 to 12 mm) were placed in the trachea and one in the left main bronchus.
Two stents had to be removed and replaced for technical reasons (one was too long and the other too narrow).
The child with syndromic TBM died when treatment was withdrawn because she could not be weaned from the ventilator.
The remaining children are alive at a median follow-up of nine months (range 1 to 17 months).
The granulation tissue response was similar to that seen after placement of metal stents.
The stents were observed to absorb gradually over a period of approximately three months, requiring serial stenting in two children.
Conclusions: Bioabsorbable airway stents are more difficult to insert than metal stents.
They cause similar early complications, especially granulation tissue formation, but appear to avoid potential long-term complications of metal stents, including vascular erosion and growth limitation.
Disclosure: Dr. McClaren has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: Renal angiomyolipomas (AMLs) in tuberous sclerosis complex (TSC) grow at a faster rate, exhibit a wider and more problematic range of symptoms, and hemorrhage more frequently than sporadic AMLs.
We examined the efficacy of prophylactic embolization of renal AMLs in TSC in decreasing tumor size, alleviating symptoms, and preventing hemorrhage while preserving renal function.
We retrospectively reviewed the charts and imaging studies of 47 consecutive patients who underwent transarterial, transcatheter embolization of 52 AMLs.
Tumor volume was measured from available CT or MRI imaging before and after embolization.
Pre-and postembolization symptoms and creatinine levels were documented.
Results: 37 patients had available follow-up imaging at a mean of 63 months post-embolization.
The mean preembolization tumor volume was 581 mL and postembolization was 284 mL; median decrease in volume was 76%.
Using the Schwartz method, the mean glomerular filtration rate before embolization was calculated to be 95.75 mL/min/1.73 m2.
After embolization the mean value was statistically unchanged at 101.97 mL/min/1.73 m2.
None of the patients experienced renal hemorrhage or symptom recurrence during the follow-up period.
Conclusions: Selective embolization of renal AMLs in patients with TSC decreases tumor volume, relieves symptoms and reduces the risk of future hemorrhage while preserving renal function.
ETOH was injected percutaneously with 25 G needle; transductal ablation performed through a 4 F micropuncture sheath.
Drug volumes, technical difficulties, percentage reduction in saliva production, family reported clinical significance, and complications were recorded.
Results: Salivary gland ablation (SGA) included bilateral SMG and SLG ablation without parotid gland ablation in 20 cases, and with unilateral parotid gland ablation in 4 cases.
One case of bilateral parotid gland ablation following surgical resection of bilateral SMGs.
Mean ETOH dose for SMG04.2 ml, and 3.1 ml for SLG.
One case of focal skin necrosis was noted; no other complications.
Patient families reported response to SGA in 24/25 cases (96%) with mean saliva production of 66%.
Greatest health and family impact was reported with elimination of hospitalizations for recurring aspiration pneumonia (2 cases), elimination of choking in bed (3 cases) , and improved patient sense of self-hygiene in 8 cases.
One complication occurred with temporary marginal mandibular nerve paralysis (resolution in 6 months).
Conclusions: Percutaneous and transductal SGA is feasible, safe, and effective in this small patient series, offering an alternative to surgical salivary gland resection, or treatment option following failed surgical intervention.
Paper #: PA-159 MR-Guided Procedures in Children: Initial Experience Joao Amaral, MD, Diagnostic Imaging, The Hospital for Sick Children, joao.amaral@sickkids.ca; Michael Temple, Dimitri Parra, Philip John, Bairbre Connolly Purpose or Case Report: The primary purpose of this study was to review our initial experience with MR-Guided procedures in children.
Our secondary objective was to share some aspects on how to start an MR-Guided program in a tertiary pediatric center.
Patients with lesions identified only on Magnetic Resonance (MR) imaging were selected to undergo an MR-Guided procedure.
Patients' demographic data, primary diagnosis, referring team's clinical suspicion, lesion's anatomical location, tissue adequacy for pathology, final diagnosis and clinical follow up were reviewed.
Aspects of starting a program of MR-guided procedures, safety concerns, imaging and technical challenges, and MR compatible materials were also addressed.
Results: To date, 7 procedures (5 bone biopsies, 1 soft tissue biopsy and 1 pre-surgical needle localization) were performed in 6 patients during 9 months.
There were 4 girls and 2 boys with a mean age of 10.2 years (3y5mo-17 yrs).
One patient had a Nasopharyngeal Carcinoma, 1 Cardiofacial Syndrome, 1 Wilm's tumor and 3 had no previous medical issues.
The clinical suspicion for 2 procedures in 2 patients was metastatic disease and for 5 procedures in 4 patients was primary malignancy or infection.
Lesions were located in the tibia (2-metaphysis and diaphysis), femur (2 -metaphysis and epiphysis), thigh (1-soft tissues), sacrum (1) and retroperitoneum (1).
All biopsies provided adequate tissue for diagnosis.
Needle localization and hook deployment was also accurate.
Malignancy was excluded in all patients.
Final diagnosis included 1 Chronic Recurrent Multifocal Osteomyelitis (CRMO), 3 Osteomyelytis, 1 fibrous tissue, 1 Osteoid Osteoma, and 1 scar tissue.
Mean follow up was 6.6 months.
No patient required a second procedure to confirm the diagnosis.
Conclusions: MR with its unique soft tissue resolution and lack of ionizing radiation is an excellent method to guide interventions in children.
One of the greatest advantages of this method is the precise target localization especially in lesions located in the bone marrow or lesions better identified on MR. Special safety measures, specific MR compatible material (needles, surgical instruments), dedicated imaging techniques to reduce or increase material/needle artifact and careful technique are paramount.
2006-2011.
11 M, 15 F; 2 .5-59 months, mean 8.3 months, median 5 months, mode 5 months.
Sonographic approach expanded as our experience grew over 71 months.
25 studies performed by a single pediatric radiologist.
Bilateral sonography included: interscalene and supraclavicular neck, nerve roots at neural foramina, cervical spinal canal, diaphragm during spontaneous respiration, rhomboid muscle, serratus anterior muscle, posterior shoulder, all performed and interpreted blind to other imaging.
Results: Interscalene and supraclavicular neck evaluated in all patients.
All exhibited echogenic interscalene portion of brachial plexus.Size and extent of traction neuroma varied.
Nerve roots at foramina noted in axial and coronal planes.
In 11 cases enlarged root(s)noted.
Cervical spinal canal studied in 19 patients: cord oscillated normally, no syrinx, cord concentric in canal.
Intracanalicular traction pseudomeningoceles on concurrent CT myelography or MRI were not apparent on US.
In 2 cases a "clumped" retracted nerve root on the cervical cord was later found to correspond to a pseudo-meningocele on CT myelogram.
Otherwise, cervical spinal canal US was unremarkable in 24 cases.
Diaphragm motion was evaluated in 23 patients during spontaneous respiration; no phrenic nerve palsy.
Rhomboid muscle was evaluated for atrophy in 16 patients; 4 had atrophy.
The rhomboids are innervated by the dorsal scapular nerve which arises solely from C5, prior to C5 joining the brachial plexus.
Intact rhomboid indicates that the central C5 root is intact.
Serratus anterior muscle, innervated by the long thoracic nerve (C5,C6,C7), was evaluated for atrophy in 12 patients; 6 had atrophy.
Dynamic evaluation of the posterior shoulder looking for posterior laxity was evaluated in 10 patients; 4 had laxity.
Posterior shoulder dislocation or subluxation is a known sequela of brachioplexopathy which sometimes requires muscle transfer when the child is older.
Conclusions: Comprehensive US evaluation of perinatal brachioplexopathy detects: extent of traction neuromafibroma from the interscalene region peripherally toward clavicles (important for neurosurgeon), thick nerve roots, phrenic nerve diaphragm palsy, muscle atrophy from denervation, and posterior shoulder subluxation.
US misses: intracanalicular traction pseudomeningoceles.
Paper #: ALT-001 Impact of the Image Gently Campaigns in Adult-Focused Hospitals: A Survey of Practice Leaders Brett Bartz, Duke University Medical Center; Donald Frush, Kimberly Applegate, Michael Callahan, Laura Coombs, Marilyn Goske Purpose or Case Report: The Alliance for Radiation Safety in Pediatric Imaging is an organization that uses social marketing to promote radiation protection for children and effect change across radiology practices.
The impact of the Alliance's Image Gently campaigns on practice patterns in radiology practices has yet to be assessed, especially outside of freestanding children's hospitals.
The purpose of this investigation was to assess the impact of the Image Gently campaigns on academic and private practices/institutions that treat children but primarily serve adults.
A web-based survey was emailed to leaders in radiology practices (n01186) who do not practice at freestanding children's hospitals utilizing the ACR's PRED database.
The survey consisted of 18 questions designed to measure the recognition and impact of the Image Gently campaigns, including the impact on practice patterns.
Results: A total of 186 practice leaders in 41 U.S. states and territories responded for a response rate of 15.7%.
The majority (94%) of sites image pediatric patients in their practices.
Respondents consisted of department chairs (60%), group presidents/CEOs (33%), and division chiefs (13%).
The majority (52%) of respondents described their practice as a hospital-based private practice without a dedicated pediatric radiology division.
The vast majority (95%) of respondents was familiar with the Image Gently campaigns; 55% of respondents reported that Image Gently had effected a change on how they imaged children.
Specifically, respondents (%) reported that the campaign caused a modification to lower dose protocols for head CT (57%), chest CT (66%), and abdominal/pelvic CT (69%).
Slightly more than half of respondents (55%), however, estimated that the Image Gently campaign resulted in no modification of pediatric fluoroscopy exposure.
Conclusions: To our knowledge, this is the first survey evaluating the impact of the Image Gently campaigns.
There is near universal recognition of the campaigns, which have impacted practice patterns beyond the freestanding children's hospital in CT, but not in fluoroscopy.
Reliability of Shear-wave Velocity Using Different Frequencies in Acoustic Radiation Force Impulse (ARFI) Elastography Mi-Jung Lee, Radiology, Severance Children's Hospital, mjl1213@yumc.yonsei.ac.kr; Suyon Chang, Myung-Joon Kim Purpose or Case Report: Although there are many studies about acoustic radiation force impulse (ARFI) measurement, standard protocol has not been established.
And a new probe with high frequency has been developed which can be applied for pediatric patients.
The purpose of this study was to assess the reliability of shear-wave velocity (SWV) at various depths using different frequencies to suggest standard measurement in ARFI elastography.
Methods & Materials: ARFI elastography of both the elasticity phantom and normal liver was performed at different depths (2-5 cm) with convex (1-4 MHz) and linear (4-9 MHz) probes.
Ten valid SWV measurements at each depth were performed.
It was repeated ten times with the phantom and it was done in 8 healthy volunteers (M:F03:5, age 20-34 years; mean 25.5).
The mean value and standard deviation of SWV were calculated.
Results: In both the elasticity phantom and the liver, variability of SWV was different between the depths in both probes.
The depth with lower variability in the phantom was 4 and 5 cm with the convex probe and 2 cm with the linear probe.
In the liver, the depth with lower variability was 4 cm with the convex probe and 3 and 4 cm with the linear probe.
In comparison of two probes, the linear probe showed lower variability at 2 and 3 cm depth in the phantom and at 3 cm depth in the liver whereas the convex probe showed it at 4 cm depth in both the phantom and the liver.
Conclusions: In ARFI elastography, measurement of depth shows different variability in both low and high frequency probes.
To obtain the most reliable measurement of SWV, using high frequency probe is recommended for 2-3 cm depth and using low frequency probe is recommended for 4-5 cm depth.
Disclosure: Dr. Lee has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Imaging 100-Year-Old Fetuses Sabah Servaes, Children's Hospital of Philadelphia; Teresa Victoria, Ann Johnson, Sandra Kramer, Richard Markowitz, Diego Jaramillo Purpose or Case Report: To demonstrate normal anatomy and pathology of medical museum specimens without disturbing the specimens.
Methods & Materials: Nine fetal specimens from a medical museum were imaged with CT and MRI (1.5 T and 3.0 T) when possible with the specimens in their preserving fluid and containers.
Results: The 9 fetal specimens are estimated to be approximately 100 years old.
One specimen is from the first trimester, seven are from the second trimester, and one is from the third trimester.
Normal anatomical structures at various stages of development including the brain (and varied sulcation pattern), lungs (lobar anatomy), and skeletal structures (several developmental features such as the ossification centers, perichondrial structures, and marrow cavitation) can be evaluated using imaging without causing harm to the specimens.
Pathologic entities including anencephaly and sirenomelia are also evaluated demonstrating features of these entities.
Conclusions: Imaging historical fetal specimens provide an opportunity to evaluate normal developmental changes and pathological entities and also to gain a better understanding of the museum pieces without damaging the museum specimens.
Pediatric CT Interpretations: Does a Tertiary Care Radiologist Make a Difference?
Wendy D. Ellis, Monroe Carell Jr. Children's Hospital at Vanderbilt University; Sumit Pruthi, David Johnson, Christopher Eakins, Chang Yu, Marta Hernanz-Schulman Purpose or Case Report: To determine whether a substantive difference exists between the pediatric imaging reports of community radiologists and reinterpretations by tertiary care radiologists at a free-standing children's hospital; and how those interpretations were related to the final diagnosis.
Methods & Materials: This retrospective review examined the computed tomography (CT) reports of all pediatric patients referred to our tertiary care children's hospital over a 17 month period (1/1/2009-5/31/2010).
The outside reports and the requested second interpretation reports were compared and their content categorized as "agreement" vs. "disagreement: major or minor".
A representative sample of 92 major disagreements in which there was reliable followup information was correlated with the final diagnosis to determine if there was added value provided by the reinterpretation.
Results: CT scans from 732 patients were submitted for reinterpretation.
Disagreements were found in 301/732 cases (41.1%); with 50.5% (152/301) classified as major disagreements.
Among the 427 neurologic cases, major disagreements occurred in 53 patients (12.4%) and minor disagreements in 92 patients (21.5%).
Among the 305 body scans, major disagreements occurred in 99 cases (32.5%) and minor disagreements in 57 cases (18.7%).
In the cohort of cases reviewed for final diagnosis, the second read interpretation was more accurate in 90.2% of cases with a p-value of <0.0001 (neurologic 84.4%, p0<0.0001; body 95.7%, p0<0.0001).
Conclusions: In our review, discrepancy rates between community and tertiary care radiologists in interpretation of pediatric CT scans were substantial, with discrepancies occurring in more than 40% of cases.
Further review of the cases for final diagnosis, showed that a significant number of the tertiary care interpretations were more accurate.
Possibilities that may account for this discrepancy include subspecialty training and elapsed time since performance of the study, which might provide additional clinical data in some cases.
Diagnostic CT scans performed at outside institutions should not be repeated considering added radiation burden to the child and additional expense.
Our data indicates there is added value to the reinterpretation which impacts the accuracy of the report (as assessed by the final diagnosis), and should be recognized by payors as integral to optimal patient care.
Ionizing Radiation Exposure from Radiography in the Neonatal Intensive Care Unit-Per-Patient Cumulative Effective Doses Amaya Basta, Radiology and Biomedical Imaging, UCSF; Jesse Courtier, John MacKenzie Purpose or Case Report: To better understand the levels of exposure to ionizing radiation for infants in the neonatal intensive care unit (NICU).
We retrospectively collected the number and types of radiographs performed per infant in our NICU by searching our radiology information system database over a five-year period.
We focused on the most common examinations (98% of all radiographs) and assigned each an estimated equivalent dose based on published literature: chest and abdomen021.3 micro Sieverts (μSv), one-view chest013.3 μSv, abdomen013.5 μSv, twoview chest026.6 μSv, two-view abdomen027 μSv.
We then calculated a cumulative equivalent dose (CED) for each infant based on the number of each type of examination they received.
Descriptive statistics were generated to depict the distribution of number of examinations and CED.
Results: Over five years, 2,626 infants cared for in our NICU received at least one radiograph of the chest and/or abdomen.
The number of examinations obtained on these infants was 9.6, 4, 1, 137 (mean, median, minimum, maximum).
The 1st quartile was 1 and the 3 rd quartile was 11 examinations.
The cumulative equivalent dose these infants received was 157.9, 61.2, 13.3, 2, 092.2 μSv (mean, median, minimum, maximum) .
The 1st quartile was 21.3 and the 3 rd quartile was 61.2 μSv.
Two hundred infants (7.6% of the study population) received a CED of over 500 μSv.
Conclusions: Descriptive statistics provide a valuable assessment for the broad range of radiation that infants receive in the NICU.
Although the distribution is skewed towards a low level of exposure, a subset of patients (7.6%) received a CED of over 500 μSv.
Identification of factors that cause infants to enter this group will be important for future dose reduction strategies.
Poster #: CR-001 Congenital Cardiac Fibroma: A Case Report Earic Bonner, Meharry Medical College, ebonner07@email.
mmc.edu; Seth Crapp, David Parra Purpose or Case Report: A 5-week-old male presented to his pediatrician with a II/VI systolic ejection murmur along the left sternal border.
He had mild tachypnea without cyanosis.
His oral intake was adequate with no evidence of failure to thrive.
He was referred to a pediatric cardiologist who performed an ECG and a transthoracic echocardiogram.
The ECG showed normal sinus rhythm at 135 beats per minute with no abnormalities.
The transthoracic echocardiogram showed a 25 x 25 x 14 mm homogeneous mass originating from the anterior free wall of the right ventricle, and mild dilation of the right ventricle.
Mild dynamic subpulmonary stenosis and a secundum atrial septal defect were also noted.
Although the murmur was significantly louder at one month follow-up, a repeat echocardiogram did not reveal any increase in the size of the mass.
At 2 months of age, a cardiovascular magnetic resonance imaging (CMRI) study under general anesthesia was performed.
CMRI revealed a 16 x 21 x 22 mm cardiac tumor that was causing narrowing of the right ventricular outflow tract.
The tumor was hypointense on T2-weighted imaging and hyperintense on T1weighted imaging, with positive delayed enhancement.
These findings, along with the size and location of the mass, are consistent with a diagnosis of a cardiac fibroma.
Chest MRA, that was also performed, showed normal extracardiac vascular anatomy with no evidence of peripheral branch pulmonary stenosis.
Cardiac fibromas do not usually increase in size; however, the concern is the child's risk of arrhythmias.
Frequent Holter monitoring was recommended for this patient.
Considerations were also made for an electrophysiology study in the next 1-2 years to determine the risk of ventricular ectopy.
At that point, the patient can be assessed for the possibility of resection of the fibroma.
Purpose or Case Report: Treatment of pulmonary atresia is complex and demands intricate solutions.
One solution is the creation of a conduit between the right ventricle and the main pulmonary artery.
The lifespan of these conduits is limited by progressive occlusion over time, which can be treated with endovascular stent placement in lieu of surgical re-intervention.
However, these stents are at high (40%) risk for fracture, typically at the stent waist.
The radiologist should be aware of this complication, as they may be the first to identify it on chest radiograph.
The purpose of this electronic poster is to familiarize radiologists with this entity by presenting 3 cases of stent fracture and migration.
Methods & Materials: Over a 6 month period, we identified three children with RV-PA stent fractures and associated stent migrations on chest radiography.
Imaging analysis was focused on the appearances of these fractured stents.
Patient management and outcomes were reviewed.
Results: Three children, 2 males, 1 female (ages 4, 3, and 3 years) were found to have asymptomatic RV-PA conduit stent fractures with fragment migration.
One chest xray was performed in the ER for fever and cough; one was pre-op for GI surgery; one was done to confirm abnormal findings seen on a routine cardiac echo.
The time between stent placement and fracture detection ranged from 1 to 22 months.
Two patients had stent fractures and embolizations to the right ventricle that required open surgery to remove stent fragments.
The third patient had embolization to both pulmonary arteries, but did not require treatment.
All patients did well.
Conclusions: Stent fractures and migrations are a relatively common complication of RV-PA conduit stent placement.
Pediatric radiologists need to be aware of this complication in order to provide value-added interpretations.
Purpose or Case Report: We describe the case of a 23 week stillborn fetus with a 5.5 cm diameter craniopharyngioma detected by ultrasonography.
A G1P0 woman in her third decade had ultrasonographic examination showing hydrocephalus, polyhydramnios and an intracerebral mass.
The nature of the mass was uncertain and intracerebral hemorrhage was considered.
The pregnancy was terminated at 23 weeks gestation.
At postmortem examination the decedent was a 650 g male fetus with a head circumference of 24.5 cm and a crown-rump length of 21.8 cm.
Anterior and posterior fontanelles appeared large.
No other external abnormality was found.
The placenta was unremarkable and cytogenetics on placental tissue showed a normal male karyotype.
Examination of fetal viscera was remarkable for mildly underweight adrenal glands (0.75 g, expected 1.5 g) and hepatomegaly (66.4 g, expected 21.7 g).
Intracranial CSF was increased in volume.
There was a suprasellar 5.5 cm diameter somewhat gritty, but smooth-surfaced tumor.
The brain and tumor together weighed 135 g. The floor of the cranium and sella turcica were grossly normal.
Histologic examination of the tumor showed an adamantinomatous type craniopharyngioma with characteristic epithelium, stellate reticulum, focal keratinizing squamous epithelium and calcification.
Pre-and Postnatal MRI of Caudal Regression Syndrome Claire B. Beaumont, MD, University of Arkansas for Medical Sciences, cbbeaumont@uams.edu; Nafisa K. Dajani, Leann E. Linam Purpose or Case Report: Caudal regression syndrome is a rare form of caudal dysplasia characterized by a spectrum of findings including agenesis of the lumbosacral vertebra, multiple orthopedic deformities in the lower limbs, as well as anomalies of the gastrointestinal and genitourinary tracts.
The mechansim of caudal regression syndrome is not completely understood but is believed to be secondary to a defect in the induction of caudal elements.
MRI is a valuable tool for identifying the specific anomalies involved with caudal regression syndrome on a case-by-case basis.
The following is a case from our institution which includes both pre-and postnatal MRI.
Unsuspecting Tuberous Sclerosis Diagnosed on Neonatal Cranial Ultrasound Vikas Menghani, MD, Pediatric Radiology, Women's and Children's Hospital, drvikasmenghani@gmail.com; Puneet Gupta, Richard Thomas, Vaseem Iqbal, Jan Najdzionek.
Purpose or Case Report: Tuberous sclerosis (TS) is a rare autosomal dominant genetic disorder causing hamartomatous proliferation in number of organ systems.
Because the classical triad of epilepsy, mental retardation and adenoma sebaceum is not commonly seen on clinical examination, imaging plays a central role in the diagnosis and treatment of tuberous sclerosis.
Central nervous system features of TS include subependymal nodules, cortical tubers, subependymal giant cell astrocytoma, white matter bands and cysts.
In patients with TS, cerebral involvement in the form of subependymal nodules is seen in 95% to 100% and white matter abnormalities are noted in 40% to 90% of cases.
Knowledge of expected radiological features is thus important in making the correct diagnosis.
Recent studies have indicated that earlier appearance of brain lesions indicate a greater risk of mental retardation and a more severe clinical course.
We present a case of a 23-day-old neonate who was referred to us with concerns for hydrocephalus.
The cranial ultrasound demonstrated multiple echogenic subependymal nodules of varying sizes and mild asymmetry of the ventricles.
The differential diagnosis included TS, TORCH infections, and X-linked subependymal heterotropia.
Areas of increased echogenicity were noted within the white matter of the left frontal lobe, which favored TS.
Subsequently, an MRI was performed to validate these findings and assess for additional white matter lesions.
The MRI showed classic manifestations of TS that included periventricular lesions and streaky, linear, wedge-shaped hyperintensities on FLAIR imaging.
A noncontrast CT scan was also performed which revealed classic calcified subependymal nodules.
Cardiac rhabdomyoma and renal angiomyolipoma are the other recognized manifestations of TS and were respectively excluded by subsequent echocardiogram and renal ultrasound.
Pyloric Atresia with Epidermolysis Bullosa: Fetal MRI Diagnosis with Postnatal Correlation Arnold C. Merrow, MD, Radiology, Cincinnati Children's Hospital Medical Center, carl.merrow@cchmc.org; Jason S. Frischer, Anne W. Lucky Purpose or Case Report: Pyloric atresia (PA) is an uncommon disorder, accounting for 1% of congenital gastrointestinal atresias.
Up to 55% of cases have associated anomalies, the most common of which is epidermolysis bullosa (EB).
Prenatal findings have been reported sonographically for each of these anomalies, both in isolation and in the rare case of association.
A case of isolated PA has been reported by fetal MRI.
We present the first reported case of PA with EB diagnosed by fetal MRI with corroborative postnatal imaging and surgical findings.
The mother of this child was initially referred to the Fetal Care Center of Cincinnati at 21 weeks gestation for a possible myelomeningocele diagnosed by prenatal ultrasound at an outside facility.
These ultrasound images were not available for review at the time of our workup.
A fetal MRI was the first study to be obtained at our institution.
The MRI showed no myelomeningocele or brain anomalies.
The stomach was moderately enlarged throughout the exam and did not empty.
Subjective polyhydramnios was also noted.
No duodenal dilation was seen, and there was minimal fluid in the distal bowel loops.
This constellation of findings raised concern for pyloric atresia, resulting in a careful search for any sign of epidermolysis bullosa due to a known association of these disorders.
Prominent debris was seen layering dependently in the amniotic fluid and in the dilated fetal stomach, and the external ears were abnormally small and misshapen.
The PA-EB association was proposed as the underlying diagnosis based on our MRI findings.
It was also postulated that skin blistering over the lumbosacral spine at the time of the prior outside ultrasound could have mimicked a myelomeningocele, thus prompting the referral to our center.
At delivery, the baby had numerous skin defects, and the ears were malformed.
An abdominal radiograph obtained after nasogastric tube placement and air injection showed no gas beyond the stomach.
A pyloric ultrasound showed a distended stomach without a patent pyloric channel to the duodenal bulb, consistent with pyloric atresia.
A skin biopsy confirmed epidermolysis bullosa, and the patient underwent a resection of the PA with gastroduodenostomy.
The baby subsequently expired less than two weeks later, most likely due to sepsis based on wound cultures and autopsy results.
Our case demonstrates the ability of fetal MRI to diagnose this rare condition and highlights the key imaging manifestations of the PA-EB association.
Disclosure: Dr. Merrow has indicated that he is an author for Amirsys and receives a royalty accordingly.
Purpose or Case Report: We demonstrate a case where the changing position of the contrast filled appendix lead to the diagnosis of malrotation, with review of the embriology of intestinal rotation.
A newborn preterm female presented with a golf ball sized umbilical mass, that reduced by itself, thought to represent an umbilical hernia vs omphalocele.
She was unstable to undergo an upper GI exam under fluoroscopy, therefore a limited contrast study was performed at bedside and was inconclusive for malrotation.
Subsequent NICU radiographs showed changing position of the appendix filled with residual contrast, visiting all quadrants of the abdomen in a random pattern over a few days period.
This confirmed our suspicion for malrotation.
It is well know that in malrotation the position of the cecum can be variable, most commonly located in the right upper quadrant or left lower quadrant.
To our knowledge it has not been described yet that the changing position of the appendix can lead to the diagnosis of malrotation.
Through this case we display the embriology of the intestinal rotation and the radiologic signs of malrotation.
Poster #: CR-008 MR Imaging Patters of Liver Transplant Complications in the Pediatric Population Edward Richer, MD, Emory University, richerej@gmail.
com; Adina Alazraki, Jonathan Loewen Purpose or Case Report: Pediatric liver transplantation is a relatively common surgery, with more than 500 transplants in the United Status annually.
The spectrum of post transplant complications has been previously described, primarily utilizing ultrasound.
As MRI has become a more widely used technique in pediatric imaging, and ultrasound findings may be non-specific, knowledge of MR imaging patterns is an important adjunct in the post-transplant evaluation.
We present a spectrum of complications, including vascular, biliary, hepatic parenchymal, and systemic complications.
Methods & Materials: Using an electronic record system, we identified pediatric patients with prior liver transplantation who subsequently underwent abdominal MRI at our institution and were found to have a post transplant complication.
Patient management and outcomes were reviewed.
Results: Our review of a subset of the available patients shows vascular complications to be the most commonly encountered abnormality at our institution, including hepatic artery stenosis/thrombosis, and portal vein stenosis/ thrombosis, cavernous transformation of the portal vein.
Biliary complications were relatively common, including bilary stenoses and bilomas.
Hepatic parenchymal and systemic complications, such as PTLD, were less common.
We demonstrate the MR imaging patterns of these complications.
Conclusions: Pediatric liver transplantation is a relatively common surgery, and the MRI appeance of post transplant complications warrants illustration as abdominal MRI becomes more widely used in pediatric imaging.
We present a pictorial review of common patterns of complication.
Imaging of Progressive Familial Intrahepatic Cholestasis (PFIC) Matthew D. Dobbs, MD , Radiology, Vanderbilt University Medical Center, matthew.dobbs@vanderbilt.edu; Sumit Pruthi, Stephanie E. Spottswood Purpose or Case Report: Progressive familial intrahepatic cholestasis (PFIC) is a relatively rare pediatric liver disease due to a genetic mutation (ABCB11 gene on chromosome 2q24-31) in a bile salt export protein causing cholestasis leading to chronic inflammation within the biliary system.
The diagnosis is made clinically with detection of a low GGT in the face of an elevated bilirubin and alkaline phosphatase.
Genetic testing confirms the diagnosis.
One of the 3 subtypes, Type 2, was shown in 2006 to be highly related to the development of hepatocellular carcinoma.
The vast majority in children in this study developed HCC at less than 2 years of age.
Radiological contribution to the management of these chronic liver disease patients is to perform surveillance imaging to detect HCC.
Due to the rarity of this condition, almost no reports exist in the radiological literature describing the imaging features or management of this condition.
Our presentation will review the imaging findings in our small population of PFIC Type 2 patients on US, CT, and MRI.
We will also review suggested surveillance imaging techniques and imaging algorithms.
Renal Rhabdoid Mimics Wilms Tumor Vikas Menghani, MD, Pediatric Radiology, Women's and Children's Hospital, drvikasmenghani@gmail.com; Paul Montgomery, Jan Najdzionek, Vaseem Iqbal Purpose or Case Report: In the past most pediatric renal tumors have been classified together under the umbrella of Wilms tumor.
However, over the last decade with advancement in imaging, several distinctive imaging features specific to renal tumors have been recognized which aid in their classification as being distinct pathologically.
We present a case of Rhabdoid Tumor where in the primary tumor arose from the kidney.
It had classical imaging features of Wilms tumor.
We want to highlight that even with the most sophisticated imaging techniques, specific renal tumors cannot always be diagnosed with preoperative imaging and how this alters the management and prognosis for child with a renal mass.
In our case, the postoperative findings, pathology and immunohistochemical techniques confirmed a Rhabdoid Tumor.
Differentiation of these two tumors is essential since in patients with Rhabdoid Tumor survival is poor with 4-year overall survival rates of 42% for stages I and II and 16% for stages III, IV, and V. On imaging, there are several features that suggest the diagnosis of rhaboid tumor.
These include subcapsular fluid collections, linear calcifications outlining tumor lobules, and vascular invasion.
Also, a pertinent feature of rhabdoid tumor due to its aggressive nature is the presence of lung metastasis (83%) and synchronous malignant brain lesions (15%).
These findings were not present on our case, which led us in formulating a diagnosis of Wilms.
Our patient is unusual in the fact that the local renal findings and absence of metastasis, synchronous malignant lesions, and vascular invasion led us to an incorrect diagnosis of Wilms tumor.
In conclusion, we would like to stress that diagnosis of rhabdoid tumor of the kidney on imaging presents a challenge because of its imaging similarity to Wilms tumor.
Ectopic Ureters in Young Infants: MRU Findings Shin-Lin Shih, MD, Department of Radiology, Mackay Memorial Hospital; Yi-Fang Chen, Chun-Chao Huang, Fei-Shih Yang Purpose or Case Report: To localize the terminations of ectopic ureters by MRI Methods & Materials: MR urography (MRU) was conducted in four female patients with hydroureter and a suspected ectopic orifice.
MR imaging was performed with a 3 T MR scanner (Achieva; Philips).
The imaging protocol mainly consisted of a single-shot T2-weighted turbo spin echo sequence with a slice thickness of 4 mm and multiplanar reformations.
The ages of the four patients were 1 day, 3 days and 2 months (for two).
The latter two patients presented with urinary tract infection.
The newborn patients presented with abnormal prenatal examination.
The pertinent findings and descriptions of a variety of renal anomalies were described.
Results: The locations of the ectopic ureters were two in the vagina, one in the uterus and one in the bladder neck.
The associated renal anomalies were a right duplex kidney in four, a left duplex kidney in one, a left ectopic dysplastic kidney in one and vesicoureteral reflux in one (confirmed by VCUG).
Conclusions: MRU may demonstrate the exact point of termination of an ectopic ureter and also the associated renal anomalies.
Poster #: CR-012 Acquired Polycystic Kidneys in Neuroblastoma Survivors Richard Bellah, , Radiology, The Children's Hospital of Philadelphia, BELLAH@email.chop.edu; Bernard Kaplan, Camilo Jaimes, Yael P. Mosse, Jill P. Ginsberg, Kevin E. Meyers Purpose or Case Report: Neuroblastoma (Nbl) is the most common extracranial solid malignancy of childhood.
With current therapy, the prognosis and long term survival of patients affected by this condition has dramatically improved.
Nevertheless, the treatment for Nbl may account for some complications further in life.
In patients with neuroblastoma, acute renal failure can occur usually as a result of a thrombotic microangiopathy associated with bone marrow transplantation.
In addition, end-stage renal disease has been reported in long-term survivors of Nbl.
This exhibit describes and illustrates the first case series of five patients with treated Nbl in whom the imaging features of polycystic kidney disease (PKD) developed over time, and in some cases, as progressive renal failure ensued.
Methods & Materials: Medical and imaging records were reviewed (IRB approved) of patients with treated Nbl in whom PKD became apparent during the course of followup imaging.
Results: Five patients displayed findings of PKD on US and/or CT. Three of the five patients (where images were available) had normal renal imaging at time of Nbl diagnosis.
The mean age at Nbl diagnosis was 2.4 years (range 1.3-3.3 yr).
The mean age at time PKD was detected was 14.6 years (range 8-18 yrs).
None of the patients had a family history of PKD, or had previously undergone dialysis.
All patients received chemotherapy and total body irradiation prior to bone marrow transplantation.
Four patients survived Nbl therapy but eventually developed end-stage renal disease.
Conclusions: An association between acquired PKD and Nbl has not been previously reported.
The etiology of this observation is still unclear, but a toxic insult is likely to account for the renal changes.
Further research is needed to establish the epidemiology, prognosis, and etiology of this association.
Abnormal Migration of the Retention Anchor Suture in a Case Following Gastrostomy Tube Insertion Surendra Narayanam, MBBS, DMRD, DNB, Division of Image Guided Therapy, Department of Diagnostic Imaging, The Hospital for Sick Children, nrssbabu@gmail.com; Joao Amaral, Luke Toh, Bairbre Connolly, Vicente Deoliveira, Dimitri Parra Purpose or Case Report: During percutaneous gastrostomy tube placement, retention anchor suture(s) are deployed into the stomach to tack the anterior gastric wall to the abdominal wall.
In our practice the thread of the retention anchor suture is cut at 14 days and the metallic portion passes pre rectum.
We report an interesting and very rare migration of the metallic portion of the retention anchor suture in post-primary gastrostomy tube insertion.
An 8-month-old girl, with a mitochondrial disease and severe hypotonia underwent percutaneous gastrostomy placement.
During the procedure the retention anchor suture thread snapped and the metallic portion of the suture remained within the stomach.
Day1 post procedure, the child became uncomfortable, so a gastrostomy tube check was performed.
The suture was not visible in the abdomen on abdominal x-ray or fluoroscopically.
On close review of the images, the suture was found projected over the distal esophagus.
Initial impression was the anchor suture had refluxed into the esopahgeal lumen.
Careful attempts were made to remove it along with the nasogastric tube, from above under fluoroscopic control.
However on withdrawal of the nastogastric tube, the retention anchor suture moved enbloc with the nasogastric tube.
Once removed the retention anchor suture was confirmed to be within the nasogastric tube.
This case illustrates the importance of examining the chest X-ray carefully before assuming a retention anchor suture has passed.
To understand the appropriate post procedural radiographic workup and its technique for timely diagnosis.
3.
To learn the potential complications of delayed diagnosis.
Pediatric Retroperitoneal Synovial Sarcoma Ahmad Aouthmany, University of Toledo Medical Center, ahmad.aouthmany@utoledo.edu; Asif Abdullah Purpose or Case Report: Pediatric synovial sarcoma most commonly affects the extremities, especially the lower thigh and knee region; other primary sites such as the retroperitoneum have been only infrequently reported.
We report an extremely rare case of a retroperitoneal synovial sarcoma masquerading as retroperitoneal hematoma in a 16-year-old white female with non-traumatic back pain and non-contrast enhanced CT findings of right quadratus lumborum and psoas region presumed hematoma.
Coagulation studies revealed Factor XI deficiency also known as Hemophilia C. However, on follow-up imaging, the presumed retroperitoneal bleed persisted and a subsequent MR examination revealed a solid enhancing mass.
CT, MR, and FDG-PET findings as well as a brief histopathology are discussed.
Our case is rare in the regards that the tumor occurred in an uncommon retroperitoneal location in a pediatric patient and was mimicking a retroperitoneal hematoma which posed a significant diagnostic challenge.
Despite a rare entity, synovial sarcoma among other sarcomatous lesions maybe considered in the differential consideration of a spontaneous retroperitoneal hematoma even in hemophiliac patients.
Longitudinal Bracket Epiphysis Michael Jubang, Geisinger, mjjubang@geisinger.edu; Farzad Sedaghat, William J. Malone, George Wu, William Mirenda Purpose or Case Report: Longitudinal bracket epiphysis is a rare anomaly with multiple synonyms such as delta bone, triangular bone, and congenital angular deformity.
The purpose of this case report poster is to discuss an 11-month-old male born with an adducted right great toe with a broad nail and a notch in the center of the distal phalanx.
The review will discuss radiographic findings, the natural progression of the disease, the treatment options, the MRI findings used for pre-surgical planning, and associated pathology.
Whole Body MRI in Pediatric Non Oncologic Diseases: Pictorial Review Ramy El Jalbout, MD, Radiology, CHU Sainte Justine, ramy.jalbout@yahoo.com; Vijay Moorjani Purpose or Case Report: With the advances in scanning techniques and the scanning sequences, the role of WBMRI is expanding.
MRI has a great role in the pediatric population owing to its inherent advantages namely lack of radiation, high tissue specificity, and high diagnostic yield at the level of the entire body under a single sedation.
Unlike the application of WBMRI in the assessment of metastasis and bone marrow involvement in leukemia, its role in systemic diseases is yet to be further investigated.
Certain diseases such as CRMO are very often multifocal.
The extent of osteonecrosis in patients on steroids, dermatomyositis and the lesions related to child abuse are very often wide spread in the skeleton.
We intend to present some of the findings of these pediatric systemic and multifocal diseases on WBMRI.
Chronic Relapsing Multifocal Osteomyelitis (CRMO): CRMO can be acute or chronic and is multifocal.
The abnormality manifests as high signal intensity.
WBMRI can guide for the best site for biopsy and provides monitoring for response to treatment.
Osteonecrosis: Only few small studies evaluated the usefulness of WBMRI in the diagnosis of both the symptomatic and asymptomatic sites of osteonecrosis in ALL patients on steroid therapy.
WBMRI is more sensitive than conventional radiographs.
The abnormalities are typically geographical areas of high STIR signal intensity.
Myopathies: WBMRI has also the role of detecting the extent of idiopathic inflammatory myopathies such as dermatomyositis in the entire skeleton.
Child abuse: WBMRI has a low sensitivity for the highly specific fractures that are pathognomonic for child abuse.
Conclusions: WBMRI is a useful examination in the pediatric patient that is radiation free, quick and allows imaging of the entire body.
It is an adjunct to dedicated MRIs to look for multifocality and extent of systemic diseases such as CRMO, osteonecrosis in patients on steroids and dermatomyositis.
It has a great potential as a screening examination but at the same time can detect both the symptomatic and the asymptomatic lesions in the bone marrow and muscles that are otherwise not seen on conventional radiography.
It also allows guidance for biopsy and monitors response to treatment.
Mobile "Cerebroliths" in Hemihydranencephaly: A Case Report Usha D. Nagaraj, MD, The Ohio State University Medial Center, usha.nagaraj@osumc.edu; Brent Adler Purpose or Case Report: Hydranencephaly is a congenital central nervous system disorder manifested by the replacement of the cerebral hemispheres with a thin membranous sac filled with cerebrospinal fluid and necrotic debris.
Hemihydranencephaly is an extremely rare brain condition in which the vascular anomaly is unilateral, with fewer than 10 cases previously reported in the literature.
This is a case of a 4-month-old male who presented to the ophthalmologist for evaluation of possible leukocoria of the right eye.
The patient had a history of a difficult vaginal delivery that required forceps delivery with possible associated trauma to the right eye.
Dilated fundoscopic exam revealed retinal calcifications.
This caused a clinical concern for retinoblastoma and CT and MRI of the orbits were obtained.
CT demonstrated profound dilatation of the left lateral ventricle with only a thin rim of cortex surrounding it.
There was some midline shift to the right with mild dilatation of the right lateral ventricle.
The thalami and brainstem were spared.
There were multiple soft tissue bodies that layered in the dependent portion of the left lateral ventricle, which were isodense to grey matter.
MRI revealed similar findings consistent with hemihydranencephaly involving the left cerebral hemisphere.
There were multiple round soft tissue masses that measured up to 1 cm in size that layered posteriorly in the left lateral ventricle.
These masses were isointense to grey matter on T2 and hyperintense on T1.
When the patient was placed with his head turned to the left, these masses moved to the dependent portion of the left lateral ventricle.
The orbits were normal on both CT and MR.
These soft tissue collections are presumed to be mobile collections of infarcted brain tissue.
This unusual appearance has not been described in the radiology literature.
We review the CT and MR findings and review the relevant literature.
Purpose or Case Report: Citrullinemia type I is a rare inborn error of urea cycle metabolism resulting in hyperammonemia.
In the classic form, the newborn presents with poor feeding, vomiting, progressive lethargy and signs of increasing intracranial pressure 3-7 days after birth, rapidly progressing to apnea, coma and death if left untreated.
We present a case of a term infant who presented to the hospital on the 5th day of life with a typical history of poor feeding and profound hypotonia.
Upon admission he had multiple episodes of apnea and hemodynamic instability prompting intubation and intensive support.
Laboratory evaluation revealed multiple abnormalities, most notably, hyperammonemia (910umol/L) and elevated citrulline (>800umol/L).
MRI of the brain performed on the 7th day of life showed findings consistent with term hypoxic ischemic encephalopathy with restricted diffusion in bilateral rolandic cortex and subcortical white matter, bilateral caudate heads and lenticular nuclei, bilateral insular cortex, and bilateral cerebral peduncles.
The genu of the corpus callosum, bilateral deep frontal white matter, and the left parietal white matter also demonstrated restricted diffusion suggesting infarction secondary to thrombosis of deep intramedullary veins.
An area of restricted diffusion in the right parietal cortex was suspicious for superficial venous infarct.
Review of the literature reveals that this case of neonatal citrillunemia has unique MRI findings.
While our patient had diffusion changes with some shared similarities to the previous two cases in the literature, there are also findings consistent with deep intramedullary venous thrombosis and infarction.
Poster #: CR-020 Duplicated Internal Auditory Canal: A Rare Anomaly of the Temporal Bone Ahmad Aouthmany, University of Toledo Medical Center, ahmad.aouthmany@utoledo.edu; Asif Abdullah Purpose or Case Report: Duplicated internal auditory canal (IAC) is a rare anomaly of the temporal bone, which is usually associated with sensorineural hearing loss.
Only a few cases have been previously described in literature.
We describe an extremely rare case of duplicated right internal auditory canal in a six month-old patient with a history of Down syndrome.
A six month-old male with Trisomy 21 presented with profound bilateral sensorineural hearing loss.
The patient failed the newborn hearing screening tests.
Past medical history was unremarkable for recurrent ear infections.
On focused physical examination, the auricles were normal appearing.
External auditory canals were patent bilaterally revealing clear and translucent tympanic membranes.
Patient did not reveal a facial palsy.
Subsequently, a high resolution computed tomography (HRCT) of the temporal bone was performed.
Duplicated appearance of the right internal auditory canal with separation of facial and vestibulocochlear segments was noted.
The facial nerve canal demonstrated normal caliber while there was significant narrowing of the cochlear canal near the fundus.
Significant stenosis of the vestibulocochlear segment of the duplicated IAC was identified at the porus acousticus.
Dehiscent right posterior semicircular canal was also seen.
An enlarged right vestibule was also noted.
A single IAC was identified on the contralateral side with significant stenosis at the porus acousticus.
High-resolution magnetic resonance imaging of IAC was recommended which revealed normal appearance of the bilateral cochlear and vestibular nerves.
Duplication of the IAC is an extremely rare anomaly involving a redundant osseous canal extending from the cerebellopontine angle through the otic capsule bone toward the labyrinth or cochlea.
A duplicated IAC may or may not be associated with congenital sensorineural hearing loss secondary to aplasia or hypoplasia of the vestibulocochlear nerve.
To evaluate for structural abnormalities that may preclude cochlear implantation, it is important to evaluate pediatric patients with sensorineural hearing loss radiologically.
Although HRCT is the best imaging modality for evaluation of osseous IAC, the IAC contents are best viewed on MRI in oblique sagittal planes of the IAC using a 3-D volumetric steady state sequence.
Neuroimaging in Hemiplegic Migraine: Cases and Review of the Literature Nicholas V. Stence, MD, Children's Hospital Colorado-Radiology, nicholas.stence@childrenscolorado.org; Sita Kedia, John A. Maloney, Jennifer Armstrong-Wells, Timothy Bernard Purpose or Case Report: Hemiplegic migraine (HM) is a rare variant of migraine with aura.
It is characterized by a motor deficit lasting up to 24 h that is fully reversible.
Little neuroimaging data for HM exists in the literature.
We report our experience with two pediatric cases of hemiplegic migraine.
We also review published cases of pediatric HM with abnormal findings on neuroimaging.
Methods & Materials: Cases 1 and 2 presented to our institution with severe headache (HA), acute right-side weakness, aphasia, and altered mental status (AMS), which did not resolve after 24 h. Magnetic resonance imaging (MRI) and genetic testing are reviewed for these cases.
The literature was reviewed for pediatric cases with neuroimaging changes during HM attacks.
Results: Initial MRI, including diffusion-weighted imaging (DWI), was negative in both patients within 24 h of onset.
Repeat MRIs at 93 h (Case 1) and 75 h (Case 2) were both positive for mild hyperintensity on trace diffusion images, and corresponding reduced diffusion on ADC maps, involving regions of the cortex and juxtacortical white matter in left middle cerebral artery distributions.
These findings completely resolved at 3 months in both cases.
MR angiograms (MRA) were negative in both cases.
Case 1 had a family history of migraines and was found to have an unreported mutation in ATP1A2 gene at a highly conserved location in vertebrates.
Case 2 had a family history of HM and was found to have an indeterminant mutation in the CACNA1A gene.
Infectious, metabolic and hypercoagubility work up was negative.
Case 1 required inpatient rehabilitation and at 1 year follow up was requiring speech therapy.
Case 2 resolved completely.
In the literature, 6 cases of hemiplegic migraine with neuroimaging changes were reported.
All cases had prolonged hemiplegic migraines (symptoms>24 h) and showed cerebral edema with or without restricted diffusion.
Conclusions: All eight HM cases in the literature with abnormal findings on neuroimaging had prolonged attacks.
MRIs for our two cases and two cases reported in the literature were initially normal at admission.
Mild swelling and restricted diffusion developed in our two cases after 24 h, and resolved on follow up MRIs.
Subtle findings on diffusion and T2 imaging may lag behind the clinical picture in HM, therefore serial neuroimaging may be useful in individuals with prolonged symptoms.
Most cases eventually show resolution clinically and on MRI.
Correlation of Neurosonographic Anatomy with Matching MR Scan Planes Denise Castro, Hospital for Sick Children, denisecastro22@ gmail.com; Pam Rasalingham, Omar Islam, Don Soboleski Purpose or Case Report: New high-resolution MR sequences have allowed for exquisive anatomic detail and enables reconstruction of images in any scan plane desired.
This ability allows for precise matching of MR image planes with the standard oblique coronal, sagittal and axial images obtained during routine neurosonography.
The purpose of this poster is to correlate the morphology demonstrated on neurosonography with the MR image, utilizing this ability in order to enhance our understanding of the neuroanatomy distinguishable on sonographic imaging.
We believe this will allow a better appreciation of the subtle differences in echotexture of neuroanatomic structures which are often ignored or overlooked on neurosonography and help improve our detection of subtle sonographic abnormalies.
Ectopic Cerebellum in the Posterior Cranial Fossa: Report of a Case and Review of the Literature Usha D. Nagaraj, MD, The Ohio State University Medical Center, usha.nagaraj@osumc.edu; Daniel Boue, Lisa Martin Purpose or Case Report: Cerebellar heterotopia is a common congenital anomaly frequently encountered in the form of cell rests around the fourth ventricle.
However, isolated well-differentiated cerebellar ectopia is extremely rare.
Of the 8 previously reported cases in the literature, only 4 have presented as a discrete, extraaxial mass and none have been described in the posterior cranial fossa.
We present a case of a 5-year-old male who initially presented with persistent daily headaches.
Physical exam including a detailed neurologic exam was within normal limits.
Non-contrast computed tomography (CT) of the brain was initially performed, demonstrating no abnormalities.
Further work-up with magnetic resonance imaging (MRI) was performed, which revealed a well-defined, extra-axial mass superior to the cerebellum and inferior to the tentorium, immediately beneath the vein of Galen.
The mass was isointense to grey matter on T1 and T2 sequences and there was no significant enhancement on post-contrast images.
There was mass effect on the vermis and the cerebellar tonsils were displaced 3 mm below the foramen magnum.
Neurosurgery was consulted and the mass was removed for diagnosis and treatment of the patient's symptoms.
The mass was easily identified intra-operatively and gross total resection was accomplished successfully.
Pathologic analysis of the mass revealed well-formed cerebellar tissue without evidence of neoplasia.
To the best of our knowledge this is the only case of ectopic cerebellum presenting as a discrete extra-axial mass in the posterior cranial fossa.
Our case shows that an extra-axial mass that parallels grey matter on all sequences can be a presentation of ectopic cerebellum.
We describe the CT and MRI findings, surgical and histopatholgical results and review the relevant literature.
Pediatric Isodense Acute Subdural Hemorrhage Jeffrey S. Kao, MD, MSEE, University of Kansas-Wichita, run4boston@gmail.com; Debbie Desilet-Dobbs Purpose or Case Report: The density (attenuation coefficient) of subdural hemorrhage (SDH) in computed tomography (CT) is important in assessing the acuity of SDHs.
An acute SDH is traditionally described as hyperdense and then becoming isodense in approximately 3 weeks when entering the subacute phase.
In this report, we document the case of a pediatric patient with the new appearance of an acute SDH within 40 h of the prior CT that was isodense.
Greater than 95% of the collection was isodense, with a small focus of hyperdensity.
Acute SDHs are known to be isodense to gray matter in patients with anemia (WP Smith, Am J Neurorad 1981).
However, the hemoglobin and hematocrit was within normal limits.
In addition, acute SDHs that are only a few hours old can have a mixed hyperdense and hypodense appearance because of uncoagulated blood before clotting takes place (J Provenzale, AJR 2007) .
Thus, an acute SDH can have an isodense appearance in a non-anemic patient.
Radiologists should consider the possibility of an acute SDH with an isodense appearance, especially in case of possible non-accidental trauma where timing of an injury is important.
Undifferentiated Sarcoma of the Esophagus in an 11year-old Male: Case Report and Radiologic/Pathologic Correlation Michael E. Daniel, MD, UT Southwestern / Children's Medical Center Dallas, michael.daniel@utsouthwestern.
edu; Lisa Sutton, Sandy Cope-Yokoyama, Neil J. Fernandes Purpose or Case Report: Mesenchymal neoplasms of the gastrointestinal (GI) tract occur infrequently in the adult and are extremely rare in the pediatric population.
The occurrence of these lesions in the esophagus is limited to a collection of case reports in the available literature.
Most esophageal mesenchymal tumors in the pediatric GI tract are benign leiomyomas.
The vast majority of malignant mesenchymal tumors in children are categorized as either sarcomas or gastrointestinal stromal tumors (GIST).
We report a case of a high grade undifferentiated sarcoma of the distal esophagus in an 11year-old male.
While this tumor most closely resembles a GIST, the immunohistochemical profile of the lesion is not typical of any distinct mesenchymal neoplasm.
A review of the literature demonstrates a single case report of a likely benign undifferentiated mesenchymal neoplasm of the distal esophagus in an adolescent.
To our knowledge, this is the first reported case of an undifferentiated esophageal sarcoma in a pediatric patient.
We provide radiologic and pathologic features of the above lesion, and review the typical imaging and pathologic characteristics of mesenchymal GI neoplasms.
Potential Airway Management Issues in Sedated Children Kimberly Fagen, MD, MS, Children's National Medical Center, kfagen@childrensnational.org; Nadja Kadom, Ira Cohen Purpose or Case Report: Many pediatric imaging studies require sedation.
It has been shown that a variety of health care professionals other than anethesiologists may provide sedation, including advanced practice registered nurses, nurse practitioners, physician assistants, fellow level trainees, emergency medicine physicians, intensivists, pediatricians and, last but not least, radiologists.
Moderate sedation, also called "conscious sedation", does generally not require an anesthesiologist as there is usually adequate spontaneous ventilation and no airway intervention required.
However, in case of a complication during the imaging study intubation may become necessary.
For patients with certain congenital or acquired conditions emergent intubation may be very difficult and should be brought to the attention of an anesthesiologist prior to inducing moderate sedation.
The four "D's" is a quick way to assess potentially difficult airways that necessitate consultation with anesthesia prior to moderate sedation: Dentition (incisor/tooth size, dental alignment, and macroglossia), Distortion (swelling from infection, tumor, or trauma), Disproportion (hyoid-chin ratio, such as with micrognathia), and Dysmobility (jaw or cervical spine movement issues, i.e.
trauma or atlanto-occipital instability).
Presence of some of these features may be an indication to consider general anesthesia for sedation; at the very least, anesthesiologist's awareness of a potentially difficult intubation adds to patient safety during moderate sedation.
Purpose or Case Report: Lymphangiomatosis describes the presence of multiple lymphangiomas often with multiorgan involvement; typically bones, spleen, mediastinum and lungs.
Although lymphangiomatosis has been described in patients ranging from birth up to 80 years, it most frequently presents in childhood.
The lesions can occur in any tissue in which lymphatics are normally found, with a predilection for neck and chest involvement.
The clinical presentation is variable including pleural or pericardial effusion, hemoptysis, protein wasting enteropathy, peripheral edema, hemihypertrophy and disseminated intravascular coagulopathy.
The coexistence of lytic bone lesions and chylothorax serves as an important diagnostic clue.
We describe typical radiographic, CT and MRI findings in the appropritate clinical setting that narrow the differential diagnosis and raise concern for this rare entity as the etiology for the patient's symptoms.
We report a 12-year-old girl and 2year-old boy with pulmonary lymphangiomatosis with typical presentation and imaging findings.
Results: Bilateral interstitial infiltrates, pericardial and pleural effusions are evident on chest radiograph.
Sampling of the pleural fluid demonstrates chylous effusion.
CT scans of the thorax reveal diffuse smooth thickening of interlobular septa and bronchovascular bundles with extensive infiltrative involvement of mediastinal fat.
Osseous and splenic lesions are demonstrated both on CT and MR.
Differential diagnosis includes interstitial edema, lymphoma and sarcoidosis.
Conclusions: The natural history of pulmonary lymphangiomatosis is characterized by progressive growth and compression of adjacent structures.
Therapy should aim to decrease the compressive effects, to control chylous effusions, and to maintain cosmesis.
The success of surgical resection is limited by inability to separate lymph collections from normal structures.
Characteristic clinical and radiographic presentation, chylothorax, and extrathoracic lymphatic dysfunction should prompt a consideration of lymphangiomatosis and prevent delay in diagnosis.
Aortic Arch Congenital Anomalies: What the Radiologist Needs to Know Luana Stanescu, Radiology, Seattle Children's Hospital, stanescu@u.washington.edu; Stephen Done Purpose or Case Report: 1. Review classic imaging findings in congenital aortic arch anomalies which can improve detection on radiographs and barium esophagogram 2.
Describe pertinent embryologic basis of the radiologic findings 3.
Describe correlative imaging findings on CT and/or MRI in dedicated cases 4.
Describe common diagnostic pitfalls Methods & Materials: After obtaining institutional IRB approval we reviewed various patients presentations with this condition and analyzed images to characterize this particular entity and it's manifestations for better definition of diagnostic criteria.
Results: Radiographs and barium esophagogram: algorithmic approach in reviewing chest radiographs in order to improve detection of aortic arch anomalies; classic findings and common pitfalls.
Cross-sectional imaging (CT and MRI): What the surgeons need to know before surgical repair; detection of associated cardiac anomalies.
Sample cases: double aortic arch, double aortic arch with complete or partial atresia of one of the arches.
Conclusions: Major teaching points of this exhibit are: 1. Review of classic features of congenital aortic anomalies on radiographs, esophagogram, CT and MRI with pertinent embryologic basis 2.
Describe the utility of various imaging modalities in congenital aortic anomalies, emphasizing common pitfalls.
Cardiovascular and Mediastinal Imaging in Children with Unexpected Clinical Presentation Shunsuke Nosaka, MD, Radiology, National Center for Child Health and Development, nosaka-s@ncchd.go.jp Purpose or Case Report: Children with cardiovascular and mediastinal diseases can be congenital or acquired in etiology.
They usually present with straightforward clinical course.
In certain situation, however, some of the children show unexpected clinical presentation predominantly with those of neighboring organs such as respiratory tract, hepatobiliary system, and gastrointestinal tract.
These unexpected presentations can be the cause of delay in proper diagnosis and treatment.
The purpose of this exhibit is demonstrate a variety of imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation.
This exhibit is case based presentation of cardiovascular and mediastinal imaging in children including tips, pitfalls and lessons learned among patients presented with unexpected clinical presentation.
Diagnostic imaging modalities for cardiovascular disease usually consist of various combinations of plain radiography, ultrasound, CT, MR imaging, fluoroscopy, nuclear medicine, and angiography.
The general concept of ALARA-as low as reasonably achievable-should always be utilized when radiation-producing modalities are indicated in children.
The diseases included will be double aortic arch found during workup for the cause of aspiration pneumonia, unilateral pulmonary vein atresia presented with recurrent episodes of pneumonia, severe mitral regurgitation secondary to chordal rupture mimicking fluminant hepatic failure, myocarditis initially present as acute abdomen, cardiomyopathy as unusual initial presentation of neuroblastoma, and thymolipoma mimicking gradual development of cardiomegaly.
Conclusions: It is important for radiologist to be familiar with imaging findings of cardiovascular and mediastinal diseases in children with unexpected clinical presentation.
Cardiac Embryology Made Easy: A Novel Teaching Approach Using Claymation Andrew Phelps, Children's Hospital Boston, aphelpsmd@ gmail.com; Purpose or Case Report: Congenital heart disease can be an intimidating subject for radiology residents, and cardiac embryology is key to its understanding.
However, this can be an equally intimidating topic to teach!
Various diagrams and animations are available in textbooks and online, but much like advanced origami, many of these resources suffer from being visually too complex for the first-time learner.
To overcome this teaching obstacle, I created my own cardiac embryology animations using modeling clay and incorporated them into a comprehensive didactic lecture on congenital heart disease.
Methods & Materials: Cardiac embryology animations were created using modeling clay, a digital camera, and Microsoft PowerPoint.
Surface and cross-sectional views were generated, depicting the key events in cardiac embryology: heart tube formation, cardiac looping, chamber division, truncus arteriosus division, and pulmonary venous connection.
Example models are shown in Figure 1 .
Results: In this lecture, the animations are presented alongside actual embryonic heart photographs.
The lecture then uses the embryology knowledge as a basis to explain the common congenital heart diseases and their MRI appearances.
Examples of septal defects, ventricular hypoplasia, and transposition of the great arteries are presented, among others.
Conclusions: Understanding cardiac embryology is required in order to approach congenital heart disease in a logical fashion.
Modeling clay animations are a cheap and easy way to simplify this complex topic.
Arterial Tortuosity Syndrome: An Introduction to the Clinical and Radiologic Manifestations in the Pediatric Population Neal Desai, UMKC SOM, neal540@gmail.com; Suchit Patel, Ayushi Gupta, Marius Hubbel, Doug Rivard Purpose or Case Report: 1.
To describe the clinical findings of Arterial Tortuosity Syndrome and give a brief discussion of the disease process.
2.
To describe the radiologic manifestations of Arterial Tortuosity Syndrome.
3.
To give a brief discussion of Loeys-Dietz Syndrome-a disease with similar arterial findings, but with unique molecular characteristics from Arterial Tortuosity Syndrome.
4.
To use this knowledge to help establish the diagnosis and reduce mortality.
Methods & Materials: Arterial Tortuosity Syndrome Overview • Epidemiology • Molecular basis • Pathophysiology • Review of signs, symptoms and presentation • Brief discussion of treatment Differential Diagnosis of Arterial Tortuosity Syndrome • Loeys-Dietz Syndrome-similarities and differences Radiologic Findings and Discussion • Chest Radiograph • Computed Tomographic Angiography • Magnetic Resonance Angiography-Neck • Magnetic Resonance Angiography-Head • Conventional Angiography Making a Diagnosis • Sample case report • Review questions Conclusions: Arterial Tortuosity Syndrome is a rare disease whose chief manifestation is severe cardiovascular connective tissue defects.
Due to the nature of these defects and the significance of rapid intervention, it is important to be aware of and recognize the radiologic manifestations associated with Arterial Tortuosity Syndrome in the presence of appropriate clinical history to help offer a better prognosis to the patient.
Dynamic Pulmonary Computed Tomography for Evaluation of Cardiopulmonary Disease Shilpa V. Hegde, MD, Arkansas Childrens Hospital, University of Arkansas, shilpavhegde@gmail.com; S. Bruce Greenberg Purpose or Case Report: Dynamic pulmonary computed tomography (DPCT) is a wide-detector CT technique that allows for continuous chest imaging during respiration.
When combined with intravenous contrast, the technique is a unique tool for evaluation of cardiopulmonary abnormalities in children with cardiopulmonary abnormalities.
The purpose of this poster is to illustrate the technique of DPCT for evaluation of cardiopulmonary disease in children with congenital heart disease and persistent respiratory distress.
Methods & Materials: Methods and Materials: 8 DPCT exams with intravenous contrast were performed on 5 infants with a history of congenital heart disease and palliative surgery.
Four continuous 350 msec gantry rotations were obtained with respiratory rates set at 40/minute.
The imaging was accomplished during the time of a single respiratory cycle.
80 kVp and low mA resulted in effective dose of≈1.5 mSv.
Eight respiratory phases were reconstructed to create 3D and MPR cine loops for evaluation of cardiopulmonary abnormalities.
Results: Cardiopulmonary abnormalities were detected in all patients.
Patency of Sano shunt, Blalock Tausig shunt or patent ductus arteriosus stent was established.
Intimal thickening was identified in one Sano shunt.
Hypoplastic branch pulmonary arteries were present in 3 infants and pulmonary vein thrombosis in 1 infant.
Left bronchomalacia was identified in four of five infants and best or only identified on the expiratory phase of respiration.
Left lung air trapping was present in two patients.
Conclusions: DPCT with intravenous contrast is the ideal study for evaluation of the post-operative infant with congenital heart disease and persistent respiratory distress.
The Role of Low-Dose CT Angiography in the Evaluation of Renovascular Hypertension in Children Jessica Kurian, MD, CHOP, kurianj@email.chop.edu; Monica Epelman, Kassa Darge, Els Nijs, Jeffrey Hellinger Purpose or Case Report: Historically, the evaluation of renovascular hypertension has been accomplished via US and conventional angiography.
Based on the reported adult experience we introduced renal CT angiography (CTA) for the evaluation of renovascular hypertension in mid-2006.
Our Institution has a robust, well-established protocol, which results in reproducible, high quality images.
We aim to present our imaging strategies for the evaluation of these patients and to discuss and illustrate the role of low-dose CTA with 3-D imaging as a noninvasive alternative in the evaluation of pediatric renovascular hypertension.
Methods & Materials: We used our department information system to identify pediatric patients (< 18 years of age) who had documented renovascular hypertension confirmed either by conventional angiography and/or surgery during a 5-year period.
We present our protocol and discuss the indications, limitations and benefits of renal CTA.
CT thin slice data, obtained employing dose reduction strategies, was reviewed and reconstructed in 2D and 3D renderings.
Pertinent US and MR studies as well as demographic and clinical data were reviewed and recorded.
Several causes for renovascular hypertension were documented and relevant CT angiographic findings were selected for presentation.
Results: Radiation dose ranged 0.58-4 mSv.
Fibromuscular dysplasia was the most common diagnosis followed by neurofibromatosis type 1.
Vascular pathology included stenoses, beading, occlusions, and aneurysms.
Disease was noted in the extraparenchymal renal arteries in approximately 70% of the cases.
The choice of the imaging modality for the investigation of renovascular hypertension in pediatric patients remains controversial.
In the authors' experience, CTA with 3-D imaging is a valuable, non-invasive diagnostic tool for the evaluation of pediatric renovascular hypertension.
Low dose protocols can reduce the radiation exposure associated with CT.
This method can spare patients the complications associated with conventional angiography.
Fetal MRI: Brain, Head and Neck Malformations-A Pictorial Essay Sumit Singh, MD, Children's Hospital of Wisconsin, sumitsingh78@yahoo.com; Mohit Maheshwari, Teresa C. Gross Kelly, Tushar Chandra, Ibrahim S. Tuna, Craig Johnson Purpose or Case Report: The purpose of the exhibit is to illustrate various brain, head and neck massses/vascular anomalies on fetal MRI.
We will also briefly discuss the normal fetal brain anatomy as seen on fetal MRI.
Methods & Materials: Major indications for fetal MRI include evaluation of inconclusive sonographic findings in cases of CNS malformations.
In our institute patients are scanned on 1.5 T MR Scanner.
A body surface six channel phased array coil is used to maximize signal to noise.
All the scans are checked by a neuroradiologist to make sure adequate 3 plane imaging of the brain or other lesion in question were performed.
3 plane scanning of the fetal body is also performed for the laterality determination of the lesion and also screen for other congenital anomalies.
Results: Prenatal USG is frequently inconclusive for evaluation of complex fetal brain and head and neck anomalies.
Most studies suggest that MRI after first trimester is safe.
In addition, advent of rapid MRI sequences like single shot fast spin echo (ssFSE) have helped in reducing scan time and motion artifacts leading to availability of diagnostic quality images.
These have led to increasing use of MRI as supplemental tool to further investigate inconclusive fetal sonographic findings.
MRI provides better anatomical delineation of these complex abnormalities.
It helps in making appropriate diagnosis with high confidence and aids in appropriate obstetric and prenatal/neonatal surgical planning or intervention.
This educational exhibit will illustrate few common fetal anomalies.
These will include agenesis of corpus callosum, malformation of cortical development, posterior fossa malformations, ventriculomegaly, in-utero stroke, orbital abnormalities and some fetal neck masses/ vascular malformation.
Correlation and confirmation with the postnatal MRI will also be provided for some cases.
Conclusions: Technical and therapeutic advances have driven the development of fetal MRI.
It is an important adjunctive tool for prenatal imaging in those instances in which a complex anomaly is suspected by sonography, when fetal surgery is contemplated, or when a definitive diagnosis cannot be determined.
It has prognostic implications and may help in optimal and timely obstetric and neonatal management.
Purpose or Case Report: This educational report will provide a review of the imaging appearance of intradiaphragmatic and subdiaphragmatic pulmonary sequestrations on fetal MRI.
The proposed pathophysiology, review of sequestration subtypes, and surgical management options will also be described.
Case examples will be provided to illustrate the fetal MR imaging findings of these variants of pulmonary sequestration that help support the diagnosis.
Specifically a "triangle sign" of T2 hyperintense tissue directed toward the diaphragm will be demonstrated.
Illustrative case examples will be placed in the context of a differential diagnosis for subdiaphragmatic masses seen on prenatal imaging.
Imaging signs that help make a diagnosis of these pulmonary sequestration variants and separate this entity from other lesions will be emphasized.
Poster #: EDU-009 MRI of the Fetal Head and Neck Masses Alok Jaju, MD, Mallinckrodt Institute of Radiology, alokjaju@gmail.com; Joshua Shimony, Per Amundson Purpose or Case Report: Fetal magnetic resonance imaging (MRI) is a useful problem solving tool for abnormalities detected by prenatal ultrasound (US).
Masses of the head and neck region can vary from benign incidental lesions to devastating neurological lesions and life threatening tumors.
We share our experience in characterizing these lesions by prenatal MRI, that can have a bearing on follow up imaging, perinatal management and overall prognosis.
We did a retrospective review of all fetal MRI studies performed at our tertiary care Children's hospital between 11/2002 and 06/2011, to identify fetuses with head and neck masses.
We reviewed the maternal demographic and clinical data, prenatal ultrasound, fetal outcomes and post natal imaging (when available).
Results: Out of the 351 fetal MRI studies, 20 had dominant head and neck masses.
Majority were encephaloceles (9 occipital, 1 parietal).
The remaining included variety of masses such as nasal glioma, teratoma (3), epidermoid cyst, hemangioma and lymphatic malformation (3) .
MRI played a useful role in distinguishing encephaloceles from other masses based on underlying bone defect and intracranial extension.
It also helped in characterizing other masses based on location and signal characteristics.
The presence and degree of airway compromise was determined.
Intracranial anomalies associated with encephaloceles including callosal dysgenesis, cerebral and cerebellar hypoplasias, migrational disorders and spinal anomalies were also correctly identified.
Conclusions: We present the prenatal MR imaging findings of a spectrum of head and neck lesions, correlating with prenatal ultrasound, postnatal imaging and clinical or pathological outcomes.
Purpose or Case Report: The immaturity of the CNS in neonatal infants makes neurologic assessment difficult.
Neuroimaging plays an essential role in the assessment of brain injury by helping to indentify the injury and expected neurologic outcome.
Cranial ultrasound (US) is usually the first neuroimaging modality used since the technique is portable, does not involve radiation and can be used sequentially.
Magnetic resonance imaging (MRI), however, is the most sensitive imaging modality for the detection of hypoxic brain injury.
The goal of this presentation is to compare the US and MRI performed within a 24-hour interval, and evaluate these findings to improve the interpretation of the US which is usually the first methodology used to evaluated these patients.
We performed a retrospective review of the neonatal imaging studies with US and MRI performed within 24-hour interval on 72 preterm and term newborns with clinical history of hypoxia-ischemia.
The imaging findings of the two modalities, MRI and US, were correlated with the pattern and severity of the injury and brain maturity.
Results: Diffuse white matter abnormalities were observed in 60% of the patients by US or MRI.
The ultrasound identified diffuse increased echogenicity which did not show correlation with MRI in 30% of patients.
Focal white matter abnormalities were better identified by MRI on non-cavitary leukomalacia which is the most common PVL observed in premature neonates with low birth weight and the most difficult to identified using US.
Cavitary leukomalacia showed strong agreement in both methodologies.
The MRI identified 6% more cases of intraventricular hemorrhage, however, the corresponding increase in hemorrhage was of minimal clinical significance.
In most cases extra axial hemorrhage was better identified by MRI.
Conclusions: After viewing this exhibit, the viewer will gain a better appreciation and understanding of the neuroimaging characteristics of hypoxia-ischemia in US and MRI, and thus improving the interpretation of the US which is usually the first imaging modality used to evaluate this patient population.
Purpose or Case Report: The most common thoracic lesions found on prenatal imaging, congenital pulmonary airway malformation (CPAM), bronchopulmonary sequestration (BPS), and congenital diaphragmatic hernia (CDH), usually have characteristic imaging findings previously described in detail.
However, common entities presenting with atypical findings and rarer thoracic entities do occur and can be characterized by fetal magnetic resonance (MR) imaging.
The purpose of this educational exhibit is to show examples of atypical presentations of common thoracic lesions and more unusual thoracic entities on fetal MR.
When applicable, prenatal MR is compared with prenatal ultrasound, postnatal imaging, operative findings, or pathology.
Methods & Materials: Using a radiology information system database, the reports of all fetal MR exams at our institution from January 2005 through January 2011 were reviewed.
When unusual thoracic findings were described in the report, all prenatal and postnatal images (when available) were evaluated.
In the cases selected, medical charts were reviewed for operative findings and pathologic reports.
Results: The cases to be described, both pulmonary and extrapulmonary in location, include: hybrid lesion in a horseshoe lung, CPAM extending across the midline, bilateral BPS, BPS located within the mediastinum, BPS located within the leaves of the diaphragm, ectopia cordis and CDH as components in Pentalogy of Cantrell, CDH with herniation of liver into the pericardium, elongated esophageal duplication cyst, chest wall lymphatic malformation, and tight double aortic arch causing congenital high airway obstruction syndrome (CHAOS).
Conclusions: After studying this educational exhibit, the reader will be acquainted with a variety of unusual fetal pulmonary and extrapulmonary lesions, with emphasis on fetal MR. Prenatal and Postnatal Imaging Findings in Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome (MMIHS) Mary Kitazono, CHOP, mkitazono@gmail.com; Richard Bellah Purpose or Case Report: To review the classic constellation of findings seen in prenatal and postnatal imaging of Megacystis-Microcolon-Intestinal-Hypoperistalsis Syndrome (MMIHS), as well as to illustrate additional imaging features that are variably seen in this syndrome.
The imaging database at our children's hospital was searched for all cases of MMIHS diagnosed since 2002.
All available prenatal and postnatal imaging studies were reviewed in patients with a diagnosis of MMIHS, and representative images are provided with a description of the findings.
Results: Since 2002, 6 patients (5 girls, 1 boy) have been diagnosed with MMIHS at our institution, including 4 on prenatal MRI and US.
The characteristic prenatal imaging findings include marked urinary bladder distension, bilateral pelvicaliectasis, and dilated, tortuous ureters, as well as a diminutive colon containing no or minimal T1W-hyperintense meconium on MRI.
Postnatal imaging studies also characteristically demonstrate a massively distended urinary bladder (with no apparent mechanical cause of obstruction) as well as a small, unused colon with dilated, hypoperistaltic small bowel seen proximal to the microcolon.
Additional findings which are variably seen include intestinal malrotation, stomach and esophageal hypoperistalsis or aperistalsis, gastroesophageal reflux, and biliary stasis.
Conclusions: Although a rare syndrome, the constellation of imaging findings in MMIHS is pathognomonic, and recognition of the classic pattern of findings can allow the radiologist to make a diagnosis of MMIHS in both the in-utero and postnatal setting.
Early diagnosis is essential for allowing prenatal counseling regarding this generally fatal disorder, as well as to optimize early management options.
Purpose or Case Report: Gastric mass lesion are uncommon.
This presentation is an educational review of pediatric gastric mass lesions including gastro-intestinal stromal tumor (GIST), inflammatory myofibroblastic tumor (pseudotumor).
Burkitt's lymphoma, squamous cell carcinoma, gastric teratoma, gastric varices, gastric hamartoma, gastric polyp and hypertrophic pyloric stenosis (HPS).
Clinical presentation is varied with upper GI bleeding, feeding intolerance, pain, weight loss and fatigue manifesting.
The imaging work-up might initially have been endoscopy or ultrasound.
Cross section imaging (CT MR) can be invaluable.
The role and impact of FDG PET on the management, staging and follow up of the oncologic pathology will be emphasized.
Imaging Findings in Megacystic Microcolon Intestinal Hypoperistalsis Syndrome, A Rare Disease Kiery Braithwaite, Pediatric Radiology, Emory-Egleston, kieryb@yahoo.com; Kiery Braithwaite, Paula Dickson, Marianne M. Ballisty Purpose or Case Report: Megacystis microcolon intestinal hypoperistalsis (MMIH) syndrome is a rare congenital form of severe functional intestinal obstruction which is more commonly found in females.
The presenting clinical and imaging features of this disease can often mimic other causes of proximal bowel obstruction in the neonate.
In combination with its common association with intestinal malrotation, the clinical picture of MMIH syndrome may be confusing at times.
Awareness of additional imaging features characteristic of MMIH syndrome may help the radiologist suggest this diagnosis.
The purpose of this study is to enhance the ability of the pediatric radiologist to suggest this rare diagnosis by recognizing this unusual constellation of imaging features.
We retrospectively reviewed the clinical data and imaging studies of four patients with MMIH syndrome at our institution.
Imaging studies included plain radiography, ultrasonography, fluoroscopy, and cross sectional imaging.
The initial presentation and clinical outcome was also reviewed.
Results: The clinical presentations of our patients, who were all female, were somewhat varied but typically included symptoms of intestinal obstruction.
The diagnosis of MMIH syndrome was made in our patients from the first few weeks of life through early childhood.
The four patients demonstrated imaging features characteristic of this disease including a very large dilated bladder, severe bilateral hydroureteronephrosis, gaseous distention of the stomach and proximal small bowel, intestinal hypoperistalsis, and a very small colon.
The clinical course of these patients that we observed was also quite variable, with some patients dying in neonatal period while another patient continues to do reasonably well at 14 years old after a multi-organ transplant.
Conclusions: MMIH syndrome is a rare and frequently lethal disease.
The ability of the pediatric radiologist to recognize this constellation of imaging findings can help the clinical team arrive at a diagnosis of MMIH syndrome.
More prompt diagnosis can aid in the development of a long term management plan for the patient and in counseling the family regarding the prognostic implications of this disorder.
Pathologies of Omphalomesenteric Duct Remnant: Radiologic-Surgical Correlation Swapnil Bagade, MD, Pediatric Radiology, Mallinckrodt Institute of Radiology, bagades@mir.wustl.edu; Geetika Khanna, Rebecca Hulett Purpose or Case Report: 1.
To facilitate understanding of embryology of the omphalomesenteric(vitelline) duct and normal anatomy of the umbilicus.
2. Review the spectrum of omphalomesenteric duct malformations and diversity of clinical presentations of these remnants.
3.
Illustrate the imaging findings of omphalomesenteric remnants, from the common such as Meckel's diverticulum to the uncommon such as the omphalomesenteric duct cyst, with surgical correlation.
Methods & Materials: Cases with complications of persistent omphalomesenteric duct were collected from the joint surgery/radiology conferences at a tertiary level children's hospital.
Imaging features were correlated with intraoperative findings.
Conclusions: Preoperative diagnosis of complications related to the omphalomesenteric duct remnants can be challenging because clinical and imaging features overlap with other etiologies of acute abdomen.
Knowledge of the embryologic, clinical, radiologic, and surgical characteristics of omphalomesenteric duct remnants will aid in early and accurate diagnosis.
Neonatal Bowel Obstruction-A Pictorial Essay Tanmay Patel, University of Kentucky; Harigovinda Challa Purpose or Case Report: Bowel obstruction is the most common abdominal emergency in the newborn period and in most cases is secondary to a congenital anomaly requiring early surgical intervention.
However not every case of abdominal distension or dilated bowel is secondary to mechanical bowel obstruction or underlying surgical condition.
Radiologic imaging forms a central role in the work up of newborns with suspected intestinal obstruction.
The role of the radiologist is to identify whether or not mechanical obstruction is present; if obstruction is identified on initial radiographs, to determine the level of obstruction, and finally to identify the etiology of obstruction.
Initial plain radiographic evaluation also helps to determine the subsequent diagnostic or therapeutic approach.
Methods & Materials: A retrospective review of multiple radiographic and fluroscopic examinations in patients with diagnosis of neonatal bowel obstruction was performed at Kentucky Children's Hospital.
Multiple examples of classical imaging findings were compiled and placed into a pictorial review.
Results: Neonatal intestinal obstruction generally presents with nonspecific symptoms such as abdominal distention, vomiting, or failure to pass meconium depending on the level of obstruction and time of occurrence of underlying congenital lesion/atresia in the intrauterine life.
Initial plain radiographs of the abdomen reveal dilated bowel loops when obstruction is present.
High intestinal obstruction is suspected when only few dilated loops are identified, while multiple dilated bowel loops are seen in low obstruction.
Most cases of high obstruction may not need another diagnostic imaging test.
All cases of distal intestinal obstruction require water soluble enema to identify the etiology of obstruction.
In conditions like functional immaturity of the colon, and meconium ileus water soluble enema is therapeutic and thus surgery can be avoided in most cases.
The objective of this presentation is to present an educational exhibit of classical imaging findings of various types of neonatal bowel obstructions, and how to differentiate between them.
Conclusions: Bowel obstruction is the most common abdominal emergency in the new born period.
Most cases are secondary to a congenital surgical condition and early diagnosis and treatment significantly reduces mortality and morbidity.
Radiographic evaluation plays a central role in the diagnosis and treatment of these conditions.
Poster #: EDU-017 3D T2-Weighted MRCP in the Pediatric Population-A Pictorial Review Nathan Egbert, MBBS MPH, University of Michigan, nathaneg@med.umich.edu; Jonathan R. Dillman, Peter J. Strouse Purpose or Case Report: To demonstrate the utility of 3D T2-weighted magnetic resonance cholangiopancreatography (MRCP) in the pediatric population, and to illustrate the MRCP findings of various conditions affecting in the pediatric pancreaticobiliary system.
We identified all MRCP exams performed on pediatric patients (< 18 years of age) from January 1, 2000 through August 1, 2011 by searching institutional electronic medical records.
We then identified representative 3D T2-weighted MRCP images of various conditions affecting the pediatric pancreaticobiliary system.
Results: Representative 3D T2-weighted MRCP images (including source, maximum intensity projection, and volume rendered images) from the following conditions will be presented: abnormal biliary narrowing/stricture (including sclerosing cholangitis, anastomotic strictures following Kasai procedure & liver transplantation, and "pseudostricture"), biliary atresia, choledochal cyst (including various subtypes, based on Todani classification), choledocholithiasis & cholelithiasis, congenital anomalies of the pancreaticobiliary system (including pancreas divisum and anomalous pancreaticobiliary junction), pancreatobiliary system trauma (including main pancreatic duct transection), and other rare conditions affecting the pancreaticobiliary system (including rhabdomyosarcoma of the biliary tree).
Conclusions: 3D T2-weighted MRCP has become an extremely useful tool in the evaluation of children with suspected disorders of the pancreaticobiliary system.
Since MRCP has distinct advantages over alternative diagnostic techniques, such as endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous cholangiography, including lack of ionizing radiation and noninvasiveness, MRCP is a much preferred initial study for pediatric pancreaticobiliary imaging.
This pictorial review is intended to highlight the 3D T2weighted MRCP appearances of various pancreaticobiliary conditions occurring in the pediatric population.
Purpose or Case Report: Magnetic resonance enterography (MRE) is rapidly emerging as an important imaging tool for the diagnosis and follow-up of inflammatory bowel disease (IBD).
Its lack of ionizing radiation makes this imaging modality especially vital to the pediatric population.
Using a casebased approach, we will demonstrate the usefulness of diffusion-weighted imaging (DWI) as part of a comprehensive MRE protocol for the assessment of IBD in children.
The basics of DWI will be discussed with particular attention to abdominopelvic techniques.
The role of MRE DWI for the evaluation of pediatric Crohn disease (CD) and ulcerative colitis (UC) will be reviewed using a case-based approach.
Key images from pertinent imaging studies will be identified by searching institutional electronic medical records and presented with relevant clinical data.
Results: A review of pediatric MRE examinations suggests DWI can be used to detect the following: 1) small and large bowel segments affected by IBD (both CD and UC) 2) abdominopelvic abscesses (including within the mesentery, body wall, iliopsoas muscle, and liver) 3) abnormal lymph nodes 4) sacroiliitis 5) perianal disease (including abscesses and other penetrating complications).
Conclusions: DWI has the potential to play a very important role in the diagnosis and follow-up of pediatric IBD.
This MRE technique is particularly useful for detecting a variety of disease-related complications.
As the exact meaning of bowel wall restricted diffusion is poorly understood to date, continued investigation will be necessary to determine the clinical and histologic significance of this finding.
Cases of CF involving the GI tract were collected from clinical workflow encounters of the authors and from the main hospital medical records database.
Relevant imaging studies were reviewed for known GI manifestations of CF.
These imaging studies were correlated with clinical histories and available intraoperative and pathologic findings.
Results: CF involvement of the GI tract presents over a wide range of ages, organs involved, and associated symptoms.
These manifestations can generally be divided anatomically into those involving the alimentary tract, hepatobiliary system, and pancreas.
Alimentary tract manifestations consist of meconium ileus in uncomplicated and complicated forms (with the latter including secondary intestinal atresia, volvulus, and perforation with meconium peritonitisdistal intestinal obstruction syndrome, constipation, rectal prolapse, duodenal fold thickening, and appendiceal dilation.
Hepatobiliary disorders secondary to CF include microgallbladder, cholelithiasis, biliary ductal abnormalities, neonatal hepatitis, and cirrhosis (including complications such as portal vein thrombosis and ascites).
Pancreatic expressions of CF include fatty infiltration, calcifications, and cysts/ cystosis, frequently in the setting of malnutrition and/or stooling abnormalities.
This exhibit will demonstrate the spectrum of clinical and radiologic GI findings in this disease from the fetal and neonatal period through adolescence across a range of imaging modalities.
Conclusions: Gastrointestinal manifestations of cystic fibrosis occur frequently in the pediatric population and may be the earliest clinical expression of the disease.
Familiarity with the variety of gastrointestinal imaging findings of cystic fibrosis can expedite appropriate diagnosis and therapy, particularly in those children in whom the primary disease is not clinically suspected.
Beyond Acute Appendicitis: Imaging of Additional Pathologies of the Pediatric Appendix Kelly Dietz, MD, Cincinnati Children's Hospital; Arnold C. Merrow, Daniel J. Podberesky, Alexander J. Towbin Purpose or Case Report: Primary acute appendicitis (or appendiceal inflammation caused by a superimposed bacterial infection in the setting of appendiceal obstruction) is by far the most common pathology of the appendix, and imaging evaluations to exclude this diagnosis occur daily in the pediatric radiology setting.
The clinical and imaging differential diagnosis in a patient with right lower quadrant pain and suspected appendicitis is a broad but well-recognized list that predominantly involves structures adjacent to the appendix including the ovaries, small and large bowel, and ureters.
There are, however, less common pathologies primarily involving the appendix which can create an imaging diagnostic dilemma in the setting of right lower quadrant symptoms.
Our goal is to review the imaging and clinical manifestations of these less commonly encountered appendiceal abnormalities.
Methods & Materials: Cases of appendices that were abnormal by imaging but ultimately determined not to be due to primary acute appendicitis were collected from clinical encounters by the authors as well as through a search of the radiology and pathology report databases.
Clinical course, surgical findings, and pathology reports (if available) were subsequently reviewed through the main hospital medical records system.
Results: The collected cases demonstrate a wide range of additional pathologies of the appendix outside of primary acute appendicitis.
A variety of imaging modalities were employed in the workup of these cases.
Examples reviewed in this exhibit include Crohn's disease, ulcerative colitis, cystic fibrosis, carcinoid tumor, inguinal hernia with incarceration, retained foreign body, pinworm infestation, and ileocolic intussusception.
Conclusions: Despite the frequency of primary acute appendicitis, there is a differential diagnosis when an abnormal appendix is found by imaging.
Familiarity with these alternative diagnoses may be particularly helpful in guiding management of the patient whose clinical presentation is not typical for primary acute appendicitis.
Methods & Materials: A hospital PACS database search from the past 10 years for patients with BWS.
Selected cases, with multimodality imaging, were cross-referenced with pathology reports from patient records database.
Results: Intricate abdominal pathologies are depicted utilizing multimodality imaging, such as plain films, US, CT, MRI and PET/CT, and with pathologic correlation.
Cases with highlight the following: Liver: hepatoblastoma, nonspecific hepatobiliary cysts, multiple hemangiomas mimicking metastatic disease; adrenal: dysplastic organomegaly mimicking neoplasm; pancreas: diffuse and focal hyperplasia in the setting of hyperinsulinism, organomegaly; renal: neprocalcinosis, including medullary sponge kidney, nephroblastomatosis, organomegaly; adnexal: ectopic paraovarian adrenal tissue mimicking metastatic lymph node; urinary bladder: benign fibro-uroepithelial polyp.
Conclusions: Diagnosis of BWS can be difficult when the classic clinical and radiological findings are not present.
These few cases highlight the unusual abdominal pathologies, so when detected, a radiologist can aid in the appropriate diagnosis and help guide therapy for these young patients.
This poster will discuss pharmaceuticals the FDA considers investigational for their intended use.
Disclosure: Dr. LeCompte has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Radiologic-Pathologic Review of Pancreatic Masses Encountered at a Tertiary Pediatric Hospital Over a 10-Year Period No Kwak, MD, Radiology, Long Island Jewish Medical Center, kwak_nb@yahoo.com; Karen Naar, Jeanne Choi-Rosen, Lee Collins, Sukhjinder Singh, Anna Thomas Purpose or Case Report: Review of pathologically proven pancreatic masses in pediatric patients encountered at a tertiary pediatric hospital over a 10-year period.
Describe the key morphologic features and other pertinent findings using various imaging modalities.
Correlate pathologic and radiologic findings.
Methods & Materials: Illustrate the various imaging characteristics of pathologically proven pancreatic masses including pseudocyst, pancreatoblastoma, solid pseudopapillary tumor, acinar cell carcinoma, ductal adenocarcinoma, lymphoma, pancreatic neuroblastoma, and inflammatory myofibroblastic tumor.
Correlate pathologic and radiologic findings.
Identify the key imaging features that allow narrower differential diagnosis.
Results: Pancreatoblastoma and solid pseudopapillary tumor are the more commonly encountered pediatric primary pancreatic tumors.
Both are bulky and heterogeneously enhancing tumors with solid and cystic elements.
Pancreatoblastoma occurs more commonly in young children.
Internal hemorrhage and fibrous capsule favor solid pseudopapillary tumor which more commonly occurs in adolescent girls.
Ductal adenocarcinoma, acinar cell carcinoma and an inflammatory myofibroblastic tumor, which were pathologically proven in our pediatric patients, are exceedingly rare entities.
The imaging findings of these cases and their pathology when available will be presented, as well as a quick literature review of these rare tumors.
Illustration and correlation of the pathologic and radiologic findings.
Conclusions: Pancreatic masses in children are rare but in general have a better prognosis than in adults.
Salient imaging findings for the various tumors encountered at a tertiary care center with pathologic and radiologic correlation.
Evaluation of Hepatoblastoma with Gadoxetate Disodium-Typical, Atypical, Pre and Post Treatment Evaluation Arthur B. Meyers, Radiology, Cincinnati Children's Hospital, arthurbmeyers@yahoo.com; Alexander J. Towbin, Daniel J. Podberesky Purpose or Case Report: Gadoxetate disodium (Gd-EOB-DTPA) is a hepatobilliary MRI contrast agent that is widely used in adults for characterization of liver tumors and is being increasingly used in pediatric patients.
Hepatoblastoma is the most common primary hepatic malignancy of childhood.
The purpose of this presentation is to describe our experience with the use of this agent in the MRI evaluation both before and after initiating therapy in patients with hepatoblastoma.
Methods & Materials: The radiology report system at our institution was queried for all patients with pathology proven hepatoblastomas who underwent a liver MR with administration of gadoxetate disodium between 8/1/10 and 2/28/ 2011.
The MR imaging characteristics of the patient's primary hepatoblastoma pre-and post-therapy (when available) and post treatment findings (when available) were reviewed.
Results: 22 MRI studies in 9 different patients were reviewed.
The patients ranged in age from 4 months to 12 years.
6 patients had pre and post treatment evaluation with Gd-EOB-DTPA enhanced MRI, 1 patient had only pretreatment evaluation and 2 patients had only post treatment evaluation.
6 of the hepatoblastomas did not take up Gd-EOB-DTPA during the hepatocyte phase and were therefore low signal intensity during the hepatocyte phase of imaging.
This was useful in the pretreatment evaluation of hepatoblastoma, particularly in defining the relationship of the tumor to hepatic and portal veins.
Post treatment Gd-EOB-DTPA imaging allowed characterization of the biliary anatomy and demonstrated the communication of a postoperative fluid collection with the biliary tree, consistent with biloma.
1 atypical hepatoblastoma showed uptake of Gd-EOB-DTPA on hepatocyte phase imaging, similar to what has been described in adults with atypical hepatocellular carcinoma.
Conclusions: Gadoxetate disodium enhanced MRI is useful in the imaging evaluation of hepatoblastoma, particularly in defining the relationship of tumor to vascular and biliary anatomy and in characterizing post-treatment complications.
Disclosure: Dr. Meyers has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Imaging of the Gallbladder and Biliary Tree in Pediatric Age Group Ihsan Mamoun, MD, Cleveland Clinic, ihsanmamoun@ yahoo.com; S. Pinar Karakas, Unni Udayasankar, Neil Vachhani, Ellen Park Purpose or Case Report: Interactive educational exhibit to illustrate the embryology, anatomical variants as well as congenital and acquired diseases of the bile ducts and gallbladder in pediatric patients.
Methods & Materials: a)The embryology of the gallbladder and biliary tree will be demonstrated with diagrams.
b) Imaging techniques for gallbladder and biliary tree including US, CT, MRI, ERCP and intraoperative cholangiogram will be discussed.
c)Imaging findings of various lesions with special emphasis on key findings that can lead to accurate diagnosis will be discussed.
d)An appropriate list of differential diagnosis will be provided.
e)An algorithm for the assessment of suspected biliary pathology will be presented.
f)The exhibit will be interactive and the reader will answer questions about the discussed entity, related imaging algorithm and management.
Results: a)Discuss congenital anomalies including duplicated and septated gallbladder, choledochal cyst, Caroli disease, situs abnormalities and biliary atresia.
b)Discuss infectious and inflammatory conditions including cholecystitis, Kawasaki's disease, sclerosing cholangitis and hepatitis.
c)Discuss iatrogenic complications including post transplant biliary stricture and leak.
d)Discuss benign and malignant neoplasms invoving the gallbladder including polyps, PTLD and rhabdomyosarcoma.
Conclusions: This exhibit will demonstrate a logical approach to imaging of the congenital and acquired diseases of the gallbladder and biliary tree based on the embryology and underlying pathology.
Postnatal Work Up of Congenital Uronephropathies-A Pictorial Essay Harigovinda R. Challa, Radiology, University of Kentucky, hch229@uky.edu Purpose or Case Report: The use of obstetric ultrasound routinely in the prenatal care has lead to the discovery of many fetal anomalies.
Uronephropathies in the newborn represent one of the largest groups of anomalies amenable to neonatal management.
Since these uropathies are detected mostly in asymptomatic patients the treatment is mainly preventive.
The pediatric radiologist has a key role in the post natal work up and management of these patients with prenatally diagnosed neprhouropathies and familiarity with the congenital urinary tract abnormalities is necessary.
Methods & Materials: A retrospective review of multiple radiographic, sonographic and fluroscopic examinations performed in the newborn babies and infants with prenatal diagnosis of urinary tract abnormalities was performed at Kentucky Children's Hospital.
Multiple examples of classical imaging findings were compiled and placed into a pictorial review.
Results: Numerous anomalies can be detected in utero, including anomalies of renal number, position, morphology, collecting system dilation and bladder, urethral abnormalities.
Of these postnatal work of congenital hydronephrosis is the most common routinely encountered clinical entity.
Renal ultrasound is the initial examination in the evaluation in all cases of prenatal hydronephrosis, which is best performed around postnatal day 5.
If collecting system dilatation persists on postnatal ultrasound, further imaging work up with VCUG, radionuclide imaging may be required depending on degree of dilatation.
Conclusions: Uroneprhopathies are increasingly detected in the prenatal life with increasing use of obstetric ultrasound.
The objective of this presentation is to demonstrate in a pictorial essay of different neprhouropathies and their workup in newborns.
Isolated Fallopian Tubal Torsion: Causes, Imaging Findings, and How to Suggest the Diagnosis Jesse Courtier, MD, UCSF Dept of Radiology, jesse.
courtier@ucsf.edu; Amaya M. Basta, Rebecca Maine, Pierre-Alain Cohen, Shinjiro Hirose, John D. MacKenzie Purpose or Case Report: The purpose of this educational report is to describe the rare entity of isolated fallopian tubal torsion in the pediatric population and depict the cross sectional imaging findings that help make a diagnosis and guide management.
The proposed pathophysiology, predisposing factors, and surgical management will be described.
An illustrative case example of 12-year-old female patient will be provided with surgical correlation.
The exhibit will review imaging findings on US, CT and MRI that help support the diagnosis including, dilated tubular structure in the pelvis, normal ovaries, and corkscrewing and beaking of the proximal fallopian tube.
Isolated fallopian tubal torsion will be placed in the context of a differential diagnosis for girls presenting with pelvic pain and the imaging signs that help make a diagnosis of isolated tubal torsion and separate this entity from other causes of pediatric pelvic pain will be emphasized.
Multimodality Imaging Characteristics of Genitourinary Rhabdomyosarcoma Rhea Udyavar, MD, George Washington University Medical Center, rudyavar@gwmail.gwu.edu; Amir Noor, Pranav K. Vyas Purpose or Case Report: In this pictorial essay, we will demonstrate salient imaging features of MR, US, and CT modalities for the diagnosis of genitourinary rhabdomyosarcoma in male (N04) and female (N04) children ages 2-14 years, evaluated at our institution over the past 6 years.
Background information, including tumor biology, staging, and treatment will also be discussed.
The Swollen Scrotum: Ultrasound Technique and Differential Diagnosis Kelli R. Schmitz, MD, Oregon Health & Science University, schmitzk@ohsu.edu; Roya Sohaey Purpose or Case Report: To review the ultrasound protocol for the performance of scrotal ultrasound and illustrate the ultrasound appearance of conditions resulting in scrotal swelling in pediatric patients.
A retrospective review of the imaging database at a tertiary pediatric referral center was performed to identify pediatric patients who presented with scrotal swelling and underwent diagnostic ultrasound.
When available, surgical/pathologic correlation was obtained.
Results: A variety of pathologic processes result in scrotal swelling.
Causes illustrated include: testicular torsion, epididymitis/orchitis, hydrocele, varicocele, inguinal hernia, trauma, adrenal rest, and testicular or paratesticular neoplasm.
Conclusions: The causes of scrotal swelling are myriad, including infectious/inflammatory, developmental, traumatic, and neoplastic etiologies.
In children, the clinical presentation of a swollen scrotum is nonspecific, and ultrasound plays a key role in making the correct diagnosis.
Experiences of Starting a Functional MR Urography Program at a University Hospital: Trials and Tribulations Steven L. Blumer, BSc, Montefiore Medical Center/Albert Einstein College of Medicine, sblumer@montefiore.org; Ibrahim Tuna, Amanda North, Benjamin Taragin, Netta Blitman, Terry L. Levin Purpose or Case Report: Starting a functional MRU program can be challenging as there are numerous potential hurdles to overcome.
This presentation describes the process of starting a functional MR Urography (fMRU) program at a university hospital and discusses the difficulties encountered starting such a program.
Selecting a sufficient patient referral base, resolving common and uncommon technological issues, and education of clinicians, patients and technical staff are some of the challenges that will be discussed.
Conclusions: Awareness of the common pitfalls in fMRU imaging and close partnering with referring physicians can make establishing a functional MRU program easier.
Despite many potential obstacles, the benefit of exquisite anatomical and functional information provided by fMRU in children, without exposure to ionizing radiation, greatly outweighs any challenges.
Disclosure: Dr. Blumer has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Pictorial Review of Ultrasound Findings in Boys Presenting to Emergency Department/Urology with Acute Scrotum Teresa Liang, Faculty of Medicine, University of British Columbia, teresaliang86@gmail.com; Peter Metcalfe, William Sevcik, Michelle Noga Purpose or Case Report: Testicular torsion is a common acute condition in adolescent boys.
Rapid and accurate diagnosis is critical.
Diagnosis is currently based on history, physical findings, and ultrasound (U/S) with Doppler.
The objective of this poster is to demonstrate ultrasound findings from a retrospective review of acute scrotum over 3 years, and to demonstrate some pitfalls of the technique with regard to testicular torsion diagnosis.
We reviewed the U/S, surgical and ED records at the Stollery Children's Hospital for boys aged 1 month to 17 years, presenting with acute scrotum from July 1, 2008 to 2011.
Age, demographics, clinical symptoms, and physical findings, U/S and surgical techniques, findings, diagnoses and follow-up were also recorded.
Results: 343 patients presented to UAH Stollery ED with acute scrotum: 35 were diagnosed with testicular torsion (2 inguinal torsion), 11 were suspected of a torsion-detorsion, 3 torsion of appendix testes, 135 epididymitis/orchitis, and 159 other diagnoses including hydroceles, varicoceles, epididymal cysts, abscesses, cellulitis and hematomas.
For the 266 patients who had ultrasound,100% sensitivity and 88% specificity for testicular torsion.
The ultrasound findings including size, vascularity and echogenicity associated with both salvageable and necrotic testicles including use of color and pulse Doppler will be reviewed.
The sonographic findings and pictorial examples associated with the more common acute scrotum etiologies will be presented.
Sonographic findings from problematic cases (those with inconclusive ultrasound reports or false positive reports) will also be addressed.
Conclusions: Ultrasound imaging problem case examples and characteristic findings of common acute scrotum presentations at Stollery Hospital at the University of Alberta are reviewed in this poster.
Primary and Secondary Amenorrhea in Pediatric Patients: From the Beginning to the End Cesar Cortes, MD, Miami Children's Hospital, n4c03@ hotmail.com; Yanerys Ramos, Ricardo Restrepo, Alejandro Diaz, Lorena Sequeira, Edward Lee Purpose or Case Report: To describe the role of imaging in evaluating patients with primary and secondary amenorrhea and to illustrate the normal imaging findings of the reproductive organs in the pediatric population as well as the imaging findings of the different etiologies causing amenorrhea.
A search of the literature is done to determine the different etiologies of amenorrhea and the role of imaging in their evaluation.
First, we will focus on the normal physiologic hormonal influence and changes of the girl's reproductive organs since birth until adolescence on ultrasound and MRI.
Images of the normal appearance of the female reproductive organs as well as imaging findings of the different common and uncommon etiologies of amenorrhea will be shown.
Then, specific reference will be made to crucial related concepts such as minipuberty of infancy, latest criteria for polycystic ovarian disease and ovarian failure syndrome among others.
Finally, the treatment, either medical or surgical will be briefly discussed.
Results: Causes of amenorrhea in children range from disorders affecting the hypothalamus, pituitary gland, adrenal glands, and ovaries, as well as uterine and vaginal structural abnormalities.
Even though history and clinical exam are essential in evaluating a patient with amenorrhea, the pediatric radiologist plays a pivotal role helping guide the area to be imaged and thus the modality that should be used.
MRI and ultrasound are the main modalities in the evaluation of amenorrhea.
Conclusions: Ultrasound and MRI are the main imaging modalities used in the evaluation of amenorrhea in children and are usually part of the work up.
Amenorrhea in children can have implications in girl's fertility allowing pediatric radiologists to play an important role in helping not only the patient but also their offspring.
Imaging of Mullerian Duct Anomalies in Children Kelly K. Horst, MD, Radiology, University of Michigan, khorst@med.umich.edu; Maryam Ghadimi Mahani, Deepa Pai, Jonathan R. Dillman, Peter J. Strouse Purpose or Case Report: The purpose of this educational exhibit is to provide an up-to-date appraisal of Mullerian duct anomalies presenting in the pediatric population.
The appearances of anatomic variants on ultrasound and MRI will be used to illustrate the strengths and potential pitfalls of these imaging modalities.
Methods & Materials: Patients who have previously undergone ultrasound and/or MRI in the course of their clinical workup within the University of Michigan Health System (UMHS) were identified using electronic medical records.
Imaging reports were reviewed by a single author in order to identify relevant imaging findings (interesting anatomic variations, associated anomalies, etc.).
Pertinent images from these imaging examinations were de-identified and saved to a secure hard drive.
The medical record was accessed by a single researcher to obtain relevant information regarding the patients' clinical presentations.
In cases of corrective surgery, pathology reports were reviewed, if available, for correlation with the imaging findings.
Results: Cases of Mullerian duct anomalies were reviewed within the pediatric population.
Clinical manifestations were correlated with imaging appearances.
Conclusions: Mullerian duct anomalies represent a range of developmental variants.
Although functioning ovaries and age-appropriate external genitalia are characteristic, there may be anomalies ranging from uterine and vaginal agenesis, to duplication of the uterus and vagina, to minor uterine cavity abnormalities.
Müllerian malformations are frequently associated with abnormalities of the renal and axial skeletal systems, and pediatric patients in particular may present with these associated anomalies.
Menstrual abnormalities may represent a more typical presentation in the adolescent age group.
This is in contrast to the adult population, which may be more likely to present with infertility.
The variation in clinical presentations make Mullerian duct anomalies difficult to diagnose and, because surgical techniques for correction and treatment depend on the underlying anatomy, understanding these variants in the context of imaging studies is important to their diagnosis and management.
Patient 2 had radiographs which showed an irregular left humeral metaphysis with an associated fracture.
Patient 3 had a 3 phase bone scan that showed slightly increased uptake on the angiographic and blood pool phases and increased activity on the delayed phase in the right femur.
Radiographs showed a moth eaten appearance of the right femur with soft tissue swelling.
Patient 4 had radiographs that showed periosteal reaction in the right tibia with an associated fracture.
Patient 5, in addition to radiographs, had an MRI that showed osteomyelitis of the left humerus and scapula with an associated subperiosteal abscess.
Patient 6 had multi focal osteomyelitis that was demonstrated on radiographs by irregular cortices and periosteal reaction involving the upper and lower extremities.
Conclusions: Neonatal osteomyelitis is an uncommon entity that can have severe complications if not diagnosis and treated promptly.
It is important to review cases and to review the appearance of neonatal osteomyelitis on multiple modalities.
Radiographs will usually demonstrate periosteal reaction and possibly soft tissue swelling.
Additional studies may be obtained to evaluate for complications, such as abscesses or involvement of the joint space.
Purpose or Case Report: Review the epidemiology of DDH.
Describe the critical diagnostic imaging findings of DDH.
Understand the role of imaging accompanying treatment.
Methods & Materials: Images including radiographs, ultrasound, CT and MRI will be used to demonstrate the current and historical role of imaging in caring for patients with DDH.
Discussion of the importance of reducing radiation exposure when choosing imaging studies will be included.
Results: Radiographs and ultrasound are used primarily in making the diagnosis of DDH.
Ultrasound and MRI are most often used during the course of treatment to assess its effectiveness.
MRI is increasingly utilized without sedation for patients in spica cast.
Conclusions: Imaging is critical in the care of patients with DDH.
Pediatric Musculoskeletal Ultrasound of the Proximal Lower Extremity (Pelvis to Thigh) Julia Rissmiller, MD, Dept of Radiology, Children's Hospital Boston, julia.rissmiller@childrens.harvard.edu; Howard Christianson, Michael J. Callahan Purpose or Case Report: To review indications for ultrasound of the proximal lower extremity (pelvis, hip and thigh), and to illustrate the practical use of ultrasound in evaluation of the proximal lower extremity, emphasizing the sonographic appearance of various musculoskeletal disorders.
Ultrasound is a well-established modality for the evaluation of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children.
In general, ultrasound has a more limited role for the primary evaluation of other pediatric musculoskeletal disorders including trauma, articular and periarticular diseases and tumors or tumor-like processes.
Advantages of ultrasound, a relatively non-invasive technique, include excellent spatial resolution, low cost, lack of ionizing radiation, lack of need for sedation, and the ability to image the patient in real-time.
The major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications.
We present ultrasound examples of pathology involving the proximal lower extremity (pelvis, hip and thigh).
Cases include developmental hip dysplasia, hip effusion, osseous metastasis to the iliac bone, osteomyelitis of the hip, femoral acetabular impingement, rectus femoris hernia, vascular malformation, Ewing's sarcoma and myositis ossificans.
Results: A range of images from pediatric diagnostic ultrasounds performed of the proximal lower extremity (pelvis to thigh) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders.
Conclusions: Ultrasound is an excellent modality for evaluating the proximal lower extremity in children, beyond the current indications of painful hip, developmental hip dysplasia, soft tissue infection, palpable masses, and foreign bodies in children.
A Multi-Modality Pictorial Review of Lesions of the Epiphysis in Infants and Children Ernesto I. Blanco, MD, St. Christopher's Hospital for Children, eiblanco74@gmail.com; Jacqueline Urbine, Evan Geller, Peter Pizzutillo Purpose or Case Report: To review the imaging spectrum of epiphyseal lesions in infants and children.
A retrospective review of our imaging database was performed to identify studies with either primary lesions of the epiphysis or processes that affect the epiphysis.
Results: Multiple epiphyseal lesions were elucidated primarily by radiography, with cross-sectional imaging included where clinically necessary.
Congenital lesions include the epiphyseal dysplasias represented here by chondrodysplasia punctata.
Epiphyseal infarction may due to multiple etiologies including slipped capitol femoral epiphysis, developmental dysplasia of the hip, sickle cell disease, or idiopathic reasons.
Neoplasms may occur in the epiphysis, including chondroblastoma and histiocytosis.
Traumatic lesions include fracture and avulsion.
Osteomyelitis can occur in the epiphysis as well.
Pseudolesions that mimic pathology will also be reviewed.
Other pathologies that can affect the epiphysis include juvenile idiopathic arthritis and hemophilia.
Conclusions: A wide spectrum of congenital and acquired pathologies may affect the epiphysis in the infant and child.
Plain radiography, computed tomography, and magnetic resonance imaging all contribute to the diagnosis of these varied lesions.
Purpose or Case Report: We aim to present the spectrum of common and uncommon hip disorders in pediatric population.
We will formulate a systematic approach and present a flowchart to workup and characterize hip diseases.
Methods & Materials: Relevant imaging appearances of normal as well as pathological hip will be presented.
Normal hip anatomy will be discussed through anatomic drawings and radiological images (plain radiographs, CT, USG, and MRI).
We will illustrate the various anatomic landmarks, measurements and lines on plain radiographs and ultrasound of hip.
Results: Evaluation of limp and hip pain in the pediatric population has undergone rapid evolution.
Surgical treatment for these disorders continues to be refined, and our ability to identify patients along the spectrum of disease continues to improve.
Yet, despite our advances, obtaining an accurate diagnosis can remain challenging, especially in the setting of mild structural abnormalities.
Many imaging studies can be used to evaluate the bones and soft tissues, but conventional radiography is the primary imaging modality for most clinical conditions.
Plain radiographs usually are obtained first because they are sensitive and specific for a wide range of bone pathology.
More sophisticated imaging modalities including radionuclide scintigraphy (bone scan), ultrasonography (USG), computed tomography (CT) and magnetic resonance imaging (MRI) are reserved for specific clinical situations.
Each of these imaging modalities has specific advantages and disadvantages.
It is the aim of this review to guide in selecting and interpreting the appropriate imaging modality for a variety of common disorders.
This exhibit will illustrate imaging features of developmental dysplasia of hip, Perthes disease, Slipped Capital femoral epiphyses, hip malformations in syndromes, femoral acetabural impingement, labral disorders, septic arthrits and other disorders.
The role of various imaging modalities in evaluation of these disorders will be discussed, along with common imaging pearls and pitfalls.
Conclusions: A systematic approach is necessary for evaluation of pediatric hip disorders.
Familiarity with normal appearances, pitfalls and specific imaging of these entities is essential for proper diagnosis and management.
Osteoid Osteomas: A Pain in the "Night" Diagnosis Nancy K. Laurence, MD, The Children's Hospital of Philadelphia, nkang26@gmail.com; Monica Epelman, Richard Markowitz, Camilo Jaimes, Diego Jaramillo, Nancy Chauvin Purpose or Case Report: A common benign bone-forming lesion, osteoid osteoma comprises approximately 12% of all benign bone tumors.
The tumor is composed of a nidus of vascular osteoid tissue and woven bone lined by osteoblasts, frequently with considerable surrounding inflammation.
The radiolucent nidus surrounded by variable degrees of reactive sclerosis usually leads to a straightforward diagnosis; however, sometimes the diagnosis of osteoid osteoma can be challenging, as it may have a non-specific and misleading appearance on different imaging modalities, particularly on MRI.
The purpose of this exhibit is to review the typical and atypical features of osteoid osteomas on different imaging modalities.
We present diagnostic dilemmas of osteoid osteomas from our institution and how imaging characteristics can aid in diagnosis.
We performed a retrospective review of our imaging database to identify cases of typical and atypical osteoid osteomas, with special emphasis on cases which posed a diagnostic dilemma on imaging.
Results: When osteoid osteomas occur in atypical locations the diagnosis can be elusive.
When located in the intraarticular space there is often minimal or absent cortical thickening and there may be a joint effusion with synovial hypertrophy.
Phalangeal lesions may cause extensive bone marrow edema and surrounding soft tissue swelling.
Both of these types of osteoid osteomas can be mistaken for infection.
The recently described "CT vessel" or "vascular groove" sign, a low density vascular groove adjacent to the nidus, is highly specific for osteoid osteoma.
In the authors' experience, a rim of sclerosis surrounding the nidus may aid in diagnosis on MRI and can be identified as an outer hypointense halo on all sequences.
We illustrate the findings in cases of atypical osteoid osteomas which may be difficult to diagnose including intraarticular, phalangeal, and vertebral osteoid osteomas.
We also show examples of the newly described sign which has high specificity for osteoid osteoma.
Conclusions: Imaging findings in osteoid osteomas can be misleading and cause misdiagnosis, especially in atypical cases.
Knowledge of their appearance in atypical locations and specific findings can aid in the correct diagnosis.
Ultrasound of Normal Entheses in the Growing Skeleton Nancy Chauvin, MD, Department of Radiology, The Children's Hospital of Philadelphia, chauvinn@email.chop.
edu; Pamela F. Weiss, Monica Epelman, Diego Jaramillo Purpose or Case Report: Ultrasound is an underutilized modality in the evaluation of the pediatric musculoskeletal system.
Evaluation of tendon insertions about the elbow, knee and foot can be easily performed with ultrasonography.
A good knowledge of the age dependent normal ultrasound appearance of the entheses is crucial in order to evaluate for pathology, such as trauma or ethesitis-related arthritis.
This exhibit will serve to provide the reader with a practical approach to imaging when assessing tendon insertions.
Optimal patient positioning and transducer selection will be discussed.
In addition, important anatomic landmarks will be described to allow for reproducibility and avoiding pitfalls.
Methods & Materials: Transverse and longitudinal ultrasound images of 12 entheseal insertion sites were performed on 20 healthy girls and boys between the ages of 5 and 17 years.
Ultrasound of the elbow was performed while in full extension and the insertions of the common flexor and common extensor tendons were evaluated.
The quadriceps and patellar insertions were imaged with patients in the supine position, with the knees flexed at 30 degrees.
The Achilles tendon and plantar fascia insertion were evaluated with the patient prone, with the feet hanging off the edge of the table.
Results: Tendons demonstrated the expected fibrillar pattern with parallel echogenic lines.
The appearance of the entheses changed as the insertion matured from sonolucent cartilage to echogenic bone.
Conclusions: Using a systematic approach and knowledge of the normal anatomy, sonography of the tendons of the elbow, knee and foot can easily be performed in children.
Pediatric Musculoskeletal Ultrasound of the Distal Lower Extremity (Knee to Ankle) Howard Christianson, MD, Radiology, Children's Hospital Boston, howard.christianson@childrens.harvard.edu; Julia Rissmiller, Michael J. Callahan Purpose or Case Report: Ultrasound is a well-established technique in children for evaluation of the painful hip, developmental dysplasia of the hip, soft tissue infection, palpable masses and foreign bodies.
In general, ultrasound has a somewhat more limited role for the primary evaluation of several other pediatric musculoskeletal disorders in the setting of trauma, articular and periarticular diseases and tumors and tumor-like conditions.
Inherent advantages of ultrasound include excellent spatial resolution, a lack of ionizing radiation, a relatively non-invasive technique and lack of a need for sedation.
Real-time imaging allows problem solving not available with other modalities which is well suited for musculoskeletal applications, particularly in the setting of trauma.
The major disadvantage of ultrasound is operator dependency, which is particularly evident in musculoskeletal applications.
The purpose of this study is to illustrate the practical use of ultrasound in the evaluation of the distal lower extremity (knee to ankle) emphasizing the sonographic appearance of various musculoskeletal disorders.
Examples include: 1) Cystic lesions around the joints: Baker's cyst, synovial cyst, ganglion cyst and suprapatellar bursitis; 2) Infectious processes: pretibial, subperiosteal and intramuscular abscess; 3) Tumor and tumor like lesions: nerve sheath tumor, tumoral calcinosis; 4) Trauma related injuries: Sinding Larsen Johansson, tibialis anterior muscle herniation, hematoma.
Methods & Materials: Cases selected for presentation from a series of diagnostic musculoskeletal ultrasounds performed at our institution.
Results: A range of images from diagnostic ultrasounds performed of the distal lower extremity (knee to ankle) will be presented emphasizing the sonographic appearance of various musculoskeletal disorders.
Conclusions: Selected musculoskeletal ultrasounds of the distal lower extremity are presented to familiarize the audience with the sonographic appearance of various musculoskeletal disorders and to highlight the tremendous potential of ultrasound in evaluating musculoskeletal disease in children and adolescents.
Role of Conventional and Dynamic Contrast Enhanced Magnetic Resonance Imaging in Diagnosis of Hemihypertrophy Syndromes in Children Shrey K. Thawait, MD, PhD , Radiology, Yale University-Bridgeport Hospital, sthawai2@jhmi.edu; Gaurav K. Thawait, Sally E. Mitchell, Laura M. Fayad, John A. Carrino, Kate Puttgen Purpose or Case Report: Hemihypertrophy syndromes in children are complex and there is some overlap among these conditions.
Hence, establishing a diagnosis can be challenging.
Identification of the correct vascular anomaly associated with these overgrowth disorders helps to correctly classify the disease into one of the several syndromes, which in turn guides management.
In this educational poster, we will review the definition, clinical presentation, conventional Magnetic Resonance Imaging (MRI) and contrast enhanced Magnetic Resonance Angiography and Venography (MRA / MRV) features of hemihypertrophy syndromes in children.
Methods & Materials: 1.
Learn the diagnostic criteria for overgrowth syndromes in children such as Klippel-Trenaunay Syndrome (KTS) and Parkes Weber Syndrome (PWS) with special emphasis on associated vascular anomalies.
2.
Gain knowledge of high resolution MRI technique for evaluation of vascular anomalies associated with the hemihypertrophy syndromes.
3.
Understand the additional value of dynamic contrast enhanced MRA / MRV in the differentiation of the hemihypertrophy syndromes in the pediatric age group.
Results: 1.
MRI technique for a dedicated "vascular anomaly protocol" consisting of fat saturated T2 weighted, pre contrast axial T1 weighted, and post contrast triplanar T1 weighted fat saturated imaging will be described.
2.
Special emphasis will be provided on dynamic contrast enhanced MRA/MRV.
3.
Conventional and dynamic MRI features of clinically proven cases of hemihypertrophy syndromes will be demonstrated.
Conclusions: Systematic MRI interpretation utilizing a dedicated vascular anomaly protocol enables the radiologist to correctly identify the hemihypertrophy syndrome, and provide detailed extent of disease.
Correlative Ultrasound, MRI Imaging and Physical Examination of Elbows in Hemophilic Children Andrea S. Doria, MD, The Hospital for Sick Children-Diagnostic Imaging, andrea.doria@sickkids.ca; Frederico Xavier, Arun Mohanta, Carina Man, Ningning Zhang, Pamela Hilliard Purpose or Case Report: 1.To report a systematic ultrasound (US) protocol for assessment of hemophilic elbows.
2.
To discuss advantages and disadvantages of US and MRI for evaluating hemophilic elbows in comparison with physical examination.
3.To illustrate US and MRI findings and associated pitfalls in hemophilic joints.
Background: The value of physical examination for assessment of early arthropathic changes in hemophilic joints is unknown.
US does not require sedation in young children, but involves operator training and standardized technique.
MRI is the reference standard imaging modality for assessment of pathology in hemophilic joints.
Standardization of a systematic protocol for data acquisition and interpretation of US findings and understanding of the correlation of findings between physical examination, US and MRI in hemophilic elbows is essential for the use US as an outcome measure both in clinical practice and research.
So far such information is not available for growing elbow joints.
Methods & Materials: Eight hemophilic boys (age range/ median, 7-17/13 years) with a history of prior elbow bleeds underwent US and MR imaging, and physical examination on the same day.
Corresponding images on US and MRI were highlighted to illustrate abnormalities and pitfalls.
Soft tissues (effusion/hemarthrosis,synovial hypertrophy,hemosiderin deposition) changes were characterized as small, moderate, or large.
Erosions, cartilage and subchondral abnormalities were graded based on depth or extent of articular changes.
Results: 1.
US is helpful for discriminating synovial hypertrophy, joint effusion/hemarthrosis, and large hemosiderin deposition which otherwise generates susceptibility artifacts on gradient-echo MRI obscuring adjacent tissues.
2.
US can visualize erosions, cartilage and subchondral abnormalities at the joint periphery.
However,differentiation between subchondral cysts and erosions is usually unfeasible by US.
3.
Prior knowledge of the degree of joint maturation is essential for an accurate evaluation of cartilage loss by US.
4.
Physical examination has limitations for assessment of early joint changes in contrast to US.
Conclusions: US can be useful for assessing hemophilic elbows, with advantages over MRI in the evaluation of soft tissues.
Further development of an US-MRI atlas on normal cartilage in growing joints is needed for definition of the value of US in the assessment of minimal osteochondral abnormalities.
Digital Atlas of Skeletal Surveys of Common Skeletal Dysplasias Shawn Parnell, MBBS, MD, DNB, Radiology, Seattle Children's Hospital, shawn.parnell@seattlechildrens.org; Corey Wall, Edward Weinberger Purpose or Case Report: Skeletal dysplasias are conditions of abnormal bone and cartilage growth which result in short stature.
Developing expertise in the radiographic evaluation of skeletal dysplasias can be difficult, as more than 250 dysplasias exist.
Exhaustive description of individual dysplasias can be found in hard copy textbooks, without the ability to compare individual dysplasias side by side.
By providing radiographic images and descriptive text of thirteen common skeletal dysplasias and two comparative normal skeletal surveys, we aim to facilitate understanding of the terminology and highlight the differences in imaging appearances one may commonly encounter in interpreting skeletal dysplasias.
Methods & Materials: Initial skeletal surveys and/or follow up radiographs obtained for evaluation of skeletal dysplasias at our institution from 2005 to 2011 were compiled and reviewed for best quality images.
Selected images for each case were labeled according to body part and view, to include AP and lateral views of the spine and skull and AP views of the extremities and pelvis.
For neonates, AP and lateral babygram images were used.
The software program used for viewing the atlas, written in C#, may be freely downloaded.
It permits linked scrolling and resizing of the images, and simultaneous comparison of different cases is available.
Cases may be viewed as unknowns or in a selfteaching mode.
Results: Radiographic images for thirteen common skeletal dysplasias and two comparative normal skeletons (neonate and child) are provided within an interactive digital atlas.
Cases include achondroplasia, pseudoachondroplasia, cleidocranial dysplasia, thanatophoric dysplasia, diaphyseal dysplasia, multiple epiphyseal dysplasia, osteopetrosis, osteogenesis imperfecta, multiple hereditary exostoses, dysostosis multiplex, fibrous dysplasia, asphyxiating thoracic dysplasia (Jeune syndrome), and spondyloepiphyseal dysplasia.
Conclusions: By displaying radiographic images of several common skeletal dysplasias in an interactive and comparative format with descriptive text, understanding of basic radiographic terminology and appearances will be facilitated.
Purpose or Case Report: 1.
To classify various pediatric MSK soft tissue masses 2.
To describe pathogenesis, imaging appearances and differential diagnosis of these lesions Methods & Materials: Radiology and clinical medical records were reviewed and pediatric patients with musculoskeletal soft tissue masses were identified.
Representative images were collected as examples of each lesion.
The lesions were then classified into different groups based on the similar pathology and etiology.
Brief discussion is done for each of these masses with their multimodality imaging appearances.
Results: The search yielded pediatric soft tissue masses of multiple different etiologies, including post-traumatic (hematoma, fat necrosis, fibromatosis coli, myositis ossificans), inflammatory or infectious (cellulitis, abscess, granuloma annulare, retained foreign bodies), pseudotumors (synovial cysts, ganglion cysts, vascular malformations) and neoplastic lesions (fatty, vascular, neural, fibrous, muscular).
Multiple different imaging modalities were used to evaluate these masses, including ultrasound, CT and MRI.
Representative examples of different lesions and their appearances on different imaging modalities will be presented and an organized approach to the diagnosis of these lesions will be discussed.
Conclusions: Musculoskeletal soft tissue masses are relatively common in children.
Majority of these are benign; however, up to 6% of these lesions can be malignant "sarcomas".
Multiple different imaging modalities often provide complimentary information in the work-up of these lesions.
Despite multimodality imaging approach, tissue diagnosis or short interval follow-up is still often required when the mass does not show typical features of a benign etiology.
Pediatric radiologists should be familiar with various pediatric MSK soft tissue masses and their imaging appearances, and should be able to guide appropriate management.
Results: 199 elbow MRI examinations were reviewed on children aged 4 months to 18 years with 28 (14%) of these investigating clinical instability in 25 children.
Mechanism of injuries included congenital dislocation 10 (36%), traumatic dislocation 13 (46%), fracture or avulsion 2 (7%) and other injuries 3 (11%).
The patient's with congenital elbow dislocations most commonly presented with radial head dislocation and associated dysplasia or flattening, effusion and less frequently dysplasia of the olecranon or capitellum.
Patient's with traumatic dislocations were frequently associated with ligamentous or capsular disruption, bone oedema and epicondylar avulsion with effusion, loose osseous bodies and fractures less often.
The epicondylar avulsions and ligamentous or tendon injuries occurred equally often in those few patients with unspecified injury mechanism.
Conclusions: A number of the bony, ligamentous, articular and developmental anomalies evident on elbow MRI have been illustrated highlighting the importance of careful and systematic review of all elbow structures when presented with a child with elbow instability.
Accurate identification of these abnormalities is vital to facilitate their appropriate management.
Methods & Materials: From our computerized radiology information system, we retrieved all patients that have foot ultrasound for evaluation of vertical or oblique talus deformities in the last 6 years (10/2005-10/2011).
The US was performed by a pediatric radiologist using a high resolution linear and tight convex curve probes with foot in neutral, plantar flexion and dorsiflexion.
All medical charts, ultrasound scans and foot radiographs were reviewed by a pediatric radiologist.
Results: We identified nine patients' with foot deformities who were suspected of vertical or oblique talus and were evaluated by ultrasound.
Seven patients are male; two of them had initial foot radiographs that were not diagnostic.
Two female patients had unilateral oblique talus deformity.
There were 7 patients with vertical talus deformity; three of them had bilateral deformities.
Conclusions: US can directly visualize the unossified navicular, the talar cartilage and their alignment.
Dynamic US can.
Ultrasound Evaluation of Costal Chrondral Pathologies in Children Presented as Anterior Chest Wall Mass or Pain Nucharin Supakul, MD, Radiology, Riley Hospital for Children, tanyasupakul@yahoo.com; Boaz Karmazyn Purpose or Case Report: To summarize our experience with the use of ultrasonography (US) for evaluation of costal cartilage pathology presented as anterior chest wall mass.
Methods & Materials: From our computerized radiology information system, we retrieved all patients that have chest wall ultrasound for evaluation of a mass in the last 4.5 years (4/2007-8/2011) .
The US was performed by a pediatric radiologist using a localized scan with high resolution linear probe.
All medical charts, pathology results, ultrasound scans and other imaging studies were reviewed by a pediatric radiologist.
Results: Ten patients were found with costal chrondral pathologies.
Nine patients presented with anterior chest wall mass and one with chest wall pain.
Eight patients had angular deformity of a single costal cartilage and one patient had biopsy proven osteochrondroma, presented with anterior chest wall mass.
One patient had a non-union fracture after motor vehicle accident, presented with anterior chest wall pain.
In patients with rib deformity, the mass was non-tender.
Nine patients had prior imaging study including chest x-rays (n08), CT scan (n0 2), breast MR (n01).
All these studies were negative.
Conclusions: US optimally demonstrated costal cartilage abnormities.
Chest radiographs and cross sectional studies were negative.
We therefore recommend using high resolution chest wall US in children with negative chest radiograph and anterior hard chest wall mass.
Challenges in Pediatric Marrow Imaging-Boning Up on Current MR Techniques Srikala Narayanan, MD, Division of radiology, Children's National Medical Center, snarayan@childrensnational.org; Neha Kwatra, Nabile Safdar Purpose or Case Report: A wide range of pathologies demonstrate similar findings when imaged using conventional MR sequences.
However, pediatric musculoskeletal imagers are increasingly leveraging newer techniques to add specificity to their diagnoses when abnormal marrow signal is detected.
The purpose of this educational exhibit is to review the application of current MR techniques to pediatric marrow imaging across the spectrum of normal, variant, and pathologic processes.
Methods & Materials: Cases with potentially overlapping imaging appearances on conventional MR sequences, including hematopoetic marrow, sickle cell disease, osteomyelitis, chronic recurrent multifocal osteomyelitis, and infiltrative neoplasms, will be presented.
The basis of various MR techniques including chemical shift imaging, "whole body" marrow imaging, diffusion weighted imaging, and fat-water separation techniques such as Dixon or IDEAL (GE) will be reviewed.
The strengths and weaknesses of such techniques in differentiating between infection, neoplasm, and normal variation will be emphasized through the case examples.
Challenges and pitfalls in the imaging of these pathologies using such techniques will be discussed.
Results: Current MR imaging techniques add specificity to diagnoses of marrow pathology which are otherwise difficult to differentiate using traditional sequences alone.
The use of opposed phase imaging can be helpful in differentiate hematopoietic marrow or infection from infiltrative and neoplastic conditions.
"Whole body" marrow imaging may serve as an alternative to other modalities which involve significant radiation exposure.
The use of diffusion weighted imaging is a promising, but developing, technique being applied to marrow pathology.
Conclusions: Pediatric bone marrow MR imaging is a challenging area for a vast majority of the radiologists.
Understanding normal developmental bone marrow changes and being aware of the pitfalls is crucial to render accurate diagnosis.
Current techniques such as IDEAL, chemical shift imaging, and "whole body" MRI have a potentially important role in further characterization of marrow abnormalities.
Radiologists Beware: Unusual Imaging Manifestations in Child Abuse Eglal Shalaby-Rana, MBBS (Hons), Children's National Medical Center, erana@childrensnational.org; Allison M. Jackson, Tanya Hinds, Katherine Deye Purpose or Case Report: To present less common imaging manifestations of injuries in child abuse that may not be readily recognized as possibly abusive injury.
Methods & Materials: Through bi-monthly review of cases with the child protection team over a period of 12 years, the imaging studies of patients with suspected non-accidental trauma were recorded.
Of the 654 pts with suspected nonaccidental trauma, outcomes were available in 599 patients.
The child protection team concluded 254 (43%) were cases of non-accidental trauma with reasonable medical certainty.
This data base was reviewed for less common injuries that were found in these medically confirmed cases of child abuse.
Results: Less common manifestations of abuse identified by radiographs included Salter-Harris injuries in the proximal humerus, and proximal femur.
Pelvic fractures were rare and when present were associated with sexual abuse.
Severe chest wall injury, with associated rib fractures, causing complete or near-complete white-out of the chest was occasionally encountered.
Soft tissue injures, such as hematomas were found in various locations in the body including the buttocks and anterior abdominal wall, were imaged on ultrasound and CT. Paraor prevertebral injuries, with or without associated bone injury were identified; one infant presented with retropharyngeal soft tissue swelling.
MRI identified cervical spine injuries which included ligamentous injury and intrathecal hematomas.
Conclusions: While classic metaphyseal lesions and rib fractures are the most common, specific injuries documented by radiologic work up of suspected non-accidental trauma, less common injuries to the soft tissue and skeletal system may occur as a result of child abuse.
The ability of the radiologist to recognize these uncommon manifestations of demonstrated in axial, coronal, and sagittal planes.
The ligament of interest will be denoted by arrows.
At the conclusion of the anatomy section, there will be a self assessment exam.
The participants then will be asked to identify the ligament.
If answered correctly, a summary slide will be displayed and, if common, images of the relevant pathology will be demonstrated.
If an incorrect answer is indicated, a slide will appear denoting the incorrect answer with explanation.
Conclusions: Hopefully, with review of this educational exhibit, the participant will have a better understanding of the relevant ligamentous anatomy of the ankle and hindfoot.
Purpose or Case Report: The purpose of this educational exhibit is to demonstrate the pathologic sonographic findings, one might encounter in the pediatric ankle.
A systematic methodological approach including patient positioning, transducer orientation and sonographic technique are vital for ideal sonographic assessment of the pediatric ankle.
Using a data search program from a large academic institution, pediatric ankle ultrasounds performed in the last 10 years were reviewed.
Pathologies include trauma, inflammation/infection, masses and congenital abnormalities.
Examples of normal anatomy will be included particularly when demonstrating ligament and tendon pathology.
The normal side was often assessed for comparison purposes.
Results: Ankle sonography is a useful modality to evaluate commonly encountered pathologies in the pediatric ankle.
Radiographically occult fractures may be discovered.
Ligament and tendon pathology, such as tears of the anterior talofibular ligament, high ankle sprain and peroneus longus tendon tears, can be easily detected.
Signs of infection that can be radiographically occult such as subtle periosteal reaction or fluid collections can be identified.
Finally, "lumps and bumps" can be characterized.
For example, one of the most commonly encountered masses in the pediatric ankle is a ganglion cyst which can be well characterized sonographically.
Awareness of imaging pitfalls is also critical to avoid misdiagnosis and to guide appropriate management.
Conclusions: With basic ultrasound skills and knowledge of normal anatomy, sonography of the pediatric ankle is a useful modality to evaluate soft tissue structures and other pathologies.
It is comparable to MRI and allows for dynamic evaluation without need for anesthesia.
Resonance Angiography (MRA) using time resolved imaging is a relatively new technique that has become increasingly utilized in the diagnosis of vascular anomalies.
We will describe the technique used at our institution, Time Resolved Imaging of Contrast Kinetics (TRICKS, GE Healthcare, Milwaukee, WI), and the parameters that can be adjusted to optimize the exam.
We will review key imaging features of hemangiomas and vascular malformations in various modalities, with a special emphasis on the TRICKS appearance.
We performed a retrospective review of all the TRICKS studies performed at our institution for suspected vascular anomalies.
In addition to the MR imaging features, we specifically analyzed the T1 weighted with fat saturation post TRICKS enhancement and the temporal TRICKS enhancement pattern.
We reviewed all additional imaging including plain film, ultrasound, and CT and correlated the radiographic imaging with the available clinical and histopathologic features.
Results: We present illustrative cases of hemangiomas, kaposiform hemangioendothelioma, venous malformations, arteriovenous malformations, lymphatic malformations, and other pitfall lesions.
We propose a diagnostic algorithm that relies heavily on the post contrast T1 weighted with fat saturation post TRICKS enhancement pattern and the temporal TRICKS enhancement pattern.
Conclusions: Time resolved contrast-enhanced MRA has become an increasingly important adjunct in the diagnosis of vascular anomalies.
Optimization of the exam technique and familiarity of the TRICKS imaging appearance is essential and can often assist in accurate lesion characterization.
Purpose or Case Report: Vertical Expandable Prosthetic Titanium Rib (VEPTR) is increasingly used in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children.
In contrast to spinal fusion surgery, VEPTR allows for growth while stabilizing the deformity.
We review the indications, pre-operative imaging, normal radiographic appearance, and complications of this device.
Methods & Materials: On review of the literature, the indications for VEPTR have expanded in the past several years to include thoracic insufficiency, idiopathic and neuromuscular scoliosis, and chest wall defects.
We illustrate the normal radiographic appearance of the three common configurations of VEPTR (cradle-to-cradle assembly, cradle with lumbar extension assembly; cradle-to-ala hook assembly).
We discuss the potential complications of VEPTR, including infection, rib fracture, dislodged hardware, and neurological injury, with an emphasis on imaging diagnosis.
Results: There is a relatively high rate of reported complications with VEPTR in the literature.
Therefore, awareness of the growing number of indications, as well as the expected and unexpected appearance of this device, aids in radiographic diagnosis of complications.
Conclusions: Vertical Expandable Prosthetic Titanium Rib (VEPTR) is gaining acceptance in the treatment of thoracic insufficiency, scoliosis, and chest wall defects in children.
Recognition of the indications, normal radiographic appearance, and complications of this device will facilitate timely and accurate diagnosis.
Disclosure: Dr. Philips has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Spectrum of Pediatric Spinal Neoplasms: An Interactive Tutorial Benjamin T. Haverkamp, MD, Radiology, University of Missouri-Kansas City, haverkampbt@umkc.edu; Salvador F. Iloreta, Maha Jarmakani, Lisa Lowe, Seth Gibson Purpose or Case Report: The objective of this educational electronic exhibit is to provide the radiologist with an approach to pediatric spinal neoplasms.
Emphasis will be placed on narrowing the differential diagnosis using a combination of lesion location, characteristic imaging findings, relevant history, and associations.
The exhibit format will include a casebased review of various pediatric spinal neoplasms, radiologicpathologic correlation, and a brief discussion of imaging findings useful in guiding surgical management.
An interactive selfassessment exam will be presented at the end of the exhibit.
Results: A review of the specific etiologies will be presented with classification as intra-and extra-medullary and extradural lesions.
Radiologic images will be related to gross and microscopic pathology.
Conclusions: After viewing this exhibit, the learner will be able to: 1.
Recognize the clinical, imaging, and pathologic characteristics of pediatric spinal neoplasms.
2.
Understand relevant imaging findings useful in surgical management.
3.
Test their understanding of the presented material through an interactive exam.
Poster #: EDU-057 Craniosynostosis Jason Tsai, MBChB, Children's Hospital Boston, jason.
tsai@childrens.harvard.edu; Diana P. Rodriguez Purpose or Case Report: To review the normal developmental appearance of the cranial sutures with computed tomography (CT) and to describe CT findings of the various forms of craniosynostoses.
In this IRB-approved retrospective study we reviewed CT images of subjects diagnosed with craniosynostosis between 2006 and September 2011.
We included patients with single-suture synostosis, isolated bilateral coronal synostosis, pansynostosis, and combined craniosynotoses.
Additionally, we identified individuals with normal appearing sutures from 0 to 5 years of age imaged with head CT to describe the pattern of normal development of the cranial sutures.
Results: A description of the normal developmental CT appearance of the cranial sutures using computed tomography has been provided.
Of the group of patients with craniosynostosis the following variables were recorded: age at presentation, the pattern of sutural fusion, skull shape, presence of hydrocephalus, genetic testing, and types of surgical correction.
Conclusions: We have demonstrated the normal developmental CT appearance of the cranial sutures and the CT patterns of the various forms of craniosynostoses, with clinical, genetic and surgical correlation.
Posterior Fossa Tumours: A Pictorial Review Sam Byott, MD, Manchester Children's Hospital, sambyott@ hotmail.com; Neville Wright, Vivian Tang, Abdu Shabani, Stavros Stivaros Purpose or Case Report: Posterior fossa tumours account for 54-70% of childhood brain tumours.
The most common differentials include pilocytic astrocytoma, medulloblastoma and ependymoma.
MR imaging is crucial to diagnosis, staging and identification of complications such as hydrocephalus and haemorrhage.
Soft tissue characteristics alongside tumour location, invasion and clinical history facilitate radiological discrimination prior to surgery.
However, there is significant clinical equipoise with regards to the imaging appearances in a significant proportion of cases making definite diagnosis difficult.
The aim of this study is to evaluate the radiological findings and correlate with histological data.
This will allow identification of the key morphological features that discriminate different tumours.
These can then be presented to educate fellow radiologists.
Methods & Materials: Radiology PACS and patient notes were used to collate radiological, histological and clinical data.
Results: There were 27 patients presenting at our institution with posterior fossa tumours.
12 had pilocytic astrocytomas, 8 had medulloblastomas and 7 had ependymomas.
One patient had an atypical teratoid rhabdoid tumour (ATRT).
Traditional features alongside more advanced MR characteristics were correlated with histology, and the features allowing for discrimination of tumour types are presented in this pictorial review.
Conclusions: Posterior fossa tumours have a highly variable radiological appearance.
We present a range of appearances and describe the important morphological features that allow radiological discrimination of tumour type.
Poster #: EDU-059 3DT1 Imaging of the Pediatric Spine Teresa C. Gross Kelly, Children's Hospital of Wisconsin, tkelly@chw.org; Ibrahim S. Tuna, Mia S. Kelly, Tushar Chandra, Sumit Singh, Mohit Maheshwari, Hervey D. Segall Purpose or Case Report: Some abnormalities of the pediatric spine can be challenging.
We have discovered that in many such cases, diagnosis of spinal lesions can be faciliated by using the 3DT1 weighted sequence.
The purpose of this educational poster is to demonstrate the remarkable usefulness of 3DT1 weighted images for delineating pathology of the pediatric spine.
Methods & Materials: Lesions of the spine that will be reivewed in this educational exhibit will be categorized as: (1) vascular (2) due to infection/inflammation (3) neoplastic/ neurogenic (4) congenital (5) traumatic/iatrogenic (6) endocrine/metabolic.
The imaging characteristics of lesions found in the pediatric spine will be described and the utility of 3D T1-weighted MR sequences for the evaluation of these lesions will be discussed.
Finally the role of imaging in the treatment planning of abnormalities of the pediatric spine will be addressed.
Results: This educational exhibit will provide numerous examples of how 3D T1-weighted imaging can elucidate diagnosis of lesions involving the spine.
Examples include enhancement of the cauda equina in Guillain Barre syndrome, lipomatous malformations, spondylolysis in children with low back pain, thecal cysts, filar cysts, metastasis, hydromyelia and ventriculus terminalis.
Conclusions: 3D T1-weighted images of the spine performed in the sagittal plane with coronal and axial reformations, as well sagittal oblique reformations (scotty dog reformations) for evaluation of spondylolysis, can facilitate the evaluation of lesions involving the pediatric spine.
The Normal Pediatric Spine: A Pictorial Review of MR Anatomy and Development in the Infant, Child and Adolescent Ibrahim S. Tuna, MD, Radiology, Children's Hospital of Wisconsin, dristuna@yahoo.com; Teresa C. Gross Kelly, Tushar Chandra, Mohit Maheshwari, Sumit Singh, Hervey D. Segall Purpose or Case Report: Radiological evaluation of the pediatric spine can be more challenging in child than in the adult patient due to the wide range of normal anatomic variants and synchondroses, combined with the unique effects of trauma in children.
MRI is an excellent imaging modality for the evaluation of the pediatric spine.
However, in order to provide an accurate interpretation of acute posttraumatic changes in the pediatric spine, particularly in the setting of abusive head trauma, a fundamental knowledge of normal anatomy, variants and pathology of the pediatric spine is required.
The aim of this educational exhibit is to illustrate normal MRI anatomy of the spine in the infant, child and adolescent.
Methods & Materials: This exhibit will first describe basic spinal embryology and development of the vertebra and spinal cord, followed by MRI depiction of the developmental anatomy of the spine from infancy through adolescence.
The changing appearance of the spinal canal, spinal cord and vertebral bodies with age will be illustrated using normal cases from the radiology database.
Sagittal and transverse diameter of vertebral bodies, thickness of the dural thecal sac, dimensions of the spinal canal, normal bone marrow signal changes, vertebral body heights, level of conus medullaris, prevertebral and paraspinous soft tissues and epidural fat thickness will be described and changes according to age will be pointed out.
Results: In early life, the spinal cord extends to the inferior aspect of the bony spinal column.
Because the vertebral bodies grow longitudinally faster than the spinal cord does, the conus medullaris may change.
Ossification of the vertebral bodies and posterior elements is nearly complete by age 10, with a resultant decrease in the spinal canal diameter.
The nucleus pulposus becomes smaller after 10 years and spans approximately half the disk space in the sagittal plane.
The spinal cord is elliptical in cross section in the cervical spine and demonstrates a difference in signal between the normal gray and white matter of the spinal cord which should not be mistaken for intramedullary pathology.
Conclusions: A solid understanding of normal spine anatomy and embryological development is essential in evaluation of pediatric spine, mainly in the setting of trauma.
Familiarity with normal anatomic variants is essential to provide an accurate interpretation of pathology in the pediatric spine.
Spectrum of Intracranial Cystic Lesions in Infants and Children Ernesto I. Blanco, MD, St. Christopher's Hospital for Children, eiblanco74@gmail.com; Eric Faerber Purpose or Case Report: To review the imaging spectrum of intracranial cystic lesions in the pediatric population.
Methods & Materials: A retrospective review of our imaging database was performed to identify studies obtained in which the findings included intracranial cystic lesions.
Results: Multiple cystic lesions were elucidated primarily by computed tomography or magnetic resonance imaging.
These lesions can be divided into nonneoplastic and nonneoplastic tumor-associated cysts.
The nonneoplastic cysts, which is the largest group, include: cavum septi pellucidi and cavum veli interpositi, choroid plexus cyst, enlarged peri-vascular spaces, pineal cyst, the large spectrum of arachnoid cysts, colloid cyst, epidermoid cyst, Rathke cleft cyst, and porencephalic cyst.
Nonneoplastic tumor-associated cysts include: craniopharyngioma, optic glioma, pilocytic astrocytoma, hemangioblastoma, and ganglioglioma.
Conclusions: Intracranial cystic lesions are relatively common entities in the pediatric population.
A wide spectrum of nonneoplastic and nonneoplastic tumor associated pathologies are presented using both computed tomography and magnetic resonance imaging.
Kelly, Sumit Singh, Ibrahim S. Tuna, Hervey D. Segall Purpose or Case Report: The aim of this educational exhibit is to provide a comprehensive review of imaging features, classification and management of pediatric spinal cord tumors.
We also aim to elicit the differences between pediatric spinal cord tumors and their adult counterparts.
We will summarize the differences between the individual tumors based on histological cell types and the pertinent implications on management and outcome Methods & Materials: This exhibit will provide an overview of the common as well as uncommon tumors of the pediatric spinal cord.
Various classification systems for these tumors-anatomical as well as histological will be discussed.
We will illustrate the relevant imaging findings that can help in differentiating these tumors.
Results: Pediatric spinal cord tumors account for 1% to 10% of all pediatric central nervous system tumors.
MRI is the mainstay for the initial diagnosis as well as the post surgical evaluation and surveillance of these tumors.
Pediatric and adult spinal cord tumours differ both in terms of anatomical location as well as histology.
The disease and treatment related morbidities are also different in children as compared to adults.
Astrocytomas, ependymomas, glioneural tumors and CSF metastasis represent the vast majority of cord neoplasms in the pediatric age group.
Some of cord tumors may also be associated with inherited syndromes (like Neurofibromatosis type 2) or may have genetic predisposition.
These would also be discussed.
We will also illustrate and discuss common non neoplastic spinal masses that may mimic tumors.
Conclusions: Pediatric spinal cord tumors have varied clinical presentations, imaging appearance and outcome.
This review would improve the understanding of these tumors thereby helping in diagnosis, management and follow up of these uncommon neoplasms.
Multi-Modality Imaging of Pediatric Head and Neck Lesions Jason Au, MD, Oklahoma University Health Sciences Center, jasonmau@gmail.com; Anthony Alleman, Mahmoud Elkaissi, Roy Jacob Purpose or Case Report: The purpose of this study is to present a side by side comparison of the multi-modality imaging features of pediatric masses.
Using cases that have been imaged with multiple modalities, the exhibit will delineate the sonographic, MR, and CT appearance of congenital, infectious, and neoplastic head and neck lesions in the pediatric population.
Methods & Materials: A restrospective search of PACS was performed on studies completed at the Oklahoma University Medical Center on the Oklahoma University Health Science Center Campus from January 2008 to the present.
Ultrasound, CT, and MR examinations were selected that depicted relevant pediatric head and neck pathology.
All studies were de-identified prior to image export.
Results: Over twenty representative cases of pediatric infections, fibrous tumors, cystic neoplasms, vascular malformation, bony tumors, developmental anomalies, and other neoplasms were selected for inclusion.
Results: Pictorial review of cases including the following representative cases: myelonmeningocele associated with Arnold Chiari malformation, lipomyelomeningocele, tethered cord with spinal lipoma/fibrofatty filum, tethered cord and dermal sinus tract, and Chiari I with syringohydromyelia.
Several unique cases including the following will be presented as well: thoracic meningocele with Arnold Chiari malformation, terminal myelocystocele, diastematomyelia, and myelomeningocele without Arnold Chiari malformation.
While MRI demonstrates the cranio-cervical junction and the cervicothoracic spinal cord better than ultrasound, ultrasound often allows for improved resolution of the distal spinal cord, lumbosacral spinal canal, and spinal dysraphism structures near the skin surface in the neonate.
Conclusions: Congenital spinal malformations are complex and variable in imaging appearance.
It is important to understand the classification in order to determine the appropriate management and prognosis.
In the neonatal period imaging should be performed with ultrasound and MRI studies, as they may provide different and complementary information.
Conclusions: Hypoxic Ischemic Injury is a common condition resulting in a wide spectrum of severe neurological defects.
While in the past treatment only consisted of supportive care for HII, recent advances have yielded promising treatment options if initiated within a limited time window.
Thus due to the severity of the disease and the need for rapid intervention, it is important to recognize radiological manifestations of HII along with its clinical signs and symptoms to offer a better prognosis to the patient.
Craniosynostosis: Looking Beyond the Sutures Tushar Chandra, MD, Children's Hospital of Wisconsin, drtusharchandra@gmail.com; Teresa C. Gross Kelly, Mohit Maheshwari, Sumit Singh, Ibrahim S. Tuna, Hervey D. Segall Purpose or Case Report: The aim of this educational exhibit is to provide a framework upon which the diagnosis of the various types of craniosynostosis can be facilitated.
Our goal is to provide an efficient way to evaluate craniosynostosis for the radiologist in clinical practice.
We plan to accomplish this goal by providing a succinct review of the sutures, an overview of the various classification schemes for craniosynostosis and potential complications associated with premature sutural closure.
The role of imaging in the evaluation of craniosynostosis will be described and the features of craniosynostosis that are most important to the craniofacial surgeon will be elucidated.
Finally, surgical strategies for the repair of craniosynostosis and postoperative findings will be described.
Results: Some of the forms of craniosynostosis may have a genetic basis, but many are spontaneous in nature.
Untreated progressive craniosynostosis can lead to inhibition of brain growth, and an increase in intracranial pressure.
MDCT with MIP and 3D surface reformations is the preferred modality for diagnostic evaluation of craniosynostosis.
It is also a robust modality for post operative assessment and long-term follow up.
MRI is a useful adjunct for assessment of associated intracranial anomalies and complications.
Timely and appropriate imaging is essential to assess for potential complications of craniosynostosis which may include intracranial hypertension, anomalies of external and middle ear, hydrocephalus, chronic tonsillar herniation, cranial base deformity, impaired venous drainage, enlarged emissary foramina and veins and optic atrophy.
On the other hand, positional plagiocephaly should not be misinterpreted as craniosynostosis.
Surgical management is typical for nonsyndromic craniosynostosis, which involves correction of craniosynostosis between three to six months of age.
Conservative management is the mainstay for syndromic craniosynostosis.
Postoperative follow up imaging for surveillance for ventricular size and signs of raised intracranial pressure are necessary.
Conclusions: Craniosynostosis is a challenging area of pediatric neuroimaging.
Knowledge of the sutural anatomy, an understanding of the potential intracranial complications caused by premature sutural closure, as well as the role that imaging plays in presurgical planning, can provide a practical way for the radiologist to evaluate craniosynostosis in a fast-paced clinical setting.
Poster #: EDU-067 The Perinatal Brain and Spinal Cord-Imaging Across a Life Border: A Case-Based Approach Anand Dorai Raju, MD, Radiology University of Tennessee, araju@uthsc.edu; Harris L. Cohen, Matthew Whitehead, Asim Choudhri Purpose or Case Report: To review normal and abnormal perinatal Ultrasound (US) and Magnetic Resonance (MR) imaging findings and note their significance for the analysis of the fetal and neonatal brain as well as spinal cord and vertebral column using a case based approach.
To highlight US and MR capabilities in allowing correct perinatal diagnosis of congenital and acquired central nervous system abnormalities.
Methods & Materials: Cases will be shown of normal and abnormal anatomic findings in fetal and neonatal brain and spinal cord imaging.
Key teaching points necessary for the diagnosis of such brain abnormalities as ventriculomegaly, Chiari malformations, holoprosencephaly, and agenesis of the corpus Callosum as well as Dandy Walker malformations and AVMs will be discussed.
Intraventricular hemorrhage, periventricular leukomalacia, anoxic injuries and infectious abnormalities will be reviewed.
Abnormal anatomic findings in fetal and neonatal spine evaluations for congenital and acquired abnormalities and key teaching points necessary for the accurate diagnosis of tethered cord, myelomeninocele, caudal regression syndrome, hydromyelia, diastomatomyelia and sacrococcygeal teratoma will be reviewed.
Some diagnostic difficulties and controversies will be addressed.
Conclusions: Ultrasound aided by MRI can provide ready diagnosis to many central nervous system abnormalities involving fetuses and neonates.
Ever improving perinatal imaging experience and technique allow for better prenatal as well as postnatal diagnosis.
Cases showing such imaging and key points helping such imaging diagnoses will be reviewed.
Overview of Imaging of Pediatric Extraocular Orbital Tumors Srikala Narayanan, MD, Division of Radiology, Children's National Medical Center, snarayan@childrensnational.org; Nadja Kadom, Gilbert L. Vezina Purpose or Case Report: To show the spectrum of benign and malignant extraocular orbital tumors in children.
Methods & Materials: We reviewed the cross-sectional imaging of orbit (CT and MR) done in the last 5 years.
Specific imaging signs of extraocular tumors including benign and malignant tumors such as hemangiomas, lymphangiomas, optic nerve glioma, optic nerve sheath meningioma, pseudotumors, rhabdomyosarcoma, orbital myofibroma, eosinophilic granuloma and neuroblastoma metastases will be shown.
Important imaging features that should be considered when formulating a differential diagnosis will be described.
Conclusions: The spectrum of diseases affecting pediatric orbit is substantially different from what we see in the adults.
It is not easy always to differentiate between different tumors.
Important imaging characteristics will help us towards better differential diagnosis.
In this exhibit, we will illustrate ultrasound anatomy of the neonatal spinal cord.
Discussion of the normal anatomic variants and pathological conditions of the spinal cord will be provided.
Representative images of a variety of common and uncommon pathological conditions of the spine will be presented to illustrate teaching points.
In abnormal cases, follow up MRI images will also be illustrated for comparison.
Results: Ultrasound is a robust screening modality for evaluation of the lumbosacral spine in neonates.
It is cheaper, readily available, safer first line imaging modality in neonates suspected to have spinal malformations.
Under able and well trained operator, diagnostic accuracy of spinal ultrasound approaches MRI.
However, MRI remains the gold standard for imaging evaluation of spine.
Normal variants that simulate disease processes like ventriculus terminalis, prominent filum terminale and central echo complex will be presented.
Congenital malformations of the cord such as tethered cord, hydromyelia, lipoma, diastematomyelia, myelomeningocele, lateral meningocele and presacral masses will also be discussed.
Conclusions: Ultrasound is a very useful screening technique for evaluation of pathological conditions of lumbosacral spine in neonates.
This review would improve the understanding of utility and limitations of ultrasound in evaluation of neonatal spinal malformations.
Purpose or Case Report: Although MRI is the standard for detecting epilepsy and brain tumor abnormalities, PET-CT is performed to ascertain metabolism related to epileptogenic regions or characterize tumor metabolic activity.
Asymmetric metabolism often correlates to structural abnormalities like cortical dysplasia.
Metabolic activity often correlates with tumor aggressiveness or grade.
FDG PET is commonly used to assess seizure and tumor metabolism.
The lesser utilized amino acid PET tracers (C11 Methionine, FDOPA) show increasing value with lower grade tumors due to high tumor to normal tissue contrast.
Literature is accumulating regarding C11 methionine (CMET) in the detection of lesions like cortical dysplasia and its ability to delineate low grade seizure related tumor lesions.
Despite the established FDG and accumulating CMET literature, little information exists about the imaging seen with both in pediatrics.
As these studies are increasingly viewed as part of fusion MRI images, there is more scrutiny of focal metabolism correlating with MRI findings and less interpretative reliance on abnormality based solely on asymmetry.
Methods & Materials: Review of 110 patients who underwent CMET and FDG brain PET-CT was performed.
Each was imaged on a Philips scanner and had prior MRI.
Studies demonstrating a variety of tumors, postoperative findings of residual or recurrent tumor, and pseudoprogression were selected.
Epilepsy cases with structural cortical abnormalities or seizure-associated tumors were also selected.
CMET and FDG studies were analyzed by 3 pediatric neuroradiologists and the imaging findings correlated with prior MRI and any pathology or follow-up imaging.
Pictorial galleries of the CMET and FDG imaging patterns were created.
Results: Pathologically proven low-grade glial tumors showed increased CMET uptake and no hypermetabolism on FDG.
High-grade tumors showed increased uptake on CMET and hypermetabolism on FDG.
Patients with residual or recurrent tumors showed uptake similar to their original tumor.
Granulation tissue and pseudoprogression changes showed increased uptake on CMET and no hypermetabolism on FDG.
Epilepsy surgery patients with cortical dysplasia or low grade glial tumors showed increased uptake on CMET and FDG hypometabolism.
Conclusions: This study illustrates the variety of findings on CMET and FDG PET-CT in pediatric patients clinically evaluated for brain tumor and epilepsy.
This atlas provides readers with a guide to the appearance of these findings on an emerging imaging technique.
Pediatric Head and Neck Neoplasms: A Multimodality Pictorial Review Alok Jaju, MD, Mallinckrodt Institute of Radiology, alokjaju@gmail.com; Marilyn J. Siegel Purpose or Case Report: Neck masses are common in children and most occur in the suprahyoid region.
Knowledge of the fascial spaces involved in conjunction with imaging features can help in diagnosis.
In this pictorial review, we present a multimodality imaging approach based on anatomy of the suprahyoid fascial spaces for evaluation of pediatric neck tumors.
Methods & Materials: Radiology information system (RIS) at our tertiary care children's hospital was queried to identify patients with suprahyoid neck masses who had imaging performed between July 2004 and present.
A variety of conditions having congenital, inflammatory, neoplastic, or vascular origin were identified and the anatomic location in the neck as well as imaging and clinical findings were retrospectively reviewed.
Results: The imaging evaluation included ultrasound, CT and MRI.
Lesions arose within the following fascial spaces of the suprahyoid neck: superficial, carotid, masticator, submandibular, sublingual, parotid, parapharyngeal, visceral, retropharyngeal and prevertebral.
Key imaging features important in diagnosis included lesion vascularity, calcification, necrosis and bone invasion.
We discuss and illustrate these imaging findings and relate them to specific suprahyoid fascial spaces.
Specific lesions include vascular and lymphatic malformations, teratoma, nerve sheath tumors, thyroglossal duct and branchial cleft cysts, pleomorphic adenoma, dermoid cyst, ranula, lymphadenopathy, abscess, lymphoma, rhabdomyosarcoma, neuroblastoma and nasopharyngeal carcinoma.
Conclusions: Knowledge of fascial spaces of the suprahyoid compartment and key imaging features on multiple modalities can aid in the diagnosis of pediatric neck masses.
Pediatric Sinusitis: Spectrum of Imaging Findings with Clinicopathologic Correlation Roy Jacob, MD, University of Oklahoma, drjacobr@gmail.
com; Paul Digoy, Robert S. Glade, Anthony Alleman Purpose or Case Report: The clinical spectrum of sinusitis in children can range from uncomplicated bacterial sinusitis to invasive fungal sinusitis.
Most cases respond favorably to medical management.
However, complications occasionally occur due to the spread to adjacent structures.
Imaging plays an important role in characterizing the disease and guiding the clinical and surgical planning and treatment.
This electronic presentation outlines the following-1.
Review radiologic anatomy and unique characteristics of pediatric sinuses.
2. Review the clinical features, pathophysiology, and microbiology of sinusitis.
3. Review of CT and MRI imaging characteristics of sinusitis with representative cases such as complicated sinusitis and invasive fungal sinusitis.
4. Review the treatment approaches of sinusitis.
Methods & Materials: A retrospective search of PACS was performed on studies completed at the OU Children's Hospital in Oklahoma City for the last three years.
CT and MR examinations were selected that depicted relevant disease processes.
Corresponding nasal endoscopic pictures were obtained from cases which required surgical management.
All studies were de-identified prior to image export.
Results: Over fifteen representative cases of the clinical spectrum of sinusitis and its complications were selected for inclusion.
Conclusions: This educational exhibit provides a concise review of imaging, clinical features, and treatment of pediatric sinusitis.
Findings will be richly illustrated with radiological and clinical images.
microcephaly or hydrocephalus.
Knowing the embryology of the cerebellum and 4th ventricle is important to perceive the development of posterior fossa malformations and to further understand the imaging findings.
Several classifications schemes have been proposed from a pure embryologic to an imaging-based approach using some essential findings such as the size of the posterior fossa, the presence of CSF collection or expansion of CSF space, and the size and morphology of the cerebellum.
MR is the gold-standard for adequately access and characterize the posterior fossa structures.
This pictorial essay will review the MR findings of some of the most common posterior fossa malformations including Dandy-Walker malformation, persistent Blakes pouch, mega cisterna magna, arachnoid cyst, paleocerebellar hypoplasia, cerebellar agenesis, cerebellar and pontocerebellar hypoplasia, cerebelar cortical malformations, isolated brainstem hypoplasia/dysplasia and Chiari malformations.
We will provide a practical approach to the MR findings of posterior fossa malformations in children.
Conclusions: MR plays a crucial role in identifying and characterizing malformations of the posterior fossa structures.
It should give a logical approach to these complex malformations thus guiding the refereeing physician into the clinical approach and in determining further investigations.
Results: Neuroimaging features of abnormal thalami as encountered in the pediatric population were detailed, and wherever applicable, the relevance of additional MR imaging sequences and techniques to determine etiology was described.
While there was considerable overlap in imaging appearances, making a precise diagnosis was found to be challenging in difficult cases, and by and large, a stepwise approach was successfully formulated and used to: 1.
Diagnose the more emergent conditions and to 2.
Devise a management algorithm for the less acute abnormalities.
Conclusions: Bilateral thalamic lesions are occasionally encountered in pediatric neuroimaging and have a limited differential; a good knowledge base and adequate technique are imperative to tease out the precise diagnosis and institute appropriate management.
Cortical Developmental Abnormalities in Pediatric Seizure Patients Ibrahim S. Tuna, MD, Radiology, Children's Hospital of Wisconsin, dristuna@yahoo.com; Mohit Maheshwari, Teresa C. Gross Kelly, Sumit Singh, Tushar Chandra, Hervey D. Segall Purpose or Case Report: To describe various cortical malformations with illustrative examples.
We will also briefly discuss the embryology, genetic basis, classification schemes and characteristic imaging findings .
Methods & Materials: This exhibit will illustrate three main categories of cortical malformations: neuronal proliferation, migration and organization.
Understanding of this complex topic would be facilitated by brief discussion on the embryological basis and proposed genetic causes of some of these cortical malformations.
Classification schemes on embryology and imaging will be discussed.
Characteristic imaging findings of these malformations will be discussed and examples from the authors database will be shown.
Results: Neuroimaging in pediatric seizures is challenging.
MRI is considered the imaging modality of choice because of superior soft tissue contrast and better ability to characterize the pathologic process.
We will also discuss the dedicated seizure protocol which is used in our institute.
PET-CT imaging can also provides additional information in cases where MRI is negative, inconclusive or does not correlate with EEG/clinical findings.
Brief discussion on advanced imaging techniques will also be presented.
Malformations are frequently detected in infancy.
However, if the initial MRI scan performed in infancy is negative, a repeat scan after 2 years of age may be helpful.
Conclusions: Evaluation of cortical malformation in seizure patients still remains a challenging area of pediatric neuroimaging.
Reviewing of the embryological basis, classification schemes and characteristic imaging findings would improve the understanding the cortical malformations and interpreting the images.
Poster #: EDU-079 SPRS Best Poster 2011 Cystic Neonatal Lesions Associated with the Spinal Cord: Discussion and Differential Diagnosis for these Uncommon Lesions Jacob Pirkle, MD, jpirkle@mc.utmck.edu, James Boyd, Brian Dupree Purpose or Case Report: To review intradural cystic neonatal spine lesions and discuss the various causes and appearance of these lesions.
This poster presentation provides a brief review of neonatal cystic spine lesions, including their etiologies, and presents the targeted audience (radiology resdients, fellows, and practicing radiologists) a helpful differential diagnosis of these lesions based upon their imaging appearance.
Methods & Materials: A brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material.
Images from several cases of cystic neonatal spine lesions are presented.
Results: A brief overview of neonatal cystic spine lesions, their etiology, and imaging appearance is presented in poster format utilizing both literature search and printed reference material.
Images from several cases of cystic neonatal spine lesions are presented.
Conclusions: Neonatal spine ultrasound is often performed to evaluate for abnormalities related to the presence of sacral dimples, cutaneous stigmata, skin tags, hairy tufts, during the evaluation of other congenital anomalies, or when prenatal ultrasound/MRI demonstrates an abnormality warranting postnatal follow-up.
The identification of cystic spinal cord lesions is relatively rare in the neonate.
However, the etiology of these lesions can often be deduced or surmised based upon the location and the imaging appearance of the lesion.
The most common cause of a cystic intramedullary spinal lesion is ventriculus terminalis, with a reported incidence of 2.6%.
Additional lesions include transient dilatation of the central canal, filar cyst, syringohydromyelia, intramedullary arachnoid cyst, and myelomalacia related to in utero/birth trauma.
Extremely rare etiologies in the neonate include epidermoid/dermoid, cavernous malformation, intranatal cystic infections etiologies, neuroepithelial cysts, and cystic neoplasms.
Mimics include diastematamyelia, spinal lipomas, and intramedullary hematomas.
Numerous imaging examples of these lesions are provided in the accompanying poster.
Brain MRI in Peroxisomal Disorders: A Pictorial Essay Bruno P. Soares, MD, Radiology and Biomedical Imaging, University of California at San Francisco, bruno.soares@ucsf.
edu; Leonardo Vedolin, Guido Gonzalez Purpose or Case Report: Our presentation aims to illustrate the brain MRI patterns in peroxisomal disorders.
Peroxisomes are intracellular organelles involved in important cellular processes including beta-oxidation of very-longchain fatty acids and plasmalogen production.
Peroxisomal disorders can be categorized into disorders of peroxisomal biogenesis, in which the peroxisomes are abnormally formed and several peroxisomal functions are deficient, and in defects involving a single peroxisomal function, in which the structure of the peroxisome is intact.
Disorders of peroxisomal biogenesis include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease and rhizomelic chondrodysplasia punctata.
Numerous disorders are caused by loss of a single peroxisomal function including X-linked adrenoleukodystrophy and Acyl-coA oxidase deficiency.
Clinical findings in peroxisomal disorders include dysmorphic features, hepatic dysfunction, neurodevelopmental delay, retinopathy and hearing impairment.
Methods & Materials: Pictorial essay illustrating brain MRI patterns in peroxisomal disorders, including disorders of peroxisomal biogenesis and disorders with loss of a single peroxisomal function.
Results: Brain abnormalities in peroxisomal disorders have a wide spectrum of patterns.
Neuronal migration disorders with abnormal myelination are typically seen in Zellweger disease and neonatal adrenoleukodystrophy.
Specifically, the association of abnormal myelination with germinolytic cysts is suggestive of Zellweger syndrome.
Classic X-linked adrenoleukodystrophy typically shows posterior central white matter involvement and symmetric demyelination also involving the corticospinal tracts and corpus callosum.
A similar pattern of white matter involvement is seen in Acyl-coA oxidase deficiency and infantile Refsum disease.
Conclusions: Brain MRI helps narrow the differential diagnosis and guides subsequent evaluation in infants presenting with clinical features concerning for peroxisomal disorders.
Therefore, knowledge of the brain MRI patterns in peroxisomal disorders is important for the radiologist interpreting neuroimaging studies.
Clots in Tots: Role of Imaging in Diagnosis of Acute Stroke and its Causes in Children Asif Abdullah, C.S.
Mott Children's Hospital of The University of Michigan, asifa@med.umich.edu; Ellen Hoeffner, Augusto Elias Purpose or Case Report: Stroke is a major cause of morbidity and mortality in children.
Long-term neurologic deficits occur in 50% to 85% of infants and children after arterial ischemic stroke.
Limited awareness regarding pediatric stroke among physicians and in general community is a major concern.
Imaging plays crucial role in the diagnosis of pediatric stroke.
The goal of this presentation is to provide awareness to the reader about the role of imaging in childhood stroke and its myriad causes in children.
We will provide a case based approach to imaging diagnosis of acute pediatric stroke based on three categories: (1) arterial ischemic stroke, (2) cerebral venous thrombosis, and (3) hemorrhagic.
Arterial ischemic stroke (AIS) is classified according to the Pediatric Stroke Classification (PSC).
PSC includes eight subtypes of AIS: (1) sickle cell disease, (2) cardioembolic disease, (3) Moyamoya syndrome, (4) cervical arterial dissection, (5) stenoocclusive cerebral arteriopathy, (6) other determined etiology, (7) multiple probable etiologies, and (8) undetermined etiology.
We will describe the role of computed tomography (CT) and magnetic resonance imaging including angiography (MRI/MRA) in identifying these causes in relation to available clinical data.
The etiologies of cerebral venous thrombosis related infarction would be discussed from an imaging perspective with a case-based approach with emphasis on MRV and SWI techniques.
Finally, we will focus on hemorrhagic causes of childhood stroke such as vascular malformation, aneurysm, neurocutaneous disorders, coagulopathy, and a variety of other causes from an imaging standpoint.
Perfusion imaging in pediatric stroke demonstrates flow within the brain and can detect areas that are at risk of ischemia; however, further studies in the pediatric population need to be validated for the role of this technique in pediatric stroke.
Results: The most important factors in the diagnosis of childhood stroke are causal investigation, appropriate laboratory tests, and imaging studies.
Imaging is frequently the first step in the evaluation of an acutely ill child.
Conclusions: Pediatric stroke is a debilitating disease that requires urgent multidisciplinary approach for diagnosis and treatment.
In cases of both ischemic and hemorrhagic origin, the radiological approach to be obtained in emergency setting leads to the initial screening and the first therapeutic possibility.
Methods & Materials: This exhibit will illustrate the characteristic imaging findings of vascular anomalies in the head and neck region.
Vascular anomalies are divided into vascular tumors and vascular malformations which include slow flow malformations (capillary malformations, venous malformations, lymphatic malformations and their combinations) and high flow malformations (arteriovenous fistula and arteriovenous malformations).
Complex malformations are also seen in several syndromes including Klippel-Trenaunay Syndrome, PHACE syndrome, etc.
Cases from author's database will be used for illustration.
Results: A review of clinical manifestations, characteristic imaging findings and interventional treatment strategies in cases of head and neck vascular anomalies will be presented with pre and post treatment imaging features.
Ultrasonography and MRI are the mainstay in diagnosis of these malformations.
CT scan and catheter angiography may occasionally be needed for diagnosis and treatment planning.
Various imaging findings and main treatment options will be listed.
Conclusions: Head and neck vascular malformations are common in pediatric population.
Understanding the characteristic imaging findings and clinical presentation is essential in evaluating the vascular malformations.
Interventional procedures are generally the preferred treatment modality, either alone or in association with surgery in majority of these cases.
Isolated Cortical Diffusion Restriction in Pediatric Brain MRI Ihsan Mamoun, MD, Cleveland Clinic, ihsanmamoun@ yahoo.com; Sarah Stock, S. Pinar Karakas, Unni Udayasankar, Janet R. Reid Purpose or Case Report: Diffusion-weighted imaging continues to emerge as a powerful neuroimaging tool.
Isolated cortical restricted diffusion is a particularly striking pattern with specific differential in the pediatric population.
We aim to review this specific imaging pattern supplemented by case examples and key physiologic and imaging concepts.
Methods & Materials: Review the concept of diffusion restriction A) Pathophysiology B) Specific imaging appearances Pictorial review of pediatric conditions that lead to cortical restricted diffusion: A) Post ictal change B) Infection-i.
Meningoencephaliitis ii.
Herpes C) Hypoxic ischemic injury D) Infarct: venous and arterial E) Posterior reversible leukoencephalopathy F) Mitrochondrial Cytopathy G) Metabolic: hypoglycemia.
Discuss certain artifacts.
Summary table and differential clues Conclusions: The pattern of isolated cortical restricted diffusion has specific differential diagnosis in the pediatric population.
The radiologist should be aware of this as use of DWI continues to grow.
This exhibit with familiarize the reader with common conditions that specifically affect the cortex and produces true restricted diffusion.
Methods & Materials: High resolution CT scan and MRI are mainstay of diagnosis and assessment in patients with sensorineural hearing loss.
In this exhibit we will present a pictorial review of CT scan and MRI images of various causes of sensorineural hearing loss (SNHL) that are seen on imaging.
Reviewing the embryologic basis of these anomalies would enable better understanding of this complex subject.
Results: The new system classifies these malformations according to descending order of severity into complete labyrinthine aplasia, cochlear aplasia, common cavity, cystic cochleovestibular malformation or incomplete partition-I (IP-I), cochleovestibular hypoplasia, and incomplete partition-II (IP-II).
There is a lot of confusion in literature pertaining to Mondini deformity.
The new classification divides incomplete partition into IP-I representing cystic cochleovestibular malformation and IP-II representing the classic Mondini deformity with three components (cystic cochlear apex, dilated vestibule, and large vestibular aqeduct).
Recently a subclassification of IP-I and IP-II has been proposed (subdividing into typical and atypical subtypes)[2].
This will be discussed briefly.
Isolated large vestibular aqueduct without associated cochlear abnormalities will also be discussed.
We will discuss the relevant embryology with correlations of malformations to the timing of embryologic insult.
Conclusions: The new classification system provides precision in description of inner ear malformation.
This also helps in providing a uniform scale for comparison of effectiveness of cochlear implant for different malformations.
Purpose or Case Report: Congenital cranial nerve anomalies often present as sensory and/or motor deficits of unknown etiology in the pediatric age group.
The early recognition of a definitive cranial nerve abnormality using high-resolution imaging can focus further clinical investigation and shorten the time to diagnosis.
Methods & Materials: To promote appropriate recognition of cranial nerve anomalies, we present the imaging findings of the most commonly affected cranial nerves and provide correlation with clinical presentation.
All studies were performed on a 1.5 T magnet with dedicated high resolution imaging of cranial nerve exit zones.
Results: Ours is a tertiary care pediatric hospital with an extensive neuroimaging database.
We intend to review all known cases of cranial nerve anomalies from the prior 5 years and present interesting and representative images including optic nerve hypoplasia as part of septo-optic dysplasia, Kallman syndrome, Duane retraction syndrome, and Mobius syndrome.
Conclusions: Congenital cranial nerve anomalies present with varied symptomatology including anosmia, impaired vision, occulomotor deficits, and hearing loss.
Additionally, clinical manifestations of cranial nerve anomalies can be difficult to recognize in the pediatric age group.
Effective imaging and prompt diagnosis is crucial to initiate appropriate clinical management.
Purpose or Case Report: MR is the standard for evaluation of tumors or epilepsy.
PET-CT imaging is often performed to ascertain metabolic asymmetries related to epileptogenic regions or to better characterize the metabolic activity of tumors.
A baseline for normals with PET-CT FDG-18 and C11 Methionine does not exist.
Methods & Materials: Retrospective review was performed of the 110 pediatric patients who underwent PET-CT with C11 Methionine and FDG.
Representative studies were selected for patients imaged during infancy (<1 yr), early childhood (1-4), childhood (4-7), late childhood (7-12), teenage (13-18).
C11 methionine and FDG studies were analyzed for normal patterns of uptake and any trends identified across the stratified age groups.
Representative pictorial image galleries of the C11 methionine and FDG imaging patterns through development were created.
Results: The pattern of radiotracer uptake on C11 methionine differed from that of FDG.
The C11 uptake remained low level throughout development compared to FDG uptake, which was robust in much of the cortex.
The cortical FDG uptake within the frontal lobes progressively increased with age.
The C11 uptake within the brainstem and thalamus was equal to cortex throughout development.
The FDG uptake within the basal ganglia was equal to cortex while the brainstem and thalamic uptake was generally less than cortex.
Several anatomic structures showed robust C11 uptake not seen on FDG.
These included the lacrimal, submandibular and parotid glands.
Incidentally, the pituitary gland and hippocampus consistently showed C11 uptake equal to cortex contrary to their appearance on FDG.
Our institutional protocol regarding the performance of combination C11 methionine and FDG brain PET-CT studies is presented.
Conclusions: This study illustrated the normal appearance of brain PET-CT imaging performed with C11 methionine and FDG in a representative cohort of the pediatric patients through development.
Normal variance imaging patterns and developmental trends seen with each radiotracer was demonstrated.
The Pediatric Cerebellum: A Pictorial Review of Normal Anatomy using MRI and Diffusion Tensor Imaging Ibrahim S. Tuna, MD, Radiology, Children's Hospital of Wisconsin, dristuna@yahoo.com; Sumit Singh, Teresa C. Gross Kelly, Mohit Maheshwari, Tushar Chandra, Hervey D. Segall Purpose or Case Report: The aim of this educational exhibit is to illustrate normal anatomical and functional anatomy of the cerebellum in the pediatric patient.
The cerebellum receives sensory input from the brain and spinal cord and integrates this information to coordinate motor control.
In addition, the cerebellum also plays a role in some cognitive functions such as attention and language.
The first step toward understanding how cerebellar abnormalities can lead to neurological dysfunction, is to provide a solid understanding of the neuroanatomy and functional pathways of the cerebellum.
We will describe basic cerebellar embryology, the various cell types and gross anatomy using MR images as well as DTI fiber tractography.
Methods & Materials: This exhibit will describe the microstructure, gross anatomy and functional pathways of the cerebellum through illustrations, MR images, diffusion tensor imaging (DTI) and pathological correlation.
First embryology of the cerebellum will be described, followed by MRI depiction of the developmental anatomy of the cerebellum from infancy through adolescence.
Finally DTI tractography images will be used to delineate functional pathways to and from, as well as within, the cerebellum.
Pathological specimens will be photographed to further illustrate gross anatomy.
Results: Afferent white matter pathways travel mainly via the inferior and middle cerebellar peduncles.
The main efferent cerebellar white matter pathway is through the superior cerebellar peduncle.
Transverse fiber tracts are present in the vermis.
There are mainly two main systems of cerebellar white matter fibers which are easily visualized with DTI color mapping; however more anterior components of DTI tracts are intermixed with afferent white matter projections following the middle cerebellar peduncle.
Conclusions: Knowledge of the precise neuroanatomy and white matter tracts of cerebellum may elucidate our ability to comprehend the clinical manifestations of cerebellar diseases in children.
A solid understanding of normal cerebellar anatomy, development and functional fiber tracts in the pediatric patient can provide a baseline that may help predict the clinical outcome of various diseases or interventional procedures.
Gastroesophageal Reflux Scintigraphy: A Low Radiation Alternative to GERD Evaluation in Children Vikas Menghani, MD, Pediatric Radiology, Women's and Children's Hospital, drvikasmenghani@gmail.com; Feraas Jabi, Jan Najdzionek, Vaseem Iqbal Purpose or Case Report: Gastroesophageal reflux disease (GERD) is among the common causes for failure to thrive, recurrent cough and aspiration in children.
Early diagnosis of GERD is essential in avoiding long-term sequelae such as growth delay, chronic lung disease, esophageal stricture, and esophagitis.
Gastroesophageal reflux scintigraphy, a noninvasive imaging modality, has been applied for detection of GERD and gastric emptying in children over the past few decades.
The radiation burden is considerably small given that a very low dose of radioactivity via a short half-life radioisotope like Technetium-99 m tagged to oral sulfur colloid is administered to a patient.
This feature makes reflux scintigraphy especially attractive as the patient can be scanned for prolonged and delayed periods without increasing radiation dose permitting not only identification but also assessment of severity of GERD.
Characterizing GERD severity is essential in determining how aggressive the pediatrician should be with therapy.
Gastroesophageal reflux scintigraphy also allows a child to be fed their regular meal tagged to radiopharmaceutical without altering food taste.
Qualitative and quantitative parameters like gastrointestinal transit and gastric emptying time can be measured, respectively.
Scintigraphy is highly sensitive to low grade reflux making it very desirable for monitoring response to therapy.
While scintigraphy like all other imaging modalities,has limitations, it continues to be an excellent technique for GERD identification and characterization as well as in monitoring response to GERD therapy.
The Pediatric Kidney-A Review of Common and Uncommon Renal Anomalies Ruby Lukse, Staten Island University Hospital, drjosemorey@gmail.com; José Morey, Jeremy Neuman, Arnold Brenner, Oren Herman, Adam Bernheim Purpose or Case Report: Renal parenchymal imaging in nuclear medicine has long been performed with 99mTcdimercaptosuccinic acid (DMSA) due to its sufficient binding to the renal tubules to permit renal cortical imaging.
DMSA is of particular value when high-resolution images of the renal cortex are needed.
This poster will be a pictorial review of common and uncommon congenital anomalies evaluated on DMSA imaging, such as horseshoe kidney, pelvic kidney, sshaped kidney and crossed-fused ectopia.
The poster will also correlate planar imaging findingss with appropriate additional imaging including computed tomography (CT), magnetic resonance imaging (MRI), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (ALARA) principle set forth by the American College of Radiology (ACR).
Purpose or Case Report: In this poster we will review the differential diagnoses of congenital anomalies that give the appearance of hydronephrosis on renal imaging of the pediatric patient.
We will show a pictorial review of both common and uncommon congenital anomalies such as congenital megaureter, ureterocele, uretero-pelvic junction (UPJ) obstruction, uretero-vesicular junction (UVJ) obstruction and posterior urethral valves (PUV).
We will also review common mimickers of hydronephrosis such as multicystic dysplastic kidney (MCDK) and pseudo-obstruction secondary to bladder overdistention.
The poster will also correlate planar imaging finds with appropriate additional imaging including computed tomography (CT), magnetic resonance imaging (MRI), fluoroscopic imaging and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (ALARA) principle set forth by the American College of Radiology (ACR).
The Pediatric Bone Scan-A Review of Neoplastic Pathology Shrita Smith, Staten Island University Hospital, drjosemorey @gmail.com; José Morey, Jeremy Neuman, Arnold Brenner, Daniel Klein, Purpose or Case Report: Bone imaging continues to be the second greatest-volume of nuclear imaging procedure performed today, offering the advantage of total body examination, low cost, and high sensitivity.
The diagnostic utility, sensitivity, specificity and predictive value of 99 m-Tc bone imaging of malignant conditions have long been established.
In fact, more than 3,450,000 bone scans were performed in the United States in 2005.
In this poster we will review the current indications for planar bone imaging for the evaluation of malignant and benign neoplasms in the pediatric population, such as osteoid osteoma, Langerhan cell histiocytosis (LCH), osteoblastoma, Ewing's sarcoma, lymphoma, osteosarcoma and osseous/hepatic metastatic disease from neuroblastoma.
The poster will also correlate planar and single-photon emission computed tomography (SPECT) imaging findings with appropriate additional imaging including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (ALARA) principle set forth by the American College of Radiology (ACR).
The Many Faces of Duplex Kidneys on DMSA Scans-A Pictorial Essay Neha Kwatra, Children's National Medical Center, nskwatra@childrensnational.org; Massoud Majd Purpose or Case Report: Renal duplication is the most common malformation of the urinary tract and is often seen in children with urinary tract infections (UTI).
The purpose of this study is to learn to recognize duplex kidneys on Dimercaptosuccinic acid (DMSA) scintigraphy, review their entire spectrum of findings and correlate with other imaging modalities.
Methods & Materials: DMSA scintigraphy is routinely performed in the nuclear medicine department with a single-head gamma camera (Siemens e.cam, Schaumberg, Illinois).
About 1.5 h after injection of DMSA, posterior and posterior oblique images are obtained using parallel and pin hole collimators.
Differential renal function is also calculated.
DMSA scan reports containing the words "duplex" or "duplicated" from 2006-2011 were populated using a radiology search engine (Montage Health care Solutions Inc.).
The images were then reviewed in PACS and representative examples were selected for the poster.
The scans were evaluated for renal position, size, contour, any evidence of duplication and parenchymal damage.
Results: Patterns of duplication included non complicated duplex kidney recognized by asymmetric renal size and a prominent cortical bar separating the two moieties, complicated duplex systems with hydronephrosis, scarring or pyelonephritis of one or both moieties.
A small nonfunctioning upper moiety was sometimes evidenced by just an indentation along the superomedial aspect of the larger lower moiety.
Cases with bilateral duplex kidneys were also seen.
Illustrative examples of each will be provided.
Correlating findings on other imaging modalities will also be included.
Conclusions: Establishing the diagnosis of duplex kidney on a DMSA scan requires a careful systematic review of the images.
The findings can be subtle and it is important for the radiologist to recognize them.
Correlation with other modalities such as ultrasound or voiding cystogram can be complementary.
The assessment of parenchymal function of the upper and lower moieties separately on DMSA scintigraphy can be of immense value in patient management and in choosing surgical options.
Poster #: EDU-094 18 F-FDG PET/CT Imaging of Pediatric Brain Tumors, Neurofibromatosis 1(NF1) and Non-Lymphomatous Head and Neck Tumors.
Lisa States, MD, Radiology, CHOP, states@email.chop.edu; Purpose or Case Report: This educational poster will review the current literature and summarize the value of 18 F-FDG PET/CT in standard clinical practice in the evaluation of pediatric brain tumors, NF1 plexiform neurofibromas and malignant peripheral nerve sheath tumors, and nonlymphomatous head and neck tumors.
Normal variants and pitfalls will be reviewed.
Comparison with other PET tracers will be briefly discussed.
Case examples will be used to illustrate the value of 18 F-FDG PET/CT in grading, staging, assessment of therapeutic response and detection of residual or recurrent disease in various pathologic entities.
Results: Examples of cases will include: benign brain tumor, residual brain tumor in the post-operative bed, brain metastasis, malignant peripheral nerve sheath tumor in NF1, head and neck rhabdomyosarcoma, mandibular osteosarcoma, and infection.
Conclusions: An understanding of the value of PET molecular imaging is essential to the success of the next phase of hybrid imaging with PET/MRI which has the potential to play an important role in the development of new diagnostic and therapeutic approaches for the treatment of pediatric brain tumors, NF1, and pediatric head and neck tumors.
Disclosure: Dr. States has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
The Pediatric Bone Scan-A Review of Non-Malignant Pathology José Morey, Staten Island University Hospital, drjosemorey@ gmail.com; Jeremy Neuman, Arnold Brenner, Vinh Phan, Cheryl Lin Purpose or Case Report: Bone imaging continues to be the second most performed nuclear imaging procedure, offering the advantage of total body examination, low cost, and high sensitivity.
The diagnostic utility, sensitivity, specificity and predictive value of 99 m-Tc bone imaging of benign conditions have long been established.
In fact, more than 3,450,000 bone scans were performed in the United States in 2005.
In this poster we will review the current indications for planar bone imaging for the evaluation of non-malignant diseases in the pediatric population, such as Acute Osteomyelitis secondary to Salmonella Enterobacteriaceae and tubercle bacillus (TB), chronic osteomyelitis, reflex sympathetic dystrophy, spondylolysis, bone infarcts in the setting of sickle cell disease, fractures (occult/stress), ankylosing spondylitis, dermatomyositis and non-accidental trauma.
The poster will also correlate planar and single-photon emission computed tomography (SPECT) imaging findings with appropriate additional imaging including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and plain film radiographs when clinically warranted and in keeping with the as low as reasonably achievable (ALARA) principle set forth by the American College of Radiology (ACR).
Purpose or Case Report: We review the radiologic features of pathologic conditions linked to diesel exposure.
The hydraulic fracturing ("fracking") technique is increasingly used in many areas of the country to extract natural gas from rock formations.
Diesel fuel, or fluids containing diesel, are one component of fracking fluid and create a potential for ground water contamination and risk to air quality.
The toxic effects of diesel exhaust are described in the literature, and include asthma, hydrocarbon pneumonitis, and leukemia.
There are no scientific data currently available on the effects of chronic diesel ingestion.
Methods & Materials: Multi-modality examples of pathology were obtained from a radiology database at a tertiary care pediatric hospital.
The specific cases displayed are not known to have diesel exposure, but are intended to serve as representative examples of the type of pathology that may be encountered in the setting of chronic diesel exposure.
Results: Imaging findings of asthma include hyperexpansion, atelectasis, peribronchial thickening, and air-trapping.
Hydrocarbon pneumonitis may demonstrate low attenuation consolidation and subsequent pneumoatocoeles with CT. Leukemia may present on plain radiographs with lucent metaphyseal bands and with marrow infiltration on MRI.
Conclusions: In conjunction with other symptoms not necessarily evaluated in the radiology department, including rhinitis, laryngitis, acute coronary syndrome, and dementia, the radiologist may suggest the diagnosis of diesel toxicity, particularly in populations that may be at high risk of exposure.
Pediatric Radiology in the Philadelphia Region: A Historical Review* Richard Markowitz, MD, Children's Hospital of Philadelphia, markowitz@email.chop.edu Purpose or Case Report: The specialty of pediatric radiology in the Philadelphia region has grown and evolved over the past eight decades originating from early "visiting" radiologists to Drs.
Hope and Kirkpatrick, the "giants" of the 1950s and '60s, to over fifty practicing pediatric radiologists today.
Clinical excellence, commitment to teaching, and advancement of knowledge through research remain the goals and ideals, much as they were many years ago.
Philadelphia has been a fertile home and environment for this evolution, mostly because of outstanding leaders and role models who have trained and influenced generations of pediatric radiologists.
Developments and leadership at the Children's Hospital of Philadelphia, St. Christopher's Hospital for Children, and A.I.
duPont Institute are highlighted.
The purpose of this poster is to tell the story of the growth and development of pediatric radiology in this area and to explore the intellectual origins, professional "genealogy," and legacies left by those who created and those who have carried on this tradition.
*Note: This material is based on a previously published article: Pediatric Radiology (2009) 39:969-981 and "Addendum" (Pediatric Radiology 2010: 1454-1455), but never presented at SPR.
Superficial Lumps and Bumps Henrietta K. Rosenberg, MD, Radiology, The Mt.
Sinai Medical School, Henrietta.Rosenberg@mountsinai.org; Diane Belvin, Neil Lester Purpose or Case Report: Superficial soft tissue masses in the pediatric age range can be quite challenging to the pediatrician and the imager.
The purpose of this presentation is to demonstrate the efficacy of duplex/color Doppler ultrasound for the diagnosis and follow up of a large gamut of superficial lumps and bumps.
Methods & Materials: We reviewed our experience during the past 6 years using ultrasound to evaluate superficial soft tissue masses that had been encountered in many parts of the body, from the skull to the soles of the feet, in a large group of patients ranging in age from newborn to 21 years.
All sonograms were performed after obtaining pertinent clinical information as well information regarding the clinical characteristics of each of the masses, e.g.
location, consistency (firm [solid], compressible [cystic]), fixed or easily movable, smooth or irregular surface, tenderness.
The masses were palpated by the imaging team and duplex/color Doppler ultrasound was performed.
Comparison sonographic views of the opposite side were obtained as needed.
Clinical followup and surgical/pathological correlation was obtained in most of the patients.
Results: Most of the masses were benign and included a wide variety of etiologies.
Most often, US was sufficient for assessment of soft tissue masses if the entire mass was included in the field of view.
If the lesion was too large for the field of view or malignancy was suspected, CT/MRI were required preoperatively.
Nuclear medicine studies are reserved for midline masses likely due to ectopic thyroid and PET was used for more complete evaluation of a lesion that was likely malignant.
Conclusions: Duplex/color Doppler ultrasound (US) is the modality of choice for evaluation of superficial lumps and bumps!
This modality allows for rapid acquisition of information without the use of ionizing radiation, intravenous contrast material, or sedation/anesthesia.
Reliable information can be rapidly acquired regarding the size, shape, borders, location, internal consistency, vascularity, vascular encasement/displacement.
Correlation of the ultrasound and clinical findings helps narrow differential diagnosis.
Sonography helps to determine what is the next best step: watchful waiting (clinical observation, follow-up US), surgical resection, or US guided interventional procedure.
Present Day Imaging of Down Syndrome Rupa Radhakrishnan, MD, Radiology, University of Cincinnati College of Medicine, radhakrp@ucmail.uc.edu; Alexander J. Towbin Purpose or Case Report: Down syndrome is a common genetic condition characterized by unique physical traits and multisystem anomalies.
The purpose of this exhibit is to portray the imaging findings of Down syndrome and discuss with illustrative examples, the use of imaging in multidisciplinary management.
Methods & Materials: Published literature was reviewed to identify the multisystem imaging findings in Down syndrome.
The electronic medical record system was then searched to find illustrative case examples from our institution.
Results: In patients with Down syndrome, abnormalities can be found in the musculoskeletal, cardiovascular, respiratory, gastrointestinal, and central nervous systems.
Abnormalities can range from emergent, life threatening conditions such as malrotation with midgut volvulus to chronic conditions such as scoliosis.
Examples of abnormalities from each organ system and the modalities used for diagnosis and management are described.
Cardiovascular system: Echocardiogram and cardiac MRI and CT are useful in evaluating congenital heart disease associated with Down syndrome.
Respiratory system: Micrognathia with macroglossia and hypotonia predisposes patients to sleep apnea which can be evaluated with dynamic MRI.
Chest CT demonstrates subpleural cysts which are characteristic of this syndrome.
Gastrointestinal system: Fluoroscopy and/or radiographs are the mainstay in diagnosing many gastrointestinal disorders including duodenal atresia, malrotation, annular pancreas, imperforate anus, and Hirschsprung disease.
Central nervous system: Choroid plexus cysts may be identified on prenatal ultrasound in a fetus with Down syndrome.
Imaging is used in the evaluation of epilepsy, hearing loss and Alzheimer disease that is more common in these individuals.
Musculoskeletal system: Multiple skeletal anomalies can be present in patients with Down syndrome.
Radiographs are often used as the method of identifying and, if needed, following the anomalies.
Prenatal imaging: Increased nuchal translucency is the earliest imaging finding.
Other features of Down syndrome can be identified on prenatal ultrasound or MRI.
Prenatal imaging is helpful in determining the prognosis of the fetus and in guiding management.
Conclusions: Modern day multidisciplinary management has improved quality of life and survival in individuals with Down syndrome.
Imaging plays a critical role in guiding management in these individuals.
Imaging the Spectrum of Lymphatic Malformations in the Pediatric Patient Andrew Schapiro, MD, Radiology, University of Wisconsin, ASchapiro@uwhealth.org; Kara Gill, Bradley Maxfield Purpose or Case Report: Lymphatic malformations (LM) occur as a result of abnormal development of the lymphatic system during embryogenesis.
As 90% of LM present by 2 years of age, these lesions represent an important pediatric entity.
LM can often be suspected clinically in an infant with the classic presentation of an asymptomatic, soft mass in the head, neck, or axilla.
However, myriad presentations are possible as LM occur in numerous other anatomic locations, can be multiple, and can be a component of mixed vascular malformations.
In addition, the true extent of LM is often not apparent clinically.
Given these considerations and the implications for proper management, imaging plays an important role in the assessment of LM.
The purpose of this exhibit is to review the spectrum of radiographic, CT, sonographic, and MR imaging findings of a variety of LM presentations.
Methods & Materials: Cases of lymphatic malformation in pediatric patients identified at a single institution over the past ten years with available imaging were reviewed utilizing PACS.
Results: Images of LM involving the head and neck, chest, abdomen, retroperitoneum, extremities, and skeletal system were identified.
In addition, cases of lymphangiomatosis and mixed venolymphatic malformation were identified.
Various imaging modalities including radiography, CT, sonography, and MR were represented.
Conclusions: Adequate knowledge of the imaging characteristics of LM across multiple modalities enables proper diagnosis, assessment of disease extent, and guidance of appropriate therapy in pediatric patients.
Results: CT and MR imaging findings in nine cases will be presented.
They include 1) Congenital absence of the inferior vena cava with thrombosis of the external iliac vein secondary to venous stasis 2) Pyelophlebitis complicating ruptured appendicitis 3) Left iliac vein thrombosis in a patient with May-Thurner syndrome 4) Splenic vein thrombosis complicating pancreatitis 5) Splenic vein thrombosis following splenectomy 6) Renal vein thrombosis in an infant of a diabetic mother 7) Adrenal vein thrombosis as the presenting sign of antiphospholipid syndrome 8) Budd-Chiari syndrome associated with underlying myeloproliferative disease 9) Iliac vein thrombosis as a manifestation of Behcet's syndrome (Hughes-Stovin syndrome, a variant of Behcet's syndrome, which presents with systemic venous thrombosis and pulmonary artery aneurysms will also be discussed).
Conclusions: Thrombosis of large abdominal and pelvic veins in children and adolescents is uncommon.
Certain conditions, both congenital and acquired, predispose to the development of venous thrombosis.
CT/MR imaging defines the extent of thrombosis, and demonstrates additional findings that may elucidate the nature of the underlying condition leading to clot formation.
Purpose or Case Report: Because abnormal gait in a young child has a wide range of causes, imaging plays a critical role in establishing the definitive diagnosis.
The purpose of this exhibit is to review the clinical clues (age, duration, laboratory markers) and imaging findings of the causes of abnormal gait in a toddler and to assess the strengths and limitations of radiographs, ultrasound, magnetic resonance imaging (MRI), and computed tomography (CT).
Methods & Materials: Cases, from a single institution experience with various causes for abnormal gait in a toddler, are reviewed and categorized into congenital, traumatic, inflammatory, neoplastic, or neuromuscular etiologies.
Results: There are various causes of abnormal gait in a toddler.
The congenital causes include spinal dysraphism, proximal and distal skeletal deformities and dysplasias.
The traumatic causes include non-accidental trauma, toddler's fracture, foreign body, and soft tissue injuries.
The inflammatory causes include juvenile idiopathic arthritis, transient synovitis, and infection, including osteomyelitis, septic arthritis, discitis, cellulitis, and abscess.
The neoplastic causes include various neurogenic, bone, and soft tissue tumors.
The neuromuscular causes include cerebral palsy and spinal bifida.
The combination of clinical presentation, supporting laboratory findings, and classic imaging findings help to distinguish the possibilities and often allows confident diagnosis.
Conclusions: Knowledge of imaging findings and clinical factors can demystify the diagnosis of abnormal gait in a toddler.
Familiarity with the clinical presentation can ensure the performance of the appropriate diagnostic studies, timely diagnosis, and effective treatment.
Nonaccidental causes should never be overlooked.
Ultrasonography has become an important tool in the radiologist's armamentarium, augmenting radiography, MRI, and CT.
Approximately 20 different contrast agents for MRI and CT are now commercially available for use.
Although most of them are FDA approved in adults, information on usage and safety in children is not readily available.
The most important reason is lack of controlled studies in children, especially for the age of 0-2 years.
However, the lack of FDA approval has not limited the use of these promising agents in children.
In fact, there is widespread off-label use of these agents in most major pediatric hospitals in the country.
Based on a review of relevant literature in children, and based on a survey of radiology faculty at major pediatric hospitals, this poster will address the gap between approved use and reality in the setting of pediatrics.
Results: Using a tabular format, this poster will provide a list of MR and CT contrast agents that are available for clinical use, their relevant clinical properties (ionic or nonionic, viscosity, linear or macrocyclic, degree of relaxivity for MRI, iodine concentration for CT, cost, dosage, halftime, incidence of allergic reactions, nephrogenic systemic fibrosis and other adverse reactions), FDA approval status (for ages 0-30 days, 30 days-2 years, and 2-17 years), common pediatric applications, and contrast injection protocols for common applications.
Conclusions: To enlighten imaging personnel about usage and safety of contrast agents in children.
Disclosure: Dr. Krishnamurthy has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
A Pictorial Essay and Literature Review of the Spleen in Sickle Cell Disease David Hindson, MD, Boston Medical Center, david.
hindson@bmc.org; Heather Imsande, Philippa Sprinz, Ilse Castro-Aragon Purpose or Case Report: The morbidity and mortality of sickle cell disease (SCD) results from acute and chronic infarction events that affect almost every organ.
Repeated infarction has some of its greatest visual and physiologic impact within the spleen.
Continuous hemolysis, sequestration and vaso-occlusion within the spleen result in loss of splenic function early in life and frequently autosplenectomy thereafter.
By 2 years of age, approximately 90% of children with hemoglobin SS disease will have diminished splenic function, putting them at increased risk for infections.
Treatments for SCD have evolved over the last 20 years, and among others include penicillin prophylaxis and immunizations, Hydroxyurea and transfusion therapy (or hypertransfusion program).
Imaging findings are a reflection of the different treatments and their efficacy.
Methods & Materials: Our institution cares for a large group of patients with Sickle Cell Disease, from birth to adulthood.
This offers an unprecedented opportunity to document the imaging findings of the spleen with different treatment regimens, and over many years.
The splenic size and morphology can be followed, by ultrasound, in a very straightforward way.
We have compiled a pictorial essay of the various imaging characteristics of spleens from infants to adults.
We also performed a literature review to compare and supplement the findings of our images.
Results: There is a spectrum of imaging findings in the spleen of patients with SCD that changes from birth to childhood.
The findings range from the normal appearance of a spleen to a calcified spleen, and include regenerative nodules, fibrosis, altered parenchymal echotexture, increased echogenicity, and changes in size, including enlargement secondary to sequestration.
The ultrasound characteristics not only change with advancing age, but also appear to depend on whether or not the patient has received specific treatments, and at what age treatment was initiated.
Conclusions: The ultrasound appearance of the spleen in patients with SCD is variable.
Treatments such as blood transfusions and Hydroxyurea, patient compliance with therapy and type and severity of the disease are some of the factors that affect imaging characteristics.
Cystic Fibrosis: Not Just for Children Cindy Miller, MD, Radiology, Yale-New Haven Hospital, cindy.miller@yale.edu Purpose or Case Report: Cystic fibrosis has been recognized for hundreds of years with the first descriptions of it including such anecdotes as mothers licking the foreheads of their children and knowing that if it tasted salty, an early death could be predicted.
It was not until 1939 that the disease was first named by Dr. Dorothy Andersen, and for the following 50 years, treatment was largely supportive, and imaging was essentially done with plain films alone.
In 1989 with the elucidation of the CFTR gene, there was an explosion of knowledge which included the range of increased awareness and understanding of the suspected etiology, imaging findings and significance of the ductus bump.
The Contribution of 3D Imaging for Evaluation of the Pediatric Central Airways Jessica Kurian, MD, The Children's Hospital of Philadelphia, kurianj@email.chop.edu; Monica Epelman, David A. Mong Purpose or Case Report: Evaluation of the central airways in children has historically been accomplished by flexible bronchoscopy, an invasive technique associated with inherent risks and complications.
Multidetector CT (MDCT) with volume rendering offers a noninvasive alternative for airway evaluation.
In this educational exhibit, we will review imaging techniques and clinical applications of MDCT for the assessment of large airway maladies in children.
Methods & Materials: MDCT imaging in children with a variety of tracheobronchial disorders is reviewed.
For each entity, the characteristic clinical features are described, and key imaging features are illustrated.
Emphasis is placed on the contribution of 3D techniques for characterizing complex airway anomalies.
Dose reduction strategies are also highlighted.
Results: The entities reviewed in this exhibit include, but are not limited to, congenital anomalies of tracheobronchial branching, airway malformations associated with situs, and congenital or acquired airway compression and/or obstruction.
Conclusions: MDCT with volume visualization is a useful adjunct for evaluation of the pediatric central airways in a variety of pathologies.
As a noninvasive technique, it avoids sedation risks and spare patients from complications associated with conventional flexible bronchoscopy.
Low dose protocols should be used to minimize radiation exposure.
bronchopulmonary foregut malformation that result from abnormal budding of the primitive foregut.
Currently, many such anomalies are initially detected by prenatal ultrasound and are further delineated by fetal magnetic resonance imaging (MRI), while others may be incidentally detected on postnatal radiologic examinations or later in life in the setting recurrent pulmonary infection.
Imaging plays a very important role in the diagnosis and characterization of these lesions and assists surgical planning.
The purpose of our educational exhibit is to illustrate the common and uncommon radiologic appearances of CPAMs using various imaging modalities, including radiography, computed tomography, prenatal and postnatal ultrasound, and prenatal and postnatal MRI.
Methods & Materials: All pediatric and adult CPAM (including both sequestration and CCAM) patients were identified using electronic medical records.
Pertinent imaging reports (including radiography, prenatal and postnatal ultrasound, CT, and prenatal and postnatal MRI) were reviewed by a single author in order to identify relevant imaging findings.
Relevant images from these imaging examinations were de-identified and saved to a secure hard drive.
Medical records were accessed by a single researcher to obtain relevant demographic information as well as data regarding the patients' clinical presentations.
In cases of corrective surgery, operative and pathology reports were reviewed, if available, for correlation with the imaging findings.
Results: Cases of pediatric and adult CPAM were identified and presented in a variety of clinical contexts.
Their appearances were reviewed through multiple imaging modalities.
Conclusions: Congenital pulmonary airway malformations are varied in their clinical presentation and imaging appearance.
The purpose of this pictorial essay is to enhance understanding of their diagnosis and to use a multidisciplinary approach in order to highlight imaging aspects that may alter clinical management.
Disclosure: Dr. Horst has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
The Imaging Evaluation of Cystic Lung Disease in Children: An Evidence-Based Approach Jordan Caplan, MD, Pediatric Radiology, Lucile Packard Children's Hospital, Stanford University, caplan@stanford.
edu; Beverley Newman Purpose or Case Report: The goal of the poster is to provide a framework for use when confronted with cystic lung disease in a child.
Methods & Materials: The differential diagnosis for the types and causes of cystic lung disease in children will be presented using an evidence-based, age appropriate approach.
Categories of disease discussed and illustrated with case examples will include: A. Congenital cystic bronchopulmonary malformations B.
Infectious cysts C. Autoimmune/inflammatory/vasculitic disease with cavitating lesions D. Neoplastic conditions E. Collagen/soft tissue abnormalities F. Mimics of cystic lung disease Results: The pathophysiology, imaging appearance, and demographics of the above entities will be reviewed with attention to relevant recent literature.
Important educational points include the differentiation of bronchopulmonary malformations from neoplasm, notably pleuropulmonary blastoma (PPB), the relationship between lung cysts and PPB, and the management and surveillance of lung cysts in children.
Conclusions: An evidence-based approach to the broad spectrum of causes of cystic lung disease in children is a useful starting point in forming a concise and pertinent differential diagnosis.
An understanding of the pathophysiology, imaging appearance, and demographics of these entities is essential in guiding patient management.
Pediatric Interstitial Lung Disease (ILD): A Pictorial Review with Radiologic and Pathologic Correlation Hollie West, MD, Diagnostic Radiology, Vanderbilt University, hollie.c.west@Vanderbilt.edu; Melissa A. Hilmes, Sudha P. Singh, Jennifer Soares, Lisa Young Purpose or Case Report: While adult interstitial lung disease is a well-described and fairly well understood group of disease processes, pediatric interstitial lung disease (ILD) remains a subject of uncertainty and misunderstanding for many clinicians and radiologists.
Confusion surrounding the phenomenon of pediatric ILD stems not only from the rarity of the disease, but also from the extensive list of disease entities that can produce ILD, the existence of certain patterns that are restricted to infants and children and the fact that patterns of ILD manifest differently in a child's developing lung than in an already developed adult lung.
Imaging plays an important role in diagnostic work-up of this disease and can guide lung biopsy in specific patient populations.
Methods & Materials: The IRB approved retrospective study will show patients at our institution over a 10 year period diagnosed with various types of ILD, including pulmonary insterstitial glycogenolysis (PIG), diffuse neuroendocrine cell hyperplasia (NEHI), surfactant deficiency diseases, and lung diseases associated with other systemic processes such as Downs syndrome and inflammatory bowel disease.
We will include patients with biopsy proven ILD and will provide examples of the major ILDs, including clinical, radiologic and pathologic correlation.
Our pictorial review will describe the radiologic patterns associated with the different forms of ILD, emphasizing what the radiologist needs to know and how to be helpful to a multidisciplinary team in the diagnosis and treatment of these diseases.
Results: The study will report the frequency of ILD at our institution, including a breakdown of the various subtypes of ILD.
We will show examples of the subtypes with correlative chest radiography, computed tomography, and pathology.
We plan to highlight specific differentiating factors between the different diseases and demonstrate how a radiologist can be helpful in collborating with clinicians in diagnosing and treating these diseases.
Conclusions: Pediatric ILD can be a confusing topic for radiologists.
Increasing knowledge and awareness of these diseases, their clinincal presentation, work up, and treatment is important for pediatric radiologists who work as part of of a multidiciplinary team.
Poster #: SCI-001 CT Radiation Dose Delivered by Community Hospitals and Imaging Centers Stephen Little, Children's Healthcare of Atlanta, stephen.
little@choa.org; Damien Grattan-Smith, Bonnie Johnson Purpose or Case Report: To evaluate and compare CT radiation dose for pediatric abdominal and cranial CT examinations performed by community hospitals and imaging centers.
Methods & Materials: 148 consecutive CT examinations (49 cranial, 99 abdominal) from 41 community hospitals and imaging centers were reviewed following transfer of care.
The examinations were performed between January and July 2011.
433 consecutive CT examinations (241 cranial, 192 abdominal) performed at our own institution were also reviewed.
CTDIVOL and DLP were obtained from the dose report for each examination (32 cm-phantom for abdominal exams, 16 cm-phantom for cranial exams).
Patient age and weight were obtained from the medical record.
Results: Average CTDIVOL for abdominal CT performed by local community hospitals and imaging centers was 8.7 mGy, while average CTDIVOL was 4.3 mGy for abdominal CT performed at CHOA.
There was a wide variation in CT radiation dose delivered.
While some sites delivered a CT radiation dose comparable to our own, others delivered a substantially greater dose.
In fact, 23% of pediatric abdominal CT exams performed by local community hospitals and imaging centers exceeded the Notification Value recommended by the AAPM (10 mGy using the 32cm phantom).
Low kVp technique for imaging small children was infrequent.
Multi-phase examinations were more often performed, resulting in additional elevation in CT radiation dose when DLP is considered.
Average CTDIVOL delivered by local community hospitals and imaging centers for cranial CT was 44 mGy compared to a CTDIVOL of 30 mGy for cranial CT performed at CHOA.
8% of pediatric cranial CT exams performed by local community hospitals and imaging centers exceeded the Notification Value recommended by the AAPM (60 mGy for 2-5 years, 80 mGy for >5 years).
Conclusions: Despite ongoing efforts at education, there is wide variation in CT radiation dose delivered for pediatric abdominal and cranial CT examinations performed by local community hospitals and imaging centers.
Appropriate use of dose check software on newer scanners may help reduce the number of children subjected to excessive CT radiation dose.
Ultimately, each site performing pediatric CT must take responsibility for minimizing radiation dose while producing diagnostic quality exams.
The Impact of Adaptive Statistical Iterative Reconstruction on CT Image Quality Parameters -A Phantom Study Karen Thomas, MD, Radiology, Hospital for Sick Children, karen.thomas@sickkids.ca; Nancy Ford, Angjelina Protik, Paul Babyn Purpose or Case Report: To quantify the effect of Adaptive Statistical Iterative Reconstruction (ASIR) on CT image quality parameters.
Methods & Materials: Phantom (Catphan 600) studies were performed on a GE HD750 64-slice scanner to investigate the impact of a) 50% ASIR compared to routine filtered back projection using variable kVp (80-140) and mAs (5-200), and b) incremental ASIR % (0, 30, 50, 70, 100%), scanning at 75mAs and variable kVp (80-120).
Pitch, acquisition FOV and detector width were kept constant.
Image noise, spatial and contrast resolution, contrast noise ratio (CNR) and Wiener spectrum analysis were performed on 0.625 mm Ax, 5 mm Ax MPR and 2 mm Cor MPR series.
Results: 50% ASIR resulted in a mean decrease in noise of 30% (0.625 mm Ax), 26% (Ax MPR) and 28% (Cor MPR) and improvement in CNR of 38-49%.
Incremental advantage was seen with stepwise increase in ASIR %.
However, application of ASIR was associated with a small reduction in spatial resolution (2-8% at 50% ASIR).
Low contrast detectability (LCD) improved except at the smallest target lesion size.
Image quality effects at very low mAs and at high ASIR % will be presented.
Conclusions: Image noise reduction and improvements in CNR and LCD with ASIR hold considerable potential for dose reduction in pediatric CT.
This study provides quantitative data that may be used to design ASIR-enhanced protocols with consideration of diagnostic task, balancing image quality benefits and potential pitfalls.
Pictorial Essay on Cardiac MR for Congenital Heart Disease on 3 T MR Scanner with RF Multi-Transmit Technology (Tx) Taylor Chung, MD, Diagnostic Imaging, Children's Hospital & Research Center Oakland, taylorchung12@gmail.com Purpose or Case Report: This is a pictorial essay (e-poster) to show artifacts on cine SSFP images pre-Tx and post-Tx upgrade on congenital heart disease cardiac MR; to illustrate methods prior to Tx-upgrade to minimize artifacts.
Disclosure: Dr. Chung has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Revisiting the Relationship Between Anthropometric Parameters and Left Ventricular Mass Abdullahi Adamu, MD, PhD, Ahmadu Bello University, scorpion68kd@yahoo.com Purpose or Case Report: The purpose of this study was to find the correlation between anthropometric parameters and left ventricular mass in normal adolescents and young adults.
Methods & Materials: 147 healthy individuals in the age range 17 to 23 years (73 males and 74 females) were included in this study.
Anthropometry was performed with standard anthropometry kit and measurements of height, weight, Body Surface Area (BSA), upper arm circumference and upper hip circumference were taken.
Echocardiography was performed and the American Society of Echocardiography (ASE)-recommended method was employed for calculation of left ventricular mass (LVM).
Statistical analysis was performed using Statistica 6.0 (Stat Soft, USA).
Results: The mean value of LVM for all our subjects was found to be 124.53±2.79 g. There was significant correlation between LVM and height (r00.52, p<0.0001), weight (r00.63, p<0.00001) and BSA (r00.64, p<0.00001).
Correlation with upper arm circumference was moderate (r0 0.46, p<0.0001), while it was found to be weak with upper hip circumference (r00.23, p<0.01).
diagnostic.
Both field strengths can be used successfully for cardiac and vascular imaging.
The decision as to which to use is weighted by local availability and the relative requirement for detailed vascular vs intra-cardiac imaging.
Disclosure: Dr. Nguyen has indicated that she will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Poster #: SCI-006 Color Coded 3D Cardiac CTA of Congenital Heart Disease: A Five Year Experience Nhi Huynh, MD, Radiology, St. Joseph Hospital and Medical Center, e.nhihuynh@gmail.com; Randy Richardson Purpose or Case Report: Post-processing of cardiac computed tomography angiograms can be performed on a commercially available workstation to create color coded 3D volume rendered images of the segmented heart and great vessel anatomy in patients with congenital heart disease.
These studies optimally demonstrate complex anatomy, streamlining communication between members of the healthcare team and providing a tool for communicating complex anatomy and treatment options with families.
These studies have been ordered with more frequency over the past five years.
We retrospectively reviewed the types of congenital heart disease demonstrated by cardiac 3D CTA over the past five years at a congenital heart center.
Methods & Materials: Color coded cardiac CTA postprocessing was performed from ECG gated prospective and retrospective CTA data on a commercially available workstation for 333/395 patients over the past three years.
The anatomy was initially segmented and colored into individual parts of the anatomy of the heart and great vessels as follows RV 0 purple, LV 0 light red, aorta 0 red, pulmonary arteries 0 blue, systemic veins and right atrium 0 aqua, pulmonary veins and left atrium 0 pink, PDA or collaterals 0 green, airway 0 yellow, coronary arteries 0 neutral.
The anatomy was then reassembled and images obtained every 3°in a 360°rotation for display.
Results: 3D color coded CTA images were used in the treatment and care of congenital heart patients for the following types of congenital heart diseases: 124 cases of complex anatomy (TGA, truncus arteriosus, HLHS, tricuspid atresia, TOF…), 67 coronary artery anomalies, 68 cases of pulmonary atresia or stenosis, 44 cases of systemic and venous anomalies, 40 cases of coarctation or interruption of the aortic arch, and 56 tracheobronchial tree anomalies.
Conclusions: Color coded cardiac CTA post-processing is an effective and viable method for demonstrating anatomy in complex congenital heart patients.
It is an excellent tool for demonstrating anatomy which is difficult to see by echocardiography such as: coronary artery anomalies, pulmonic atresia, aortic arch coarctation or interruption, and tracheobronchial anomalies and/or stenosis.
Neuroimaging in the Evaluation of HIE in Term Neonates Post Hypothermia Therapy Julio M. Araque, MD, Radiology, Medical College of Georgia, jaraque@georgiahealth.edu; Jatinder Bhatia, Leann VanLandingham Purpose or Case Report: To illustrate and review the potential utility of brain MRI, CT and ultrasound in hypoxic ischemic encephalopathy in newborns treated with hypothermia.
Neuroimaging studies including brain ultrasound, CT and MRI of fifteen term newborns treated in our institution with therapeutic hypothermia, since April 2010 were evaluated retrospectively.
More relevant lesions are depicted and the diagnostic and prognostic value of the findings is discussed and compared with a review of the literature.
Results: Recent studies showed that patients treated with cooling had a more favorable prognosis than was suggested by the clinical grade of encephalopathy compared with infants treated with standard care.
Our institutional protocol includes the performance of MRI, and Ultrasound.
CT is performed when is a clinical impossibility of perform MRI.
Brain ultrasound was performed in all the 15 patients.
MRI scans were obtained in 11 neonates.
CT was obtained in 3 patients.
All MRI studies included DWI.
The utility of DWI and ADC maps as an aid in diagnosis of non-ischemic lesions is becoming increasingly established.
MRI evidence of brain injury was visible on basal ganglia in 8 cases with negative ultrasound.
Abnormal signal intensity in the posterior limb of the internal capsule coexists with lesions in the basal ganglia and thalami have been associated with abnormal motor outcome.
The remaining 3 newborns did not develop significant MRI evidence of brain injury.
It has been suggested that the ability of MRI to predict subsequent neurological impairment is unaltered by therapeutic hypothermia.
Further research is needed for defining the relation between MRI findings and cooling.
It is possible that imaging findings might be delayed in cooled infants.
Conclusions: MRI offers the highest sensitivity in detecting anoxic injury of the neonatal brain.
MR biomarkers in combination with clinical markers may identify patients with adverse outcome with therapeutic implications.
Disclosure: Dr. Araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Purpose or Case Report: To correlate bowel wall diffusionweighted imaging (DWI) apparent diffusion coefficient (ADC) values with multiple MR enterography (MRE) and clinical findings in pediatric small bowel Crohn disease.
Methods & Materials: 54 pediatric Crohn disease patients with MRE exams containing diffusion-weighted imaging and demonstrating terminal ileitis were identified.
Minimum bowel wall ADC values were tested for correlation/association with other MRI findings and clinical parameters (including laboratory values).
Results: There is negative correlation between ADC value and degree of bowel wall thickening (R0(−)0.27; p00.048).
Lower ADC values were significantly associated with striated pattern of arterial phase postcontrast enhancement (p0 0.007), greater degree of arterial phase postcontrast enhancement (p00.006), and presence of stricture (p00.005).
ADC values were not associated with diseased bowel length, degree/pattern of delayed postcontrast enhancement, degree of mesenteric inflammation or fibrofatty proliferation, or clinical markers of inflammation.
Conclusions: Restricted diffusion in pediatric small bowel Crohn disease is associated with other MRI findings of that are suggestive of active disease, including degree of bowel wall thickening and degree and pattern of arterial phase postcontrast enhancement.
Our data also suggests that DWI may be useful when attempting to characterize small bowel strictures as either predominantly inflammatory or fibrotic, although further investigation is needed.
Quantification of Blood Flow Into and Out of the Liver with 4 d Phase Contrast MRI in the Pediatric Patient Binh Huynh, MD, Radiology, Stanford, bhuynh@stanford.
edu; Shreyas Vasanawala, Albert Hsiao Purpose or Case Report: The ability to probe blood flow dynamics in the liver may aid management of children with liver disease, including shunt fractions in portal hypertension and arterial flow fraction in diffuse liver disease.
The purpose of this study is to evaluate the ability to measure blood flow into and out of the liver with time resolved volumetric (4D) phase contrast MRI in the pediatric patient.
Methods & Materials: Nineteen consecutive patients were retrospectively identified who underwent 4D flow imaging through the level of the hepatic vessels on 1.5 T and 3 T magnets.
A software enabling 4D flow program was utilized to first assess for the feasibility of measurement of flow in the hepatic artery (HA), portal vein (PV), splenic vein (SPV), superior mesenteric vein (SMV), supra (SIVC) and infrahepatic (IIVC) inferior vena cava.
If measurable, calculations were performed to evaluate for internal consistency by comparing the sum of SMV and SPV flow to PV flow.
Calculations were then performed to compare hepatic inflow (PV+HA) to hepatic outflow (SIVC-IIVC) and for the percentage of PV and HA contribution to hepatic inflow.
Results: Of the nineteen patients, all of the above mentioned six vessels were visualized and measurable in two patients, both of which were imaged on the 1.5 T magnet.
In the remaining patients, flow measurements were limited by respiratory motion artifacts obscuring the smaller vessels, and severe eddy currents, particularly in patients imaged with the 3 T magnet.
The evaluation for internal consistency demonstrated an average of 1.2% (0.06% & -1.5%) difference between SMV+SPV and PV flow.
Hepatic inflow was found to closely match the measured hepatic outflow with an average difference of 11.1% (19.1% & 3.1%).
The portal vein was found to contribute 82.9% and 87.3% to hepatic inflow, while the hepatic artery contributed 17.1% and 12.7%.
Conclusions: Measurement of hepatic flow with phase contrast MRI is more challenging than assessment of thoracic flow.
When respiratory artifacts are minimal, vessels can be identified and measurements have internal consistency and good agreement between hepatic inflow and outflow at 1.5 T. Conversely, flow measurements were limited at 3 T by eddy currents.
Thus, ongoing efforts are aimed at mitigating respiratory motion artifacts at 1.5 T. Poster #: SCI-011 MRI Findings in Post-Fontan Hepatopathy Adina Alazraki, MD, Radiology, Emory University/Children's Healthcare of Atlanta, adina.alazraki@choa.org; Pinar Bulut, Kiery Braithwaite, Miriam Vos, Rene Romero, Nitika A. Gupta Purpose or Case Report: As advances in congenital heart disease continue to improve both mortality and quality of life, associated complications are becoming more prevalent.
Amongst patients who have had Fontan repair for hypoplastic left heart syndrome, tricuspic atresia, or other right heart dysfunction, it is well known that liver disease is a complication.
We describe the MRI findings in post-Fontan patients and propose MRI as a useful tool to the hepatologist's evaluation of these patients.
Methods & Materials: IRB approval was obtained for a retrospective review of 29 patients who underwent Fontan repair and were subsequently referred for hepatology evaluation between 2010-2011.
All but one patient was scanned on a SiemensTrioTrim 3 T magnet; one patient was scanned on a GE Twinspeed 1.5 T magnet with an equivalent protocol due to orthodontics.
A standardized departmental protocol was utilized.
MRI findings were correlated with age at surgery and years since surgery.
MR images were reviewed independently by 2 pediatric radiologists and compared with the dictated report in the patients record.
Results: 17 patients underwent MRI of the abdomen.
4 patients had MRI incompatible hardware and 8 patients were not scanned secondary to insurance denial.
Patients were divided into 4 groups based on elapsed time since surgery: less than 5 years, 5-10 years, 10-15 years, and greater than 15 years.
(table 1) MR images were evaluated for the presence of fibrosis, congestion and any other hepatic abnormalities.
Fibrosis was determined based on a specific pattern of delayed reticular enhancement in combination with liver morphology.
Congestion was deemed present if there was increased T2 signal in the liver parenchyma or periportal regions in combination with cloud-like enhancement on dynamic post-contrast images.
All patients demonstrated morphologic changes in the liver with varying degrees of hepatic fibrosis and hepatic congestion.
Fibrotic changes were often non-uniform, and thus could be underdiagnosed by biopsy.
Interestingly, 4 patients, 24%, had focal arterially enhancing lesions speculated to represent vascular proliferative lesions, however, none warranted biopsy.
Conclusions: It is established that patients who undergo Fontan develop hepatic abnormalities.
MRI is a reliable, non invasive technique that accurately demonstrates these findings.
MRI may be a more sensitive method to evaluate the etiology and full extent of hepatic disease.
Poster #: SCI-012 Complications Within the Interventional Radiology Division of a Tertiary Care Children's Hospital: Initiatives for Ongoing Quality and Practice Improvement Brian Dillon, Children's Hospital Boston, brian.
dillon@childrens.harvard.edu; Pamela Sanborn, Yolanda Milliman-Richard, Darren Orbach, Stephan Voss Purpose or Case Report: Between 2004 and 2010, procedure-related complications occurring within the division of interventional radiology at our institution were recorded and classified according to level of severity.
The goals of this study were to determine rates of procedurebased complications based on severity, to establish thresholds for complications, and to determine whether measurable trends in complications over time were evident.
Methods & Materials: Between 2004 and 2010, 14,042 interventional procedures were performed within the division of interventional radiology at our institution.
Adverse events were characterized both according to level of severity (using an institutional 5 point severity scale), and with brief descriptions of individual events.
Adverse events were reviewed monthly at the division's morbidity and mortality conference, with respect to procedure type and operator.
Based on review of our interventional radiology data and benchmarks rates used for diagnostic errors, threshold complication rates were established by consensus between the department quality improvement committee and the division of interventional radiology.
For severe events (Level 4 and 5) there is no allowable threshold; all such events were subjected to both internal and institutional review.
Results: The overall complication rate was less than 1% for all procedures performed.
The complication rates for the respective severity levels were: Level 1 (0.235), Level 2 (0.3), Level 3 (0.1), Level 4 (0.249), and Level 5 (0.028).
The severity of a given complication was not associated with procedural complexity.
No operator-specific trends were identified.
Conclusions: Since 2003, the Society of Interventional Radiology has offered guidelines and strategies for improving safety and quality in interventional radiology.
However, no specific benchmark data or procedural recommendations are available for pediatric interventional procedures.
Our results demonstrate rates of complications well below published overall complication rates for interventional radiologic procedures.
This database of procedure-based complications serves as a foundation for a quality improvement program that allows review of complications with respect to specific procedure types, individual operators, and procedural complexity, in an effort to institute an ongoing and continuous process of quality improvement within interventional radiology.
Purpose or Case Report: Dysosteosclerosis (DSS), an extremely rare dense bone disease, features short stature and fractures and sometimes optic atrophy, cranial nerve palsy, developmental delay, and failure of tooth eruption in infancy or early childhood consistent with osteopetrosis (OPT).
Bone histology during childhood shows unresorbed primary spongiosa from deficient osteoclast action.
Additionally, there is remarkable progressive flattening of all vertebrae mimic PPi blocking mineralization.
During EHDP treatment for GACI, In our patient prolonged high dose EHDP resulted in severe skeletal deformity resembling hypophosphatasia which was reversable with drug stoppage.
Methods & Materials: A 7-year-old boy with GACI referred for profound, acquired, progressive skeletal deformity.
He was receiving 200 mg/day of EHDP and was wheelchair bound.
We studied him and his response to stopping EHDP.
Results: Skeletal radiographic findings resembled pediatric hypophosphatasia with pancranial synostosis, widened physes with metaphyseal osteosclerosis, "tongues" of radiolucency, along with cupping and fraying, and long-bone bowing.
In addition there were large intra and extraarticular calcifications.
Radiographic features of BP-induced OPT included femoral Erlenmeyer flask deformity and osteosclerosis (lumbar sine DXA z-score +5.7).
Biochemical parameters of mineral homeostasis were essentially normal although serum osteocalcin was low and he had markedly elevated serum levels of creatine kinase and TRAP-5b consistent with osteopetrosis (OPT).
After stopping EHDP, he improved quickly with remarkable healing of his rachitic appearing skeleton and decreased joint calcifications.
Conclusions: Our patient with GACI had profound skeletal deformities from high-dose EHDP therapy that significatly improved with drug stoppage.
Magnetic Resonance Imaging in the Evaluation of Infants with Hypoxic Ischemic Encephalopathy Julio M. Araque, MD, Radiology, Medical College of Georgia, jaraque@georgiahealth.edu Purpose or Case Report: To illustrate and review a spectrum of brain abnormalities of infants with HIE.
Defining the most useful approaches and MRI sequences, to facilitate identification and early diagnosis of lesions with the potential to predict outcome and abnormal neurodevelopment.
Methods & Materials: Reviewed available evidence on MRI strategies for evaluating infants with Hypoxic ischemic encephalopathy.
Different cases illustrating lesions are presented and discussed for proper diagnosis correlating physiopathology and imaging appearance.
More relevant findings are depicted with didactic illustrations.
Identifying studies where new techniques such as DWI, ADC, DTI, SWI, or MRS adds significant diagnostic value to the overall interpretation.
Results: MRI is routinely performed as a very sensitive method for detection of HIE lesions.
Advanced MR techniques, such as DTI, DWI, ADC, MRS, SWI offer the possibility of detecting injuries at a time when intervention is theoretically possible.
The understanding of the physiopathology allows for prediction of the location and extent of lesions, facilitating identification and appropriate classification.
The identification of infants with potentially abnormal neurodevelopment, offers the opportunity to provide therapeutic neurodevelopmental interventions in early childhood.
MRS is the best MR biomarker to predict neurodevelopmental outcome in asphyxiated full-term neonates.
Brain metabolite ratios and regional ADC values may vary between MR systems and coils.
Development of normal values for each institution is required, and support of physicists is mandatory.
Conclusions: MRI continues to evolve as a valuable adjunctive tool routinely obtained in nearly all cases of HIE.
Advanced MRI techniques increase sensitivity of conventional T1 and T2-W images and outperform computer tomography and ultrasound for confirming the diagnosis of hypoxic-ischemic brain injury or providing prognostic information for the care of patient with HIE.
Disclosure: Dr. Araque has indicated that he will discuss or describe, in the educational content, a use of a medical device or pharmaceutical that is classified by the Food and Drug Administration (FDA) as investigational for intended use.
Posterior Fossa Abnormalities in Children Amit Gupta, MBBS, Radiodiagnosis, R.N.T.
Medical College, Udaipur, Rajasthan, India, amitsensation@yahoo.co.in Purpose or Case Report: The aim of this exhibit is to demonstrate various conditions involving the posterior fossa in children with emphasis on importance of embryologic development of cerebellum in reaching a correct diagnosis.
Methods & Materials: This pictographic presentation displays the imaging features of cases encountered in our clinical practice on 1.5 Tesla magnetic resonance (MR) imaging.
Results: With the advent of MR imaging, there has been a revolution in identification and characterization of malformations of the brain This is especially true in posterior fossa, where the sensitivity and specificity of MR imaging with its multidimensional imaging capability are far superior to those of computed tomography (CT) in the detection of subtle morphologic abnormalities.
However, there is still a great deal of confusion regarding their classification, terminology, and spectrum of expression and this is where neuroembryology is of great help.
This exhibit demonstrates : 1) Review of embryology and normal anatomy of cerebellum.
2) MR appearance of spectrum of conditions involving posterior fossa in children which includes developmental abnormalities (Dandy-Walker complex, Arnold Chiari malformations, cerebellar dysplasia/ hypoplasia, Joubert's syndrome, etc.
), cysts (arachnoid cyst, giant cisterna magna etc.
), tumours (medulloblastoma, ependymoma, hemangioblastoma etc.)
and miscellaneous conditions.
significantly reduces dose (1/3 of other gadolinium based contrast agents), and doesn't require trigger imaging.
Conventional MRI provides important information regarding the anatomical extent, size, and relation to critical anatomical structures thus when combined with TWIST, MRI provides the best information without use of radiation in children.
Functional Connectivity MRI in Pediatric Brain Tumor Patients with and without Epilepsy Andrew V. Poliakov, PhD, Radiology, Seattle Children's Hospital; David Bauer, Edward Novotny, Seth D. Friedman, Dennis Shaw, Jeff Ojemann Purpose or Case Report: Functional connectivity MRI (fcMRI) is a way to evaluate cortical networks across different modalities such as motor, sensory, vision, and the default mode network using functional Magnetic Resonance Imaging.
fcMRI relies on correlation in fMRI image intensity that occurs between functionally connected regions.
This effect can be seen in awake as well as anesthetized patients.
We evaluated these pathways in pediatric patients with brain tumors.
Methods & Materials: Patients were randomly selected from our tumor database.
Inclusion criteria included age less than 18, history of brain tumor resection, and complete fcMRI data.
Imaging was performed on a 3 T Siemens Trio system.
Functional MRI data were acquired as part of a clinical imaging protocol over 6.5 -8 min using a gradient echo, echo-planar sequence.
Preprocessing of fMRI data followed by Independent Component Analysis (ICA) was performed using FSL software.
Functional connectivity analysis was performed using software provided by 1000 Functional Connectomes Project, based on AFNI and FSL software packages.
Correlation maps were produced by extracting the BOLD time course from a seed region, computing the correlation coefficient between that time course and the time course from all other brain voxels, correcting for multiple sampling and degrees of freedom and thresholded at a z value of 3.0.
Results: Fourteen patients were included in the study, eight female and six male.
Tumor types include ganglioglioma (5), pleomorphic xanthoastrocytoma (2), juvenile pilocytic astrocytoma (2), ependymoma (1), anaplastic astrocytoma (1), glioblastoma multiforme (2), and primitive neuroectodermal tumor (1).
Seven patients had tumor-associated epilepsy, and seven patients did not.
The figure shows connectivity patterns in the motor network in patients without (A) and with (B) epilepsy.
In the patients without epilepsy, functional connectivity was often displaced but not decreased or absent.
In the patients with epilepsy, we observed decreased or absent functional connectivity.
Similar results were found for default mode network: connectivity was diminished or absent in the patients affected by epilepsy.
Conclusions: fcMRI is a novel technique that may prove useful for evaluation and presurgical planning by giving us insight into how tumors disrupt function.
Functional connectivity was often displaced but relatively preserved in the patients without epilepsy.
It was disrupted or absent in the patients with epilepsy.
Poster #: SCI-022 Corpus Callosum DTI Measurements in Neurofibromatosis Type 1 and Normal Controls Nadja Kadom, MD, Radiology, Children's National Medical Center, nkadom@childrensnational.org; Amir Noor, Rhea Udyavar, Marine Bouyssi-Kobar, Iordanis Evangelou, Maria T. Acosta Purpose or Case Report: Many patients with Neurofibromatosis type 1 (NF1) have corpus callosum enlargement; pathogenesis and underlying pathophysiology are unclear.
The goal of our study is to investigate the pathophysiological basis of corpus callosum enlargement in NF1 patients through MRI diffusion tensor (DTI) measurements.
Methods & Materials: Retrospective study, IRB approved.
Patients consecutively selected from institutional data base; inclusion criteria: established diagnosis of NF1, brain imaging with DTI sequence, abnormally high corpus callosum to skull ratio; excluded were patients with complications of NF1 that could affect size of the corpus callosum.
Age and gender matched normal controls were randomly selected from the radiology data base.
ROI were placed manually over the corpus callosum for DTI measurements using DTI-Studio by two independent researchers, one blinded to diagnosis.
Results: Fifteen NF1 patients and matched controls were analyzed.
The corpus callosum to skull ratio was found to be significantly different between the experimental and control group (p00.0001).
For NF1 patients we found: a trend to lower apparent diffusion coefficient (ADC, p00.067), significantly higher radial diffusivity (p00.023), significantly lower axial diffusivity (p00.0002), and significantly lower fractional anisotropy (FA, p00.0012).
Conclusions: The significantly lower axial diffusivity in NF1 can indicate that there are more crossing fibers in the corpus callosum of NF1 patients than in normal controls.
Further studies using comparative DTI tractography may be helpful in further investigating this stipulation.
The significant increase in radial diffusivity can be explained by a variety of factors, including thinner myelin sheaths, increased interstitial fluid, smaller axons, or a combination thereof.
The trend of lower ADC may indicate low axonal diameter, as ADC has been shown to more strongly correlate with axonal diameter without the myelin sheath.
In future studies we will correlate abnormal corpus callosum DTI markers with cognitive functions in NF1 patients to see if relationships exist that can be used as predictors of cognitive deficits in NF1 patients.
Screening for Vitamin D Deficiency in Children with Suspected Non-Accidental Fracture Conor Kain, MD, Tripler Army Medical Center; Veronica Rooks, Laura Keller, Jordan Pinsker, Allyson Cordoni, Sarah Frioux Purpose or Case Report: Determine if routine screening of vitamin D levels after suspected non-accidental fracture detects vitamin D deficiency and changes clinical outcomes.
Methods & Materials: After IRB approval we reviewed all skeletal surveys performed at Tripler Army Medical Center (TAMC) in the last 10 years and selected the children who were evaluated for suspected non-accidental fracture.
We determined if 25-hydroxyvitamin D [25(OH)D] level was requested for these patients and characterized the provider's clinical suspicion of vitamin D deficiency as high or low.
Per the 2010 Institute of Medicine Report and 2011 Endocrine Society Guidelines we defined vitamin D deficiency as a 25 (OH)D level of less than 20 ng/mL.
We calculated the prevalence of children with low 25(OH)D levels whose providers had low clinical suspicion for vitamin D deficiency.
Results: 396 skeletal surveys were done at TAMC from November 2000 to July 2011.
99 were performed after identifying a suspected non-accidental fracture.
Of these patients 11 children from ages 1 to 7 months had 25(OH)D levels requested.
For children whose providers had a low pre-test suspicion for vitamin D deficiency, the prevalence of vitamin D deficiency was 12.5% (95% binomial CI 0.003-0.524, 1 of 8 cases.
These results indicate that at least one out of every three hundred children evaluated for nonaccidental fracture could have vitamin D deficiency despite a low clinical suspicion by their provider, although the actual rate is likely much higher given that we found one in eight cases.
The child we identified with a low Vitamin D level whose provider had no suspicion for rickets was treated with Ergocalciferol and continued to be evaluated for abuse.
Conclusions: Routine vitamin D level screening after nonaccidental fracture may detect vitamin D deficiency in children for whom there is low clinical suspicion.
As our population resides at a low latitude and receives greater than average sun exposure, the rate of deficiency in children with suspected non-accidental fracture may be much greater in other areas.
Comet Tails and Dirty Shadows: The Secrets Behind Artifacts in Pediatric Ultrasound Adam Edelstein, Pediatric Radiology, Massachusetts General Hospital; Anuradha Shenoy-Bhangle, Katherine Nimkin Purpose or Case Report: To review common ultrasonographic artifacts, explain what causes them, and show how they can be used to aid in diagnosis in a variety of pediatric conditions, including less common entities.
Methods & Materials: Ultrasonographic images in patients less than 18 years of age were reviewed.
Cases were selected that showed classic artifacts which helped with the diagnosis of a variety of entities.
Results: Ultrasound artifacts include comet tail, reverberation, ring down and "dirty" shadowing.
These can be used to help characterize a variety of pediatric conditions including gossypiboma, bezoar, subcutaneous foreign body, complications of NEC, and staghorn calculus.
Artifacts can also be used to confirm the presence of stool or bowel gas.
Conclusions: Familiarity with ultrasonographic artifacts is critical for tissue characterization and can help narrow the differential diagnosis in difficult pediatric cases.
Cardiac CTA: Non-Vascular Ring Tracheobronchial Compression Secondary To Enlarged Patent Ductus Arteriosus in Infants with Congenital Heart Disease.
Nhi Huynh, MD, Radiology, St. Joseph Hospital and Medical Center, e.nhihuynh@gmail.com; Todd Chapman, Randy Richardson Purpose or Case Report: Tracheobronchial compression or narrowing secondary to a vascular ring has been well documented.
The purpose of this study is to describe the frequency of airway compression secondary to an enlarged patent ductus arteriosus detected by CCTA without the presence of a vascular ring.
Methods & Materials: A retrospective study of 282 CCTA exams in infants was performed over the period between 03/ 28/2007 and 09/28/2011.
CCTA was performed with a 64-slice MDCT, with EKG gating, followed by three-dimensional reformations.
Results: Of the 282 congenital heart disease infant patients, there are 49 patients with tracheobronchial compression or narrowing.
Of these 49 patients, 20 patients reported to have patent ductus arteriosus as the primary cause of tracheobronchial compression or narrowing.
Approximately 41% of patients with airway compression in patients with congenital heart disease are secondary to an enlarged and/or tortuous patent ductus arteriosus.
None of these cases were due to a vascular ring.
Of these 20 patients, 10, 6, and 4 patients demonstrated to have mild, moderate, and severe airway compression respectively.
Conclusions: Tracheobronchial compression or narrowing secondary to vascular ring with a patent ductus arteriosus has been well documented.
In this study, we demonstrate that a significant percentage of airway compression in patients with congenital heart disease without a vascular ring is due to a tortuous enlarged patent ductus arteriosus.
Cardiac CTA is uniquely equipped to evaluate airway compression due to an enlarged patent ductus arteriosus and can help improve patient care in congenital heart disease patients with respiratory symptomatology.
Pediatric Liver MR Elastography: A Primer Suraj Serai, PhD, CCHMC, suraj.serai@cchmc.org; Daniel J. Podberesky, Alexander J. Towbin Purpose or Case Report: A wide variety of pediatric liver disorders may be complicated by the development of liver fibrosis and ultimately cirrhosis.
With early interventions, the progression to hepatic fibrosis can be slowed, halted, and in some cases reversed.
Liver biopsy has long been considered the gold standard for assessing the presence and degree of liver fibrosis.
However, liver biopsy has disadvantages, due to its potential sampling error, risk of complications, relatively high cost, intra-and inter-observer variability, and, in general, poor acceptance by pediatric patients and their parents.
MR elastography (MRE) is a relatively new, non-invasive technique that provides a safe, rapid and cost-effective method for objectively evaluating of a wide variety of hepatic diseases by quantitative stiffness evaluation of the liver-parenchyma.
The purpose of this exhibit is to review our clinical experience with this technique and illustrate the application of liver MRE in the pediatric population at our medical center.
Methods & Materials: A review of pathogenesis and staging of liver fibrosis in children and current methods available for assessing liver fibrosis will be provided.
A review of MRE physics and technique, including the specific liver MRE protocol used at our institution will be illustrated.
We will review widely-used and emerging clinical indications for liver MRE, as well as benefits and limitations to the technique, supported by brief literature review.
Results: In addition to sharing our liver MRE technique, we will illustrate clinical case examples from our institution of a variety of liver disorders including non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, storage disorders, cardiac disease, and idiopathic elevated liver enzymes.
Conclusions: This educational exhibit will review our experience with liver MRE, a safe, newly available technique which will play an increasingly important role in the noninvasive evaluation of pediatric liver disease.
Poster #: SCI-027 Spectrum of Tuberculosis in Children Amit Gupta, MBBS, Radiodiagnosis, R.N.T.
Medical College, Udaipur, Rajasthan, India, amitsensation@yahoo.co.in Purpose or Case Report: The aim of this exhibit is to present a spectrum of tuberculosis (TB) in the human body which commonly involves pulmonary, nervous, musculoskeletal, gastrointestinal and genitourinary systems.
This pictographic presentation displays the imaging features of TB cases encountered in our clinical practice with reference to plain X-rays, CT and MRI as appropriate.
Results: With the advent of the newer modalities, the utility of the plain skaigram has been largely limited a initial screening tool only.
Whereas CT scores over MRI in pulmonary TB (parenchymal disease, lymphadenopathy, pleural effusion, empyema, miliary disease) and abdominal TB (spectrum from mesenteric lymphadenitis to visceral involvement), the magnetic resonance (MR) imaging is much better in diagnosing CNS TB (tuberculoma, abscess, meningitis, subdural empyema and myelitis).
In musculoskeletal and genitourinary TB, CT and MR imaging may be preferred based on the stage of disease and the character of the lesion.
Cardiac involvement (pericarditis) is among the less common affections of TB.
Conclusions: Tuberculosis is a multisystem disease that can affect virtually any part of the body from head to toe.
TB demonstrates a variety of clinical and radiologic findings and has a known propensity for dissemination from its primary site and therefore can mimic numerous other disease entities.
Hence it is imperative for radiologists to understand the typical disease distribution, patterns and imaging manifestations of TB.
2010 Janet L. Strife, MD 2011 Carol M. Rumack MD 1987 Ole A. Eklof, MD 1987 Clement C. Faure, MD 1987 Andres Giedion, MD 1987 Denis Lallemand, MD 1987 Arnold Lassrich MD 1992 Donald R. Kirks, MD 1992 Beverly P. Wood, MD 1993 Hooshang Taybi MD 2008 Marta Hernanz-Schulman, MD, FACR 2009 M. Ines Boechat, MD, FACR 2010 Neil D. Johnson, MBBS 2011 Dorothy I. Bulas, MD *Deceased SINGLETON-TAYBI AWARD INVESTIGATOR AWARD This award is given to the author of the best paper presented by a Resident or Fellow at the SPR meeting MD 2002 Ricardo Faingold, MD 2003 Andrea Doria, MD 2004 Nina M. Menezes, PhD Anthropometric parameters are a strong determinant of LVM in healthy individuals Kiyarash Mohajer, Pierangelo Renella, Paul J. Finn Purpose or Case Report: Despite theoretical advantages of higher field strength SSFP cine imaging, time-resolved magnetic resonance angiography (TR-MRA), and high resolution contrast-enhanced MRA (CE-MRA) were performed.
Two readers independently evaluated the data for image quality, vessel and cardiac chamber definition, and presence of artifacts.
SNR and CNR were calculated.
Results: 95% of SSFP cine images at 3 T were rated as good or excellent quality with 73% having mild and 24% having moderate artifacts (k00.07) 100% of arterial and venous phase CE-MRA images were considered good or excellent Cardiac chamber definition was considered good or excellent in 95% of arterial and venous phase CE-MRA images (k00.08).
100% CE-MRA images showed good or excellent definition of the thoraco-abdominal vessels On average, both readers scored cine SSFP images higher at 1.5 T and CEMRA images higher at 3.0 T. Overall diagnostic performance was high at both field strengths.
Conclusions: MRI of pediatric patients with CHD and vascular abnormalities at 3.0 T is feasible.
Relative to 1.5 T, SNR and CNR are both improved at higher field strength and higher resolution CEMRA is achievable T are more prevalent, they rarely render cine imaging non-Poster # Exclusion criteria were lack of correlating US or follow up information.
Two pediatric radiologists blinded to US findings reviewed the MR images and analyzed the contents of abdominal wall defect, organ location and attachment; spine anomalies; umbilical cord and limb anomalies.
Results: Our search yielded 16 patients.
All fetuses had ventral wall defects, small thorax and eviscerated liver and bowel.
In two cases kidneys were in extracorporeal location.
In 12/16 there was no membrane covering extruded organs.
In five MR showed organs attached to the placenta or uterine wall (mainly bowel and liver) Mahmoud Al-Hawary and, by adolescence, paradoxical metaphyseal osteopenia with thin cortical bone.
Reports of consanguinity indicate autosomal recessive inheritance Our studies, spanning ages 11-44 mo, showed weight 50%, but length diminishing from~30% to −2.3 SD.
Head circumference was +4 SD.
She had frontal bossing, blue sclera, normal teeth, genu valgum, and unremarkable joints.
Radiographs showed orbital and facial sclerosis, basilar thickening, "bone-in-bone" appearance in the pelvis, sclerotic long bone ends, and fractures of ribs and extremities.
Progressive metaphyseal widening occurred as vertebrae changed from ovoid to flattened and became beaked anteriorly.
Consistent with OPT, serum PTH concentrations reflected dietary calcium levels.
Serum bone alkaline phosphatase, osteocalcin, and TRAP5b were sub-normal.
Iliac crest contained excessive primary spongiosa and no osteoclasts.
Splice sites and exons were intact for the genes encoding cholride channel 7, T-cell immune regulator 1, OPT-associated transmembrane protein 1 The hallmarks include stippled epiphyses, nasal hypoplasia, and hypoplastic distal phalanges and developmental delay.
Punctate calcifications are seen not only in the epiphyses but also in the paravertebral regions.
Paravertebral puncta are commonly associated with defective ossifications in the cervical spine.
The malformation of the cervical spine causes spinal canal stenosis and instability, which occasionally necessitate surgical intervention None of the cases had brain infarction.
Conclusions: Tortuousity and luminal narrowing of the cervical arteries is a common finding in CDP-BT.
This previously unknown malformation is an important factor to discriminate patients at increased risk of cerebral ischemia, particularly in patients undergoing surgical intervention.
Disclosure: Dr. Okabe has indicated that she will discuss or describe Severe Skeletal Toxicity from Protracted Etidronate Therapy for Generalized Arterial Calcification of Infancy William H. McAlister, MD, Mallinckrodt Institute of Radiology Campbell Sheen Purpose or Case Report: Generalized Arterial Calcification of Infancy (GACI) is an autosomal recessive disorder caused by deactivating mutations within the gene for ectonucleotide pyrophosphatase phosphodiesterase-1 (ENPP1).
ENPP1 on osteoblasts, chondrocytes, and vascular smooth muscle cells hydrolyzes nucleotide triphosphates to nucleotide monophosphates and inorganic pyrophosphate (PPi) Can Time-Resolved Contrast-Enhanced MRA (TWIST) Classify Soft Tissue Vascular Anomalies in the Head and Neck in Children Accurately?
Aylin Tekes, MD 28 children from 0-17 years of age were enrolled.
TWIST and conventional MRI was performed (Triplanar T2-Weighted [T2-W] imaging with fat saturation, pre-contrast axial T1-Weighted [T1-W] imaging, and post contrast triplanar fat-suppressed T1-W imaging).
TWIST was performed in coronal plane using blood-pool MR contrast agent (Ablavar-Lantheus) to enhance image quality and spatial resolution of MRA.
Two pediatric neuroradiologists evaluated all patients in two different sessions, 15 days apart: one session conventional MRI with contrast was evaluated, in the second session TWIST was evaluated.
Clinical evaluation and/or percutaneous venogram/lymphogram data were the gold standard.
Results: Our patients had diagnosis of infantile hemangioma (n04), venous malformation (n012), and lymphatic malformation (n012).
TWIST alone could accurately classify 26/ 28, conventional MRI with contrast could accurately classify 22/28.
Conventional MRI with contrast combined with TWIST could accurately classify all cases.
Conclusions: TWIST offers high temporal resolution in the order of seconds, and provides functional data about the dynamics of contrast enhancement comprising the arterial, venous and delayed venous phases Kiery CR-2, SCI-11 EDU-91, EDU-92 EDU-35, EDU-40, PA-045 EDU-86, PA-036 A-092, PA-093, PA-108, PA-109, PA-113 EDU-7, EDU-59, EDU-60, EDU-62, EDU-66, EDU-69, EDU-78 EDU-59, EDU-60, EDU-62, EDU-66, EDU-69, EDU-78 Suraj SCI-26, PA-124, PA-125 EDU-98 PA-147, EDU-86, PA-036 The Society for Pediatric Radiology gratefully acknowledges the support of the following companies in presenting the 55th Annual Meeting and Postgraduate Course: CME committee reviewers for this activity have disclosed any relevant financial relationships.
No conflicts of interest exist.abuse and offer potential mechanisms of injury may help make the diagnosis of child abuse.
The Pediatric Elbow-MRI Findings with Multimodality Correlation Michael Guandalini, MD, Royal Children's Hospital; Murray Bartlett Purpose or Case Report: To describe and illustrate elbow abnormalities identified by MRI performed in a cohort of pediatric patients with multimodality correlation.
Methods & Materials: Retrospective review of MRI elbow studies performed at The Royal Children's Hospital, Melbourne between 2003 and .
The studies were reviewed by a Pediatric Musculoskeletal Radiologist and Pediatric Radiology Fellow with patient demographics, clinical indication, findings and selected images recorded.
Results: 199 elbow MRI examinations were reviewed on children aged 4 months to 18 years (123 boys, 76 girls) with equal numbers of left and right sides examined.
Clinical indications included previous trauma in 147 cases (74%) and nontraumatic conditions in 52 (26%).
The most common traumatic indication was suspected or confirmed fractures or avulsions (21%) followed by osteochondral or cartilage injuries (18%), growth arrest (16%), loose bodies (14%) and ligament injuries (10%).
Hemophilia (38%) was the most frequent nontraumatic indication followed by neoplasm (17%).
Mild to severe arthropathy, fractures, physeal growth arrest, subluxations, osteochondral lesions and loose bodies were the most frequently demonstrated abnormalities.
Ligament strains and tears, bone oedema, neuromuscular abnormalities, infections and several neoplasms including lipomas, vascular/lymphatic malformations and bone tumors also featured.
Conclusions: This pictorial review illustrates the broad range of abnormalities one might expect to encounter on pediatric elbow MRI studies, highlighting the major features and corresponding appearances on CT and plain X-ray.
Spectrum of Patellar Tendon Avulsive Injury on MRI in Children: Differentiation Between Acute and Chronic Avulsive Injuries of the Inferior Patellar Pole and Tibial Tuberosity Zeyad Metwalli, MD, Baylor College of Medicine, metwalli@ bcm.edu; Herman Kan, Scott Rosenfeld, R. P. Guillerman Purpose or Case Report: The extensor mechanism of the knee is an intricate component of the joint and is frequently injured in pediatric athletes.
Due to the strength of the patella tendon, trauma to the anterior knee is often manifested by avulsive injuries, which may occur on an acute or chronic repetitive basis.
Purpose: This pictorial review will illustrate differentiating radiographic and MRI features of acute and chronic avulsive injuries of the pediatric knee.
Outline: 1.
Anatomy and physiology a.
Discuss the anatomic differences of the pediatric and adult knee extensor mechanism b. Pathophysiology and biomechanical basis for chondro-osseous avulsion injuries versus tendon tears in the skeletally immature.
Purpose or Case Report: The purpose of this educational exhibit is to demonstrate the magnetic resonance imaging (MRI) appearance of the ankle and hindfoot ligaments using an interactive approach.
Methods & Materials: A 3 Tesla Siemens MRI scanner with a multichannel ankle coil was utilized in the acquisition of images of ankle and hindfoot.
Three dimensional volume acquisition proton density images will be used to demonstrate the ligamentous anatomy of the ankle and hindfoot in axial, axial oblique, coronal, and sagittal planes.
Results: The exhibit will begin with an interactive review of the ankle and hindfoot ligamentous anatomy with each ligament Poster #: EDU-073 CNS Imaging Findings in Hemophagocytic Lymphohistiocytic Syndrome Rupa Radhakrishnan, MBBS, MD, DNB, Radiology, University of Cincinnati College of Medicine, radhakrp@ucmail.
uc.edu; Marcia K. Kukreja, Alexandra Filipovich, Alexander J. Towbin Purpose or Case Report: Hemophagocytic lymphohistiocytosis (HLH) is a rare, life threatening condition caused by an uncontrolled proliferation of activated lymphocytes and histiocytes with high levels of inflammatory cytokines.
The organs most commonly involved in this disorder include the liver, spleen, lymph nodes, bone marrow and central nervous system (CNS).
The purpose of this exhibit is to review the CNS imaging findings associated with HLH, its complications, and its management.
The published literature was reviewed to identify the potential imaging findings HLH.
The electronic medical record system was then searched to find illustrative case examples from our institution.
Cases demonstrating the primary imaging findings as well cases highlighting complications of the disease or its therapy were selected.
Results: CNS involvement is common in HLH with approximately 75% of patients demonstrating neurological symptoms.
CT findings of CNS involvement include diffuse parenchymal atrophy, low attenuation lesions in the white matter and calcifications.
MR findings include diffuse leptomeningeal and perivascular enhancement, T2 hyperintense lesions with nodular or rim enhancement as well as confluent white matter lesions, and diffuse parenchymal volume loss of the cerebrum and cerebellum.
Restricted diffusion has been demonstrated in some lesions.
Ring enhancing parenchymal lesions have been described representing active demyelination.
Intracranial hemorrhage may occur as a result of thrombocytopenia and coagulation abnormalities.
Sepsis with opportunistic organisms can involve the CNS and produce intracranial findings such as parenchymal abscesses.
CNS changes, such as posterior reversible encephalopathy syndrome, are also seen with the commonly used immunomodulatory regimen used in the treatment of HLH.
Conclusions: This exhibit will aid the viewer in identifying the CNS imaging findings of HLH as well as the complications of the disease and its therapy.
While the CNS imaging findings are not specific, they may help the radiologist formulate a diagnosis in association with the other clinical and imaging findings; furthermore, imaging can help the clinical team in managing the disease and its complications.
Methods & Materials: Medical records of our pediatric patients with palpable head masses over the last 5 years, were reviewed and images were collected.
Correlation of US of these lesions with other imaging modalities and/or pathologic diagnosis was done.
Results: US appearances of various head masses including congenital/developmental (encephalocele, meningocele, dermoid, occipital protuberance), traumatic (cephalhematoma, subgaleal hematoma, calvarial fracture), inflammatory/infectious (sebaceous cyst, histiocytosis, dermatitis), vascular (malformations, pseudoaneurysm) and neoplastic (benign and malignant lesions including metastases) etiologies, will be illustrated with case based approach.
MRI and/or CT or tissue diagnosis can be problem solving.
Role of ultrasound guidance for percutaneous procedures (biopsy, sclerotherapy) will also be described.
Conclusions: Ultrasound can play an important role in the delineation, diagnosis and guiding further management of pediatric palpable head masses.
US can differentiate various scalp lesions and suggest the underlying calvarial defect or involvement to some extent, helping to narrow the differential diagnosis for such lesions.
Color doppler US can be useful to detect vascularity within the lesion or vascular lesions.
Given that US is often requested for the evaluation of palpable head masses, pediatric radiologists should be familiar with their sonographic features.
Posterior Fossa Malformations-A Pictorial Review Rui Santos, MD, BC Children's Hospital, ruiradiologia@gmail.
com; Khalid Khashoggi, Angela T. Byrne Purpose or Case Report: Posterior fossa malformations are a group of central nervous system anomalies that may be detected during pregnancy or present early infancy with features that include hypotonia, developmental delay, mutations responsible for the disease and proposals as to mechanism of action of the mutation with respect to disease manifestations.
This preceded the development of hypotheses regarding the relationship between genotype and phenotype and the attempt to utilize imaging modalities that could better assess disease activity as it related to functional status.
The purpose of this exhibit is to briefly review the history pre-1989 and to focus on the numerous ways in which the understanding has improved since that time.
Conclusions: 1.
There are over 1000 different mutations of the CFTR gene responsible for cystic fibrosis with varying prevalence throughout the world.
2.
The class of mutation often dictates its particular mechanism of action.
3.
There is some relationship between genotype and phenotype-particularly with respect to pancreatic involvement.
4.
Newer imaging modalities including CT and MRI with or without hyperpolarized helium are better predictors of disease severity than is plain film.
Imaging Pulmonary Tuberculosis in Infants: What are the Most Useful Diagnostic Radiological Findings?
Handan Cakmakci, Pediatric Radiology, Dokuz Eylul University Hospital, handancakmakci@gmail.com; Nevin Uzuner, Filiz Tetik Purpose or Case Report: Early diagnosis and treatment are very important for infants with tuberculosis.
Infantile pulmonary tuberculosis is more symptomatic, and the risk of severe and life-threatening complications such as tuberculous meningitis or miliary tuberculosis is higher.
Bacteriologic confirmation of the disease in children is difficult and in younger infants (<3 months), the tuberculin skin test is frequently negative.
Therefore, radiological findings play important role in diagnosing tuberculosis in infants.
The purposes of this study are to identify chest x-ray and lung CT findings in pulmonary tuberculosis of infants and consider the most useful diagnostic findings of these age group patients.Methods & Materials: Chest radiographs and chest CT images of 7 infants who were diagnosed in our hospital from 2005 to 2011 were retrospectively reviewed.
The study group included 2 boys and 5 girls ranging in age from 2 to 12 months (mean age, 6 months).
Chest x-ray and computed tomography images were analyzed considering air space consolidation, nodular lesions, cavitating lesions, mediastinal enlargement, hyperinflation, bronchial narrowing, atelectasis pleural effusion on plain radiography and additional mediastinal calcific or caseating lymph nodes on CT images.Results: Air space consolidation was seen on 5 out of 7 chest x-ray and computed tomography images.
Nodular lesions were seen 2 out of 7 chest x-ray and computed tomography images.
Cavitating lesion was seen on 1 out of 7 chest x-ray and computed tomography images.
Mediastinal enlargement suggesting lymph node was seen 5 out of 7 chest x-ray and computed tomography images.
Hyperinflation, bronchial narrowing was seen 2 out of 7 chest x-ray and computed tomography images.
Atelectasis, pleural effusion was seen 1 out of 7 chest x-ray and 2 out of 7 computed tomography images.
Mediastinal caseating lymph nodes, mediastinal calcific lymph nodes were seen 3 out of 7 computed tomography images.
Conclusions: Frequent and the most useful diagnostic radiological findings of pulmonary tuberculosis in infants are mediastinal or hilar lymphadenopathy with central necrosis and air space consolidations.
Disseminated nodules including miliary lesions and airway complications are also detected in this age group.
CT can show detailed parenchymal lesions and tuberculous lymphnodes especially calcified ones.
The Ductus Bump: Radiographic Findings of This Normal Variant and Differential Diagnoses Anusuya Mokashi, Staten Island University Hospital, anusuya.mokashi@gmail.com; Jeremy Neuman, Cheryl Lin Purpose or Case Report: The Ductus Bump: Review of radiographic findings, differential diagnoses and current controversies.
The ductus bump was first described in 1965 by Berdon et al as a transient physiologic mass in the chest in newborn infants.
Some controversy remains as to the exact etiology and clinical significance.
Although initially thought to represent a dilated ductus arteriosus, recently it has been suggested that it actually represents a ductus arteriosus aneurysm that spontaneously resolves.
Others contend it represents dilation of the infundibulum of the closing ductus.
Regardless of etiology, the time of discovery, location, and rapid resolution are characteristic of this entity.
In this presentation we will review the radiographic and echocardiogram findings of the ductus bump, as well as discuss the differential diagnosis.
The frontal radiographic findings are a round mass to the left of the vertebral spine projecting from the mediastinum near the aortic arch.
This mass does not indent the esophagus and it cannot be seen on the lateral view.
It is classically said to resolve within the first few days of life.
The controversy regarding the etiology has also led to some disagreement involving the clinical significance and appropriate follow up, which will also be discussed.
After reviewing this educational poster, the reader will have Conclusions: Abnormalities of the posterior fossa are often difficult to differentiate solely on the basis of their radiologic appearances alone.
However, an accurate diagnosis is essential for proper treatment planning and genetic counselling.
Therefore it is imperative for radiologists to be well versed with the normal anatomy and development of cerebellum so as to correctly diagnose the various posterior fossa abnormalities.Poster #: SCI-019Imaging of Oculoauriculofrontonasal Syndrome with Low-Dose 3-Dimensional Computed Tomography Paritosh C. Khanna, MD, Radiology, Seattle Children's Hospital, pkhanna@uw.edu; Kelly Evans, Gisele Ishak, Joseph Gruss, Michael Cunningham, Anne Hing Purpose or Case Report: Oculoauriculofrontonasal syndrome (OAFNS) combines elements of abnormal morphology of the frontonasal and maxillary processes of the face.
The aim of our exhibit is to demonstrate the low-dose Computed Tomography (CT) features of this syndrome, in seven patients who have been followed at Seattle Children's Hospital (SCH) over 18 years.
We underscore the imaging features of this condition, and describe additional features including bony nasal abnormalities not previously described in the literature, to improve imaging recognition of this spectrum.
We present 3D CT imaging features of a series of eight patients with OAFNS.
In keeping with the ALARA (As Low As Reasonably Achievable) concept and the Image Gently recommendations (www.imagegently.
org), CT head and face studies were obtained on six of eight patients at SCH, while two had prior exams at outside institutions.
Using a 64-slice multidetector CT scanner (GE LightSpeed VCT, Waukesha WI), low-dose CT (120 kV, 150 mAs or lower depending on age) of the head and face was obtained.
Planar bone window and 3D surface rendered images were analyzed.
Results: Our series of patients demonstrated bifid nasal bones, uni-or bilateral mandibular hypoplasia, temporomandibular and zygomatic dysplasia and bony external auditory canal abnormalities.
One patient had an interfrontal bone with a frontal bony defect that was contiguous with the metopic suture.
We describe additional previously unidentified CT anomalies of the nasal bones, anterior nasal spine and nasal septum.
These structures are involved in all patients who had CT imaging available, although unique features are present in each case.
Conclusions: CT is the mainstay of imaging of craniofacial anomalies in the post-natal period, both pre-and postoperatively.
In addition to our low-dose CT imaging findings of OAFNS, novel nasal bone anomalies identified by our group serve to identify a new subset of patients with this syndrome and may help refine the phenotype of the OAFNS spectrum.
key: cord-010119-t1x9gknd authors: nan title: Abstract Presentations from the AABB Annual Meeting San Diego, CA ctober 7‐10, 2017 date: 2017-09-04 journal: Transfusion DOI: 10.1111/trf.14286 sha: doc_id: 10119 cord_uid: t1x9gknd nan Background/Case Studies: Zika virus (ZIKV) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (TT).
RNA persistence has been reported in whole blood (WB) long after clearance of viremia in plasma, raising concerns over the risk of TT with plasma based nucleic-acid amplification testing (NAT).
The dynamics of ZIKV persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of ZIKV infection.
We sought to characterize the dynamics of infection through prospective enrollment of ZIKV RNA1 blood donors.
Study Design/Method: Donors identified through investigational ZIKV NAT screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes.
Plasma and RBC were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks 1, 3, 6, 12 and 24 following index donations from 50 donors and detailed symptom questionnaires were administered at each study visit.
Blood compartments and body fluids were tested for Zika RNA by real time RT-PCR.
Plasma samples were tested for Zika specific IgM and IgG antibodies Results/Finding: The percent of ZIKV RNA1 samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table.
Plasma viremia declined rapidly after index donations whereas RBC-and WB-associated viral RNA persisted for up to 3 months and peripheral blood mononuclear cell (PBMC) associated virus was detected intermittently at low levels and waning by 3 months.
Urine and saliva detection decreased significantly after 2 weeks and was undetectable by 3 months.
Of donors who were enrolled in the acute pre-seroconversion stage of infection 65% (15/23) developed multiple ZIKV related symptoms 1 week post index donation, compared to only 30% (7/27) for donors detected post-seroconversion.
Conclusion: ZIKV RNA persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported.
The persistence of Zika RNA in RBCs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress.
WB testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women.
Iron Status and Novel Risk Factors for Iron Depletion in a Diverse Donor Population Bryan R. Spencer* 1 , Yuelong Guo 2 , Ritchard G. Cable 3 , Joseph E. Kiss 4 , Michael Paul Busch 5 , Grier Page 2 , Stacy Endres-Dighe 2 , Steve Kleinman 6 , Simone Glynn 7 , Alan Mast 8 and for the NHLBI Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) 9 .
1 American Red Cross, 2 RTI International, 3 American Red Cross Blood Services, 4 Blood Systems Inc., 5 Blood Systems Research Institute, 6 University of British Columbia, 7 NIH/ NHLBI, 8 Blood Research Institute, 9 NHLBI Background/Case Studies: Blood centers and regulators in the United States (US) are evaluating strategies for minimizing iron depletion in blood donors.
The logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors.
Studies donors have been conducted in predominantly Caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy.
Study Design/Method: Over 12,600 donors were enrolled from 4 US blood centers for ferritin testing.
The study population was enriched for racial minorities [1600 African-American (AA), 1600 Asian (As), 1000 Hispanic (Hisp)] and for "Super Donors" (1600, who had completed 101 donations in two years without low hemoglobin deferral).
The minority donors and the remaining 6800 non-Hispanic White (NHW) donors were an unselected population with no specific eligibility criteria.
Subjects completed questionnaires on risk factors for iron depletion.
Logistic regression was used to identify demographic and behavioral predictors of Absent Iron Stores (AIS, ferritin <12 ng/ml) and Low Ferritin (LF, ferritin <26 ng/ml).
Results/Findings: Across all subjects, 19% had AIS and 42% had LF, with a high degree of variability based on demographic factors and donation behavior.
In models stratified by race, expected patterns common to all 4 groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > 50 years old.
In models including all subjects, race was an independent predictor of both AIS and LF controlling for age, sex, body weight, donation frequency, and other factors (Table) .
AA and As donors showed %20% decreased risk for AIS compared to NHW, while Hisp donors had 25% higher risk.
Daily use of exogenous iron reduced risk for LF and AIS by 30 to 40%, respectively, while the estimated benefit from less-than-daily use was lower (5 to 19% protection).
Regular use of antacids was associated with a 20% or greater increment to risk.
Reported use of hormone supplements showed opposing effects in males and females.
Use of oral contraceptives or estrogen in females reduced risk by %15-20%, while males who reported current use of supplemental testosterone had twice the estimated risk for AIS.
Conclusion: This large study confirms the high prevalence of LF and AIS in US donors and the principal risk factors of age, sex, and donation frequency.
The diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance.
In developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population.
Data are reported as mean (6SD) *P < 0.05 compared to Batf31/1, 200uL HOD RBCs MFI, median fluorescence intensity Background/Case Studies: During storage, red blood cells (RBCs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion.
The proportion of cleared RBCs is correlated with storage duration, which may be responsible for the rapid clearance of up to 25% of transfused RBCs, reducing transfusion yield.
It has been shown, using imaging flow cytometry that a subpopulation of morphologically altered RBCs accumulates during storage.
The reduced surface area of these small RBCs (sRBCs) suggests their rapid elimination by the spleen in the hours following transfusion.
This hypothesis remains to be clarified, since the physiological mechanisms of RBC clearance remain to be precisely identified.
Study Design/Method: Murine "young" and "old" RBCs (respectively on D1 and D14 of storage) were transfused into different models including splenectomized or macrophage-depleted mice.
Flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify RBCs retention in organs.
The accumulation, during storage, and the posttransfusion disappearance of sRBCs were analyzed by imaging flow cytometry.
Results/Finding: Using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration (62% on D14 vs 100% on D1 of storage).
A clearance of the storage-damaged RBCs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized (74%) and macrophage-depleted (79%) groups.
As in humans, we observed the accumulation of a subpopulation of small RBCs (mouse small RBC: msRBC) of reduced projected surface area with altered morphology.
These msRBCs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than 50% at 2h post-transfusion.
In contrast, in macrophage-depleted mice, msRBCs are kept in circulation at 2h posttransfusion.
At 24h, these msRBCs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms.
In control mice, storage-damaged RBCs are mostly retained in the spleen but also in the bone marrow (BM) .
No retention is observed in the liver, kidney or lung.
In macrophage-depleted mice, retention is decreased in the spleen and BM.
Conversely, elevated retention is observed in the BM of splenectomized mice, associated with a transient retention in the kidney and liver.
Conclusion: During storage of murine RBCs, damaged RBCs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms.
They include, as observed in humans, a subpopulation of small RBCs which undergoes a rapid macrophage-mediated clearance.
The increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors.
Age Dependent Relapsing and Remitting Autoimmune Hemolytic Anemia in a Murine Model Andrea Sut Ling Wong* 1 , Amanda L Richards 2 and Krystalyn E Hudson 2 .
Background/Case Studies: Breakdown of tolerance to RBC antigens may result in development of pathogenic autoantibodies (autoAb) and lead to autoimmune hemolytic anemia (AIHA), a severe and sometimes fatal disease.
AIHA in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit.
However, the mechanisms behind such observations are not understood.
To gain insight into tolerance (or loss thereof) to an RBC autoantigen, we utilized the HOD mouse, which expresses an RBC-specific triple fusion protein consisting of hen egg lysosyme (HEL), ovalbumin (OVA), and, Duffy (HOD).
HOD mice were bred to a transgenic mouse that expresses a T cell receptor specific for an OVA peptide in HOD presented by MHCII (OTII mice).
Thus, HOD 1 OTII 1 mice are predisposed to have autoreactive CD4 1 T cells.
Study Design/Method: Four cohorts of HODxOTII F1 mice (16-48 mice/ cohort) were bled monthly for 15 months to assess for autoAb production.
Peripheral RBCs were stained with anti-complement (C3) and mouse immunoglobulin Ab.
Spleens were weighed and splenocytes were stained with anti-CD71 and TER119 to assess for the presence of RBC progenitors.
Statistical analysis between HOD 1 OTII 1 autoAb 1 vs. HOD 1 OTII 1 autoAbvs.
HOD -OTII 1 was performed using Kruskal-Wallis test and corrected for multiple testing with Dunn's test.
Results/Finding: OTII CD4 1 T cells were not deleted in the thymus of HOD 1 OTII 1 mice; rather, they matured to the periphery.
Despite these peripheral autoreactive T cells, no detectable autoAb were observed in HOD 1 OTII 1 .
However, as they aged, 15-50% of HOD 1 OTII 1 were positive for RBC autoAb by 6 months.
Thereafter, $50% of the autoAb 1 mice stopped producing autoAb within two months after onset and remained autoAb free throughout the study.
In 3 of the 4 cohorts, 60-100% of autoAb 1 mice were female.
HOD 1 OTII 1 autoAb 1 mice also had enlarged spleens compared to HOD 1 OTII 1 autoAband HOD -OTII 1 mice (0.42g vs. 0.21g and 0.14g, resp., p<0.04).
This may due to RBC consumption, extramedullary erythropoiesis, or both.
Consistent with increased erythropoiesis, elevated numbers of RBC progenitors (CD71 hi Ter119 inter ) were observed in the spleens of HOD 1 OTII 1 autoAb 1 mice but not in HOD 1 OTII 1 autoAband HOD -OTII 1 (2.86% vs. 0.06% and 0.05% resp., p<0.03).
Moreover, autoAb and C3 deposition were found (0.1-2% and 3-10%, resp.)
on Ter119 1 RBCs in all of the HOD 1 OTII 1 autoAb 1 mice analyzed.
Conclusion: Several features known to exist in human AIHA were observed, including age-dependant autoAb production, relapsing of autoimmunity after onset, and an increased frequency in females.
This model may serve as an experimental system to investigate the mechanisms of AIHA.
B5-A01A Reduction in Neutrophil Numbers Is a Risk Factor for RBC Alloimmunization Amanda L Richards 1 , Christopher A Tormey 2 and Krystalyn E Hudson* 1 .
Background/Case Studies: Red blood cell (RBC) alloimmunization occurs in up to 10% of transfusion recipients (excluding ABO and RhD).
The underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not.
Patients who receive multiple RBC units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (MDS) and sickle cell disease patients.
However, despite chronic transfusions, some patients never develop RBC alloantibodies.
It has been recently reported that poly (I:C)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (PMN) numbers.
Additionally, RBC transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by PMNs.
Here, we test the hypothesis that PMNs regulate RBC alloantibody generation.
Study Design/Method: Mice: C57Bl/6 (B6) mice were treated with PBS, or anti-Ly6G to deplete PMNs, followed by poly (I:C) to elicit inflammation, and finally a transfusion of allogeneic DiO-labeled RBCs expressing a synthetic antigen, HOD (HEL-OVA-Duffy).
Multiple splenic cellular subsets were evaluated for DiO fluorescence, an indirect measure of RBC consumption, at 18-24 hours post-transfusion.
Anti-HOD alloantibody generation was assessed 14 days post-transfusion by flow cytometry.
Humans:Retrospectively, mean white blood cell (WBC) and PMN counts were collected on chronically transfused MDS patients at VA Connecticut Healthcare.
For alloimmunized patients (n55), WBC and PMN counts were assessed on the day of exposure to the alloimmunizing RBC unit, whereas counts were averaged for the entirety of RBC therapy for non-alloimmunized patients (n55).
Patients were matched for numbers of RBC transfusions.
Results/Finding: Mice: The MFI of anti-HOD antibodies was significantly increased in PMN-depleted mice, compared to controls (3/3 experiments, p<0.01).
While many control mice made no alloantibody (non-responders), all PMN-depleted mice made detectable anti-HOD.
PMN depletion also led to a significant reduction in DiO1 leukocytes, suggesting a lack of compensatory mechanism(s) for RBC consumption.
Absence of PMNs also shifted RBC consumption from macrophages to immune-stimulating dendritic cell subsets.
Flow cytometric analysis revealed that PMNs with internalized RBCs upregulated expression of co-inhibitory molecules (e.g.
PD-L1), compared to PMNs (without internalized RBCs) from the same mouse; thus, PMNs may regulate alloimmunization through antigen presentation and/or inhibitory signals.
Humans:.
Alloimmunized MDS patients had a significant decrease in PMNs, compared to non-alloimmunized (p<0.05); no significant differences were detected in mean WBC counts between the two arms.
Conclusion: These data demonstrate that in both murine and human settings, PMNs may play a significant role in regulating RBC alloimmunization and may provide key insights into predicting which patients will become alloimmunized.
B9-A03B CXCR5 1 PD1 1 and CCR7 1 Expressions Characterize Responders to RBC Immunization Benoît Vingert* 1,2,3 , Marie Tamagne 1,2,3 , Sadaf Pakdaman 1,2,3 , Anoosha Habibi 2,3,4 , Philippe Bierling 1,2,3,4,5 , Rachid Djoudi 1 and France Pirenne 1,2,3,5 .
1 EFS Ile de France, 2 Laboratory of Excellence GR-Ex, 3 IMRB U955 -Eq2, 4 AP-HP, 5 Universit e Paris Est Background/Case Studies: Post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction.
In human, mechanisms responsible of RBC alloimmunization are not fully defined.
CD4 1 T cells are major for antibodies production.
We have already shown in responder patients that the majority of anti-RBC CD4 1 T cells have a Th17 profile.
In contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating CD4 1 T cells with a CXCR5 1 PD1 1 phenotype.
This phenotype is usually associated with the presence of Tfh cells, specialized in the production of antibodies.
It has been suggested that some of the activated circulating Tfh could have a CXCR5 1 PD1 hi profile, with a differentiated expression of CCR7.
CCR7 is essential for T cells domiciliation in lymph nodes where the encounter T and B cells is major for B cell differentiation and antibody production.
Others chemokines receptor like CCR6 and CXCR3 can also differentiate circulating Tfh subpopulations.
In this study, we were interested in the phenotype and function of these CXCR5 1 PD1 1 lymphocytes which were paradoxically highly represented in non-responder patients.
Study Design/Method: The membrane and functional phenotype of the circulating CXCR5 1 PD1 1 cells were compared in 2 groups of transfused sickle cell patients : alloimmunized (n514) and non-alloimmunized patients (n510).
The analysis was also performed in non-transfused healthy controls (n512).
All assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors Results/Finding: The CXCR5 1 PD1 hi subpopulation expression was identical between transfused groups and controls.
CCR6 and CXCR3 expressions show no difference between the transfused groups or the controls.
However, in non-responder patients, CCR7 expression was very strong independently of the expression of PD1.
In the aim to determine the help of the circulating CXCR5 1 PD1 1 cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for 5 days with B cells, and in the presence of SEB protein.
The levels of antibodies after SEB stimulation were identical with the CXCR5 1 PD1 1 subpopulations from transfused groups or controls.
Conclusion: The paradoxical presence of circulating CXCR5 1 PD1 1 cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response.
However, in responder patients, the high expression of CCR7 on circulating CXCR5 1 PD1 1 cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses.
In conclusion, the study of the CXCR5 1 PD1 1 profile and the CCR7 1 expression in these cells could help to differentiate responder and non-responder patients to RBC immunization.
Primed CD4 T Cells to One RBC Alloantigen Can Enhance Subsequent Alloimmunization Seema R Patel* 1 , Ashley Bennett 1 , Kathryn Girard-Pierce 1 , Connie Arthur 1 , Amanda Mener 1 , Patty Zerra 1 , Christopher A Tormey 2 , Jeanne Hendrickson 3 and Sean Stowell 4 .
1 Emory University, 2 Yale-New Haven Hospital, 3 Yale University, 4 Emory University School of Medicine Background/Case Studies: While red blood cell (RBC) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion.
However, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion.
However, how immunity to one RBC alloantigen primes immunization to a completely distinct alloantigen remains unknown.
Though CD4 T cell help classically occurs through direct recognition of a peptide that resides within a target B cell antigen, individuals who develop antibodies toward one RBC alloantigen experience increased rates of antibody formation against completely distinct RBC alloantigens.
These observations suggest that CD4 T cells that respond to one alloantigen may directly facilitate immunity to a completely distinct RBC alloantigen.
Study Design/Method: B6 recipients were transfused with KEL RBCs in the presence or absence of poly I:C (PIC), followed by transfusion of HOD RBCs, KEL RBCs, RBCs expressing HOD and KEL (HOD x KEL), or a mixture of HOD and KEL RBCs (HOD 1 KEL).
To examine the role of CD4 T cells, PIC/KEL primed B6 recipients were CD4 T cell depleted prior to transfusion.
In addition, B6 recipients were adoptively transferred with CD4 T cells from na€ ıve or PIC/KEL primed donors, followed by transfusion of HOD RBCs or (HOD x KEL) RBCs.
Anti-HOD and anti-KEL alloantibody formation was evaluated using indirect immunofluorescence staining.
Results/Findings: KEL RBC transfusion in the presence of PIC (PIC/KEL) not only enhanced anti-KEL antibody production through a CD4 T celldependent process, but this same priming event directly facilitated anti-HOD antibody formation following subsequent (HOD x KEL) RBC transfusion (p < 0.001); PIC/KEL primed recipients transfused with (HOD 1 KEL) RBCs or HOD RBCs alone failed to impact anti-HOD antibody formation.
The ability of immunity to KEL to boost a humoral response to the HOD antigen following (HOD x KEL) RBC transfusion required KEL priming in the presence of PIC.
CD4 T cell depletion prevented PIC/KEL primed recipients from boosting an anti-HOD antibody response (p < 0.0001) and transfer of CD4 T cells from PIC/KEL primed recipients likewise directly facilitated anti-HOD antibody formation following a (HOD x KEL) RBC transfusion (p < 0.001).
Conclusion: These results demonstrate that CD4 T cells primed to one RBC alloantigen can directly enhance the immune response to a completely distinct RBC alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody Background/Case Studies: Platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood.
Immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of HLA-matched platelets.
Still, many patients are refractory even after receiving HLA-matched platelets.
It was shown previously that CD8 T cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within 24 hours post transfusion in B celldeficient mMT recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of CD8 T cells prevents such clearance.
Since minor antigenic differences still exist between donor HLA-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise HLA-matched platelets.
Study Design/Method: To test whether minor antigens can stimulate CD8 T cell-dependent platelet clearance we examined platelet refractoriness using mOva and OTI transgenic mice.
Leukoreduced donor platelets from mOva mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine MHC class I H2Kb, were labelled in vivowith the fluorescent dye CFSE and transfused into wildtype (WT, C57BL/6) mice or OTI mice, whose CD8 T cell receptors recognize a specific ovalbumin peptide in the context of MHC class I H2Kb.
In some experiments OTI mice were primed with mOva or WT splenocytes one week prior to mOva platelet transfusion, and in others WT mice were adoptively transferred with OTI splenocytes 48 hours before mOva platelet transfusion.
Platelet recovery was measured immediately after transfusion as well as after 2, 4, 8, 16, and 24 hours and on days 2-5.
Results/Finding: Transfusion of mOva platelets into OTI mice results in significant platelet clearance as compared to transfusion with WT platelets.
Clearance kinetics demonstrate platelet loss starting after 8 hours and peaking at 24 hours, and are similar whether OTI mice are na€ ıve or previously primed with mOva splenocytes.
Specifically, mOva platelet recovery in OTI recipients is 28-35% versus >60% in WT recipients at 24 hours (p<0.05), whereas transfusion of WT platelets into either OTI or WT recipients is approximately 60% at 24 hours after transfusion.
Adoptive transfer of OTI CD8 T cells into WT mice recapitulates the effect, with significant mOva platelet clearance at 24 hours compared to WT platelet clearance (p<0.05).
Conclusion: This work extends the ability of CD8 T cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving HLA-matched products.
This study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of T cell responses may prove beneficial.
Background/Case Studies: Alloimmunization against major histocompatibility (MHC) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants.
We have previously demonstrated that pathogen reduction with riboflavin and UV light (UV1R) is effective both at rapidly killing donor white blood cells (WBCs) and at blocking their ability to stimulate an allogeneic response in vitro.
Furthermore, UV1R treatment of allogeneic platelet rich plasma (PRP) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions.
As cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by UV1R, as well as evaluate the immunogenicity of PRP containing WBCs killed by other methods.
Study Design/Method: WBC-rich PRP was prepared from C57Bl/6 mouse blood and treated with UV1R, and WBCs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis.
Membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry.
BALB/c recipients were transfused with either UV1R treated WBC-rich PRP, or UV1R treated WBC-poor PRP either alone or with added untreated, apoptotic, or necrotic WBCs, all generated from allogeneic C57Bl/6 donor blood.
A second transfusion of untreated WBC-rich C57Bl/6 PRP was given 2 weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion.
Results/Finding: UV1R treated WBCs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation.
Alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p<0.01) or necrotic (p<0.05) WBCs, but not those given UV1R treated or apoptotic WBCs.
Ex vivo cytokine responses to stimulation with C57Bl/6 WBCs were reduced in recipients of either UV1R or apoptotic WBCs, and enhanced in recipients of untreated or necrotic WBCs.
Conclusion: The mechanism of WBC death following UV1R treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis.
However, both UV1R treated and apoptotic WBCs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures.
Background/Case Studies: In mitochondria-less red blood cells (RBCs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of RBC storage lesion.
Oxygen saturation (SO 2 ) of venous blood is generally assumed to be around 70-80% as measured from a central venous line.
However, a recent investigation of SO 2 levels in freshly prepared leukocyte-reduced red cell concentrates (LR-RCCs) revealed unexpectedly wide SO 2 distribution (mean 45.9%617.5% [Yoshida et al.
2017; Blood Transfusion 15, 172] .
The present study was undertaken to determine the distribution of SO 2 in LR-RCC produced at a medium-size blood center using a novel non-invasive SO 2 probe.
Additionally, quantitative metabolomics were carried out to examine the redox status of the stored RBC under various SO 2 levels.
Study Design/Method: The SO 2 from 977 units of LR-RCC were examined on five consecutive days representing 78% of the collected units during the period at a regional blood center where all the units were processed at room temperature within 8 hours of blood collection.
SO 2 was measured noninvasively through the PVC bag immediately prior to refrigeration by employing a Resonance Raman spectrometry (Pendar Microvascular Oximeter A3U11; Pendar Technologies, Cambridge MA).
In addition to SO 2 , process methods, RCC volumes, blood types, gender and process times were recorded for analyses.
In a separate study, LR-RCC (n54) from human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions for up to 42 days (SO 2 ranging from <3 to >95%) prior to UHPLC-MS metabolomics analyses in presence of 13 C, 15 N or deuterated internal stable-isotope labeled standards for absolute quantitation.
Results/Finding: Measurements of SO 2 carried out non-invasively at a blood center yielded a similar wide distribution as previous study from 497 units of LR-RCC procured and sampled invasively within 24 hours after blood collection [Yoshida ibid].
The shape of the SO 2 distribution appeared near normal with the mean of 47.0%621.0%, median 45.2%, range < 5% to > 95% and inter-quartile range (IQR) of 31.4%-61.9%.
Male donors showed higher SO 2 compared to female donors (p<0.04).
No correlations were observed between SO 2 levels and processing time, donor age or blood types.
Metabolomics workflow indicated that lower SO 2 levels ameliorate the energy and oxidative metabolic lesion.
Lower SO 2 levels yielded higher rate of GSH synthesis, higher NADPH concentration, higher GSH / GSSG and NADPH/NADP 1 ratios, lower supernatant urate consumption and lower purine oxidation.
The surprisingly wide distribution of starting %SO 2 levels was observed from LR-RCC manufactured at a blood center using 8-hour room Background/Case Studies: Cellular prion protein (PrP C ) is a GPI-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (BM), and lymphoid tissue.
PrP C can be converted post-translationally into scrapie-PrP (PrP Sc ), which is involved in the pathogenesis of neurodegenerative diseases including Creutzfeldt-Jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals.
However, biological functions of PrP C have yet to be conclusively elucidated.
Study Design/Method: In this study, PrP C knockout mice (KO) are utilized to investigate the role of PrP C in the hematopoietic system with controls of age and sex-matched PrP C transgenic mice harboring a slightly augmented PrP C expression.
Peripheral blood was examined by hematology analyzer to establish counts.
Bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages.
Histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy.
Results/Finding: Complete blood count (CBC) showed a significant increase of WBC in KO mice.
Closer analysis of WBC differential revealed that the elevated number of WBC in KO mice was due to lymphocytosis.
Specifically, KO mice had a 3-fold increase in the absolute lymphocyte count (KO 7.59 6 4.63 x10 9 /L vs. WT 2.90 6 1.32 x10 9 /L, p 5 0.0303), as well as a higher lymphocyte percentage compared to controls.
KO mice also had a trend toward higher hemoglobin, RBC, and hematocrit compared to WT mice.
Additionally, platelet count in KO mice was higher than control mice.
Of interest, the mean platelet volume indicating platelet size was significantly increased in KO mice compared to controls (KO 6.00 6 0.29 fL vs. WT 5.24 6 0.56 fL, p50.0140).
A comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of RBC and megakaryocyte in BM, and of lymphocytes in the thymus, spleen and lymph nodes.
Histological analysis of BM, thymus, spleen and lymph node tissue from KO and WT animals failed to show morphological differences between the two groups.
Conclusion: Absence of PrP C resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis.
Our findings indicate that PrP C might be critical in the survival and trafficking of lymphocytes in peripheral blood.
The molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies.
Potential Role of CD81FOXP31 Regulatory T Cells Derived Exosomes in Their Immune Modulation Yiming Yang*, Rufeng Xie and Jie Yang.
Blood Engineering Laboratory, Shanghai Blood Center Background/Case Studies: Exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process.
Human periphery blood CD8 1 FOXP3 1 Tregs cells are reported as more stable regulatory cells with greater inhibition effects.
However, CD8 1 FOXP3 1 Tregs derived exosomes and their functions involved in CD8 1 Tregs mediated immune-modulation were seldom reported.
Study Design/Method: CD8 1 T cells were freshly purified from PBMCs, cultured with anti-CD3/CD28 antibody packaged beads and IL-2, and then polarized with TGF-b and rapamycin into CD8 1 FOXP3 1 Treg cells.
The harvest cells were co-cultured with CD3/CD28 beads stimulating CD41CD25effector cells in the transwell plate.
The supernatant derived from CD81 Tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with PEG.
The harvest precipitation was resuspended in PBS and exosomes were analyzed by SEM and NTA.
Exosome surface marker CD63, CD81, TSG101 and other proteins expression were evaluated by flow cytometry and western blot.
microRNA was isolated with miRcute miRNA kit and miR-155, let-7b, let-7d were measured by qPCR.
The precipitated exosomes were further purified by CD63 immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation.
Results/Finding: As compared with direct contact co-culture, separated CD8 1 Treg cells could suppress the proliferation of effector cells with a small decline (p>0.05), which means some non-contact factors involved in the CD8 1 Treg mediated immune modulation.
A total number of 4.57 6 0.52 3 10 8 /10 6 cells exosomes were harvest.
Electron microscope analysis demonstrated a kind of round-shaped membrane vesicle 50-150nm in diameter (145.16 6.7nm by NTA).
CD63 and CD81 were expressed on these Background/Case Studies: Regulatory T cells (Tregs), containing CD4 1 and CD8 1 subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (AIDs).
Unfortunately, due to the instability of natural CD4 1 Foxp3 1 regulatory T cells (nTregs) in inflammation conditions (including instability of Foxp3, conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate CD81 regulatory T cells stability both in vitro and in vivo.
In our previous works, we found that CD8 1 Treg has an effective therapeutic function on CIA mice.
In this study we aim to investigate the stability of induced polyclonal human CD8 1 regulatory T cells in inflammation and transfusion.
Study Design/Method: Human CD8 1 Tregs were induced with TGF-b1 and rapamycin from CD8 1 T lymphocytes in vitro.
Collagen-induced arthritis (CIA) mice were induced with type-two collagen as an autoimmune disease model.
In vitro the stability of CD8 1 Tregs when encountering with inflammation were test by Foxp3 expression, Th1 and Th17 cells conversion in inflammations conditions (IL21TGF-b11IL211IL23 and IL21TGF-b11IL1b1IL6) on day3, day6 and day9.
In vivo, CD8 1 Tregs were transfused into CIA mice and then their survival in mice and Foxp3 express were evaluated to reveal the stability of CD8 1 Tregs in an inflammation condition model.
Additionally, we also investigate the stability maintenance of CD8 1 Tregs when induced factor TGF-b1 and rapamycin were removed by testing the Foxp3 expression on day3, day6 and day9.
Results/Finding: Ex vivo induced human CD8 1 Treg were Foxp3 1 (90.40 6 1.40%) and did not secret IL17A (both in supernatant and % of cells).
Foxp3 express in CD8 1 Tregs were maintained after induced factor TGF-b1 and rapamycin were removed on day3, day6 and day9.
In vitro, Foxp3, IL2 and IFN-c expression has no significant difference when compared with controlled Tregs on day3, day6 and day9 and did not secret IL17A when encounter with inflammation conditions (IL21TGF-b11IL211IL23 and IL21TGF-b11IL1b1IL6).
In vivo, CD81 Treg cells were transfused into CIA mice on the peak of disease onset (35 days after the first Collagen immunization, has inflammation condition in vivo) to test CD8 1 Tregs survival.
CD8 1 Tregs were found in CIA mice foot (27.4 6 2.03%), blood (4.55 6 1.03%) and spleen cells (1.90 6 0.05%) 72 hours after transfusion and their % of foxp3 1 were remained.
Conclusion: The results revealed that ex-vivo induced and expanded human CD8 1 Tregs are stable in inflammation and transfusion and can maintain Foxp3 expression when induced factor were removed, these make CD8 1 Treg a novel and stable cell for potential cell therapy on AIDs.
This research can provide some instructive reference and improve the utilization of blood components.
Tolerogenic Dendritic Cells Induced By mTOR Suppression and Control Inflammation in Chs Model through S6K Related Proteins Translation Inhibition.
Li GAO*.
Shanghai Blood Center Background/Case Studies: Tolerogenic dendritic cells (tDCs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA),et al.
However, the traditional strategy base on the tDCs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tDC adaptive cell therapy in future clinical use.
Study Design/Method: Human tDCs were derived from fresh purified monocytes from PBMNCs isolated from buffy coat and induced by mTOR inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated.
The mature markers and endocytisis were detected by flow cytometry.
The production of cytokines and chemokines was measured using ELISA.
Mechanism investigation was analysis by Real-time PCR and Western blotting.
Contact hypersensitivity (CHS) model, an atopic dermatitis animal model, was treated with tDCs induced via mTOR suppression and analyzed by ear thickness and tissue leukocytes number calculating.
Results/Finding: Human tDCs treated with mTOR inhibitors had a lower mature marker CD83/CD80/CD86 expression after TLR signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the LPS absent group.
Moreover, mTOR suppression extremely reduced the capacity of LPS treated human DCs to stimulate autogenic na€ ıve T cell proliferation, which is one of the most important characteristics of tDC.
Beyond expectation, the common signal transduction pathway, MAPK and NF-jB pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tDC when exposure to LPS stimulation.
However, the P70S6K and its downstreanm proteins, especially the protein S6, which controls the protein translation, were shown in charge of the tolerogenic induction mechanism.
The data were also supporting the suggestion that rare difference on mRNA transcription of the related functional proteins in tDCs induced by mTOR inhibitors when exposure to LPS stimulation from the non-induced cells, although there was more transcription of IDO induced by mTOR inhibitors.
More important, edema responses of ears were clearly weakened in the CHS model and recruited less leukocytes to the tissue when co-sensitized with mTOR inhibitors or with tDCs induced by mTOR inhibitors suggested that the tDC induced by mTOR suppression were able to control hypersensitivity inflammation response in vivo.
Conclusion: Accordingly, tDC induced by mTOR suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mTOR-S6 related protein translation inhibition.
Xiaoyun Fu* 1,2 , Mikayla Anderson 1 and James C Zimring 1,2 .
1 BloodworksNW Research Institute, 2 University of Washington School of Medicine Background/Case Studies: Red blood cells (RBCs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion.
Bioactive lipids generated during RBC storage have been implicated in certain adverse outcomes.
Recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) accumulate during RBC storage despite leukoreduction.
To evaluate the extent of membrane lipid degradation and oxidation in stored RBC units among the donor population with different blood groups, we quantified PUFAs and lysophospholipids (LPLs) in 405 leukoreduced RBC units.
Study Design/Method: RBC units from different donors were acquired and processed on day 43 (one day past their expiration).
35 bioactive lipids including 10 common Fatty acids, 10 oxylipins, and 15 LPLs were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM).
Total fatty acid concentrations of selected units were also analyzed.
A One-way ANOVA test was used to determine significant difference of analytes amongst the different blood groups.
Results/Finding: We observed a wide distribution in concentration of major PUFAs in 405 stored RBC units.
For example, arachidonic acid (AA) ranges from 0.5 -10.7 mM, linoleic acid (LA) (1.4-28 mM), dihomo-c-linolenic acid (DGLA) (0.1-0.8 mM), eicosapentaenoic acid (EPA) (0.03-3.1 mM), docosahexaenoic acid (DHA) (0.2-3.0 mM), and alpha-linolenic acid (ALA) (0.06-2.3 mM).
Ten oxylipins including HETEs, HODEs, and DiHOMEs, and 15 LPLs including LPCs, LPSs, and LPEs all showed a large variation in concentration among donors.
Of 35 analytes quantified, 25 showed a significant difference in concentration among different blood types by one-way ANOVA testing (FDR<0.05).
The AB Rh1 blood group consistently exhibited the lowest concentration of major PUFAs, while the O Rh-blood group showed the highest, averaging a two-fold difference in concentration (O Rh-/AB Rh1).
The fold increase of O Rh-/O Rh1 among PUFAs ranges from 1.3 to 2.1, suggesting the Rh blood group, independent of the ABO blood group, correlates with donor to donor variation in lipid metabolism.
Conclusion: The wide distribution in the concentration of bioactive lipids among 405 stored RBC units suggests that lipid degradation is highly donor-Background/Case Studies: To ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality.
In the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions.
microRNAs (miRNAs) levels are modulated by these storage-related damages, which makes miRNAs ideal candidates as potential biomarkers of quality monitoring.
Lately, nanoparticles have been widely studied and used for biosensing applications.
The objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals.
Study Design/Method: Gold nanoparticles (GNPs) surrounded by a fluorescent silica shell were prepared using a wet chemistry method.
miRNA-223 was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions.
Custom RNA and DNA molecular beacons were designed and used as a probe for the specific detection of miRNA-223 targets in PBS and human plasma.
These fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-GNPs using an EDC/NHS cross-linking reaction.
The hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence.
Results/Finding: GNPs (49 6 6 nm) surrounded by a thick fluorescent silica shell (22 6 2 nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability.
Conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis.
The fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (R 2 RNAprobe 5 0.96 and R 2 DNAprobe 5 0.98) with miRNA-223 concentration, down to a 10-nM limit of detection.
Hybridization assays in 1% human plasma appear to demonstrate denaturation of RNA probes and targets.
Conclusion: Biocompatible fluorescent GNPs were prepared and used as tools for blood product characterization.
The conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques.
The functionalization of the probe is still being optimized.
The fluorescence response of the molecular beacon was characterized for the detection of a model miRNA target in PBS and in 1% human plasma.
Energy Metabolism Profile of Erythrocytes during Storage Suping Ren*, Qun Yu, Yanbing Wang, Changlan Li and Yu Wang.
Background/Case Studies: The moment the mature red blood cells (RBCs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues.
Red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell.
Glycolysis is the only energetic metabolic pathway for mature RBCs to obtain ATP which is the energy for RBCs to maintain a number of vital cell functions.
Generally, the current methods used to measure RBCs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.XF technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, RBCs have no nucleus, mitochondria and other organelles, so application of the XF technology in erythrocytes and exploration of the assay conditions are necessary.
6.7 6 0.0 a 6.9 6 0.0 6.7 6 0.0 a 6.6 6 0.0 6.7 6 0.0 a 6.9 6 0.0 Total ATP,lM/gHb 7.6 6 0.3 a 5.2 6 0.3 7.3 6 0.2 a 5.5 6 0.3 7.3 6 0.4 a 4.9 6 0.5 Extracellular Lactate,mM 5 6 1 a 6 6 1 7 6 0 8 6 0 6 6 0 6 6 1 Extracellular Glucose,mM 32 6 1 a 50 6 3 2 9 6 1 a 38 6 1 2 5 6 0 a 28 6 1 Extracellular Na 1 ,mM 142 6 2 143 6 2 138 6 1 a 137 6 1 144 6 1 141 6 1 Extracellular K 1 ,mM 1 6 0 a 4 6 0 1 6 0 a 5 6 0 1 6 0 a 4 6 0 a p<0.05, paired t-test b INTERCEPT Blood System for Red Blood Cells is not approved for commercial use.
c This project has been funded in whole or in part with Federal funds from the DHHS; ASPR; BARDA; Contract No.
HHSO100201600009C.
Background/Case Studies: Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer.
In vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported.
Study Design/Method: Swirling of distributed products was monitored one year before and one year after implementation of Intercept pathogen inactivation.
Metabolic parameters like pH, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products.
Furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling.
Results/Finding: In an experimental phase on a limited number of products (n56) to validate the Intercept pathogen inactivation method, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products.
Once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former.
Furthermore, the number of apheresis products with premature low swirling increased by 50% (63/12,492) compared to the previous year without pathogen inactivation (42/12, 931, P50.030, Chisquare) .
In contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased (2/15,286 vs 10/ 13,488).
Of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5.0x10 11 ) than unaffected controls (3.5x10 11 ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects.
These signs included lower pH, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation.
Conclusion: The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above 5.0x10 11 .
In Vitro Quality of Single Dose Amotosalen/UVA Treated Platelets in 35% Plasma/65% PAS-3 after 5 Days of Storage Crystal Stanley 1 , Marguerite Kelher 2 , Nero Evero 1 , Melissa VonGoetz 3 , Betsy Donnelly 3 and Anna Erickson* 3 .
1 Belle Bonfils Memorial Blood Center, 2 University of Colorado, 3 Cerus Corporation Background/Case Studies: The INTERCEPTV R Blood System for platelets is FDA approved for the ex vivo preparation of pathogen-reduced Amicus O apheresis platelet components (PC) in PAS-3 to reduce the risk of TTI, including sepsis, and to potentially reduce the risk of transfusion-associated GVHD.
Registration studies (ClinicalTrials.gov NCT02298842) are in progress to support approval of the Trima O apheresis platform for collection of platelets components (PC) suspended in PAS-3 and plasma.
The objective of this study was to evaluate in vitro function of platelets suspended in 35% plasma/65% PAS-3, collected using the Trima platform, after treatment with the INTERCEPT Blood System for platelets.
Study Design/Method: Double dose apheresis PC, 7.5 6 0.6 310 11 platelets in 602 6 52 mL, were collected on the Trima apheresis platform in 35% plasma/65% PAS-3.
A sample was taken from each donation prior to dividing the donation to produce INTERCEPT treated apheresis PC (T), using the small volume (SV) set, and an untreated control PC (C).
Input volumes for replicates, n56, were 302 6 26mL (T) and 300 6 27mL (C) with doses of 3.8 6 0.3 3 10 11 (T) and 3.7 6 0.3 3 10 11 (C).
All PC were stored under the same conditions and evaluated on Day 5 and Day 7 for physical/metabolic characteristics.
Results/Finding: On Days 5 and 7 all T and C PC had pH228C !6.2.
The dose recovery for T was 87%63%.
On Day 5, T had lower count, volume, dose, bicarbonate and glucose compared to C PC; however, parameters predictive of in vivo function (ATP, morphology score, HSR, and ESC) were equivalent between T and C (Table 1) .
Conclusion: Trima PC in 35% plasma/65% PAS-3 treated with the INTER-CEPT Blood System for platelets using the SV set and stored for 5 days retained in vitro metabolic and functional properties consistent with in vivo functionality.
Induction of Pluripotent Stem Cell-Derived Cardiomyocyte Toxicity By Supernatant of Long Term-Stored Red Blood Cells in Vitro Feng-Yan Fan 1,2 , Yang Yu 1 , Li-Ping Sun 1 , Shu-Fang Wang 1 , Rui Wang 1 , Lei-Ying Zhang 1 and Deqing Wang* 1 .
1 The Department of Blood Transfusion, The PLA General Hospital, 2 The Department of Blood Transfusion, Air Force General Hospital, PLA Background/Case Studies: Recently, multi researches have reported that longer term-stored red blood cells(RBCs) units were associated with increased risks of clinically adverse events, especially in critically ill patients.
However, other studies have concluded the negative results.
Whether RBCs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic.
To study the adverse effects of longer term-stored RBCs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(SSRBCs), and study the possible mechanism.
Study Design/Methods: 1 Five doses of leuko-reduced RBCs were prepared, and supernatant was isolated by centrifugation on d0, d14 and d35.
We looked at the cardiotoxicity of SSRBCs on human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs).
HiPS-CMs were treated with SSRBCs in 17% final volume simulating the large volume blood transfusion.
Using real-time cellular analysis (RTCA) technology the beating of hiPS-CMs was recorded in real time in detail.
2 Levels of K and lactic acid (LA) were tested using automatic biochemical analyzer.3 K and LA solution with concentrations being consistent with SSRBCs were prepared and cocultured with hiPS-CMs.
We analyzed the cardiotoxicity of K and LA solution on hiPS-CMs.
4 Treated hiPS-CMs with d35 SSRBCs, d35 K and cell culture media for 48h.
The nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence.
Total RNA of hiPS-CMs was isolated and mRNA analysis microarray was implemented.
Screened for toxic effects related signaling pathways through bioinformatics analysis.
Results/Findings: 1 d0 SSRBCs had no obvious influence on beating state of HiPS-CMs-HiPS-CMs treated with d14 SSRBCs stop beating, but beating patterns restored at 48h.
HiPS-CMs treated with d35 SSRBCs stop beating, and beating patterns did not restored at 48h.
2 Levels of K and LA in SSRBCs changed most obviously.
3 Only d35 K solution made hiPS-CMs stop beating and can restore in 48h; d0 K, d14 K and LA solution did not influence the beating pattern in At the end of the treatment for 24h, hiPS-CMs treated with d35 SSRBCs show obvious shrinkage.
At the end of the treatments for 48h, cells treated with d35 K and d35 SSRBCs both show obvious shrinkage, the shrinkage in d35 SSRBCs group was more serious.
The immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected.
5 Gene Expression Array results show a total of 140 genes were differentially expressed in d35 SSRBCs group compared with naive group.
There was no consistent separation within the d35 K and naive group.
Fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect.
Under the condition of simulating the large volume blood transfusion, SSRBCs of long term-stored RBCs have toxic effect on myocardial cells.
2 In addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects.
3 Further study should be applied to signal pathways on SSRBCs induced cytotoxicity.
4 Large volume transfusion of long term-stored RBCs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it.
Background/Case Studies: Processing thawed, deglycerolized red cell concentrates (RCC) in a functionally closed system allows for a prolonged storage after thawing.
Thawed cells are better maintained in AS-3 as compared to SAGM.
The presence of citrate in AS-3 seems to be necessary to prevent hemolysis of thawed cells.
During storage in AS-3, ATP and 2,3-DPG levels rapidly decline.
Recently developed additive solutions like PAG3M and AS-7 have shown to better maintain 2,3-DPG and ATP levels during storage of normal, unfrozen, RCC.
However, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells.
We therefore designed PAG3C in which the mannitol of PAG3M was replaced by citrate.
The aim of this study was to investigate the in vitroquality of thawed, deglycerolized RBC during storage at 2-68C in PAG3C.
Study Design/Method: Leukoreduced RCC (n56) in PAG3C (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at 2-68C.
On day 8, RCCs were glycerolized using ACP215 (Haemonetics V R , Braintree, MA) to a final concentration of 40% (w/v), frozen and stored for at least two weeks at -808C.
After thawing and deglycerolization using ACP215, RCC were resuspended in PAG3C.
During storage at 2-68C, stability (hemolysis), ATP and 2,3-DPG levels were determined.
Results were compared with thawed RCC (prefreeze storage in SAGM, n58) resuspended in or SAGM (n54).
Results/Finding: Pre-freeze storage in PAG3C resulted in increased 2,3-DPG levels at day 8 as compared to storage in SAGM, resp.
9.1 6 7.6 mmol/g Hb and 1.9 6 0.7 mmol/g Hb.
Hemolysis during post-thaw storage in PAG3C remained below 0.8% for 35 days and was comparable with storage in AS-3.
In SAGM, hemolysis remained below 0.8% for 7 days.
During the first 2 weeks of post-thaw storage in PAG3C, both ATP and 2,3-DPG levels increased, followed by a gradual decline during prolonged storage.
During the whole postthaw storage period, RCCs in PAG3C showed significantly higher ATP and 2,3-DPG levels compared to AS-3 or SAGM.
While in SAGM and AS-3, 2,3-DPG levels were undetectable after 7 days post-thaw storage, in PAG3C, 2,3-DPG levels only decreased to 6.1 lmol/g Hb after 35 days of storage.
Conclusion: Pre-freeze storage in PAG3C resulted in increased 2,3-DPG levels.
As compared to AS-3, post-thaw storage in PAG3C showed comparable hemolysis while ATP and 2,3-DPG levels were much better maintained.
Based on a maximum allowed hemolysis of 0.8% and an ATP content of >2.7mmol/g Hb, thawed RCC can be stored at 2-68C for 35 days in PAG3C.
Background/Case Studies: Platelets (PLTs) are vital for effective treatment of hemorrhage.
Cold (48C, 4C) storage of PLTs in platelet additive solution (PAS) is a promising alternative to conventional storage at room temperature (RT) due to a lower risk of bacterial concerns, preservation of PLT function, and mitigation of PLT activation.
Currently only 2 apheresis (AP) and PAS systems are FDA-approved for use in the US: Trima and Isoplate-PAS (ISO; Terumo) and Amicus and Intersol (INT; Fenwal) .
The goal of this study was to assess the adhesive function of long-term cold-stored PLTs collected by FDA-approved AP/PAS methods.
Study Design/Method: PLTs were collected (n54-5) in 65% ISO using a Trima or in 65% INT using an Amicus and stored for 15 days at RT and 4C.
Samples were tested on Day 1 (baseline, BL), 5, 10, and 15 of storage to assess PLT adhesion under shear flow (Bioflux).
ACD vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (RBCs) for all Bioflux runs.
Simulated whole blood was created by combining PLTs labeled with calcein-AM with RBCs at 40% Hct.
Labeled blood was perfused through microfluidic channels (Fluxion) coated with 100 ug/ml type-1 collagen at 720s -1 shear rate.
Images were acquired every 30 sec for 10 min using a fluorescent microscope and % surface coverage was reported.
Data were analyzed using two-way ANOVA and posthoc Tukey test with significance at P<0.05.
Results/Finding: Both RT-INT and RT-ISO PLTs showed significantly decreased adhesion by Day 5 of storage compared to BL (BL: 11.6 6 1.7%, RT: 4.9 6 1.2%; p<0.005).
4C-INT samples showed no difference in adhesion at any timepoint compared to BL-INT but significantly enhanced adhesion compared to both RT-INT and RT-ISO.
In contrast, 4C-ISO PLTs showed significant enhancement of surface coverage compared to BL-ISO by Day 5 (P50.03) and compared to 4C-INT by Day 10 (P<0.01).
Conclusion: Our work suggests that 4C storage of PLTs collected with a Trima AP system in ISO for up to 15 days offers a significant enhancement in adhesive function compared to PLTs collected with an Amicus system in INT and stored at 4C.
These results are surprising since both 4C-INT and 4C-ISO have been shown to express similar levels of CD62P, PAC-1, and phosphatidylserine and may suggest differences in PAS PLT intracellular signaling.
As expected, storage at 4C of PLTs collected on either platform demonstrated superior function to RT storage.
A PLT product with superior hemostatic function and a shelf-life 3x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster PLT availability for trauma care in the US.
TABLE 1.
Comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after 2-week storage between unirradiated and irradiated groups (n 5 29) TANG, is an anti-inflammatory agents and has a good safety records in clinic.
It could reduce the severity of experimental autoimmune encephalomyelitis (EAE), asthma, colitis, systemic lupus erythematosus(SLE) and other immune diseases.However,its potential in inducing transfusion tolerance remains to be explored.The aims of our study are to find if baicalin could inhibit red blood cell (RBC) immunization and to elucidate the possible mechanism of YIN-CHEN-TANG in preventing HDN.
Study Design/Method: We used human red blood cells with adjuvant Lipopolysaccharide (LPS) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on RBC immunization.
Mice were divided into a normal control group, a human RBC transfused positive control group receiving human RBC and LPS intravenously weekly for five weeks, a control group receiveing dexamethasone (10mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin (250mg/kg/day) intraperitonealy daily for five weeks.
Assessment of human RBC immunization was performed by measuring serum immunoglobulin G (IgG) and immunoglobulin M (IgM) against human RBC weekly.
And the lymphocyte changes in spleen are also monitored by flow cytometry.
Results/Finding: We found that baicalin treatment decreased serum IgG but not IgM production significantly since the second week, with a concomitant reduction in Th17 cells and increase in CD4 regulatory T cells in both spleen and mesenteric lymph nodes.
And there are no significant differences in the percentage of Th1,Th2,Tfh and Tfr CD4 subpopulation among all groups.In addition, baicalin treatment didn't decrease the size of spleen and the percentage of CD4 positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse.
Our results indicate that baicalin could inhibit RBC immunization especially IgG production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.Considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events.
In addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for YIN-CHEN-TANG as a widely used Chinese herbal medicines in preventing HDN.
Comparison of Immucor's Pak Plus and Pak Lx Assays for the Detection of Human Platelet Alloantibodies Randy M Schuller* 1 , Sarah Kloss 1 , Sara Crew 1 and Sandra J Nance 2 .
1 American Red Cross, 2 American Red Cross and American Rare Donor Program Background/Case Studies: Alloantibodies directed against human platelet membrane glycoproteins (GP) Ia, IIa, IIb, IIIa, Ib, IX, IV, and CD109 have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (FNAIT), post-transfusion purpura (PTP), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma.
Polymorphic epitopes on these GPs give rise to 28 unique human platelet antigens (HPA).
Identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders.
Currently the only 510k FDA approved test permits the identification of these HPA antibodies to the glycoprotein level.
Immucor has recently released Pak Lx, a research use only (RUO) assay in the United States that has the ability to identify HPA antibodies to a single nucleotide polymophism (SNP).
We compared the performance of Pak Lx to the FDA approved Immucor PakPlus.
Study Design/Method: We compared PakPlus and Pak Lx results from 40 plasma and serum clinical specimens.
Group 1 contained a single HPA alloantibody specificity with or without HLA antibodies (n526).
Group 2 included 5 specimens with HLA antibodies alone and Group 3 consisted of 9 patient samples that were negative for both HPA and HLA antibodies.
Pak Lx utilizes a Luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, GPIV) and HLA Class I. PakPlus uses an ELISA method and results can only be reported to the glycoprotein location (GPIIb/IIIa, GPIa/ IIa, GPIb/IX, GPIV) along with HLA Class I.
However, based upon the pattern of reactivity observed in the PakPlus and Pak Lx assays it is possible to determine the most probable HPA antibody specificity to the HPA SNP.
Results/Finding: Conclusion: When analyzing HPA antibody specificity, there is 100% concordance observed for HPA-1a, HPA-1b and HPA-5b antibodies.
The Pak-Plus assay had difficulty discriminating HPA-5b from HPA-5a antibody when HPA-5a antibody was present (3 false positive samples) although the Pak-Plus signal OD to cutoff OD ratio was significantly higher for HPA-5a when compared to HPA-5b in these samples.
The discordant HLA Class I antibody results between the assays was isolated to very weakly positive antibody (within 10% of the cut-off for PakPlus and <2.0 adjusted ratio for Pak Lx).
We conclude that Pak Lx is an easy to use platelet alloantibody screening method that has the ability to differentiate HPA antibodies to the allele level.
Histo-Blood Group Antigen Lewis Y Promotes Cell Migration Via Regulation of Microtubule Acetylation Huijun Zhu* and Ping Lu.
Shanghai Blood Center Background/Case Studies: Blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction.
Beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes.
Lewis Y is a histo-blood group antigen belonging to ABH family.
LeY consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology.
LeY is demonstrated to affect cell mirgration via various mechanisms.
However.
although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and LeY.
As microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of LeY in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice.
Study Design/Methods: We first manipulate LeY expression in breast cancer cells by overexpression or siRNA knockdown of fucosyltransferases, and block LeY activity in MDA-MB231 cells using anti-LeY antibody, to verify the effect of LeY on cell migration.
Then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability.
To establish the role of LeY in cell migration via microtubule modification, we use HDAC6 specific (Tubacin) and nonspecific (TSA) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of FUT1 overexpression on cell motility.
Results/Findings: FUT1 overexpression increases both LeY expression and cell migration, while FUT1 knockdown leads to the opposite.
LeY activity blockade by anti-LeY antibody also significantly inhibits cell migration.
Western Blot and Immunostaining results show a-tubulin acetylation level is negatively related with LeY expression.
Tubacin or TSA treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of FUT1 overexpression in promoting cell migration is eliminated.
Conclusion: It can be concluded from the results above that LeY can promote cell migration via regulation of a-tubulin acetylation, wherein LeY may have interaction with deacetylase HDAC6.
As tumor promoter, HDAC6 becomes the target of many anti-cancer drugs.
We demonstrated the potential association of LeY and HDAC6 function in this study.
Many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to LeY; they can even be components in a network with other important molecules and contribute to the destiny of diseases.
Transfusion of blood products is frequently needed by tumor patients.
Most attention is focused on the search of compatible blood for reducing transfusion reaction.
However, it may lower the chance for the disease to advance to take account Background/Case Studies: Reducing the risk of bacterial contamination in platelet (PLT) products is of great concern since PLT storage occurs at room temperature (RT).
Pathogen reduction technologies (PRT) were developed to inactivate pathogens prior to transfusion; however, studies have shown that PRT may damage PLTs over the course of extended storage at RT resulting in a greater loss of function than what is normally concomitant with platelet storage lesion.
Storage of PLTs in platelet additive solution (PAS) at 48C helps to preserve PLT function and reduces the risk of contamination.
In this study, we established the impact of PRT performed after long-term coldstorage of PLTs in PAS, instead of before storage, on PLT function, mitochondrial respiration, and cell death parameters.
Study Design/Method: PLT units were collected in PAS (n53) and stored at 48C for up to 10 days.
After this time period, the bag was treated using Mirasol PRT (riboflavin and UV).
Samples were obtained and tested on the day of collection (baseline, BL), pre-Mirasol (PRE), post-Mirasol (POST), and 30 minutes post-Mirasol .
Aggregometry (ADP, Collagen, TRAP), ROTEM, flow cytometry (CD62P [P-Selectin] , lactadherin [PS] , , and GPIb), high-resolution respirometry (Oroboros), and imaging flow cytometry (Amnis) were used for analysis.
Data are reported as means6SEM, and paired student's t-tests were used to determine statistical significance (p<0.05).
Results/Finding: On Day 10, P-Selectin levels were significantly higher in PRE than BL (p50.03).
Mirasol treatment caused a significant increase in PAC-1 expression compared to PRE (PRE: 10.5 6 3.1%, POST: 28.1 6 4.7%; p50.04), which remained after incubation.
A significant drop in both Collagen and TRAP aggregation was observed in POST samples compared to PRE, but ADP aggregation response was preserved.
No differences in P-Selectin, GPIb expression, and mitochondrial respiration were observed between PRE and POST samples.
POST-30 samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to PRE and POST.
Conclusion: PRT treatment of PLT units in PAS after 10 day storage at 48C presents a unique alternative to PRT treatment of PLTs prior to RT storage.
In addition to providing a lower risk of bacterial contamination, 48C-stored PAS PLTs may provide better preservation of hemostatic function than standard-of-care RT PLTs, even after Mirasol PRT treatment.
However, we show here that Mirasol PRT of Day 10 4C-stored PAS PLTs followed by incubation (30 minutes or more) results in widespread cell damage and should be avoided.
Safety Evaluation of Lyophilized Canine Platelets in a Model of Coronary Artery Bypass Graft (CABG) Todd M. Getz* 1 , Arthur P. Bode 1 , Anne S Hale 2 , Michael Stanton 3 , Mark Johnson 4 and G. Michael Fitzpatrick 3 .
1 CellPhire, 2 BodeVet, Inc, 3 Cellphire, 4 Background/Case Studies: Cellphire has completed a micro dose clinical safety trial using lyophilized human platelets.
Cellphire also evaluated the safety of Lyophilized Canine Platelets (LCP) in comparison to Liquid Stored Canine Platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (CABG) in the canine.
This safety study was in support of a future Phase II human clinical trial in cardiac patients.
Study Design/Method: Three groups of eight mixed breed hounds underwent CABG to create an anastomosis and were administered LCPs equal to 33, 10, and 3.3% of the Total Circulating Platelet Count (TCPC).
One group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control (2-day old liquid-stored platelets).
Safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the 4 hr evaluation period.
Full necropsies with complete tissue analysis were also performed.
Efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (PT, APTT, Fibrinogen, and TEG).
The results demonstrated that administration of the test article at doses up to 33% of the TCPC was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group.
The mortality noted on study was considered to be related to the surgical model and not a result of test article administration.
The results also demonstrated that administration of doses of 10% and 33% of the TCPC produced a significant decrease in blood loss.
The LCPs at 10% and 33% TCPC were as effective in mitigating blood loss as 2-day old liquid-stored platelets and trended towards being more effective.
No appreciable differences in coagulation parameters were observed between groups.
Conclusion: The results of the study demonstrate that administration of LCP up to 33% of the TCPC was safe in a canine CABG model.
The data also demonstrate that administration of LCP at doses of 10% and 33% of the TCPC reduced blood loss.
These results suggest a starting dose above 3.3% TCPC may be required to achieve an effective dose in future human Phase II trials in cardiac patients.
Although the study was not powered for efficacy, these data indicate a level of safety, as 10% TCPC had similar efficacy signals as 33% TCPC with no observable severe adverse events.
The starting effective dose may vary depending on the clinical indication.
Future studies will be required.
This study was funded under BARDA contract HHSO100201300021C.
The Study on PCR-SSP Technique for the Genotyping of CD36 329-330del.AC Mutation and the Genetic Polymorphism of CD36 329-330del.AC in Chinese Population Lilan LI* and Guoguang WU.
Nan-Ning Institute of Transfusion Medicine Background/Case Studies: CD36 (Platelet glycoprotein IV, SCARB3) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in Chinese population.
Except anti-HLA, anti-CD36 is the most common antibody of clinically relevant platelet antibodies in Chinese population, which is associated with the high frequency of CD36 deficiency in China.
CD36 gene mutation is the main reason that leads to CD36 deficiency.
CD36 329-330del.AC (frameshift at AA110) mutation is one of the CD36 mutations that causes CD36 deficiency.
have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood.
The recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions.
This places an already highly vulnerable pediatric population at risk for acquiring preventable infections.
The primary aim of this project was to determine MMR vaccination immunogenicity in patients chronically transfused with RBC.
Study Design/Method: Medical charts were reviewed for vaccination and transfusion histories.
MMR-specific antibodies were quantified in 28 pediatric patients who received both doses of the MMR vaccine at 12 and 18 months of age while they were on a chronic RBC transfusion program for sickle cell disease, B-thalassemia major, Diamond-Blackfan anemia or pyruvate kinase deficiency.
There was no formal control group; long-term immunity rates in the literature are !90% for all MMR components.
Results/Finding: Table 1 shows immunogenicity to vaccine components.
Delays between vaccination and serology testing averaged 6.8 years (0.5 to 16.5 years).
Thirteen patients (46%) were chronically transfused at the time of serology.
Twenty-three patients (82%) seroconverted to at least one of the vaccine components.
Conclusion: To the best of our knowledge, this is the first study designed to measure the effect of RBC transfusions on MMR vaccine immunogenicity.
Although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the MMR vaccine in chronically transfused patients immunized prior to 6 months posttransfusion.
Weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post RBC transfusions is called for in chronically transfused infants.
Post-vaccination serology should be considered.
Cold Stored Uncrossmatched Whole Blood Can be Safely Administered to Pediatric Trauma Patients Christine M Leeper 1,2 , Franklyn Cladis 2 , Richard Saladino 2 , Darrell Triulzi 3 , Barbara A Gaines 2 and Mark Yazer* 1 .
1 University of Pittsburgh, 2 Children's Hospital of Pittsburgh of UPMC, 3 Institute For Transfusion Medicine Background/Case Studies: The use of uncrossmatched cold stored whole blood (WB) is becoming increasingly popular in the initial resuscitation of trauma patients without a current ABO group.
WB has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood.
This report details the initial use of WB in pediatric trauma patients.
Study Design/Method: Pediatric trauma patients !3 years old and !15 kg with evidence of hemorrhagic shock were eligible to receive up to 20 cc/kg of cold stored, leukoreduced group O negative WB during their initial resuscitation.
All WB units had a low titer of anti-A and -B (<50) to reduce the likelihood of hemolysis in non-group O recipients.
Biochemical markers of hemolysis were measured on the day of WB transfusion and the following two days.
Admission thromboelastograms were obtained and repeated as necessary during the resuscitation.
After receipt of the maximum quantity of WB, conventional components were utilized.
Results/Finding: In approximately 11 months, 15 trauma patients received WB: 7 group O and 8 group A recipients, 53% male, median (IQR) age was 11 (4.5-14) and 73% blunt trauma mechanism.
Patients were severelyinjured with a median (IQR) Injury Severity Score of 36 (22-51) and 47% mortality rate.
The median (IQR) quantity of WB transfused to group O recipients was 21.9 (14.8-24.3) ml/kg versus 13.4 (9-18) ml/kg to non-group O recipients.
No transfusion reactions were reported.
The mean 6 standard deviation haptoglobin concentrations for non-group O recipients was 51.3 6 14.4 mg/dl on day 0, 86.3 6 36.8 mg/dl on day 1, and 126.9 6 45.8 mg/dl on day 2; the corresponding haptoglobin concentrations for group O recipients were 51.4 6 38.0 mg/dl, 84.7 6 61.5 mg/dl, and 134.8 6 68.3 mg/dl, respectively (p>0.42 for all comparisons).
Similarly there were no significant differences in total bilirubin, LDH, creatinine, and potassium at any time point.
Regarding evaluation of cold platelet function, we compared the subset of patients who received WB but no warm platelets (n57) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n514).
The mean 6 standard deviation platelet volume administered was 112 6 24 cc for whole blood recipients versus 147 6 68 cc for warm platelet recipients.
When pre-and posttransfusion TEG and platelet counts were analyzed, there was no difference in median platelet count or TEG maximum amplitude (MA) between cold and warm platelet groups.
Conclusion: Use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe.
Receipt of low titer group O WB did not lead to detectable hemolysis amongst the non-group O recipients.
Given this finding, the maximum quantity of WB per patients will be increased to 30 ml/kg.
Identification of Red Blood Cell Antibodies in Human Breast Milk By Novel Adaptation of Serological Method Philippe P Pary*, Alexis Leonard, Lauren Hittson, Naomi LC Luban, Deepika S Darbari, Yunchuan Delores Mo, Cyril Jacquot, Valli Criss and Jennifer Webb.
Children's National Medical Center Background/Case Studies: Human breast milk contains immunoglobulins that are present in maternal serum and secretions.
Data in mice has demonstrated the potential for Kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused Kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking.
A two week old infant with a history of Rh-D hemolytic disease of the fetus and newborn (HDFN), previously treated with Intravenous Immunoglobulin and phototherapy, was referred for anemia and reticulocytosis.
Patient was O positive, positive Direct Antiglobulin Test (DAT) 41 with anti-human IgG only, and a 31 positive antibody screen by gel method.
Antibody identification showed anti-D in both the plasma and eluate.
Patient was transfused O negative red cells and discharged.
Over several weeks, the patient returned twice for persistent anemia requiring additional transfusions.
At eight weeks of age, evaluation showed a persistent DAT IgG reactivity concerning for continued antibody exposure.
Maternal breast milk was evaluated as a potential source.
Study Design/Method: Based on similar properties of human breast milk and plasma, testing to identify IgG antibodies using a stantard tube saline method was performed with a 60 minute 378C incubation, followed by 3 automated washes prior to the addition of anti-human IgG reagent.
As a control, breast milk from an O positive, antibody screen negative mother was used to assess for interference by milk proteins.
Antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method.
Antibody identification and titers were also performed when indicated.
Only freshly collected breast milk stored at room temperature for less than 3 days was found suitable for this technique.
Results/Finding: The patient's mother showed plasma anti-D with a titer 4096 and the breast milk showed anti-D with a titer between 16 and 64.
The patient had a consistent plasma anti-D titer of 8.
The patient's mother chose to stop breast feeding after 8 weeks, and the patient's hemoglobin was improved at 12 and 16 weeks of age.
Using this method, we identified two additional cases of breast milk induced hemolysis: another anti-D and an anti-Jka.
Conclusion: Testing showed that it is possible to identify red cell IgG antibodies in human breast milk using a standard tube saline method.
We identified implicated antibodies in the breast milk received by infants with persistent anemia due to HDFN.
Breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding.
Cessation of breast feeding correlated with improved hemoglobin in affected infants.
Background/Case Studies: Red blood cell (RBC) transfusion is lifesaving for patients with sickle cell disease (SCD), but is commonly complicated by RBC alloimmunization.
Despite transfusion protocols serologically matching for C,E, and Kell antigens, alloimmunization to Rh antigens continues.
SCD patients often exhibit a hybrid RHD-CE-D gene which is often characterized by the production of a partial C antigen.
It has been previously documented that 30% of C1 SCD patients from the West Indies and West and Central Africa are partial C and at (30%) risk for alloimmunization to the C antigen through transfusion of C1 RBCs.
This study sought to determine the prevalence within a cohort of children with SCD at a U.S comprehensive SCD center.
Study Design/Method: RBC genotyping results performed on all SCD patients using PreciseType HEA array (Immucor, Norcross, GA) at Children's Healthcare of Atlanta were reviewed and compared to the serologic type for Rh (C/c, E/e) antigens.
The prevalence of C-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial C antigen based on the detection of the RHCE*ce(733G,1006T) allele in the absence of an RHCE gene encoding a conventional C antigen in trans, since this allele is commonly linked to the hybrid RHD*DIIIa-CE(4-7)-D gene which encodes the partial C antigen.
Review of the blood bank information system was performed to identify the number of C-antigen positive transfusion exposures and frequency of alloimmunization to the C antigen.
Results/Finding: Out of a total of 255 patients with genotype/Rh phenotype data available, 78 (30.6%) were C antigen positive serologically.
The allele frequency of RHCE*ce(733G,1006T) was 0.071.
In total, 15 (5.9%) patients possessed RHCE*ce(733G, 1006T) in the absence of conventional C gene in trans.
Of the 78 C antigen positive patients, 15 individuals (19.2%) were predicted to be partial C based on four molecular profiles [RHCE*ce(733G, 1006T)/RHCE*ce:12; RHCE*ce(733G, 1006T)/RH*cE:1; RHCE*ce(733G, 1006T)/RH*ce(733G):1; RHCE*ce(733G, 1006T)/RH*ce(733G, 1006T):1].
In these 15 partial C patients, no anti-C alloantibodies (or other Rh antibodies) were detected after 60 transfusion exposures (57 C-antigen negative units; mean: 4, range: 0-36), likely from placement of a C-negative RBC restriction upon detection of the RHCE*ce(733G, 1006T) allele.
Conclusion: This report confirms previous data of a high prevalence of the partial C antigen in SCD patients historically typed as C-positive serologically, and demonstrates the benefits of RBC genotyping to prevent alloimmunization to a highly immunogenic Rh antigen by identifying individuals who should receive C-negative blood.
All patients with SCD should have RBC genotyping performed for determination of their RBC phenotype, preferably prior to receiving transfusions.
Investigational Detection of Zika Virus RNA in US Blood Donors Paula P Sa a* 1 , Megan L Nguyen 1 , Melanie C Proctor 1 , David E Krysztof 1 , Gregory A Foster 1 , Erin K Sash 1 , Sandy S Dickson 1 , Joua Yang 1 , Jeffrey M Linnen 2 , Kui Gao 3 , Jaye P Brodsky 4 and Susan L Stramer 1 .
1 American Red Cross, 2 Grifols Diagnostic Solutions Inc., 3 Grifols Diagnostic Solutions, Inc, 4 Quality Analytics, Inc Background/Case Studies: Zika virus (ZIKV), an emerging flavivirus, is primarily transmitted by infected Aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus.
Acute ZIKV infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders.
The large proportion of asymptomatic cases, high numbers of returning travelers from ZIKV-active areas, severe clinical consequences to developing fetuses, the detection of RNA in asymptomatic donors during the French Polynesia epidemic, and 4 suspected cases of transfusion transmission in Brazil led FDA to release guidance documents to minimize the risk of ZIKV transmission via blood/ blood components.
Study Design/Method: Investigational testing by mini-pool (MP)-NAT using the Procleix Zika Virus Assay (TMA) was implemented on collections from five presumed high-risk US states on 6/20/16 (FL, GA, SC, MS, AL).
Following revised guidance on 8/26/16, testing was extended to all blood donations; conversion from MP-NAT to individual donation (ID)-NAT was implemented in phases and completed on 12/12/16.
Travel history questions were discontinued on 1/23/17.
Confirmatory testing included repeat TMA; in addition, RT-PCR, serology and red cell (RBC) TMA were performed.
Estimates of viral loads were performed by end-point TMA on plasma and RBCs.
Results/Finding: As of 4/8/17, 2,288,855 donations were tested including 393,713 (17%) in 24,611 MPs.
No reactive donations were identified by MP-NAT.
Of the 1,895,142 ID-NAT donations, 72 were initial reactive (IR) of which 8 (11%) confirmed positive (CP) by subsequent testing (CP rate of 1:286,107; positive predictive value of 11%; specificity of 99.997%).
Five (62%) CP donations were ID-NAT repeat reactive (RR); 3 (38%) donations were ID-NAT IR only, IgM positive and RNA positive in RBCs.
CP donors resided in MA, TX, CA, NY, WV and 3 in FL, 2 of which were local transmissions.
Six donors had traveled to a ZIKV-active area returning to the US from 2 to 73 days prior to donation.
Two donors with a travel risk reported clinical symptoms; 6 CP donors (75%) remained asymptomatic.
ZIKV RNA was detected in RBCs from all CP index donations with estimated levels varying from less than 40 copies (c)/mL to about 8Ê 5 c/mL.
At the time of writing, the longest period of detection in RBCs was 91 days vs. 17 days in plasma from the same TMA-RR donor.
ZIKV RNA levels in plasma were obtained from 1 IR and all RR donors, ranging from 12 to 2000 c/mL.
Study Design/Method: Plasma from blood donors were screened by individual donation (ID-NAT) for the presence of ZIKV RNA with the cobasV R Zika test.
ID-NAT1 samples were repeated in duplicate and further tested by a second NAT to confirm infection and estimate VL, and for anti-ZIKV IgM.
Simulated MPs of 6 were prepared by diluting NAT1 plasma 1:6 and tested to discriminate ID-NAT only detectable donations.
NAT yield samples for which simulated MP and conclusive IgM results were available (n5308) were sorted into 4 categories corresponding to sequential stages of acute ZIKV infection: IgM-/low VL; IgM-/high VL; IgM1/high VL; IgM1/low VL.
Results/Finding: Of 52,942 donations collected April 3-December 31, 352 were reactive for ZIKV RNA.
IgM-index donations had higher VLs (mean 1.1 x 10 6 vs 8.3 x 10 4 IU/mL) and higher proportions of simulated MP-detectable results (93% vs 23%) than IgM1 donations.
The distribution by stage of infection was evaluated as the epidemic evolved.
Over the course of the epidemic, the rates of ID-NAT only detectible and IgM1 donations increased (Table 1) .
Conclusion: This study demonstrates how the viral and immunological profiles of ZIKV infection in the index donations shifted through the course of the 2016 PR epidemic.
Categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of MP vs ID-NAT screening likely correlate with VL and serological stages of infection.
Staging of infections also has implications for diagnostic testing and understanding the durations of ZIKV viral and immunological markers in blood and persistence of ZIKV in body fluids and tissues.
cobasV R Zika is not commercially available for blood screening.
Data generated under the cobasV R Zika IND is preliminary and has not been reviewed by FDA.
This project has been funded in whole or in part with Federal funds Detection of Zika Virus RNA in United States Blood Donations Using cobas V R Zika on the cobas V R 6800/8800 Systems Lisa Lee Pate* 1 , Phillip C Williamson 2 , Michael Paul Busch 3 , Susan Rossmann 4 , Scott Jones 5 , Ann Butcher 1 , John Duncan 1 , Jean Stanley 1 and Susan A Galel 1 .
1 Roche Molecular Systems, Inc., 2 Creative Testing Solutions, 3 Blood Systems Research Institute, 4 Gulf Coast Regional Blood Center -Sugar Land, 5 QualTex Laboratories Background/Case Studies: In February 2016, the US FDA recommended that all blood donations in areas with active Zika virus (ZIKV) transmission be tested with an FDA approved nucleic acid test (NAT) for ZIKV RNA or treated with an FDA approved pathogen reduction technology.
The cobasV R Zika test was approved under an investigational new drug application on March 30, 2016 and testing of Puerto Rico donations began on April 3, 2016.
As a precautionary measure some blood centers in the US states also began NAT testing for ZIKV.
In August 2016, the FDA recommended universal screening of all blood donations.
The aim of this study is to describe the detection of ZIKV RNA in blood donations collected in US states between April 3, 2016 -February 28, 2017 using the investigational cobasV R Zika for use on the cobas V R 6800/8800 Systems.
Study Design/Methods: Donations were screened with cobasV R Zika by individual donation testing.
All initial reactive (IR) results were repeated in duplicate.
Supplemental testing included an alternative NAT (AltNAT) assay which is less sensitive than cobasV R Zika and serology testing for anti-Zika IgM and IgG.
Reactive donors were invited to enroll in follow-up, which included cobasV R Zika and serology testing.
A donor was considered to be Zika confirmed positive if at least one replicate of the repeat testing by cobasV R Zika was reactive on index donation or follow-up, reactive by AltNAT on the index donation, or positive for anti-Zika IgM on index or follow-up.
All IR donations were also retested at a 1:6 dilution to simulate mini-pool testing.
Results/Findings: A total of 1,776,190 blood donations were screened using cobasV R Zika.
Of 56 IR donations, 12 were repeat reactive (RR), 39 non-RR and 5 had no repeat testing.
Of the 12 RR donations, 7 were positive by AltNAT; 3 of these were IgM positive.
All 4 AltNAT negative donors were IgM positive.
One donor was Alt-NAT equivocal and IgM negative.
Of the 5 RR donors that were not IgM positive on index, 3 enrolled in follow-up and all seroconverted.
Of 39 non-RR donations, 38 were AltNAT negative and 1 is pending supplemental testing.
8/38 donors were IgM positive on index.
30 donors were IgM negative on index; 15/30 enrolled in follow-up; 14 remained IgM negative and 1 was 1gM inconclusive.
Of 5 donations without repeat testing results, 2 met criteria for positive (1 was AltNAT positive, IgM negative and 1 AltNAT negative, IgM positive).
1 donation is pending additional testing.
Altogether, 22/56 IR donations met the criteria for true positive on the index donation.
9/22 (41%) true positive donations were reactive when retested in a simulated minipool.
16/22 were IgM positive.
Conclusion: 0.001% of the 1,776,190 donations in US states screened for ZIKV RNA were confirmed as true positives.
cobas V R Zika is not commercially available for blood screening use.
Using Monte Carlo Simulation Luiz Amorim* 1 , Marc Germain 2 , Gilles Delage 3 , Maria Esther Lopes 1 and Yves Gr egoire 3 .
1 HEMORIO, 2 HemaQuebec, 3 H ema-Qu ebec Background/Case Studies: Zika virus was implicated in very large and recent outbreaks, in French Polynesia (2013) , and in Brazil (2015/2016), which was followed by outbreaks in South America, Central America and Caribbean.
Four probable transfusion transmitted cases were reported in Brazil; since 80% of Zika cases are asymptomatic, the actual transfusion rates can be much higher than reported.
In this study, we used a Monte Carlo simulation for risk estimation during the Brazilian outbreak.
Study Design/Method: The data feeding the Monte Carlo simulation were collected from January 1 st , 2016 through November, 26 th , 2016, from Brazil (the whole country) and for Rio de Janeiro state, one of the outbreak epicenters.
The data came from Brazilian epidemiologic bulletins and from Brazilian blood donation figures.
The risk assessment was performed separately for whole blood (WB) donation and for apheresis platelets (AP).
The model took into account the following parameters: Zika incidence in Brazil and in Rio; lognormal distribution symptomatic viremia (period: 5 days, with 99% of the values lower than 18 days); 20% of infected donors with symptoms lasting 2 days; 1.2 donation/donor/year for WB and 1.75 for AP.
The formula for transfusion risk calculation was: incidence X infectious period X average donation number per donor per year (WB, x/y; aph, z/y) X (1 -proportion of refused donors) X (1proportion of discarded donations due to post donation -PD -information).
Results/Finding: The table below shows the results.
The estimated risk of transfusion transmitted Zika is very important in Brazil and in Rio de Janeiro, where it can attain 1:13,598, for apheresis platelets.
The severe consequences of Zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in Brazil Dengue (DENV) arboviruses in the population are not available in Brazil.
The objective of this study was to assess the contemporaneous incidence of these agents in donors at 4 large geographically dispersed blood centers located in the southeast and northeast of Brazil.
Study Design/Method: In the Brazil public blood bank system, NAT screening for HIV, HCV and HBV is performed on minipools (MP from 6 donations).
The residual volume of MP plasma, 0.35 -0.45 mL, is routinely discarded.
Beginning in April 2016 each blood center saved $67 MPs/week for retrospective testing using the triplex ZIKV, CHIKV, DENV Transcription Mediated Amplification (TMA) assay developed by Grifols/Hologic.
MPs were shipped to the USA and batch tested at Grifols.
In the first two weeks (April 3-15) 3 MP6 were combined into pools of 18 donations; thereafter MP6 were tested without additional pooling.
To estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and 95% confidence intervals (CI) calculated using the method developed by Biggerstaff.
Results/Finding: The triplex assay performance was shown to have very high sensitivity (95% limit of detection <20 copies/mL for ZIKV/CHIKV/ DENVs) and to accurately discriminate each of the arboviruses.
Testing of the first 6 months of samples is complete for 6,292 MP, comprised of 37,752 donations collected from April 3 to October 9, 2016.
A total of 77 pools were positive, with 76 detected between April-June 2016.
The table summarizes the highest monthly estimated percent positive donors for each virus in each city.
Months with highest percent postive donors were April or May.
At the peak over 0.6% of donors in Belo Horizonte and Rio were viremic for ZIKV, whereas ZIKA was not evident in donors in Recife, but over 0.4% of donors in that city were viremic for CHIVK during the peak.
Conclusion: During the latter part of the arbovirus outbreak season in Brazil in 2016, ZIKV, CHIKV, and DENV were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in Brazil were extensively exposed to viremic blood components.
The use of donor MPs for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections.
Universal Zika Screening for Blood Donors in Singapore Sally Lam* 1 , Sze Sze Chua 1 , Mars Stone 2 , Michael Paul Busch 2 and Ai Leen Ang 1 .
1 Health Sciences Authority, Blood Services Group, 2 Blood Systems Research Institute Background/Case Studies: Singapore reported its first locally transmitted Zika case on 26 August 2016.
The numbers rose rapidly to 386 cases by the end September, with eight clusters (hotspots) of cases island-wide.
Zika virus (ZIKV) shares the same mosquito vector, Aedes aegypti, as the Dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and Guillan-Barr e syndrome, which hastened Singapore's Blood Services Group (BSG) to look into securing the safety of blood supply from the Zika threat.
We aimed to assess the assay performance of USA-FDA investigational (IND) Procleix ZIKV nucleic acid technology (NAT) assay for universal blood donation screening in Singapore to prevent transfusion-transmitted Zika infection.
Study Design/Method: All blood donations were screened for Zika with the Procleix ZIKV NAT assay since 1 October 2016.
Zika NAT reactive samples were tested at Blood System Research Institute (BSRI) for Zika RNA in plasma and red cells by PCR and for Zika and Dengue IgM and IgG antibodies.
A Zika confirmed case was defined by the presence of Zika RNA by PCR and/or Zika antibodies.
The analytical sensitivity was evaluated using 300 blinded frozen samples consisting of 25 replicates of 11 half log dilutions of the WHO International Standard for ZIKV and 25 replicates of negative controls prepared by BSRI.
Probit analysis was performed to determine the 50% and 95% limits of detection (LOD) .
Clinical performance of the Procleix ZIKV assay was also assessed with local patient samples obtained from Institute of Infectious Disease and Epidemiology, Singapore and a 14 member blinded ZIKV reference panel from the USA-FDA.
Results/Finding: A total of 63,144 donations were screened from 1 October 2016 to 31 March 2017, with 1 false positive case and 1 Zika confirmed donation detected.
Alternative ZIKV PCR tested positive in both the plasma and red cells with an estimated plasma viral load of 9.54x10 5 copies/ml.
Zika IgM was negative in the index donation sample but present in the 10-day post-donation follow up sample..
The donor reported no clinical symptoms.
The analytical sensitivity for the Procleix ZIKV assay was determined to be 2.1 copies/ml at 50% LOD and 10.0 copies/ml at 95% LOD.
The Procleix ZIKV assay detected RNA in 6 out of 9 patient samples and provided 85.7% agreement to the results of the USA-FDA ZIKV reference material.
Conclusion: The investigational Procleix ZIKV assay showed good analytical sensitivity and clinical performance, suitable for blood screening of Zika infection especially in asymptomatic donor populations.
BSG commenced universal Zika NAT screening by individual donation testing following the Zika outbreak with 1 confirmed Zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of 1 in 25,888 donations in Singapore.
Background/Case Studies: A CAP/AABB Work Group suggested that steps be taken to phase in RHDgenotyping for patients with a serologic weak D phenotype.
Weak D types 1, 2 and 3 express all the major RhD epitopes and these patients can be managed as RhD-positive, which may lead to a reduction in unnecessary Rh immunoglobulin (RhIG) administration and conservation of RhD-negative RBCs.
Study Design/Method: RHD genotyping was performed on all patient samples with weaker than expected or discrepant RhD typing results, utilizing a commercially available genotyping kit manufactured by Immucor (RHD BeadChip).
Initially, testing was performed at a reference lab while the RHD BeadChip was validated and implemented at this institution.
A serologic weak D phenotype is defined as weak to 21 reactivity on initial gel testing.
If genotyping demonstrated weak D types 1, 2 or 3, the intent was to manage the patient as RhD-positive.
If weak D types 1, 2 or 3 were NOT detected, the patient is considered at risk for alloimmunization and treated as RhDnegative.
While RHD genotyping results were pending, RhD-negative RBCs were used and if pregnant, the patient was eligible for RhIG.
Results were generally available in 2 to 4 weeks.
Results/Finding: RHDgenotyping was performed on 22 patient samples over 15 months.
Of these 22 patient samples, 13 (59%) were weak D types 1 or 2.
The remaining samples demonstrated a variety of alleles including known partial D variants (see Table) .
One patient identified as weak D type 1 required multiple transfusions over the study period, and refused RhD-positive RBCs.
The remaining weak D types 1 and 2 patients have not received transfusions at this institution since they were genotyped.
Four of 4 obstetric weak D types 1 and 2 patients received RhIG while genotyping was pending.
Conclusion: Testing and management of patients with serologic weak D phenotypes is not standardized.
RHD genotyping may lead to more consistent, personalized patient care and appropriate management of resources.
In this 15 month study period 13 serologic weak D patients were identified who could be managed as RhD-positive, however this did not result in withholding any doses of RhIG nor conservation of RhD-negative RBCs.
Genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies.
These outcomes highlight the limitations of current genotyping processes, including long turn-around-time Background/Case Studies: The RH blood group is highly immunogenic and the most clinically significant blood group secondary only to ABO.
Currently, in the United States, blood donors who type RhD-negative by serology undergo weak-D testing to identify some weak and partial states of RhD expression.
However, not all RhD expression can be detected serologically.
It has been suggested that investigation of serologic RhD-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in RhD-negative recipients.
Study Design/Method: RHD genotyping of all serologic RhD-negative blood donors presenting to our blood donor center was implemented to identify units with altered RHD alleles that should be characterized as RhDpositive.
Repeat donations were not tested.
Initial serologic testing of blood donors was performed using 3 FDA approved anti-D reagents.
When reactivity with all 3 reagents was negative, RHD genotyping was performed using a commercially available genotyping kit manufactured by Immucor (RHD BeadChip).
This assay detects over 80 RHD variant alleles and additional DNA sequencing was performed in selected cases.
To maximize efficiency samples were batched for testing; testing was generally performed once a month.
If an RHD variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to RhD-positive recipients.
Results/Finding: Over a period of 8 months we tested 509 RhD-negative blood donors.
There were 3 (0.6%) partial-D, 1 weak D (0.2%), and 3 (0.6%) DEL donors.
In one donor sample a novel RhD allele was identified through DNA sequencing (RHD*IVS5-46_42delTCTC).
The phenotype associated with this allele variant is unknown.
Investigation of previous donations from these 8 donors showed that 6 RhD-negative recipients received RBCs from 4 of these donors.
Five of these recipients underwent antibody screening after an average follow-up period of 5 months; anti-D was not detected in any sample (See Table) .
Conclusion: Serologic testing occasionally fails to identify some RhDpositive donor units, which could place RhD-negative recipients at risk for alloimmunization.
DNA-based testing can be used to identify donors who have the potential to sensitize RhD-negative individuals.
In this limited study period a small number of serologic RhD-negative donors, whose genotype indicated potential to sensitize recipients, were found.
However, review of recipient transfusion records indicated that prior exposure to these donors' RBCs did not lead to detectable immunization to date.
Future potential sensitizing events will be avoided by restricting these units to RhD-positive recipients.
Grifols Diagnostic Solutions Labs, 5 Grifols Immunohematology Center Background/Case Studies: Pregnant women with RHD variants may be candidates for RhIG prophylaxis if molecular analysis reveals a genotype associated with possible anti-D formation.
Proposed testing algorithms advocate molecular characterization of weak D types but if a patient types as RhD-positive, no further action is proposed.
Women with partial D variants who may also be at risk of anti-D formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as D-positive but who may be candidates for RhIG prophylaxis.
Our hospital is in an urban setting in which 63% of deliveries are to African-American patients.
We initiated routine, full-gene RHDsequencing for obstetric patients whose serology demonstrated not only weak D, but also those who were categorized as "D1" with 31 reactivity to determine the prevalence of partial D patients in an ethnically-mixed population who may be at risk of anti-D formation.
Study Design/Methods: From October 2016 to March 2017, we performed routine D typing (NEO, Immucor) on 1875 obstetric specimens followed by RHD sequencing on samples with either a serologic weak D phenotype or anti-D testing strength of 31 using at least 1 antibody.
Solid phase and manual testing used the series 4 and series 5 reagents.
Four additional anti-D reagents manufactured by Grifols (DG Gel Anti-D), Quotient (Anti-D blend), Biorad (Anti-D (RH1) blend), and Ortho (BioClone Anti-D) were also used for supplemental testing.
RHD sequencing was performed by Sanger methodology using routine clinical protocols.
Results/Findings: RHD polymorphisms or variations were identified in all 13 samples.
Two of 13 (15.4%) were D1 with an RHD gene with only common, known intronic variants that is predicted to produce the "reference" RhD protein (IVS1-29C, rs2301153; IVS3 1 117C, rs28521909; and IVS3 1 124A, rs28562109).
Two (15.4%) were D1 and heterozygous for two apparently new RHD coding variations which we are confirming by further testing.
Four (30.8%) patients had RHD alleles with known potential to make anti-D (RHD*DOL2, RHD*DAR1.2, and 2 with weak D type 4.0).
One had weak D type 96, which has uncertain susceptibility to alloimmunization and one was weak D type 1, which has not yet been associated with anti-D. Interestingly, two (15.4%) had variable D expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning.
One patient Background/Case Studies: A weak D type 2 is a variant of the RhD protein that comprises an amino acid substitution located in the 12th transmembrane segment and expresses a reduced amount of the D antigen.
This variant is known to be associated with the missense mutation c.1154 G>C which is the first nucleotide of the exon 9 of the RHD gene and thus could be implicated in exon 9 skipping when it is mutated.
When performing NGS (Next Generation Sequencing) analysis to fully genotype known patients, we identified an additional variant.
Study Design/Method: DNA samples were studied by Beadchip technology (Immucor/Bioarray solutions) and NGS using the SureSelect Human All Exon V6 (Agilent) and the Nextseq500 platform.
In silico analysis with different bioinformatic tools was used to predict splicing events.
Furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon 9 skipping of RHD gene.
This study was completed by the comparative modeling between the wild type and the weak type 2 RhD proteins.
Results/Finding: By a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of 10 patients previously characterized by BeadChip technology.
Interestingly, 4 out of 10 carry the c.1154-31C>T intronic variation on the RHD gene, already described and associated with a Del allele.
Among these last 4 patients, one has been previously characterized as RHD weak type 2 carrying the c.1154G>C (p.Gly385Ala).
Independently, Sanger sequencing on 50 unrelated RHD weak type 2 samples pinpoint to a linkage disequilibrium between c.1154G>C (ExAC, MAF 5 0.001145) and the c.1154-31C>T (ExAC, MAF 5 0.2496).
In silico analysis of both mutation located close to the splice acceptor site of the exon 9 does not predict a significant reduction of its strength score.
With minigene vectors harboring RHD wildtype exon 9, mutant RHD c.1154G>C, mutant RHD c.1154-31C>T and double RHD mutants c.1154G>C plus c.1154-31C>T, we showed no influence on skipping of exon 9 due to these mutations.
Comparative modeling of RhD proteins pointed out an additional hydrophobic interaction on the RhD weak type 2 between Ala385 (transmembrane helix 12) and Val183 (transmembrane helix 6) hampering membrane insertion.
Conclusion: The c.1154-31C>T variation is always associated in cis with the missense mutation c.1154G>C on the allele RHD weak type 2.
The c.1154-31C>T can be found alone on the RHD gene as a neutral polymorphism.
We assess that these two mutations isolated or combined do not lead to abnormal RHD transcripts.
Our results clearly demonstrate that the weak D antigen reactivity observed with RhD type 2 red blood cells is due to the substitution of alanine at amino acid position 385 to glycine.
Topology of Jk-Weak or Jk-Negative Single-Nucleotide Missense Variants in the Kidd Protein Glenn Ramsey*.
Northwestern University Background/Case Studies: The human urea transporter-B (HUT-B) protein carrying the Kidd blood group has 10 transmembrane (TM) and 2 tilted ureapore a-helices, a long extracellular connector segment, and 2 cytoplasmic segments at each end.
Numerous single-nucleotide missense variants (SNMVs) weaken or abolish expression of Jk a/b antigens determined at p.280.
We mapped all reported Jk-weak or Jk-negative (Jk-neg) SNMVs onto the HUT-B structure to explore topological correlates of Jk antigen expression.
Study Design/Methods: JK*A and JK*B SNMVs affecting Jk expression were compiled from dbRBC and ISBT registries, literature searches and 2010-2016 AABB, ISBT and British Blood Transfusion Society meeting abstracts.
SNMV locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian UT-B derived for analysis of UT function (Levin EJ, 2012) .
Results/Finding: Seven SNMVs located within 1 amino acid (aa) from the exofacial or internal end of a TM helix are mostly weak variants (Table) .
All 3 at the exofacial ends (p.A93T, p.W240R, p.V333D) are Jk-weak; the two Jkneg exceptions p.G298E and p.G299E are at the internal end of the TM helix bearing Jk a/b .
Four SNMVs in the cytoplasmic N-terminal segment are mostly weak variants.
In contrast, 13 SNMVs within membrane helices are mostly Jk-neg variants.
Three Jk-weak SNMVs (p.V10M, p.E44K, p.V76I) have been associated with allo-anti-Jk a/b to the antigen on their alleles ("weak partial").
Six of the 13 Jk-neg variants are within 19 aa (p.270-p.299) of Jk a/b at p.280.
None of these SNMVs are in the long extracellular connector region or the cytoplasmic C-terminal segment.
Jk-neg variants p.N289S and p.S291P are adjacent to p.288F and p.292L which line part of the urea transporter pore.
Conclusion: In the transporter-structured RhD and RhCE proteins, SNMVs with weak D, C, c, E or e expression are mostly within the RBC membrane, and non-canonical antigen-negative SNMVs are unusual.
In the structurally similar Kidd HUT-B, most Jk-weak SNMVs are at the ends of the TM helices or in the N-terminal cytoplasmic segment.
Among 13 Jk-neg SNMVs, most are in membrane helices.
However, whether a variant appears Jk-weak or Jk-neg may depend on the extent of testing.
Next-generation sequencing may provide more complete structure-antigen correlations.
Background/Case Studies: The Kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the SLC14A1 gene, which encodes the urea transporter UT-B1.
The Kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine.
The cause of the identical Kidd-null phenotype with dominant inheritance [In(Jk)] has not yet been defined, though it was first described in 1965.
In contrast to recessively inherited Kidd-null phenotype, this is not associated with mutations in the SLC14A1 gene.
The aims of the studies was to Identify and characterize the causative gene for Dominant Kidd-Null red blood cell phenotype (InJk).
Jk-weak (bold)/ Jk-neg expression n Within 1 aa from TM a-helix end V76I, A93T, W171R, W240R, G298E, G299E, V333D* Cytoplasmic N-terminal V10M, G40S, E44K, L45P In membrane TM and urea-pore a-helices R64W, R64Q, G65D, I117T, A183V, L246R, A248T ‡, A270A §, L272F, N289S, S291P, T319M 2/10 * Second nucleotide variant in this allele is synonymous (p.P196P).
‡Reported as Jk-neg but considered Jk-weak by ISBT.
§Near splice point.
Study Design/Method: We identified several families with dominant inheritance of the Kidd-null phenotype in multiple kindreds in Spain.
We performed whole-genome linkage analysis, exome sequencing, expression (RT-PCR and Western) analyses, and Urea lysis using patients' cells.
In addition, two probands underwent urine concentration tests.
Results/Finding: Using molecular approaches, we mapped the affected locus to a 5 Mbp region in 19q13.11-13.2 with an LOD score of 9.6.
Using deep sequencing, we identified a potential deleterious mutation in the ZNF850 gene, which deletes 84 bp resulting in loss of an entire zing finger domain.
The identical del84-ZNF850 mutation is present in all affected individuals, and is absent from all controls tested (n>2000).
In addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the ZNF850 locus, thus completely lacking the common allele, were identified.
We also obtained DNA from an unrelated InJk individual reported from Japan.
In this individual, there was a similar, though not identical, ZNF850del84.
None of the other potential genetic variants identified in the Spanish kindreds was present in the DNA from the InJk individual from Japan.
Consistent with the fact that the Kidd antigen, encoded by the SLC14A1gene, is a urea transporter that has been associated with renal function, we found that people with the ZNF850del84 in Spain had an inability to concentrate their urine.
Conclusion: A predicted zinc finger deletion at ZNF850, prevalent in Southern Spain due to a founder mutation, leads to UT-B1 dysfunction and underlies the dominantly inherited Kidd-null blood phenotype.
The phenotype associates subnormal urine concentrating ability.
In Background/Case Studies: Di-(2-ethylhexyl) phthalate (DEHP) makes PVC film flexible and useful for blood products.
During storage, DEHP can leach from the bag film into solution and be metabolized.
Studies in rodents have suggested that exposure to DEHP may be associated with adverse health effects, albeit at high dosages.
Attempts to find DEHP alternatives for blood bags have been difficult due to the RBC membrane-stabilizing effect of DEHP.
Bis(2-ethylhexyl) terephthalate (DEHT) a non-ortho-phthalate is structurally and functionally similar to DEHP, but distinct from a metabolic and toxicological standpoint.
DEHT can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor.
The study objective was to evaluate the quality of fresh frozen plasma (FFP) stored in DEHT containers versus FFP stored in DEHP containers at 30 days and 1 year.
Study Design/Methods: Thirty-six WB units were collected into CPD solution, leukoreduced, centrifuged, and separated into RBC and plasma.
ABO identical plasma units were pooled together in groups of three.
The 12 pools included 5 group A, 6 group O and 1 group AB.
Each plasma pool was weighed, mixed, sampled, divided into DEHP and DEHT pairs, and frozen at less than -208C within 8 hours of collection.
In vitro plasma testing (PT, aPTT, Factor V, Factor VIII, Fibrinogen, Protein C, and Protein S) was done on Day 0 (Pool), Day 30, and 1 Year of storage.
DEHP and DEHT paired plasmas were thawed and tested at the same time.
Plasticizer concentrations were determined on Day 0, Day 30, and 1 Year of FFP storage.
DEHP and DEHT and their monoesters were analyzed by liquid chromatography-mass spectrometry.
Internal standards were deuterated-DEHP, MEHP, DEHT and MEHT.
The lower limits of quantification (LLOQ) were: DEHP 5 2.9 ppm; MEHP 5 0.3 ppm; DEHT 5 0.9 ppm; and MEHT 5 0.2 ppm.
Results/Findings: Mean and standard deviation (SD) for key clotting factors and plasticizer results are summarized in the table.
There was no statistical difference in any plasma parameter between DEHP and DEHT bags at the same time period.
Factor VIII retained greater than 80% of its initial value.
Plasma stored in DEHT bags had an average plasticizer content 90% lower than that of the DEHP bags.
Background/Case Studies: Plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (RBCs) and/or crystalloid solutions.
Spray-dried plasma (SpDP) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (FFP).
The spray-drying process preserves coagulation proteins, but high molecular weight multimers (HMWM) of von Willebrand factor (VWF) are decreased.
The objective of this study was to compare SpDP and FFP in reconstituted whole blood (rWB) to test the hypothesis that SpDP is not inferior to FFP in facilitating platelet adhesion and thrombus formation.
Study Design/Method: Under an IRB-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at 100 g to separate RBCs from platelet-rich plasma (PRP).
PRP was diluted 3-fold in PIPES-Saline with 1.4mM PGE1 and centrifuged at 1900 g. The platelet pellet was resuspended in either SpDP or FFP and recombined with the packed RBCs to create rWB with hematocrit of 34-40% and 150,000-250,000 platelets/mL.
In addition, two rWB pairs were reconstituted with SpDP diluted 1:1 (SpDP50%) with plasma from a patient with Type 3 VW disease (T3VWD).
Samples were fluorescently labeled with a GPIIbIIIa-specific antibody and the sample was flowed through a Type I collagen-coated microchannel at a shear rate of 1600 s -1 for 180 seconds.
Still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (SA) along the length of the channel.
Ratio paired t-test was used to compare SA in samples reconstituted with SpDP vs. FFP.
The margin of noninferiority was 20% (SpDP/FFP > 0.8).
Results/Finding: Six batches of SpDP/FFP were evaluated using 17 subjects.
There was no statistical difference between the SpDP/FFP pairs (P50.7558).
The mean ratio of SpDP/FFP was 1.21 with a 95% CI of 0.84 -1.57.
Comparing SpDP vs. SpDP50%, there was no difference (median ratio 5 1.045, range: 0.95-1.14) in SA.
Two-way ANOVA demonstrated that batch did not significantly affect ratio of SA in SpDP vs. FFP.
Conclusion: SpDP, despite a decrease of VWF HMWM, was not inferior to FFP in ability to support platelet adhesion and thrombus formation.
On average, SA in samples reconstituted with SpDP was 20% greater than in samples reconstituted with FFP.
The lower limit of the 95 th % CI is a difference of 16%, which is less than the a priori determined margin of noninferiority of 20%.
Even with 50% dilution with T3VWD plasma, there was no reduction in platelet adhesion and thrombus formation in the SpDP rWB samples.
These data support the development of in-human studies to evaluate the efficacy and safety of SpDP in preventing and reversing trauma-related coagulopathy.
Spray-Dried Plasma Deficient in High Molecular Weight Multimers of Von Willebrand Factor Retains Hemostatic Properties Michael A. Meledeo* 1 , Qiyong Peter Liu 2 , Grantham C. Peltier 1 , Ryan C. Carney 2 , Ashley S. Taylor 1 , Colby S. McIntosh 1 , James A. Bynum 1 and Andrew P Cap 1 .
1 U.S. Army Institute of Surgical Research, 2 Velico Medical Inc Background/Case Studies: Restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs.
A single unit spray dried plasma (SpDP) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von Willebrand factor (vWF) and an increase in low molecular weight multimers.
vWF is critical in platelet adhesion and thrombus formation.
Following work demonstrating enhanced function with use of glycine-based reconstitution solutions for SpDP, this study examines two different SpDP pretreatment conditions.
Study Design/Method: The samples were: (1) FFP; (2) FFP with 70mM glycine; (3) regular SpDP without pretreatment (rSpDP), rehydrated with glycine-HCl:glycine; (4) SpDP pretreated with glycine-HCl (20mM); and (5) SpDP pretreated with glycine-HCl:glycine (20mM:50mM; both pretreated were rehydrated in water).
Six donor-matched plasmas of each type were tested.
vWF activity was measured by ristocetin cofactor assay.
Fibrin polymerization kinetics were analyzed by turbidimetry.
Thrombin generation (TG) was observed by thrombogram.
Chemistry was evaluated by i-STAT.
Residual cell material was quantified by flow cytometry.
Coagulation properties were measured by thromboelastography (TEG) in plasma and reconstructed whole blood (40% Hct with 200 platelets/nl from typematched donors).
Platelet adhesion to collagen under shear was measured by BioFlux.
Results/Finding: Pretreated SpDP showed enhanced vWF activity over rSpDP (p < .05).
Fibrin polymerization density was slightly diminished in rSpDP vs. FFP (0.879 vs. 0.742 O.D., p < .01), but TG was unchanged.
Bicarbonate/base excess were lower in SpDP samples vs. FFP (p < 0.001).
Residual cellular material (especially platelet-derived) was reduced threefold in rSpDP vs. FFP (p < .01) and an additional twofold in pretreated SpDPs vs. rSpDP (p < .05).
TEG results were unchanged in plasma-only samples; in reconstructed WB there was a reduction in amplitude (clot strength) in all SpDP samples vs. ; p < 0.01).
Platelet adhesion was equivalent in pretreated SpDPs and FFP, while rSpDP was improved vs. all other samples (71.53% surface coverage vs. 30.26-43.87%, p < .05).
Conclusion: SpDP has a longer shelf life and easier storage requirements than FFP and was equivalent or superior to FFP in most of these in vitro assays.
SpDP pretreated with glycine solutions was similar to FFP in most assays and showed superior vWF activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated SpDP.
Clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted.
The Interaction between Red Blood Cell Transfusion and Lung Injury: The Influence of Blood Component Manufacturing Methods Mathijs Wirtz* 1 , Anita Tuip-de Boer 1 , Ruqayyah Almizraq 2 , Jason P. Acker 3 , Philip J. Norris 4 , Jennifer A Muszynski 5 and Nicole Juffermans 1 .
1 Academic Medical Center, 2 University of Alberta, 3 Canadian Blood Services, 4 Blood Systems Research Institute, 5 Nationwide Children's Hospital Background/Case Studies: Red blood cell (RBC) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation.
The causative factor is not known but may include residual cells or extracellular vesicles (EVs) .
In this study we investigated the functional effect of different manufacturing methods of RBC products on the response of pulmonary cells in an in vitro model of mechanical ventilation.
Study Design/Methods: Groups of RBC products (whole blood filtered [WBF] , red cell filtered [RCF] , apheresis derived [AD] and whole blood derived [WBD]) were manufactured from 8 donors (blood type A or B).
Supernatants were prepared after 4-5 (fresh) and 41-42 days of storage (stored) for measurement of thrombin generation and EV analysis.
A549 type II alveolar cells were seeded onto flexible membranes and incubated with RBC supernatant.
Cells were subjected to 25% stretch using a cellstretcher.
Control cells were not stretched.
After 24 hours, IL-8 and IL-6 production were measured.
Results/Findings: Both fresh and stored supernatants from AD products significantly increased pulmonary cell IL-6 and IL-8 production compared to incubation with other RBC products and non-incubated controls, which was further exacerbated by cell stretching.
AD products also had significantly increased thrombin generating ability compared to other RBC products, as well as a significantly increased number of RBC-derived EVs compared to RCF and WBD products (p<0.05) .
Incubation of stretched cells with stored WBF products resulted in higher IL-8 production compared to other blood products and stretched controls.
RCF products did not activate pulmonary cells, had an absence of TG and had low levels of EVs compared to other products.
Conclusion: Manufacturing methods markedly influence the interaction of RBC products with lung cells.
AD products activate lung cells, which is further aggravated by cell stretching.
This may in part be mediated by RBC-Background/Case Studies: Investigators previously demonstrated immunosuppressive effects of RBC supernatant on monocytes in vitro, with greater effects seen in response to older units.
Recent clinical data suggest that RBC manufacturing method may influence immunomodulatory potential, but this has not been directly measured.
We used in vitro models to test the hypothesis that RBC supernatants obtained by different manufacturing methods will have differential effects on monocyte function.
Study Design/Method: RBC products were manufactured by 4 different methods from 5 individual donors, each: (whole blood filtration [WBF] , red cell filtration [RCF] , apheresis, and whole blood derived [WBD] ).
RBC products were stored in SAGM (WBF and RCF) or ADSOL-containing preservative solution (apheresis and WBD).
Supernatants were obtained after 4-5 days (fresh) and 41-42 days (expiry).
Monocytes were co-cultured in media plus 20% RBC supernatant or media only (control) followed by LPS stimulation.
Experiments were performed in 5 replicates, each with a distinct monocyte donor.
Comparisons between groups by ANOVA with Dunnett's post-test for multiple comparisons.
Data are mean 6 SD of % of control values.
Results/Finding: Exposure to apheresis or WBD RBC supernatants suppressed monocyte LPS-induced TNFa production capacity compared to controls (Table 1) .
This was true for fresh units and those at expiry.
For monocytes exposed to RBC supernatant alone without LPS, interleukin-8 production was higher after exposure to fresh WBF (248 6 115 % control, p 5 0.02) or WBD at expiry (292 6 111 % control, p 5 0.0005).
Conclusion: Manufacturing method and/or storage solution significantly alters immunomodulatory effects of RBC supernatant on monocytes in vitro and may confound analyses of clinical effects of RBC storage duration, particularly within international multi-center studies.
A Magnetic Levitation System to Study the Impact of Donor Gender, Age and Blood Storage Conditions on Red Blood Cell Density Profile Gozde Durmus* 1 , Alessandro Tocchio 1 , Anita Howell 2 , Kaushik Sridhar 1 , Jason P. Acker 3 and Utkan Demirci 1 .
1 Stanford University, 2 Canadian Blood Services, Centre for Innovation, 3 Background/Case Studies: The amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males.
It is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population.
It is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (RCC) units.
As red blood cells (RBCs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells.
Study Design/Method: Our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average RBC age and density of red cell units.
We have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations.
This label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of 10 -4 g/mL.
First, to determine the effect of RCC storage on the density profile of RBCs, levitation and single-cell density profiles were measured at 7, 14, 21, 28, 35 and 42 days.
In addition, to determine the effect of donor age and sex on the RBC density profile, blood samples from 24 volunteers with four different age and sex categories (Male, 18-40 years; Male, >60 years; Female, 18-40 years; Female, >60 years) were profiled.
Results/Finding: First, we observed that the levitation and density profiles as well as morphology of RBCs within RCC units change significantly during storage.
In addition, RBC density was significantly different between young (1.098 g/mL) and older female donors (1.109 g/mL) (p < 0.01).
Moreover, RBCs from young males (1.096 g/mL) were significantly less dense compared to RBCs profiled from older female donors (1.109 g/mL) (p < 0.05).
Conclusion: We have developed a magnetic levitation system for the point-of-care, real-time evaluation of RBC and red cell concentrate (RCC) quality.
We envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes.
Cytokine production of pulmonary cells IL-6 (pg/mL) IL-8 (pg/mL) Background/Case Studies: Oxidation Reduction Potential (ORP) or Redox is the ratio of activity between oxidizers and reducers.
Redox imbalance caused by a higher production of reactive oxygen species (ROS) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (OS).
While OS can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease.
In the present study we investigated changes in hemoglobin, free heme, and ORP as red blood cells (RBC) age and the effects of red blood cell age on ICU patient morbidity and mortality.
Study Design/Method: 120 ICU patients were enrolled in this prospective observational trial investigating the effect of transfused RBC age on ICU patient morbidity and mortality.
All RBCs were pre-storage leukoreduced and ABO identical.
Citrated blood samples were collected from each RBC unit prior to issue.
The RBC supernatants were tested for free hemoglobin/ heme and ORP.
The patients were followed prospectively.
Results/Finding: A total of 426 RBC units were transfused.
Patients and RBC characteristics are shown in the table.
Significant reductions were detected in ORP values over storage duration (p<0.001).
Substantial correlations were also found between ORP and free hemoglobin (p<0.05) and ORP and free heme (p<0.05).
Interestingly, there was a statistically significant difference between the average ORP values of the transfused RBC in patients who developed infection with higher ORP values measured in RBC units given to patients who developed post-transfusion infections 132 6 10 vs 127 6 13 (p<0.05).
No significant differences were observed between ORP and patient mortality, hospital/ICU days, or thrombosis.
Also, no correlations were detected between free heme/hemoglobin or RBC age and infection development.
Conclusion: These data demonstrate that older blood has lower ORP values as well as increased free heme/hemoglobin.
There were no differences in ORP values between the different blood groups once RBC age was controlled for and there were no statistically significant differences in patient mortality associated with ORP, free heme/hemoglobin, or RBC age.
The decreased ORP values observed in the older blood are likely attributable to the "storage lesion".
Higher transfused RBC ORP values were associated with subsequent development of infection, and younger RBCs were found to have higher ORP values.
Thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients.
Further studies are needed.
Background/Case Studies: No randomized trials in humans have addressed whether only exposure to red blood cells (RBCs) that have been stored for a long time is associated with harm.
We explore the effect on inhospital mortality of transfusing RBCs stored for more than 35 days compared to RBCs stored for 7 days or less.
Study Design/Method: Data from a multi-national randomized controlled trial were used for this exploratory analysis.
The patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest RBCs in inventory or the oldest (standard issue) RBCs providing a large cohort of patients receiving RBCs with storage durations along the entire RBC storage continuum of 1 to 42 days.
Using a time dependent variable patient exposure was defined by the maximum storage duration of RBCs received.
This was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to RBCs less than or equal to 7 days storage duration -reference group), medium age (at least 1 RBC of 8-35 days storage), and oldest (at least 1 RBC greater than 35 days storage).
The primary outcome was all-cause in-hospital mortality.
Cause-specific Cox regression models of in-hospital death assessed the effect of exposure of RBCs in each category to exclusive exposure to RBCs stored for 7 days or less.
The effects of fixed and time-dependent confounders were dealt with through stratification and regression.
Sensitivity analyses were conducted with a) weekly partition with cut-points every 7 days, and b) a finer partition using cut-points every 3 days.
Results/Finding: 24,726 patients receiving 90,530 RBCs were included in the analysis.
Exposure to RBCs stored for more than 35 days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest RBC units (stored for 7 days or less) after adjusting for several fixed and time-dependent potential confounders (HR 5 0.91; 95% CI: 0.72, 1.14; p 5 0.400).
Exposure to blood stored for at most 8-35 days yielded a similar hazard ratio (HR 5 0.90; 95% CI: 0.73, 1.10; p 5 0.295).
In the sensitivity analyses using weekly partitions, exposure to RBCs stored for greater than 35 days compared to exclusive exposure to RBCs stored 7 days or less was not significant (HR 0.90; 95% CI 0.72, 1.14; p 5 0.381).
The confidence intervals around the hazard ratios for the other 7-day intervals all include 1.
Similar findings were obtained with partitioning exposure data into 3 day intervals where exposure to RBCs stored for 40-42 days was not associated with increased risk of death compared with exclusive exposure to RBCs stored for 1-3 days (HR 0.82; 95% CI 0.37, 1.83; p 5 0.635).
The confidence intervals around the hazard ratios for the other 3-day intervals all include 1.
Conclusion: Individuals exposed to RBCs stored for more than 35 days were not at increased risk of in-hospital death compared to individuals exposed exclusively to RBCs stored for 7 days or less.
Transfusion of Anaerobically Stored Red Blood Cells Improves Recovery in Experimental Rat Hemorrhagic Shock Model Alexander Williams* 1 , Cynthia Walser 1 , Tatsuro Yoshida 2 , Andrew Dunham 2 and Pedro Cabrales 1 .
1 University of California San Diego, 2 New Health Sciences Inc. Background/Case Studies: Hemorrhagic shock (HS) severely decreases oxygen (O 2 ) delivery and induces cardiovascular collapse.
In parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (RBCs) to restore blood volume, O 2 carrying capacity, and hemodynamic stability.
The quality of the transfused RBCs determines the recovery from HS, and extent of clinical sequelae prompted by the HS.
This study compares the ability to recover from HS with conventionally stored RBCs, anaerobically (O 2 saturation <10%) stored RBCs, or anaerobic/hypercapnic (O 2 saturation <10% and pCO 2 (@378C) $70mmHg) stored RBCs.
Study Design/Method: Packed red blood cells (pRBCs) stored in AS-3 after leukorfiltration were created from donor Sprague-Dawley rats.
pRBC units were randomly stored under either 1) conventional; 2) anaerobic; or 3) anaerobic/hypercapnic conditions.
Rats (150-200g) were hemorrhaged to 50% of blood volume, held in hypovolemia for 30 minutes, and resuscitated to recover blood pressure to 90% pre-hemorrhage with pRBC stored for either 1 or 3 weeks.
Systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation.
Data were analyzed using two-way ANOVA, followed by the appropriate post hoc analyses.
1(11%) NEG patient showed short term response and 6(67%) patients showed progressive disease.
At the NEG group standard eval 1(11%) patient showed response and 3(33%) had progressive disease.
1(11%) NEG patient had long term response compared to 11(21%) POS patients.
At the POS short term eval 22(42%) patients showed response and 20(38%) patients had progressive disease.
At the POS group standard eval, 20(38%) patients showed response and 6(11%) patients had progressive disease.
Overall, 28(53%) POS patients responded compared to 2(22%) NEG.
Conclusion: There is a trend in lower response rate in patients with negative antibody screens compared to positive controls.
These findings suggest that an anti-CD38 neutralizing substance could play a role in treatment response.
Alternatively, reduced CD38 expression may also contribute.
The low response rates seen in both groups may result from biased selection.
The need for repeat T&S and presumed repeat transfusions may be preselecting patients with more aggressive disease.
Also, only a small number of patients were suitable for review.
A larger prospective study that controls for such variables is needed.
A Review of Blood Utilized during Provider-Activated and Critical Administration Threshold-Triggered Massive Transfusion Events Patrick Ramos* and John Hess.
Division of Transfusion Medicine, Harborview Medical Center Background/Case Studies: Traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (RBCs) in a 24-hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event.
To address these issues, a level I trauma center adopted the critical administration threshold (CAT) as an additional indication for activating its massive transfusion protocol (MTP).
This study reviewed blood utilized during massive transfusion events based upon whether the MTP was provider-activated versus CAT-triggered.
Study Design/Method: All massive transfusion events between January and April 2017 were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused.
The transfusion of three or more blood components in an hour defined CAT.
A massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the MTP or blood components were transfused at a rate that met CAT criteria.
The massive transfusion start time is based on either the time the provider activated the MTP or the time the first blood component was transfused, whichever came first.
Unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components.
This information was tabulated to determine the monthly number of provider-activated MTPs, CAT-triggered MTPs, and average blood component transfused per massive transfusion.
Conclusion: Blood utilization is lower within the CAT-triggered MTPs even though it outnumbered provider-activated MTPs.
However, the mode for both groups suggests that most massive transfusion require less blood components than the average rate.
Using the mode provides an approximate 24% replacement of blood volume.
This should be enough to counter the early signs and symptoms of hemorrhagic shock.
Though this study did not review the appropriateness of provider-activated MTPs, using CAT as an indicator ensures clinicians are prepared for a potential massive transfusion.
Further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused.
The mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently.
If this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused.
The Long Term Storage Effect of 0.2M Dithiothreitol on Red Cell Antigen Integrity in Reagent Red Blood Cells Heike Carrel* 1 , Laurie Sutor 1,2 , Germ an Leparc 3 , Marjorie Doty 3 and William Crews 1 .
1 Carter BloodCare, 2 UT Southwestern Medical Center, 3 OneBlood Background/Case Studies: Anti-CD38 drugs, such as daratumumab, pose a problem for the transfusion service.
They may cause a number of false positives, including positive direct antiglobulin tests (DAT), indirect antiglobulin tests (IAT), and panreactivity in eluates.
Such results can prolong compatibility testing and delay delivery of blood products for patients.
Treating reagent red cells (rRBCs) with 0.2M dithiothreitol (DTT) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies.
This study aimed to investigate the effect of DTT-treatment on rRBC antigen integrity over a 28 day period.
Study Design/Method: Twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-D, -C, -E, -c, -e, -M, -S, -s, -Fy a , -Fy b , -Jk a , and -Jk b ), were tested against untreated and 0.2M DTTtreated rRBCs (Immucor Panoscreen I, II, III; DTT from Acros Organics).
DTT treatment of rRBCs was performed using the methodology described in the AABB Technical Manual (18 th edition).
Each of the 12 plasma aliquots was further separated into 28 aliquots and stored at -208C until day of use.
Fresh aliquots were thawed each day to avoid unintended antibody integrity degradation.
A Polyethylene Glycol (Immucor) enhancement technique was used and reactions were read at the IAT phase.
Hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the Haemonetics color comparator chart.
Serological antibody reaction strengths were observed and documented each day.
(25) A monthly breakdown for both groups also displayed a downward trend in the average use of blood components.
Results/Finding: There was noticeably more hemolysis with the DTTtreated cells over time compared to the untreated cells.
Red cell antigens remained serologically detectable on the DTT-treated cells throughout the study, despite a greater degree of observed hemolysis.
There was minimal difference in reactivity strength between untreated and DTT-treated cells for antigens not affected by DTT.
In most instances, the DTT-treated cells reacted slightly more strongly.
None of the antibodies produced reactivity strengths of less than 11 with the untreated or DTT-treated cells during the study.
Conclusion: Long term storage of 0.2M DTT-treated rRBCs does not compromise antigen integrity.
Advance DTT-treatment and storage of a large aliquot of rRBCs may serve to increase efficiency in the transfusion service.
Background/Case Studies: Monocyte monolayer assay (MMA) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of RBCs for alloimmunized patients.
The requirement for fresh auto/allogenic monocytes for MMA is highly restrictive due to tedious processing of fresh peripheral blood (PB).
Our previous study described processing and cryopreservation of buffy-coat (BC) derived and fresh PB-monocytes for MMA assay.
The aim was to evaluate the functional properties of cryopreserved BC-monocytes as substitute for fresh PBmonocytes in MMA in evaluation of previously reported clinically significant RBC alloantibodies.
Study Design/Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy-coats (Histopaque-10771), pooled, suspended in cryopreservation media (20% DMSO; 1:1) and stored in liquid nitrogen.
PBMC membrane integrity post-thaw was determined by trypan blue exclusion.
PBMCs were cultured on poly-L-lysine-treated coverslips (378C, 5% CO 2 , 1 h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (O1) RBCs sensitized with either anti-D (positive control), anti-Scianna-2 (Sc2) or anti-AnWj or lipopolysaccharide stimulated for 2 h. Aliquots of the sensitized RBCs were tested for opsonization by indirect antiglobulin test (IAT).
Phagocytosis index (PI) was determined microscopically as the number of fully phagocytosed RBCs/100 monocytes.
Supernatants were analyzed for cytokines using Luminex technique.
Results/Findings: Cryopreserved PBMCs showed 96.2 6 1% viability postthaw.
We report no significant difference in phagocytosis of anti-D sensitized RBCs by cryopreserved monocytes vs fresh monocytes.
We show a significant increase in TNF-a, IL-1b, IL-6, IL-8, MIP-a (p < 0.01), MIP-b and GRO (p < 0.05) secretion from cryopreserved BC monocytes vs both fresh BC and PB-monocytes.
Sc2-and AnWj-sensitised RBCs resulted in a PI of 9.2 6 2% and 60.2 6 6.4% respectively vs anti-D sensitized RBCs (PI: 72 6 8.7%).
A weak (11) reactivity by IAT was observed for anti-AnWj sensitized RBCs while anti-D sensitized RBCs resulted in 41 IAT reactivity.
These results correlated with previously reported results for clinical significance and MMA when using freshly obtained autologous or healthy donor monocytes.
Conclusion: This study shows that cryopreservation preserved monocyte viability and phagocytosis function for MMA.
As previously reported with fresh monocytes MMA assay, the two alloantibodies tested with cryopreserved BC monocytes were shown to have a phagocytic index of clinical significance (PI>5%).
The use of cryopreserved BC-monocytes has the ability We describe antigen typing discrepancies in 5 patients, involving 3 antigens (C, Jk a , S), revealed when serologic results differed from the phenotype predicted by DNA testing.
All 5 patients had 3-41 positive DAT with anti-IgG and warm autoantibodies identified in the plasma.
Investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents.
Study Design/Method: Standard tube hemagglutination methods were used for antigen typing.
RBCs were treated with EDTA glycine-acid (EGA) using Gamma EGA Kit.
Genomic DNA was isolated from WBCs and HEA PreciseType performed.
Results/Finding: The RBCs of patients 1 and 2 typed C-on initial testing with Immucor Gamma-clone anti-C, but were predicted C1 by HEA Precise-Type.
EGA-treated RBCs gave 31 reactions with the same anti-C reagent.
Patient 1 RBCs gave variable reactivity (vw-11) with Bio-Rad Seraclone and Ortho BioClone anti-C.
Patient 2 RBCs gave 11 reactivity with all 3 anti-C reagents when incubated for the maximum incubation time allowed.
Patient 3 RBCs were Jk(a1) with Immucor Gammaclone anti-Jk a , which the manufacturer states is suitable for testing DAT1RBCs, but predicted Jk(a-) by HEA.
EGA-treated RBCs tested Jk(a-) with the same reagent.
RBCs from patients 4 and 5 tested S1 with Bio-Rad Seraclone anti-S (3-41), but predicted S-by HEA.
Further testing with Immucor Gammaclone anti-S showed RBCs from both patients were S-.
EGA-treated RBCs from both were non-reactive with both anti-S reagents.
Conclusion: Commercial monoclonal reagents are valuable resources, especially when phenotyping DAT1 RBCs but not all manufacturers include reagent limitations regarding testing of DAT1 RBCs.
We describe 2 cases of false negative tests with monoclonal anti-C due to antigen blocking by IgG, and 3 cases with false positive tests with anti-S (n52) and anti-Jk a (n51) typing.
False positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected.
Extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved.
Results concordant with DNA testing were obtained with EGA-treated RBCs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent.
These cases caution the potential for both false negative and false positive results for samples with 3-41 positive DAT and supports testing to dissociate IgG from RBCs strongly DAT1 before antigen typing.
In addition, this report highlights the benefits of DNA testing as part of the routine reference laboratory workup.
Background/Case Studies: Sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions.
Accepted US clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management.
To mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable.
Giblett, long ago, introduced a relative scale relating the RBC antigen immunogenicities to (an assumed) immunogenicity of "K" (http://bit.ly/2opqFew ).
Here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen.
Study Design/Method: We define immunogenicity, or sensitization risk, r, for any antigen ("Ag") of interest, as the conditional probability of alloantibody ("Ab") formation, given allo-exposure to Ag, i.e.
r :5 prob(Ab|al-loExp), so that prob(Ab) 5 prob(Ab|alloExp)*prob(alloExp) and 0 r 1; rewriting prob(alloExp) 5 prob(Recipient, "R", lacks Ag)*prob(Donor , "D" has Ag); and estimating prob(Ab) 5 nAb/nR, nAb denoting the number of Ab in nR recipients, we obtain: nAb/(nR*prob(R lacks Ag)) 5 r * prob(D has Ag), the left-hand side representing the observed sensitized fraction, U, i.e.
the number of observed Ab in relation to the number of recipients at risk.
Conclusion: Several antigens, though corresponding antibodies may be rare (e.g.
"Jsa", "e", "U"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk.
In conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available.
Our approach may be readily extended to additional RBC antigens and other antigen systems.
Background/Case Studies: AABB and FDA require a 12 month deferral of donors with a tattoo applied using non-sterile needles or reusable ink.
We review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink.
We recently added two large states in which we collect blood to the Acceptable States List (ASL).
We compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations.
Study Design/Method: We analyzed allogeneic interview responses to the screening question, "In the past 12 months have you had a tattoo?"
and if 'yes', whether the tattoo was applied by a state regulated entity.
Blood centers in 2 states were selected for the analysis before and after state tattoo regulation.
In State A, a comparison period of similar 3 months before (2/ 2015 -4/2015) and 3 months after (2/2016 -4/2016) was selected; for State B, a similar 4 months before (12/2015 -3/2016) and 4 months after (12/2016 -3/2017) was selected.
Frequency and rate of responses were compared in before and after periods.
Among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including HIV, HBV and HCV.
Results/Finding: A higher proportion of donors presenting to give blood admitted to having a recent (<12 months) tattoo in the post period in both states.
This increase occurred immediately following the addition of States A and B to the ASL (data not shown).
Among those who responded yes to having a tattoo, in States A and B respectively, there was a 13-and 3-fold increase in accepted donors (Table) .
The absolute number of accepted donors with tattoos increased from 13 to 567 (State A) and 151 to 1,496 (State B), which annualized, represents a potential gain of 2,216 (State A) and 4,035 (State B) additional donations.
All donors who had a tattoo in regulated states (ASL) tested negative for HIV, HBV and HCV.
Conclusion: To counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base.
The immediate rise in the number of donors reporting a tattoo following the addition of the 2 states may reflect a decline in self-deferrals based on having had a recent tattoo.
We demonstrated an increase in the potential number of donations without compromising safety.
Background/Case Studies: Transgender donors represent a small fraction of blood donors.
Determining their eligibility to donate has been challenging for blood centers.
To assess behavioral risk, the donor is required to answer gender specific questions.
The same is true when assessing TRALI risk where the donor is asked about a history of prior pregnancies.
Prior to the implementation of the FDA's Final Rule, blood centers asked donors for their birth gender and determined eligibility based on that gender.
If the donor changed their gender they were asked to answer both the male and female questions.
The Final Rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender.
In order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center.
and tracked.
Donors were contacted to resolve any descrepancies.
Donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified.
HLA antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative.
Results/Finding: From 2013 -2015 , there were 181 donors identified who had changed their gender from their birth gender; 121 female donors changed their gender to male and 60 male donors changed their gender to female.
There were 7 (6%) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations.
Three of these donors were apheresis donors who had been tested for HLA antibodies.
One tested positive and the other two tested negative for HLA antibodies.
The four other donors were whole blood donors and had not been tested.
An HLA test was added to these donors' records so that the test could be performed the next time they presented to donate.
Conclusion: Transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male.
Six percent of transgender males that we identified reported a prior history of pregnancy.
At our center, when a donor requests a gender change from female to male, an HLA test is requested for the next donation.
First time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information.
Consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that HLA antibody testing can be performed.
Effect of Variable Volume Scale Introduction in a Large Multi-Site Blood Center Ralph R Vassallo*, Marjorie D Bravo and Hany Kamel.
Blood Systems, Inc. Background/Case Studies: Regulations allow whole blood donation [WBD] of up to 10.5 mL/kg or 15% of estimated blood volume [EBV] .
Traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the 15% limit.
Variable volume scales [VVS] can be programmed to vary unit volume (up to 550 mL) by donor EBV.
This maximizes transfusable RBCs and plasma and recovered plasma [RP] volume.
RP from WBDs is a small but important source of derivatives and blood center cost recovery.
We report the effect of introducing the Hemoflow VVS on donor reaction rates and RP volume in a large blood center.
Compared to previous fixed settings, variable collection volumes were expected to decrease by 10 mL at EBVs <3.5L in donors !23 yo, but increase by 5-40 mL for all others.
Study Design/Method: Donor vasovagal reaction [VVR] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [LOC]) for successful WBDs were obtained from the center's hemovigilance database for the 18 mos.
before a 6 mo.
phased implementation of the VVS, and the subsequent 24 mos.
Multivariable analysis [MVA] by 6-mo.
periods was performed in a model incorporating donor sex, age, first-time [FTD] vs. repeat status, EBV and donation site.
Both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse.
Results/Finding: Compared to the baseline period, a significant increase in prefaint reaction rates were noted in Pre-Implementation (Impl) periods 1 & 2, continued during Impl and Post-Impl periods 1 & 2, returning to the baseline rate in Post-Impl periods 3 & 4 (Table) .
More severe reactions showed an increasing trend that only became significant in Post-Impl periods 3 & 4.
The MVA showed the VVS as independent factor contributing to the increased prefaint and more severe reactions.
However, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low EBV, FTD status and collection site (not shown).
Plasma unit volume increased an average of 3.8 mL during Post-Impl periods 1 & 2 from the temporally matched baseline & Pre-Impl period 1.
Conclusion: Following an initial increase in mild VVRs during and immediately after implementation of the VVS, VVR rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend.
The subsequent increase in prolonged/offsite reactions and LOC after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the VVS.
Small but significant increments in RP volume improve derivative availability and offset the cost of the VVS.
Comparison of Vasovagal and Citrate Reaction Rates in Donors According to Type of Apheresis Procedure Pierre Robillard* 1 and Yves Gr egoire 2 .
1 Hema-Quebec, 2 H ema-Qu ebec Background/Case Studies: Apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used.
Citrate reaction (CR) results from various degrees of hypocalcemia in donors.
Blood volumes taken from donors vary according to type of procedure and use of volume replacement.
Loss of blood volume is in part responsible for the occurrence of vasovagal reactions (VVR).
This analysis was conducted to estimate the incidence of CR and VVR according to various types of apheresis procedures performed at our blood center.
(YFV) were reported in Some Brazilian states -Rio de Janeiro, Sao Paulo, Minas Gerais and Espírito Santo, mainly.
The vectors of those cases were mosquitoes from the Haemagogus and Sabethes genders, whose habitat is the tropical forests.
Since many Brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the Aedes mosquitoes.
In order to minimize this risk, Rio de Janeiro health authorities decided to promote a mass vaccination in late March, 2017.
The vaccine is produced with live and attenuated YFV, which can circulate for at least 4 weeks after vaccination.
In some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases.
Due to that, Brazilian blood regulation authority established a 4 week deferral period after YFV vaccination.
This action could dramatically affect the availability of blood donors.
This study shows the measures taken by Rio de Janeiro blood center to circumvent this risk and attract more donors.
Study Design/Method: The strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against YFV.
There were no financial advantages to the donors, since YFV vaccine is completely free of charge for any Brazilian citizen.
The vaccine was administrated by trained nurses, in an office close to the donors session.
If, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications.
The blood center annnounced just before the mass vacination campaign launching that it would vaccinate 600 people who came to the blood center to donate blood.
If, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications.
Results/Finding: During the five days of campaign, we received 3,351 blood donors candidates; from those, 2,449 were accepted as a blood donor, after medical interview.
The deferral rate was 26.9%.
At the same period of the year 2016, there were 1,215 prospective donors, and 883 blood donations.
The deferral rate was 27.3%.
The "Get vaccinated against YFV .
.
.but give blood before" campaign was able to attract, in a five day period, 1,566 additional donors, compared to 2016 same dates.
That represents a 177.34% increase in the number of blood donations, without deferral rate increment.
There was a slight increase in the proportion of first-time donors, from 42.7% in 2016 to 45.8% in 2017.
Conclusion: The strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against YFV.
dose loss which must be accommodated when collecting plt donations to ensure the US plt dose of !3.0x10 11 is met.
Currently, Triple Set kits for PR are only approved in Europe.
Plt loss, and adjusted apheresis targeting parameters may impact split rate (SR) or products per apheresis procedure.
Inventory suitable for PR without impacting US blood center SRs warrants evaluation and optimization.
Study Design/Method: 1,000 apheresis collections from 4 centers with different SRs were analyzed.
A baseline SR for conventional PC was calculated assuming i) a minimum dose (allowing for production loss) of 3.1 x10 11 for single (S), 6.3x1011 11 for double (D), and 9.5x10 11 for triple (T) conventional PCs, ii) concentration and volume requirements from apheresis device manufacturer were used.
For each collection, dose, volume, and concentration were assessed for PR kit compatibility, based on storage medium (PAS or 100% plasma) assuming i) a minimum dose (allowing for production loss) of 3.5 x10 11 for S and 6.7x10 11 for D for PR units, ii) removing small quantities from units with excess volume or dose to meet PR specs., iii) if all or part of an out of parameter D or T collection could be divided into one or more kits for PR, eligible parts undergo PR, and the remainder treated conventionally, iv) collections unsuitable for PR specs.
or would decrease SR if treated would be counted as conventional PCs.
Results/Finding: Conclusion: Blood centers today can adopt PR for a significant percent of their current supply (as high as 99%) without affecting their SR. Compatibly increases further by dividing T and large D donations.
Percent achievable depends on their current S, D, T proportion of collections and practices.
Changes to D and T collection parameters, optimized donation and counting accuracy, and volume reduction will improve PR compatibility further.
Individual analysis is warranted for each blood center.
RBC 2RBC PLT/P PLT PLT/RBC/P PLT/RBC 2PLT 2PLT/RBC 2PLT/P #donations 47990 63 1775 10832 968 476 67 1150 142 10252 Citrate exposure (mls) 41-85 71 138 263 266 300 322 349 478 Study Design/Method: A randomized (2:1), placebo-controlled, single blind, 15 subject, single-site study of ascending microdoses of autologous (apheresis-derived) Thrombosomes was conducted.
Subjects were divided into 5 cohorts, receiving increasing doses, ranging from 1/1,000 -1/10 of the lowest effective dose found in the above rabbit model.
Cohorts 4 and 5 received the 1/10th dose, but cohort 5 received two 1/20th doses one hour apart.
The primary end points were safety and tolerability.
Subjects were monitored in-hospital for 24hrs post infusion and followed for up to 60 days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests.
Results/Findings: There were no serious adverse events (SAEs) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline.
There were a total of 68 AEs: 40 were treatment emergent (TEAE), of which 8 were treatment-related (6 Thrombosomes and 2 Control).
All TEAEs were mild or moderate in severity.
In cohorts 4 and 5, 3/4 Thrombosomes subjects had treatment related adverse events.
One cohort 4 subject developed an upper respiratory infection and elevated WBCs within 8 hours post infusion, which resolved by 24 hours, and an elevated D-dimer at 24 hours post infusion, which resolved by Day 7.
This subject also had an elevation of Prothrombin Fragment 1 1 2 at baseline, which increased post transfusion and peaked at 24 hours with resolution by Day 14.
One cohort 5 subject developed non-specific T-wave changes at 1 and 2 hours following her 2nd infusion that resolved by Day 21 without clinical symptoms.
Troponin levels and ECHO stress tests were normal.
EKGs were considered possibly a normal variant or related to placement of the EKG leads.
Another cohort 5 subject developed an IgG platelet autoantibody on Days 7-21, which was undetectable on Days 42-60; there was no change in platelet counts.
The Thrombosomes autoantibody assay was positive at baseline, Days 7-14, and negative on Days 21-60.
Background/Case Studies: Cryopreservation of platelets (PLTs) could extend the shelf life from 5-7 days to over two years.
Cryopreserved PLTs (CryoPLTs) appear to have a greater in vivo hemostatic effect than liquidstored PLTs.
PLTs have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses.
This study was designed to assess whether reconstituted cryo-PLTs carry out protein synthesis upon thawing and short term storage.
Study Design/Methods: Apheresis PLTs were cryopreserved with 5% DMSO and stored at 2808C.
After thawing, the unit was reconstituted in thawed FFP spiked with either 500 lM puromycin (Pm) or 250 nM biotinlabeled Pm.
PLTs were stored at room-temperature with agitation.
Samples were drawn immediately after reconstitution as well as after 2, 4 and 24 hours to assess Pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by CD62P binding, phosphatidylserine exposure by annexin-V binding and microvesicle count in the supernatant.
PLT microvesicles (PMV) were prepared from the supernatant by ultracentrifugation.
PLTs and PMV were lysed in a Triton X-100containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads.
Results/Findings: In vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of CD62P and phosphatidylserine.
PMVs were generated during cryopreservation and the count increased by 11-fold during 24 hour storage.
Immunoblot analyses of the PLTs showed a 2-and 4-fold increase in Pm incorporation after 4 and 24 hours of storage, respectively.
Massspectrometry revealed 23 unique proteins that were synthesized after 4 hours of storage, which was confirmed for GTPase and GTPase-regulatory proteins Rac1, Rap1 and RhoGDI by immunoblot analyses.
Analyses of the PMV translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage.
This finding suggests that a defined panel of proteins is packaged into PMVs upon freezing and thawing.
Additionally, the PMV translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-PLT translatome, including the proteins Rac1, Rap1 and RhoGDI.
Conclusion: This study has demonstrated that cryo-PLTs can synthesize proteins upon reconstitution in FFP and subsequent storage.
Discovery of a subset of these proteins in the PMV suggests their encapsulation, possibly in a selective manner.
This observation provides novel insights into the capacity for protein synthesis in cryo-PLTs and the potential regulation of protein packaging into PMV.
Background/Case Studies: In 2015, the authors' hospital-based blood bank received variances from the FDA and AABB for the use of cold stored platelets (CSPs) with a shelf life of 3 days.
These group A CSPs, stored in a refrigerator in the Emergency Department, were used to support the trauma program for use in massively bleeding patients.
The placement of the CSPs on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients.
Study Design/Methods: Eight double unit CSPs were collected using the Trima AccelV R .
Two double CSPs were pathogen reduced using the Inter-ceptV R pathogen reduction system.
Half of the CSP pairs were subjected to flat storage in a refrigerator; the other half were loaded into a CredoV R 4-496 cooler with 2 units of FFP, 2 units of RBCs, and 1 unit of whole blood.
Three to 5mL of platelets were collected via syringe from each unit at 0 min (before storage in cooler or refrigerator) and after 0.5, 1, 3, 5, and 6 hours of storage for functional validation of platelets.
The platelet count, agonists (thrombin receptor agonist peptide (TRAP), adenosine diphosphate (ADP) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (PS, annexin-V binding), P-selectin, fibrinogen receptor (PAC-1 binding) were measured by Coulter counter, 2 channel aggregometer, and digital flow cytometer.
Paired Wilcoxon rank sum tests were used to analyze differences in degradation rates with p<0.05 deemed significant.
Conclusion: Platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for 6 hours.
Therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival.
C49-A03H Molecular Sieving: Beyond Genotyping Ghazala Hashmi 1 , Reinhard Klemm 2 and Michael Seul* 1,2 .
1 BioMolecular Analytics, 2 ImmunoInformatica Background/Case Studies: More than a decade after its commercial introduction (Hashmi2005 http://bit.ly/2oHLehE), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g.
the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing.
Here, we introduce Molecular Sieving as an alternative to the current approach of managing special donor unit inventories.
This novel process for DNA analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns.
Study Design/Method: Molecular Sieving is a special format of LeanSequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding 401 RBC antigens in MNS,RHCE, LU,KEL,FY,JK, DI,YT,DO and CO, including the identification of 30 RHCE alleles.
Molecular Sieving extends these capabilities to the analysis of pools to attain large scale.
Thus, in one format of the process, 4*4*96 samples are accommodated in a single run.
Following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes.
Here, MolecularSieving was used to identify suitable donor units for 135 sensitized sickle cell anemia ("SCA") patients (Tb1 in Cas-tro2002, http://bit.ly/2oplxHr, excluding Le and e(variant) and assuming 1 request per patient), presenting with up to 9 allo-antibodies ("Ab") in multiple combinations.
Proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests.
Results/Finding: Sieving of only 1 =2 plate holding 4*48 candidate units from actual black donors, followed by profiling of 44 samples selected to enrich for "e Neg" and "c Neg" and "C-E-K-Fya Neg", produced assignments for 127 of 135 requests (594.1%), as indicated by colors, and shown in the row "assigned" below: Thus, the number of assignments substantially exceeded the number of wells processed.
Moreover, the remaining 66 pooled samples produced 44 additional assignments to a second set of 135 requests, for a total of 171 assignments from only 92 wells.
In another scenario, sieving of a full plate of 4*96 samples, produced $250 assignments for two successive batches of 271 requests from SCA patients, a yield exceeding 2.5x.
Sieving alone typically fills 65-75% of requests of moderate complexity ( 5 Ab).
Conclusion: MolecularSieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies.
Molecular Sieving for Identifying Red Blood Cells with Special Phenotype Attributes Kristopher Fernandez 1 , Monica Kalvelage 2 , Ghazala Hashmi* 1 and Michael Seul 1 .
1 BioMolecular Analytics, 2 LifeShare Blood Centers Background/Case Studies: Providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization.
The allelic diversity of the predominantly black patient population, especially at the RH locus which encodes a variety of "partial" phenotypes further complicates the problem (Chou2013 http://bit.
ly/2ppVfEq ).
Study Design/Method: Molecular Sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including 30 at the RHCE locus) that encode 401 MNS, RH, LU, KEL, FY, JK, DI, YT, DO and CO antigens.
Based on sieving, samples may be grouped by molecular attribute patterns ranging from single "Ag2" (e.g.
E2,C2,e2,c2) to specific combinations of "Ag2" (e.g.
C2E2K2Fya2 and C2E2Jsa2) or combinations of alleles such as those encoding partial RH phenotypes.
Sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns.
Genomic DNA from 384 (predominantly) black donors, independently genotyped by one of two commercial methods were provided by LBC.
At bmx, 96 pools were prepared prior to amplification, and analyzed by a novel LeanSequencing method.
Results/Finding: All pool genotypes were consistent with available individual sample genotypes.
Antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous.
Illustrative of the former pattern type are these: Appropriate pool queries revealed that sieving alone identified, among the 124 C2 samples, 24 that were also V2 and VS2 and, among the E2 samples, 12 that were also negative for any partial_e phenotype.
Illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence.
By segregating het pools into subpopulations, we were able to select 6 specific "eE" pools of which 4 were demonstrated (in subsequent profiling) to contain an e-sample.
We also identified pools "het" for alleles indicating the possible presence of a rare donor, for example Yta|b (8 pools), Co a|b (8) and others.
Conclusion: MolecularSieving of a single 96-well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "Ag-neg" patterns including "partial:" RH phenotypes and combinations of C2, E2 and Jsa2.
These samples are thus confirmed "ag Neg" and available for assignment.
Sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand.
"antigen-neg" pattern partial_c partial _c, _e samples available after sieving 124 132 100 100 360 208 192 40 28 92 52 Background/Case Studies: Sequence information generated from next generation sequencing (NGS) is often computationally phased using haplotype-phasing algorithms.
Utilizing experimentally derived haplotype information improves this prediction, as routinely used in HLA typing.
Among the 36 blood group systems, however, experimentally derived haplotypes are known for short genes only, such as ICAM4 (Landsteiner-Wiener) and ACKR1 (Duffy).
For longer genes, such as ABO of >20 kb, most haplotypes are only statistically derived.
We recently established a large dataset of long ERMAP haplotypes, which code for the Scianna blood group system.
Study Design/Methods: The nucleotide sequence of >21 kb each was used for all physically confirmed 48 ERMAP alleles that we previously published.
Full-length sequences were aligned and variant sites were extracted manually.
The Bayesian coalescent algorithm implemented in BEAST v1.8.3 was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny.
Results/Findings: We found at least 4 clades representing clusters of 5 to 11 alleles.
For each clade, one observed allele was identified as the ancestral allele for its cluster of alleles.
Using the 4 alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant.
Conclusion: We explored the phylogenetic structure and evolutionary events underlying the origin of different ERMAP alleles and predict ancestral alleles.
In the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles.
The probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence.
The alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of NGS data.
The new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode Group O.
Rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of A and B and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression.
ABO genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate ABO genotypes with phenotypes.
Background/Case Studies: Evolutionarily related ABO and GBGT1 genes encode A and B glycosyltransferases (AT and BT) and Forssman glycolipid synthase (FS), which catalyze the biosynthesis of A and B, and Forssman (FORS1) oligosaccharide antigens responsible for the ABO and FORS blood group systems, respectively.
Human AT and BT possess LeuGlyGly and MetGlyAla, respectively, at codons 266-268, and these tripeptides are important in determining the sugar specificity of enzymes, N-acetyl-D-galactosamine (GalNAc) for AT and galactose for BT.
Functional FSs possess Gly-GlyAla at the corresponding codons, and exhibit GalNAc specificity.
It has been recently shown that human AT gained weak FS activity when the Leu-GlyGly was substituted by GlyGlyAla, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates.
Study Design/Methods: We have searched for additional mechanisms that might enable human AT to express FORS1.
A variety of amino acid substitution constructs of human AT were prepared.
Additionally, exon deletion constructs of AT mRNA transcripts were also prepared.
DNA from those expression constructs was transfected into COS1(B3GALNT1) cells, and cell-surface expression of FORS1 antigen was immunologically monitored with a monoclonal anti-FORS1 antibody.
Results/Findings: We found that Met69Thr/Ser substitutions also conferred human AT with weak FS activity.
We also found that the deletion of exon 3 or 4 of human AT transcripts bestowed weak FS activity.
Because altered RNA splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of FORS1 antigen on certain cancer cells and tumors in Forssman antigen-negative human species.
Furthermore, the co-introduction of one of those changes together with the GlyGlyAla substitution synergistically conferred strong FS activity, in addition to strong AT and BT activities.
Conclusion: The substitution of the GlyGlyAla tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme.
Met69Thr/ Ser or exon 3/4 deletion may alter the intra-Glogi localization of the enzyme.
And those mechanisms function in synergy.
The overlapping usage of acceptors by glycosyltransferases encoded by ABO and GBGT1 genes is reminiscent of common ancestral origin of alpha 1,3-Gal(NAc) transferase genes.
The finding that AT can synthesize FORS1 implicates that the boundary between ABO and FORS systems may not be as strict as was previously delineated due to the crosstalk in-between.
Rh typing is required by the FDA and FACT/AABB for identity testing.
Since most antibodies in CB plasma are maternal in origin, the ABO/Rh phenotype relies only on the red cell typing.
A and B antigens are not fully developed at birth, presenting about one third of A or B antigen expression levels compared to adult cells.
This can result in indeterminate ABO results for some CB products.
We evaluated the use of DNA-based methods for ABO typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results.
Study Design/Methods: A total of 29,308 CB units (CBU) were typed for ABO/Rh (Beckman Coulter PK System Blood Grouping and Phenotyping) during the period 7/1/2008 -4/1/2017.
ABO genotyping targeting specific SNPs for Groups A, A2, B, O1, and O2 and, if needed, gene sequencing was conducted in cases with indeterminate results, and in 4 CBU that were provided for transplantation with ABO discrepancy found at the transplant center.
Results/Findings: Sixty-two (0.21%) CB samples had no reportable ABO/ Rh phenotype on initial testing, and therefore the CBU could not be used clinically.
Molecular ABO/Rh typing resolved all but one.
All cases were heterozygous (A/O, B/O, or A/B); in 53% the predicted ABO phenotype was A Rh neg (Table 1a ).
The predominant donor race was Caucasian (65%).
Four CBU with ABO discrepancy were also evaluated by genotyping (Table 1b) .
In 3 of those, ABO typing performed at the hospital on the day of transplant differed from that reported by the CB bank; the fourth was identified by posttransplant ABO typing of the recipient.
Molecular genotyping resolved the discrepancies.
CBU identity was always verified by confirmatory HLA typing.
Conclusion: There is currently no FDA approved DNA-based ABO assay.
However, ABO genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" CBU that could not be used otherwise.
Further, genotyping can help resolve ABO discrepancies.
ABSTRACT cobas V R HEV for use on the cobasV R 6800/8800 Systems is a qualitative PCR test for the detection of HEV RNA in human plasma.
The purpose of this study was to evaluate the prevalence of HEV RNA among US blood donations collected in the Midwest, a region reported to have a higher prevalence of HEV infection, and the Eastern US.
Study Design/Methods: 30,695 fresh and 20,029 frozen EDTA plasma samples from American Red Cross donors, collected from February 2015-2016, were de-identified and screened by individual donation testing (ID-NAT) using cobasV R HEV for use on the cobasV R 8800 System under a research protocol.
Samples were primarily from Midwestern and Eastern regions of the US.
Samples reactive on cobasV R HEV were further tested by an alternate HEV NAT, HEV RNA quantitation, HEV genotyping, and for HEV antibodies.
Results/Findings: Of 50,724 valid results, a total of 3 donations were reactive on cobasV R HEV and all were confirmed positive.
The confirmed donations were from a 65-year old male in Indiana, a 21-year old male in California, and a 55-year old female in Kentucky.
All 3 donations were positive by hemi-nested PCR and alternative HEV NAT; however, only the Kentucky donation had a high level of HEV RNA (1440 IU/mL), and was strongly positive for both IgM and IgG HEV antibodies.
The Indiana donation was genotyped as 3a, the California donation genotype 3b, and no genotype determined for the Kentucky donation (see Table) .
The clinical specificity for the cobasV R HEV test in ID-NAT was 100% (95% exact CI: 99.993% to 100%).
Conclusion: Based on the 3 confirmed-positive donations of 50,724 tested, the HEV prevalence was 0.006% (95% exact CI: 0.001% to 0.017%) with a detection rate of 1:16,667 (95% CI, 1:588-1:100,000).
To date, no cases of TT-HEV have been documented in the US.
However, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted HEV.
Background/Case Studies: Monitoring the epidemiology of TTIs within the donor population is critical to provide an ongoing assessment of infection risks associated with FDA policy changes such as the MSM deferral criteria.
TTIMS is a multi-center, federally-funded program intended to derive HBV, HCV and HIV prevalence, incidence, viral genotypes, and donor risk factors for greater than 50% of blood collected in the US.
TTIMS is supported by two distinct coordinating centers (laboratory and risk factor, LRCC, and donation database, DDCC).
Here we report 13 months of prevalence along with demographic trends from the DDCC.
Study Design/Methods: Four blood providers and their respective testing laboratories participated.
Standardized consensus-positive (CP) monitoring definitions were established for donor test results for HBV, HCV and HIV.
These results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set.
Rates of nucleic acid test (NAT) yield (seronegative) and concordant positives (serologic plus NAT positives) were combined to comprise CPs, were computed overall for donors and donations and by demographic, geographic and temporal characteristics.
Where appropriate, rates were compared for differences using 95% confidence intervals.
This analysis contains data from 9/1/15-9/30/16.
Results/Findings: Among 7,578,462 donations reported (16.2% from firsttime and 83.8% from repeat donors), there were respectively 483, 1489 and 194 CP results for HBV, HCV and HIV with corresponding rates of 6.37,19.63 and 2.56 per 100,000 (pht) donations.
Prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of 23:1, 24:1 and 5.4:1 for HBV, HCV and HIV.
Rates (pht) among males were higher than among females for all markers (HBV 8.3 vs 4.2; HCV 23.5 vs 15.2; HIV 3.9 vs 1.0).
In general, higher rates for all markers were seen among minority donors, those in the 25-39-year age group (also 18-24 year for HIV), and those from the southeast (and south central for HIV and HCV, and southwest for HBV).
No trends were noted over time when 3-month periods were compared.
Conclusion: Data from 4 major US blood systems were successfully combined and are a baseline for monitoring purposes.
Demographic trends are similar to those observed in other donor studies and generally agree with community trends.
Changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population.
Screening Donated Blood from Babesia Endemic Regions of the United States Using a Transcription-Mediated Amplification Assay on a Fully Automated System Vanessa Bres* 1 , Melanie C Proctor 2 , Deanna Self 1 , Monique Portugal 1 , Adrian Gurrola 1 , Laura Tonnetti 2 , Sonia Bakkour 3 , Cheryl Lobo 4 , Michael Paul Busch 3 , Susan L Stramer 2 and Jeffrey M Linnen 1 .
1 Grifols Diagnostic Solutions Inc., 2 American Red Cross, 3 Blood Systems Research Institute, 4 New York Blood Center Background/Case Studies: The Procleix V R Babesia assay on the Procleix Panther V R system is a qualitative in vitro nucleic acid test currently under development.
The assay, which is based on Transcription-Mediated Amplification (TMA), detects four clinically relevant Babesia species (B. microti, B. divergens, B. duncani, and B. venatorum) in human whole blood specimens.
This test is intended to screen blood donations individually and in pools of up to 16 donations.
Whole blood samples are lysed and then pooled on the automated Procleix Xpress V R system prior to testing on the Procleix Panther system.
These studies evaluated the preliminary analytical and clinical performance of the Procleix Babesia assay on the Panther system.
Study Design/Method: Analytical sensitivity was determined by diluting in vitro synthesized RNA transcripts for the four Babesia species.
Fresh B. microti-infected hamster whole blood, cryopreserved B. duncani-infected hamster whole blood and fresh B. divergens-infected human erythrocytes were tested to determine the limit of detection (LOD) of parasites/mL (p/mL) by probit analysis.
Clinical sensitivity and specificity were determined by screening 32,274 unlinked whole blood donations collected from August 25 th 2016 to April 7 th 2017 in the northeastern United States.
Initial reactive donations were confirmed by repeat testing, PCR, and/or IgG immunofluorescence assay (IFA).
Reactive individual donor lysates were tested in pools of 16.
Results/Finding: The Procleix Babesia assay detected all four Babesia species with a 95% LOD ranging from 7.10-13.51 copies/mL.
The preliminary 95% LOD in parasites/mL ranged from 0.64-3.61 p/mL for B. microti (n59), from 0.92-1.52 p/mL for B. duncani (n52), and from 0.62-4.95 p/mL for B. divergens (n52).
Of the 32,274 donations screened, 17 initial reactive and 14 confirmed positive donations were identified for specificity of 99.991% (95%CI: 99.972-99.997%).
Of the confirmed positive specimens, 8 were reactive by both IFA and PCR, 5 by IFA only and 1 by PCR only.
All confirmed positive samples were reactive in lysate pools of 16.
Donors of reactive donations resided in CT (11), NJ (1), NH (1) and ME (1) for an overall incidence of 1:2,305, and 1:1,433 in CT.
Conclusion: The Procleix Babesia assay on the Procleix Panther system demonstrated high clinical specificity and sensitivity and detected all four Babesia species with similar sensitivity.
All confirmed positive donations were also detected in pools of 16 thus demonstrating the effectiveness of pooled lysate screening.
Conclusion: Use of the LAg avidity assay shows that in both first-time and repeat HIV-positive US blood donors, newly-acquired (i.e., incident) HIV infections are more frequent in younger donors.
The use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type.
Epidemiology of Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus in United States Blood Donors Lauren A Crowder* 1 , Whitney R Steele 1 , Ed P Notari 1 , James Haynes 1 , Roger Y Dodd 2 and Susan L Stramer 1 .
1 American Red Cross, 2 American Red Cross (retired) Background/Case Studies: From 2004 -2012 , the prevalence of HBV and HCV in US blood donors decreased, while HIV rates remained constant.
However, incidence has not been recently calculated.
Here we report the prevalence, incidence and residual risk (RR) of HBV, HCV, and HIV in a large US blood system from 2008-2015.
Study Design/Methods: Prevalence was calculated in 2-year intervals.
Incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (PY).
RR was calculated using the window periods of 18.5, 7.4 and 9.1 days for HBV, HCV and HIV, respectively.
Linear regressions were calculated with p<0.05 (*) as significant.
Results/Findings: From 1/1/08-12/31/15, there were more than 48 million donations from 13,204,447 donors (51.4% female, 33% First-Time (FT), 81.4% Caucasian).
There were significant decreases in donation prevalence for HBV and HCV (p50.014 and 0.044), but no significant decrease in HIV during the 8 years (see Table for F and R 2 values).
A significant decrease was seen in FT donor prevalence for HBV and HCV (p50.026 and 0.042).
Prevalent FT donors were significantly more likely to be male (68.3% -HBV, 59.8% -HCV, 79.7% -HIV; p<0.001).
Incidence for all agents declined (significant only for HBV; p50.035).
The decrease in HCV incidence was not significant, but there were fewer incident donors in the last 2-year period (74 in 2012-2013 vs. 19 in 2014-2015) .
HCV incident donors in 2014-2015 were more likely to be male (79.0% vs 46.0% in 2012-2013, p<0.001) and were younger (84.2% vs. 67.6% in 2012-2013 <40 years, p50.011).
Overall, incident donors were more likely to be Caucasian males (p<0.01).
RRs for all 3 agents decreased over time with RRs in 2014-2015 of 1 in 1,565,000; 1 in 2,680,000; and 1 in 2,435,000 for HBV, HCV and HIV, respectively.
Conclusion: Prevalence, incidence and RR of HBV, HCV and HIV have generally decreased within this blood system over the 8-year time frame.
As donor screening and deferral regulations evolve, it is important to monitor these risks.
It is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates.
Furthermore, in 2015, MAYV was isolated from a patient in Haiti, suggesting the virus is already circulating in the Caribbean.
The extent of MAYV transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for MAYV emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion.
Study Design/Method: Platelet components (PC) prepared in PAS were spiked with MAYV and treated with amotosalen and UVA illumination.
Samples were collected pre-UVA and post-UVA illumination for infectious titer determination.
AS-5 RBCs were spiked with MAYV, mixed with glutathione (GSH)/Processing solution, dosed with 200lM amustaline, and incubated for 18hrs at room temperature.
Samples were collected prior to the addition of amustaline (pre-treatment) and following the 3hr incubation (post-treatment) to determine infectious titers.
Infectious titers for all samples were determined by plaque assay on Vero76 cells.
The extent of inactivation was determined by comparing the infectious titers (plaque forming units (PFU)/mL) in pre-vs. post-treatment samples.
Results/Finding: MAYV was inactivated to the limit of detection in both PC and RBCs.
In platelets, >6.9 log 10 , or >6.2 log 10 PFU/mL, inactivation of MAYV was achieved.
In RBCs, inactivation of MAYV was >6.2 log 10 , or >5.5 log 10 PFU/mL.
Conclusion: This study demonstrates robust inactivation of MAYV by both amotosalen/UVA treatment in PC and amustaline/GSH treatment in RBCs.
These systems are efficient at inactivating Alphaviruses that have demonstrated or have the potential for transfusion-transmission, including MAYV, CHIKV and RRV.
PRT offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple Alphaviruses are epidemic or endemic, and testing is not feasible.
(Data have not been submitted for FDA review and INTERCEPT for red blood cell is not approved for commercial use).
Thrombotic Thrombocytopenic Purpura with High Adamts-13 Inhibitor May Represent a Distinct Disease Subset in Response to Therapy Based on Immature Platelet Count (A-IPC) Dynamics Hamza N Gokozan* 1,2 , Hollie M Reeves 1,2 and Robert W Maitta 1,2 .
1 Case Western Reserve University School of Medicine, 2 University Hospitals Cleveland Medical Center Background/Case Studies: Thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues.
Early therapeutic plasma exchange (TPE) initiation has improved survival.
Absolute immature platelet count (A-IPC) has been found to aid in diagnosis and follow-up of TTP patients.
A-IPC changes in response to therapy in patients with low ADAMTS13 activity and high inhibitor have not been analyzed in a patient cohort.
We analyzed A-IPC response to therapy in five patients with ADAMTS13 deficiency and high inhibitor at a large tertiary academic medical center.
Study Design/Method: Patients had ADAMTS13 activity of <5% and high inhibitor (1.4-8).
Mean age of cohort 22.8 years (range 17-64).
Four patients were female and one was male.
Patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean 15.2 x 10 9 /L, range 9 -27 x 10 9 /L) and low A-IPC (mean 1.5 x 10 9 /L, range 0.5 -3.6 x 10 9 /L).
Patients were initiated on daily TPE and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab 375 mg/m 2 (4 patients) and cyclophosphamide 400 mg/m 2 (one patient).
TPE continued until platelet count reached 150 x 10 9 /L for at least two consecutive days.
Immature platelet fraction (%-IPF) and A-IPC (%-IPF x platelet count) were obtained with daily pre-TPE CBC.
A-IPC ratio was calculated from baseline.
Results/Finding: Patients responded rapidly to daily TPE (mean of 2.4 days [range 1-4 days]) when they achieved a three-fold increase in A-IPC from baseline (mean 11.1 x 10 9 /L, range 2.2 -25.3 x 10 9 /L) and a rapid improvement in platelet count.
However, this improvement in platelet count was not accompanied by expected decreases in A-IPC, suggestive of recovery from disease.
All patients experienced platelet (mean 217.6 x 10 9 /L, range 200 -294 x 10 9 /L) and A-IPC (mean 19.4 x 10 9 /L, range 13 -28.5 x 10 9 /L) decreases that occurred concurrently while receiving daily TPE so that after a mean of 11.6 days (range 8-14 days) mean platelet count was 65.4 x 10 9 /L (range 14 176 x 10 9 /L) and mean A-IPC 3.2 x 10 9 /L (range 0.7 -6.6 x 10 9 /L).
Patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with TPE after a mean of 20.8 days of A-IPC and platelet count instability.
A-IPC trended to levels indicative of restoration of a negative feedback after this time.
Conclusion: Rapid decreases in platelet counts after a good response in TTP patients may raise suspicion for presence of high ADAMTS13 inhibitor.
Patients with a high inhibitor have similar A-IPC dynamics during which initial high A-IPC production is followed by unexpected decreases in A-IPC concurrent with platelet counts.
Recovery occurs once negative feedback between platelet and A-IPC production is re-established.
Patients with a high inhibitor may represent a distinct subset of TTP as suggested by A-IPC responses.
Benchmarking the Centralized Urgent Plasma Exchange Service for Patients Admitted with a Diagnosis of Thrombotic Thrombocytopenic Purpura at a Multi-Hospital Healthcare System Jansen N Seheult* 1 , Michelle N Stram 1 , Joan Sevcik 2 , Alesia Kaplan 2,3 and Joseph E. Kiss 2,3 .
1 Department of Pathology, University of Pittsburgh Medical Center, 2 Blood Systems Inc., 3 University of Pittsburgh Background/Case Studies: Consensus guidelines recommend that therapeutic plasma exchange (TPE) must be started as early as possible and within 4-8 hours after the diagnosis of thrombotic thrombocytopenic purpura (TTP) has been made; however, there are limited data documenting actual practice.
There are several operational facets of delivering a centralized urgent TPE program in a multi-hospital healthcare system, including: central venous (CV) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure.
This study analyzes the time elapsed between major steps from diagnosis to initiation of TPE in patients admitted with TTP.
Study Design/Method: A retrospective review of the electronic medical record and laboratory information systems from January 1, 2013 to November 1, 2016 was conducted to identify all TTP patients undergoing urgent TPE.
Demographics, comorbidities, and other pertinent laboratory tests (such as ADAMTS-13 activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients.
Temporal data for TPE request, CV access placement, plasma product release (which usually happens after CV access), arrival of TPE team and initiation of the procedure were extracted from procedure notes and the blood bank information system.
Descriptive and summary statistics were generated using Stata version 14 (Statacorp, TX).
Group comparisons were made based on hospital location, level of care and history of TTP using a Wilcoxon rank-sum test.
Results/Finding: Of the 96 TTP patients identified, 22 were excluded due to missing temporal data for important variables.
The majority (85%) of patients were treated at central academic centers, with the remainder being treated at peripheral sites.
Fifteen patients (20%) had a prior history of TTP and 26% had severe ADAMTS13 deficiency on admission.
The median time from TPE request to initiation was 5.6 hours (interquartile range: 4.7-7.2 hours).
There were non-significant trends to shorter time intervals from request to CV access and request to TPE initiation in patients admitted to the intensive care unit (ICU) versus non-ICU patients (table 1) .
Treatment was not started within an 8-hour window in 13 patients; the median time to CV access was significantly longer in these patients (5.8 vs 2.47 hours, p<0.001).
Two of these patients had a prior history of TTP and only four patients had severe ADAMTS-13 deficiency.
The majority (more than 70%) of the time interval between TPE request and TPE initiation was spent obtaining CV access and plasma products.
There were no significant differences in time intervals comparing patients with a new diagnosis of TTP versus patients with recurrent/ relapsed disease (table 1) or between patients treated at a central academic center versus a peripheral hospital.
Conclusion: The consensus 4-8 hour target window from TPE request to initiation appears feasible for a centralized TPE program servicing a multi-48A TRANSFUSION 2017 Vol.
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Addressing limitations in availability of CV access would likely yield the greatest improvement in timeliness of urgent TPE.
Cytoreductive Therapy for Cellular Hyperviscosity: Utility of Cytapheresis Treatment for Chronic Myelogenous Leukemia and Essential Thrombocythemia.
Jan C Hofmann* and Dobri D Kiprov.
California Pacific Medical Center Background/Case Studies: Several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (CML-AT) or essential thrombocythemia (ET) may improve short-term outcomes.
However, no randomized controlled trial (RCT) assessing the efficacy of cytapheresis treatment in this group of pts has been performed.
Study Design/Method: From January, 2006 through January, 2017, we performed cytapheresis (Cy) treatments (txs) for 123 pts with either CML-AT or ET, and clinical and/or laboratory evidence of cellular hyperviscosity.
84 pts (68%) had CML-AT and received 319 leukapheresis (Lp) txs; 39 pts (32%) had ET and received 124 thrombocytapheresis (Tc) txs.
CML-AT pts presented with median WBC 398 x 10 9 /L (range 193-689 x 10 9 /L), of which 63% had blast percent >75% or blast count >100 x 10 9 /L.
Median age was 42 years (8-79 years); 62% were male.
CNS symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates.
22% of CML-AT pts had no sxs of lks; 40% pts had sxs of either CNS or pulm lks (1 sxs), and 38% pts had sxs of both CNS and pulm lks (2 sxs).
ET pts presented with median platelet (plt) count of: 1738 x 10 9 /L (642-3510 x 10 9 /L)and 71% pts had sxs of thrombosis (evidence of CVA or TIA, MI, or DVT).
Median age was 66 years (31-89 years); 58% pts were male.
Results/Finding: All pts received a course of Cy tx with following objectives: 1) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and 2) stabilizing CML-AT pts for induction chemotherapy (ind chemo).
WBC (or plt ct) tx goals were: WBC count (ct) <100 x 10 9 /L for CML-AT pts, and plt ct <450 x 10 9 /L for symptomatic ET pts and <750 x 10 9 /L for asymptomatic ET pts.
CML-AT pts received median of 3 Lp txs (mean 3.9 txs/pt; range 2-8 txs).
ET pts underwent median of 2 Tc txs (mean 3.4 txs/pt; 1-7 txs).
Outcomes were evaluated by percentage of pts who: 1) reached WBC (or plt ct) tx goal, and 2) received ind chemo.
"Improved" outcome was defined as pts who reached their WBC (or plt ct) tx goal during CY tx; "stabilized" were pts who achieved >50% reduction in WBC (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither.
In CML-AT cohort, 76% pts improved, 21% pts stabilized; and 3% pts worsened.
In ET cohort, 85% improved, 14% stabilized, and 1% were unchanged.
For CML-AT pts, median final WBC ct 5 96 x 10 9 /L (range 66-307 x 10 9 /L); 94% pts received ind chemo.
For ET pts, median final plt ct 5 705 x 10 9 /L (263-1087 x 10 9 /L); 95% pts had resolution of thrombotic 49A TRANSFUSION 2017 Vol.
57 Supplement S3 symptoms.
4% of CML-AT pts and 0% of ET pts expired within 1-4 days after course of CY tx.
Of 3 expired pts, 2 pts had both blast crisis and sxs of CNS/ pulm lks; 1 pt had intracranial hemorrhage or CVA; and 2 pts were hypotensive, intubated, or unable to tolerate ind chemo.
Conclusion: Pts with CML-AT or ET and evidence of impending thrombosis may benefit from cytoreductive therapy.
A limited number of cytapheresis treatments (median 2-3 txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes.
A RCT to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful.
Background/Case Studies: Partial normal saline replacement during plasma exchange procedures is common practice.
Benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations.
However, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions.
The goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution.
Study Design/Method: A four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used 100% albumin or 80% albumin-20% normal saline (80/20) as replacement.
Patients who received plasma entirely or partially as replacement were excluded.
The procedure type ordered (100% albumin vs 80/20), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases.
Repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where 100% albumin was used versus those that used 80/20.
Covariates included were fluid types, age and gender.
Odds ratios (OR) and 95% confidence intervals (CI) were used as a measure of risk.
We used the term significant for a two-sided p-value < 0.05.
Results/Finding: During the study period, 3650 procedures were documented for 414 subjects (46% female), age range 0-93 years, of which 2,470 (67.7%) received 80/20.
The type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity.
Replacement with 100% albumin had a significantly lower risk of having either event than by using 80/20, [p50.002, OR (CI): 0.40(0.22, 0.72)] , and also had a significantly lower risk of causing hypotension [p50.023, OR (CI):0.45 (0.22, 0.89)] in addition to a lower risk of causing citrate toxicity [p50.042, OR (CI): 0.
24 (0.06, 0.95)].
Age had a significant effect on having a hypotensive event [p50.04, OR (CI):1.1 (1.0, 1.1)] but no effect on citrate toxicity or the combined outcome.
Gender had no effect on frequency of any event.
Conclusion: Partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure.
Age also increases the risk of hypotension.
Use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients.
Background/Case Studies: Therapeutic apheresis (TA) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (AEs).
There are few studies published on AEs associated with TA but they lack uniformity of data.
Moreover, there is no common database in the United States (US) to report TA-associated AEs.
We evaluated the annual incidence rates of AEs associated with TA at a large tertiary academic medical center over a 10 year period and compared it to published literature.
Study Design/Method: We conducted a 10-year retrospective study of TA procedures performed and AEs were classified according to criteria described in Table 1 .
During the study period, TA were performed using COBE Spectra (Software versions 4.7 and 6.1) and since 2013 the Spectra Optia apheresis system (version 8.0).
Literature search was conducted for data published on AEs associated with TA.
Four studies from US and 13 non-US studies (Canada, Europe and Japan) were analyzed.
Trend for AE rates from 2007-16 was also analyzed.
Statistical analysis was performed using Chi square and Spearman rho tests.
Results/Finding: The overall AE incidence was 6.9% (396 of 5,684 procedures) during 10 year period.
Frequency of AEs associated with therapeutic plasma exchange (TPE) was significantly higher (8.5%, p<0.00001) compared to other TA procedures.
We found significant correlation between number of TPE and AEs (Spearman rho 0.7, p50.002) over the 10 years and significant down trend of moderate and severe AEs with a Spearman rho of -0.64 (p50.04) and -0.83 (p50.003) respectively.
There were no fatalities during the study period.
Majority of AEs were Grade I (60%) and Grade II (28%): 32/5684 (0.6%) procedures were not completed due to AEs.
Comparison of AEs [6.9% (396/5,684)] to both European [11.2% (N513, 12, 256/ 109, 842) ] and other US studies [13.6% (N54, 860/6,324)] showed a statistically significant difference (p<0.0001).
Conclusion: Overall incidence of AEs was significantly lower than current published literature.
Incidence of AEs published in other countries is significantly lower than rates published in US.
Differences in incidence of AEs in literature emphasizes need for uniform reporting and stratification of AEs and development of a common database to report TA-associated AEs.
We propose a grading rationale in order to standardize reporting of AE (Table 1) .
Variations in Biochemical Markers of Bone Metabolism during Plateletpheresis: Impact of Socio-Demographic and Lifestyle Factors?
Markus Dettke*.
AKH Vienna University Hospital Background/Case Studies: Plateletpheresis is associated with short-term variations in biochemical markers of bone turnover.
Socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health.
In the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors.
Study Design/Method: Altogether 200 platelet donors with a donation activity of up to 150 platelet donations participated in the study.
After a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism.
Blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (OC) and the bone resorption marker cross-linked telopeptides of type I collagen (CTX), among other parameters.
The effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors.
Results/Finding: Plateletpheresis resulted in an increase in the serum levels of the bone resorption marker CTX and the bone formation marker OC.
Both parameters returned to base levels within 2 hours after the end of the collection.
Multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in CTX or OC.
There was no association between individual donor career or the number of previous donation and the observed increase in bone turnover.
The only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis.
Increase in serum CTX, showed an inverse correlation to changes of serum ionized calcium.
Continuous IV supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for CTX compared to OC.
Conclusion: The amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism.
Known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover.
Transfusion with Optimized Blood Products Versus Transfusion with Standard Products in a Trauma-Transfusion Rat Model Mathijs Wirtz* 1 , Jordy Jurgens 1 , Jacoline Buchner-Doeven 1 , Joris Roelofs 1 , Philip Spinella 2 , Jennifer A Muszynski 3 , Carel Goslings 1 and Nicole Juffermans 1 .
1 Academic Medical Center, 2 Washington University School of Medicine, 3 Nationwide Children's Hospital Background/Case Studies: Transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products.
We hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction.
Study Design/Methods: Blood products were prepared from syngeneic rat blood according to blood bank standards.
Soluble mediators were removed from red blood cells (14 days old) and platelets (5 days old) by washing.
Plasma was filtered through a 0.22um filter.
Rats ($350 grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $30% of their estimated blood volume, which was calculated to be 57mL/kg.
Hemorrhage continued until a mean arterial pressure of 40mmHg was reached.
Rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham.
Blood samples were taken up to 4h after trauma to assess biochemistry and coagulation status.
Ex vivo whole blood stimulation tests with LPS were performed after sacrifice, and organ damage was assessed by histopathology.
Blood products were sampled to assess for biochemical changes.
Comparisons between groups was done by ANOVA and Dunnett's post-test for multiple comparisons.
Results/Findings: Filtering or washing of blood products significantly stabilized pH, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products.
Both resuscitation groups received an average of 17mL/kg of blood products in a 1:1:1 ratio.
However, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, ASAT and ALAT.
The coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care.
Immune response to LPS was decreased following trauma compared to healthy controls but did not differ between groups.
Conclusion: Filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response.
These results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model.
Safety and Efficacy of Tranexamic Acid during Cardiovascular Surgery: A Single Center before-and-after Study Takuma Maeda* and Shigeki Miyata.
National Cerebral and Cardiovascular Center Background/Case Studies: Tranexamic acid (TXA), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of TXA is effective in reducing blood loss after cardiovascular surgery.
However, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes.
Consequently, we stopped using TXA in April 2013, which enabled us to conduct a before-and-after study.
The present study aimed to examine the association between TXA and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model.
We also assessed the association between TXA and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and 30-day mortality).
Study Design/Method: This single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between January 2008 and July 2015 (n53535).
Because of missing data on patient characteristics, 257 patients were excluded.
The incidence of adverse effects associated with TXA and other clinical outcomes were evaluated before (January 2008 to March 2013, n51987) and after (April 2013 to July 2015, n51291) using a propensity score model.
We estimated propensity scores using a logistic regression model for TXA use as a function of 18 baseline variable, generating 969 pairs of patients who received or did not receive TXA.
We also evaluated the adverse effects of TXA using segmental regression analysis.
Results/Finding: Propensity-matched analysis showed that seizures were more common (8.7% vs 3.7%, p<0.001) and ventilation time was longer (15 h vs 13 h, p50.04) significantly in the TXA group than in the non-TXA group.
In contrast, transfusion volume and blood loss were significantly lower in the TXA group than in the non-TXA group (2000 ml vs 2200 ml, p50.009; and 1265 ml vs 1460 ml, p<0.001, respectively).
However, 30-day mortality was not statistically different between the groups (1.6% vs 1.4%, p50.82).
None of the other outcomes were significantly different.
Segmental regression analysis yielded similar results.
Conclusion: Even though TXA may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality.
The use of TXA is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of Japanese society.
It seems to be advantageous to use TXA because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time.
Sustained Impact of Blood Management Strategies in Orthopedics: Continuous Quality Improvement Linda Levinus* and Michele Deeney.
New England Baptist Hospital Background/Case Studies: Transfusions are one of the most over-utilized treatments performed in any hospital setting (Choosing Wisely Campaign, April 2014, www.choosingwisely.org/societies/american-association-of-bloodbanks).
Costs and risks associated with transfusions are high and may have a significant impact on patient safety.
In our institution we perform over 12,000 joint replacements and spine surgeries per year, making transfusion-associated costs very high.
Since our last formal evaluation of the metrics used post implementation of Patient Blood Management (PBM) Strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians.
The objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard Table 1 ).
The data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through FY16.
Length of stay has also shown a continued reduction, which is an indicator that the PBM strategies implemented have not compromised quality outcomes.
Further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our PBM program.
Going forward, these practices, along with investigating use of additional PBM strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics.
Safety and Efficacy of 4-Factor Prothrombin Complex Concentrate: A Retrospective Review of Outcomes at an Academic Hospital Stephanie Jalaba*, Hollie Benson, Nan Zhang, Jill Adamski and Theresa Kinard.
Mayo Clinic Arizona Background/Case Studies: 4-factor prothrombin complex concentrate (PCC) contains factors II, VII, IX, X, Proteins C and S and is used for reversal of vitamin K antagonists in acute major bleeding or urgent, invasive procedures.
Occasionally, it is used off-label when plasma is not optimal for achieving hemostasis.
This study compares the efficacy of on-label and off-label use of PCC in correcting coagulation parameters and reducing allogeneic blood transfusion.
Study Design/Methods: A retrospective chart review was performed for PCC use at our institution in 2015.
Marginal modeling (GEE method) was used to account for within patient correlation and assess changes in lab values and products transfused.
Logistic regression (GEE method) was used to evaluate potential risk factors for unsuccessful hemostasis (UH5 rate of transfusion after PCC !
rate before PCC) or thrombotic complications.
Results/Findings: The reduction in PT (p5.005) and PTT (p5.05) was significantly greater in on-label than off-label use.
Interestingly, transfusion reduction in RBC (p5.03) and plasma (p5.04) after off-label use was significantly greater than on-label use.
20 cases, both on-label and off-label, with UH were associated with cell saver, acute normovolemic hemodilution (ANH), or cardiopulmonary bypass (CPB).
The odds of having UH were 5.5 times (p5.0072) more with cell saver or ANH, and 5.3 (p5.0130) times more with CPB.
Post-PCC thromboses were identified in 6 cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin K, Factor VIIa, or extracorporeal support.
Background/Case Studies: When a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (RBC) at all times by always having an in-date type and screen specimen.
Per current AABB Standards, this necessitates a new sample every 3 days.
This can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a PICC line may be placed.
In order to mitigate these issues, we chose to extend the type and screen specimen to expire after 7 days in patients without RBC alloantibodies other than passively acquired anti-D due to Rh immune globulin administration.
Study Design/Method: Patients expected to have an antenatal hospitalization of at least 4 days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to 7 days.
The transfusion service medical director reviews the case and gives final approval.
We observed Only 1 patient did not have an in-date specimen when the extended out-dating was requested.
Thirty-eight (38) patients were in-patients continuously until delivery.
Five patients were discharged prior to delivery-1 moved to another state, 1 was admitted later at another local hospital, and three were readmitted for later deliveries.
The mean interval from approval to delivery was 17 days (range 0-63).
Six (6) patients delivered within 3 days of approval.
After approval, the mean number of additional specimens per patient was 2.1 (range, 0-9).
No patient required transfusion prior to delivery.
Five patients received transfusion of at least 1 RBC at the time of delivery, and none had evidence of transfusion reaction.
Conclusion: Since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe.
With only 6 patients delivering within 3 days of approval for extended specimens, 37 patients avoided collection of at least 1 specimen each, and 16 patients avoided at least 4 collections each.
Since new antibodies are not detectable for at least 10 days after immunization, even longer extension of pre-transfusion specimen out-date may be considered.
Although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to 7 days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient.
Iron Metabolism in Critically Ill Patients Developing Anemia of Inflammation Margit Boshuizen* 1,2 , Jan M. Binnekade 1 , Benjamin Nota 2 , Pieter R Tuinman 3 , Kirsten van de Groep 4 , Olaf L Cremer 4 , Janneke Horn 1 , Marcus J Schultz 1 , Robin van Bruggen 2 and Nicole P Juffermans 1 .
1 Academic Medical Center, 2 Sanquin Research and Landsteiner Laboratory, 3 VU University Medical Center, 4 University Medical Center Utrecht Background/Case Studies: Anemia due to inflammatory processes (anemia of inflammation, AI) frequently occurs in critically ill patients.
In AI, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis.
Knowledge on changes in iron metabolism during the course of AI is limited, hampering the development of strategies to counteract AI.
This study aimed to investigate the dynamics of parameters of iron metabolism during the development of AI in critically ill patients.
Study Design/Methods: A case control study was performed in 2 tertiary ICUs in The Netherlands comparing 30 patients who developed AI during ICU stay with 3 control groups: 30 non-anemic patients with sepsis, 30 non-anemic patients without sepsis, and 10 patients with anemia due to acute blood loss.
Patients were matched on age and sex.
A linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time.
Results/Findings: In patients with AI, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) .
Ferritin and hepcidin were increased in AI compared to controls.
In the course of AI development, erythroferrone decreased.
Differences in iron metabolism between groups were not influenced by disease severity.
Patients with AI differed from patients with anemia due to acute blood loss, the latter was characterized by high iron (15.4 vs. 2.9 mmol/L, p<0.001) and transferrin saturation (53 vs. 9 %, p<0.001), and low ferritin (104 vs. 645 mg/L, p<0.001).
Conclusion: In critically ill patients with AI, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy.
Iron metabolism in AI is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that AI is not solely determined by severity of inflammation.
Iron metabolism in AI patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic ICU patients should take the cause of anemia into account.
Clinical Oral Abstract Session: Novel Approaches to Processing and Assessing Cell Therapy Products A Paradigm Shift in Stem Cell Isolation and Storage Jeffrey Drew*.
Cells4life Group LLP Background/Case Studies: Widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells.
The recommended therapeutic cell dose is approximately 2.5 x 10 7 cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals.
Cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (WCF).
However, all current methods result in significant loss of the WCF, some up to 65%, whilst leaving 25% of the starting volume of erythrocytes.
Additionally, there is an almost total loss of potentially important, low abundance cellular subsets.
The use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery.
Study Design/Methods: We have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy.
On combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within 30 minutes.
The WCF remains in solution and can be easily separated from the erythrocyte sediment.
The WCF can then be concentrated by gentle centrifugation into a small volume containing less than 1% of the original erythrocyte content.
The addition of DMSO for cryogenic storage and controlled freezing using standard procedures then completes this simple process.
Results/Findings: We have clearly demonstrated that this method allows almost the entire WCF to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined.
In addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately 65% of the CD341 fraction post separation and freeze thaw (Table 1) .
Possibly more important, the CFU assay results reproducibly yield higher counts of CFU-GM, CFU-GEMM and BFU colonies (Table 1) which is a strong indicator that this method will improve patient outcomes.
In addition, our separation method isolates and preserves the megakaryocyte-like cells (CD451CD611) and early projenitor cells expressing Oct4 and Nanog (markers for VSELs) which are two examples of cellular subsets usually lost using current separation techniques.
Conclusion: These results demonstrate that our method achieves: 1.
Routine recovery of the WCF at levels higher than current methods, independent of volume.
2.
Higher percentage recoveries of all cell types tested than can be achieved with existing methods.
3.
Markedly higher post-thaw recovery of viable nucleated cells than any current methodology.
4.
Almost complete removal of hematocrit.
As a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units.
Therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods.
Effects of Implementation of an Absolute Lymphocyte Count Target, in Addition to CD341 Target, for Hematopoietic Progenitor Cell Collection Edwin A Burgstaler*, Luis F Porrata, Dennis A Gastineau, Eapen K Jacob and Jeffrey L Winters.
Mayo Clinic Background/Case Studies: Lymphoma patients receiving >0.5x10 9 lymphocytes(lymph)/Kg during peripheral blood stem cell transplant have superior survival.
In addition to a CD341 cell target of 4.0x10 6 /Kg, a lymph target was also implemented.
Fifty patients before (No ALC) and after (ALC) implementation were retrospectively evaluated.
Study Design/Method: Lymph and CD341 yields, number of collections, lymph target reached, and days to engraftment were examined.
Mobilization was G-CSF (G) or G-CSF 1 plerixafor (G1Pl).
Consecutive No ALC and ALC procedures were examined.
The Mann-Whitney and Chi Square tests were used for statistical comparison, p< 0.05 considered significant.
Results/Finding: 110 No ALC and 159 ALC collections occurred among the 50 patients.
Fenwal Amicus was used for 91% of the No ALC and 99% of the ALC collections (TerumoBCT Spectra Optia CMNC used for remaining).
Diagnosis was 5 Hodgkin's and 45 non-Hodgkin's lymphoma (No ALC); 7 Hodgkin's and 43 non-Hodgkin's lymphoma (ALC).
Pre procedure WBC and lymph counts were significantly higher for No ALC (WBC 49.3, Lymph 2.0x10 9 /L) than ALC (WBC 39.1, Lymph 1.2x10 9 /L).
Equivalent whole blood (corrected for AC) was processed for No ALC (16.4L) and ALC (17.1L).
For ALC group, extra collections beyond CD341 target were: 0 days: 24%, 1 day: 36%, 2 days: 22%, 3 days: 16%, and 5 days: 2%.
Significantly more patients were mobilized with G1Pl in No ALC group (N581) than ALC group (N560) and 42 collections in ALC group had mobilization discontinued after CD341 cell target reached.
There was no significant difference in G (13.2x10 9 lymph) compared to G1Pl mobilized collections (13.0x10 9 lymph); both were significantly higher than the collections where mobilization had been discontinued (5.9 x10 9 lymph).
Days to WBC engraftment (13.5 No ALC vs 13.0 ALC) and platelet engraftment (13.0 NO ALC vs 12.0 ALC) were not significantly different.
Median number of collections for No ALC (2) and ALC (3) were not significantly different.
Data (medians) in the table.
Conclusion: Not all patients achieved the 0.5x10 9 lymph/Kg or even the 0.3x10 9 lymph/Kg targets.
Implementation of a lymph target increased patients obtaining 0.5x10 9 lymph/Kg from 40% to 54%.
Only 12% had <0.3x10 9 lymph/ Kg.
Discontinuation of mobilization once CD341 cell target was reached significantly reduced lymph yield.
The median increase of one collection per patient following implementation was less than had been expected.
Extended Preprocessing Storage Impairs Cord Blood Hematopoietic Stem Cell Activity Suria Jahan* 1,2 and Nicolas Pineault 2,3 .
1 Canadian Blood Services, 2 University Ottawa, 3 Canadian Blood Services, Centre for Innovation Background/Case Studies: Large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (UCB) unit.
Current NET-Cord-FACT standards specify that units can be stored for almost 48 hours at room temperature (RT) as long as units are cryopreserved by 48-hours post-collection.
The impact of such delay on hematopoietic stem cell (HSC) function is unclear since most studies have not used transplantation assays that measure HSC key properties and activities.
We hypothesized that such processing delay reduces the engraftment activities of UCB units.
We set out to measure the loss in engraftment activities associated with preprocessing storage.
Study Design/Method: UCB units (n53) were split with one half processed immediately (baseline 8-12 hours) and the second after 43 hours storage at RT.
UCB were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use.
Viability was assessed post-thaw, and thawed UCB buffy coat cells were transplanted into NSG mice.
Serial transplantation was used to test the self-renewal and differentiation activities of HSC, while limiting dilution (LD) assay and Poisson statistic were used to estimate the frequency of Scid repopulating cells (SRC) in thawed units.
Results/Finding: Storage before processing had no significant impact on the recovery of viable post-thaw CD451 cells and CD341 cell (n53).
Primary NSG mice were transplanted with a UCB cell dose that contained a total of 7,500 annexinV NEG viable CD341 cells.
The latter was done to avoid any bias towards one group or another.
Short term platelets (190 vs. 140 hPlt/mL, p50.06) and leucocytes (1.2% vs. 0.2% hCD451, p<0.02) engraftment at 4-weeks were significantly reduced in stored mice vs. baseline (n53), and similar results were observed long-term at 16-weeks.
Long-term human bone marrow (BM) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups.
BM cells from primary mice were transplanted into secondary recipients and human engraftment investigated 3 months post-transplant.
Strikingly, the frequency of human CD451 BM cells was 10-fold greater in baseline vs. stored mice (p<0.01, n52).
Hence, storage at RT of UCB units is associated with a deficit in engraftment activity likely due to a loss in HSC activity and/or numbers.
To distinct between both possibilities, the net number of SRC in baseline and stored samples for two units were calculated by LD transplantation assay.
The net number of SRC measured 22-weeks post-transplants were reduced by 30% in unit 1, and by 80% in unit 2.
Conclusion: Prolonged preprocessing RT storage significantly impairs the engraftment activities of UCB units.
The reduced engraftment in secondary transplants coupled with the results from the LD assays suggest that this engraftment deficit origins from loss of HSC numbers.
Our results stress the importance of rapid UCB processing to avoid loss of engraftment activity.
Acoustic Microfluidic Separation of Blood Components Charles Lissandrello, Ryan Dubay, Kenneth Kotz and Jason Fiering*.
Draper Background/Case Studies: New cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing.
While apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process.
Continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood.
It has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density.
Meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells.
It has been shown previously that acoustic separation can separate blood components including purification of lymphocytes.
However, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges.
In contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with RBC and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel.
Study Design/Method: Acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($1mm).
This results in an acoustic force across the channel that drives cells toward the axial center stream.
Because the force increases with a cell's size and density, lymphocytes experience a weaker force than RBCs and other classes of WBCs.
Thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet.
Likewise, platelets can by separated from lymphocytes.
Initial and output cell counts are measured by a standard hematology analyzer.
Results/Finding: In our acoustic system, lymphocyte purity (% of total WBCs) was enriched up to 97%, using leukapheresis product as the starting material.
This enrichment was achieved in a single pass through the device (residence time of 1sec).
Total lymphocyte recovery was 43% and monocyte concentration was reduced 76%.
Furthermore, in a two-pass process platelets were reduced by 75%.
In a 12-fold parallel system we tested RBC separation from plasma and achieved 90% separation at 72ml/hr.
Conclusion: Acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels.
Such disposable devices are suitable for scale up to clinical bioprocessing systems.
Lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument.
Background/Case Studies: The use of natural killer (NK) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies.
NK cells have been used at our institution for the past 15 years.
Most patients have a reaction with NK cell infusion with some reactions being quite severe.
We retrospectively analyzed the reactions associated with NK cell infusions to help address why some patients have more severe reactions than others.
Study Design/Method: Retrospective chart review of NK cell infusions performed at our institution from 9 clinical protocols from 2008-2016.
An infusion reaction was defined as any symptom from the time of NK cell infusion up to 4 hours afterwards.
A severe reaction was defined as any symptom with Grade 3 or higher severity (graded on Common Terminology Criteria for Adverse Events-CTCAE).
Preliminary data was analyzed using R 3.3.1.
Two major endpoints of interest were: 1) infusion reaction with any symptom and 2) severe infusion reaction.
To numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (IQR) were used.
A Wilcoxon test was performed to test the association between the continuous variables and our end points.
A Chi-Square test was used to test the association between categorical variables and our endpoints of interest.
Results/Finding: There were a total of 127 NK cell infusions.
There were 119 (94%) patients with an infusion reaction of any symptom and there were 37 (29%) patients with a severe reaction.
Infusion rate (mL/min) was similar among those with any reaction (median52.55, p50.42) and those with severe reaction (median52.52, p5 0.42).
Infusion rate (mL/min/kg) was also similar among those with any reaction (median50.03, p50.43) and those with severe reaction (median50.03, p50.15 respectively).
Incubation of NK cell product overnight in IL-2 vs IL-15 had similar reaction rates for those with any symptom (88% had reaction with IL-2, 86% had reaction with IL-15, p50.94) and those with severe reaction (28% had severe reaction with IL-2, 24% had severe reaction with IL-15, p50.80).
Patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the NK cell product (median52.44 x 107) versus those without (median51.92 x 107, p50.02).
Conclusion: Our preliminary data analysis reveals that a higher number of monocytes in the NK cell product may contribute to severe infusion reactions, causing patients to have a grade 3 or higher symptom.
Limitations to this study include this was a retrospective review at a single institution.
A Streamlined Mixed Lymphocyte Reaction (MLR) Assay for Evaluation of Human Mesenchymal Stem Cell Immunomodulation Activity Christopher P Delavan 1 , Maryanne C Herzig* 1 , Barbara A Christy 1 , James A. Bynum 2 and Andrew P Cap 2 .
1 US Army Institute of Surgical Research, 2 U.S. Army Institute of Surgical Research Background/Case Studies: Mesenchymal Stem Cells (MSC) have been investigated for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma.
A consensus is building that the immunomodulation by MSCs is key to their therapeutic potential.
MSCs suppress peripheral blood mononuclear cells (PBMC) proliferation in vitro, suggesting a correlation for suppressing PBMC inflammatory responses in vivo.
Current mixed lymphocyte reaction (MLR) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs.
In the study detailed here, MSCs are analyzed in a direct co-culture with PBMCs using a luminescent ATP assay.
Study Design/Method: Blood was obtained from an in house blood bank and PBMCs were separated by centrifugation over Ficoll-Paque in Leuco-Sep tubes as specified by the manufacturer.
The pooled donor PBMCs were stored at -80.
MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) or human umbilical cord endothelial cells (HUVEC) were serially diluted starting at 50-60,000 cells/ well and cultured in 96 well plates for 4-48 h in their respective medias.
On Day 0, MSCs were washed, resuspended in PBMC media and incubated with or without 150,000 freshly thawed PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0-5 lg/ml).
Proliferation of both MSCs and PBMCs was assessed in triplicate wells by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega).
Results/Finding: PBMC proliferation in response to PHA gave a robust ATP signal by 72 h, with >6 fold increase over control PBMCs.
No increase in ATP response or proliferation was seen in the absence of PHA.
Co-culture with MSCs inhibited PBMC proliferation dependent upon MSC passage, source, MSC media additive.
Intra-assay variance of triplicate samples was 10.0%.
Inter-assay variation of MSC preps run under identical conditions was 7.5%.
Inhibition of PBMC proliferation was graded from 0-100% over the range MSC concentrations therefore an EC50 of MSC cell number resulting in 50% suppression of PBMC could be determined for each MSC prep.
This EC50 however was dependent upon PBMC donor pool.
Conclusion: Direct co-culture of live MSCs with freshly thawed PBMCs give a robust determination of immunosuppression by MSCs.
Graded responses can be determined, allowing comparison of potency between MSC preparations.
This streamlined assay can be performed within 72 h, without irradiating cells and with minimal equipment outlay.
Background/Case Studies: A high prevalence of iron depletion (ID) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute 10% or more of the US blood supply.
Differences between donors 16-18 years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion.
Study Design/Method: Donors aged 16-49 were eligible for ferritin testing if they donated at a high school (HS) blood drive at the start of the 2015/16 academic year at two blood centers.
Samples from return donations over the remainder of the school year were also tested.
The prevalence of Absent Iron Stores (AIS, ferritin <12 ng/ml) and Low Ferritin (LF, ferritin <26 ng/ml) were estimated for 16, 17, 18 and 19-49yo groups separately for both genders.
Linkage to operational databases established first-time (FT) vs repeat (RPT) donor status.
Linear regression analysis tested for differences in natural log of enrollment ferritin values by age.
Multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors.
Results/Finding: A total of 4265 donors contributed 6219 donations.
Donors were evenly split by gender, 66% were FT donors, and 87% were 16-18yo.
FT and RPT 16-18yo donors had on average lower ferritin values at enrollment (p<.0001), and a greater percentage were iron-depleted than donors 19-49yo (Table) .
In repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both AIS and LF.
Controlling for these covariates, donors aged 16-18 have sharply higher risk for iron depletion than donors 19-49yo.
Odds for LF were 4 to 6 times greater in the younger donors, and for AIS were 3-to 4fold higher.
Preliminary statistical models indicate 16yo donors may have greater risk for LF than 17 or 18yo by 4 to 5 percentage points, controlling for other factors (p5.06).
Conclusion: The prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in 16-18yo donors than in adult controls.
Logistic regression analysis confirms lower age as an independent risk factor for iron depletion.
Blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors.
Mitigation of Iron Deficiency in Young Donors -a Preliminary Report Ralph R Vassallo*, Marjorie D Bravo, Mary Townsend and Hany Kamel.
Blood Systems, Inc. Background/Case Studies: Iron deficiency is observed in blood donors who meet regulatory hemoglobin (Hb) requirements for blood donation.
Frequent donations result in negative iron balance and eventually lead to anemia.
Young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs.
Study Design/Method: Serum ferritin testing was implemented on 12/19/ 2016 by a large blood collector.
Testing was performed on successful 16-18 y/o whole blood and apheresis donations.
Low ferritin (LF) was defined as a value < 20 ng/mL in females (F) and < 30 ng/mL in males (M).
Donors with low ferritin were notified of deferral from red blood cell (RBC) donations (12 months for F and 6 months for M) and counseled to take 18-28 mg of elemental iron daily for 60 days.
For M and F, a ferritin < 12 ng/mL indicated absent iron stores (AIS) and < 26 ng/mL indicated iron deficient erythropoiesis (IDE).
Ferritin levels !
20 ng/mL in F and !
30 ng/mL in M were considered as indicating an iron-replete state.
Conclusion: Ferritin testing of young donors identified individuals with LF who would benefit from risk mitigation, e.g., delaying subsequent RBC donations and/or taking iron supplements.
LF is more common in F than in M donors.
LF is more prevalent in M and F donors with any RBC donations in the prior 24 months.
An appreciable number of donors with no RBC donations in the prior 24 months presented with LF.
These data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for M than for F, e.g., universal iron replacement in teen male donors may not be warranted above a certain Hb value.
Ferritin Blood Screening in Minor or Young Adult Donors Jennifer L Ritter* 1 , Joan Williams 1 , Michelle Humphries 1 , Nancy Haubert 1 , Ben Reynolds 1 , Michael Phillips 1 , Randall Spizman 1 , Ralph R Vassallo 2 , Hany Kamel 2 , Sally Caglioti 1 , German Leparc 1,3 and Phillip C Williamson 1 .
ABSTRACT completely investigated.
The adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors.
1 New studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors.
2 Study Design/Methods: Over 28,000 serum samples from donors aged 16, 17 and 18 years were analyzed for ferritin levels using the Beckman Coulter AU680 instrument and reagent kit.
The anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody.
Immune complexes formed in solution scatter light in proportion to their size, shape and concentration.
The decrease in light intensity is measured spectrophotometrically.
3 Results/Findings: Background/Case Studies: The risk of cardiovascular (CV) disease in adults can often be identified during adolescent years.
The presence of even borderline levels of multiple risk factors increases the likelihood of a CV event.
Our blood program routinely provides a total non-fasting cholesterol (TC) and blood pressure (BP) measurement for all blood donors.
We added glycated hemoglobin (HbA1c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among 21,007 adolescents (ages 16-19; 61.5% female) who donated blood from 2015 to 2016.
Study Design/Method: Abnormal risk factor levels were defined as HbA1c !
5.7%, SBP/DBP !
120/80 mm Hg and TC !170mg/dL, as suggested by the American Heart Association for adolescents.
The presence of isolated risk factors was defined as one single abnormal risk factor per individual.
Clustering of risk factors was defined as the presence of 2 or more abnormal risk factors in the same individual.
Donor sex was recorded at the time of donation.
Results/Finding: Table 1 shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort.
Overall, 11,283 (53.7%) adolescents had at least one abnormal risk factor (61.8% of males, 48.6% of females).
Of these, 8,709 adolescents had isolated abnormal risk factors, and 2,574 adolescents had clustering risk factors.
Higher proportions of males were in the abnormal BP alone, Background/Case Studies: Pre-donation determination of hemoglobin (Hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality.
However, a variety of Hb testing strategies are used across blood services to satisfy this selection criterion.
This study aimed to identify how Hb screening practices vary across blood donation services and to what extent they influence deferral rates for low Hb.
Study Design/Method: An online survey was performed among members of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.
Additionally, data from literature were used to extend the dataset.
The survey involved a detailed assessment of Hb screening practices, numbers of donations and low Hb deferrals for male and female donors separately.
Multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, Hb cutoffs (high/low with high defined as !13.5 g/dL for men and !12.5 g/dL for women), iron monitoring (Y/N), iron supplements (Y/N providing or prescribing), and geographical location on deferral rates due to low Hb.
Results/Finding: Data were included from 52 blood services worldwide and complete data were available for 25 blood services.
Deferral percentages for low Hb varied from 0.01% to 8.81% among male donors and 0.03% to 46.73% among female donors.
Hb deferral rates were notably higher in Asian blood services.
Overall, iron monitoring was associated with 53% lower Hb deferral rates in men (95% Confidence Interval [CI] 11% to 75%) and 61% lower rates in women (95%CI 15% to 82%).
Iron supplementation was associated to 57% lower Hb deferral rates among women (95%CI 22% to 76%) but there was no evidence of such an effect among men (p50.680).
Each one-week increase in minimum donation intervals resulted in 8% lower Hb deferral rates among women (95%CI 1% to 14%) but not among men (p50.454).
At the 5% level of significance, higher Hb cutoffs do not appear to have an effect among men or women.
Conclusion: The variation in Hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in Hb screening and deferral practices.
Mitigation strategies should consider the variable response among men and women.
These insights can help improve both blood service efficiency and donor care.
were: characteristics of donors (age, sex, size, weight, region); Hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the 5 previous years and the lifetime).
Data were analyzed using logistic regression stratified by sex.
Results/Finding: 9.15% of all candidates for WB donation were deferred in continental France in 2015.
Deferral was significantly more frequent in women (11.16%) than in men (7.29%), due to anemia in 24.41% of deferred women and 9.79% of deferred men.
Plotting mean Hb recovery against time showed mean recovery times ranging from 20 to 30 weeks.
Analysis (Table) identified 3 main factors associated with a higher likelihood of Hb recovery: higher logarithm of time since previous donation, lower levels of Hb at previous donation, higher number of blood donations in the 5 previous years.
Conclusion: The 3 main factors associated with higher likelihood of Hb recovery after WB donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors.
Mean times required for Hb recovery were long enough to require further studies to assess interdonation intervals in France.
Background/Case Studies: Red blood cell (RBC) transfusion has been related to thrombo-embolic events.
Microvesicles in the RBC product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration.
Study Design/Method: We investigated whether transfusion of RBCs containing microvesicles promotes coagulation in human recipients.
As transfusion is mostly administered to ill patients, we used a model of mild endotoxemia.
Eighteen healthy volunteers were randomized to receive either saline, 2 days stored or 35 days stored autologous RBC transfusion two hours after infusion of lipopolysaccharide (LPS, from E.coli, 2 ng/kg).
Blood was sampled every 2 hours up to 8 hours after LPS infusion.
Results/Finding: LPS resulted in a mild increase in thrombin generation.
During storage, the total number of microvesicles increased from 1.4e108 (IQR 8.3e107-1.9e108) /ml in the fresh product to 1.7e110 (IQR 7.9e109-2.3e110/ml; p<0.01) in the stored product (p <0.001), which were mostly RBC derived vesicles.
After transfusion, microvesicles from stored RBC products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within 6 hours.
However, infusion of stored RBC microvesicles did not augment thrombin generation.
Levels of D-dimer and thrombin-antithrombin complex were also unaffected.
Conclusion: Transfusion of autologous RBCs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia.
Background/Case Studies: Transfusion-associated circulatory overload (TACO) is characterized by hydrostatic pulmonary edema related to blood transfusion.
We sought to examine contemporary risk factors and outcomes for TACO during a period where patient blood manaement has led to declines in blood utilization.
Study Design/Methods: At four academic hospitals, cases of TACO were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity.
TACO incidence was calculated, and clinical characteristics were compared with control patients.
Odds ratios (OR) were calculated using multivariable logistic regression.
Hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression.
Results/Findings: 200 cases of TACO and 405 matched controls were enrolled from 20,845 transfused patients who received 128,263 blood components from May 2015 until July 2016.
TACO incidence was 1 case per 100 patients transfused.
In addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of TACO: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (See Table) .
Compared to controls, TACO cases were more likely to require mechanical ventilation (71% vs. 49%; p < 0.001), experienced longer intensive care (4 vs. 3 days; p50.04) and hospital length of stay following transfusion (10 vs. 7 days; p< 0.001), and had higher mortality (21% vs. 11%; p50.02).
Conclusion: The incidence of TACO was lower than what has been reported by prior active surveillance studies.
Despite declines in its incidence and the number of blood components transfused per case, TACO remains a complication of transfusion with significant associated morbidity and mortality.
In addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with TACO after controlling for other covariates in the model.
Additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for TACO.
Background/Case Studies: The residual risk of bacterial contamination of single-donor apheresis platelets (AP) was recently addressed by the March 2016 FDA draft guidance to enhance the safety of platelet transfusion.
This document also describes an existing pathway for AP outdate extension from 5 to 7 days using an FDA cleared rapid test (RT).
Our hospital based transfusion service has used this RT to enhance the safety of AP transfusion since July 2008 and to routinely extend AP outdate to day 7 since February 2016.
This study reports a 103 month experience of secondary screening of AP using a RT.
Study Design/Methods: All AP were obtained from our hospital-based donor center or one of four external suppliers.
AP were screened by culture based methods post-collection and prior to entry into our inventory.
From July 2008-January 2016, AP underwent RT on day 4.
Day 6 and 7 units were transfused with physician approval when deemed medically necessary.
Any units remaining in inventory on Day 8 had a second RT performed.
From February 2016-January 2017, AP underwent RT on day 5 with routine outdate extension to 7 days by performing a second RT on day 6 and a third RT on day 7, as per manufacturer instructions.
Any positive RTs were repeated in triplicate.
Repeat RT positive units were quarantined and cultured to identify true positives.
False positives (FP) were defined as repeat RT negative (type 1) or repeat RT positive with negative confirmatory culture (type 2).
All RT results were reviewed during both study periods.
AP transfusion and outdate rates were also summarized.
Results/Findings: Since July 2008, 20,010 AP were entered into inventory.
Of these, 11,840 (59%) were transfused prior to RT testing.
The remaining 8170 (41%) underwent RT on day 4 or day 5.
Of these 43 (0.5%) were RT positive (29 type 1 FP, returned to inventory; 14 type 2 FP, discarded), leaving a total available inventory of 8156 units tested by RT.
Of these, 5631 (28% of original inventory) were transfused before the end of day 5 and the remaining 2525 (13% of original inventory) reached a day 5 outdate.
A total of 1561 (8% of original inventory) were transfused on day 6 or day 7.
Of these, 768 underwent a second RT on day 6 (2 RT positives; 1 FP type one and 1 FP type 2) and 233 underwent a third RT on day 7 (no positive results).
A total of 964 (5% of original inventory) outdated on day 7.
Of these, 754 underwent a second RT on day 8 (no positive results).
Conclusion: To date we have performed 9925 RTs on AP at our hospital.
No true positives have been identified.
Use of RT over the study period decreased our outdate rate from a predicted 13% to only 5%.
A total of 1522 AP have been tested twice by RT (768 on day 5 and 6; 754 on day 4 and 8) with 2 (0.1%) positive results, both of which were deemed FP by repeat testing or culture.
A total of 233 units have been tested 3 times (day 5, day 6 and day 7) with no additional positives identified.
We have not yet identified any units with an initial negative RT result that subsequently converted to a true positive.
There is a low FP rate which should also be expected when performing repeat testing on the same unit.
These data suggest that the yield for repeating the RT every 24 hours, as currently specified by the manufacturer instructions, is quite low.
Additional studies are needed to clarify how RT can optimally be used to enhance detection of AP bacterial contamination.
Survival of Trypanosoma Cruzi in Human Blood Components Laura Tonnetti*, Aaron Thorp and Susan L Stramer.
American Red Cross Background/Case Studies: Trypanosoma cruzi, the agent of Chagas disease, is associated with 8 to 10 million infections worldwide, mostly in Latin America.
Despite the extensive immigration from endemic areas, only 5 cases of transfusion-transmission (TT) T. cruzi have been reported in the US, before blood donor screening was implemented in 2007.
Contributing factors to the low number of TT cases are a possible association between parasite lineage and TT, and high numbers of unreported cases.
Platelets are almost exclusively involved in T. cruzi TT cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (RBCs).
We investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in TT of T. cruzi.
Study Design/Method: Whole blood (WB) units were spiked with T. cruzi trypomastigotes to a final concentration between 10-10,000 parasites/mL.
Each parasite concentration in WB was tested x2.
An aliquot of contaminated WB was used to prepare hemocultures to detect live parasites before preparation of components.
RBCs were separated and half of the components leukoreduced (LR) by filtration.
Platelets and plasma were separated, along with one aliquot of plasma collected before LR.
RBCs were stored at 48C for up to 42 days; platelets were stored at 228C (RT) under agitation for 5 days and plasma was frozen at -208C.
Aliquots for culture were removed weekly from RBCs, daily from platelets and after 30 days from frozen plasma.
All samples were cultured in Liver Infusion Tryptose (LIT) media at 278C for detection of live parasites for up to 16 weeks.
Results/Finding: Hemocultures from spiked-WB were positive at all concentration of parasites.
LR'd and non-LR'd RBCs cultured before storage were positive at all concentrations.
After storage at 48C, RBCs from all units spiked with 10,000 parasites/mL were positive for up to 21 days; all further times yielded negative results.
At lower concentrations, only non-LR'd RBCs spiked with 1000 parasites/mL were positive for up to 7 days.
Plasma samples cultured before freezing were positive at the highest concentration in one non-LR'd sample, while all others were negative.
Platelets obtained from WB spiked with 10,000 and 1000 parasites/mL were positive up to 5 days at RT.
No parasites were observed in plasma or platelets prior to storage at lower concentrations.
Molecular analysis to determine the presence of parasite DNA in each component is on-going.
Conclusion: Platelet storage conditions offer a suitable environment for T. cruzi survival; however, high concentrations of parasites also survived in RBCs at 48C for up to 3 weeks.
Leukoreduction offers partial protection, while freezing conditions appears unsuitable for T. cruzi survival.
Hemovigilance Monitoring of Platelet Septic Transfusion Reactions (STR) after Treatment with INTERCEPT TM Pathogen Reduction or Large Volume, Delayed Bact/ALERT TM Bacterial Culture Screening Richard Benjamin* 1 , Marion Lanteri 2 and Larry Corash 1 .
1 Cerus Corporation, 2 Scientific Affairs Department, Cerus Corporation Background/Case Studies: Amotosalen/ultraviolet A (UVA) light (INTER-CEPT TM Blood System, Cerus Corporation) pathogen reduction (PR) and delayed, large volume, bacterial culture with the BacT/ALERT TM System (DLVBC) (BioMerieux, Inc) represent respective best-in-class systems to reduce the risk of STR associated with platelet concentrates (PC).
Where implemented, hemoviligance (HV) programs continue to receive reports of suspected STR, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure.
Study Design/Methods: United Kingdom (2006 -2015 ), French (2006 -2015 , Swiss (2011 -2015 ), and Belgium(2009 -2015 HV reports, and Cerus Corporation's adverse event records were reviewed to assess the residual risk and imputability of STR with amotosalen/UVA-treated or DLVBC-screened PC.
Results/Findings: Approximately 1.35 million DLVBC-screened were issued with a 7 day outdate after release into inventory 3 days after collection, and $2.3 million amotosalen/UVA-treated PC were released into inventory on day 1 or 2, with a 5 to 7 day shelf-life.
No septic fatalities were reported with either technology.
The French, Belgium and Swiss HV programs monitored >2.83 million conventional, non-DLVBC-screened PC and recorded 58 STR and 9 fatalities.
Concurrently, zero definite and 2 possible STR were reported with 607,871 amotosalen/UVA-treated PC, significantly fewer than with conventional PC (Table 1 ) (20.5 STR per million vs. 0.0 per million, P<0.001).
One definite, 1 possible, 7 undetermined/indeterminate non-fatal STR and 5 contaminated "near miss" PC were reported with 1.35 million DLVBC-screened PC between 2010 and 2015, for a reduced falsenegative rate compared with the prior five years (3.7 STR per million vs. 16.3 per million, P <0.05).
HV programs highlight a major weakness when reporting STR.
Stringent criteria are used to determine definite imputability, including evidence of patient infection, PC contamination and irrefutable evidence of a donor source, with confirmation of strain identity.
Reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis.
Some of these cases are almost certainly due to bacterial contamination of PC, suggesting that the actual rates of sepsis are considerably higher than that reported by HV programs.
Conclusion: Best-in-class pathogen reduction and bacterial culture systems reduce STR risk, although underreporting and inadequate clinical data may result in underestimation of the true rates.
Pathogen reduction of Background/Case Studies: Despite extant mitigation measures (e.g.
diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the United States (US).
Consequently, the US Food and Drug Administration has recommended adoption of additional measures such as point of release testing (PORt) and/or pathogen reduction to safeguard against transfusionassociated sepsis.
However, PORt poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention.
We evaluated a second bacterial culture to contend with residual risk.
Study Design/Method: Phased implementation of secondary bacterial culture testing (BacT/ALERT TM ,BioMerieux, Inc., Durham, NC) was initiated in October 2016 for all platelets received at our institution.
At time of receipt at the blood bank (day 3 post collection), products were sampled using a sterile connection device (TSCD TM , Terumo, Elkton, MD) and a sampling kit (Sam-pLok TM Sampling Kit, 10 mL, ITL BioMedical, Malaysia).
Five mLs of product was transferred aseptically to BacT/ALERT BPA (aerobic) culture bottles using the same sampling device.
Inoculated culture bottles were loaded into the BacT/ALERT incubator modules and incubated at 35C for three days.
Results/Finding: A total of 9473/11,066 (85.6%) platelet products were successfully cultured (934/1373 [68.03%] and 1842/1912 [96.3%] in October 2016 and March 2017 respectively).
Over the 6-month period, two true positive cultures were obtained (incidence of 1 in 4736 platelet products).
The cultures grew Acinetobacter species (Case A) and coagulase negative Staphylococcus species (Case B); both positive results were obtained four days following collection.
Repeat testing of Cases A and B grew the same organisms identified in the initial cultures.
There was a co-component in our inventory (Case A) with negative initial and repeat cultures.
None of the products were released for transfusion.
The initial post-collection product cultures remained negative at the collection facility.
Over the same time period, no false positives were detected.
Implementation required hiring one additional dedicated FTE; the total cost (technologist time, equipment and related supplies) was calculated to be $US16.83 per product tested.
The cost per averted case was $US79,707.
Conclusion: We demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions.
This presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions.
Importantly, it offers a viable alternative to PORt in high volume institutions where logistic (e.g.
time and personnel) constraints impede practical adoption of PORt.
An increase in cases of blood culture positive transfusion reactions (BCPTR) was noted at our hospital; BCPTR was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction.
We sought to characterize the risk and clinical presentation of BCPTR at our institution.
Study Design/Method: An analysis was conducted of all reported transfusion reactions at Johns Hopkins Hospital (JHH) between January 2009 and December 2016.
The data, extracted from hemovigilance records, were evaluated to determine the incidence of BCPTR; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction.
Bacterial culture results were evaluated for both patients and associated blood products (i.e.
partially transfused or residual product in blood bag).
Results/Finding: In the 7-year study period, a total of 3280 transfusions reactions were reported, 18 of which were BCPTR (0.55% of transfusion reactions).
Of the 18 BCPTR, 15 (83%) were associated with apheresis platelets, 2 (11%) with red blood cells, and 1 (6%) with plasma.
Recipient diagnoses spanned hematologic/oncology (n512), renal (n53), cardiac (n51), autoimmune (n51), and obstetrics (n51).
An organism was identified in both the blood product and recipient in 10 (56%) cases; in 6 (33%) cases an organism was grown in the blood product but not the recipient; and in 2 (11%) cases an organism was isolated from the recipient only, due to inability to culture the product.
The transfusion recipients in 5 of the 6 cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion.
Symptoms of BCPTRs included fever (83%), chills (67%), nausea and vomiting (50%), pain (27%) and dyspnea (22%).
Blood pressure (BP) decreased in 22%, increased in 17%; 61% of reported BCPTRs had no change in BP.
Conclusion: The signs and symptoms of BCPTRs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting.
Furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions.
Hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions.
Furthermore, excessively stringent criteria (CDC/NHSN Blood Safety Surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics.
Clinical Oral Abstract Session: Immonohematology and Genetics --Sickle Cell Disease and Beyond Blindspots and Cross-Reactivities of Anti-Human Globulin Specific for IgG Subtypes Heather Howie 1 , Jenna Lebedev 1 , Linda Kapp 1 , Xiaohong Wang 1 , Meghan Delaney 2 , Lay See Er 1 and James C Zimring* 3 .
1 BloodworksNW Research Institute, 2 Bloodworks NW, 3 University of Washington School of Medicine Background/Case Studies: There are four different subclasses of human IgG (IgG1-IgG4), each with different effector function.
Essentially all existing data on the effect of IgG subclass on hemolytic transfusion reactions and HDFN, were generated using AHG specific for IgG subclasses.
In recent decades, it has become appreciated that there are at least 29 natural human variants of IgG.
In this study, the reactivity of IgG specific AHG was tested against all 29 known variants.
Study Design/Methods: The heavy and light chain variable regions of an anti-K1 monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known 29 IgG variants.
Plasmids were expressed by co-transfection into CHO cells.
The resulting panel of antibodies were pre-incubated with K11 RBCs and were then subjected to testing with currently available IgG subtype specific AHG (monoclonal AHGs from Southern Biotech and Sanquin, polyclonal AHGs from Sanquin and The BindingSite).
All testing was carried out by flow cytometry.
Results/Findings: Polyclonal reagents against IgG2, IgG3, and IgG4 had cross-reactivity with variants found in other IgG subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table 1).
Titrations of the AHGs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected.
However, cross-reactivity could be neutralized by pre-incubating AHG with the crossrecognized IgG forms (against a third party antigen); the remaining reactivity recognized the intended IgG subtype without detectable cross-reactivity.
No cross-reactivity was detected for polyclonal anti-IgG1 or for any of the monoclonal AHGs tested.
Monoclonal anti-IgG3 had a blindspot for IgG3-04, due to the shorter hinge region on IgG3-04.
No blindspots were detected in other monoclonal or polyclonal AHG.
Conclusion: The relative quantitation of different IgG subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology.
Herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots.
As such, the existing data regarding IgG subtype biology may have some inaccuracies as a result of these defects in IgG specific AHG.
Genotype Matching for Pediatric Sickle Cell Disease Patients Nancy Robitaille* 1 , Yves Dominique Pastore 1 and Maryse St-Louis 2 .
1 CHU Sainte-Justine, 2 Hema-Quebec Background/Case Studies: Among the different treatment modalities available for sickle cell disease (SCD), blood transfusion is frequently used.
However, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for C, E and Kell antigens.
This is partly explained by different antigen frequency among Caucasian blood donors and African-American recipients, and by variants in the Rh blood group of people of African-descent.
Blood group genotyping has been proposed as a potential way to alleviate this problem.
The SCD cohort of a pediatric academic hospital was genotyped for RHD, RHCE and FY genes.
The primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar Rh variants could be identified.
Study Design/Methods: Since 2008, our local blood provider intensified recruitment of African-descent blood donors.
These donors were phenotyped and genotyped for clinically relevant antigens by different means: GenomeLab SNP Stream, laboratory-developped assays and IDCoreXT.
As of 2014, 205 SCD children were genotyped by sequencing RHD, RHCE and FY cDNAs after obtaining informed consent.
Extended red blood cell phenotypes were done at diagnosis at the hospital.
Patients' genotypes were compared to H ema-Qu ebec's donor database to attribute blood donors to specific patients.
Results/Findings: From diagnosis until September 2016, 117 (57%) patients had been transfused and 14 had antibodies with known blood group antigen specificity: anti-C, anti-E (2), anti-hrb, anti-Fya, anti-Jka, anti-Jkb (2), anti-S, anti-M, anti-Sc2, anti-Leb (2).
Seventeen patients (8.3%) were either D2 or partial D. RHCE results showed that 163 patients expressed a normal c antigen and 32 expressed partial c. As for e antigen, 163 had a normal antigen, 38 bore a partial antigen and 3 were weakly expressed.
Fy(a2b2) phenotype was found in 182 (89%) patients.
A total of 2606 genotyped blood donors of African-descent were available.
The table below indicates the compatibility with these donors.
Conclusion: This study shows that several patients have RHCE variants difficult to match, even with available genotyped blood donors from their community.
Although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs.
The continued effort put towards recruitment and pheno/genotyping should improve the situation.
Using Genetic Markers to Select Responders and Non-Responders Sickle Cell Disease (SCD) Patients for Transfusion with RH Haplotype Matching Red Blood Cell (RBC) Units Tamires Delfino dos Santos 1 , Emilia Sippert 1 , Mayra Dorigan de Macedo 1 , Sheila Fatima Perecin Menegati 1 and Lilian Castilho* 1,2 .
1 Hemocentro Unicamp, 2 University of Campinas Background/Case Studies: RBC alloimmunization has been associated with several factors and with individual characteristics of each patient.
We recently found that TNFA-308A, IL1B-511T cytokine polymorphisms, RHAG 808G>A and HLA-DRB1*15 alleles may predict a good responder phenotype (Sippert et al, Transfusion 2017) and that RHAG 808A and HLA-DRB*15 alleles are closely linked to RH alloimmunization.
Based on this and considering the challenge to fulfill the transfusion needs of the patients with RH variants, we used these genetic markers to select responders and nonresponders SCD patients for transfusion with RH haplotype matching RBC units and evaluated the risk of alloimmunization.
Study Design/Method: Our study included 96 non-alloimmunized patients with SCD, homozygous for HbS, receiving a range of 5-289 RBC units.
RBC antigen phenotypes of each patient and history of RBC antibodies were obtained from the medical records and transfusion service computerized database.
RBC genotyping was performed using wHEA, wRHD and wRHCE BeadChip arrays (BioArray Solutions, Immucor) in accordance with the manufacturer's instructions.
Cytokine gene polymorphisms (TNFA-308G>A, IL1B-511C>T) and the RHAG 808G>A gene polymorphism were analysed by PCR-RFLP and TaqMan assays.
HLA class II genotyping was performed using PCR-SSO.
Results/Finding: Among 96 non-alloimmunized patients, 21 were homozygous or compound heterozygous for RH variant alleles.
From those, 6 had RHAG 808A and/or HLA-DRB*15 alleles and at least one cytokine polymorphism (TNFA-308A or ILB1-511T) associated with risk of alloimmunization and were transfused with extended and RH haplotype matching RBC units.
The other 15 patients with no risk factors associated with RBC alloimmunization were considered non-responders and were not transfused with extended and RH matching units.
All patients were followed for one year and did not develop RBC antibodies.
Conclusion: These findings contributed to the development of a transfusion strategy for non-alloimmunized SCD patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder SCD patients, allowing blood with high level of compatibility to be Five discrepant samples required sequencing.
ID CORE XT identified three RHCE*ceAR samples encoding a partial c, and a partial e (predicted phenotype: Vweak, VS-) and 2 were confirmed by sequencing.
The third sample was found to be RHCE*ceVS.01,RHCE*ceBI on sequencing (predicted phenotype V1,VS1).
The 3 samples were typed as V1 (or ce S ) and VS1 (or e S ) by HEA.
In addition, ID CORE XT accurately identified RHCE*ce[712G]in 2 samples.
This SNP has been linked to various allelic variants affecting c and e antigenic expression.
Both samples were predicted to be c1 by HEA.
Conclusion: Blood group genotyping platforms vary depending on the specific SNPs that are included in each assay.
Such variations may be clinically significant when genotyping is used as a tool for providing matched blood.
Discrepancies leading to differences in the predicted phenotype could affect unit selection.
Despite the discrepancies between the 2 methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes.
Background/Case Studies: Over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (wAIHA) on the basis of DAT scores of agefractionated RBCs: Type I wAIHA, comprising 80% of patients, showed increased binding of autoantibodies to aged RBC, whereas Type II wAIHA autoantibodies (20% of patients) bound young and old RBCs with no apparent prejudice.
Band-3 is a ubiquitously expressed RBC transmembrane protein which plays a vital role in maintenance of RBC structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with wAIHA.
Band-3 is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal RBC senescence, which causes band-3 to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band-3 aggregates forming new epitopes which are recognized by natural IgG autoantibodies causing phagocytosis and destruction of senescent RBCs.
Type I wAIHA has been postulated to be caused by an exacerbation of normal RBC senescence.
Study Design/Methods: In an effort to confirm and characterize the two wAIHA subtypes we age-fractionated whole blood samples from 22 patients with wAIHA on discontinuous PercollV R gradients and looked for differences in DAT results between less (young RBCs) and more dense (aged RBCs) fractions, fractionation patterns and band-3 tyrosine phosphorylation.
Results/Findings: We confirm that two distinct types of wAIHA can be identified based on autoantibody reactivity with the youngest and oldest autologous RBCs.
Further, comparing 5 Type I and 5 Type II patients, we found that Type I is characterized by 5 PercollV R fractions (similar to healthy storage-matched controls) but increased band-3 tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of RBC development.
Type II patients were characterized by 3-4 PercollV R fractions, lacking the fraction containing the oldest RBCs, and showed a complete lack of, or dramatic decrease in, band-3 tyrosine phosphorylation compared to healthy storage-matched controls.
Conclusion: These results confirm the two distinct types of wAIHA.
In Type I wAIHA, the increased binding of autoantibodies to older RBCs coupled with increased tyrosine phosphorylation of band-3 suggests that RBCs from Type I patients are aging faster than RBCs from normal healthy controls; this may represent an accelerated and pathogenic form of normal RBC senescence.
In contrast, Type II wAIHA where autoantibodies bind strongly to either young or old RBCs coupled with a lack of fractionated bands that represent the oldest RBCs and a dramatic diminution in tyrosine phosphorylation of band 3 suggests faster destruction of RBCs, consistent with the early published data, and metabolic changes that could affect RBC function.
Microbial Pathogen Primary Sequence Correlates with Blood Group Antigen Immunogenicity Ian Baine* 1 , Burak Bahar 1 , Jeanne Hendrickson 2 , Krystalyn E Hudson 3 and Christopher A Tormey 1 .
1 Yale-New Haven Hospital, 2 Yale University, 3 Background/Case Studies: It is known that specific groups of patients immunologically respond more readily than others to RBC antigens.
While RBC antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved.
Studies have shown that there is significant primary sequence identity between common RBC antigens and microbes, and that cross-reactivity is possible between antigens in experimental models.
We hypothesize that responder populations may be immunologically primed to form RBC alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity.
Study Design/Method: We performed peptide homology searches of the most immunogenic RBC antigens, based on previously published antigenicity findings.
Thirteen amino acid peptides containing the polymorphic residues of K, Jk a , Lu a , E, c, M, C, Fy a , and S antigens were queried for identity with microbial peptides using the BLAST database (blastp, PAM30 ABSTRACT algorithm, E value51x10 -6 , Word Size5 6, Gap Costs: Existence59 Exten-sion51).
Search results were restricted to bacteria and fungi, with a selective threshold of >80% identity set for inclusion criteria.
To corroborate with observed patient data, we also examined preceding cultures from 162 alloimmunized patients to explore agreement between specific pathogens and RBC alloantibodies.
Results/Finding: Significant peptide identity was found between RBC antigens and pathogenic organisms including B. fragilis, P. aeruginosa, Candida spp.
among others.
Linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when Fy a (an outlier) was removed (b5-0.0017, r 2 50.624 & p50.0197); that is, lower immunogenicity antigens were associated with larger number of kingdoms.
k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (C), moderate (E, c, S, M) and high (K, Jk a , Lu a , Fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence.
Of 162 alloimmunized patients reviewed, 105 were culture-positive.
Of these, 76% of the anti-C/c group (13 of 17 patients) and 16% of the anti-K group (7 of 43 patients) had microbe-antibody agreement.
Remaining microbe-RBC antibody agreements ranged from 0 -11.1%.
Overall, 21.9% (23 of 105 patients) demonstrated agreement.
Interestingly, we observed a particularly strong agreement between infection with Klebsiella species and anti-K, despite the lack of >80% sequence identity.
While 27.6% (29 of 105) patients reviewed had positive cultures for Klebsiella species, 62.1% of these (18 of 29 patients) demonstrated an anti-K.
Conclusion: Our study highlights the potential connection between microbial infection and RBC alloimmunization, based on shared epitopes.
We speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between RBC antigen immunogenicity and prevalence of microorganisms.
Longitudinal studies of microbial carriage (or acute microbial infection) and RBC alloimmune responses in larger patient cohorts may be informative.
Background/Case Studies: Thromboelastogram (TEG) has been incorporated into many hospital armories to manage transfusions during cardiovascular (CV) surgeries.
Some institutions use well-defined protocols for TEG utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative).
On the other hand, at some institutions TEG utilization is driven mainly by clinical judgment.
When TEG is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before TEG is performed.
There is no published literature on how pre-TEG transfusions impact TEG results and guide further transfusion requirements during CV surgeries.
In this study, we have tried to address this issue.
Study Design/Method: We retrospectively reviewed 109 TEGs performed on 76 patients undergoing CV surgeries at our institution from Jan 1 to Dec 31, 2016.
No specific TEG protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period.
Only the first TEG performed during surgery was included in the analysis.
We excluded the patients that received only red blood cell (RBC) transfusions during the surgery because RBC transfusions are usually not based on TEG results.
For the 56 TEGs analyzed, TEG results were divided into three categories: "normal" (reaction time (R), kinetics (K), angle (a), maximum amplitude (MA), and lysis at 30 minutes (min) all within reference range), "hypocoagulable" (R>10 min, K>3 min, a<53 degrees, MA<50 mm) and "hypercoagulable" (R<5 min, K<1 min, a>72 degrees, MA>70 mm).
Fisher's exact tests and Z-scores for two population proportions were used to identify statistically significant differences in TEG results and blood product utilization.
Results/Finding: Out of 56 TEGs analyzed, 37 patients (66%) received pre-TEG transfusions.
We found significantly fewer hypocoagulable TEG results in pre-TEG transfused patients than nontransfused patients (8% vs. 32%, p50.02).
The data also reflected a trend suggesting that there may be more normal TEG results in pre-TEG transfused patients compared with nontransfused (32% vs. 11%, p50.07).
There was no statistically significant difference in transfusions after obtaining TEG results in both groups.
However, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-TEG compared to nontransfused (100% versus 33%, p50.06).
Conclusion: Pre-TEG transfusions impact TEG results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during CV surgeries.
The decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-TEG transfusions may be due to more clinical significant bleeding in these patients to begin with.
Background/Case Studies: Orthotopic liver transplantation (OLT) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion.
In this setting, cell salvage autotransfusion (CS) is been used as an alternative to decrease allogeneic red blood cell transfusion.
However, as long as some studies have shown that CS in OLT decreases allogeneic blood transfusion, others reported that CS presented little benefit or might have been associated with increased blood loss through fibrinolysis.
In this study, we evaluate CS efficacy in reducing allogeneic blood transfusion in the intraoperative period.
Study Design/Method: We retrospectively evaluated data from 670 liver transplants, performed from 2011 to 2015 in a single-center.
Patients were divided in two groups: one with cell salvage (CS) and another without CS (NCS).
Study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups.
CS was used in all liver transplant recipients but patients with malignancy and sepsis.
Blood transfusions were indicated based on clinical and hemodynamic criteria.
Clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and Model for End-Stage Liver Disease (MELD) score.
Statistical analyses were performed using t-test, Chi-square test, Mann Whitney test.
Results/Finding: In this study period, 670 OLTs were performed.
A total of 345 patients was submitted to CS.
The median age was 51 years (range 10-78 yo).
Cirrhosis caused by chronic hepatitis C virus infection was the main etiology of liver disease.
Hepatocellular carcinoma (HCC) was found in 31,6% of the patients.
The average MELD score was 29,6 6 9,4 and it was slightly higher in the CS group (31,3 vs 27,9, p<0,001) .
There was no statistically significant difference in other variables such as body weight, height and cold ischemic time.
The mean salvaged blood volume was 8856 6 4503 ml and mean reinfused blood volume was 914 6909 ml.
Allogeneic blood transfusion was required in 71,8% patients in the CS group, compared to 46,7% patients in the NCS group.
However, average red blood cells (RBC) and fresh frozen plasma (FFP) units transfused were lower in the CS group.
The threshold for RBC transfusion was significantly lower in the CS group (2,4 units vs 3,39 units, p<0,001 Background/Case Studies: Hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsADDIN EN.CITE.DATA.
Massive transfusion protocols (MTP) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geADDIN EN.CITE.DATA.
Blood product wastage benchmarks are loosely established, and data on wastage associated with MTPs especially sparse.
With a redesign of MTP and Obstetric Massive Transfusion Protocols (OBP) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols.
Study Design/Method: Following Institutional Review Board approval, a retrospective study on blood product wastage associated with the MTP and OBP between July 2015-December 2016 was performed.
Data on numbers of products dispensed and wasted were manually collected from Transfusion Service paper and electronic records and an automated data report from the electronic medical record.
Results/Finding: The MTP resulted in higher total number of wasted products than the OBP (27 and 15 products, respectively) however, OBP wastage occurred more frequently in the 18 month period.
This reflects automatic thawing of cryoprecipitate in the first round of deployed products in the OPB.
MTP-trauma activations contributed higher wastage than non-trauma activations (23 versus 4 products).
This is skewed by one month when 20 products were wasted due to expiration of product on the floor.
Cooler-related issues (6) and products dwelling too long out of a controlled environment (5) were common reasons reported for wastage.
The overall product wastage rates for MTP: trauma, MTP: nontrauma, and OBP were 1.7%, 0.3%, and 2.3%, respectively, with a total exsanguination protocol waste rate of 1.33%.
The difference between the overall proportion of waste between the MTP and OBP protocols was insignificant (p50.176).
Conclusion: Wastage associated with both protocols was low and there is no statistical difference between MTP versus OBP wastage.
Coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods.
Better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols.
A 17 year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group O, Rh negative (D-) red blood cells (RBCs) through a rapid infuser during resuscitation.
Transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample.
The current FDA guidance and AABB standard of two samples for determination of blood type to prevent cases Wrong Blood in Tube (WBIT) or electronic identification systems do not always catch or clarify these errors.
Study Design/Methods: Patient was tested by manual tube method.
Two different technologists using two different reagent racks performed initial testing with matching results.
Results/Findings: Two samples were collected during resuscitation from the patient and typed as O D-.
Patient was transfused with 5 units of O D-RBCs before stabilizing.
Two days later another sample was collected and typed as O Rh positive (D1) with mixed field being seen on the anti-D. A weak D testing was performed to see if the negative result with anti-D could be strengthened through incubation.
Both original samples still resulted as D(table A) .
After consulting the patient care team it was discovered the samples were collected above the IV site after one unit had been completed and while the second unit was being transfused.
It was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate.
The transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected.
Conclusion: The initial samples were collected above the IV site and were contaminated with the D-blood product being rapidly transfused during resuscitation.
The samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the IV site.
Because both samples were collected while the unit was being transfused, contamination was in both.
Use of a handheld barcode system would not have caught this error because the patient had been correctly identified.
Future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen.
Facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site.
Impact of Cell Saver Usage during Solid Organ Transplants at a Major Institution Holly Ross* 1 , Edward Smith 2 , Thomas Brown 2 , Foeks Jeremy 2 , Metcalf Suzanne 2 , James Johnson 2 , Peter Davis 2 , Karafa SW Badjie 1 and Abba Zubair 1 .
1 Department of Laboratory Medicine and Pathology, Transfusion Medicine, Mayo Clinic, 2 Department of Anesthesia, Mayo Clinic Background/Case Studies: Our institution performs an average of 398 solid organ transplants (SOTs) yearly.
Transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (RBC) transfusions annually.
Even the best practices for allogeneic transfusion are not without risk.
Transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization.
The advent of intraoperative blood recovery has reduced the need for allogeneic donor RBCs during surgeries expected to bleed heavily.
With the Cell SaverV R (HaemoneticsV R , Braintree, MA), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support.
This study sought to examine the amount of allogeneic donor RBC units saved during SOTs through the use of the Cell Saver for intraoperative blood recovery.
Study Design/Methods: Data was collected for SOTs which utilized the Cell Saver.
These included liver, liver/kidney combination, lung, and heart transplants.
Data A 29 y.o.
female was admitted to the trauma department after a motor vehicle collision (MVC) and transfused 2 O(1) RBC units from the kiosk.
Her blood type was determined as O(-) with a negative RBC antibody screen (AS).
She was transfused 10 more units of O(-) RBC.
Two months later, a repeat AS identified two new RBC alloantibodies, anti-D and anti-E.
The anti-D formation resulted from the 2 O(1) RBC transfused from the kiosk, but the source of the anti-E was undetermined since E antigen is expressed in 1% of Rh(-) individuals.
The trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a 29 y.o.
female patient should not have received O(1) RBCs.
Study Design/Method: An investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (EMS).
Results/Finding: The trauma unit was busy with 10 admissions during the 5 hours preceding the patient's arrival.
The chart review found the following physical attributes; patient was overweight (107 kg) with obvious facial deformities from the MVC, that compromised age assessment.
It was determined that the kiosk was fully stocked with both O(-) and O(1) RBC units.
One clinical provider recalls that the patient identification (ID) might have been unknown.
Review of the EMS communication states "patient is a 50 y.o.
female."
Conclusion: Use of visual examination to determine age was significant in the selection of O(1) RBC for this patient.
The trauma staff proposed and implemented a change in policy to prevent future incidents.
Any female patient that arrives without ID or written confirmation of age will be transfused O(-) uncrossmatched RBC until a blood type can be determined.
After being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution.
This is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched RBC transfusions.
Rate of ABO/Rh Confirmation in Outpatient Pelvic Organ Prolapse Surgery Alexis R Peedin*, Taylor Brueseke, Yara Park and Jay S Raval.
University of North Carolina Background/Case Studies: Approximately 375,000 surgeries for urinary incontinence or pelvic organ prolapse (POP) are performed annually.
For abdominal pelvic floor disorder (PFD) surgeries, transfusion rates historically range from 6-16%, whereas transfusion rates for vaginal and robotic PFD surgeries range from 0.2-1.6% and 0.3-1.4%, respectively.
Since the implementation of College of American Pathologists (CAP) requirements for ABO/ Rh confirmation, approximately 15% of patients who receive a transfusion in our hospital required a second ABO/Rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative Type & Screen (T&S).
The primary objective of our study was to assess the rate of ABO/Rh confirmation in women who underwent outpatient POP surgery.
Study Design/Method: This was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing POP surgical repair from May 2015 -May 2016 in our academic tertiary care institution.
Among this sample, patients were excluded if their first T&S was drawn before our institution implemented the ABO/Rh confirmation requirement.
Fisher's Exact Test was used, and statistical significance was defined as p<0.05.
Results/Finding: We identified 66 patients for analysis, of whom 65 (98.5%) had a preoperative T&S ordered.
Two (3.1%) of these 65 patients had positive antibody screens; one patient had an anti-K and one had a warm-reacting autoantibody.
Fifty-nine (90.8%) of the 65 patients required a second ABO/Rh specimen per hospital protocol; 51 (86.4%) of these actually had a second specimen drawn.
In patients for whom ABO/Rh confirmation was indicated, there were no differences between those who did and did not have ABO/Rh confirmed when comparing age, body mass index (BMI), pre-operative hemoglobin (Hgb), or surgical approach (Table 1) .
No ABO/Rh discrepancies were identified.
One patient received 1 unit of red cells after abdominal POP surgery.
Conclusion: The rate of requiring ABO/Rh confirmation before POP surgery was markedly higher than that seen in all patients receiving transfusions at our institution (90.8% vs. 15%, respectively).
Because the vast majority of women undergoing vaginal or robotic POP surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative T&S for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent ABO/Rh confirmation.
Volume Reduction of Red Cells to Reduce Transfusion-Associated Adverse Events Related to Hyperkalemia Maressa T Pollen*, Laura Knicks, Linda Van Tol and C. Michael Knudson.
Background/Case Studies: One attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia.
This problem is exacerbated in conditions of massive transfusion and in patients with renal failure.
Washing RBCs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time.
Here, we estimate the amount of potassium that is removed by volume reduction of red cell units.
We also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia.
Study Design/Method: Expired or over temperature units (n527) that had been removed from inventory were utilized for these studies.
Each unit was weighed and a volume reduction procedure was performed.
The supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays.
For all formulas, weight was converted to volume using a specific gravity of 1.05 g/ml.
The Hematocrit (Hct) of the volume reduced RBC was measured using a Sysmex xs-1000i instrument.
The percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (RBC Hct X RBC volume) by the total supernatant prior to the procedure (residual supernatant 1 removed supernatant).
The remaining free potassium (mEq) was calculated as the (concentration of potassium in the supernatant (mmol/L) x the estimated red blood cell residual supernatant volume.
To simulate the process that would occur in the setting of a massive transfusion protocol (MTP), 5 units were subjected to the volume reduction while recording the time needed to process all 5 units.
This was performed twice for a total of 10 Units processed in this manner.
Results/Finding: The volume reduction procedure reduced the supernatant volume by an average of 72% (range 49%-87%).
In units between 21 and 42 days (n510), the estimated mean residual K1 was 1.89 mEq (Range 0.61 to 2.21).
In the two mock MTP trials, the time to complete the procedure was approximately 50 minutes and we estimate an additional 5-10 minutes would be required to modify and issue the units in our LIS/EMR.
Conclusion: A manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells.
This procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia.
The procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion.
processing, cryopreservation, and non-specialized hospital collection.
Preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (NS) alone, NS plus antibiotic/antimycotic (AB/AM) and a dry container.
Prolonged exposure to AB/AM solution retarded outgrowth of MSCs, but control of microbial growth in cultured tissue samples was needed.
These findings were used to construct a validation study.
Study Design/Methods: A validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth.
Collected UC tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal (3) and caesarian (2) births.
UC collections were divided into 3 segments to test 3 conditions.
Segment explants were placed on 0.1% gelatin-coated gridded tissue culture plates (32 explants per plate) in enriched medium specified for MSC outgrowth containing antibiotic only with an endpoint of 21 days.
Growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants.
One segment (fresh control) was dissected and planted without further processing.
The 2 remaining tissue segments were soaked in (AB/AM) saline solution for 1 hr and 24 hrs at 48C, respectively.
Tissue segments were frozen in cryo bags with a proprietary 10% DMSO/large molecular weight sugar solution.
Background/Case Studies: It has been the practice in our institution to process 3 or 4 times the total blood volume (BV) of the patient, up to a maximum of 25 liters (L) per procedure, to obtain peripheral blood CD341 stem cells.
As a consequence, a patient often would need to spend 6 hours or more on the machine.
It would be desirable to be able to specify the exact volume of blood to process to achieve the desired CD341 cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage.
Study Design/Methods: Our institution recently implemented the new Spectra Optia CMNC collection protocol, a continuous flow and continuous collection procedure that uses the Automated Interface Management (AIM) system to precisely manage the separation interface.
An analysis of our 2016 collection data suggested a highly reliable collection process, so a prediction algorithm (PA) based on the linear regression between the patient's CD341 pre-count and CD341 yield, normalized per liter of blood processed, was derived utilizing the patient's CD341 pre-count, the patient's weight in kilograms (Kg), and the target CD341 dose/Kg.
This PA calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral CD341 stem cells.
The initial equation was modified to add an additional 15% to the predicted volume, to account for the natural variability of the process.
This PA was then tested prospectively in the clinical setting.
Results/Findings: In 8 patients, representing both allogeneic and autologous donors, the average blood volume processed was 14.8 L. The range was 4.9 L -21.6 L. The target dose was achieved in all patients.
Our previous practice for these 8 patients would have required, assuming a standard 4 BV procedure, processing an average of up to 28 L per patient, with a range of 20-62 L. To quantify how well the new PA works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected CD341 yield.
The result was a high correlation between these two ratios (R 2 5 0.92), indicating that the algorithm produces very consistent results.
Conclusion: The predictability of our collection process during the time period analyzed was a robust R 2 5 0.92, confirming the findings in the first data analysis.
The blood volumes processed and patient time on the machine decreased substantially, with some patients only needing 2 hours or less to achieve their target dose.
Nurses and lab medical technologists have seen a dramatic change in their workflow.
The number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day.
All in all, implementation of this PA has produced huge increases in patient and provider satisfaction.
Important factors that likely contributed to the success of the protocol included the precision and consistency of the AIM system of the apheresis device, as well as the small number of nurses (1-2) who performed the procedures, resulting in less variability.
The economic impact of this PA has not been quantified, but might be an interesting area for future studies.
Background/Case Studies: ZarzioV R , a biosimilar granulocyte colonystimulating factor (G-CSF) has recently been introduced into clinical practice.
Its use has stimulated a certain debate regarding their possible less efficacy and security on CD341 mobilization.
The aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as G-CSF is used.
Study Design/Method: We retrospectively evaluated autologous mobilization processes performed between June 2015 and March 2016.
Patients (n525) evaluated were diagnosed with malignant lymphoma (n515), multiple myeloma (n59) and Primary Amyloidosis (n51) and were mobilized according to standard protocols.
Collection CD341 cellularity target was established !
2x10E6/kg.
Two groups, good and bad mobilizers, have been determined.
Predictors of unsuccessful mobilization were defined by >65 years old, previous fludarabine, lenalidomide, or bendamustine treatments or !2 previous regimens, present peripheral cytopenias, active disease and previous mobilization failure.
Mann-Whitney U test was used to compare means and comparisons of medians were performed by the median test.
CD341 count was performed according ISHAGE protocol.
Adverse events (AE) were analysed according to CTCAE v4.0.
Results/Finding: The media (range) general collection parameters were: CD341 (day 5) 27.50/mL (4.5-157.5/mL), blood volume processed 23204mL (9718-39618mL) and 4.96 (2-7.30) exchanged volemias.
Seventeen patients were considered bad mobilizers, 7 needed plerixafor and 5 had to undergone a collection procedure twice.
There were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (SD) ); p50.071].
There were no significant differences on mobilization characteristics and product cellularity between both groups.
Five mobilization AE were observed [muscle pain (n52), fever (n52) and flu syndrome; all grade 1].
Two patients could not undergo hematopoietic stem cell transplantation due low CD341 cellularity.
Conclusion: There are differences between products collected from the good mobilizer (rich in GM and CD34) versus poor mobilizer (with plerixafor) rich in CN and CMN.
The mobilization with ZarzioV R could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting.
Background/Case Studies: Mesenchymal stem cells (MSCs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma.
However, MSCs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common.
Before applying MSCs in a preclinical animal model, we sought to determine the procoagulant properties of rat MSCs in vitro.
Study Design/Methods: Bone marrow and adipose derived MSCs (BMSC and AMSC) were isolated from bones (femur and tibia) and visceral fat tissue in normal young Sprague Dawley rats respectively.
Both BMSCs and AMSCs were cultured and passaged using DMEM medium with 20% fetal bovine serum.
BMSC and AMSC at passage 2-5 were used in this study.
The tissue factor expression of MSCs was determined by immunohistochemistry.
Citrated whole blood collected from normal rats was treated with rat BMSCs and AMSCs at low, medium and high doses (1.5 3 10 4 /ml, 7 3 10 4 /ml and 1.5 3 10 5 /ml respectively).
The prothrombin time (PT), coagulation properties and platelet aggregation (response to ADP, collagen and PAR4) were measured by hemostasis analyzer, rotational thromboelastometry (ROTEM) and impedance aggregometry (Multiplate) respectively within 30min and 2hr after incubation.
Results/Finding: Tissue factor was significantly expressed among both BMSC and AMSC at all passages in vitro.
BMSC and AMSC at any dose and time of treatment neither shortened nor elongated PT in whole blood.
However, both BMSC and AMSC significantly shortened the clotting time (CT) (None: 334 6 35 seconds, versus low, medium and high doses of AMSC (145 6 2, 111 6 6, and 75 6 12 seconds), and BMSC (155 6 2, 90 6 10, 80 6 7.0 seconds), p<0.05), clot formation time (CFT, p<0.05) and increased alpha angle (p<0.05) by NATEM measurement, but did not significantly affect the CT, CFT and alpha angle by EXTEM.
Maximum clot firmness (MCF) and fibrinolytic index were not affected by MSCs.
There was no significant impact of both BMSC and AMSC on platelet aggregation simulated by ADP, collagen and PAR4.
No significant differences of hemostatic and platelet function were found between the treatments of BMSC and AMSC.
Conclusion: Consistent with reports from human derived MSC, both rat BMSC and AMSC significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment.
However, MSCs had no direct impact on platelet aggregation in vitro.
As considering the procoagulant capability of MSCs, future study will be necessary to determine the optimal dose and safety of using MSCs for systemic application in vivo.
Comparison of the Terumo BCT MNC and Cmnc Protocols for Peripheral Blood Stem Cell Collections Lindsey Westbrook* 1 , Neil Bagamasbad 2 , Reynold Dilag 2 , Melissa Nasser 2 , Nicole Bauer 2 , Jennifer Wheeler 3 and Mary Berg 1 .
1 Department of Pathology, University of Colorado -Anschutz Medical Campus, 2 Department of Medicine, Division of Hematology, University of Colorado Hospital, 3 Scientific Support, Terumo BCT Background/Case Studies: Terumo BCT recently offered a new method of peripheral blood stem cell (PBSC) collection using the Spectra Optia, an apheresis instrument.
The new protocol, Continuous mononuclear cell collection (CMNC) collects cells continuously as opposed to the older protocol, the Mononuclear cell collection (MNC) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from MNC within a cell separation chamber.
Our institution has used both protocols and the purpose of this study was to compare PBSC product characteristics and run times between the CMNC and the MNC protocols.
Study Design/Method: A retrospective review and comparison of parameters from 120 collection procedures using the MNC protocol and 173 collection procedures using the CMNC protocol was done using the t-test.
Data from patients/donors (including 36 allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (CD34)-positive (CD341) throughput, CD341 collection efficiency (CE%), platelet loss 71A TRANSFUSION per total blood volume processed (plt loss/TBV), and collection product characteristics were included in the analysis.
Results/Finding: Numerical results are summarized in the Table.
The MNC and CMNC donor groups included 14 and 22 allogeneic donors, respectively.
Donor weight was not significantly different between the two groups.
Pre-procedure WBC values were also similar between the two groups.
Run time was found to be significantly shorter using the CMNC protocol compared to the MNC protocol.
Product volume was also significantly lower in the CMNC group compared to the MNC group.
Although the volume was lower, the CMNC product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the MNC product.
The CD341 throughput was significantly higher in the CMNC group than the MNC group.
The CD341 CE% was found to be slightly increased in the CMNC group, though not significantly.
The platelet loss was not significantly different between the protocols when normalized for total blood volume.
Product hematocrit (HCT%) was significantly higher using the CMNC protocol; however, the red blood cell volume never exceeded 20 mL due to the lower product volume with the CMNC protocol.
The CMNC protocol collects a smaller volume of a purer product when compared to the MNC protocol with comparable platelet and red blood cell loss.
Staff members who perform apheresis procedures are pleased by the shorter run time.
Background/Case Studies: Hematopoietic stem cell (HSC) donors and their recipients need not have a matching blood type.
Eventually, the HSC recipient will become the blood type of the HSC donor.
This scenario can become quite a conundrum if the HSC recipient becomes a patient in need of an organ transplant.
In order for a patient to receive a donor organ, the patient and donor's blood type and HLA typing must be compatible.
Study Design/Methods: Blood type was determined using gel test cards.
HLA typing was determined by using sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP), and sequence based typing (SBT) technologies.
HSC sources were bone marrow and umbilical cord blood.
Results/Findings: Patient #1, originally typed as an A2, had 1 bone marrow donor and 2 cord blood transplants.
One of the cord blood transplants successfully engrafted.
The engrafted unit was from a type O donor.
Patient #1 is now typing as type O.
Patient #2 was originally typed as A2 and received a bone marrow transplant from a type B donor.
Patient #2 is now front-typing as a B and backtyping as an AB.
Since the patient's ABO front and back-type do not match, a note must be made, that when confirming ABO during crossmatch, the ABO will not match.
The patient now has an HLA and ABO identical kidney match (his father who is a type B).
Previously, the patient and his father were ABO incompatible.
The ABO and HLA results on both patient #1 and patient #2 indicate that the HSC transplants have engrafted.
Results also indicate that the ABO and HLA now match that of the donor and differ from the recipient's original ABO and HLA type.
Due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant.
Both patients will be entered into the UNet system according to their "new" ABO and HLA types, as UNOS regulations require patients to be listed as per the results of two separate ABO typing tests.
The patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial.
Background/Case Studies: Mesenchymal stem cells (MSC) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma.
MSCs express tissue factor (TF) that activate the clotting cascade and interfere hemostasis.
Hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including MSCs.
In this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat MSC in vitro.
Study Design/Method: Bone marrow and adipose derived MSCs (BMSC and AMSC) were isolated from bones (femur and tibia) and visceral fat tissue in normal young Sprague Dawley rats respectively.
Both BMSCs and AMSCs were cultured using DMEM medium with 20% fetal bovine serum under either normoxia (20% O 2 ) or hypoxia (3.5% O 2 ).
MSC growth curves were measured by cell counter.
The TF expression was determined by immunohistochemistry.
CD90/CD29 and CD45 were measured as positive and negative markers of MSC respectively by flow cytometry.
The citrated rat whole blood was treated with MSC (1.5 3 10 5 /ml) either from normoxia or hypoxia.
The coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (ROTEM).
Results/Finding: Hypoxia potentiated the growth of BMSC by 15%, but depressed the growth of AMSC by 30% at day 5 in comparison to normoxia.
Both BMSC and AMSC equally expressed CD90 and CD29 but not CD45 under any culture condition.
Tissue factor was significantly expressed among BMSCs and AMSCs from both normoxia and hypoxia.
Whole blood treated with BMSCs and AMSCs from normoxia significantly shortened the clotting time (CT: 468 6 64 (control), versus 170 6 13 (BMSC), and 195 6 60 (AMSC) seconds) by NATEM.
Hypoxia also significantly shortened CT (165 6 20 (BMSC), 169 6 50 (AMSC) seconds, p<0.05 as compared to control), but the changes in CT were not significantly different between BMSCs and AMSCs.
Maximum clot firmness (MCF) and fibrinolytic index did not change after treatment with BMSC and AMSC regardless of the normoxia or hypoxia conditions.
Conclusion: Tissue factor is constitutively expressed in rat BMSCs and AMSCs.
Adjustment of the MSC culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of MSC (BMSC and AMSC).
This study also suggests that the procoagulant properties will not be affected if MSCs are recruited into injured tissues with hypoxic environments.
Future study will be necessary to determine the optimal dose MSC and whether it is safe to use MSCs for systemic application in trauma.
Effect of Double-End Cryopreservation on Gene-Transduced Human Hematopoietic Stem and Progenitor Cells Sandeep K Srivastava*, Jiaqiang Ren, Steven Highfill, Narda Theobald, Suksee DeRavin, Andre Larochelle, David F Stroncek and Sandhya R Panch.
National Institutes of Health Background/Case Studies: Current early-phase clinical gene therapy trials use freshly collected or cryopreserved CD341 cells as the starting fraction prior to gene manipulation.
Following gene-transduction and culture, the end product is infused fresh into recipients.
For wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (DEC) of CD341 cells during manufacture (i.e.
immediately post-collection and again, post-gene modification).
DEC helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion.
Our objective was to study the effects of DEC on gene transduced mobilized peripheral blood (MPB) CD341 cells.
Study Design/Method: Cryopreserved CD341 cells from 2 healthy adult donors were thawed and transduced (TR) in RetroNectin coated tissue culture bags with an EF1-alpha-YFP lentivirus (2.5% concentration) and media (X-VIVO-10, human serum albumin(HSA), 100 ng/mL each of cytokines (SCF, TPO and FLT3-L) over 2 days.
Untransduced (UTR) cells were cultured as controls.
TR and UTR fractions were re-cryopreserved.
A standard freeze-mix of 5% DMSO, 6% Pentastarch, HSA, plasma-Lyte A was used for cryopreservation.
Viability, Hematopoietic stem cell (HSC) (CD34 1 CD38 -CD45RA -CD90 1 CD49f 1 cells) phenotyping and CFU assays were done following first thaw (PT1), post-transduction (PTxn) and second cryopreservation-thaw (PT2).
Results/Finding: TNC recovery decreased gradually in the donor samples at each step.
Transduction efficiency, CD34%, CFUs were similar before and after PT2.
HSCs ranged from 824 to 1655 cells/10 6 CD341 cells in the PT2-TR arm compared to a range of 286 to 1416 /10 6 CD341 cells after PT1.
Viability, % CD341 and CFUs were lower in the TR compared to the UTR arm.
This difference was not altered after PT2 (Table) .
Conclusion: DEC of MPB human CD341 cells decreases TNC recovery, but has minimal effects on CD341 cell phenotype, transduction efficiency and cell function.
HSC numbers were within acceptable range after recryopreservation.
Lower viability and CD34% in the TR arm compared to the UTR arm is likely due to vector toxicity.
This was unaffected by recryopreservation.
Additional studies to assess DEC mediated changes on CD341 cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in NOD/SCID mice will inform clinical trials.
Background/Case Studies: Autologous peripheral blood stem cell (PBSC) transplantation has been used as a powerful resource during the treatment of some hematological malignancies.
Cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy.
In some cases, PBSC are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial.
Our work presents retrospective data on PBSC infusion after long-term storage.
Study Design/Method: All products were harvested after patient mobilization with G-CSF by apheresis with COBE Spectra V R .
Flow cytometry analysis of CD341 cells was performed prior to cryopreservation.
The cryoprotective solution was freshly prepared by addition of 20% hydroxyethyl starch, 18% human serum albumin and 10% DMSO at final concentration.
PBSC were cryopreserved by direct immersion on -808C mechanical freezer (dump freeze) and stored until transplantation.
Post-thaw viability was determined from stored cryotube samples by Trypan Blue exclusion minutes prior to infusion.
Cells were thawed and infused on bedside.
Engraftment was defined as the first day of 03 consecutive days of neutrophil count >0.5 x10 9 /L and platelet count >20 x10 9 /L after 07 days.
with G-CSF for four days and patients with G-CSF for five days with use of Mozobil when CD341 was below 10 x 10 6 cells/L on the fourth day.
HPC collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients.
All procedures were realized based on a prediction algorithm using pre-CD341 on the day of the collection and estimating WBC liters to be processed to obtain sufficient stem cells for the transplant.
This algorithm was designed using linear regression of peripheral blood CD341 on the day of the collection versus collected CD341 per liter of blood processed.
There was no distinction between patients and donors, once the efficiency coefficient was used for both.
Collected material was sent to analysis and total CD341 was calculated.
Final laboratory count of CD341 per kilogram was compared with the number predicted by the algorithm with Spearman's correlation to evaluate whether the formula is effective.
Calculations were made using IBM SPSS 23 Software.
Results/Findings: Among patients collecting HPC for autologous transplantation, 69,23% needed only one day of HPC harvesting, while 25,64% needed two days and 5,13% needed three or more days.
Our collection efficiency (CE) and standard error of the mean (SEM) was 49 1-2,91%.
After comparing predicted values with CD341 collected in the final product, we found a very strong correlation of 0.873 (p<0.01) for patients and a strong correlation 0.653 for healthy donors (p<0.01).
Conclusion: The use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate WB liters to process and avoid unnecessary procedures in both patients and healthy donors.
This study evaluated the phenotypic characteristics of UC-MSCs derived from fresh and cryopreserved cord tissues (CT), as described in ISCT's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; !95% CD90, CD105, CD73 and 2% CD14, CD19, CD34, CD45, HLA-DR) Study Design/Method: Umbilical cord tissue (N510) was washed, blood vessels removed, cut into 0.5-3mm pieces, and washed twice in saline.
Fresh tissue was immersed in 0.9% saline for same day culture, while frozen tissue was cryopreserved for at least 24 hours prior to culture.
For colony forming unit (CFU) testing tissue was plated directly in a 25cm 2 tissue culture flask following a wash in PBS with antibiotic/antimycotic.
The tissue was allowed to adhere for 10 minutes prior to the addition of cell culture media.
Media was changed several times a week.
Cells were passed when robust colony growth was observed and in subsequent cultures >80% confluence.
All cells were tested on an MSC flow panel at passage 2 just prior to confluence.
Results/Finding: Both fresh and cryopreserved tissue showed excellent colony forming capabilities.
Average time for cellular emergence of 8 days (Fresh 5 7.8, Frozen 5 8.1), and 13 days (total) for the MSC's to reach passage 1 (Fresh 5 12.6, Frozen 13.4).
All cells were ready for flow analysis in approximately 3 weeks time.
There was no statistical difference between fresh and frozen tissue in their colony emergence (p 5 0.81), or their growth rates (p > 0.05 for all).
Flow cytometry showed average !95% for positive markers and 2% negative markers.
There was no statistical difference between fresh and frozen flow result (p > 0.05).
Conclusion: UC-MSC's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology.
Flow cytometry analysis showed strong MSC phenotype in both fresh and frozen samples.
The data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain MSC colonies.
Studies have shown that HSCT improves survival and disease-free survival rates when compared to conventional chemotherapy treatments.
The increase in the number of HSCTs over the last years has demanded quality and safety improvements of cell processing and cryopreservation services.
Cell recovery and viability are crucial parameters to assess UCB quality as a viable HSCT graft source.
Study Design/Method: Twenty-five UCB units cryopreserved for periods of 2 up to 11 years (2004 to 2017) were analyzed.
Units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using DMSO (dimethylsulfoxide) cryoprotectant with 10% concentration.
Informed consent and the unit discard terms for all units were obtained.
Units were thawed in a 378 C water bath and 0.5ml aliquots were diluted at a 1:1 proportion with 5% human albumin solution and plasmin were prepared, enabling DMSO stabilization and concentration reduction.
The following analysis were performed: nucleated cell count (TNC) in an automated hematologic counter and cell viability using flow citometry.
Post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using 7-AAD marker through flow cytometric analysis.
Results/Finding: UCB storage period was 7.24 years (mean) and cell recovery was 86.31% (mean).
There was no statistically significant correlation between storage period and post-processing cell recovery (p 5 0.11).
Post-thaw cell viability of 63.13% (mean) showed no statistically significant correlation with unit storage period (p 5 0.07).
Post thaw cell viability results are within parameters defined in other studies.
Background/Case Studies: Umbilical cord (UC) tissue is a rich source of mesenchymal stem cells (MSCs) that can be collected noninvasively at birth and stored for potential future use.
As such, a growing number of stem cell banks have established UC storage programs based on mounting preclinical evidence of its therapeutic potential.
However, little has been reported on the ability to isolate MSC-like cells from UC tissue after extended periods of cryopreservation.
This work describes and characterizes the isolation of MSCs from UC tissue cryopreserved as a composite material at a family stem cell bank for 5 years.
Study Design/Method: Donated UC units from consenting mothers were evaluated.
Units had been cryopreserved as composite tissue pieces in LN 2 vapor in a DMSO-based cryoprotectant for 5yrs.
(5.49 6 0.431; n54).
Units were rapidly thawed and rinsed in DPBS, then 25 pieces were excised from each using a biopsy punch.
Pieces from each unit were explanted in a 5x5 grid pattern in MSC-supportive medium and incubated for 7 days, after which the tissue was discarded and media exchanged.
Cells were isolated on the 14 th day, counted, and subcultured for two passages.
At the end of each passage, cells were collected, counted and population doubling time was calculated.
Isolated cells from each unit were also evaluated for MSC immunomarkers.
Results/Finding: Small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a 100% success rate.
Cells were positive for the MSC markers CD73, CD90, and CD105 (98.8 6 0.7%, 98.7 6 0.6%, and 97.8 6 0.6%, respectively) and negative for the hematopoietic markers CD34/45 (1.1 6 0.7%).
Passage 1 and passage 2 doubling times were 1.92 6 0.47 days and 2.07 6 0.43 days, respectively, which are in line with values reported for MSCs isolated from fresh UC tissue.
Conclusion: Due to their immature status, ease of collection, and potential therapeutic value, UC MSCs are an appealing candidate for future clinical 75A TRANSFUSION 2017 Vol.
57 Supplement S3 research and treatment.
The present work demonstrates that the long-term cryopreservation of UC tissue does not disrupt the ability to isolate functional MSCs from the tissue at a later date.
Importantly, growth characteristics of isolated MSCs appear to be comparable to those reported for MSCs from fresh UC tissue.
Based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion.
These results support the feasibility of storage of UC as a composite material for future potential cell isolation and expansion to clinically relevant doses.
Large Volume Leukapheresis with Spectra Optia Cmnc Protocol in Adult and Pediatric Patients: Performance and Determination of CD34 Yield Prediction Algorithm Ines Bojanic* 1 , Nelly Besson 2 , Ivana Vidovic 1 and Branka Golubic Cepulic 1 .
1 Department of Transfusion Medicine and Transplantation Biology, University Hospital Centre Zagreb, 2 Terumo BCT Background/Case Studies: Large volume leukapheresis (LVL) have shown to enhance CD341cell yield collected.
This study evaluated performance and safety of the Spectra Optia CMNC protocol (version 11) in adult and pediatric LVL.
A prediction algorithm for CD341cell yield was also tested.
Study Design/Method: We evaluated retrospectively 67 LVL performed in 46 adult patients, and 14 LVL in 11 pediatric patients treated in UHC Zagreb from March 2016 till September 2016.
Mobilization regimen combined chemotherapy and filgrastim; 2 poor mobilizes received plerixafor additionally.
A combination of ACD-A and heparin was used as anticoagulant (ACD-A:whole blood ratio 1:24).
In patients weighting 25kg (n59), a RBC prime was performed.
CD34, lymphocyte(Ly) and monocyte(Mo) collection efficiencies (CEs) were calculated.
A customized prediction algorithm was determined on linear regression between pre-CD341cell count and CD341cells collected / blood volume processed.
Prediction accuracy was evaluated by comparing predicted CD34 values to real CD34 yield.
Results are presented as median (IQR).
Results/Finding: In both groups, CD34, Ly and Mo CEs were high.
Target CD34 dose was successfully reached in 1 procedure in 30 (65,2%)adults and in 9 (81.8%) children.
All procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in 5 (7.5%) adults, while all pediatric apheresis went uneventful.
No bleeding episodes occurred, and no transfusion was needed.
Product and procedure characteristics* A high correlation between preCD341cells and CD341cells collected/ blood volume was observed in both groups (r 2 50.97 and 0.83 in adults and children respectively, p<0.0001) suggesting CD34 yield could be predicted based on preCD341cells and blood volume to process.
Linear regression equations served as prediction algorithm.
The high correlation between predicted CD34 yield and observed CD34 yield (r 2 50.95 and 0.82 in adults and children respectively, p<0.001) showed accuracy of the algorithm.
Implementation of the algorithm could have allowed sparing a median of 10.1(8.9-12.9)L of blood in 20 adult procedures, and 5.9(3.5-7.8)L in 7 pediatric procedures.
Conclusion: LVL performed using Spectra Optia CMNC protocol is safe and efficient in adults and in low body weight children.
High CD34, Ly and Mo CE1 were observed in both groups.
Implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort.
Mesenchymal Stem Cell Therapy in Steroid Refractory Graft-Versus-Host Disease (GVHD) Emese Molnar* 1 , Aniko Barta 2 , Arpad Batai 2 , Zoltan Csukly 2 , Zita Farkas 2 , Laszlo Gopcsa 2 , Gabor Tatai Background/Case Studies: Steroid refractory acute graft-versus-host disease (GvHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (HSCT).
More experience accumulates in the immunomodulatory effect of mesenchymal stem cell (MSC) infusion in numerous immunopathological disorders -such as GvHD -and signals.
MSCs have a HLArestrictive and non-immunogenic nature.
Study Design/Method: We have evaluated the efficacy of MSC transfusions in cases of acute GvHD refractory to conventional immunosuppressive treatment.
The patients with steroid-resistant GvHD had received third-party MSCs (derived from Wharton's jelly and bone marrow) 4 times per case weekly at a dose of 1 million cells/kg.
Clinical response was assessed 28 days after administering the first dose.
Complete remission was defined as the complete disappearance of symptoms.
Partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition.
Results/Finding: In all 12 patients had received 13 cycles of MSCtreatment (4 dose per cycle).
The median age was 47 years old (19-56) with a male/female ratio of 1:2.
Distribution of the original malignancies (n): acute myeloid leukemia: 6; acute lymphoblastic leukemia: 2; myelofibrosis: 1; myelodysplastic syndrome: 1; multiple myeloma: 1; T-cell lymphoma: 1.
Nine patients had undergone allogeneic HSCT with matched unrelated donors, the other three had stem cells derived from HLA-identic relatives.
The first episode of GvHD after HSCT was started on the median 63rd day (7-455).
The involved organs were skin (2), gut (4), skin and gut combined (7) and even lung in 3 cases.
The median time of MSC's first infusion was 274 days after the stem cell transplantation (HSCT) and 165 (19-1974) days after the first episode of GvHD.
4 of the 13 cycles of MSC-treatment led to complete remission (30.8%) and 7 resulted inpartial remission (53.8%).
Conclusion: We have evaluated MSC-therapy as an effective treatment of GvHD in the majority of the observed cases with 83% overall cumulative response rate.
The application of third-party MSCs offers a promising alternative in the therapy of GvHD and other GvHD-associated complications after HSCT.
Further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety.
Background/Case Studies: Stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team.
Whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment.
A typical goal for most adult procedures is 2 million CD341 cells/kg.
If a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal.
Given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections.
Measuring a patient's CD341 cells/mL in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the 2 million cells/kg goal.
The ideal minimum CD341 cells/mL that will lead to successful harvest has not been conclusively identified.
Study Design/Methods: We analyzed the collection data from 55 patients to evaluate the predictive value of the CD341 cells/mL level.
Data was collected over 6 months from every patient who underwent a stem cell collection.
Four patients were allogenic donors and 51 were autologous donors.
The patients' weight, diagnosis, and pre-procedure CD341 cells/mL level were all collected.
The run time, amount of volume processed, and the absolute viable CD341 cells collected were recorded.
The collection efficiency and the CD341 cells/kg were calculated for each patient.
Results/Findings: Our data showed a strong linear correlation between pre-procedure CD341 cells/mL and post-procedure CD341 cells/kg (r50.95).
Any patient who had a pre-procedure CD341 cells/mL count of 29 or greater had a collection of at least 2 million cells/kg.
Any patient who had a pre-procedure CD341 cells/mL count of 16 or less collected less than 2 Conclusion: The pre-procedure CD341 cells/mL level in the peripheral blood has a very strong predictive value for the post-procedure CD341 cells/kg level.
To confidently know that a patient will be able to produce the desired 2 million cells/kg, a pre-procedure CD341 cells/mL count of at least 29 should be obtained.
For any patient with a count below 16, they should be counseled that their collection is likely to take at least a second day and a second procedure.
Further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between 16 and 29 CD341 cells/mL should be conducted.
Heidi Elmoazzen 1 , Antonio Giulivi 1 , Michael Halpenny* 1 , Lisa Martin 1 , Donna Perron 1 , Chris Bredeson 2 , Lin Yang 1 , Locksley McGann 1 , Paul Birch 1 and Jason P. Acker 1 .
1 Canadian Blood Services, 2 Ottawa Hospital Background/Case Studies: A critical aspect of Hematopoietic Progenitor Cell processing is the cryopreservation method.
Our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing DMSO (5% final concentration) and HES (Hydroxyethyl Starch).
Pentastarch (HES source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor.
This required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality.
Study Design/Method: The validation study consisted of 3 phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant.
Phase I -Samples from four different cryoprotectant formulations were tested for TNC, CD34, viability and CFU at three points during manufacturing (fresh, post processing and post thaw).
Phase II -Mock HPC, Apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations.
Phase III -Five clinical transplants were performed with HPC, Apheresis products cryopreserved using the recommended replacement (Hetastarch).
Results/Finding: Phase I -Results indicate that aliquots cryopreserved in 5% DMSO and 1.7% HES (Hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (CFU).
Phase II -The majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations.
Phase III -Transplants performed resulted in a mean engraftment time of 12.6 days for ANC500 with no adverse patient reactions observed.
Engraftment times using the new Hetastarch formula were compared to the previous engraftment times with no significant difference.
Conclusion: A change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality.
In addition, maintaining the current 5% DMSO final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation.
This study demonstrated the acceptability of the Hetastarch formulation using 5% DMSO and 1.7% Hetastarch to replace Pentastarch in the cryoprotectant formulation used for cryopreservation of HPC, Apheresis products.
Background/Case Studies: Autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (PBSCs).
Traditional mobilization regimens include granulocyte colony stimulating factor (G-CSF) with or without chemotherapy, but have failure rates ranging from 5% to 40%.
Plerixafor is an adjunct agent used to improve mobilization in many clinical settings.
However, its high cost is a significant concern.
The manufacturer-recommended dose is 0.24 mg/kg, therefore patients weighing >100 kg would require a second vial, thus doubling the drug cost.
In 2013 we implemented a policy of capping plerixafor at 24 mg for patients weighing >100 kg.
This retrospective study compares the mobilization of patients >100kg who received capped doses (2013) (2014) (2015) (2016) , with historical control patients (2010-2013) who received full or uncapped doses.
Study Design/Method: Patients weighing >100 kg with CrCL >50ml/min who received capped and full doses of plerixafor were identified in the pharmacy database.
Electronic medical records were used to collect baseline characteristics and cell collection data.
Results/Findings: A total of 47 and 40 consecutive patients were included in the capped and full dosing groups, respectively.
They showed comparable baseline distributions of age, weight, gender and diagnoses.
Plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups.
In the capped dosing group, fewer patients received chemomobilization or plerixafor upfront.
When compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of CD341/cells kg and achieved a comparable collection success rate.
The strategy dose capping plerixafor at 24 mg for patients >100 kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half.
Mean and range of %CD34 in peripheral blood were calculated.
The data show that in the Non-Hispanic group, the youngest donors (<30yrs) have a higher pre-apheresis %CD34 level than any of the other groups, reaching statistical significance when comparing the %CD34 pre-apheresis between the youngest group (<30 yrs) and the oldest group (>540 yrs).
Hispanic donors show statistically similar %CD34 pre-apheresis levels over all age groups.
Moreover, the Hispanic older age group (>540yrs) had a statistically higher %CD34 pre-apheresis level than the Non-Hispanic older age group.
Conclusion: In this analysis of 121 sequential unrelated PBSC donors, Hispanic donors maintain a similar pre-apheresis %CD34 level even as the donor ages, while Non-Hispanic donors show a decreasing pre-apheresis %CD34 level as they age.
If proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups.
This small data set would suggest that people of Hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age.
Further studies of larger cohorts are needed to validate this observation.
If proven, this has far reaching implications within the stem cell research and therapy arena.
Background/Case Studies: An update in HPC apheresis collection software led to higher collection volume in the organization's human progenitor cell (HPC) products without a corresponding increase in total cellular counts.
Incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger DMSO load to the recipient, often resulting in the need to transfuse over several days.
The objectives of this study were to develop suitable mock HPC (mHPC) products and evaluate the effectiveness of the BioSafe PeriCell volume reduction technology on white blood cell (WBC) recovery and viability.
Study Design/Method: HPC products are not readily available for development.
mHPC were created from whole blood buffy coats (BCs).
Fresh ABO compatible BCs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells.
The mHPC products were then diluted in plasma to produce an appropriate concentration and volume.
HPC collection data from last 3 years was analyzed to determine the 95 th percentile, median and 5 th percentile values for both HPC volume and WBC concentration.
Six mHPC products were tested; three high WBC (234 x 10 6 cell / mL) and three low WBC (114 x 10 6 cells / mL) concentrations, each at high (505 mL), low (265 mL) and median (355 mL) volumes.
Each unit was processed sequentially from high, median and low volumes.
Hence, the highest mHPC volume was processed for volume reduction first with a Sepax 2 (PeriCell Protocol, CS.430.1 kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth.
One additional mock product was prepared for a reproducibility study and was volume reduced three times.
WBC concentration and 7-AAD viability was determined before and after each volume reduction.
A control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time.
Results/Finding: Mock HPC products had a mean starting 7-AAD viability of 76 6 8% [range 64-85]% and a hematocrit of 14 6 5% [9-19] which is well below the maximum allowable limit of the PeriCell.
No significant differences in WBC recovery or change in viability were seen between the 6 mHPC products.
Aggregate data showed that the mean WBC recovery of the volume reduction process was 97 6 8% [64-105] with a 3 6 3% [-2-11] change in viability.
The recovery protocol used to salvage product after each volume reduction gave a recovery of 99 6 4 [92, 104] % and a change in 7-AAD viability of 2 6 2 [0, 11] % from the input product.
The method was found to have a CV of 2.0%.
The change in WBC concentration and WBC viability of the test products was not significantly different from the unprocessed control samples.
Conclusion: Mock BC products are a suitable alternative where HPC products are not available for development and are a good use of product otherwise directed for rejection and disposal.
The volume reduction protocol evaluated had minimal impact on the WBC concentration and WBC viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with HPCs and will facilitate transfusion of HPC products into the recipient.
The protocol is now in use with patient HPC products and engraftment kinetics will be tracked in a postimplementation study.
Validating 78A TRANSFUSION clinical assessment.
In the first phase, 2 cryopreserved PBSC products were tested.
Two aliquots were thawed simultaneously for each product: One was passed through a pre-set infusion pump and a second control aliquot was drained by gravity.
Each aliquot was tested for baseline total nucleated cell (TNC) count and viability, and for final TNC recovery, Trypan Blue (TB) viability, CD34 7-AAD viability, and potency (CFU).
The effect of longterm exposure to DMSO was assessed by visually inspecting the product for aggregates and measuring viability up to 3 hours post thaw.
The second in vivo phase included use of an infusion pump for 10 consecutive autologous patients, with comparison of infusion and transplant outcomes to 18 previous infusions by gravity drip.
Comparison variables included infusion rate, adverse events (AE), and engraftment time.
Results/Finding: No significant differences were observed between infusion pump and drip for the 2 products tested in vitro, including TNC recovery, cell viabilities, and potency.
For both methods TNC TB viability decreased by more than 20% within 1 hour, while CD341 cell viability remained stable up to 3 hours post thaw.
Small aggregates appeared after 1 hour for both methods and increased by a similar rate over time.
Comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables.
Engraftment time was similar for both groups.
ANC days to engraftment for pump and drip were 10.8 6 1.3 and 11.6 6 1.0, respectively (p-value50.075).
Platelet days to engraftment for pump and drip were 17.9 6 2.2 and 20.2 6 5.0, respectively (p-value50.207).
Infusion rates were slightly higher for the pump group.
For control patients, 2 required transfer of products to syringes due to slow infusion rate and 2 others experienced allergic and hypotension infusion adverse events.
Conclusion: No significant in vitro or clinical differences were observed between thawed PBSCs infused by gravity or an infusion pump.
These results demonstrate that the use of a pump for PBSC infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times.
Donor racial distribution among the 278 ZIKV ineligible CBUs was: Caucasian 52%, Asian 9%, Black/AA 20%, and Multi-race 21%.
Racial distribution of all clinical CBU donors was Caucasian 49%, Asian 15%, Black/AA 20%, and Multi-race 17%, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel.
There were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility.
Conclusion: Our study indicates that currently the leading risk factor for ineligible CB donors is potential exposure to ZIKV: 78% of all ineligible CBUs and 21% of all banked CBUs in the study period.
We anticipate the number of cases to decrease following maternal education and travel warnings.
Recognizing the importance of ZIKV in public health, and its potential transmission via HCT/P products, an FDA approved screening test for HCT/ P donors becomes a timely necessity.
Acknowledgments: Funded by Zimmer Biomet, a Zimmer Biomet company, IBGRL Red Cell Reference and NHSBT Reagents Background/Case Studies: During storage, red blood cells (RBCs) become less deformable, deplete 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium.
Longer RBC storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation.
A sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (Citra Labs, LLC, Braintree, MA), is approved by the U.S. Food and Drug Administration for the rejuvenation of stored RBCs.
The solution acts by restoring 2,3-DPG and ATP in stored RBCs to levels equivalent to those in the circulation.
The aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored RBCs.
Study Design/Method: A 10 mL aliquot was removed from ABO/Rh grouped, leucocyte depleted RBC units (n 5 20), which were stored in SAGM for 22 days, to act as untreated controls.
The remainder of each unit ($270 mL) underwent treatment with the rejuvenation solution (50mL, 60minutes at 37 o C), followed by cell washing twice in SAGM ('manual' centrifuge-based process).
To represent current transfusion laboratory practice, units were crossmatched against plasma from 39 random donors, using both Diamed gel column and glass tube technique.
Phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on RBC surface antigens (A, B, D, C, c, E, e, K, M, N, S, s, P 1 , Lu a , k, Kp a , Kp b , Le a , Le b , Fy a , Fy b , Jk a , and Jk b ), including whether it exposed crypt antigens (T, Tn, Tk*, Th, Tx*, and CAD).
Crossmatch and phenotype agglutination scores observed for the untreated and treated RBCs were then compared.
Results/Finding: Crossmatch findings were defined as compatible, suitable, and incompatible.
The study identified no difference between the crossmatch reaction profiles of untreated and treated RBCs.
Furthermore, no difference was observed in the phenotypic state between untreated and treated RBCs.
Conclusion: Treatment of 22 day old stored RBCs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples.
Background/Case Studies: Cryopreserved platelet production is burgeoning worldwide.
Currently, there are no automated platelet cryopreservation methods.
By contrast, red blood cell cryopreservation using the ACP 215 (Haemonetics Corp., Baintree, MA) has automated the processing within a closed system, increased labour productivity and provided high quality blood components.
Purpose: To automate platelet cryopreservation procedure.
Study Design/Method: Apheresis platelet concentrates (PC) were collected on the Trima Accel system.
Platelet counts were performed using an ABX Micros 60.
PC were centrifuged at 1250g in a Sorvall RC3C1 Centrifuge (Sorvall, USA) for 10 min.
The combination cryoprotectant DMSO1Dextran (CryoSure Dex40, Germany) was used for PC cryopreservation.
Cryopreserved PC (CPC) were frozen and stored in a Kelvinator chest freezer.
CPC were thawed at 37 degrees C (Barkey plasmatherm) for 10 min.
CPC osmolality was measured with an Osmomat 030 osmometer.
Results/Finding: Staged platelet cryopreservation technology has been developed.
Platelets were cryopreserved in a closed system (Patent No.
: RU 169287 U1).
During the first stage, CPC were spun to separate a plateletrich plasma (PRP) fraction from platelet-poor plasma (PPP).
The second step was to resuspend the PRP by adding a combination of DMSO1Dextran (CryoSure Dex40) , as a cryoprotectant, to obtain a final concentration of 5% DMSO in the platelet suspension.
The Injectomat MC Agilia and NPBI Compomixer M3 were instrumental in automating that phase.
PC to be frozen had an osmolality of no less than 1500 mOsm/L.
PRP and PPP were frozen at a cooling rate of 1-38C/min and stored at -85 0 in the chest freezer for up to 24 months.
Pre-transfusion defrosted platelets were also processed in a closed system (Patent No.
: RU 167874 U1).
Our transfer set made it possible to automate platelet resuspension in plasma through the agency of the Exadrop V R .
Post-thaw PRP was resuspended in plasma, which lowered the osmolality to 380 mOsm/L.
Freeze-thaw recovery of platelets was 80% or more of the original population.
Defrosted PC were stored at 20-24 0 with continuous gentle stirring from a Helmer platelet agitator for no longer than 4 hours before transfusion.
It took no more than 30 min to cryopreserve PC and process pre-transfusion thawed platelets.
The automated processing accounted for the bulk of the time (over 20 min).
Conclusion: The automated technique developed reduced the workload while offering reproducibility of the procedure and high CPC quality.
The use of closed systems ruled out bacterial contamination.
Employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring.
Bacterial Detection in Leukoreduced Apheresis Platelets on Day 4 and Day 5 Evelyn C. Oyler*.
SunCoast Blood Bank Background/Case Studies: The recently published FDA draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to 7 days.
This evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets.
Study Design/Method: A large community blood center and transfusion service collects leukoreduced apheresis platelets (LRAP) using Amicus Separator System (Fenwal, Lake Zurich, IL) and Trima Accel System (Terumo BCT, Lakewood, CO).
Previously-cultured LRAP units were sampled on day 4 for secondary culture using BacT/ALERT (BioMerieux, Durham, NC) and rapid bacterial tests using BacTx (Immunetics, Boston, MA) and PGD (Verax, Marlborough, MA).
If LRAP unit is still available, it is also sampled and tested for rapid testing on day 5.
A total of 60 LRAP units were tested over a 3-month period: 50 were cultured and rapid tested on day 4; 10 were rapid tested on day 5.
The rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use.
Results/Finding: Of the LRAP units evaluated for this study, there were 59 True Negatives (TN) and 1 False Positive (FP) on day 1 when tested by BacT/ALERT, with 60 TNs on day 4.
BacTx testing results showed 50 TNs on day 4 and 10 TNs on day 5.
Testing using the PGD kit showed 50 TNs on day 4; and 8 TNs and 2 FPs on day 5.
FP results were confirmed by performing a secondary culture, which were found to be negative.
BacTx requires a specific analyzer and 30 minutes are required for result interpretation.
There is no instrument requirement for PGD and reactions can be read within 20 minutes.
Conclusion: The results of this evaluation makes PGD the best fit for this blood center based transfusion service.
PGD offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for LRAP in 100% plasma and LRAP in PAS/plasma.
Its ease of use allows for testing of LRAP on day 4 and day 5 during the night shift to be accomplished without additional staffing and allows to extend outdate to 7day storage of LRAP.
Change in Growth Factor Content of Human Serum for Use As Eye Drops during Frozen Storage for 1 Year Jos Lorinser 1 , Pieter F van der Meer 1 , Hans van der Heiden 2 and Dirk de Korte* 1 .
1 Department of Product and Process Development, Sanquin Blood Bank, 2 mu-Drop Background/Case Studies: Growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome.
Stability of growth factors during frozen storage in mini containers (140 mL) is unknown.
If these products can be stored at -188C it will be feasible to store this product in 3-star household freezers, making the product available for patients in need of serum eye drops.
The purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at -188C or <-25 to -358C packed in a new micro dose device for single use as eye drops.
Study Design/Method: Serum produced from 500 mL whole blood donations from non-remunerated healthy donors was quickly frozen.
After frozen storage at <-258C for 3-12 months and controlled thawing, six different sera were used to fill a large number of mini (140 lL) containers, which were refrozen and stored at either -188C or <-258C.
During storage at 3 months intervals, samples were tested for several growth factors, using MagpixV R Luminex Multiplex assays and compared to control samples stored at <-808C.
Growth factors tested were PDGF-AA&AB/BB, TGF-ß1/2/3, VEGF, 80A TRANSFUSION 2017 Vol.
57 Supplement S3 EGF, FGF2.
The study was a fact-finding study, without preset acceptance criteria.
Results/Finding: PDGF-AB/BB and TGF-ß1 were the most abundant growth factors, on average 35, resp.
40 ng/mL.
Also PDGF-AA was detected at relatively high concentration in human serum, on average 11 ng/mL.
TGF-ß2, EGF and VEGF were detected at relatively low values, resp.
3 ng/mL, 0.5 ng/mL and 0.3 ng/mL.
Average levels of FGF2 and TGF-ß3 were close to detection limit (< 0.2 ng/mL).
The controls stored at <-808C showed for all growth factors close to 100% of the initial values in samples at T50 (moment of filling mini containers).
For serum stored at <-258C for up to 12 months, most factors showed less than 2 % decrease, except for PDGF-AA and TGF-ß2, showing 6% resp.
3% lower values.
For serum stored at -188C the values for TGF-ß1, EGF and VEGF were stable, whereas PDGF-AB/BB, PDGF-AA and TGF-ß2 showed a decrease of resp.
9, 17 and 3%.
Conclusion: Human serum eye drops can be stored in the new micro dose device at -188C (3-star household freezers) or <-258C (professional freezers) for at least one year after preparation without large decreases in growth factor content.
The maximum decrease was found for PDGF-AA in serum stored at -188C.
It is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness.
Further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond 1 year.
Ruqayyah Almizraq* 1 , Heather Inglis 2 , Phillip Norris 2,3 , Jennifer A Muszynski 4 , Nicole Juffermans 5 , Jelena Holovati 1 and Jason P. Acker 1,6 .
1 University of Alberta, 2 Blood Systems Research Institute, 3 University of California, San Francisco, 4 Nationwide Children's Hospital, 5 Academic Medical Center, 6 Canadian Blood Services Background/Case Studies: Different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components.
The aim was to identify, quantify and characterize residual cells and extracellular vesicles (EVs) in stored RBC products produced by different blood manufacturing methods.
Study Design/Methods: Thirty-two RBC units produced using whole blood filtration (WBF), red cell filtration (RCF), apheresis, and whole blood derived (WBD) methods were examined (n58 per method).
Residual platelets and white blood cells (WBCs) were measured on day 5 using flow cytometer (FC).
On storage day 5 and 42, number and cell of origin/surface markers of EVs were assessed with FC, and concentration and size-profile of EVs were examined using tunable resistive plus sensing (TRPS).
Results/Findings: On day 5, apheresis and WBD units had significantly greater residual platelets in comparisons to RCF (vs: apheresis p<0.01, WBD p<0.05) and WBF (vs: apheresis p<0.0001, WBD p<0.01) methods.
While RCF units yielded the lowest count of Platelet-EVs (CD41a1) on day 5 and 42, the highest number of Platelet-EVs were in apheresis (day 5) and in WBD (day 42).
Similarly, there was significant difference among methods in the number of WBC-EVs (CD31, CD141, CD161, CD191, CD66b1) and RCF contained the smallest concentration.
Moreover, both TRPS and FC showed an increase in the total number of EVs on day 42 vs day 5 in all of the processing methods.
Noteworthy, TRPS showed that the number of small EVs/exosomes (< 200 nm) was greater than large EVs (!
200 nm) in all of the products on day 5 and 42, and the highest level of EVs < 200 nm were in apheresis units.
TRPS results also showed a significant difference in the EVs size-profile amongst all RBC products (p<0.05).
Conclusion: This study shows that the method of manufacturing significantly affects RBC and non-RBCs EVs characteristics throughout storage, which has the potential to impact quality and safety of RBC products.
The differences in the EVs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences.
Coagulation and Complement Assays in Whole Blood Stored at 48 Centigrade Maryanne C Herzig* 1 , Crystal Lafleur 2 , Chriselda G Fedyk 1 , Sherrill J. Slichter 3 and Andrew P Cap 2 .
1 US Army Institute of Surgical Research, 2 U.S. Army Institute of Surgical Research, 3 University of Washington Background/Case Studies: Whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least 2 weeks of storage at 48C without agitation.
It may be possible to extend the preservation of platelet function by agitating WB.
In order to more fully characterize the quality of WB stored at 48C with or without agitation, we evaluated complement activation as a marker of inflammatory potential.
Study Design/Method: Subjects donated one unit of WB collected in CPD-A2 (citrate phosphate dextrose anticoagulant with adenine).
The WB was not leukoreduced nor was it separated into components.
Units were stored under refrigerated conditions for 10, 12, 15, or 22 days after collection.
Units were stored for 12 days without agitation.
Units stored for 10, 15 or 22 days were agitated during storage with a Model 400 Hybridization Incubator at 48C set for end over end rotation at 2-3 rpms.
At the appropriate time point, platelet free plasma was obtained from the WB sample and stored at -808C.
The frozen plasma was analyzed by ELISA assays to determine: thrombinantithrombin complex (TAT) as a marker of coagulation; soluble CD40L as a measure of platelet activation and granule release; plasmin anti-plasmin complex (PAP) as a marker of fibrinolysis; plasminogen activator inhibitor (PAI-1) as another fibrinolytic measure; and complement activation markers C3a, C4d, C5a and C5b-9.
Data was analyzed by one way repeated measure ANOVA.
Results/Finding: Only 49 6 12% of the platelets were recovered in units stored for 12 days without agitation.
These levels did not meet FDA requirements of 5.5 x 10 10 platelets per WB unit.
Subsequently, WB was agitated and platelet recovery was 71-76%.
No difference was seen in ELISA analysis for agitated or non-agitated samples.
No change was seen in TAT or PAP levels between T0 (day of collection) and T10, 12, 15, or 22 measurements.
Significant elevations of PAI-1 and sCD40L indicate activation of platelets and inhibition of fibrinolysis (p<0.001).
Activated complement peptides C3a, C5a, and C4d were all elevated over time (p<0.001) while sC5d-9 was not.
However, only C3a and C4d levels at T22 were above normal reference ranges at 1.30 and 1.41 times maximum reference, respectively.
Conclusion: Whole blood agitation appeared necessary to recover platelets at or above FDA requirements.
Whole blood stored at 48C for 10-22 days did show some activation of complement proteins.
In contrast to studies in stored red blood cells with elevations of sC5d-9 reported, WB showed elevation of C3a, 5a and C4d and not sC5d-9.
Complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life.
Meredith Lummer* 1 and Christian Todd 2 .
1 Cerus Corporation, 2 Community Blood Serivces Background/Case Studies: The INTERCEPTV R Blood System for Platelets (Cerus, Concord CA) is used for the pathogen reduction (PR) of platelet collections, and replaces irradiation, CMV testing, bacterial culture and point of issue bacterial testing.
To better understand PR compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly 1.1x10 10 6 5.6x10 9 9.8x10 10 6 5.6x10 10 310 6 330 430 6 440 900 6 260 28000 6 33000 3 6 3 186 31 30 6 22 110 6 97 RCF 1.9x10 10 6 7.4x10 9 4.2x10 10 6 1.1x10 10 13 6 4 316 14 530 6 160 5100 6 2000 3 6 3 96 7 176 7 346 11 Apheresis 2.4x10 10 6 2.0x10 10 1.0x10 11 6 6.1x10 10 520 6 320 700 6 310 2200 6 1900 9800 6 4100 14 6 17 7 6 5 466 15 120 6 24 WBD 6.4x10 9 6 3.1x10 9 4.6x10 10 6 1.5x10 10 350 6 140 760 6 360 1000 6 180 4400 6 2400 3 6 2 426 23 57 6 24 120 6 56 Platelet collections must meet specific volume, concentration, and dose ranges to qualify for INTERCEPT PR.
Changes made to apheresis devices included adding the following 4 collection targets: 4.4x10 11 in 350mL, 6.6x10 11 in 400mL, 6.8x10 11 in 400mL, and 7.0x10 11 in 400mL.
Study Design/Methods: Four months of collections were retrospectively analyzed.
Platelet collections were evaluated to determine eligibility for PR treatment, and all products meeting PR processing specifications (unless intended for an HLA matched recipient at a hospital not able to accept PR products) underwent PR treatment regardless of potential impact to split rate.
A minimum post-treatment dose of 3.0x10 11 or 6.0x10 11 was required to classify collections as singles or doubles respectively.
Volume/dose mitigation (removal of volume to increase the number of products eligible for PR) was not utilized during this study.
Thus units were treated conventionally if volume, dose, and/or concentration did not meet PR specifications without further manipulation.
Results/Findings: 64% of all single and double collections were eligible for and underwent PR treatment.
Split rate for single and double collections was 1.34.
Conclusion: It is possible to treat 64% of single and double platelet donations with INTERCEPT PR at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices.
Platelet collections that fall outside of the specifications for PR are processed and distributed as conventional products.
Strategies to increase eligibility toward 100% while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation.
Further evaluation is needed to determine the additional quantity of PR eligible products resulting from such changes.
Monique P Gelderman* 1 , Andrey Skripchenko 1 , Fei Xu 1 , Ying Li 1 , Stephen J Wagner 2 , Pamela H Whitley 3 and Jaroslav G Vostal 1 .
1 FDA/CBER/ OBRR/DBCD/LCH, 2 American Red Cross Holland Laboratory, 3 American Red Cross Mid-Atlantic Research Facility Background/Case Studies: Platelets (PLTs) stored at room temperature (RT) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur.
Storing PLTs at cold temperature (4-6 o C [CT]) limits bacterial growth but results in rapid clearance upon transfusion.
The development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies.
Thus, an animal model of PLT circulation that could predict performance of human PLTs in human volunteers would positively impact the development of alternate storage conditions.
Study Design/Method: We designed an immunodeficient (SCID) mouse model to evaluate recovery of human PLTs and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new PLT storage condition: thermocycling PLTs (11 hrs CT: 1 hr 37 o C [TC]).
Autologous apheresis PLTs stored for 7-days at RT, TC and CT were radiolabeled and infused into healthy human volunteers (n59) and the same non-labeled PLTs were also infused into mice (n590).
Blood samples from humans and mice were collected over time to generate survival and clearance curves of the PLTs in circulation.
Flow cytometry was used to detect and analyze the human PLTs in the mouse samples to generate such curves; counts <5% were considered background.
Results/Finding: The mean recoveries of infused PLTs were 51.2 6 16.7% for RT, 37.7 6 12.3% for TC and 23.1 6 8.8% for CT in humans.
In mice, mean recoveries of the same PLTs were 24.9 6 10.3% for RT, 19.1 6 9.8% for CT and 16.2 6 6.9 for CT (mean6SD).
To compare performance of the PLTs in humans and mice we expressed all recoveries as a percentage of the RT recoveries.
In humans TC was $74% and CT was $45% of RT.
In mice TC was $76% and CT was $64% of RT.
The area under the survival curve (AUC) was calculated for the individual mouse study and human trial data sets.
The results of both AUC were normalized to 100% for RT PLTs.
Human TC PLTs had 26% AUC while CT PLTs had 11% AUC compared to RT PLTs in humans.
In comparison, the same TC PLTs had 39% AUC and CT PLTs had 26% AUC of the RT AUC in the mice.
The calculated ratios of the AUC between the TC PLTs and CT PLTs of the human data set and mouse model data set are 2.4 and 1.5, respectively.
Conclusion: The SCID mouse model differentiates between RT PLTs and CT PLTs similar to humans based on AUC and PLT recovery data.
However, the mouse model cannot differentiate between CT PLTs and TC PLTs as occurs in humans.
Even though the mouse model cannot differentiate between CT PLTs and TC PLTs, it may still be a useful tool to screen other novel storage conditions for human PLTs.
Converting the Component Manufacturing from a Manual Process to Automation Nicole Peters* 1 and Geeta Paranjape 1,2 .
1 Coastal Bend Blood Center, 2 Carter Blood Care Background/Case Studies: Initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes.
Our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation.
The CompoMat G5 was evaluated using a multi-team approach including component manufacturing staff, equipment management, QA, Regulatory Affairs and IT.
Study Design/Method: After a comprehensive evaluation, the team decided to purchase the CompoMat G5 with the CompoMaster Net Software for data management.
Implementation was planned for a November 2014 go-live.
.
To centralize processing, new work counters were installed.
Fresenius Kabi installed the CompoMat G5s and CompoMaster in June 2014.
Training and validations were successfully completed and a full launch occurred mid-March 2015.
Device and SOP training was performed.
Training Qualification Checklists were completed for each technician with a required number of successful units processed and completed December 2014.
Validation was completed and signed off in March of 2015.
Manufacturing data was collected using the CompoMaster Net data management system and our Quality Control Software for Platelet (PLT) parameters, including PLT count, PLT weight, and PLT yield from before implementation (BI) and after implementing (AI) of the CompoMat G5 system.
Data points were collected from 210 units BI and 302 units AI.
Results/Finding: Upon initial implementation, staff training and use, the CompoMat G5 was found to be easy.
PLT weight spread was reduced from an average of 22gm to an average of 15 gm.
Actual PLT weights were reduced from an average of 63gm to 59gm, resulting in an average increase in recovered plasma of 3.78ml per unit.
PLT count on average increased from a count of 1435 to 1506 (10 3 /mm 3 ) with a negligible change in PLT Yield.
Conclusion: PLT weight spread was reduced by 31.8% after implementation of the CompoMat G5 and our PLT concentrations increased on average by 5%.
We were able to consistently produce a smaller volume PLT (average 59 gm), which gave us 3.78ml more plasma per unit for recovered plasma.
The team intends to review a dryer Cryo as a next step for potential additional plasma yields for recovered plasma.
Deglycerolization of Manually Glycerolized, Frozen Rccs Using a Closed System Cell Processor Anita Howell 1 , Angela Hill 1 , Brandie Dennis 2 and Jason P. Acker* 2,3 .
1 Canadian Blood Services, Centre for Innovation, 2 Canadian Blood Services, 3 University of Alberta Background/Case Studies: Upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (RCCs), many rare RCCs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory.
A study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (RBC) in vitro quality.
As the closed cell processor uses a fixed centrifuge bowl for deglycerolization and RBC resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product.
Study Design/Methods: 13 ABO/Rh matched LR SAGM RCCs were pooled and split to produce 6 large (354 mL) and 6 small (244 mL) RCCs.
The RCCs were stored to 14 d and glycerolized manually by mixing 400 mL of glycerol with the RCC in a 2000 mL freezing bag.
Units were frozen at -658C for !
72 h before being removed from frozen storage and thawed in a 378C water bath.
3 large RCCs and 3 small RCCs were deglycerolized using the organization's current procedure on the COBE 2991 cell processor prior to re-suspension in 0.9% saline, 0.2% dextrose.
The remaining RCCs were transferred into a 1L bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of 75 6 5%, and deglycerolized in a 275 mL centrifuge bowl on the ACP-215 with re-suspension in AS-3.
RBC quality was tested at 24 6 2 h post-deglycerolization.
Results/Findings: Large RCCs had significantly higher hemoglobin per unit (COBE: p50.006, ACP215: p50.007) and lower cell recovery (COBE: p50.002, ACP215: p<0.001) post-deglycerolization than smaller RCCs on both cell processors.
Large RCCs deglycerolized on the COBE 2991 had higher hemolysis (p<0.001) and supernatant potassium (p50.001) than did small volume RCCs.
Large COBE 2991 RCCs had higher hematocrits (p50.033), hemoglobin (p50.006), and recovery (p50.001) than did large ACP-215 RCCs.
However, all COBE 2991 RCCs had higher (p<0.001) hemolysis (0.99 6 0.24 %) levels than did ACP-215 RCCs (0.31 6 0.02 %).
COBE 2991 RCCs failed to meet regulatory hemolysis standards of 0.8%.
Conclusion: Addition of a 400 mL bolus dose of glycerol to RCCs of different volumes results in different concentrations of glycerol in the frozen RCC product and may lead to differences in frozen RCC quality.
Additionally, the size of the RCC impacts quality for RCCs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits.
Use of the closed cell processor with resuspension in AS-3 and storage for 24 6 2 h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in RCCs glycerolized manually.
The ACP-215 cell processor can therefore be used to deglycerolize RCCs glycerolized using a manual, open system glycerolization method.
Background/Case Studies: Washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions.
Platelet washing process is time-consuming which may delay transfusion.
This study was conducted to evaluate the manual platelet washing method (MM) using 0.9% saline and centrifugation and the semi-automated washing method (SAM) using the COBE 2991Blood Cell Processor.
Study Design/Method: In this study, 20 units of single donor platelets were evaluated (10 washed using the MM and 10 washed using the SAM.
The collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate.
The platelet counts were measured on the Sysmex EXN and the total plasma protein samples were measured on the Roche Cobas 6000.
Results/Finding: Table 1 shows that the average platelet recovery for the SAM (92%) was significantly higher compared to the MM (82%).
The MM had a slightly higher average protein removal compared to the SAM.
No platelet clumps were observed in either the MM or the SAM.
It was observed that the hands-on time for the MM took 10-15 minutes longer than the SAM.
Background/Case Studies: The INTERCEPTV R Blood System for platelets is currently licensed for pathogen reduction (PR) of Amicus platelets in Inter-Sol (PAS-3) for input platelet doses of 2.9 to 8.0 3 10 11 platelets in 255 to 420 mL of 47 to 68% plasma and 32-53% PAS.
A new platelet processing set was designed with three storage containers (TS) to process apheresis platelet components in PAS-3 containing doses of 6.0 to 12.0 3 10 11 platelets in a volume of 420 to 650 mL.
Study Design/Methods: Apheresis PCs (Amicus V R ) were collected in 35% plasma and 65% PAS-3.
One study was performed at the nominal dose (9.2 -10.0 x10 11 platelets), volume (558 -629 mL) in 65% PAS/35% plasma using single donor apheresis collections.
Two studies were performed to evaluate the high dose and high volume condition (9.7 -11.8 x 10 11 platelets in 593 -659 mL) using either single or pooled donations.
Input PCs (n520) were treated with the INTERCEPT TS set by the end of Day 1 post collection; the incubation time in the Compound Adsorption Device (CAD) container ranged from 4 to 16 hours and the INTERCEPT treated PCs were stored in 3 containers (n560).
Day 5 and 7 post-donation PCs were evaluated using a panel of in vitro platelet function assays Results/Findings: In vitro function data for apheresis PCs in PAS-3 treated in the INTERCEPT TS set demonstrated acceptable in vitro function (Table 1 ).
All INTERCEPT treated PCs had pH(228C) !6.2.
Platelet dose and volume recovery post-treatment ranged from 82% to 99% and 88% to 92%, respectively.
Conclusion: Pathogen reduced platelet components processed using the INTERCEPT TS set from either single or pooled apheresis donations maintained acceptable in vitro quality through 7 days of storage.
INTERCEPT Blood System for Platelets TS set is currently not approved for use in the US.
Background/Case Studies: The possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern.
While current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats.
The Pathogen Reduction Technology (PRT) represents a proactive strategy to further reduce transfusion-transmitted infectious risk.
However, the scientific community broadly agrees over the fact that PRT has negative impacts on the product's quality markers.
This study aims at evaluating the impacts of the Mirasol PRT on platelet (PLT) quality and PLT processing.
Study Design/Method: Two ABO-compatible platelet concentrates (PCs) containing 100% plasma obtained from either apheresis or SAGM whole blood (WB)-derived processing were paired, pooled and then split into two equal units.
One unit was used as a non-treated control (CTRL) (n56).
Riboflavin was added to the other PC unit and then exposed to UV light according to the manufacturer's instructions for the Mirasol PRT (Teru-moBCT) (Test) (n56).
Numerous in-vitro quality markers (PLT concentration, ATP, pO2, pCO2, pH, glucose, lactate, sodium, and potassium) were measured for both Mirasol-treated and non-treated PCs on days 1, 3, 5 and 7 for apheresis PCs, and on days 2, 3, 5 and 7 for WB-derived PCs.
Two flow cytometry assays were used to evaluate CD62p expression with and without thrombin activation, and to measure the percent annexin Vpositive PLT.
TRANSFUSION 2017 Vol.
57 Supplement S3 Results/Finding: Platelet recovery was 92 6 5% and 81 6 10% for apheresis and WB-derived PCs, respectively.
Mirasol-treated PCs showed higher levels of annexin V-positive cells (3% 6 1 (Test), vs. 1.7% 6 0.5 (CTL) on day 5) and a higher rate of CD62p expression than control PC units (58% 6 7 (Test), vs. 23% 6 6 (CTL)) on day 5).
The Mirasol treatment generates changes in pH, glucose and lactate for PCs during storage.
Conclusion: The Mirasol treatment induces a loss in the net number of PLTs/unit and elevated platelet activation.
Changes in pH, glucose and lactate suggest that PRT affects PLT metabolism.
Finally, PRT has numerous impacts on logistic, storage and processing time constraints of blood bank operations.
Nevertheless, the Mirasol PRT is routinely used in Europe with acceptable clinical outcomes.
Evaluation of a Test Method to Detect Bacterial Contamination in Platelets; Bactx TM Assay Ji Hye Park Sexton* 1 , Lorraine Blagg 2 , Christi E Marshall 1 , Herman Woodson 1 , Sean Erony 1 , Krishna Patel 1 and Eric Gehrie 3 .
1 The Johns Hopkins Hospital, 2 Johns Hopkins Hospital Transfusion Medicine Dept, 3 Johns Hopkins University School of Medicine Background/Case Studies: Bacterial contamination of platelets (PLTs) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality.
Therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical.
The BacTx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria.
Here, we report an analysis of the BacTx assay at our hospital.
Study Design/Method: We aimed to determine the sensitivity and specificity of the BacTx assay.
340 intact leukoreduced apheresis PLT (LRAP) units were tested by BacTx at storage day 4.
As a control, each intact LRAP was also cultured by an automated bacterial detection system (BacT culture) on storage day 3.
The results of the BacTx test were compared to the results of the BacT culture system.
Results/Finding: A total of 340 LRAP were tested.
335 LRAPs initially tested negative by BacTx, while 5 LRAPs initially tested positive by BacTx.
All 5 initial positive BacTx tests were negative when subjected to repeat testing.
In contrast, all LRAPs tested negative with the BacT culture system.
The specificity of the BacTx test was 98.5%.
We did not have any true positive test results; therefore, the sensitivity of the BacTx could not be determined.
Conclusion: This is a small study of only 340 platelet units.
The expected rate of bacterial contamination of platelets is less than 1 per 2000 units.
The 1.5% initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the BacTx assay.
In Vitro Quality of Rejuvenated and Washed CPD/As-1 and CP2D/As-3 RBC Alan D. Gray* 1 , Matt Landrigan 2 , Pamela Whitley 3 , Michael Wellington 3 , Sherrie Sawyer 3 , Shalene Hanley 3 , Emily Rondeau 4 , Louise Herschel 4 , Neeta Rugg 5 , Patricia A.R.
Brunker 3 , Shawnagay Nestheide 5 , Jose Cancelas-Perez 5 , Larry Dumont 6 and Zbigniew M. Szczepiorkowski 7 .
and 2,3-DPG to fresh levels.
The objective was to demonstrate that in vitro quality measures are maintained for RBC when stored for >24 hours after treatment with an FDA approved rejuvenation solution.
Study Design/Method: Whole blood (530-550 mL) was collected and processed at 3 sites into leukocyte-reduced RBC (a total of n563 CPD/ AS-1 and n564 CP2D/AS-3).
50 mL of rejuvenation solution (Citra Labs) was added to each RBC on Day 35 (D-35), incubated for 60 minutes with agitation at 378C water bath (Helmer DH4), washed (Haemonetics ACP215), and stored in AS-3 at 1-6 oC for 7 days (D-36 through D-42).
In vitro recovery (%) was calculated and hemolysis, ATP, and 2,3-DPG were determined on Day 0, D-35, D-35 after rejuvenation and washing (postRJV), D-36, D-38, D-40, and D-42.
All units were cultured on D-35 postRJV and on D-42, and then concentrated by centrifugation on D-42.
Results/Finding: In vitro RBC recoveries were 95.7% and 95.5% (AS-1 and AS-3, respectively) and no bacterial growth was observed.
Hemolysis on D-42 was maintained <1% in 58/63 (92%) AS-1 units and 63/64 (98.4%) AS-3 units.
All AS-1 and AS-3 units (100%) had hemolysis <1% following concentration by centrifugation.
Morphology score was reduced to 77% (AS-1) and 74% (AS-3) by D-35, restored after rejuvenation (91%, 92%, respectively) and maintained through D-42 (>90%).
ATP was restored and maintained above fresh levels after rejuvenation.
2,3-DPG was restored above fresh levels and was maintained !80% of fresh levels through D-38.
All values were significantly different compared to D-35 except as noted (p<0.001, paired ttest) ( Table 1) .
Conclusion: Rejuvenation of stored RBC restores ATP and 2,3-DPG above fresh values and morphology to near fresh levels while maintaining improved in vitro RBC quality measures through D-42 when compared to nonrejuvenated RBC on D-35.
This study is funded by Zimmer Biomet.
Storage >24 hours is not FDA approved for use at the time of this publication.
Liposomes and Rejuvenation: New Approach for Improving Quality of Stored Red Blood Cells Luciana da Silveira Cavalcante 1 , Jason P. Acker* 1,2 and Jelena Holovati 1 .
Background/Case Studies: Liposomes have been shown to minimize RBC membrane damage occurring during 42-day hypothermic storage (HS), while rejuvenation solutions have been shown to restore RBC metabolism by maintaining ATP and 2,3-DPG levels.
This study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored RBCs.
Study Design/Methods: Five leukoreduced packed RBC units obtained were pooled and split.
The units produced were segregated into four experimental groups: sham control (S), liposome-treated (L), rejuvesol-treated (R) and liposome 1 rejuvesol-treated (L1R).
The pRBCs were incubated for 1 h at 378C with HEPES-NaCl (sham), liposomes (DOPC:CHOL, 7:3 mol%, 2 mM lipid), Rejuvesol or liposomes plus Rejuvesol.
The in vitro quality was accessed by hemolysis, deformability, aggregation, ATP and 2,3-DPG at day 42 HS.
Results/Findings: Hemolysis was significantly decreased in all treatments compared to sham control (0.60 6 0.06%): L (0.53 6 0.01%, p50.042), R (0.43 6 0.02%, p50.004), L1R (0.48 6 0.06%, p50.020).
Ektacytometry analysis showed an increase in maximum elongation (EI max ) in R (0.55 6 0.01, p50.010) and L1R (0.55 6 0.01, p50.010) treatments compared to S (0.53 6 0.01) but not L (0.53 6 0.01, p50.936).
RBC rigidity (KEI) increased in all treatments compared to sham (1.19 6 0.07): L (1.28 6 0.06, p50.025), R (1.44 6 0.17, p50.010) and R1L (1.44 6 0.06, p50.004).
Aggregation amplitude was significantly increased by R treatment only (24.07 6 1.67 au vs. 19.12 6 1.38 au, p50.004).
ATP levels were significantly higher in all treatments compared to sham (1.64 6 0.14 mmol/g Hb): L (2.00 6 0.21 mmol/g Hb, p50.010), R (4.70 6 1.20 mmol/g Hb, p50.004), L1R (5.00 6 1.56 mmol/g Hb, p50.004).
The levels of 2,3-DPG were no longer detectable in S and L treatments at day 42.
The combined treatment was comparable to R (2.38 6 3.26 mmol/g Hb vs. 2.62 6 2.20 mmol/g Hb, p50.868).
Conclusion: Both rejuvenation and liposome treatments improved the quality of stored RBCs compared to sham control.
The combined treatment (L1R) did not have a greater impact in improving in vitro quality of stored RBCs compared to rejuvenation alone.
Step Toward a Unique and Adaptable Thermoregulation System Lucie Boyer 1 , Eric Ducas 1 , Patricia Landry 1 , Nathalie Dussault 1 , Jacques Bernier 1 , Danny Brouard* 1 and Anne Maltais 2 .
1 H ema-Qu ebec, 2 Institut de technologie des emballages et du g enie alimentaire Background/Case Studies: H ema-Quebec (HQ) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area.
In collaboration with the Institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the 500-mL whole blood Leukotrap RC System (Haemonetics Corp.).
The objective is to design a packaging system for the rapid cooling (T < 108C) of one to six 500-mL whole blood units (WBU) within 8h from collection.
Moreover, the insulating and thermoregulation system must maintain the internal temperature of WBU between 18C and 108C for 24h under extreme external conditions (-308C to 408C), including the initial blood cooling period.
Study Design/Method: The proposed packaging design is based on an external Coroplast box containing six Vacuum Insulated Panels (VIP) for increased insulating efficiency.
Preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned 58C Phase Change Material (PCM).
The number of PCM, their position and conditioning were optimized and tested in order to meet the expected performance criteria.
Preconditioned PCM were stored into VIP boxes for 24h at 20-248C before each test to mimic a worst-case scenario for remote blood drives.
For the experimental testing, 500-mL WB bags were filled with 555 mL saline 0.9% at T 5 308C to mimic freshly collected WB.
Probes were positioned inside the saline-filled bags to monitor temperature profiles of WBU under extreme winter (-308C) and summer (408C) conditions.
Shipping boxes were filled with either one or six bags (n5 2).
Results/Finding: The results showed that the thermoregulation box prototype is able to cool WBU bags under 108C in 4.55 6 0.62h and maintain their internal temperature between 18C and 108C for 24h with final values ranging between 6.38C and 9.38C for the extreme summer scenario.
Similar results were obtained for the extreme winter scenario; units reached the 108C threshold value in 2.4 6 0.2h and the bags' internal temperatures were within the acceptable range for 24h.
Conclusion: The insulating and thermoregulation system met HQ performance criteria.
Preliminary results showed that PCM could be conditioned at temperatures higher than -138C without any significant impact on the system performances.
HQ is currently validating the shipping box prototype performances.
Additionally, we are working on reducing the PCM conditioning time to optimize logistic operations.
As this packaging has many advantages in terms of durability, price and convenience, HQ intends to evaluate this system for the packaging and transport of other lines of blood products.
Stuart Weisberg* 1 , Christopher C. C Silliman 2 , Beth Shaz 1 , Marguerite Kelher 2 and Claudia S. Cohn 3 .
1 New York Blood Center, 2 Bonfils Blood Center, 3 Department of Laboratory Medicine and Pathology, University of Minnesota Background/Case Studies: Platelets collected and stored in platelet additive solution (PAS) reduce recipient exposure to donor plasma components.
To better define the effects of PAS on platelet supernatant composition, we compared total protein, isohemagglutinin titers, HLA antibodies and in vitro neutrophil (PMN) priming activity in supernatants of PAS-C platelets to plasma platelets.
Study Design/Methods: Apheresis platelets from group O blood donors were collected into either 100% donor plasma (n550) or 65% PAS-3 / 35% donor plasma (n550).
Within 12 hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-A and anti-B titer, and PMN priming activity within the total and lipid extractable fractions.
All samples were screened for HLA antibodies.
Screen-positive samples were tested using Luminex single bead assays for antibody strength and specificity.
Soluble CD40 ligand (sCD40L) was measured using solid-phase ELISA.
Results/Findings: Supernatants of PAS-C platelets had significantly lower total protein concentration, anti-A and anti-B titers compared to plasma platelets.
There was no significant difference in the number of HLA-antibody screen positive PAS-C (3/50 products) compared to plasma platelets (2/50 products); however, the HLA-antibody screen-positive supernatants of PAS-86A TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT C platelets had fewer HLA specificities (2 specificities) compared to those of the plasma platelets (18 specificities).
PMN priming activity was significantly increased in the supernatant of PAS-C platelets.
The lipid extractable fraction was not affected; however sCD40L levels were increased in the supernatant of PAS-C compared to plasma platelets (Table 1) .
Conclusion: Decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of PAS-C platelets.
Decreased anti-A and anti-B titers may prevent hemolysis from minor ABO mismatch.
Lower HLA-antibody specificities may mitigate transfusion related acute lung injury (TRALI).
Increased PMN priming by PAS-C platelets is likely due to platelet membrane release of sCD40L and not bioactive lipids.
Although sCD40L has been associated with TRALI, only PMN priming with lipid -not cytokine -agents has been causally linked with TRALI.
The mechanism and clinical impact of increased sCD40L in PAS-C platelets remain to be elucidated.
Background/Case Studies: Current guidelines require a reduction of residual white blood cells (rWBC) below 5x10 6 WBC in US and 1x10 6 WBC in Europe, per unit.
The established reference method for testing rWBC in platelet (PLT) and red blood cell (RBC) products is flow cytometry.
Alternative technologies have been developed including hemocytometry and microfluorometry.
Study Design/Methods: This study compared performance and workflow efficiency of the FACSVia, a flow cytometer with a simplified workflow and automated loader to the ADAM automatic microscopic cell counter based on imaging technology.
Nonfiltered whole blood (WB) samples, apheresis platelet units (n52) and leukoreduced (LR) RBC units (n52) were used to generate spiked samples.
Apheresis platelets and LR RBC were filtered to deplete WBCs and were used as a diluent.
Nonfiltered WB samples were the source of WBCs to prepare a sample of 1000 WBC/uL.
The spiked samples of 5, 12.5, 5, 25, 50 and 100 WBC/uL were prepared from the source sample of 1000 WBC/uL and filtered platelet and RBC units.
To evaluate linearity, WBC concentrations (0, 12.5, 5, 25, 50, 100 WBC/uL) were measured using ADAM and FACSVia.
Samples were stained and run in triplicate on each analyzer.
Data was analyzed using linear regression.
The results were proportional to the WBC concentration in the spiked samples.
Reproducibility of the two systems was measured by running spiked samples (0, 5, 25, 50 WBC/uL).
10 tubes of each sample were stained and run per system.
The %CV and %Diff were calculated.
A batch of 20 samples (PLT and RBC) were run on both analyzers, repeated for 5 days.
Workflow efficiency was assessed observationally by measuring the time of tasks performed.
Tasks recorded were Instrument QC, assay controls and sample testing and analysis.
Results/Findings: The WBC concentration results for PLT and RBC samples on FACSVia correlated well with ADAM (r-PLT50.996, slope50.972), (r-RBC50.999, slope50.992).
The %Diff-PLT at 5, 25, 50 WBC/uL were 7.8, 4.7 and 10, respectively.
The %Diff-RBC at 5, 25, 50 WBC/uL were 10.8, 3.2 and 14.7, respectively.
The average total testing time was similar on both instruments; 89 min for the FACSVia and 92 min for the ADAM.
Of the total testing time, ADAM required continuous hands-on time, while FACSVia demonstrated 62% (56 of 89 min) hands-off time.
Conclusion: Both instruments showed comparable precision, linearity and accuracy.
While the average total testing time was similar on both instruments, FACSVia offered a significant workflow efficiency advantage.
Users saved an average hands-on time of 56 minutes that could be used on other tasks.
Platelet Rich Plasma and Quality Control: Is There a Role for the Blood Bank?
Claudia S. Cohn* 1 and Mickey Koh 2 .
1 Department of Laboratory Medicine and Pathology, University of Minnesota, 2 St George's Hospital and Medical School Background/Case Studies: Autologous Platelet-rich plasma (aPRP) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases.
PRP isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aPRP is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment.
Thus, the consistency and quality of aPRP is questionable and the lower yielding PRP may have decreased efficacy.
Study Design/Methods: A survey was designed to assess aPRP manufacture, usage and quality control (QC) measures taken prior to its use.
A survey was developed with input from content experts.
The survey was sent to members of BEST and ISBT.
Survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown.
A total of 62 completed and partially completed surveys were received.
Results/Findings: Responses came from 13 countries, but the majority of responses came from the United States (US).
Of the respondents, 35% reported aPRP use in their hospital.
aPRP was used predominantly for outpatients, though >40% of hospitals also used aPRP in the in-patient setting.
In most hospitals, aPRP was used by 1-5 MDs; however, 3 hospitals had >10 MDs using aPRP.
The aPRP was used for orthopedics, wound/incision repair, rheumatology and other indications.
In the US the aPRP was manufactured outside of the blood bank, while outside the US aPRP was isolated by blood bank personnel.
Nearly all the aPRP manufacturing was done with no quality control (QC) measures (97%); however, 3 respondents assessed the final product prior to release.
These QC measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing.
In some cases, if the aPRP failed QC it could still be used, pending an MD's approval.
In the 3 hospitals conducting QC on the final aPRP, the testing was done by the blood bank.
A subset of respondents from African nations also used allogeneic PRP (allPRP).
In contrast to the patterns of use with aPRP, allPRP was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'.
The allPRP was manufactured in the blood bank or the donor center with no QC other than a regular check of the centrifuge used to isolate the PRP fraction.
Conclusion: PRP is used in hospitals throughout the world for a wide variety of indications.
The blood bank is involved in its manufacture in some countries, but in the US aPRP is made outside of the blood bank.
Quality control of aPRP production and the final product is not done in most hospitals.
To improve the consistency and efficacy of PRP, more stringent QC measures need to be in place.
Background/Case Studies: The morphology of donated red blood cells (RBC) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (PS), and decreased intracellular ATP.
These changes have been associated with increased RBC clearance within hours of transfusion.
Analysis of morphological alterations of stored RBC with imaging flow cytometry (IFC) has identified a subpopulation of small RBC that accumulates upon storage.
This RBC subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (Roussel, Dussiot et al, 2017) .
Some of the storage alterations are reversible when the RBC metabolism is reestablished.
As such, treatment with a rejuvenation solution (Citra Labs) before transfusion is expected to restore some of the RBC properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion.
Study Design/Methods: A multi-parametric analysis of RBC alterations was performed to evaluate the effect of rejuvenation on RBCs stored in SAGM (n56) under blood bank conditions at Day 3 (D3), at Day 42 (D42), after rejuvenation (R), and after rejuvenation and washing (RW).
Morphological alterations of stored RBCs were evaluated with IFC (Imagestream X Mark II, AMNIS V R ).
Results/Finding: Rejuvenation increased the level of intracellular ATP, confirming the metabolic effect of this process.
Population distribution as per RBC projected surface area measured by IFC depicted a well-demarcated subpopulation of small RBC that increased with storage from 2.1-8.8% at D3 to 8.3-68.1% at D42.
Rejuvenation markedly reduced this storage-induced spherocytic shift (1.7-29.3%) and partially restored RBC morphology, an effect confirmed by differential interference contrast microscopy.
The restoration effect of the rejuvenation process did not correct the storage-related loss of RBC elongation but was associated with a decrease in PS exposure (Table) .
Conclusion: Our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation.
The impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation.
Red Cell Concentrate Volume and Manufacturing Method Impact Post-Thaw Quality in Cryopreserved Products Processed Using a Closed Cell Processor Anita Howell 1 , Angela Hill 1 , Tracey Turner 2 , April Xu 2 , Brandie Dennis 2 and Jason P. Acker* 2,3 .
1 Canadian Blood Services, Centre for Innovation, 2 Canadian Blood Services, 3 University of Alberta Background/Case Studies: The blood service uses both top/top with whole blood filtration (WBF) and top/bottom with red cell filtration (RCF) methods to prepare CPD/SAGM LR red cell concentrates (RCCs).
Mean volume (mL) is higher in WBF units (314 6 15) than in RCF units (275 6 16), with similar hematocrits.
A closed system cell processor is currently being implemented for cryopreservation of RCCs.
Post-deglycerolization re-suspension in AS-3 additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size.
The impact of the resulting variation in hematocrit on post-thaw in vitroRBC quality was evaluated to ensure that regulatory standards can still be met for RCCs at the extreme edge of the input volume range.
Study Design/Methods: 12 small RCF (252-263 mL) and 12 large WBF (322-353 mL) RCCs were stored for 21 d before being glycerolized and frozen at -658C for !72 h. Large RCCs whose red cell mass exceeded the capacity of the 275 mL deglycerolization centrifuge bowl were volume reduced prior to glycerolization.
RCCs were thawed in a 378C water bath, deglycerolized and re-suspended in AS-3.
RCCs were stored 14 d and then tested for in vitroRBC quality.
Results/Finding: Small RCF RCCs had lower (p<0.05) hematocrit, specific gravity, hemoglobin per unit, supernatant K 1 and Na 1 concentration, deformability (EI MAX ), and higher (p<0.001) recovery than did large WBF units.
No significant differences in hemolysis, ATP, 2,3-DPG, p50, RBC indices, RBC morphology, or residual glycerol were seen between groups.
The majority of units met acceptance criteria (Table 1) , however 8 of 12 large WBF units had RBC recoveries < 80% due to pre-glycerolization volume reduction, and 2 of the small RCF units had hemoglobin values < 35 g per unit.
When the recovery and hemoglobin failure rates are analyzed against the organization's RCC production volume distribution, the mean recovery is projected to be well above 80% and the hemoglobin failure rate would be below 10% of units tested; compliant with regulatory standards.
Conclusion: The differences between groups in the cryopreserved RCC physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass.
The lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality.
0.03 6 .02 0.33 6 .28 0.83 6 .3 0.32 6 .14 Elongation index (30Pa) 0.602 6 .008 0.585 6 .017 0.580 6 .017 0.578 6 .017 This study is funded by Zimmer Biomet.
(Hasan 1994) .
The objective was to determine the effect of RBC rejuvenation on RBC oxygen release capacity (ORC) and estimated oxygen consumption (VO 2 ) after simulating a single unit transfusion of either standard or rejuvenated RBC stored for 42 days.
Study Design/Method: Oxygen dissociation curves (ODC) (Hemox Analyzer, TCS Scientific) were generated from fifty-two (52) RBC units (leukocyte-reduced), CPD/AS-1 or CP2D/AS-3, on Day 0, Day 42, and after rejuvenation and washing (PW).
The ODC for each sample was used to determine ORC (mL O 2 /g Hb) and Total Releasable Oxygen (TRO) of the unit (mL O 2 ).
ORC was determined by assessing the change in % O 2 saturation from 100 mm Hg PO 2 (e.g., lung) to 40 mm Hg PO 2 (e.g., venous blood) multiplied by 1.34 mL O 2 /g Hb (Li 2016).
A simulated baseline pretransfusion VO 2 of 115 mL O 2 /min was estimated using the Day 0 ORC and assuming a 7 g/dL transfusion trigger with a cardiac output of 5 L/min and 5 L blood volume.
Paired Student's t tests were used for comparative statistical analyses.
Results/Finding: RBC rejuvenation on day 42 restored ORC and TRO to levels greater than Day 0 ( Table 1) .
ORC of the rejuvenated unit was 1.5 6 0.2 times and 3.4 6 0.5 times greater than RBC on Day 0 and Day 42, respectively (p<0.001).
VO 2 increased after a simulated single unit transfusion of RBC (Day 0, Day 42, and PW) by 19.3%, 8.9%, and 28.8% over the pre transfusion VO 2 , respectively (p<0.001).
Conclusion: These results suggest a transfusion with rejuvenated RBCs has the potential to release 3.3 times the volume of O 2 compared to standard, untreated RBCs stored for 42 days.
Inferior oxygen delivery to tissues (VO 2 max) has been observed during exercise in healthy human volunteers after transfusion of two autologous RBC units stored for 42 days vs 7 days which seem dependent on genetic variability and storage time (Bennett-Guerrero 2017).
Therefore, transfusion practices to correct anemia may be less effective than intended due to the variable ORC of standard stored RBC units.
Transfusion strategies should consider whether the use of RBC with increased ORC may be physiologically advantageous.
Disclosure: This study was funded by Zimmer Biomet.
Rejuvenation Solution as an Adjunct Storage Solution Maintains Physiological Hemoglobin Oxygen Affinity during RBC Unit Storage Andrea Ansari* 1 , Jay Srinivasan 1 , Gustaaf de Ridder 2 , Alan D. Gray 3 , Matt Landrigan 4 , Keaton Charles Stoner 5 , Angela Crabtree 6 , Jessica Poisson 7 and Ian Welsby 8 .
1 Duke University School of Medicine, 2 Duke Health Pathology, 3 Citra Labs, a Zimmer Biomet Company, 4 Zimmer Biomet, 5 Duke University, 6 Department of Pathology, Durham Veterans Affairs Medical Center, 7 Duke University Hospital, 8 Duke University Medical Center Background/Case Studies: Deleterious changes develop during the storage of packed red blood cells (RBCs) collectively called the "storage lesion".
These include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of ATP, Snitrosohemoglobin, vasodilatory capacity, and cell surface PS expression, and depleted 2,3-diphosphoglycerate (2,3-DPG).
The loss of 2,3-DPG increases the oxygen affinity of hemoglobin, resulting in lower p50 (partial pressure of oxygen at 50% hemoglobin saturation).
Decreased p50 may negatively impact the ability for transfused RBCs to release oxygen to peripheral tissues.
An FDA-approved rejuvenation solution (Citra Labs) can restore normal levels of ATP and 2,3-DPG, normalizing membrane function and oxygen affinity, respectively.
This process requires incubation at 378C for an hour, an impractical step in time-sensitive situations, followed by washing of the RBCs.
We tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of RBCs.
Study Design/Method: Eight units of group A1, leukoreduced PRBC stored in AS-1 were obtained from our local blood center.
After 3 days of storage, units were divided into 4 separate aliquots: control (CTL), wash (W), standard rejuvenation (SR), and cold rejuvenation (CR).
The rejuvenation solution (50ml) was added to the CR group, and all groups were then stored for another 12 days at 1-68C.
On day 15 of storage, the SR group was incubated for 1 hour at 378C with rejuvenation solution, after which the W, SR, and CR groups were separately washed on a C.A.T.S V R (Fresenius Kabi) using the High Quality Wash setting.
Hemoglobin p50 was measured by tonometry using a Hemox Analyzer (TCS Scientific).
Deformability (Elongation Index or EI) was measured by ektacytometry (LoRRca Mechatronics).
Supernatant plasma free hemoglobin (PFHb) was measured using visiblelight spectrophotometry.
Cell surface PS expression (PS1) was measured by Annexin V flow cytometry.
All group results were compared using nonparametric Wilcoxon signed-rank tests with a 5 0.05.
Results/Finding: Significant differences in p50 were noticed between all groups (Table 1) .
EI, PS1, and PFHb did not differ between groups.
Conclusion: Cold rejuvenation prevents the increased oxygen affinity (lower p50) seen over 15 days of RBC storage without adverse effects on deformability or hemolysis.
This offers an alternative to incubated rejuvenation to provide clinicians with ready access to RBCs with a high/normal p50 that may better release oxygen to the tissues.
cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function.
Reactive antibodies and other inflammatory agents in RBCs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (TRALI), anaphylaxis, and even death.
In this study, a multifunctional bead-based filter was evaluated for removal of K 1 , along with free hemoglobin (Hb) and other pRBC contaminants that can contribute to transfusion related adverse events.
Study Design/Method: Ten leukocyte-reduced pRBC (300mL) units stored in AS-1, obtained from a regional blood donor center at expiration (42 6 2 days), were passed by gravity through sorbent-devices containing 50 mL of multifunctional polymer bead, at a flow rate of 20 mL/min.
Supernatants were analyzed for K 1 removal as well as free Hb, antibodies and cytokines (27-plex, BioRad).
RBCs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively.
Results/Finding: Filtration of the aged pRBC units through the sorbent device reduced [K 1 ] from 54.2 6 5.0 to 1.98 6 1.3 mEq/L; equivalent to an 84.6% reduction.
Free Hb was reduced by 96.3% from 2.5 6 1.0 to 0.39 6 0.2 mg/mL.
Antibodies, specifically IgG, IgA, and IgM decreased from 9.91 6 3.1 to 2.40 6 1.1 mg/mL (77.7%), 0.48 6 0.1 to 0.25 6 0.01 mg/mL (48.9%), and 0.73 6 0.2 to 0.49 6 0.1 mg/mL (31.5%), respectively.
Inflammatory cytokines were significantly reduced, specifically: IP-10 from 144.27 6 16.2 to 18.43 6 2.7 pg/mL (87.2%), MIP-1b from 37.37 6 5.7 to 7.23 6 2.5 pg/mL (80.7%), and PDGF from 1348.3 6 291.9 to 77.91 6 22 pg/mL (94.2%).
Filtration had no significant impact on cell surface markers of RBC viability (<0.1% decrease) or sensitivity to osmotic changes.
Values listed represent mean 6 SEM (P < 0.01 for all analytes tested).
A paired ttest was used to assess significance.
Conclusion: The sorbent filter was highly effective in reducing the levels of extracellular K 1 as well as free Hb, antibodies, and cytokines from pRBCs without impact on RBC viability or integrity.
This study demonstrates the viability of a multifunctional sorbent filter for removal of K 1 along with other detrimental components from stored pRBCs that can readily be incorporated into transfusion practices to minimize adverse effects.
Background/Case Studies: Platelets carry no Rh antigens, but residual red blood cell (RBC) in platelet products can immunize D negative recipients if the donor is D positive.
Current recommendation is to give Rh immunoglobulin (RhIG) to Rh negative patient if they receive Rh positive platelet unit to avoid potential alloimmunization to D antigen.
A recent study has shown a very low frequency (1.5%) of D alloimmunization when a Rh mismatch platelet is transfused.
Restricting D negative patients to receive only D negative platelets could create shortage and cause inventory challenges.
Higher yields of platelets with minimum to none residual RBCs are obtained with new generations of apheresis machines.
As a consequence, the need for prophylactic Rh immunoglobulin (RhIG) may be unnecessary with the use of apheresis derived platelets.
The accurate determination of residual RBC in a platelet unit is important for patient safety to prevent Rh alloimmunization.
Hemocytometer is considered the gold standard for cell counting.
However, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers.
Currently there are no standardization and/or guidelines to advise what system to use for RBC quantification in platelet products.
Study Design/Method: We designed this study to quantify the residual RBC in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods.
We measured the amount of red blood cells per microliter in 50 apheresis and 50 whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, Sysmex (Sysmex America, Lincolnshire, IL) and Advia 2120 (Siemens Healthcare Diagnostics, Tarrytown NY).
The whole blood derived platelet units were produced using Acrodose TM system technology.
We conducted non-parametric permutation test based on 10000 permutations to compare Sysmex and Advia between apheresis and whole blood derived groups.
ABSTRACT collection) RBCs to RBCs stored for 42 days and after treatment with an FDA approved rejuvenation solution.
Study Design/Method: The addition of a rejuvenation solution to stored Red Blood Cells (RBCs) has been shown to increase ATP and 2,3-DPG profiles to fresh levels.
The objective was to compare 50% hemoglobin-oxygen saturation (p50) and morphology profiles of fresh(day of collection) RBCs to RBCs stored for 42 days and after treatment with an FDA approved rejuvenation solution.
Results/Finding: In vitro RBC recovery (overall) was 97.2 6 2.2%.
Hemolysis (%) was similar on Day 42 before and after dry-air incubation with the rejuvenation solution (0.34 6 0.14% vs 0.35 6 0.14%).
Percent hemolysis (%) decreased after washing (0.24 6 0.07%) and was maintained below <1% for all units during storage for 24Hr (0.51 6 0.19%).
Average ATP and 2,3-DPG were restored above the average fresh values.
The morphology score decreased $25% by Day 42, which was restored to near fresh values following rejuvenation and washing and storage 24Hr (93.7% and 95.1%, respectively).
RBC oxygen affinity, as assessed by p50, was restored above fresh values.
All values were significantly different compared to Day 42 (p<0.001, paired t-test) ( Table 1) .
Conclusion: RBC morphology was restored to near fresh and average ATP, 2,3-DPG, and p50 were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process.
RBC Morphology, ATP and 2,3-DPG were maintained during storage 24Hr.
Rejuvenation of refrigerated RBCs may offer avenues to improve RBC quality prior to transfusion.
Vandi Ly*, Dimath Alyemni, Warren R Korn, Matthew J Brune and Julie Katz Karp.
Thomas Jefferson University Hospital Background/Case Studies: Blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana.
Therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion.
Donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, D9tetrahydrocannabinol (THC) and 11-OH-D9-tetrahydrocannabinol (11-OH-THC).
Study Design/Method: De-identified donor plasma segments were sequestered and stored frozen until time of testing.
Testing for THC and 11-OH-THC was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based on a method modified from Lacroix and Saussereau.
In summary, this method used dabsyl chloride derivatization of THC and 11-OH-THC to produce samples for LC-MS/MS analysis.
LC used a C18 column.
Post-column detection by MS/MS used positive ion electrospray with Q1:Q3 ion pairs of m/z 5 605.3:225.3 (internal standard (IS), d3-THC), m/ z 5 602.2:225.2 (THC), and m/z 5 618.3:256.1 (11-OH-THC).
Quantitative results for THC and 11-OH-THC were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from 0-50 ng/ mL for both THC and 11-OH-THC.
Limits of quantitation, defined as 5 standard deviations above background, were 0.7 ng/mL for THC and 7 ng/mL for 11-OH-THC.
Results/Finding: A total of 424 donor plasma samples were tested for THC and 11-OH-THC.
No samples tested positive for either THC or 11-OH-THC.
Theoretical calculations according to statistics of a Poisson distribution indicated that there would be a 50% probability of one or more positives at a prevalence of 0.16% positive samples, and a 95% probability of one or more positives at a prevalence of 0.71% positive samples.
Results thus indicated a boundary of prevalence of the presence of active THC-metabolites in plasma samples to be less than 1% among this donor population.
Standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than 12 hours for post-exposure detection of THC and/or 11-OH-THC in plasma.
Conclusion: Testing of 424 donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives.
Statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than 1%.
Probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics.
Elisabeth Maurer-Spurej* 1 , Ruqayyah Almizraq 2 , Daniel Millar 3 and Jason P. Acker 4 .
1 University of British Columbia, 2 University of Alberta, 3 LightIntegra Technology Inc., 4 Canadian Blood Services Background/Case Studies: The controversy around the quality and clinical impact of aged red blood cell concentrates (RCC) is ongoing.
Current studies are limited by the lack of quality measures suitable for routine screening of RCC.
Based on evidence that fragments called microparticles (MP) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of MP screening to characterize the effect of RCC production methods and storage.
Study Design/Method: Red blood cell concentrates were prepared by whole blood filtration (WBF; Top/Top) or red cell filtration (RCF; Top/Bottom) methods, centrifuged to prepare a supernatant and tested for MP content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, ATP and red cell deformability on days 7, 21, and 42 of storage.
One RCF RCC was tested on days 1, 5, 14, 21, and 43 and six 10 mL aliquots were stored in parallel and tested on days 14, 21, and 43.
All samples were tested for MP Content and compared to the other quality indicators.
Results/Finding: MP Content showed a linear increase with storage time with statistically significant differences between days 14, 21 and 43 (p<0.001) and correlated with supernatant hemoglobin, and inversely with ATP or RBC elasticity.
Both MP testing methods agreed with respect to total MP Content.
Starting levels of the quality indicators varied between donations, preparation methods (WBF RCC contained much higher levels of MP), and storage time.
MP Content in the 6 aliquots were consistent at each time point but statistically higher than in the original RCC on and after day 21 of storage.
Conclusion: MP Content correlates with measures of hemolysis and other RBC quality indicators and could be implemented as a routine screening tool.
Differences in MP content between donors, processes and age could be monitored and used to inform component production decisions.
Measuring MP Content would allow 100% screening of RCC products in studies and pragmatic QC initiatives which are needed to settle the controversy about the clinical effect of RCC age.
Single Donor Spray-Dried Plasma: The Future of Plasma Therapy?
Qiyong Peter Liu*, Jihae Sohn, Ryan C. Carney, Sruthi Sundaram and Mark A Popovsky.
Velico Medical Inc Background/Case Studies: Frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time.
Spray-dried plasma (ODP, On Demand Plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care.
The objective of this study is to determine if ODP can be consistently manufactured at a blood center with key proteins and coagulation function comparable to FFP.
Study Design/Methods: Units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into ODP using Velico's spray dryer.
ODP (n 5 60) and paired FFP aliquots were stored for 31-33 days at 2-68C and -188C, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance.
The volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted ODP and FFP for direct comparison.
Results/Findings: Compared to FFP, ODP had !
80% levels of functional clotting factors (fibrinogen, factors II, V, VII, VIII, IX, X, XI and XII), plasminogen, and protease inhibitors (antithrombin III, protein C, protein S; plasmin, C1 esterase and alpha 1-proteniase inhibitors).
The level of factor XIII in ODP was slightly lower, about 70% of FFP by both activity and antigen assays.
ODP was identical to FFP in the levels of albumin, immunoglobulins (IgA, IgG and IgM), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor XIII.
The levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments I1II and Ddimer) and complement (C3a and C5a) activation in ODP remained similar to FFP.
ODP was equivalent to FFP when assessed by aPTT, PT and thrombelastography.
TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT Spray-drying fragmented a substantial number of high molecular weight von Willebrand factor (vWF) multimers into smaller ones, leading to a net increase of vWF multimers in ODP.
The size re-distribution reduced the vWF ristocetin cofactor activity (vWF:RCo) to 62% in ODP relative to FFP, but had no impact on vWF antigen and factor VIII function (stabilized by vWF).
vWF-specific studies have shown that ODP retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by Meledeo et al/US Army Institute of Surgical Research and Bercovitz et al/Blood Center of Wisconsin).
Conclusion: ODP can be manufactured at a blood center with a quality comparable to that of FFP.
Future studies will determine if the product is bioequivalent to FFP and comparable in safety and efficacy.
Background/Case Studies: The collection time of whole blood is, according to European Guidelines, limited to 15 minutes.
In addition, donations with collection times between 12 and 15 minutes should not be used for preparation of platelet (PLT) concentrates (PC) because of the chance of too much activation of PLT.
It seems justified to re-evaluate the quality of PLT from these donations because new generations collection systems and mixers were introduced, including a more efficient needle.
The aim of this study was to investigate the in vitro quality of PC prepared from 12-15 minutes buffy coats (BC) with the aim to prevent unnecessary discarding of BC and to simplify the total blood bank process.
Study Design/Method: Single-donor PC (sPC, n56) were prepared from one 12-15 minutes BC and 60 mL of autologous plasma in a 600 mL PVC-DEHP container.
As a reference, sPC from donations with collection times of <12 minutes were prepared (n55).
In addition, PC were prepared from 5 BC, of which at least 4 BC were from 12-15 minutes donations (n55).
After pooling of the BC, 300 mL of PAS-E was added and a standard pooling set with a PVC-BTHC storage container was used for storage of PC.
All PC were stored for 8 days at 22 6 28C and sampled at regular intervals for determination of the in vitro quality.
Aggregation tests were performed with Chronolog (ADP or collagen) and Multiplate (arachidonic acid) aggregometers.
Thromboelastography (TEG), using kaolin as an activator, was applied for assessment of the overall clotting capacity.
Values are expressed as mean6 SD.
A non-paired t-test or a Mann-Whitney U test was applied for statistical analyses of normal or non-normal distributed data respectively.
Results/Finding: Volume (67 6 5 vs. 66 6 16 mL) and platelet content (74 6 11 vs. 71 6 15 x10 9 ) were similar in both groups.
At the end of storage, both groups showed comparable in vitro quality (Day 8, pH(378C): 6.84 6 0.16 vs 6.83 6 0.17, other data not shown).
No differences in aggregation response after stimulation with arachidonic acid, ADP or collagen were measured.
TEG parameters in both groups were also comparable.
The five-donor PC fulfilled all requirements of European Guidelines, aside from occurrence of small aggregates at Day 6 and/or 8 in 2/5 PC (possibly because sometimes AB0 incompatibility was accepted).
On Day 8, PLT showed low CD62P expression (17.1 6 1.8%) and phosphatidylserine exposure (Annexin V binding, 8.9 6 1.9%).
Hypotonic shock response of platelets was comparable with historical data.
Conclusion: Single-PC in plasma as well as five-donor PC in PAS-E, prepared from 12-15 minutes whole blood donations had a normal composition and showed good in vitro quality during 8 day storage.
To substantiate that the exclusion of 12-15 minutes donations for PC preparation could be stopped, further studies will be performed.
The Effects of a Pneumatic Tube System on Red Blood Cell Units Amy Mata* 1 , Jessie Miller 1 , Ranee Marie Wannarka-Farlinger 1 , Sandra Bryant 1 , Scott A Hammel 1 , Sherry Stern 1 and Camille van Buskirk 2 .
1 Mayo Clinic, 2 Mayo Clinic Rochester Background/Case Studies: The use of Pneumatic Tube Systems (PTS) has become commonplace in many healthcare facilities throughout the world.
The purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care.
A downfall of PTSs is that they have the potential to play a role in increased hemolysis.
While several studies have been published on the effects of PTSs on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (RBC).
The objective of this study was twofold: to determine if the PTS that is in use at our facility contributes to an increase in hemolysis of RBC units and to evaluate how the PTS system affects red cell microparticle (RMP) levels.
Study Design/Method: Forty-one units of AS-3 RBCs, 20 irradiated and 21 non-irradiated, were selected for the study.
The units varied in age, ranging from 2 to 42 days old.
Specimens were obtained from each unit both prior to and after being transported through the PTS, which runs underground and spans the length of a mile and a half.
Specimens were spun down and the plasma supernatant was removed.
All specimens were evaluated for plasma hemoglobin (hgb), potassium (K), hemolysis index (HI), and RMPs.
The Wilcoxon signed-rank test and p value were used to compare the pre and post values.
Additional statistical analysis was performed to compare the values after adjusting for age and irradiation.
Results/Finding: After sending the RBC units through the PTS, hgb, HI, and RMPs were statistically (p< 0.05) higher than before.
When adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and HI.
The K values did not significantly change.
RMPs significantly increased, but only if the units were irradiated (p50.02).
(Table) Conclusion: The use of a PTS provides an effective means to transport blood products; however, it can contribute to biological changes within RBC units.
It is uncertain at this time how those changes can affect the outcome of patients who receive these products.
Each PTS system is different in its specifications and should be validated prior to being used to transport blood products.
Validation of Factor VIII Levels of Thawed Fresh Frozen Plasma after 5 Days of Storage Pei Lun Karen Lim* 1 , Erma Sofia Sumardi 1 , Isamar Eduardo Ancheta 1 , Susan Lim 2 , Christina Yip 1 , Lip Kun Tan 2 and Shir Ying Lee 3 .
1 National University Hospital Singapore, 2 National University Hospital, 3 National University Hospital, Singapore Background/Case Studies: Plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure.
Fresh Frozen Plasma (FFP) has to be placed in the freezer within 8 hours of processing and stored at -188C or colder in order to preserve its coagulation factors.
Thawed FFP has an expiration period of 24 hours hence to reduce wastage, this study aims to investigate Factor VIII (FVIII) activity in thawed plasma stored for 5 days and kept at 1 to 68C.
FVIII was chosen as it is an important coagulation factor in correcting coagulopathies.
Arbitrary FVIII level acceptance limit was set as not less than 50 IU/dL.
Study Design/Method: Randomly selected units of FFP (n510) were measured for FVIII concentration based on clotting assay (STAV R -DEFICIENT 92A TRANSFUSION 2017 Vol.
57 Supplement S3 VIII Diagnostica Stago).
FVIII levels were measured at five time points: prefreezing, 0, 24, 72 and 120 hours post-thawing.
FFP were thawed using Helmer Plasma Thawer (Helmer Scientific) at 30 to 378C for 35 minutes.
An aliquot of thawed FFP from each unit was removed and measured for FVIII before refrigeration (0 hours post-thaw).
Thawed plasma (TP) units were kept in a refrigerator at 1 to 68C for 5 days for subsequent testing.
Results/Finding: Results obtained were listed in Table 1 .
Units 7 to 9 were not tested for FVIII at post thaw-24 hour due to operational issues.
The overall FVIII concentration decreased at an average of 13% from pre-freezing to post thaw 0 hour.
After further storage of TP post thaw-24 hour and -72 hour, residual FVIII level remain to be above 50 IU/dL except unit 10 which had a lower initial FVIII concentration.
At post thaw-120 hour, 7 out of 10 units tested had residual FVIII activity within the pre-set standard of 50 IU/ dL.
The average decline from 0-hour post-thaw to 24-hour, 72-hour and 120hour post-thaw was 36.5%, 42.7% and 47.9% respectively.
There was no observed trend of any blood group having higher or lower pre-freezing FVIII and this is likely due to small sample size.
Conclusion: Decrease of coagulation factor such as FVIII in FFP is expected due to its diminishing stability.
Nevertheless, our data showed that majority of the TP retained at least 50 IU/dL of FVIII.
Typically patients with factor levels below 30 IU/dL may start to show abnormal coagulation profile.
While TP is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding.
Further study extending to measurement of other labile factor such as FV may add value to the validation study.
Validation of the Pathogen Reduction Method Using Amotosalen/ UVA: Comparing Pathogen-Reduced Pooled PRP-Platelets and Conventional Single PRP Platelets for Quality and Bacterial Inactivation Efficacy Lubna Ahmed Almenawi 1 , Ayman Mohamad Sabri 1 , Ali Abdullah Alajeafi 1 , Ashwaq Hasan Alhekri 1 , Saleem Bin Mahfouz 1 , Ali Hasan Alkhodari 1 , Rawya Saeed Shealy 1 , Marcus Picard-Maureau* 2 and Hussain Bana Almalki 1 .
1 King Abdulaziz Hospital and Oncology Center, 2 Cerus Europe BV Background/Case Studies: The growing number of transfusiontransmitted infectious (TTI) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in Saudi Arabia.
While for a limited number of these pathogens TTI risk can be reduced using blood screening assays, alternative solutions are anticipated.
Pathogen reduction (PR) technology was identified as a potential solution.
Validation of amotosalen/UVA photochemical treatment in our blood center was performed by comparing the platelet component (PC) quality of the standard "control" single-donor PRP-concentrate in 100% plasma over a 5 day storage period and the new "test" pathogen-reduced, pooled (pools of 5) PRP PC in 100% plasma over a 7 day storage period.
The efficacy of the bacterial inactivation was also assessed in our setting.
Study Design/Method: The quality parameters of 4 leucoreduced test PCs were assessed at day 7 of storage and compared to leucoreduced control PC at day 5 of storage.
The test PCs were pathogen-reduced with the INTERCEPT Blood System (Cerus Corporation, Concord, U.S.A.) at day 0; the process was completed by day 1 post-collection.
Samples were taken daily for quality analysis from test and control PC until day 5 and day 7, respectively.
For bacterial spiking, additional PC were spiked with each receiving 4 mL of 1 McFarland ($ 1.2x10 9 CFU) S. aureus, S. epidermidis, E. coli, P. aeruginosa or S. viridans, respectively, to challenge PR efficacy.
Results/Finding: The average platelet loss in the test PC post PR treatment was 4.7% 6 2.0, the total average platelet loss at day 7 was 11.2% 62.8.
The average platelet loss in the control units at day 5 was 9.5% 61.4.
The average pH of the test units at day 7 was 6.64 60.04 and in the same range as the control PC, pH 5 6.89 60.09.
Glucose concentration in test PC at day 7 (13.8 63.0 mmol/L) was lower than in the day 5 control units (18.32 61.06 mmol/L).
Lactate levels increased during the course of storage; lactate levels at days 5 and 7 were outside the range of the assay (>15 mmol/L).
Cultures inoculated with pathogen reduced, bacterially spiked units were negative after 7 days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth.
Conclusion: The quality parameters of the pathogen reduced test PC were within specifications and comparable to the conventional control PC.
The high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/UVA pathogen reduction is safe and efficient to enhance PC transfusion safety.
Keaton Charles Stoner* 1 , Jay Srinivasan 2 , Jessica Poisson 3 and Ian Welsby 4 .
1 Duke University, 2 Duke University School of Medicine, 3 Duke University Hospital, 4 Duke University Medical Center Background/Case Studies: The coagulation cascade relies on a complex interaction between proteins known as clotting factors.
Cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients.
However, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation.
My study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation.
Study Design/Method: The Duke Proteomics Core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry TRANSFUSION developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses.
A tissue factor-activated test on the ROTEMV R delta hemostasis analyzer (EXTEM) was performed on each condition.
For each source, dose-response curves for clotting time (CT), alpha angle, and maximum clot formation (MCF) were generated using linear regression models.
Inter-source unit variability was determined by ANOVA and Tukey's HSD post-hoc analysis (RStudio Inc.).
Results/Finding: LC-MS/MS identified 256 proteins in cryo; of the 10 most abundant, only fibrinogen was relevant to coagulation.
Notably, the American Red Cross (ARC) single donor source had the steepest slope for MCF (4.44 mm/dose), indicating a greater per dose potency than the other sources.
The ARC single donor source had the highest mean MCF across all dosing levels, but also the highest standard deviations and response variability.
The ARC single donor source was significantly more potent than the Australian source.
Conclusion: Paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for CT, MCF, and alpha angle can provide physicians with more information regarding cryo dosing.
Future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy.
Viral Inactivation and Enrichment of Factor VIII, Factor XIII, Fibrinogen and Von Willebrand Factor (VWF) Multimers from Fresh Frozen Plasma (FFP)Using, "VIPS Plasma, Virus Inactivation Treatment System".
Background/Case Studies: The solvent/detergent (SD) process used for plasma can safely inactivate all lipid-enveloped viruses.
The method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact.
This study is done to assess viral inactivation and, Factor VIII, Factor XIII, Fibrinogen and von Willebrand factor (VWF) multimers enrichment capacity of, "VIPS Plasma, Virus Inactivation Treatment System".
Study Design/Method: "VIPS Plasma, Virus Inactivation Treatment System" comprise of interconnected bag system where the S/D reagents are removed by filtration and the final products subjected to bacterial (0Á2 lm) filtration.
Cryoprecipitate mini-pools (400 6 20 mL) were subjected to doublestage S/D viral inactivation, followed by one oil extraction and a filtration on a S/D and phthalate [di(2-ethylhexyl) phthalate (DEHP)] adsorption device and a 0Á2 lm filter.
The initial and the final products were compared for visual appearance, blood cell count, factor VIII, Factor XIII, Fibrinogen and Von Willebrand factor (VWF) multimers.
Initial and final products were also checked for HIV, HBV, HCV, dengue, malaria and bacterial contaminations.
Results/Finding: Our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor VIII, Factor XIII, Fibrinogen and von Willebrand factor (VWF) multimers were well conserved (Table 1) .
Kit ensured bacterial sterility (Table 3 ) and most importantly, final product was free of HBV, HCV and HIV (Table 2) .
Conclusion: It's the first time, "VIPS Plasma, Virus Inactivation Treatment System", is used in South Asia for product enrichment and viral inactivation.
Results showed effective product enrichment and viral inactivation in our conditions.
But further investigation is needed to characterize functional activity of the enrich component.
Irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries.
Background/Case Studies: Buffy coats (BC) from donors who used pain medication like aspirin and ibuprofen up to 4 days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (NSAIDs) is inhibition of platelet (PLT) aggregation.
These NSAIDs inhibit the enzyme cyclooxygenase-1, thereby blocking synthesis of thromboxane A 2 from arachidonic acid.
However, the quality of platelet concentrates (PC), prepared from this BC is not known.
The aim of the study was to investigate the in vitro quality of PC prepared from NSAID-BC and autologous plasma during storage.
Study Design/Method: Single-donor PC (sPC, n518) were prepared from a NSAID-BC and 60 mL of autologous plasma.
Information about the type of pain medication was extracted from the anamneses form.
The sPC were stored for 8 days at 22 6 28C and sampled at regular intervals.
Aggregation tests were performed with Chronolog (ADP or collagen) and Multiplate (arachidonic acid) aggregometers.
Thromboelastography (TEG, kaolin) was applied for assessment of the overall clotting capacity.
sPC in plasma from normal controls (n55) were investigated as a reference.
Values are expressed as mean6SD or as median & IQR.
A non-paired t-test or a Mann-Whitney U test was applied for statistical analyses of normal or nonnormal distributed data respectively.
Results/Finding: Volume (69 6 4 vs. 66 6 16 mL) and PLT content (67 6 14 vs. 71 6 15x10 9 ) were similar in both groups.
On Day 8, both groups showed comparable pH and changes in PLT content (data not shown).
Phosphatidylserine exposure on Day 8 was significant higher in a subset of donors who had used ibuprofen (n55).
Aggregation tests with arachidonic acid revealed in general a low or absent response for sPC with aspirin (0,0-30, p<0.05), diclofenac (31,1-76) and naproxen (0,0-24, p<0.05), compared to normal controls (76, .
No differences were detected in aggregation with ADP or collagen.
With TEG, slightly longer R-times (initiation phase) were measured on Day 1 in sPC with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen).
These differences disappeared during storage.
Conclusion: Storage properties of sPC prepared from NSAID-BC were comparable with sPC from normal controls.
Main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen.
PLT from donors who used ibuprofen showed little or no deviations.
This is most likely caused by the fast (<24 hour) disappearance of ibuprofen from the blood circulation and the reversible binding to PLT.
The use of BC from donors who used ibuprofen will be further investigated in a 'worst case' (PC in plasma) and 'best case' (PC in additive solution) scenario.
The effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to PLT.
Background/Case Studies: Previously it was shown that donors could be classified as having platelets (PLT) with good, average or poor storage properties [Bontekoe, Transfusion, 2014] .
A main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of pH during storage of 'poor' PLT concentrates (PC).
This might be caused by a different functionality of the PLT mitochondria and there are indications that donors with a history of 'poor' PCs are more likely to have health issues, pointing towards Metabolic Syndrome and Type 2 diabetes (T2D).
Because of the strong rise of people with T2D in the Dutch population, the aim of this study was to characterize PLT from whole blood donors diagnosed for T2D, but accepted as donor.
Study Design/Method: Twelve whole blood donors with T2D, not using insulin, were selected and buffy coat (BC) and plasma were, after overnight hold, used for preparation of a single-donor PC (sPC).
An equivalent number of sPC was prepared from age and sex matched control donors, derived from the same collection sessions.
sPC were stored for 8 days at 22 6 28C and sampled on Day 1, 4 or 5 and 8.
The diabetic marker HbA1c was determined in red cells and cholesterol and triglyceride levels in plasma.
From both groups 3 'good' (pH day8 >6.6) and 3 'poor' (pH day8 <6.3) storing sPC were selected and analysed in more detail.
Results/Finding: Donors were of age 57 6 10 year and primarily men (75%).
Donors with T2D had a higher mean BMI (30.3 6 4.6 vs.25.4 6 3.4 kg/m 2 ) and higher HbA1c than controls.
The sPC of both groups had the same volume (70 6 5 vs 726 2 mL) and PLT content (71 6 9 vs 73 6 11x10 9 ) but on Day 1 glucose concentration was higher in the diabetic group (20.5 6 1.7 vs 18.9 6 1.4 mM, p<0.05).
On Day 8, the average in vitro quality was comparable in both groups (data not shown).
When combining 94A TRANSFUSION 2017 Vol.
57 Supplement S3 the selected 'good' and 'poor' storing PLT from both groups, a large difference in lactate production was observed (0.14 6 0.04 vs 0.36 6 0.03 mmol/ day/10 11 PLT).
The 'poor' PLT showed a faster decline of the mitochondrial membrane potential (as measured with JC-1) during storage than 'good' PLT.
Remarkably, a difference in triglyceride levels was detected on Day 1 ('poor':2.2 6 0.7 vs 'good':1.1 6 0.2, p<0.01).
Conclusion: BC from donors with T2D who did not use insulin and fulfilled all donor criteria, were comparable with BC from age and sex matched controls, and seem suitable for preparation of PC.
When selecting the 'good' and 'poor' storing PLT from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria.
Metabolic Syndrome and T2D are still suspected as health issues involved in 'poor' storage of PLT because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored PCs.
Whole Blood Leukoreduction Failures --Following Manufacturer's Instructions May Not be Enough Karen Klinker*, Nancy M. Dunbar and Zbigniew M. Szczepiorkowski.
Background/Case Studies: Our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation.
The manufacturer recommends minimum wait time of 30 minutes prior to filtration.
Anecdotally, the vendor states waiting an hour improves the leukoreduction.
We experienced leukoreduction failures in January and February of 2017 detected by our routine QC.
We initiated an investigation as to the cause of these unexpected failures.
Study Design/Method: For each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past.
Hemoglobin S determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin S positive.
Results/Finding: A relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure.
We found that shorter wait times increased the percentage of leukoreduction failures (see table 1).
All units that failed had wait times less than one hour.
A similar trend was noticed for the previous year.
The investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure.
Staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues.
Platelet counts on the donors involved were available and none were outside of the normal range.
Various lot numbers of the collection sets were involved, and no donors were repeat failures.
Conclusion: In our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures.
We extended our minimum wait time to 60 minutes based on our data.
We have not experienced any leukoreduction failures after this change.
Absolute Immature Platelet Count in Diagnostic Algorithm and Management of Pediatric Thrombotic Microangiopathy Hamza N Gokozan* 1,2 , Katharine A Downes 1,2 , Hollie M Reeves 1,2 and Robert W Maitta 1,2 .
1 Case Western Reserve University School of Medicine, 2 University Hospitals Cleveland Medical Center Background/Case Studies: Prior studies highlighted the utility of absolute immature platelet count (A-IPC) and A-IPC ratio once therapeutic plasma exchange (TPE) is initiated to differentiate thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies.
This can be helpful to determine those who may benefit from prompt initiation of TPE when tests such as ADAMTS13 are not readily available.
We report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for TTP in which A-IPC measurement was clinically useful.
Study Design/Methods: Previously healthy 12 month old unvaccinated girl presented with history of diarrhea for 5 days which was bloody at onset, accompanied by fever and dehydration.
Laboratory results showed: white blood cell count: 32 x 10 9 /L, platelets: 62 x 10 9 /L, BUN: 77mg/dL, creatinine: 2.4mg/dL, lactate dehydrogenase 1940 U/L.
Hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis.
Peripheral blood smear revealed schistocytes.
On third day of hospitalization, platelet count decreased to 44 x 10 9 /L, ADAMTS13 sample was sent out and TPE was initiated for clinical suspicion of TTP versus hemolytic uremic syndrome, atypical versus Shiga-toxin mediated.
Immature platelet fraction (%-IPF) and calculated A-IPC (%-IPF x platelet count) were obtained with daily pre-TPE CBC.
A-IPC ratio was calculated from baseline.
Results/Findings: Platelet count began to increase prior to TPE initiation (74 x 10 9 /L and A-IPC of 4.7 x 10 9 /L).
Two consecutive TPE were completed which resulted in a platelet count decrease to 54 x 10 9 /L and A-IPC of 5.1 x 10 9 /L.
A-IPC ratio was 1.1 below the ratio of 3 which has been reported for TTP patients.
Similarly A-IPC count was not below 5 x 10 9 /L threshold reported in setting of TTP with severe ADAMTS13 deficiency.
At this time stool culture obtained prior to start of TPE came back positive for E. coli O157:H7 toxin.
Testing of C3, C4, Factor H, Factor H autoantibody, Factor I and Factor B were normal.
ADAMTS13 activity was 93%.
Patient was treated for the infection and platelet count improved within 10 days to 315 x 10 9 /L, with resolution of her renal failure: BUN: 42 mg/dL, creatinine: 0.65 mg/dL.
No additional seizures were observed during follow-up.
Conclusion: Measurement of A-IPC can be used to aid clinical decisions in pediatric patients suspected of TTP especially when ADAMTS13 testing and those for other etiologies are still pending.
TPE did not seem to have a significant effect in A-IPC but decreased platelet counts in this patient.
A-IPC is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time.
Background/Case Studies: Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by low ADAMTS13 activity.
Many patients with severe autoantibody-mediated ADAMTS13 deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years.
It is unclear if disease course and characteristics of recurrent/relapsed TTP may be different from that seen at initial presentation.
Since absolute immature platelet counts (A-IPC) have been shown to be useful in the diagnosis and to follow response to therapy of TTP patients, we proceeded to evaluate if A-IPC pattern was different in relapsed verse initial presentation.
Study Design/Methods: Our study cohort consisted of three patients (two female and one male) with acquired TTP (ADAMTS13 activity <5%) who underwent daily therapeutic plasma exchange (TPE).
Clinical course and laboratory values were reviewed.
Platelet count (PLT), immature platelet fraction (%-IPF) and A-IPC (%-IPF x platelet count) were analyzed during treatment course.
A-IPC values at presentation and peak, A-IPC peak time (days), and PLT count recovery time (days) were compared between initial onset and relapse episode for each patient.
A-IPC percent change in relapse episodes compared to initial presentation was calculated.
Results/Findings: All patients had an increased %-IPF, and decreased A-IPC and PLT count at presentation in both initial and recurrent episodes.
Once TPE treatment was initiated, A-IPC rapidly increased and reached a peak value 2-3 days prior to PLT count recovery, consistent with that previously described in TTP patients.
However, compared to first onset, recurrent episodes featured lower A-IPC at presentation (results shown as percent decrease, column 1), increased peak A-IPC value (results shown as percent increase, column 2), delayed A-IPC peak, and delayed PLT recovery (Table 1) .
Moreover, recurrent episodes required more procedures compared to initial presentation (Table 1) .
Conclusion: Recurrent/relapsed TTP demonstrate lower A-IPC at presentation and a delayed and increased A-IPC peak value in response to TPE compared to initial presentation.
A longer treatment course was observed in recurrent patients.
Future studies of more relapsed TTP patients are needed.
Donors Undergoing Frequent Plateletpheresis and Its Effect on the Hematological Parameters Sweta Nayak*, Poonam Coshic and R.M Pandey.
All India Institute of Medical Sciences Background/Case Studies: Frequent plateletpheresis donors are assets for the blood banks.
The well-being of these donors has been a matter of concern.
In our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure.
We also try to compare the effect 3 cell separators used for plateletpheresis on the post donation hematological parameters.
Study Design/Method: The study was conducted during February 2016 to March 2017 on all the repeat plateletpheresis donors coming to the Department of Transfusion Medicine for the 2 nd time within a month of the first plateletpheresis.
The values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated.
The plateletpheresis were done either on Hemonetics MCS1 separator (Hemonetics Corporation, Braintree, Massachusetts, USA), Fresinius separator (COM.TEC), DN (Fresinius HemoCare GmbH, Bad Homburg v.d.H, Germany) and Gambro Trima Accel, software version 5.0 after taking consent from the donors.
The target collection of each procedure was a dose of 3 x 10 11 platelets in 200-250 ml of plasma.
To compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at 2 consecutive donations within 7 days were considered.
Data was analyzed by Stata 14.
Within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or Wilcoxon Rank Sum test.
The comparison among the cell separators was done by Kruskal-Wallis test or one way ANOVA.
Results/Finding: Of the 98 donors, 35 repeated the plateletpheresis within a week (group I) and 63 underwent 2 nd plateletpheresis within 8-30 days (group II).
No significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the 2 groups (p50.025).
Though above the eligibility cutoff of 1.5 lakhs/ml, platelet counts were lower than baseline in group I donors whereas it was higher at 2 nd plateletpheresis in group II donors.
There were 49 donors who presented to us for the 3 rd time for plateletpheresis with a mean gap between 1 st and 3 rd plateletpheresis being 46 days.
No significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p50.000).
Plateletpheresis through all the 3 cell separators had similar effects on the hematological parameters.
Conclusion: There was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis.
Post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis.
Efficacy of Therapeutic Plasma Exchange on Angiotensin II Type 1 Receptor Antibodies in Two Kidney Transplant Recipients Chisa Yamada*, Silas P. Norman, Milagros Samaniego and Laura Cooling.
Background/Case Studies: Some kidney transplant recipients develop antibody mediated rejection (AMR) without detected HLA donor specific antibodies (DSAs) in sera.
In recent years, angiotensin II type-1 receptor antibody (AT1RAb) has been reported to cause AMR, especially refractory AMR, possibly by contraction of renal arteries.
At our institution, therapeutic plasma exchange (TPE) followed by IVIG every other day has been applied to reduce AT1RAbs in kidney transplant recipients, and we here report efficacy of TPE treatments in two cases.
Study Design/Methods: Two kidney transplant recipients who received TPE treatment followed by IVIG to decreased AT1R Ab are reviewed.
Results/Findings: Case 1: The patient is a currently 43-year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age 22, and a second deceased donor transplant due to a rejection of the transplanted kidney at age 38.
Three years post-transplant, her creatinine (Cr) started to rise from 0.7 to 1.35 mg/dl and a biopsy showed Banff criteria grade 2 AMR, grade 2A T-cell mediated rejection (TCMR) and grade 3 interstitial fibrosis and tubular atrophy.
HLA DSA had been negative in serum, but high level AT1RAb was identified at >40 U/ ml (high: >17 U/ml, intermediate: 12-17 U/ml, negative: <12 U/ml).
She received 6 TPE treatments every other day and started losartan.
After a course of TPE, AT1RAb decreased to 32 U/ml and histology showed improvement of AMR and TCMR, however, Cr kept increasing slowly to 1.9 ml/dl.
In one month, her AT1RAb increased again to >40 U/ml, therefore, she received 3 more TPE treatments with a decrease in her AT1RAb to 16 U/ml.
Although AT1RAb level increased slightly to 20 U/ml after 3 months, her Cr has been stable at 1.3-1.6 ml/dl.
Case 2: The patient is a 25-year-old Mean 1/-SE -54.71/-12.9% * 183.1%1/-12.8%* * p<0.05 96A female with malignant hypertension who received a deceased donor kidney transplant at age 24.
Her Cr started to rise 2 weeks post-transplant from 1.4 to 2.68 mg/dl without detectable HLA DSA.
Although biopsy showed no AMR or TCMR, there was focally severe arteriopathy.
She was found to have high AT1RAb level at 18 U/ml.
She received 6 TPE procedures every other day and AT1RAb decreased to 8 U/ml with a decrease of Cr to 1.98 mg/dl and improved arteriopathy in histology.
Because her AT1RAb level slightly increased to 12 U/ml over the next 2 weeks, she started weekly TPE treatment.
After 5 weekly TPE, TPE treatment was stopped because her AT1RAb level remained relatively unchanged.
Her Cr has been stable at around 1.5 ml/dl to date.
Conclusion: We present 2 kidney transplant recipients who received TPE treatments for high AT1RAb levels.
A course of TPE procedures followed by IVIG every other day was effective to decrease AT1RAb levels; however, weekly TPE had no effect on reducing AT1RAbs.
TPE treatment may be also beneficial to improve histological AMR and clinical kidney function.
Experience in Management of Thyroid Storm By Plasmapheresis Tatiana Belousova*, Vanya Jaitly, Brian Castillo, Hlaing Tint, Kimberly Klein and Yu Bai.
University of Texas Health sciences center at Houston Background/Case Studies: Thyroid storm (TS) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with Graves' disease.
Clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately.
We report two cases where TS with severe cardiac complications was managed by plasmapheresis (PLEX) with excellent effect.
Study Design/Method: A 36 year old man (Patient A) with a medical history of hyperthyroidism present with TS complicated with cardiogenic shock [Ejection Fraction (EF) < 10%], renal and hepatic dysfunction as well as coagulopathy.
Patient was persistent tachycardic while being intubated, sedated and requiring Tandem heart support.
A 33 year old man (Patient B) with a medical history of hypothyroidism (on Synthroid for 2 years), end stage renal disease and non-ischemic cardiomyopathy (EF of 20-25%) presented for evaluation of dual kidney-heart transplant.
He subsequently developed TS with multiorgan failure.
Standard steroid medication treatment showed little response.
Results/Finding: Both patients underwent urgent PLEX along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised.
A 1-1.5 plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed.
Both patients showed significant clinical improvement within 12 hours of the procedure completion.
Their total T4, T3 and free T4 levels trended to normal or near normal range within 24 hours (Table) .
In addition, the PLEX effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated PLEX unnecessary.
Both patients had total thyroidectomy 3-4 weeks after the event with great clinical outcome.
Conclusion: Our cases demonstrate that PLEX is a safe, effective treatment option in managing TS patient with severe cardiac dysfunction.
The procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications.
Extracorporeal Photopheresis in S ezary Syndrome Treatment: Hospital-Based Blood Bank Experience Sandra Ortega S anchez* 1 , Laura Martínez Molina 2 , Cristina Muniesa Montserrat 2 , Octavio Servitje Bedate 2 , Silvia Cosano Navarro 1 and Maria Isabel Gonz alez Medina 1 .
1 Banc de Sang i Teixits, 2 Dermatology service.
Background/Case Studies: Extracorporeal photopheresis (ECP) is an immunomodulatory therapy widely used since 30 years in cutaneous T cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last 20 years, also used in graft versus host disease treatment.
The use of ECP in cutaneous T cell lymphoma (CTCL), mycosis fungoides (MF) and S ezary Syndrome (SS) in their erytrodermic form are recently categorized by the American Society for A pheresis (ASFA) 2016, as first line treatment alone or in combination with other therapies, with a strong recommendation: grade IB, category 1.
Since MF and SS are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression.
The objective of this observational study is to assess outcomes of 10 patients diagnosed with SS and compare them in their first evaluation once the 20 th procedure is been performed.
Study Design/Method: ECP is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( 8-methoxypsoralen, 8-MOP) and UVA light, and subsequent reinfusion of the treated cells which are now induced to apoptosis.
Volume treated varies from 1.5 to 2 total body volume (TBV) and the schedule for SS disease is one cycle (two daily ECP procedures) twice per month.
The venous access was peripheral in all cases except in 2 where central catheter was needed.
The procedures were performed with OPTIA or Amicus devices for the aphaeresis and external UVA irradiation for off-line system (in 7/10 patients) and with online system (Therakos) just in 3.
Main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ECP treatment.
Results/Finding: Global response rate is 77'7% (partial remission 66.6% and complete remission 11.1% with maintained response).
No severe side effects related with the procedure were found.
The patient outcomes analyzed are similar to results in published literature.
Conclusion: Cases treated in our hospital confirm the efficacy of ECP in SS treatment, with a good safety profile.
Another great advantage of ECP is the relative lack of immune suppression.
Many questions remain still unanswered about ECP: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results.
All these questions and more make prospective studies necessary to be performed.
: 3835 U/L) requiring transfusions, mild thrombocytopenia (144 x 10 9 /L), acute kidney injury (BUN 175 mg/dL, creatinine 2.51 mg/dL).
By the third hospitalization day Hgb improved to 10 g/dL, however with worsening thrombocytopenia (16 x 10 9 /L) that led to clinical concern for TTP.
Peripheral smear showed many red cell fragments.
Patient was transfused with platelets day prior to first TPE.
Immature platelet fraction (%-IPF) and A-IPC (%-IPF x platelet count) were obtained with daily pre-TPE CBC.
A-IPC ratio was calculated from baseline.
Results/Finding: Four TPE in five days were performed (hospital days 6-10).
Platelet count and A-IPC improved to 52 x 10 9 /L and 6.6 x 10 9 /L respectively just prior to first TPE.
Response to four TPE led to a decrease in both platelet count (30 x 10 9 /L) and A-IPC 1.98 x 10 9 /L.
These dynamics did not resemble those which had been described for TTP patients with ADAMTS13 deficiency.
ADAMTS13 obtained prior to TPE initiation was resulted at this time and was 67%.
No causative organism or toxin was identified after urine, blood, and stool examination and culture.
Based on these results, TPE was discontinued which led to an immediate increase in A-IPC (2.64 x 10 9 /L) that preceded platelet count increase to 80 x 10 9 /L three days later when patient was discharged.
Other laboratory values at this time were LDH of 635 U/L, Hgb: 11.2 g/dL in the setting of recovery of renal function.
Conclusion: Timely diagnosis of TTP is essential to start of TPE.
A-IPC dynamics differ in TTP compared to other thrombotic microangiopathies.
In our patient A-IPC failed to improve despite TPE and improved once procedures were discontinued and were followed by increases in platelet counts three days later.
When TTP is not the causative etiology, A-IPC can help adjust therapy and lead to clinical improvement.
Further research is needed to characterize immature platelet dynamics in non-TTP microangiopathies.
Infection and Its Role in the Clinical Course of Idiopathic Thrombotic Thrombocytopenic Purpura Associated with Severe ADAMTS13 Deficiency Eiman Hussein* 1 and Jun Teruya 2 .
1 Department of Clinical Pathology, Cairo University, 2 Texas Children's Hospital Background/Case Studies: TTP is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient ADAMTS13.
Since the introduction of therapeutic plasma exchange (TPE) as a treatment modality for TTP, its prognosis has improved dramatically.
Nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes.
Despite the notable progress that has been made with studies that emphasized the pivotal role of ADAMTS13, the epidemiology of TTP remains uncertain.
Previous studies have suggested that many factors appear to influence its pathogenesis.
Some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course.
One of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against ADAMTS13.
The aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic TTP.
Study Design/Method: Patients with idiopathic TTP who underwent TPE from January 2008 through March 2017 were studied.
Sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered.
We only included patients with ADAMTS13 activity of less than 10%.
Data on infections that occurred at or within a week prior to the development of TTP were analyzed.
Results/Finding: Thirty-two patients were categorized as idiopathic TTP with severe ADAMTS13 deficiency.
Eight patients (25%) were associated with suspected bacterial infection.
Four of the 8 patients (50%) showed acute relapse coincident with bacterial infections.
Central line associated Staphylococcus aureus infections occurred in three patients and Acinetobacter urinary tract infection was reported in one patient.
One patient had symptoms of respiratory infection before the development of TTP, on his initial as well as his relapsing episode.
Refractoriness to treatment was demonstrated in 3 patients.
It was associated with dental abscess in one patient.
The other two were associated with Mycoplasma Pneumonia.
TPE sessions were continued in all refractory patients until their death.
Conclusion: In patients with idiopathic TTP refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes.
Sandra Satoe Kayano*, Marcos Paulo Colella, Rafaela Guerra Maciel, Ingrid Priscila Ribeiro Paes Ferraz and Rafael Colella.
A C Camargo Cancer Center Background/Case Studies: Therapeutic Leukapheresis (TL) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion.
Leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis.
The objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than 10 kilograms using a single apheresis procedure.
Study Design/Method: In October 2015 and June 2016, two children with possible leukemia were submitted to TL procedure.
They were 6 and 9 months old, and weighted 7,0 and 9,1 kilograms.
central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system.
The device was primed with 285 ml of ABO, Rh and Kell compatible, leukocyte-reduced, irradiated, 64% hematocrit packed RBCs, and the anticoagulant used was ACD-A plus heparin (750 mL of ACD-A and 7,500 units of heparin), at a blood to anticoagulant ratio of 25:1.
A complete blood count was determined before and after apheresis.
The room was heated to avoid hypothermia, and ionized calcium was measured every 30 minutes to prevent hypocalcemia.
During the collection, changes in blood pressure, oxygen saturation and heart rate were observed.
Net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume.
When the procedure was completed, the blood that filled the apheresis tubing was discarded.
The patients were in the intensive care unit (ICU) under the supervision of a pediatric physician and ICU nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner.
Results/Finding: The white blood cell (WBC) in blood was counted immediately before apheresis in both subjects, and were 120.000 and 150.000/ mm 3 .
The formula "Collection Pump Flow 5 0,0003 x Inlet Flow x Preapheresis WBC count" was used with the goal of removing up to 3 x 10 9 leukocytes/mL.
A single leukapheresis procedure was performed with 2 total blood volume processed per patient.
Immediately after the 2-hour procedures, WBC count were 74.000 and 92.000 WBC/mm3, and 12-hour post TL, WBC count were respectively 45.000 and 70.000/mm3.
Net fluid balance was zero in both procedures, and the patients required no transfusion.
Conclusion: TL was safe and efficient.
Experience with leukodepletion in infants is limited, and a procedure in children weighing 10 kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection.
However, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases.
Background/Case Studies: Nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures.
Data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice.
The Korean Society for Apheresis (KSFA) has launched an online web based registry system for apheresis procedures since 2006.
We report the data from the year 2016.
Study Design/Method: The registry is consisted of two sub-registries.
One addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures.
Data is registered by voluntarily participating hospitals in Korea.
Results/Finding: A total of 13,302 apheresis procedures were performed at 37 hospitals.
Therapeutic plasmapheresis was the most frequent procedure (50.4%) followed by autologous peripheral blood stem cell (PBSC) collection (23.9%), allogeneic PBSC collection (11.0%), donor leukapheresis (4.0%), and therapeutic leukapheresis (3.9%).
COBE Spectra (37.4%) and Amicus (16.8%) were the most widely distributed instruments.
Centrifugation was the dominant technique (92.2%) for therapeutic plasmapheresis.
Detailed information was given for 4,199 therapeutic plasmapheresis procedures performed on 786 patients (some items were not completely filled out).
Spectra Optia (42.7%) and COBE Spectra (26.6%) were the most frequently used instruments for therapeutic plasmapheresis.
Fresh frozen plasma (FFP) was used most frequently (47.2%) as the replacement fluid followed by 5% albumin (26.3%), 4% albumin (13.3%), and 5% albumin 1 FFP (11.1%).
Most of the procedures were performed for 1 plasma volume (72.4%).
ACD (88.4%) and heparin (11.5%) were used for anticoagulation.
Central venous catheter (91.9%) was the dominant type of vascular access.
Major clinical indications were desensitization for ABO incompatible renal transplantation (24.1%), antibody mediated rejection in renal transplantation (19.9%), thrombotic microangiopathy (11.5%), desensitization for ABO compatible renal transplantation (4.7%), neuromyelitis optica spectrum disorders (4.6%), and hyperviscosity in monoclonal gammopathies (4.6%).
Adverse reactions were observed in 8.5% of the procedures.
Allergic reaction (55.2%), hypocalcemic symptom (20.4%), and hypotension (6.9%) were frequently reported.
Therapeutic effect was achieved in 86.5% of the patients.
Our apheresis registry has been well run for 10 years.
Recent data reflects the increase of ABO incompatible transplantation in Korea.
Revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region.
Plasma Exchange May Not Always be Necessary in Patients with Severe Hypertriglyceridemia and Acute Pancreatitis.
Jan C Hofmann* and Dobri D Kiprov.
California Pacific Medical Center Background/Case Studies: Hypertriglyceridemic pancreatitis (HP) is characterized by severe hypertriglyceridemia (sHTG: triglyceride >1000-2000 mg/dl), acute pancreatitis (AP), and absence of other causes.
HP is a potentially fatal complication of acute pancreatitis with an incidence of $18 deaths/100,000 cases/year.
Complications of sHTG include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy.
We report on the effective use of plasma exchange (PE) to treat patients (pts) with HP refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions).
Study Design/Method: We reviewed the medical records of 41 pts who were diagnosed with HP from January, 2009 through January, 2017, and referred for immunotherapy evaluation.
27/41 (66%) pts received conventional therapy (CT) and PE (PE group), and 14/41 (34%) pts received CT alone (CT group).
Mean age was 36 years (range 16-79), and 56% were female.
Baseline mean triglyceride level (normal <150 mg/dl) for PE group was 6,728 mg/dl (4,652-12,486) versus 3,142 mg/dl (1,697-5,120) for CT group.
Baseline mean lipase level (normal <393 U/L) for PE group was 1,798 U/L (797-2,745) versus 923 U/L (472-1,796) for CT group.
Results/Finding: All pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving 2-3 medications.
24/27 (89%) of PE group and 11/14 (79%) of CT group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis.
20/27 (75%) of PE group and 6/14 (43%) of CT group received heparin therapy to stimulate lipoprotein lipase release.
The PE group underwent an average of 2.85 PE treatments (txs) (median of 2, range 1-4 daily txs) using 5% albumin; 7/27 (26%) required FFP to treat dilutional coagulopathy.
In most cases, we did not perform PE txs when baseline triglyceride levels were <3000-4000 mg/dl and lipase <950-1375 U/L (2.5-3.5 X upper limit of normal).
Mean triglyceride levels after 2 PE txs were 1,976 mg/dl (627-3,968) for PE group (mean decrease 72%); mean triglyceride levels after 48 additional hours of ongoing CT were 1,576 mg/dl (487-2,873) for CT group (mean decrease 50%).
While the PE group achieved a greater mean decrease in triglyceride levels after 2 PE txs (compared to the CT group after 48 hours of CT), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p>0.05).
Limitations of the retrospective cohort study include lack of long-term follow-up.
Conclusion: This small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia.
It suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms.
Randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis.
Role of Plasma Replacement in Therapeutic Plasma Exchange for Hypertriglyceridemia: A Single Patient Study Geoffrey Wool* and Angela Treml.
University of Chicago Background/Case Studies: Our apheresis service performs chronic therapeutic plasma exchanges (TPE) for a 47-year-old man with a chronic history of hypertriglyceridemia >1000 mg/dL, diabetes mellitus type II, and chronic abdominal pain.
His abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing.
Targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia.
Hypertriglyceridemic pancreatitis is a category III indication for TPE by ASFA 2016 guidelines, in a patient unresponsive to optimal medical management.
ASFA 2016 guidelines for this disorder state that "Some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (TG) removal.
No direct comparisons of replacement fluids have been reported".
There are three apheresis physicians on our service and use of partial plasma replacement has been variable.
We undertook a retrospective study of the efficacy of partial plasma replacement in this patient.
Study Design/Method: We have performed 39 TPE on this patient.
We performed a chart review to capture replacement fluid use and pre-and post-TG levels, if drawn.
TPE was performed using Spectra Optia (Terumo, Lakewood, CO) exchanging approximately one plasma volume, using entirely 5% albumin for exchange fluid (100% albumin procedures) or partial plasma replacement (2-3 units of thawed plasma).
Twenty-six TPE had pre-and post-procedure TG values available.
We determined the percent TG reduction achieved by the TPE.
We also determined the daily rate of TG increase until the next TPE appointment (to assess any long-term effects of plasma preventing TG rebound).
Significance was assessed by Student's t-test (one-tailed, heteroscedastic).
Results/Finding: Twelve TPE were performed with partial plasma replacement, while 27 were performed with 100% albumin replacement.
Table shows that partial plasma replacement was associated with significantly greater % TG reduction.
The rate of subsequent daily TG increase was also lower with partial plasma replacement, but this did not meet significance.
One mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional IV diphenhydramine.
Conclusion: We have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in TPE for hypertriglyceridemia.
In this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % TG reduction, but not with prevention of post-TPE TG rebound.
Safety and Efficacy of Local Albumin Replacement for Therapeutic Plasma Exchange Phandee Watanaboonyongcharoen* 1,2 , Metha Apiwattanakul 3 , Sompis Santipong 2 , Jutaluk Jaipian 2 , Jettawan Siriaksorn 2 and Ponlapat Rojnuckarin 1 .
1 Chulalongkorn University, 2 King Chulalongkorn Memorial Hospital, 3 Prasat Neurological Institute Background/Case Studies: Therapeutic plasma exchange (TPE) with albumin replacement has been used to treat a variety of diseases.
However, there had been rising cost and supply shortage of imported albumin in our country.
To solve the problem, our National Blood Centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin.
The objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for TPE.
Study Design/Method: All TPEs using local albumin as a replacement from two tertiary care hospitals performed from June 2016 through February 2017 were included.
Complete blood count and serum calcium were tested before TPE.
Serum albumin was tested before and after TPE.
Local albumin is available as a 20% solution.
Before using, it was diluted to a 4% albumin concentration with normal saline.
All the patients were hospitalized and received oral calcium before TPE to prevent hypocalcemia.
The adverse effects were recorded.
Results/Finding: The total of 156 TPEs in 38 patients were included as shown in the Table.
Neurologic disorders were the most common indication for TPE, followed by autoimmune diseases.
The median total plasma volume was 3,000 (range 1,750-4,200) mL.
Although the corrected calcium level was low (<8 mg/dL) in 3.2% (5/156) before the procedure, no clinical manifestation of hypocalcemia was detected.
Adverse effects were observed during the TPE procedure in 2 patients.
The first patient had 2 events of mild symptomatic hypotension.
He previously took angiotensin converting enzyme inhibitor.
The second patient complained nausea after finishing TPE.
All reactions were mild.
The incidence of adverse effects was 1.9% (3/ 156).
In 2014, the incidence of TPE adverse effects was 1.6% (2/125) when commercial albumin was used.
The difference was not statistically different (P 5 1.000).
Median serum albumin levels pre-TPE and post-TPE were 3.6 (1.9-4.4) and 3.9 (2.4-5.0) g/dL.
The increase in serum albumin after TPE was statistically significant (p<0.001).
Eighty-two percent of pre-TPE serum albumin levels were lower than 4.0 g/dL explaining the rises of albumin after the procedures.
We demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing TPEs.
Safety, Efficancy and Cost-Effectiveness of Mononuclear Cell Collections for Autologous Immunotherapies: Experience from a Private Outpatient Collection Facility within the EU Markus Dettke*.
AKH Vienna University Hospital, Cyto-Care.eu Background/Case Studies: Within the EU the collection of mononuclear cells (MNC) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers.
We report about the challenges to perform the leukapheresis procedure (LA) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness.
Study Design/Method: We reviewed the records of altogether 60 outpatients who underwent a total of 100 LA procedure at Cyto-Care, a private held medical practice/ certified cell collection facility located in Vienna, Austria.
All patients participated in various industry-sponsored clinical P I-III trials; the study sponsors were responsible for the manufacturing of the active cell product.
Disease entities were mainly prostatic cancer (75%) and ovarian cancer (20%).
Based on differences in the study protocols LA was performed either one-time (41%), two-times (27%) or three-times (32%), with an interval of at least 2 weeks between repeated collections.
Results/Finding: All patients successfully completed the apheresis course.
Because of poor venous access, 3 out of 60 patients (5%) required a shortterm femoral catheter insertion.
There were no serious side effects in patients who required a femoral catheter, or in patients with repeated LA procedures.
Side effects of the LA procedure mainly consisted on mild hypocalcaemia-related symptoms in 16% of patients.
A follow-up survey one week after completion of the LA revealed no infectious complications, and no patient required hospitalization.
Median cell yield collected per single apheresis was 1.4x10 10 WBC consisting of 1.1x10 10 MNC.
MNC cell yields remained stable even in repeated LA collections.
All cell products were successful transformed into an active cellular product.
Analysis of the cost structure showed that the total cost of care was 32% lower in the setting of a private collection center compared to hospital-based apheresis centers.
Conclusion: Leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction.
This service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products.
Although typically associated with monoclonal gammopathies (e.g.
Waldenstrom's macroglobulinemia and multiple myeloma), HVS has rarely been reported in patients with disorders of immune system such as rheumatoid disease, Sjogren's syndrome, HIV and IgG4-related diseases.
Therapeutic plasma exchange (TPE) is indicated in HVS due to monoclonal gammopathy (ASFA category 1 indication).
However, there are limited data for the utility of TPE in HVS due to polyclonal gammopathy.
Study Design/Methods: A 70 year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months.
Fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns).
Pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-Ro antibodies.
Serum rheumatoid factor was markedly elevated to 57,000 IU/mls (ref.
range < 35) and anti-cyclic citrulline peptide antibody was elevated to 34,339 units (ref.
range < 20) .
Serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes.
Serum IgG, IgM and IgA were 4610, 2890 and 1320 mg/dL respectively.
A cryoglobulin screen was negative.
Serum free kappa to lambda ratio was 1.74.
Peripheral blood flow cytometry did not identify any monoclonal Bcell population.
Plasma viscosity was noted to be 8.5 centipoise (cp) at admission (ref.
range 1.6 -1.9).
PET-CT imaging was negative.
The patient was treated with high dose steroids; a single TPE procedure was performed using the following parameters: volume treated -1 total plasma volume; replacement fluid -5% albumin and normal saline in a 50:50 ratio; replacement fluid volume: 110% of the total volume processed.
The procedure was tolerated without complication.
Results/Findings: Immediately post-TPE her plasma viscosity level dropped to 2.4 cp.
Serum IgG, IgM and IgA levels decreased to 2040, 1510 and 672 mg/dL respectively.
Her RF had decreased to 19,900 IU/ml.
The patient reported subjective improvement in strength.
She subsequently received two infusions of rituximab separated by two weeks.
Her plasma viscosity has remained less than 3 cp since TPE.
Conclusion: Polycolonal gammomathy (e.g.
secondary to RA) is a rare cause of HVS.
TPE can provide transient relief of symptoms in unusual cases of HVS and may facilitate therapy to prevent recurrent HVS episodes.
Therapeutic Plasma Exchange in Neuromyelitis Optica Spectrum Disorders -Experience from Tertiary Care Centre in North India Ratti Ram Sharma*, Rekha Hans, Satya Prakash, Naveen Sankhyan and Neelam Marwaha.
Postgraduate Institute of Medical education and research Background/Case Studies: Neuromyelitis optica spectrum disorder (NMOSD) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis.
TPE is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase.
Study Design/Method: To assess the efficacy of plasma exchange in patients of NMOSD not responding to high dose intravenous steroids.
we did a retrospective review of TPE records for patients with NMOSD over a period of three years (Jan 2013 -Dec 2016).
TPE was done using, Cobe spectra (Terumo BCT, Lakewood Co. USA), replacing one to one and half patient plasma volume with 5% human serum albumin or Fresh frozen plasma on alternate days.
The improvement in clinical signs and symptoms was recorded after each TPE procedure and at the end of the therapy.
Adverse reactions if any were also recorded Results/Finding: Eleven patients of NMOSD between 4 to 35 years age (M: F; 1:2) underwent 62 TPE procedures with an average of 5.6 per patient.
All the patients were on high dose immunosuppressant therapy without much clinical improvement.
Three (27%) patients had only visual symptoms, 5 (46%) had both visual as well as muscular symptoms whereas 3 (27%) patients had muscular symptoms only.
Three (27%) out of the seven tested, were positive for AQP4-IgG.
All the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients.
Post exchange recovery in the muscle power was observed in 8 patients with grade-1, in 1 patient, and by grade-2, in seven.
Adverse events were observed in 8% (5/62) of the procedures with allergic reactions to replacement fluid as most common event (n-3) followed by hypotension (n-2).
Follow up was available in 55% (6/11) of patients and are doing well on immunosuppressive therapy.
One patient died due to respiratory failure after 3 months and another had relapse for which he underwent second TPE cycle and continue to do well.
Conclusion: TPE is a safe and effective adjunct therapy to high dose immunosuppression in NMOSD.
Trima Accel Software Upgrade from 6.0 to 6.4 for Platelet Collections Rachel M Beck*, Kimberly J Duffy, Sandra Bryant, Audrey E Traun, Mary M Benike, James R Stubbs and Justin D Kreuter.
Mayo Clinic Background/Case Studies: TerumoBCT released Trima Accel software version 6.4 as an enhancement to allow for the collection of platelets (plt) with Platelet Additive Solution (PAS) and provide additional improvements to increase overall reliability.
Additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates.
This software was expected to function similarly to version 6.0.
The objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected.
Study Design/Methods: Prior to 1/16/2016, plt collections were performed on nine Trima Accel machines operating with version 6.0.
Upgrading and validating all nine machines to version 6.4 occurred from 1/16/2016 to 4/30/ 2016.
The Trimas were programmed with the same plt configurations both before and after software update.
Platelet collection data from version 6.0 (5/1/2015 to 9/30/2015) was compared to version 6.4 (5/1/2016 to 9/30/ 2016).
Incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period.
Generalized estimating equations (GEE) were used to assess differences between plt collections with version 6.0 and 6.4, adjusting for multiple visits per donor, with significance defined as p-value < 0.05.
Results/Findings: Following the upgrade to version 6.4, staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products.
Version 6.4 of the Trima Accel showed a statistically significant increase in possible leukocyte contamination from 3% to 5% of collections as compared with version 6.0.
Both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version 6.4.
Conclusion: Due to FDA limitations not allowing for the implementation of Trima Accel PAS plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time.
Subsequently, the version 6.4 software is no longer required.
With the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version 6.4 currently does not provide added value over version 6.0 for plt collection.
pulmonary and neurologic symptoms due to leukostasis.
Therapeutic Leukocytaphersis (TL) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis.
TL procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density.
There are two programs in the cell separator, a mononuclear (MNC)program which has greater centrifuge speed and efficiency for the collection of MNCs and a polymorphonuclear (PMN)cell program with lower centrifuge speed for the collection of PMNs.
Hydroxyethyl Starch(HES) is preferred for the collections of granulocytes for transfusion from healthy donors.
Use of HES facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection.
Though use of HES in TL was not associated with adverse events with its use as a volume expander (Pagano) its use in TL varies and no reports are available on the efficiency of leukodepletion using HES for TL.
Study Design/Method: We received a request for leukoreduction in 32 yearold lady with chronic myelogenous leukemia (CML) who had a good response to Imatinib.
She is 30 weeks pregnant with an increased WBC count due to the discontinuation of Imatinib.
We performed TL with the COBE Spectra using a replacement fluid of 500 ml 5% albumin.
WBC counts were monitored pre and post TL in the patient and in the collected product.
We modified the collection based on these results using the MNC program with ACD-A or the PMN program with ACD-A .
As leukodepetion was not adequate with these programs we elected to use HES after discussion with the patient and her physician.
TL was performed using 500 ml of HES with citrate and the PMN program.
WBC pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated.
Results/Finding: The efficiency of % WBC depletion was calculated by product WBC to patient WBC based on blood volume and also Pre to Post WBC The patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures Conclusion: Therapeutic leukocytapheresis in CML patients is safe and more effective in reducing the WBC count with the use of 500 ml of hydroxyethyl starch with anticoagulant.
Post procedure patient WBC counts sometimes may not provide the data on the efficiency of leucodepletion.
Background/Case Studies: Early recognition of hypertriglyceridemia (HTG) in the setting of acute pancreatitis (AP) is critical to initiate effective therapy.
The role of plasmapheresis as an early/adjuvant approach in acute HTG-induced pancreatitis is controversial.
Currently, there are no consensus guidelines in optimal therapy and is ASFA category III.
Reported here is a case where the TG level as well as clinical symptoms improved after one therapeutic plasma exchange (TPE).
Study Design/Method: A 45 years old male with history of hypertension, HTG, and Diabetes Mellitus (DM) presented to our emergency department with excruciating abdominal pain.
The patient was diagnosed with HTG at 20 years old.
He was treated initially with diet and lifestyle modification.
However, his clinical course has been compromised after developing pancreatitis with 3 acute episodes requiring prolong hospital admission of approximately 2 months each which were successfully treated medically.
However, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency.
Since the first episode of pancreatitis, he was then medically managed with fenofibrate, Lovaza, Lisinopril, Levemir and NovoLog.
During evaluation on current admission, he was found to have a TG level of 4980 mg/dl, lipase 92 U/L, glucose 250 mg/dl, Bicarbonate 24 mmol/L, anion gap 12.
CT findings were consistent with AP without evidence of necrosis and stable pancreatic pseudocyst.
Medical therapy was started with Omega 3 fatty acid, fibrate, statin, hydration as well as pain control.
Statin therapy was suspended on day 3 of hospitalization, because he was noted to have elevated liver function tests (LFT) and TPE was requested and started on day 3 after admission.
Results/Finding: The patient TG decreased by 52% (2365 mg/dl) with medical therapy, followed by additional 67% (767 mg/dl) after one volume of TPE.
His symptoms significantly improved and was discharged with medical treatment on day 6 after admission.
Compared to previous episodes, his hospital stay was significantly decreased.
TG levels remained below 1000 mg/dl at 20 days follow up after discharge.
Conclusion: Early TPE may be of value in treating patients with elevated TG associated with recurrent pancreatitis.
Plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety.
Background/Case Studies: From 2009 to 2013, a national blood donor center in Southeast Asia conducted a program to monitor the ferritin levels of platelet blood donors.
The aim of this study was to explore the trend of changes in ferritin.
Study Design/Method: In this study, we collected 5,129 cases whose ferritin levels have been monitored more than twice with an interval of detection in 150-160 days.
The collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay.
Inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over 50 lg/L.
And the upper limit was set to be 244 lg/ L in male and 158 lg/L in female as described in manufactures insert.
The impact on ferritin from gender, age, and the blood donation frequency were examined with ANOVA test.
The blood donations frequency was categorized into five groups: 0 times, 1 to 3 times, 4 to 6 times, 7 to 9 times and more than 10 times.
The high frequency (more than 10 times group) blood donors were analyzed ferritin changes in longitudinal data.
Results/Finding: There were 5,129 donors included in the study, of which 4,944 were male (96.4%) and 185 were female (3.6%).
The mean ferritin was 82.0 lg/L in male (95% CI: 80.7-83.2 lg/L) and 66.5 lg/L in female (95% CI: 60.9-72.0 lg/L).
The result of ANOVA indicates that the group with the highest frequency (more than 10 times) has the significant lowest ferritin level (p<0.05).
The average change of ferritin if donation over 10 times would up to 13.4 and 14.1 lg/L in younger and elder 50 y/o male and 18 and 23 lg/L in female.
And then for high frequency (half a year more than 10 times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin.
The average change about ferritin in high frequencies donors (over 10 times in 150$160 days) was reduced from 21.5 lg/L in the first period to 4.1 lg/L in the third period (1 period5150$160 days).
Along with the more and more period, the decline of ferritin decreased.
Conclusion: This analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors.
But the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down.
A Rare Case of Blood Donation Precipitating Acute Delirium Joseph Griggs* 1 , Mary Townsend 2 and Lizabeth Rosenbaum 3 .
1 University Of New Mexico Hospital, 2 Blood Systems, Inc., 3 Blood Systems Inc. Background/Case Studies: We report a case of whole blood (WB) donation that precipitated a transient agitated delirium.
A 22 year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation.
Approximately 10 minutes after an uncomplicated WB donation, the donor had an observed, brief loss of consciousness in the post-donation area.
No fall or injury was seen.
Shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding.
The donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained.
He ultimately was sedated and intubated, and transferred to the intensive care unit.
Study Design/Method: An extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and PCR-based studies for multiple organisms including West Nile, herpes, HIV, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse.
Radiographic imaging was performed including a chest x-ray, and a CT and MRI of head and spine.
In addition, an EEG was performed.
The inpatient neurology and psychiatry services were consulted for this patient.
Results/Finding: After the sedation was discontinued, the patient was successfully extubated and rapidly improved.
He completely returned to baseline within 24 hours of onset of the event.
Laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse.
Radiographic imaging and EEG studies showed no abnormalities.
In addition, infectious disease marker testing performed by the blood center laboratory was negative.
Investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers.
A week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes.
Conclusion: To our knowledge, this is the first report of blood donation precipitating a transient acute delirium.
At the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient.
The health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor.
However, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed.
Although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood.
A Targeted Approach to Increasing the African American Blood Donor Pool Arnethea Sutton* 1 , William Korzun 1 , Teresa Nadder 1 , Susan Roseff 2 and Elizabeth Ripley 1 .
1 Virginia Commonwealth University, 2 Virginia Commonwealth University Medical Center Background/Case Studies: A continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from African American donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations.
One population in particular, African Americans, only account for 1% of blood donors in the United States.
Literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community.
Study Design/Method: African Americans in Richmond and Norfolk, Virginia were recruited through churches and local universities.
The study's aims were to develop, implement, and assess a targeted educational approach incorporating the Theory of Planned Behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate African Americans non-donors to attempt to donate blood.
Participants attended a 1-hour educational session where they were educated on the importance of red blood cell donations from African Americans.
Participants completed three surveys -one before the session, one directly after the session and one, two months after the session.
A two-proportion z-test was used to compare the known proportion of African Americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables.
Results/Finding: A total of 142 subjects were included in the data analysis.
Sixteen percent of the study participants presented to donate as a result of attending the educational session.
This resulted in a statistically significantly higher proportion of African Americans presenting to donate than the current proportion in the areas of the state where this study was conducted.
Results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor.
The educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session.
Conclusion: This study shows that a targeted educational program can change attitudes toward blood donations in African Americans resulting in an increase in new blood donors.
Additional studies are needed to see if this behavior will continue and whether African Americans can influence their community to increase awareness and motivation for life-long blood donation.
were from female basic trainees Conclusion: The significant increase in hemoglobin deferrals at Basic Training Site A from 2015 to 2016 could be a result of a change in the blood drive timing of the training schedule of that location.
In 2015, Basic trainees at site A were scheduled at day 57 of 70.
In January 2016, the blood drive date changed to day 60 of 70.
The extra three days in the Basic Training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population.
At Basic Training Site B, the significant increase from 2015 to 2016 of hemoglobin deferrals can be attributed to a larger male population presenting at this site for Basic Training.
Additionally, the percentage of female recruits donating at the blood drives decreased in 2016.
These observations support the hypothesis that the increase in hemoglobin deferrals in 2016 resulted from the implementation of the male hemoglobin standard change from 12.5 to 13.0 g/dl at Basic Training Site B.
When planning for blood drives at basic training site B, screening of an additional 24% of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard.
Blood Donation in the Donor with Spinal Cord Injury Joan-Ramon Grífols* 1 , Eva Alonso 1 , Oscar Bascuñana 1 , Monica Romero 1 , Teresa Vich 1 , Elena Castaño 1 , Laura Carbonell 1 , Eva Palomas 1 , Saray Almerge 1 , Francesc Carpio 1 and Xavier Curia 2 .
1 Banc de Sang i Teixits, 2 Institut Guttmann Background/Case Studies: Donation of blood components (BC) in donors with spinal cord injuries (SCI) is poorly studied.
Paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of BC donation.
The literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their SCI it's obvious.
In daily practice these potential donors are often rejected for donation with no specific criteria related to their SCI.
The objectives of this study are to establish the selection criteria for BC donation in people with SCI based on medical criteria.
To evaluate the rate of adverse donation blood reactions of these donors against a donor control group without SCI.
Study Design/Method: Our organization regularly organizes a donation campaign at a rehabilitation center for patients with SCI.
In this campaign some donors with SCI as donors without (professionals of the center, relatives, etc.)
donate blood.
From January 2015 to December 2016 we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had SCI and number and typology of adverse reactions to the donation detected in both groups.
Donors with SCI higher than T5 due to the high risk of autonomic dysreflexia were excluded for donation.
Donors with SCI below T5 and less than one year of evolution were set as temporary exclusion criteria.
The presence of neurogenic bladder was not considered a reason for exclusion.
Results/Finding: In the analyzed period, 219 donors came to give blood, of these, 15 (7%) were excluded for donation for various reasons.
Two of the donors excluded suffered SCI higher than T5 excluding them due their high risk of dysreflexia.
Another one donor excluded suffered SCI lower than T5 but his hemoglobin levels were lower than our selection criteria.
Of the 204 donors selected for donation 16 (7.8%) had SCI lower than T5 and T6.
Adverse reactions to donation (1.4%) were recorded in our haemovigilance program, none of them in donors with SCI.
Conclusion: According to our experience donors with SCI lower than T5 have not had any type of adverse reaction to the blood donation.
There should be selection / exclusion criteria based on the donor's paralytic conditions.
The vagal syndrome that could appear as a complication to the donation in these SCI donors should be approached differently to the usual protocols that we use.
Blood Donor Center's Experience with Changing from Manual to Automated Blood Pressures Kimberly J Duffy*, Sandra Bryant, Audrey E Traun, Kristine I Borth, Mary M Benike, James R Stubbs and Justin D Kreuter.
Mayo Clinic Background/Case Studies: Blood pressure (BP) is important for determining the health and suitability of blood donors.
The manual method of reading BP can result in variability due to minor variances in the way staff perform the manual procedure.
Automated BP devices are able to reduce the variability in BP determination.
In December of 2013, automated BP devices were validated and replaced the manual BP method in our blood donor center.
The objective of this retrospective study is to determine if the change from a manual to an automated BP process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed.
Study Design/Methods: Data for the manual BP process was accumulated for an 11 month period from January 2013 to November 2013.
The same information was assembled for the automated BP process for the 11 month period of January 2014 to November 2014.
The automated BP process implemented in mid-December 2013; so the December data for both 2013 and 2014 has been excluded from the study.
BP, BP deferrals, reactions, donor weights and demographics were evaluated for each time period.
A donor may be included multiple times in each year and could be in both sets of data.
Generalized estimating equations were used to assess differences between automated and manual BP with significance defined as p < 0.05.
Results/Findings: Significantly more people were deferred using automated BP compared to manual BP readings (p50.006).
Both systolic and diastolic BP measured significantly higher by automated BP method than by manual method.
Although donors in the automated BP group experienced fewer reactions than those in the manual BP group, the reduction was not large enough to reach statistical significance.
Even after adjusting for gender, weight and age at donation, BP deferrals, systolic and diastolic BPs all remained significantly higher (all p < 0.03) with the automated BP while and reactions remained non-significantly lower (p 5 0.086).
Conclusion: Automated BP devices have improved convenience for both staff and donors.
With a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated BP devices may play a minor role in the safety of blood donors.
For the purpose of this study, only the hemoglobin values that were below 12.5 g/dl will be compared as a surrogate for deferral.
To adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet A and lancet B, using the appropriate distribution for the data type, defining statistical significance as p-value < 0.05.
Results/Findings: The average hgb was slightly lower with lancet B but there was a larger change with the number of donors under 12.5.
Statistically more visits with hgb less than 12.5 g/dl used lancet B than lancet A. Additionally, fewer first time donors were seen during the lancet B time than during the lancet A time.
After adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under 12.5 was 16.5% higher with lancet B than with lancet A.
Conclusion: Donor's hgb was slightly lower with lancet B than lancet A, but not clinically different.
Slightly more lancet Bs were used per visit than lancet As.
In addition, more hgb deferrals were obtained using lancet B than lancet A.
Even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet B than lancet A.
The slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels.
Prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended.
Blood Donors' Acceptance and Response Towards Implementation of Automatic Appointment Booking Yi Lin Ang*, Ching Lian Toh and William Choon Hong Sim.
Health Science Authority Background/Case Studies: With surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management.
Disliking the obligation imposed by appointments, Singaporean donors generally prefer "walk-ins" as opposed to appointment bookings.
Blood Services Group (BSG) Singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling.
This paper aims to study donors' level of acceptance towards this initiative.
Study Design/Method: To determine the donors' acceptance rate, data was collected from 1 January to 31 March 2017.
After completing their donation, donors were automatically given the next earliest eligible date for their next donation.
Those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (Donor-Care) to make changes to the appointment offered.
A reminder will be sent to their phone via SMS and/or email to their account three days before the appointment date.
Data was collected from Donor-Care and was used to measure the number of appointments made and declined over the three months period.
Donors who declined appointment scheduling were verbally interviewed for their reasons.
Results/Finding: A total of 6680 donors who has donated blood in the blood bank's main branch were used as the baseline for this study.
85% of donors (N55678) accepted automatic appointment booking, whereas some donors (N51002) were not comfortable with it.
77% of those who declined still preferred walk-ins (N5771) based on their own time schedule, the rest decided that variable situations (N5112), donation frequency (N569) and choice of preferred donation locations (N550) were reasons for declining automatic appointment booking.
Prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through Donor-Care was 19%.
A comparison was made and found that this study shown a significant increase of acceptance rate by 66%.
Conclusion: Generally, the results were positive and the automatic appointment booking system enabled BSG to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention.
BSG is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate.
Currently BSG has 4 collection centers, each managing its own appointment system.
The eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site.
4) , 3.28 0.4940 4 1 poisson distribution, 2 normal distribution, 3 logistic distribution, 4 lognormal distribution 105A TRANSFUSION (p>0.05) in donor and reference populations except in younger (20-44 yrs) male donors (p<0.0021; donor 4.9%, reference 10.0%).
Mean donor SBP, DBP, and pulse were 125 6 14.7 mmHg, 75.1 6 9.6 mmHg, and 75.9 6 11.2 bpm, respectively.
Screening blood pressure levels consistent with hypertension (29.4% male; 16.6% female) in the 20-44 year donor group, significantly (p<0.0001) higher than the reference population (11.2% male; 8.7% female).
No differences were observed in the 45-64 year groups.
Conclusion: Normal Source donor demographic and physiologic characteristics often paralleled those of the reference USA populations.
However there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in 20-49 year old females.
Developing Blood Donor Educational Materials Gay Wehrli* 1 , Susan Rossmann 2 , Louis M. Katz 3 and Dan A Waxman 4 .
1 University of Virginia Health System, 2 Gulf Coast Regional Blood Center -Sugar Land, 3 Americas Blood Centers, 4 Indiana Blood Center Background/Case Studies: Donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating.
Donor education (DE) materials must address mandates set forth by regulatory agencies.
These materials must be accessible and understandable by the general population.
The goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized DE materials.
This study was IRB approved as an exempt protocol.
Study Design/Method: We developed a DE document written at an 8 th grade comprehension level.
A convenience sample of volunteers was identified for this two-part study.
A focus group (FG) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page DE document.
The quiz was followed by a group discussion for feedback.
The preand post-quiz contained the same 10 multiple choice questions with single best answers including the option to answer, "I don't know."
The DE document was revised based upon the FG feedback and quiz results.
The revised, 3.5 page, DE document was then tested using the same pre-and post-quiz during individual interviews (II).
Results/Finding: Demographics and quiz results are summarized in table 1.
Results from the FG and II revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain HIV testing.
Post-quizzes from the II group revealed an improvement in knowledge acquisition for all four areas.
Feedback from both groups reiterated that the document was too long.
Conclusion: Developing DE materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level (8 th grade).
Testing DE materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population.
The next steps for this group will be to pilot the further revised, two-page DE document at donation sites.
Effect Analysis of the 'Rh(-) Blood Supply Program' Establishment HyeSung Han*, DeokJa Oh, BuJa Hur and ChulYong Kim.
Korean Red Cross Blood Services Background/Case Studies: The Rh(-) blood supply program was developed in 2011 for the purpose of prompt and stable blood supply.
Based on the computerized system, the program operates the emergency contact/ communication.
This program has 4 major functions such as the request of the emergency blood, the recruitment and management of the Rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program.
The aim of the research is to validate the effect of Rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the Rh(-) blood supply program.
Study Design/Method: Researchers investigated the database from 2011 to 2016 after the Rh(-) blood supply program was developed.
Investigators analyzed and compared the recruitment and blood donation of the Rh(-) blood donors for the emergency blood donation and securing the blood supply upon request.
Results/Finding: The data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from 5.6% to 21.4% in 2011 and 2016, respectively.
Also, the actual participation rate of Rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from 26.8% in 2011 to 57% in 2016.
Moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request.
Conclusion: The result showed that the Rh(-) blood supply program was effective for the recruitment/management of the Rh(-) blood donors for the emergency blood donation.
This system contributes to recruiting and managing Rh(-) blood donors who pledge to donate blood and securing Rh(-) blood in emergency situation .
The institution that needs to meet the demand of rare blood type could possibly use the Rh(-) blood supply program which leads to securing special type blood.
Hanwei Chen*.
Wuhan blood center Background/Case Studies: In China, volunteer blood donors can donate platelets by apheresis (AP) up to 24 times per year.
However, the awareness and knowledge of AP donation is much lower than whole blood donation among the Chinese population.
There are approximately 1.3 million doses of AP transfused within 1.375 billion people each year In China; It is one challenge to recruit new AP donors and retention them as frequency AP donors in China.
Study Design/Method: One stratified recruitment and retention strategy established and applicate at Wuhan blood center since 2006.
Firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; Secondly, group message for permanent AP donors and had not donated with an interval of more than 180 days in low inventory.
Thirdly, specific recruiter telephone for those AP donors who had donated APs for more than 4 times and had not donated for more than 90 days or less than 4 times with an interval of more than 60 days from the last donation; The last one is preparing one letter of thanks for those AP donors who gave more than 8 times annually which advise them to voluntarily come to the blood center for AP donation when they were available.
Results/Finding: Over the past decade, the overall donation time of AP donors increased by 7.46 times from 5550 to 41420 and the doses of AP increased by 7.41 times from 7363 to 54553 within 10 years.
The APs collected fulfilled the clinical needs.
According to the donation frequency, AP donors were divided into 5 groups: those who donated AP once, those who donated 2-4 times, 5-9 times, 10-29 times, and those who donated more than 30 times, respectively.
It was found that the number of permanent AP donors who donated AP more than 30 times was only 965 (2.1%), but they denoted a total of 76432 doses of AP (29.2%) from 2006 to 2016.
Conclusion: APs increased at a rapid and steady pace in Wuhan blood center from 2006 to 2016, which not only met the clinical needs but also were supplied to other region outside Wuhan.
And in addition, the permanent AP donors who gained more attention donated the greatest percentage of platelets.
In conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region.
years were evaluated at 4 sites on 2 consecutive donations for finger stick (fs) hemoglobin (HB) per site policy.
Venous (ven) and capillary (cap) ZPP and ven ferritin (Fer) were performed per manufacturers' direction.
Donors were assessed for subclinical iron deficiency using ranges (Fer <26 ng/mL and ZPP levels >100 umol/mol heme) at 3 HB levels.
Participants completed an online survey between donations to collect data on symptoms of anemia.
Univariate linear regression analysis was used to determine relationship between tests.
Results/Finding: Subclinical iron deficiency was present among first-time and repeat blood donors at all 3 HB levels with both genders and all age groups.
(Table) There was a highly significant correlation between fs ZPP and ven ZPP 87.8% (R50.937) at first and 86.5% (R50.93) at second donations.
At first donation when compared to fs HB, only 10.4% (R50.323) of variation could be explained by variation in fs ZPP, 12.3 % (R50.35) by ven ZPP and 9.4% (R50.307) by ven Fer.
At second donation, when compared to fs HB, only 9% (R50.30) of variation could be explained by variation in fs ZPP, 14.4% (R50.38) by ven ZPP and 20.1% (R50.448) by ven Fer.
For each donation, variation among tests (fs HB, ven Fer, ven ZPP and fs ZPP) was significant (p<0.001) suggesting strong evidence against correlation.
55% (181) responded to the survey of which 4% (13) reported not feeling well after donation.
It should be noted that noted that 1% (3) female study participants reported feeling unwell after the first donation and had ferritin levels below 26ng/mL but the ZPP levels were less than 100 umol/mol heme.
Of the 3% (10) male participants that reported not feeling well none had ferritin levels below 26 ng/mL nor ven or fs ZPP levels above 100 umol/mol heme.
Conclusion: Subclinical iron deficiency was present at all hemoglobin levels.
There was insufficient correlation with fs HB and ven Fer to support use of fs or ven ZPP analysis as measurement of iron stores for blood donors.
Symptoms reported by study participants were not consistent with laboratory results.
The minimum male Hb was raised from 12.5 to 13.0 gm/dL.
FDA imposed specific VS ranges for acceptable pulse (P) and blood pressure (BP), removing center-by-center discretion.
A survey of members of America's Blood Centers (ABC) was performed to assess the impact on donor deferrals resulting from these changes.
Study Design/Method: Online survey software (SurveyGizmo, Boulder, CO) was used to solicit collections and deferral information from 59 blood centers over two intervals, July-Dec. 2015 and July-Dec. 2016 (i.e., before and after the implementation deadline for the final rule respectively).
Information on deferral at presentations for whole blood (WB) donations and apheresis platelet (AP) donations was requested for Hb thresholds and VS.
The information was stratified by gender (male5M, female5F), and ABO type.
Statistical analysis included t-tests for numerical and chi-square for categorical data (Minitab 17.0, Chicago IL).
P <.05 was considered significant.
Results/Findings: Data were provided by 40 of 59 centers invited, representing 2,420,886 and 2,945,802 WB donations and 272,094 and 319,161 AP donations in aggregate during the two intervals respectively.
Gender and ABO distributions appeared representative of the US donor base.
Among M WB donors the rate of deferral rose from 1.5% to 2.9% in the two intervals among aggregated donation attempts (p<.001), and for M AP from 1.8 to 3.5% (p<.001).
The mean "by center" deferral rates (table) were similar to that and significant (p<.001).
Mean by center Hb deferral rates among F donations during the two intervals were 11.6 and 11.9% (p50.241) for WB, 11.8 and 13.0% (p5.041) for AP, respectively, absent any change in their acceptable Hb thresholds.
Data on VS deferrals were much sparser.
For P deferrals, only 12 centers could provide specific high vs. low vs. irregular pulse deferrals; 27 provided only a summary (i.e total pulse deferrals), and 1 could provide none.
For BP, 8 provided detail (high vs. low), 28 summary and 4 none.
P deferrals increased in the successive intervals among F WB donors from a center mean of 0.57 to 1.49% (p5.018) and for M WB donors from 0.78 to 1.16% (p5.006).
Where details were available, high and irregular pulses were responsible for most of the changes for both genders.
BP deferrals were not significantly increased among WB donors, regardless of gender.
The data sets and deferral rates re: VS in AP donors were quite small, possibly reflecting culling during their prior donation experience.
Conclusion: Substantial additional donor deferrals attended the increased Hb thresholds for M in the final rule, for both WB and AP.
Changes were more modest among female donors, consistent with the absence of changes in allowable Hb levels.
Modest but significant changes attended more stringent requirements for VS, though data limitations restrict this aspect of the analysis.
Background/Case Studies: Diabetes mellitus is reaching potentially epidemic proportions in India.
Given the disease is now highly visible across all sections of society within India, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years.
Due to its ease of use, several studies have found that HbA1c testing can identify patients in the community who might otherwise go undiagnosed.
We took an initiative to find out the incidence of diabetes by random blood sugar (RBS) measurement among Indian blood donors and measure the HbA1c levels among those with RBS >180 mg/dL Study Design/Methods: A prospective study was done at department of Transfusion Medicine and department of Biochemistry from 1 st March 2017 to 31 st March 2017.
Total of 1,861 blood donors were tested for RBS.
Those with RBS > 180 mg/dl were further tested for HbA1c by gold standard HPLC method using variant II Biorad.
Blood donors with >180 mg/dl RBS and HbA1c > 6.5% were advised to consult a physician for further evaluation.
Results/Findings: Of the 1,861 donors tested, 44 (2.36%) donors showed a RBS of > 180 mg/dl.
Forty two (95.45%) were males and 2 (4.54%) females with a mean age of 40.55 years (26-56 years).
Of these, 14 (31.81%) were known case of Type-II diabetes mellitus (DM) on oral medications and were excluded.
Of the remaining 30, 8 (26.66%) of them had a family history of DM.
Of these 30 donors, 8 donors did not give a consent for testing for HbA1c.
Among the 22 donors tested for HbA1c levels, 16 (72.72%) had HbA1c > 6.5%.
All the 16 donors were counselled and referred to a physician for further management.
The overall incidence of donors having DM in the population is 0.87% (16 of 1839 donors).
Conclusion: Screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society.
Incidence of Low Ferritin Levels in Regular Male Blood Donors with Acceptable Hemoglobin Levels in Singapore Ramir Alcantara* 1 , Hwee huang Tan 1 and Ai Leen Ang 2 .
1 Health Sciences Authority Blood Services Group, 2 Health Sciences Authority, Blood Services Group Background/Case Studies: Iron deficiency is a known complication of regular blood donation.
In order to protect the donor's health and prevent iron deficiency, AABB increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from 12.5 to 13.0 g/dl last May 2016.
The current minimum acceptable hemoglobin for male donors in Singapore is 12.5 g/dl.
The aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels (12.5-12.9) and in donors with hemoglobin 13 g/dl and above.
Study Design/Method: During a 4 month period, serum ferritin testing was performed on 350 regular male whole blood and 250 regular male apheresis donors who made at least 3 donations in the last two years with an acceptable hemoglobin level.
The donors were divided into groups according to donation type and hemoglobin range; Group A (whole blood with hemoglobin 12.5-12.9) Group B (whole blood with hemoglobin !13, Group C (apheresis with hemoglobin 12.5-12.9) and Group D (apheresis with hemoglobin !13).
The serum ferritin levels of the four donor groups were compared and analyzed.
A ferritin level below 30 ug/L is considered low and levels below <12 ug/L are considered having absent iron stores.
Results/Findings: 55.1% of donors in the study have ferritin levels below 30 ug/L.
There were more donors with low ferritin in group A compared to group B, 80% and 53% respectively (p<0.05).
In apheresis donors, low ferritin rates were higher in group C donors compared with group D, 49% and 30% respectively (p50.001876).
Ferritin results for the 4 groups can be seen in table 1.
Conclusion: More than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or 54.3% have absent iron stores.
Donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after 4 months or more.
Since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented.
Due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin 12.5-12.9 g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in Singapore be increased to 13.0 g/dl.
Other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media.
Background/Case Studies: Safe blood is a crucial and irreplaceable component in the medical management of many diseases.
The Voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than 15 % of the demand of the blood in Pakistan.
Motivation among the youth, particularly students, is essential to make voluntary blood movement more successful.
To assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of Karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in Pakistan Study Design/Method: A cross sectional prospective study was conducted among 600 students from different universities and colleges of Karachi.
A well-structured and pre-tested questionnaire, in English, was used to access the knowledge, attitudes and practices about voluntary blood donation.
A scoring mechanism was used to understand overall knowledge level.
Obtained data was analyzed.
Results/Finding: The sample population consisted of 54% male and 46% female students in the age group of 18-28 years.
Only 65 % of the students have heard about voluntary blood donation and 28 % of the students have given blood once in their lifetime and among them 19 % are blood donors at the moment.
42 % of the participants believed that there is a specific reason why they don't donate blood and 59 % believed that there is a risk involved for the donors, when donating blood.
80 % students wanted to promote voluntary blood donation.
Fear and lack of awareness on blood donation are the reasons for not donating blood.
Students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends.
(21); miscellaneous effects were reported in 23 courses.
Side effects led to interruption of supplementation in 55 instances.
Ferritin levels (MGT6SD) at entry into the program and at the last visit were 48.9 6 2 and 65.4 6 1.7 mg/L in participants, vs 64.1 6 2.2 and 56.3 6 2.2 mg/L in controls.
The positive impact of iron supplementation on ferritin levels was observed only in those who took !75% of the tablets.
Ferritin levels<26mg/L were found in 4,8% of participants and 14.7% of controls.
Deferral for low hemoglobin was below 1% in both groups.
Conclusion: An iron supplementation program in a DRBCD program is feasible.However, when taking into account acceptance to participate and compliance with supplementation, only 50% of donors obtain full benefit from such a program.
Using an iron preparation which is better tolerated may increase compliance.
Background/Case Studies: Hereditary Hemochromatosis (HH) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under 21CFR630.10 and the collection is a physician-ordered therapeutic phlebotomy.
Blood collections Establishments do not need an exception or alternative under § 640.120 to make a collection under this provision if the requirements set forth in § 630.15(a)(2) are met.
The objective is to describe current HH donors and long-term contributions of to our hospital-based donor center and hospital blood supply.
Study Design/Method: In 2001, an IRB protocol was approved for the enrollment and therapeutic phlebotomy of HH patients/subjects.
This required filing an FDA variance to permit HH donor blood for use in our allogeneic supply without disease labeling.
The frequency of therapeutic bleeds are guided by routine clinical assessment, MCV/hemoglobin, serum ferritin, and transferrin % saturation monitoring.
Serum ferritin levels of 50 -75 ng/ mL are targeted for maintenance phlebotomy.
Operationally, a custom, computerized database application is employed to ease phlebotomy management.
Results/Finding: Since inception, the cumulative number of HH subjects enrolled in the hemochromatosis protocol reached 547, of whom 365 (67%) are C282Y homozygotes.
Without active recruitment, accrual rate is about 7 per quarter, with 69% of subjects qualifying as allogeneic donors.
The mean current age is 59.7 years, 65% male, 96% Caucasian.
The majority of HH donors (276 of an active cohort of 318) are in the maintenance phase of therapy with an average of 2.6 donations/year and a 4% deferral rate.
Over the last 5 years, HH donors contributed approximately 8-11% of the hospital's allogeneic blood supply, averaging 475 whole blood units for transfusion per year.
Moreover, HH donor's whole blood (WB) donations provided 30-40% of blood for in vitro research at our institution with an average of 180 WB research donations/year.
There have been no HH donor-derived transfusion-transmitted infections over 16 years.
Since 5/23/16, with an increase in male Hgb deferral threshold to 13g/dl, there has been only 1 HH male deferral from blood donation.
Conclusion: A simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from HH subjects was established.
Blood donated by HH donors remains an important resource at our hospital.
HH donors benefit from careful medical follow-up of their iron status.
This mutually beneficial relationship is feasible and sustainable.
Testing for Accuracy of Non-Invasive Blood Hemoglobin Methodology in a Blood Donor Setting Michele Walker*, Sharon Garcia and Mythili Ram.
Gulf Coast Regional Blood Center Background/Case Studies: The objective of the study was to assess the accuracy of hemoglobin (Hb) levels measured on the OrSense NBM-200 non-invasive occlusion spectroscopy device by comparing them to Hb levels measured on venous samples with a laboratory hematology analyzer.
In addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method.
Study Design/Method: Study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (SD) of the difference between the NBM-200 non-invasive sample results and the Sysmex hematology analyzer venous sample results.
Staff were provided training on the use of the NBM-200 non-invasive occlusion spectroscopy device.
Over a span of 7 days, 200 eligible blood donors, both male and female, were first screened by the NBM-200 non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method.
A venous sample was collected from each of the 200 blood donors for the performance of Hb measurement on the Sysmex hematology analyzer within 1-3 hours of collecting the venous samples.
Results/Finding: The SD of the difference between the NBM-200 non-invasive sample results and the Sysmex hematology analyzer venous sample results was not to exceed 1.1g/dl.
The Hb measurements obtained from the NBM-200 and the Sysmex hematology analyzer were analyzed using the statistical software Minitab and the SD of the difference was reported to be 0.978 g/dl.
The precision of the NBM-200 yielded a co-efficient of variation of .02 g/dl and a standard deviation of .33 g/dl.
Conclusion: The operators found the NBM-200 easy to install, maintain, and operate with minimal training.
The NBM-200 non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results.
It was comparable to the capillary finger stick method and deemed suitable for screening donors.
Donors were satisfied with the process and appreciated the safe, painless methodology.
Ronel Swanevelder 1 , Ravi Reddy 1 , Dhuly Chowdhury 2 , Don Brambilla 2 and Edward L. Murphy* 3 .
1 SANBS, 2 RTI International, 3 UCSF/BSRI Background/Case Studies: To maintain an adequate blood supply, South African blood centers need to collect more blood from their majority Black African population.
Success in recruiting first-time Black blood donors has been tempered by lower suboptimal return rates.
Study Design/Method: We performed a prospective cohort study of firsttime, Black blood donors donating during a four-month period in 2014 and followed them for one year.
Within 56 days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone.
Questions used 4-point Likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (Muthivhi et al.
2015) .
Linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year.
Results/Finding: We included 2,902 first-time Black donors with median age 23 and female predominance (59%).
Within one year, 1,786 donors (62%) attempted at least one additional donation.
When Likert scales were analyzed as an ordinal variable (45 strongly agree to 15 strongly disagree), donor return was associated with the following motivators "Blood donation is an easy way to make a difference" (odds ratio for each Likert increment (OR) 5 1.16, 95% CI 1.06-1.28), "I donated in response to adverts/campaigns on the radio, TV or newspapers" (OR51.11, 95% CI 1.00-1.23).
Responses to altruism-associated statements were not associated with return.
Among deterrents, donors were less likely to donate if they agreed with the statement "I am afraid of the sight of blood" (OR50.83, 95% CI 0.72-0.96) and "I wasn't treated well by the <blood center> staff" (OR50.85, 95% CI 0.74-0.97).
Surprisingly, donors were more likely to return if they agreed with the statement "I was afraid of finding out about my HIV status" (OR51.19, 95% CI 1.03-1.37).
A secondary analysis treating the Likert scales as 4-level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "If I give blood then blood will be available when I need it" and "I don't know where the nearest blood collection point is" were more likely to return.
Conclusion: This novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation.
It is interesting that self-esteem and marketing predicted return better than altruism.
Fear and poor customer experience are recognized deterrents which could be addressed.
We plan to use these data to construct Black donor recruitment interventions which may be tested using randomized trial designs.
Willingness to Donate Blood during the Summer Christopher D Bernard 1 , Ramya Ghantasala 1 , Obhijit D Hazarika 1 , Nicole Leonard 1 , Cori A Polonski 1 , Zachary B Wunrow 1 , Michelle Heleba 2 , Jan K Carney 1 and Mark K Fung* 1 .
1 University of Vermont Larner College of Medicine, 2 American Red Cross Blood Services Background/Case Studies: Each year donation rates fall in the summer months straining blood banks' capacities to meet local demands.
In hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits.
Study Design/Method: An anonymous 16 question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region.
Questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation.
Results/Finding: A total of 292 surveys were received.
Survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "Too busy" (27.5 %) and "Traveling is a time for me to relax."
(30.6 %).
Of the respondents who travel in the summer, very few reported donating while traveling (3.4 %).
Summer donation rates between summertime travelers (36.5 %) and non-travelers (36.4 %) were essentially equivalent.
The most preferred methods of scheduling appointments were via the regional blood donor center website (45.6 %) and phone (28.4%).
Willingness to use a regional blood donation smartphone app was highest among respondents ages of 18 to 34 (45-55%) and lowest among ages 55 and older (13-15%).
Of respondents with no prior knowledge of summer seasonal shortages (22 %), 2/3rds indicated newfound motivation to donate.
Background/Case Studies: Viral infections (Adenovirus, EBV, CMV, BK, HHV6, and RSV etc.)
have been implicated as major contributors to posttransplant morbidity and mortality in Hematopoietic stem cell transplantation (HSCT) from unrelated donors.
Investigators have shown that in-vitro expanded virus specific cytotoxic T lymphocytes (CTLs) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the HSCT setting in recent clinical trials.
Present clinical trials have shown that CTLs can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin-4(IL-4) and IL7.
Others have used banked third party Epstein Barr Virus (EBV)-specific CTLs generated from third party EBV-seropositive blood donors with encouraging results.
Study Design/Methods: Eligible and consented Blood donors were tested for CMV antibodies by serology.
CMV-seropositive Whole Blood (WB) units underwent buffy coats processing from non-leucocyte reduced WB units collected in Fenwal triple Blood-Packs TM that underwent 2 hard spins at 3800 rpm for 7 minutes with separation after each spin on a CompoMateV R G5.
Plasma and buffy coat was separated from red cells after the first spin.
The second spin lead to the separation of the buffy coat from plasma.
The buffy coats were submitted to the GMP Stem Cell Lab for processing of Cytomegalovirus-specific CTLs.
HLA typing at high resolution for HLA-A/-B/-DRB1 loci was obtained for all donors.
Results/Findings: Forty five eligible healthy blood volunteers (13M [29%]: 32 [71%] F); median age 42 years (range 21-70) donated a unit (500 mL) blood from which buffy coats (average volume 56 ml) were processed.
The buffy coat process was previously validated on 20 WB units.
The mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures 1 and 2.
All of the buffy coats received by the GMP Stem Cell Lab were adequate in cell numbers to be processed.
The processing of buffy coats from whole blood is a viable option for the concentration of PBMCs specifically for production of viral specific CTLs as third party off the shelf products as well as use in other research projects that require PBMCs from healthy adults.
Background/Case Studies: The goal of this presentation is to describe the journey and challenges towards TJC, Patient Blood Management (PBM) Certification.
Transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy.
Providence Holy Cross Medical Center (PHCMC), as the Providence California Region alpha site, has embarked on this journey.
Our goals are PBM certification and reduction of the number of unnecessary transfusions by 20% within 12 months of the program launch while improving patient outcomes.
This paper will discuss our journey toward certification and the various hurdles being overcome.
Study Design/Method: TJC, AABB, and the Society for the Advancement of Blood Management have served as our primary resources for identifying current evidence-based transfusion practices and management methods.
We needed to identify our organizational gaps in data gathering and analysis.
Then we could determine baseline performance and set improvement targets.
From our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff.
We took the following steps to develop our PBM program: Formed an interdisciplinary PBM team consisting of physicians, nurses, blood bank staff, and data analysts Constructed a report on RBC transfusions to help identify outliers and opportunities Background/Case Studies: The maximum surgical blood ordering schedule (MSBOS) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and RBC allocation before each surgery.
The extent to which hospitals have an MSBOS and its design was explored in this survey.
Study Design/Methods: The survey was designed, piloted and refined by members of the BEST collaborative and invited colleagues.
It was then encoded in online survey software and the link distributed to BEST members and colleagues who were encouraged to respond and to further distribute it.
The survey was open for 34 days.
Results/Findings: There were 158 completed responses, of which 73 (46%) indicated that their hospital had an MSBOS and 85 (54%) did not.
The majority of hospitals without an MSBOS were academic centers (36/85, 42%) from Oceania (26/85, 31%) or Europe (23/85, 27%), had between 500-999 beds (30/85, 35%); the majority of these hospitals transfused between 1,001-4,999 RBCs (21/85, 25%) per year.
15/85 (18%) are going to implement an MSBOS in 2017.
Of those with an MSBOS, the majority 23/73 (32%) were from North America.
The majority were academic hospitals (39/ 73, 53%) with 500-999 beds (43/73, 59%) that transfused !20,000 RBC units per year (21/73, 29%) offering a wide range of surgical services.
On average there were 207 6 577 procedures listed in the MSBOS'.
The MSBOS recommended no pre-transfusion testing for a mean of 30% of the procedures listed, a pre-operative type and screen for 38%, crossmatching RBC units for 28%, and for 4% of procedures a different recommendation was made.
Most (32/73, 44%) of the MSBOS' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only 5/73 (7%) of MSBOS' were created solely by using procedure-specific data, and most (35/73, 48%) do not use patient-specific data in making a testing recommendation.
Most MSBOS' are updated less frequently than annually (30/73, 41%), and the hospital transfusion committee is often (39/73, 53%) involved in updating it.
The MSBOS' are generally available electronically in both the operating rooms and in the blood banks.
It was the opinion of the majority of respondents (30%) that the MSBOS was used regularly by only a limited number of surgeons and anesthesiologists, 23% of respondents felt that it was regularly used by all surgeons and anesthesiologists; 10% felt that it was not used at all at their hospital, 36% did not respond.
Conclusion: An MSBOS was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used.
However, 18% of the hospitals currently without one indicated that it would be implemented in 2017 suggesting that these hospitals perceive the value of having one in place.
Implementing and following an MSBOS can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing.
Blood Management -One Hospital System Experience Leana Serrano Rahman*, Mallika Gupta, Susan Solometo, Ronald Walsh and Joan Uehlinger.
Montefiore Medical Center Background/Case Studies: Our system, a Pioneer ACO, is a 1490-bed tertiary-care referral center dedicated to serving patients from across the New York City area and beyond.
The comprising four hospitals see 93,000 hospital admissions and nearly 300,000 emergency department visits annually.
We have active programs in high risk OB, stem cell transplant, solid organ transplant (heart, liver, and kidney), CT surgery, ECMO, oncology and critical care.
Transfusion Medicine plays a key role in the support of these services.
Blood product spending in 2015 was approximately $15.8M.
In Nov. 2015, an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures.
The Vice President-sponsored multidisciplinary committee was composed of representatives of: Surgery, Anesthesia, Blood Bank, Pediatrics, Perfusion, Cardiothoracic Surgery, Critical Care, Medicine, and Emergency Department.
Study Design/Method: First Important Step: "Know your numbers"-Although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information.
Baseline numbers were imperative to the committee's ability to effect change.
A home grown one time only report revealed which services and clinicians were the highest volume users.
The initial plan was to target their use with education.
An initial goal was set to reduce expenditure by $1.2M.
The journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization.
Utilization was analyzed using a home grown crystal report "Transfused Patients by Location".
This report was further compared to utilization patterns (2014 and 2015), by "dollars spent" and "total units per patient" by the project manager using excel.
Key Initiatives developed by the committee 1.
Development of evidence based transfusion triggers.
2.
Education on evidence based transfusion triggers across multiple campuses, specialties and resident programs 3.
Clinical Information System (CIS) "soft stops" when ordering blood products outside guidelines.
RBC order set defaulting to "1" unit instead of "2" units.
4.
Updated guidelines posted to easy to find internal intranet spots Results/Finding: Despite higher patient volumes and a more complicated patient mix in 2016, we were still able to reduced blood product expenditures by $933,874 when compared to 2015.
Conclusion: In spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management.
Cord Blood Pathway to Reduce Iatrogenic Blood Loss in Neonatal Intensive Care Patients Tracy Shachner* 1 , Anna W Rains 2 and Christopher T Clark 3 .
1 University of Tennessee Graduate School of Medicine, 2 Univeristy of Tennessee Medical Center, 3 Univeristy of Tennessee Graduate School of Medicine Background/Case Studies: Anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population.
Methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia.
At a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established.
The cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (NICU), preventing need for additional blood draw.
The blood tubes are saved for 1 week post-delivery, with cost of $1.10 per delivery tray for sterile tubes.
With an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age.
Study Design/Method: Labor and delivery data from our facility in 2016 was analyzed, and the gestational age and birth weight of all infants transferred to the NICU was collected.
From this data, we were able to calculate the total blood volume of these infants using MedCalc 3000 system.
By using the blood volume values, and assigning a value of 1.5 mL as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established.
TRANSFUSION Results/Finding: In 2016, there was a total of 3,331 infants delivered at our facility.
Out of all the deliveries, 487 (14%) infants were transferred to the NICU.
Of those infants, 27% received at least one red blood cell transfusion and 7% received at least one platelet transfusion.
Of the 487 infants transferred to the NICU, 98 (20%) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to 1% (which we considered to be significant), had the cord blood pathway not been in effect.
The percentage of blood volume preserved in these infants ranged from 1.0% all the way up to 3.9%.
In those 98 infants, the birth weight ranged from 400-1650 grams, and the gestational age ranged from 22 weeks to 36 weeks and 4 days.
Conclusion: The established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of NICU infants who are most susceptible to iatrogenic anemia.
The infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than 2500 grams).
Development of a Standardized Response Team for Massive Hemorrhage Events Outside of an Operating Room Setting James Burner* 1 , Shannon Davis 2 , Suzan New 2 , Vaishali Patel 2 and Oren Guttman 2 .
1 University of Texas Southwestern Medical Center, 2 UT Southwestern Medical Center Background/Case Studies: Managing a massive transfusion protocol (MTP) in an operating room (OR) is a relatively frequent occurrence with team members well trained in their specific roles.
However, in the event of MTP activation outside of an OR, sufficient and/or appropriately trained individuals may not be present.
This can lead to a scene of confusion and chaos with potential for patient harm.
Study Design/Method: A Failure Mode Effects Analysis was performed to develop a standardized process for managing MTP outside of an OR setting.
With participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's MTP was analyzed for potential errors.
The results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-OR patient.
Results/Finding: Code Hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring).
Our Code Hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (EMR) coordinator.
Their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during MTP and enable enhanced closed loop task performance.
The hemorrhage coordinator establishes reliable 1:1 communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination.
The EMR coordinator enters all orders into the EMR, sends/communicates laboratory results and ensures blood products are available to the resuscitation team.
The primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.
), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices).
Additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments).
Conclusion: The code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an OR setting.
Future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation.
Background/Case Studies: Preoperative anemia affects up to 50% of surgical patients and increases the risk of red blood cell (RBC) transfusion.
Both preoperative anemia and perioperative RBC transfusion are associated with increased risk of adverse outcomes following surgery.
Preoperative treatment of anemia includes oral and intravenous (i.v.)
iron and erythroid stimulating agents (ESA) such as erythropoietin (EPO); however, the optimal treatment strategy for preoperative anemia remains to be established.
Our objectives were to evaluate the efficacy and safety of ESA and iron therapy based on their effects on the prevalence of RBC transfusions and adverse thrombotic events.
Study Design/Method: We searched the Cochrane Central Register of Controlled Trials, MEDLINE and EMBASE from inception to July 2016; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings.
No language restrictions were applied.
We included randomized controlled trials in which adult patients undergoing surgery received either an ESA and/or iron before surgery, versus iron or no intervention.
Three authors independently reviewed the studies and extracted data from included trials.
Risk of bias was assessed for all included studies.
Where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models.
Our primary outcome was the number of patients transfused with red blood cells.
Secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism).
Results/Finding: A total of 79 randomized controlled trials (8, Conclusion: Amongst patients undergoing surgery, the administration of an ESA in addition to oral or i.v.
iron was associated with a reduction in patients requiring RBC transfusion.
Intravenous iron was less effective at reducing RBC transfusion.
Neither treatment was associated with any clear increase in risk of adverse thrombotic events.
Additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia.
Evidence Based Blood Therapeutics Scott Neeley* 1 and Stephanie Rogers 2 .
1 Dignity Health St Joseph's Medical Center, 2 Dignity Health Background/Case Studies: Over 12 million units of packed red blood cells (PRBC) are transfused annually in the United States and there is no clinical basis for as many as half of these transfusions.
No randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence.
Study Design/Methods: A system wide goal was launched across 39 hospitals to decrease the number of PRBC transfusions given to clinically stable patients with hemoglobin (Hgb) levels >5 7.0 g/dL.
The numerator consisted of all PRBC units transfused to patients with a Hgb of 7.0 g/dL or greater prior to transfusion and the denominator consisted of all PRBC units transfused.
Exclusions included cardiac surgery, Nursery, NICU, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which 4 or more PRBC units were transfused in one episode.
Data was extracted directly from the Electronic Medical Record and hospitals received patient level detail every month for all PRBC units transfused to patients with a Hgb of 7.0 g/dL or higher prior to transfusion.
An extensive educational campaign re: evidence-based transfusion practice was launched for Physicians and Nurses, including the development of a Blood Therapeutics toolkit, development of standardized Dignity Health Blood Therapeutics Guidelines, a one day Blood Therapeutics Advanced Training Symposium, on-site visits to 21 hospitals including 16 CME presentations, online Physician and Nursing educational videos, communication tools including infographics and "7 is the new 10" buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers.
Additionally, the EHR powerplans were revised to ensure available selections for "Transfusion Indication" (required field) were aligned with evidence based guidelines.
Facilties were encouraged to develop multi-disciplinary Blood Therapeutics Committees to review all transfusions given to patients with pre-transfusion Hgb >5 7.0 g/dL on a routine basis, providing feedback to Providers whose transfusions were deemed not in accordance with current evidence-based guidelines.
Results/Findings: From FY2015 to FYTD2017, there was a 26% reduction in PRBC units transfused to patients with Hgb >5 7.0 g/dL, starting at a baseline of 67% down to 41%.
This represents an FY17 annualized savings of $9.732M, from a baseline of 82 units per 1,000 patients days down to an average 71 units and approximately 2,000 fewer units transfused per month.
Conclusion: Blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk.
Transfusions to stable, non-bleeding patients with Hgb levels >5 7.0 g/dL are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm.
Furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of PRBC should be transfused rather than two.
Three AF studies (SDM5-0.258) reduced RBC units and two studies decreased the percentage of patients transfused (OR5 0.700).
Forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (OR 50.264) and RBC units transfused (SDM5-0.553).
Qualitative/meta-analyses were translated into recommendations by an Expert Panel and approved by the LMBP Workgroup for reducing RBC transfusion.
Recommendations are: Early assessment and effective AM; RT, Hb alerts in CPOE/CDS; reduction of blood loss and AF assessing the percentage of patients and RBC units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement.
Conclusion: Conclusion: The LMBP A-6 method led to evidence-based recommendations for reducing transfusion.
Critical laboratory support is needed to achieve continuous quality and patient safety.
Background/Case Studies: Reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management.
The use of electronic decision support tools such as best practice alerts (BPAs) to enforce red blood cell (RBC) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse.
The tools in use to date have not provided a dose of RBCs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of RBC given.
A therapeutic hemoglobin/hematocrit (Hgb/HCT) targeted approach to RBC indications/ orders allows for the calculation of a dose of RBCs to achieve the desired target and could further reduce the use of RBC units.
Our group has developed a computer algorithm to calculate RBC dose based on patient specific data drawn from the electronic medical record (EMR) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice.
This study describes our initial experience with the use of this algorithm in non-surgical RBC transfusion.
Study Design/Method: The Blood Utilization Calculator (BUC) is a mathematical formula that draws patient specific information including index Hgb/ HCT and calculates a dose in number of units of RBCs to transfuse in order to achieve a selected target Hgb/HCT.
Hgb/HCT target based indications for RBC transfusion were designed and used as the basis for RBC order set with in the EThe BUC was embedded within the EMRs RBC order set to provide a recommended transfusion dose in number of units when any nonsurgical RBC indication was selected.
The target Hgb/HCT for these indications was 7g/dL/21% or 8g/dL/24%.
The number of RBC units ordered and transfused were tracked prospectively for each of the orderable indications.
Comparison of units transfused per month before and after the BUC implementation was performed using Student's t-test.
Results/Finding: Historically, the three non-surgical RBC indications represented approximately 42% of the total RBC transfused.
Prior to the BUC the mean number of non-surgical RBC units transfused was 590 1 24 units/ month.
After the first 5 months of BUC activation the mean number of units was 439 1 50 units/month a reduction of 151 units/month or 26% of nonsurgical blood use (P50.003 by t-test).
Non-surgical RBC use now represents approximately 29% of the total RBC use hospital wide a 13% reduction.
This change represents a significant cost savings in RBCs over time.
Conclusion: The use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical RBC transfusion rate providing enhanced patient blood management and potential cost savings.
Implementation of Patient Blood Management at a Community Hospital -30 Month Report Card Richard Gammon*.
Oneblood, Inc. Background/Case Studies: A collaboration between blood center between (BC) as consultant and three hospital (4001 beds) healthcare system (HCS) to implement a patient blood management (PBM) program was undertaken.
This is a review of the first 30 months.
Study Design/Method: During year one PBM working group was established.
Achievements included physician engagement programs, creation of transfusion committee and providing nursing education.
Auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved.
In year two, IT created best practice alerts (BPA) when an order did not meet transfusion threshold criteria.
BPA showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (RBC)) and allowed ordering physician to cancel order after review.
A blood administration video was created.
It was mandatory that all physicians granted privileges complete within six months.
Low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (WG) to address knowledge and practice gaps.
In year three, as historically at this HCS very few Jehovah's Witness patients (JWP) presented, PBM WG was involved with implementation of a bloodless medicine program.
All steps of care were addressed including identifying JWP at registration, creating 115A TRANSFUSION special arm bands, forming a bloodless medicine physician group, implementing nursing BPA in the electronic medical record, creating advanced directives and marketing to the public.
Results/Finding: The following were monitored for compliance (2Q14 vs. 1Q17): present and completed consents (66 vs. 94%), present and completed nursing flow sheets (19 vs. 96%), transfusion thresholds supported (73 vs. 100%), discharge instructions provided (17 vs. 86%); (3Q15 vs.1Q17) vital sign compliance (39% vs.71%).
JWP increased from 27 to 225 (04/16-03/17).
Cost savings were realized by decreased utilization and implementation of BPA.
(Table 2016 -1Q17) Conclusion: PBM implementation at a HCS is a continuous and multiyear process.
Even with a robust program challenges such as vital sign compliance remain.
Improving Patient Outcomes in the Golden-Hour Beatrice LeBeuf*.
Medical City Plano Background/Case Studies: In emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival.
Nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging.
Rapid administration of blood products is vital to the survival of these patients.
We implemented BloodTrack Emerge (Haemonetics, Braintree, MA) in our Trauma emergency department (ED) as part of a quality improvement initiative to more efficiently provide group O RBCs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource.
Study Design/Methods: We treat approximately 30-40 trauma patients monthly.
An assessment of our current blood supply chain revealed a multistep, manual process that took about 8 minutes to prepare and physically transport a cooler from the blood bank to the ED.
Coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not.
This practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only 10 percent of coolers fully used.
It also consumed valuable staff time as technologists typically made 20-45 trips per month from the blood bank to the ED.
Plus, there was no effective way to maintain traceability, control access to coolers or monitor usage.
Results/Findings: Since our November 2016 implementation, BloodTrack Emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the Medical City Plano's verification as a Level 1 trauma center.
Rather than preparing coolers of blood in case they may be needed in emergency situations, BloodTrack Emerge provides ED staff ready access to emergency units whenever they're actually needed -and frees up an estimated 6-10 hours of tech time per month during which they can perform other tasks.
Audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response.
Plus, by stocking emergency blood supplies in the ED, the blood bank isn't unnecessarily tying up group O RBC units.
Today, the blood bank stocks and maintains 2-4 units of group O RhD negative, 4 units of O RhD positive, and 4 units of group A thawed plasma/ liquid plasma in BloodTrack Emerge.
Conclusion: Implementing BloodTrack Emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations.
Background/Case Studies: Platelets are a limited resource for which the benefits of transfusion must be weighed against the risks.
In 2015, the AABB published platelet transfusion guidelines to assist providers.
At our academic medical center, a computer provider order entry (CPOE) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions.
Discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g.
Count < 10 K/ml [prophylaxis]) with the option to add a free-text comment.
The order is placed and data is stored for later review.
Study Design/Method: Override platelet orders placed from June 2015-October 2016 were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department.
One of five "codes" was assigned to each order: I-Indicated or NI-Not indicated (based on institutional/AABB guidelines); NMI-Need more information; P-Protocol (e.g.
liver transplant), and NIC-Non-indication comments (e.g.
reserve for OR).
Free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides.
Results/Finding: Over a 17-month period, 1,270 CPOE override platelet orders occurred.
The percentages of code assignments by month are provided in Table 01 below.
Overall, 532 (42%) were assigned as not indicated (NI).
The top keywords assigned to free-text comments were "platelet count less than.
.
."
(325), "active bleeding" (303), "platelet count of .
.
."
(173), and "downtrend" (92), many with specified platelet count goals.
Certain platelet count goals and reasons for transfusion (e.g.
"downtrend," "anticipate drop," or "per service,") are not included in institutional or AABB guidelines.
Of note, 618 (49%) of overrides were placed by Hematology-Oncology providers.
Conclusion: A majority of override platelet orders were determined to not be indicated based on institutional and AABB guidelines.
Of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions.
It is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns.
This review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on Hematology-Oncology) in order to improve blood product utilization practices.
Background/Case Studies: early diagnosis of iron deficiency anemia (IDA) by clinical laboratories (CL), with effective prevention and treatment in primary care may have an impact on packed red blood cell (PRBC) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers.
They all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population.
Results are described after implementing a process to prevent IDA, its early detection and treatment for years 2014-2016.
Study Design/Methods: performance measure after educational and organizational intervention.
Setting: public integrated healthcare system located in north Africa bordering Morocco, isolated by 207 km sea distance to nearest continental Spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production.
CL involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments.
Process: guidelines for first step CL diagnosis of IDA and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose).
Transfusion was avoided for stable IDA patients without active bleeding or coronary heart disease, with a safety hemoglobin (Hb) threshold of 5,5g/dl.
Severely anemic patients were closely followed to asses Hb increase and referred for etiology studies when Hb> 9 g/dl.
Background/Case Studies: Bedside nurses are critical in safeguarding the delivery of appropriate patient care.
More recently, nurses have also begun to play an important role in patient blood management (PBM) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside.
The goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice.
Study Design/Method: A short electronic survey (12 questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion.
The survey was distributed to all registered nurses via email from floor leaders.
Responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift.
Results/Finding: There were a total of 32 complete responses (16%).
The nurses had a range of experience from less than one year to forty years.
Ninety percent stated they discussed transfusion necessity with patients, 81% with physicians, and of these, 59% reported doing so proactively before an order was placed.
Ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only 3% responded that they would suggest product was not needed.
Nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of 7-8 g/dL (56%), platelet count of 20-50,000 (38%), and INR of greater than 2.0 (69%).
Conclusion: This study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity.
The limited number of survey responses suggests a discomfort with their level of education in transfusion practice.
This, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of PBM programs.
Background/Case Studies: The use of red blood cell per 1,000 inhabitants may vary 3 folds between European countries, revealing that there may be substantial room for blood optimization strategies.
Patient Blood Management (PBM) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes.
The objective of our study was to assess the effect of a nationwide PBM program on public health in Portugal.
Study Design/Method: The first phase of this research project involved a group of 18 key opinion leaders (KOL) in a stated preference inquiry to assess the relative value of specific PBM strategies, grouped in PBM pillars, to highlight the need for strategy prioritization in the implementation of a nationwide PBM policy.
Adaptive conjoint analysis techniques were used to elicit KOL preferences.
In the second phase a decision analysis model was used to estimate the impact of PBM implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy.
Model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from Portuguese national health databases and literature review.
The public health value of PBM implementation in Portugal derives from the comparison of two scenarios: "current clinical practice" and "with PBM implementation".
Results/Finding: KOL elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred PBM strategies (14.4% and 14.0%), for the remaining strategies weights varied between 7.0% and 10.6%.
We estimate that 384,704 patients would be eligible for PBM strategies in one year time horizon, resulting in 594 premature death avoided (3.8% reduction) corresponding to a gain of approximately 1,500 life years and a reduction of 3,660 (6.0%) disability adjusted life years (DALY) relative to the current clinical practice.
A decrease of 233,141 in-hospital days is expected mainly due to a 8.4% reduction in hospital length of stay and a 37.3% reduction in 30-day readmission rate.
In this population the overall transfusion rate could decrease to 4.3% from the current 8.7% (51.2% reduction) implying 17,202 blood transfusion avoided and 65,214 red blood cells units spared.
Conclusion: We anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources.
Results/Finding: 237 adult liver transplants were performed during the evaluation period.
Preoperative hemoglobin, creatinine, MELD score, spontaneous bacterial peritonitis (SBP), preoperative hemodialysis, gender, and portal vein thrombosis (PVT) gave the strongest model predicting RBC usage.
If the model predicted <1250ml of RBCs, all cases with 0ml transfused were captured and only 7.8% of the time >1250ml were used.
If 1250-2000ml RBCs were predicted to be transfused, >2000ml were used 25% of the time.
If predicted usage was >2000ml, 53% of the time it exceeded 2000ml.
Conclusion: A model using specific preoperative factors can be used to predict intraoperative RBC usage.
Patients at risk for >1250ml of RBC transfusion can be identified with reasonable accuracy using this model at our institution.
Use of this model might help improve preparation and utilization of the blood bank.
Review of Blood Ordering Practice for Elective Surgeries in a Maternity Hospital Qi Raymond Fu*.
KK Women's and Children's Hospital Background/Case Studies: Pre-operative over-ordering of blood is common, resulting in waste of blood bank resources.
Blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients.
The Cross-match to Transfusion (CT) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service.
According to the American Association of Blood Banks (AABB), a CT ratio of less than 2.0 is favorable, and anything above indicates over-ordering and cross-matching of blood.
To achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital.
Study Design/Methods: Data on elective surgeries requiring blood for standby was collected retrospectively over a 3 month period (Jan to Mar 2017).
Details of total blood cross-matched, issued, transfused and returned were analyzed along with the CT ratio.
Results/Findings: During the 3 month period, there were 274 patients undergoing obstetrics and gynecology procedures requiring blood on standby.
A total of 494 units of blood were requested.
154 units were crossmatched, of which 138 units were sent to the Operating Theatre (OT).
Only 33.3% of blood issued to OT were transfused (n546) while the rest were unutilized.
The observed CT ratio was 3.35.
Conclusion: Although only 31% of total blood requested was crossmatched, the CT ratio remains above the recommended guideline of !2.0, with almost 70% of cross-matched blood unutilized.
There is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload.
Establishing and adhering to a maximum surgical blood order schedule (MSBOS) could help in conserving blood and prevent over-ordering of blood.
Background/Case Studies: Total knee arthroplasty (TKA) is a major orthopaedic procedure with increased perioperative blood loss.
This perioperative blood loss could be more significant in patients undergoing bilateral TKA in a single stage.
The increased blood loss in bilateral TKA often requires blood transfusion which results in high post-operative morbidities.
Study Design/Methods: In this retrospective study 35 patients who received tranexamic acid (TXA) (study group) and 31 patients who did not receive TXA during surgery (control) were evaluated for blood loss and transfusion requirement.
The study group received a single bolus dose of TXA 1gm IV before tourniquet deflation on first side knee.
Statistical Background/Case Studies: Blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks.
One strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products.
We investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior.
Study Design/Method: This retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital.
Each time a crossmatch for packed red blood cells (pRBCs) was ordered via the hospital's electronic ordering system, an indication (e.g.
"hemodynamically stable with hemoglobin < 7.0 g/dL") must be selected.
If the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated.
Ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications.
An alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion Results/Finding: Over seven months, there were 1732 unique alert encounters.
Of these, 1531 (88.4%) led to a crossmatch being ordered while 201 (11.6%) led to the order being canceled.
Providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds (7.0 g/dL) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds (8.0 or 9.0 g/dL Background/Case Studies: The maximum surgical blood ordering schedule (MSBOS) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins.
With improved patient data management systems it is now possible to create an MSBOS based on actual red blood cell (RBC) utilization data on a per-patient basis.
This study investigated the transfusion patterns at 4 academic hospitals with data-derived MSBOS.
Study Design/Method: The 4 hospitals were in 2 groups, with one shared MSBOS for each group.
Three of these hospitals were large academic centers while one was a children's hospital.
At each center the MSBOS recommended no pre-transfusion testing if 5% of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (T&S) if 5-24% of the patients had been transfused, and a crossmatch of the median number of RBCs transfused if !25% of the patients had been transfused.
Data were collected at each center over a 1 month period between January to March 2017 and included a maximum of 400 cases per hospital during that one month to ensure equal representation between centers Results/Finding: Between these 4 centers there were a total of 1599 cases analyzed.
Some of the more frequently performed surgeries included orthopedics (23% of cases), general surgery (16%) and cardiac surgery (11%).
There were 1362 T&S ordered for these cases, of which 5 were positive for antibodies on the day of surgery.
Of all the T&S ordered, 52% were ordered in accord with the MSBOS recommendation, 26% were ordered when the MSBOS did not recommend one, and in 0.2% a T&S was not ordered when the MSBOS recommended one.
Background/Case Studies: Peripartum blood transfusion is more common in South Africa than in the USA and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio 5 6.12 for prenatal hemoglobin (Hgb) 8-8.9).
We therefore analyzed the etiology and characteristics of antenatal anemia according to HIV status at a large hospital with a HIV prevalence of 29% among obstetric patients.
Study Design/Method: We studied a sample of anemic (Hgb<10.0 g/dL) pregnant women who were referred to an antenatal anemia clinic at a large hospital in South Africa.
Clinical information was abstracted and blood was sent for laboratory studies.
T-tests were used to compare continuous variables between groups.
Results/Findings: A total of 301 women were enrolled, with median age 27 (interquartile range 23-32) years, median gravida 2 / para 1 and median gestational age 28 weeks.
Mean Hgb before referral was 7.5 g/dL and most were already taking oral iron therapy.
A total of 169 women were HIV positive with mean CD41 lymphocytes counts of 394 cells/uL; 29 (12%) of HIV positive subjects were on anti-retroviral therapy (ART) prior to the pregnancy and 156 (92%) were on ART during the current pregnancy.
Iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in 292 (97%) of women.
There was concurrent chronic disease (n52), infection (n52), vitamin B12 deficiency (n52) and antenatal hemorrhage (n56); 10 had other/unknown/missing causes of anemia.
There were few pregnancy related complications.
HIV positive women had higher levels of C-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and RBC folate than HIV negative women (Table) .
Conclusion: Iron deficiency is the overwhelming cause of antenatal anemia among South African pregnant women.
Compared to HIV-negative women, HIV-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate.
A high proportion of HIV positive women were receiving ART, consistent with national guidelines.
Future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion.
Background/Case Studies: A 2 month old boy presented to our institution after a 1 month hospitalization in Japan.
He was admitted there, several weeks after his unremarkable term birth to an AB Rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count.
He was found to be anemic and thrombocytopenic and required multiple transfusions.
Also, he had a diffuse, scaling, erythematous rash over his inner thighs.
Study Design/Method: Initial workup was suspicous for an allergic/necrotizing enterocolitis.
The patient had an elevated LDH and potassium, and concern was raised for leukemia with possible tumor lysis syndrome.
A sample sent to our blood bank showed an anti-E, with a positive DAT (IgG and complement), and was positive for E, e, and C antigens.
Concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the Japanese hospital; both possibilities were excluded.
Further workup revealed no infection or hematologic proliferation.
Biopsy of his rash showed spongiotic dermatitis.
His clinical course deteriorated, and he developed hepatomegaly and jaundice.
A concern for Wiskott-Aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated IgE (15270 kU/L; RR: 0-2.9).
Anti-platelet antibodies were identified.
Three days after admission, testing was sent for genetic alterations of FOXP3, while a Japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of FOXP31 CD41 lymphocytes.
The majority of these indications are seen in adults and for which a reported plasma wastage is $1.8%.
Fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population.
During the utilization review process at our primary pediatric institution, we noted a mean wastage of 8.2% over the last 5 years.
With recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (MTP) outcomes, our facility decided to implement the use of thawed plasma and benchmark MTP plasma wastage.
Study Design/Method: Blood Utilization Review revealed an increase in the overall percentage of plasma wastage from 2014 to 2016, with a peak of 11.6% (range 3.2%-11.6%).
A single cause could not be readily identified prompting us to query Children's Hospital Association (CHA), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for 2016 MTPS.
Results/Finding: In 2016, MTP was activated 28 times.
In 6 cases the patient did not receive any blood product and in 11 cases plasma was already available at the time of RBC allocation/issue.
This left 11 cases to evaluate.
The median time to plasma availability was 29 minutes (range 4 minutes -61 minutes).
The mean plasma wastage for MTP activations was 32% (range 0-100%).
Of the 9 CHA replies, 3 were using thawed plasma and their wastage was </5 5%.
Conclusion: Increasing clinical evidence supports the prompt transfusion of plasma in the setting of MTP to decrease morbidity and mortality.
Our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of 29 minutes.
Our implementation began with thawing a single unit of plasma for "Level 1 Neuro Trauma" alerts, and we are now keeping a single unit of AB plasma thawed at all times.
Plasma is now available at the same time as RBCs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are 9.5 %) to a level consistent with external benchmarking.
Prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the Trauma Team.
Ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice.
Background/Case Studies: Bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT).
Granulocyte transfusions (GTx) have been used for over 40 years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children.
To contribute to this discussion we demonstrated 5 cases of GTx in pediatric patients with age from 8 months to 16 years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing HSCT and chemotherapy.
Study Design/Method: Donors: Data concerning a total of 56 granulocytes donations from 2015 to 2016 were collected from 43 health donors (21 male/ 22 female).
Donors had matched blood type and serology status for cytomegalovirus when the patient was negative.
Granulocytes were mobilized with a single dose of rHu-G-CSF (Filgrastim) subcutaneously, 5mcg/kg/dose (donors up to 70kg, maximum of 600mcg) and oral dexamethasone (8mg), 12h prior to apheresis.
Apheresis was performed using Cobe Spectra V R or Spectra Optia V R with citrate as anticoagulant.
All products were irradiated with 40Gy.
Results/Finding s: Granulocytes number increased 5 times from basal levels and the median of cells collected were 4.89 x10 10 (range 1.09-9.85).
In general donors reported no significant adverse reactions.
From 56 donations, only in 4 was reported light paresthesia during the procedure.
Patients: All patients were medicated prior to transfusions with diphenhydramine and acetaminophen.
The product was transfused within 24 hours after collection, with efforts to transfuse as soon as possible.
Details about the transfusions are described on Table 1 .
Adverse reactions for patients were minor and not frequent (4 in 66 GTx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that GTx is well tolerated.
Conclusion: Our study provides further evidence that GTx transfusions can be safely performed on pediatric patients.
Irina Kumukova* 1 , Elena Kurnikova 1 and Pavel Trakhtman 2 .
1 Russian Institute for paediatric haematology, oncology and immunology, 2 InstitutefFor Pediatric Hematology, oncology and immunology Background/Case Studies: Infections cause major mortality among persons with prolonged neutropenia related to HSCT and chemotherapy and among patients with granulocyte disorder.
The granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics.
We want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections Study Design/Method: We analyzed the retrospective data for the period 04/2012-02/2016.
Donors underwent granulocyte mobilization with G-CSF in a dose 3-5 mg/kg s.c. 12-16 hrs prior to donation; granulocytes were collected with COBE Spectra, CaridianBCT.
The analysis was performed through Microsoft Excel, nonparametric Mann-Whitney's and Spearman tests Results/Finding: Was performed 169 granulocytes collections in 153 donors (82 f, 71 m), ages 18 to 58.
The mean increment of WBC after stimulation was 22,6x10 9/L (9,2-41,9 x10 9/L; f-23,2 x10 9/L; m-21.9 x10 9/L); in the age groups 18-39 years (n5121) and over 40 years (n532), the mean increment of WBC was respectively 22,2 x10 9/L (9,2-41,9x10 9/L) and 24,02x10 9/L (12,6-35 x10 9/L).
The mean volume of treated blood was 566, a50,05) .
The mean number of total WBC in the product was 38,43x10 9/L (13.38-77x10 9/ L; f-34,8x10 9/L, m-42,7x10 9/L).
Only three donors (1,96%) had apheresisrelated adverse effects The analysis included 244 granulocyte transfusions in 35 cases of infectious complications in 32 patients.
The mean number of transfusions was 6.9 (1-60).
Each patient received transfusions from mean 5 donors (1-32); the granulocyte transfusions started at the mean 8 days (2-30) after infection; the mean dose of WBC on the transfusion was 11.6x10 8/kg (2-30.7x10 8/kg).
WBC increment was able to estimate after 144 transfusions.
WBC increment was recorded after 96 transfusions (66.7%); the mean increment was 1,12x10 9/L (0,01-11,38 x10 9/L).
Efficacy was determined by the number of patients who survived an infection episode and amounted to 80.6%.
In the cases of severe sepsis, the efficacy of transfusions was 76.2%.
The frequency of post-transfusion reactions was 22%, (n 5 8): febrile nonhemolytic reactions 8,3% (n 5 3); pulmonary complications 11,1% (n 5 4); the anti-HLA antibodies formation 2,7% (n 5 1) Conclusion: No significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline WBC and their increment after stimulation.
The mean total WBC in the collected product and the mean volume of treated blood from men and women were significantly different.
Perhaps the reason for lower cell products from women is to treat smaller blood volume.
The lack of increment WBC after transfusion is not equivalent to lack of its efficacy.
We found a significant association between transfused dose and increment of WBC.
We deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics CP150 Hemolytic Disease of the Newborn Due to Anti-Rh17 Keegan Barry-Holson* 1 and Jennifer Andrews 2 .
1 Stanford University, Dept of Pathology, 2 Stanford University, Dept of Pathology & Pediatrics Background/Case Studies: Anti-Rh17 is a rare antibody against a high incidence red blood cell (RBC) antigen (Ag) present in people with common Rh phenotypes.
This Ag is missing in individuals who are Rh null or have Rh deletion phenotypes, including D--/D--.
D--individuals strongly express D and lack C, c, E, and e Ags.
The clinical relevance of anti-RH17 has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (HDN).
We present a rare case of HDN due to anti-Rh17 alloimmunization during pregnancy in a D--mother.
Study Design/Methods: An O-positive full term infant was born to an Opositive G3P1 > 2 mother with a negative 1 st trimester antibody screen and no prior transfusions.
She had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the 1 st trimester.
Father's blood type is unknown but presumably he has Rh antigens.
The infant was transferred to our institution at 6 hours of life because he was found to have anemia (hemoglobin 12.0 g/dL), severe hyperbilirubinemia (total bilirubin (t Bili) 9.0 mg/dL), reticulocytosis (8%) and a positive direct antiglobulin test (IgG 21).
He was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for HDN.
He was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis.
T bili rose to a maximum of 16.7mg/dL on day 8 of life and phototherapy was restarted.
His t bili subsequently stabilized and he was discharged home and followed in clinic.
Meanwhile, his mother donated blood given there were no compatible red blood cells available in the United States via rare donor query.
Nine days after discharge, he was readmitted for worsening anemia (hemoglobin 6.3 g/dL) and was given steroids and washed maternal red blood cells.
He was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia.
Results/Findings: At delivery, the mother's antibody screen was positive and anti-Rh17 was identified; no other alloantibodies were detected.
Antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement.
Maternal serum was pan reactive against panel cells and non-reactive against D--cells.
Anti-Rh17 sera did not react against maternal RBCs.
Phenotyping of the mother revealed that she was D1 C-E-c-e-.
Molecular testing confirmed her D--genotype; Molecular BeadChip Test yielded no type due to low signal for e, E, V and VS Ags.
Genotyping for Rh variant and targeted genomic RHCE testing failed to detect several RHCE exons.
Father was unavailable for further testing.
Conclusion: We report a rare case of HDN due to anti-Rh17 antibody in a D --mother.
We hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population.
These studies have important implications for genetic counseling for mother's sisters.
Management of Severe Autoimmune Hemolytic Anemia: A Case Report of an Infant Treated with Manual Whole Blood Exchange with Rapid Clinical Improvement Yunchuan Delores Mo* 1 , Cyril Jacquot 1 , Valli Criss 1 , Philippe P Pary 1 , Jay Greenberg 1 , Naomi LC Luban 1 and Edward CC Wong 2 .
1 Children's National Medical Center, 2 Quest Diagnostics Background/Case Studies: Management of severe autoimmune hemolytic anemia (AIHA) presenting with life-threatening anemia is challenging, particularly in the pediatric population.
Mortality rates in AIHA are typically low; however, in children, the rate may be as high as 4-11%.
Although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (WBEX) to successfully treat AIHA in older children and adults refractory to first line treatment.
To our knowledge, this is the first case report in which an infant with severe AIHA has been successfully treated with manual WBEX in an acute care setting.
Study Design/Methods: Case report format.
Results/Findings: A 2 month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (Hb)/hematocrit (Hct) of 1.6 g/dL/4.9%.
WBC counts (19 x 10 9 /L) were mildly elevated and platelet counts (410 x 10 9 /L) were within normal limits.
Her history was notable for upper respiratory tract infection 6 days prior to the onset of anemia.
Laboratory studies on admission showed hyperbilirubinemia (total 7.1 mg/dL, direct 1.4 mg/dL), normal LDH (318 U/L), and undetectable haptoglobin (<7 mg/dL) indicative of ongoing hemolysis.
Clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis.
She was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access.
The patient's blood group was O, Rh (D)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin (3-41 reactivity) with positive autocontrol.
DAT was 41 positive for anti-IgG and negative for C3 despite a positive cold antibody screen.
The patient weighed 6.9 kg with an estimated total blood volume of 620 mL.
She initially received simple transfusions totaling 20 mL/kg of least incompatible group O Rh(D)-negative RBCs with no incremental response.
Manual WBEX was then performed with 463 mL of reconstituted whole blood consisting of O, Rh(D)-negative RBCs and AB fresh frozen plasma (FFP) to an Hct of 40%, utilizing the central venous catheter.
No adverse events took place over the course of the 2 hour exchange.
Her one hour post-exchange Hb was 9.5 g/ dL and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin (21).
After initiation of steroid therapy (methylprednisolone, 2 mg/kg/day), she continued to improve clinically.
One week later, the patient was discharged home with a Hb of 11 g/dL.
One month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal IgM and IgA levels with markedly elevated IgG levels (3168 mg/dL).
At a subsequent follow-up visit 3 months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids.
Conclusion: We demonstrate a case of severe neonatal AIHA successfully treated with manual WBEX.
The main advantages of WBEX include removal of both autologous RBCs and plasma as well as infusion of allogeneic RBCs.
In this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation.
In summary, manual WBEX is a potentially safe procedure that may be performed in young children with severe AIHA.
ABSTRACT operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury.
Clerical and serologic investigations revealed no cause for hemolysis.
Mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered.
Study Design/Methods: In vitro simulated transfusions were performed via syringe.
Measurements included hematocrit (Hct), free hemoglobin, and visual hemolysis index.
Washed and unwashed red blood cells (RBCs) were tested with or without a one-way valve, using a 24 or 16 gauge (G) intravenous (IV) catheter.
Each one-way valve was used to test three identical samples.
Constant pressure was applied manually (rapidly, 1.431/-0.49 ml/ second) or with a mechanical syringe pump (slowly, 2 ml/min).
A subset of the manual transfusions was timed.
Control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter.
Results/Findings: The one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed RBCs (see Table) .
With the 24G catheter, the mean change in Hct was -3.531/-0.69% with the one-way valve and 0.221/-0.13% without (p<0.00001).
Comparing the one-way valves tested, differences in hemolysis were observed (change in Hct; p<0.0001).
During rapid manual transfusion with a 24G catheter and unwashed RBCs, hemolysis was greater for samples that took longer to transfuse 4.5ml when using a one-way valve (change in Hct versus time: r5-0.75, p<0.0001) compared to a significantly different (p50.0085) slight increase in hemolysis for samples that took less time to transfuse 4.5ml when not using a one-way valve (change in Hct versus time: r50.58, p50.23).
Correlations between time and hemolysis were similar, but insignificant using 24G with washed RBCs and the 16G IV catheter.
Conclusion: Mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve.
During rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis.
Background/Case Studies: Gerbich (Ge) antigens expressed on glycophorin C are present in 99.9% of the population.
Ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).
Ge antibodies also suppress erythropoiesis resulting in late-onset anemia.
We report a case of HDFN due to anti-Ge3.
Study Design/Methods: A woman of Paraguayan origin with prior terminated pregnancies presented at 24 weeks gestation with passive anti-D and an anti-Ge3 titer of 256.
She was D-and GE:-2,-3, 4 by antigen typing.
Her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at 37 weeks.
Cord blood was DAT positive for IgG; the eluate confirmed anti-D and anti-Ge3.
The birth hemoglobin (Hgb) was 12.6 g/dL, reticulocyte (retic) was 8.6%, bilirubin (bili) was 2.8 mg/dL; the infant was discharged.
On day 7 of life, the infant was referred to Pediatric Hematology for lethargy and poor feeding, with Hgb 7.6 g/dL, retic 2.6%, and bili 6.6 mg/dL.
Ge3-blood was not available from the blood center or rare donor registry.
The mother was B Rh-and baby was B Rh1.
Obstetrics had to authorize maternal blood donation due to her Hgb of 10.9 g/dL.
Maternal blood collection and RBC washing was expedited and the infant received 40mL of maternal RBCs within 24 hours, at which time his Hgb was 6.1 g/dL.
Post-transfusion Hgb was 10.8 g/dL.
One week later, the infant was symptomatic with Hgb 7.1 g/dL, retic 1.0%, bili 2.1 mg/dL.
A 2 nd aliquot of 60mL washed maternal cells was transfused.
Two weeks thereafter, the infant had Hgb 7.8 g/dL, retic 0.7%, anti-Ge3 titer 8, and needed another transfusion.
The maternal blood stored for just 3 weeks had hemolyzed necessitating a 2nd maternal donation for baby's 3 rd transfusion.
At 6 weeks, the infant's anti-Ge3 titer was 2, Hgb 9.2 g/dL, retic 1.7%; no transfusion was necessary.
At 8 weeks of life, Hgb was 10.2 g/dL, retic was 3.3%, and the baby was thriving.
Results/Findings: Serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-Ge3 HDFN.
Molecular analysis revealed that the mother was homozygous Ge3-negative GE*01.-03, the father had homozygous wild type GE*01, and the infant was heterozygous GE*01/GE*01.-03.
Conclusion: The infant had HDFN due to antibodies to the high prevalence Ge3 antigen.
The continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-Ge3.
Hemolysis of stored maternal blood was consistent with the absence of glycophorin C. This case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of HDFN resulted in a successful neonatal outcome.
Background/Case Studies: Patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes.
In pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice.
In addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult.
Because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution.
We designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions.
Study Design/Methods: A data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders.
We focused on RBC orders given the TRIPICU randomized clinical trial results (1) supporting a hemoglobin trigger of 7 g/dL in stable critically ill children and FFP since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (INR) values without bleeding.
Results/Findings: In 2016, 14, 247 RBC orders occurred and the top three patient groups were: 34% in congenital heart disease patients, 25% in hematology/oncology patients and 14% in neonates in the neonatal intensive care unit (NICU).
Average hemoglobin of every patient was 9.85 g/dL as measured in the 72 hours prior to RBC order placement.
In 2016, 3105 FFP orders occurred and the top three patient groups were: 46% in neonates in the NICU, 28% in congenital heart disease patients and 13% in pediatric intensive care patients.
Average INR of every patient was 2.09 as measured in the 72 hours prior to FFP order placement.
Conclusion: We have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital.
This serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses.
Background/Case Studies: Bacterial contamination of PLTs remains an ongoing threat to transfusion recipients.
Recently, a psoralen-based PR technology that reduces the replication potential of pathogens in stored PLTs was FDA approved.
We describe our approach to phasing PR-PLTs into our inventory, including preliminary results of an ongoing QA study of neonatal and pediatric (PEDS) recipients of PR-PLTs.
Study Design/Methods: Before the arrival of PR-PLT, we undertook an educational campaign for hospital administrators, IT staff, laboratory staff, clerical/clinical aides, nurses, and physicians.
We also contacted risk management and the hospital ethics committee.
Phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based PR-PLT product.
Shortly following the arrival of PR-PLT, we introduced day 5 bacterial "safety measure" testing of our conventional (C-PLT) supply.
A PEDS QA study monitored PLT utilization and adverse transfusion event reporting relating to both PR-and C-PLT transfusions.
This study evaluated neonates (0-4 months of age), infants (>4-12 months of age) and children (>12 months-18 years of age) who received at least one transfusion of PR-PLTs.
Results/Findings: Risk management and the ethics committee agreed that both PR-PLTs and bacteria tested C-PLTs would be the hospital standard of care.
PR-PLTs were phased in and transfused to patients based on ABO compatibility and expiration date, per routine, without regard for patient age or medical condition.
After 4 months, PR-PLT represented 30% of our platelet inventory (average daily PLT inventory: 45 units).
We encountered no complications with the PR platelet phase-in, either from a clinical, informatics or logistical perspective.
Due to the dual inventory, many PEDS patients in all age groups were transfused with both PR-and C-PLTs (Table) .
Two potential transfusion reactions (TRs) were reported over the study period in teenage recipients, one associated with a C-PLT and the other with a PR-PLT.
In both cases, the symptoms were ultimately attributed to an underlying medical condition.
No rashes were observed among 16 transfused neonates (0-4 m) who received any PR-PLTs and phototherapy.
Background/Case Studies: Packed red blood cell (PRBC) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children.
However, evidence shows that hemoglobin (Hgb) in PRBCs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased 2,3 DPG levels.
Standardization of PRBC transfusion practices in this population and the scientific evidence on which current practice is based is limited.
Additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events.
Study Design/Method: Medical records of 60 pediatric patients receiving PRBC transfusion over a 12 month period were retrospectively reviewed.
A total of 44 patients were identified as receiving allogeneic PRBC transfusion.
16 patients who received autologous blood (cell salvage) were excluded.
Patient characteristics, length of stay, PRBC transfusion volume, pre-and post-transfusion Hgb, and adverse events were collected.
Results/Finding: The average pre-transfusion Hgb was 10.6 g/dL with post-transfusion Hgb rising to 14.5 g/dL.
The mean PRBC volume transfused was 46.3 mL using a dose of 15mL/kg for all patients.
Complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (NEC), and death (Table) .
Conclusion: Evidence based transfusion guidelines are lacking in neonates and infants.
A typical dose of 10-15 mL/kg in a 2 kg patient, for instance, would translate into 3 full PRBC units (about 1000 mL) in an average size adult.
The current standard dose of 10-15 mL/kg yields very high increases in Hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity.
Additionally, many of these patients received volume reduced products which delivers a higher Hgb concentration per transfusion.
Dosing should be based on goal Hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear.
pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only 3 case reports suggesting this association.
However, each of these cases is confounded by other findings in addition to a Mycoplasma infection.
We describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a Mycoplasma infection without a detectable cold agglutinin.
Study Design/Methods: The patient is a 7month-old, previously healthy female infant who presented to the hospital with a 1-week history of fever, fatigue, decreased appetite, and pallor.
She was only treated with acetaminophen.
She also developed clear rhinorrhea the day before hospital admission.
At the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of 2.7 g/dL and 9.1%, respectively, platelets of 635,000, and a reticulocyte count of 10.3%.
All other elements of the complete blood count were within the normal reference range for age.
A complete metabolic panel revealed no abnormalities except for a total bilirubin of 4.9 mg/dL with a direct fraction of 0.43 mg/dL.
A FilmArray Respiratory Panel (BioFire Diagnostics; Salt Lake City, UT) detected Mycoplasma pneumoniae, while all other pathogens (19 total) were non-detectable.
The patient was started on a 5-day course of azithromycin (Zithromax).
Results/Findings: Prior to RBC transfusion, blood bank evaluation revealed that the patient was O-positive and had a stronglyreactive antibody screen.
Further testing demonstrated an antibody reactive with all reagent red blood cells.
The DAT was strongly reactive for IgG but very weakly reactive for C3.
An eluate was reactive with all reagent red cells tested.
Finally, a cold agglutinin study was negative with undiluted serum.
In addition to starting azithromycin, the patient was given IV methylprednisolone.
During her 8-day hospital course, the patient received 2 RBC transfusions on the day of admission and several RBC transfusions thereafter (see Table 1 ).
Despite transfusion, her hemolytic process persisted, so she was infused with a dose of IV immunoglobulin on hospital day 6.
Her hemoglobin rose to 8.4 g/dL on hospital day 7 and increased to 9.5 g/dL on hospital day 8.
At that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next 2 weeks.
She was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to 11.2 g/dL on day 57 after her hospital admission) with no recurrence of her hemolytic process.
Conclusion: M. pneumoniae infection is a typical cause of CAD and has only rarely been associated with warm autoimmune hemolytic anemia.
Our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant.
With the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a M. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. )
and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia.
The difficulty in eliminating the cord blood testing is the neonatologists' reliance of using ABO incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing.
Currently the TS requires all positive DAT tests to be communicated to the nursing staff immediately.
Given that the DAT strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the TS staff may also consider notifying nursing staff only for those patients whose DAT is 3 or 41.
Platelet and Leukocyte Immunohematology, Testing and Genetics Table 1 .
Of 53 pairs, 7 pairs were complete match (2/2), 26 pairs were partial match (1/2), 20 pairs were complete mismatch (0/2).
The matching rate of HLA-DPB1 in our study is 13%.
Conclusion: The matching rate of HLA-DPB1 in 10/10 HLA matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of HLA-DPB1 in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between HLA-DPB1 and unrelated hematopoietic stem cell transplantation.
This work was sponsored by National Science Foundation of China (81401732) Background/Case Studies: Thrombotic thrombocytopenic purpura (TTP) is caused by severely reduced activity of the von Willebrand factor-cleaving protease ADAMTS13.
Therapeutic plasma exchange (TPE) as well as immunosuppression minimize the morbidity and potential mortality of this presentation.
Absolute Immature platelet counts (A-IPC) have been shown to help diagnose and follow TTP patients' responses to therapy.
We report the case of a man with relapsing TTP, low ADAMTS13 with high inhibitor, treated with mycophenolate mofetil in which A-IPC-indicated an unexpected response to therapy.
Study Design/Method: A 56 year old male with a 7-year history of TTP, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing TTP.
Patient had been treated in prior admissions with TPE, prednisolone, rituximab, and cyclophosphamide with clinical improvement.
He was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, ADAMTS13 <5% and inhibitor of 3.6.
On day of admission platelet count was 95 x 10 9 /L which decreased within five days to 14 x 10 9 /L leading to initiation of daily TPE along with mycophenolate mofetil discontinuation just prior to TPE start.
Immature platelet fraction (%-IPF) and calculated A-IPC (%-IPF x platelet count) were obtained with daily pre-TPE CBC.
A-IPC ratio was calculated from baseline.
ABSTRACT Results/Finding: A-IPC and platelet count were 1 x 10 9 /L and 14 x 10 9 /L respectively.
Counts improved rapidly post-TPE initiation and after one TPE his A-IPC tripled to 3.2 x 10 9 /L achieving the ratio of 3 previously shown to be diagnostic of TTP.
On day 5 his A-IPC and platelet counts had improved to 7.5 x 10 9 /L and 218 x 10 9 /L respectively.
Absence of anti-PF4 antibodies ruled out heparin-induced thrombocytopenia at this time.
On day 6 he had an unexpected decrease in both A-IPC and platelet count to 4.8 x 10 9 /L and 132 x 10 9 /L respectively, worsening by day 8 to 1.7 x 10 9 /L and 40 x 10 9 /L respectively despite daily TPE.
Patient received 25 additional TPEs that failed to improve A-IPC or platelets which on day 32 were 0.4 x 10 9 /L and 13 x 10 9 /L respectively.
A-IPC had remained at this level for 16 days suggesting that the observed decrease was irreversible.
ADAMTS13 activity remained <5% low with a high inhibitor.
Patient's clinical condition continued to deteriorate and family placed patient on comfort care.
Conclusion: TTP patients have low A-IPC and PLT counts at presentation, with the former improving first post-TPE initiation.
Despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low A-IPC.
In the setting of TTP, or relapsing TTP use of immunosuppression should be closely followed and A-IPC may aid in establishing early if therapy is affecting platelet production.
Application of Luminex Bead Technology to Detect HPA-1a, HPA-3a, and HPA-5a Antibodies Su-dan Tao*, Ying Liu, Yan-min He, Ji He and Fa-Ming Zhu.
Blood Center of Zhejiang Province, Key Laboratory of Blood Safety Research, Ministry of Health Background/Case Studies: Detection of antibodies against human platelet antigens (HPAs) is crucial for patients' refractory to platelet transfusion therapy.
In the text, Luminex bead coupled with anti-GPIIb/IIIa and anti-GPIa/IIa monoclonal antibody was implied to detect HPA-1a, HPA-3a, and HPA-5a antibodies, and the sensitivity of Luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (MAIPA) assay.
Study Design/Method: Monoclonal antibodies P2 and Gi9, specific for platelet glycoproteins GPIIb/IIIa and GPIa/IIa, were separately coupled to Luminex xMAP beads.
Four standard sera, containing anti-HPA-1a, anti-HPA-3a, anti-HPA-5a and anti-HPA-5b respectively, were bought from NIBSC; three negative sera without HPA antibodies were prepared from AB type blood donors.
Platelets (containing HPA-1aa, HPA-3ab and HPA-5aa) were collected and reacted with anti-HPA-1a, anti-HPA-3a, anti-HPA-5a and anti-HPA-5b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins.
The beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a Luminex100.
The HPA-1a serum was diluted to 10 serial dilutions (from neat to 1/502) to test the sensitivities of MAIPA and Luminex beads assay.
The two methods were then used to test five blinded samples which were collected from FMAIT patients.
Results/Finding: Luminex bead technology showed that the MFI values of HPA-1a, HPA-3a, HPA-5a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( .08 vs 37.05), which implied that the Luminex bead technology could specifically identify negative and positive sera of anti-HPA-1a, anti-HPA-3a, anti-HPA-5a.
Furthermore, because the platelet was HPA-5aa, the HPA-5b serum did not react with the coupled beads with MFI was comparable to negative control (286.59 vs 127.25).
The sera were re-tested by MAIPA and the results of which were comparable to Luminex bead technology, illustrating that detecting HPA antibodies by Luminex beads technology was successful.
The sensitivity of Luminex bead assay and MAIPA to detect anti-HPA-1a was 1/128 (0.78IU/ml) and 1/64 (1.56IU/ml), respectively.
No cross-reactivity was observed with the samples containing HLA, ABO or other platelet antibodies.
All results of five blinded samples tested by Luminex assay showed that four sera were positive for GPIIb/IIIa antibodies which were consistent with MAIPA results.
Conclusion: The Luminex beads coupled with GPIIb/IIIa and GPIa/IIa monoclonal antibodies could be successfully used to detect HPA-1a, HPA-3a and HPA-5a antibodies via the epitopes on platelet glycoproteins.
The sensitivity of Luminex technology was higher than MAIPA technology.
(aHUS) is a thrombotic microangiopathy (TMA) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin.
The literature suggests the presence of pathogenic mutations in complement proteins in 50% of cases of aHUS.
There is a lack of well-defined recommendations regarding testing for genetic aHUS.
Complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs.
We reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population.
Study Design/Method: We performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from 1 January 2014 to 31 December 2016.
Clinical history was solicited by genetic counselors.
Cases were classified by the authors as primary aHUS (TMA and renal failure without identifiable cause), secondary TMA (TMA and renal failure with identifiable cause previously associated with TMA) or non-TMA.
The test panel identified variants in complement proteins (CFH, CFI, MCP, Factor B, C3, C4BP, THBD, DGKE, CFHR3, CFHR1, CFHR4 and CFHR5) that were classified as VUS (variances of uncertain significance), pathogenic or benign by the American College of Medical Genetics.
Chi square analysis/Fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary aHUS versus secondary TMA.
Independent sample t-test was used to compare differences in continuous variables between primary aHUS and secondary TMA.
Results/Finding: Of 134 patients tested, pathogenic mutations were detected in 13% (18/134) and VUS in 35% (47/134).
20% (27/134) of patients did not fulfill criteria for TMA; no pathogenic mutations were found in this group and 9 (33%) had VUS.
31% (42/134) of patients had primary aHUS; of these, 28% (12/42) had pathogenic mutations and 40% (17/42) had VUS.
48% (65/134) of patients had secondary TMA; of these, 9% (6/65) had pathogenic mutations and 32% (21/65) had VUS.
In patients with pathogenic mutations, 39% (7/18) were children, 22.5% (4/18) had a positive family history of aHUS and 28% (5/18) had recurrent disease.
Patients with primary aHUS had a significantly lower age at presentation (22 6 18 vs. 33 6 20 yrs; p-value: 0.005) and a higher proportion of pathogenic mutations (28% vs. 9% p-value: 0.009) compared to patients with secondary TMA.
Gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups.
Conclusion: We found a lower frequency of patients with pathogenic mutations compared to reported literature.
Our data suggests that patients with secondary TMA should be carefully evaluated prior to ordering genetic testing and those without TMA should not undergo this test.
Counting of Platelets in Platelet Concentrates on Hematology Analyzers PentraXL80 and Sysmex XN9000 Compared with a Flow Cytometric Method Farshid Ezligini 1 , Kjersti Roen Eriksen 1 , Annette Vetlesen 1 , Thomas Larsen Titze 1 and Geir Hetland* 1,2 .
1 Oslo University Hospital, 2 University of Oslo Background/Case Studies: Hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (PLT) concentrates (PCs).
A flow cytometric method for counting of PLT in PCs has been developed as validation tool (van der Meer et al, Transfusion 2012).
Therefore, it is pertinent to evaluate PLT counting in BCs on hematology analyzers with this validation method in a flow cytometer.
Study Design/Methods: Samples from ten apheresis PCs and 33 buffy coat-derived PCs were subjected to PLT counting on hematology analyzers PentraXL80 (Horiba ABX, Montpelier, France) and XN9000 Sysmex TOA (Kobe, Japan) (both impedance score), and additionally, diluted and stained with anti-CD41a FITC in TrueCount tubes (BD Biosciences)(internal bead standard) for measuring in a Gallios flow cytometer (Beckman Coulter, Indianapolis IN, USA).
Results were analyzed by Paired Samples Test and shown in Bland-Altmann plots.
Results/Findings: Mean PLT values x10 9 /L 6 SD were 819 6118, (<) 1106 6137, (<) and 1195 6176 for counting by Sysmex TOA, PentraXL80, and the Gallios flow cytometer, respectively.
Sysmex count was the very lowest 129A TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT (31.4% less than for flow cytometry), but all PLT counts were significantly different (p<0.001), although least so (7.4%) between Pentra and flow cytometry.
Conclusion: As validated by the flow cytometric method, PentraXL80 seems suitable for routine quality control of PCs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting.
The much lower PLT count on Sysmex may reflect its optimization for PLT counting in whole blood rather than in PCs.
Fast, Precise & Easy HPA Typing with Real-Time PCR Jonathan Downing 1 , Arishma Lata 1 , Roland Russnak 2 , Zachary Antovich 2 , Heather Dunckley 1 and Thierry Viard* 2 .
1 New Zealand Blood Service, 2 Linkage Biosciences Background/Case Studies: The interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis.
Human Platelet Antigens (HPA) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible HPA.
Thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders.
The New Zealand Blood Service performs HPA typing on a pool of platelet donors to provide compatible transfusions where the need arises.
The molecular basis of most HPAs has been characterized as generally caused by a single-nucleotide polymorphism (SNP).
HPA typing has typically been performed using PCR-SSP, a method that utilizes time-consuming post PCR analysis steps.
The aim of this study was to evaluate the use of real-time PCR-based techniques in a transfusion laboratory setting.
Study Design/Method: We evaluated a commercially available solution which consists of 24 reactions that identify both variants of 12 relevant SNPs located within HPA genes (HPA-1 through HPA-11, and HPA 15).
Genomic DNA purified from 48 blood samples, previously genotyped for HPA-1,-2,-3,-4,-5 and -15 by our in house PCR-SSP method were used in this study as validation samples.
Results/Finding: Results of the validation samples were 100% concordant with typing obtained by PCR-SSP.
The Real-Time PCR approach overcomes the major challenges of HPA molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time.
The analysis is facilitated by a software which generates the results.
With less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately 90 minutes.
Further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced.
The Real-Time PCR approach with automated analysis was implemented by the New Zealand Blood Service Tissue Typing laboratory in late 2016 and to date has tested 749 DNA samples from 400 blood donors (349 donors were tested in duplicate).
Concordance between the sample replicates was 100%.
There were 24 occasions where the assay had to be repeated, giving a repeat rate of 3.2%.
Occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required.
This occurred most commonly with the HPA-3 (4.7%) and HPA-5 (1.2%) assays.
Conclusion: Real-Time PCR with automated analysis provides an effective, robust an accurate method for molecular HPA genotyping.
With its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting.
Genetic Variation of CD36 Antigen Deficiency Expression in Jiangsu Chinese Han Population Qing Chen* 1 , Jianyu Xiao 1 and Chengyin Huang 2 .
1 Jiangsu Province Blood Center, 2 Jiangsu province Blood Center Background/Case Studies: CD36 has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-Caucasian.
CD36 deficiency varies widely among different ethnic populations, with the frequency of 3-11% in Asians and 2.4% of African Americans, respectively.
However, there is little information on the molecular basis of individuals with CD36 deficiency in Jiangsu Chinese Han population.
Study Design/Method: To investigate platelet CD36 expression levels and to determine the molecular basis of CD36 deficiency on the platelet surface of the Han population in Jiangsu region.
CD36 expression levels on platelets were detected by flow cytometry among 243 blood donors in Jiangsu region.
Donors without CD36 antigen expression on their platelet surface were further to be determined the expression of CD36 antigen on their peripheral blood monocyte cells.
The coding exons of CD36 gene and adjacent introns were amplified and sequenced in CD36 deficient individuals.
Results/Finding: Among these 243 blood donors, CD36-deficient and CD36-expression individuals were 2.47% (6/243) and 97.53% (237/243), respectively.
The frequencies of Type I and Type II CD36 deficiency among the study population were 0.41% (1/243) and 2.06 % (5/243), respectively.
Among 237 individual with platelet CD36 expression, according to mean fluorescence intensity (MFI) value, 45, 141 and 51 individuals showed low, moderate and high expression levels of CD36, respectively, and their MFIs were 1725.9 6 343.6, 3876.1 6 788.5 and 8431.6 6 529.9 (P<0.05), respectively.
The type I CD36 deficiency individual were heterozygous for 1200-13A>G and 430-14C>G, respectively.
Among Type II CD36 deficiency individuals, two harbored a T insertion at position 560 in exon 6 which caused frameshift at codon 187; one has a T>C exchange at position 538 in exon 6 which resulted in a tryptophan to arginine substitution at codon 180; one has a A insertion before the 17th bp of the start codon ATG in the promoter region; one were heterozygous for 748 1 2T>C and 1006 1 2T>G, respectively.
Conclusion: Platelet CD36 surface expression levels were diversified in the Jiangsu Chinese Han population.
The frequency of the Type II CD36 deficiency was higher than that in Type I.
The study findings indicated that the frequency of CD36 deficiency in the Chinese population is slightly lower than that in other Asian countries.
Background/Case Studies: CD36-deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders.
The frequency of platelet CD36-deficient individuals widely varies among ethnic groups, with 3% to 11% in Japanese, 8% in sub-Saharan Africans, 2.4% in African Americans, and 0.3% in Caucasians.
Although some studies of CD36 deficiency are focused on the Asian populations, relatively little information has been reported in the Chinese population.
Here we investigated the CD36 expression on platelets in large samples of the Eastern Chinese donors.
Study Design/Methods: Peripheral blood samples were collected from 1282 unrelated platelet-apheresis donors in the Eastern China.
The expression of CD36 antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (FITC-anti-CD36 and PEanti-CD41).
The isotype control (FITC-mouse IgG) was also analyzed to calculate a reference range of CD36-nagtive phenotype.
For those donors with CD36-negative platelets, CD36 antigen expression on monocytes was analyzed further to distinguish between CD36 type I and type II deficiency.
Flow cytometric parameters were statistically analyzed by Mann-Whitney test.
The work was supported by National Natural Science Foundation of China (81570170) and Zhejiang High-Level Innovative Health Talents.
Results/Findings: The MFI (mean fluorescence intensity) of platelet CD36 in all 1282 samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype.
On account of this limitation, we classified the CD36 phenotypes using the (mean 1 3SD) of the background MFI observed in isotype controls.
Forty-three samples were detected as CD36 deficiency on platelet, in which one sample was CD36 negative both on platelet and monocyte.
The frequency of CD36 type I and type II deficiency in the Eastern Chinese donors was 0.08% and 3.3%, respectively.
The average MFI of CD36 deficiency samples was significantly lower than CD36 positive samples (15.2 6 7.9 vs 79.8 6 37.8, P< 0.0001).
Conclusion: The frequency of platelet CD36 deficiency in the Eastern Chinese donors was close to Japanese and African Americans.
It means that the possibility of CD36 antibody occurred by pregnancy and transfusion in this population is existed.
It is useful to find and register CD36-deficient donors by large-samples screening for potential immune thrombocytopenia patients with CD36 antibody.
Background/Case Studies: CD36 (GPIV, chromosome 7q11.2) is an 88 kDa glycoprotein expressed on multiple cell types including platelets (PLTs), monocytes (MONO), & erythroblasts.
Although rare among whites, CD36 deficiency (CD36-n) is observed in 3-10% of Africans (T1264G) & is classified as either type I (CD36-n PLT, CD36-n MONO) or type II (CD36-n PLT, CD361 MONO).
An acquired type II CD36-n phenotype can also be observed in the setting of myelodysplastic syndrome (MDS).
Type 1 CD36-n individuals can develop anti-CD36 alloantibodies with PLT refractoriness & neonatal alloimmune thrombocytopenia.
We report a case of profound PLT refractoriness caused by anti-CD36 in a patient with newly diagnosed MDS.
Study Design/Method: HLA antibody testing was performed with a commercial bead-based fluorescent assay.
CD36 phenotyping (PLT, MONO) of patient & family members was performed by flow cytometry (FC).
CD36 staining of bone marrow was performed by immunohistochemistry.
PLT crossmatching (PLT-XM) was performed by the American Red Cross.
PLTspecific alloantibody testing & CD36 DNA sequencing were performed at a commercial reference laboratory.
Results/Finding: The patient was an 80 year-old, group O1 African-American male who presented with blurry vision & lightheadedness.
Complete blood count findings were significant for hemoglobin 4.4 g/dL & PLT count 5K/mL.
Bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, 5-10% blasts & a complex karyotype with del(7)(q22q34) consistent with MDS.
PLT refractory work-up was initiated after repeated PLT transfusion failures with corrected count increments (CCIs) < 5.
HLA antibody testing was negative (class I panel reactive antibody (PRA)50%).
The patient was PLT-XM-incompatible with most donors (10/14).
A trial of 4 group O, PLT-XM-compatible PLTs was unsuccessful (CCI 1).
Subsequent testing for PLT-specific alloantibodies identified anti-CD36.
FC-phenotyping showed no CD36 on patient's MONO or PLT, consistent with type I CD36-n.
Preliminary DNA results show that the patient is heterozygous for T1264G.
Because CD36-n apheresis PLT were unavailable from blood suppliers, the patient's 3 children & grandson were screened as possible donors: All showed normal CD36 expression on PLTs.
Trial of eltrombopag & romiplostim was attempted with no improvement in PLT count.
Repeat HLA antibody testing (day 16) demonstrated new class I alloantibodies (PRA 5 55%) in response to transfusion (21 apheresis PLTs, 5 RBCs).
Given his PLT refractoriness & poor prognosis, the patient opted for hospice.
Conclusion: We describe a patient with CD36-n & severe PLT refractoriness in the setting of new MDS, and 7q-chromosomal abnormalities.
The absence of CD36 on PLT & MONO support congenital type 1 CD36-n although a contribution by the patient's underlying MDS cannot be excluded.
Rapid Platelet Donor Classification: HLA & HPA Profiles By "Leansequencing" without DNA Purification Dipika Patel 1 , Kristopher Fernandez* 1 , Eric Senaldi 2 , Pascal George 2 , Michael Seul 1 and Ghazala Hashmi 1 .
1 BioMolecular Analytics, 2 Central Jersey Blood Center Background/Case Studies: Prophylactic platelet transfusion is the standard of care for managing thrombocytopenia.
In the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy.
While the benefits of customizing transfusion therapy have long been recognized (Gmur1978 http://bit.ly/2q51heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of DNA analysis.
To address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined HLA class I and HPA signature without DNA purification using a novel "LeanSequencing" process.
Study Design/Method: Under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $100 adult donors who had made !
6 donations in the previous 12 months.
Samples (labeled with study barcodes) were shipped weekly to BioMolecular Analytics ("bmx") for preparation of "crude extracts" for LeanSequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps.
Briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for 2, 3, 4, 5, 6, 7, 8, 9, 11, 15 and for HLA Class 1 (A,B,C) , the latter using a proprietary design that limits analysis to informative alleles in the HLA sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software.
A subset of crude extracts was purified and analyzed side by side with positive and negative controls.
Results/Finding: Crude extracts from buccal swabs produced viable profiles for HPA as well as HLA class I with significant savings in time-to-result.
As an illustration, the table reports allele frequencies for platelet-antigens ("HPA") that are consistent with a predominantly Caucasian or Hispanic platelet donor population (http://bit.ly/2pDplF8) in HW equilibrium.
Similarly, HLA-class I haplotype frequencies were determined.
Conclusion: LeanSequencing lends itself to the rapid determination of HLA-class I and HPA signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project.
The process could be readily implemented to another site using the elements and process developed.
The "Pool & Plex" process and the early donor recruitment enables economies of scale for matched donor procurement.
The Serological Characteristics and Heritage Background of a Novel HLA Allele, HLA-a *26:82 Chuan-fu Zhu*, Yong-hong Song, Xiang-min Nie and Wen-ben Qiao.
Blood Center of Shandong Province Background/Case Studies: There are 16,429 HLA alleles documented according to the IMGT / HLA Sequence Database in Janury2017, And more than 80% of them were identified in the last 10 years.
Besides sequences many of the novel HLA alleles have not been analyzed their serological reactivities.
HLA-A *26:82 allele was fist detected in our laboratory during our HLA typing for China Bone Marrow Donor Program(CMDP).
For further study, the serological characteristics and heritage investigation were performed.
Study Design/Methods: The routine HLA tying for the potential donors from CMDP were performed by bi-allelic Sequence-Based Typing method,using a commercial kit (ROSE Europe GmbH, Frankfurt, Germany).
In the case of no full matched HLA typing results, group specific HLAssure-SE SBT Typing Kit (Texas Biogene Inc., Taipei, Taiwan) was employed to identify the nucleotide sequences of the novel allele.
Fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele.
The HLA serological specificity was indicated by One Lambda(ASN72D)HLA kit.
Results/Findings: No full matched result was obtained at HLA-A locus in HLA typing for a donor,which suggested the possible existence of a novel allele.
The latter nanalysis indicated that the proband have a nove1 nucleotide sequences at HLA-A locus, the new sequences was most close to those of HLA-A *26:01:01:01, but 1 nucleotide substitution in exon 4, by nt 746 C-A (codon225 ACC-AAC), which resulted in one aminoacid substitution ,Thr-Asn.
The novel HLA-A allele was officially named as HLA-A Background/Case Studies: Anti-D is a frequent cause of hemolytic disease of the fetus and newborn (HDFN).
As a rule, immunization occurs in D negative pregnant women, but occasionally anti-D is also observed in carriers of D variants.
Currently, maternal plasma analysis for determination of the fetal RHD status became an exciting new tool for the management of D-negative pregnant women, but one of the challenges in non invasive fetal RHDgenotyping is the presence of D variants in the pregnant women.
We present a case of a 13 year-old pregnant woman typed as AB1, who delivered a baby affected by severe HDFN.
The newborn was typed as B1 and presented a positive direct antiglobulin test (DAT) with an anti-D identified in the eluate.
The baby was treated by exchange transfusion and the mother's sample was investigated.
Study Design/Method: Serologic testing was done by hemagglutination in gel cards.
Genomic DNA was extracted from whole blood by spin column and all RHD exons were sequenced by Sanger sequence method.
Results/Finding: The mother's RBCs reacted 41 with the four monoclonal anti-D used (IgM clones P3x61 and RUM 1 and the blends clones TH281MS26 and D1751D415) and were typed as C-c1E-e1.
An anti-D was identified in her serum.
Molecular analysis showed the 410C>T and 455A>C in exon 3, the SNP 509T>C changes in exon 4 and the 667T>G nucleotide change in exon 5.
The set of SNPs found is similar to the molecular background of DOL3, except for 455A>C change.
Conclusion: This novel set of SNPs found in this mother is related to a novel RHD allele leading to a partial D antigen involved in the production of an anti-D that can cause severe HDFN.
This finding shows the need to elucidate the clinical significance of different RHD genotypes in various ethnic backgrounds.
The and Erytra V R (the routine reference platform) was performed.
A total of 1089 immuno hematological tests (465 ABO/D grouping (including 33 newborn samples), 12 extended erythrocytic phenotype, 562 antibody screening, 14 antibody identification, 16 DAT) and 20 crossmatches were performed on patient's whole blood samples.
The Erytra Eflexis V R performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations.
Concordance between systems was assessed and discrepancies were analyzed.
The following performance metrics were assessed: time to first result (TTFR), turn-around time (TAT) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system.
For the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements.
A threshold for in vitrodetection of anti-D gamma globulin was also determined.
V R Analyzer and the reference method were obtained in 99.2% of the ABO/D tests (n5265), 99,7% of the antibody screening tests (n5377), 88,8% of the antibody identification tests (n59) and 100% of the DAT tests (n510).
There were 4 discrepancies (2 ABO/D for the same patient, 1 for antibody screening and 1 antibody identification: In both cases, the Erytra Eflexis V R could conclude whereas Erytra could not due to a poor reaction.
Use of the STAT mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than 10 min).
Detection threshold of the D antibody was assessed at 2.5 ng/ml (0.0125 UI/ml) whereas the French recommendations are 20 ng/ml.
The possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation.
The impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility.
V R results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory.
Erytra Eflexis V R meets both the requirements for French regulatory in immunohematology and for ISO 15189 accreditation.
Background/Case Studies: Kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (HDFN).
We report a case of HDFN secondary to anti-Kpb that resulted in multiple intrauterine transfusions of Kp (b-) donor cells and hemolytic anemia upon birth.
Case: A 31 year old G5P3 presented during her fifth pregnancy with anti-Kpb with an initial titer measured of 64.
By history, the anti-Kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated.
The patient's antibody titers peaked at 16 during the fourth pregnancy which resulted in a healthy male without anemia or jaundice.
.
In the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery Doppler exams (1.7 MoMs) peaking at 27 weeks.
This resulted in three intrauterine transfusions.
Due to potential labor and the finding of reversed diastolic flow on middle cerebral artery Doppler studies, a finding that has been associated with impending intrauterine fetal demise, Caesarean delivery was performed at 35 weeks gestation.
The baby boy required phototherapy for hyperbilirubinemia.
The indirect bilirubin at birth was 3.4 mg/dL with 13.6 g/dL hemoglobin.
The baby typed as O Positive, Kp (b1) with a micro positive DAT.
The antibody workup revealed an anti-Kpb.
Continued hemolysis required one more transfusion at 6 weeks of age.
The positive DAT and passively acquired anti-Kpb were no longer detected by 8 weeks of age.
His hemoglobin recovered to 9.0 g/dL with an indirect bilirubin of 1.4 mg/dL at 9 weeks of age.
All clinical signs of hemolytic anemia were resolved.
Study Design/Method: Serologic testing included PEG IAT by tube methods.
Acid elution was performed using Immucor Gamma ELU-KIT II.
Molecular testing was performed using Immucor Bio-Array HEA platform.
Results/Finding: Antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies.
A new weakly reacting anti-S was detected on the day of the delivery.
The baby typed as S positive however the anti-S was not detected in an eluate prepared from the baby's red cells.
All of the intrauterine transfusion units were S negative.
Conclusion: To our knowledge only five case reports have been described for anti-Kpb which resulted in moderate to severe HDFN.
Pregnant mothers with anti-Kpb detected should be monitored closely.
Background/Case Studies: In some clinical cases, the C3d-specific DAT may be too insensitive to detect low, but significant levels of C3d, or it may be inconclusive due to spontaneous red cell (RBC) aggregation.
Further, the DAT is not well suited to quantify the number of immunoprotein molecules on RBCs, since a "1111" reaction corresponds to about 500 molecules/ cell.
A number of flow cytometric methods for the detection of RBC-bound C3d have been published.
However, these are mainly designed to quantify the fraction of RBCs with C3d-sensitization.
The aim of this study is to present a flow cytometric method for the quantification of the level of RBCbound C3d.
Study Design/Method: Ten microliters (uL) of 1:80 (after documenting experimentally that this amount ensured maximum binding of anti-C3d) mouse monoclonal anti-human anti-C3d (Abcam, clone 7c10) were added to 5 uL of a 2.5% RBC suspension.
After incubation for 60 minutes at 4C, samples were washed x3, and 25 uL of 1:10 diluted anti-mouse-F(ab)2-PE (RO480, DAKO) were added.
After incubation at 4C, samples were washed and resuspended before being acquired on a flow cytometer (Becton Dickinson FACSCanto II).
To enable calibration of fluorescence signals in antibody binding capacity (ABC), a calibration standard (DAKO QIFIKIT) stained with RO480 was run in parallel with all experiments.
Background fluorescence (in ABC) was subtracted to yield net ABC values corresponding to specific staining with anti-C3d.
The assay, in parallel with our routine DAT (DC-Screening I, ID-Card, gel card, BioRad) was applied to a series of A1 RBCs stained with 10 levels (2fold dilution, 1:1 -1:512) of O serum with high titer anti-A.
To estimate the normal range of RBC-bound C3d, EDTA-stabilized samples from 4 healthy donors were tested.
Finally, the assay was applied to a sample from a patient with clinical AIHA with an inconclusive DAT due to unspecific DAT polyreactivity.
Results/Finding: The correlation of the net level of RBC-bound C3d (values ranging from 0 to 3,393 ABC) with level of 0-serum dilution (used to sensitize A1 RBCs) proved to be highly linear (logarithmic vs. logarithmic plot; r2 5 0.97, p < 0.0001).
Compared with DC-Screening 1, the sensitivity of the flow cytometric assay was superior.
It detected C3d sensitization at least 4 dilution steps further.
The median normal level of RBC-bound C3d was 11 ABC (range 7-20 ABC, n54).
The assay enabled demonstration of specific C3d-sensitization in the patient; the level of RBC-bound C3d in the sample was significantly elevated (1,907 ABC).
Conclusion: The presented flow cytometric assay is capable of quantifying the level of RBC-bound with a high degree of linearity and analytical sensitivity.
Further, it is capable of quantifying the level of RBC-bound C3d in DAT polyreactive samples.
Background/Case Studies: ABO blood group system of red blood cells (RBCs) consists of A and B oligosaccharide antigens and anti-A and anti-B antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(Landsteiner's Law).
Because of the expression of those antigens on some epithelial and endothelial cells in the body, the ABO matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation.
In spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of ABO.
Forssman (FORS) system is another RBC blood group system which consists in a glycosylation polymorphism specified by the GBGT1 gene.
In humans, the ABO and GBGT1 genetic loci are located on chromosome 9q34, and the functional alleles encode A and B glycosyltransferases (AT and BT) and Forssman glycolipid synthase (FS), which catalyze the last biosynthetic steps of A and B, and Forssman (FORS1) oligosaccharide antigens.
The molecular genetic bases for allelism of those two systems in humans have been well-elucidated.
The ABO and GBGT1 genes are also present in some other species in addition to humans.
However, the presence/absence and functionality/non-functionality are species-dependent.
Molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown.
Study Design/Methods: Utilizing genomic information available from Gen-Bank and Ensembl databases, the gene maps of the chromosomal region surrounding the ABO and GBGT1 genes have been constructed of 88 vertebrate species.
Results/Findings: Extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations.
However, numerous differences were also identified.
These include chromosomal rearrangements, as well as the insertions and amplifications of specific genes.
Interestingly, the ABO and GBGT1 genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution.
Conclusion: Genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments.
Therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes.
Alloimmunization Despite Phenotype Matching in a Patient with Sickle Cell Disease and a Complex RHCE Genotype Jessica Kneib* and Emily Coberly.
University of Missouri Health Care Background/Case Studies: Red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease.
Sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least C, E, and K1 antigens to reduce this risk.
Unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial RHCE variants.
Study Design/Method: A 14 year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion.
TRANSFUSION 2017 Vol.
57 Supplement S3 The patient's blood type was O positive and her red cells had been previously phenotyped as C-, c1, E-, e1 and K1-.
An antibody screen was positive, and antibodies against C and e antigens were identified in the plasma.
The patient had only received phenotypically matched units negative for C and E antigens for all previous transfusions at our institution, based on her known red blood cell phenotype.
Blood samples were sent to a reference laboratory for molecular testing to look for partial RHCE variants that might explain the antibody development.
Results/Finding: Molecular testing was performed to reveal the presence of two different partial RHCE alleles, resulting in a predicted phenotype of D1, C-, E-, partial c1, partial e1.
The probable RHCE genotype, RHCE*ce-JAL/RHCE*ce733G, results in partial expression of both c and e antigens.
In addition to the known risk of alloimmunization against the absent C and E antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens.
Based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody.
Conclusion: Although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial RHCE genotypes.
In this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against C, c, E, and e antigens.
As the patient had already made alloantibodies against C and e antigens, it was determined that she would require units that were molecularly matched to her RHCE variants for all future transfusions.
This case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial RHCE variants.
Altered Splicing in the RHD*Weak D Type 2 Allele Associated with the Skipping of Exon 9 in a Pregnant Woman and Her Newborn Carolina Bonet Bub* 1 , Maria Giselda Aravechia 1 , Thiago Costa 1 , Marilia Sirianni 1 , Eduardo Bastos 1 , Leandro Santos 1 , Lilian Castilho 2 and Jos e Kutner 1 .
1 Hospital Israelita Albert Einstein, 2 Hemocentro Unicamp Background/Case Studies: RHD*weak D type 2 is a variant commonly found in Caucasians associated with a weak D phenotype.
As previously reported (Vege et al, Transfusion 2007) the c.1154G>C change (p.G385A), which characterizes the RHD*weak D type 2 allele is a splicing variant that induces skipping of the whole RHD exon 9.
We report an altered splicing in the RHD*weak D type 2 allele associated with the skipping of exon 9 in a pregnant women and her newborn with weak D expression.
Study Design/Method: The D antigen expression was evaluated with 4 commercially available monoclonal anti-D reagents: 1 blended IgM/IgG (clones TH-28/MS-26), 2 IgM (clones MS201 and P3x61) and 1 IgG (MS26) in tube and on gel cards.
C, c, E and e phenotyping were performed in gel.
RHD genotyping was performed with the RHD BeadChip platform from Immucor.
Direct automated sequencing of the 10 RHD exons and flanking intron regions was performed by the Sanger dideoxy method.
Results/Finding: Both pregnant women and newborn samples were phenotyped as D1 w C-c1E1e1.
The samples showed weak hemagglutination reactions (11/21) with all anti-D clones used.
RHD Beadchip results showed the LS* signal indicating a possible deletion of exon 9 in both DNA samples.
Sequencing showed the c.1154G>C change and the intronic c.1154-8T>A and c.1154-31T>C substitutions, which are associated to the RHD*weak D type 2 allele.
Conclusion: Our results showed that c.1154G>C associated with c.1154-8T>A and c.1154-31T>C variations had probably a functional impact on splicing inducing exclusion of exon 9 in both DNA from mother and newborn.
This finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-D IgG prophylaxis for women with weak D type 2.
Background/Case Studies: Sickle cell disease (SCD) patients require red blood cell (RBC) transfusions to minimize disease-specific symptomatology.
Previous studies have shown that more than 50% of children with SCD receive at least one RBC transfusion in their lifetime.
Both simple transfusions and erythrocytapheresis are associated with increased risk of RBC alloimmunization.
Published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies.
Therefore, we looked at the alloimmunization rates of pediatric patients with SCD in the Unites States (US) and other countries.
Study Design/Method: A literature search was performed for studies published on alloimmunization rates of SCD pediatric patients including HbSS, sickle beta-thalassemia and HbSC.
We evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per 100 transfused units across world literature and compared them using chi-square analysis.
Results/Finding: Fourteen studies reporting data to derive alloimmunization rates of pediatric SCD patients were found.
These included eleven US studies with 1,057 patients and 3 studies from other regions (Brazil, Egypt and France) with 641 patients.
Majority of patients included in the studies had HbSS disease.
Patients received either episodic, chronic simple transfusions or erythrocytapheresis.
Age range for the US studies was 0 to 26 years and for the other countries 0 to 20 years.
Available data from 5 US studies included a total of 91 alloantibodies, the most frequent of which were antibodies to C, E, Kell, M, S and Kidd antigens (18.7%, 16.5%, 15.4%, 7.7%, 7.7% and 6.5% respectively).
Alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per 100 transfused units in other studies.
We evaluated rates using both approaches as per available data.
US had an alloimmunization rate of 16.5 % (14.1 to 19.2, 95% CI) vs. 9.4% for non-US studies (7.3-11.8, 95% CI) (p50.0008) and 134A TRANSFUSION 2017 Vol.
57 Supplement S3 more alloantibodies per transfused patient (0.25 vs. 0.096, p50.0001).
Similarly, the number of alloantibodies per 100 transfused units in the US, evaluated from five studies, was higher compared to a large French patient cohort (0Á68 vs. 0.33, p50Á0005).
Average number of RBC units transfused per patient in the US was also higher compared to data from France (77 vs. 45, p50.0001).
Conclusion: Despite limited studies available to compare alloimmunization rates in pediatric SCD patients in the US and other countries, the overall rates are higher in the US.
Though no definitive reasons could be concluded from the available data, limiting the number of RBC exposures, i.e.
units transfused in non-critical conditions could lead to lower alloimmunization rates.
Results/Findings: A post-transfusion sample was referred to the IRL for a TRXN investigation.
There were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum.
ABO/Rh and crossmatches using LO-ION TM were repeated on the pre-and post-transfusion samples with no discrepancies.
The post-transfusion DAT was positive with a negative eluate.
The hospital requested another unit before the investigation was complete.
Antibody identification on the post transfusion sample with LO-ION TM was negative.
Suspecting a weak antibody, additional investigation using PeG TM on both samples revealed an anti-C. No additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using PeG TM .
Conclusion: The patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-C in the patient's plasma/serum against C antigen on the transfused cells.
Anti-C was not detected by our routine antibody identification techniques.
The MMA confirmed anti-E, -M and -C were clinically significant.
Laura Bailey* 1 , Melissa Grohotolsky 2 , Lisa DeBlass 1 , Bala Carver 1 and Kip Kuttner 2 .
1 Health Network Laboratories, 2 Miller Keystone Blood Center Background/Case Studies: The En a antigen is a high prevalence antigen in the MNS blood group system.
The antigens of the MNS system are carried on glycophorin A (GPA) and glycophorin B (GPB).
Anti-En a is a rare immune IgM/IgG antibody made by individuals who lack all or part of the GPA protein.
Anti-En a has been implicated in fatal HTR and HDFN.
The En(a-)phenotype can result from either a rare deletion of the GPA protein or the even rarer M k phenotype.
Because individuals with the M k phenotype lack both the GPA and GPB protein their red blood cells type as M-, N-, S-, s-, U-, En(a-), Wr(b-) and have reduced sialic acid.
Study Design/Method: 23 year old white Mennonite female G1,P0 presented to her midwife for prenatal care with the intent of home delivery.
She had a positive antibody screen by solid phase at the hospital transfusion service.
An antibody identification panel was done in gel.
Testing for antibodies against selected cells (U-and U var ) in tube with PEG enhancement and phenotyping was done.
Based on MNS phenotype, anti-En a was suspected.
The specimen was referred to an immunohematology reference laboratory (IRL).
The testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration.
Following identification of anti-En a by the IRL the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home.
The midwife was also advised to consider autologous blood donation and /or testing of siblings.
Results/Finding: Testing by the hospital blood bank demonstrated positive reactivity in the antibody screen.
The gel antibody panel AHG phase resulted in 21 panagglutination and a negative autocontrol, suggesting a high prevalence antibody.
The phenotype was performed and determined to be M-, N-, S-, s-, U -.
Outdated U variant reagent cells reacted in PEG IgG phase ruling out anti-U.
Anti-En a was suspected and the sample was referred to the IRL.
Allogeneic adsorptions were performed to rule out antibodies to common red cell antigens.
Lack of reactivity on a ficin panel eliminated the presence of anti-U,-Wr b .
Phenotyping with unlicensed anti-U was negative and unlicensed Glycine Soja demonstrated 11 reactivity, suggesting that the patient is En(a-).
The patient's phenotype is consistent with the M k phenotype.
Based on the lack of reactivity on the ficin panel, the antibody was identified as anti-En a FS.
Since anti-En a is extremely rare, this specificity could not be confirmed due to the lack of En(a-) cells and appropriate antisera.
The baseline antibody titer was 2 at IgG phase without enhancement.
Conclusion: This case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital.
Studies performed after the patient was transferred closer to home confirmed the anti-En a (FS) and genotyping was performed.
Three titers were performed for the remainder of the pregnancy and held at 2.
Although anti-En a has been implicated in HDFN, a healthy infant was delivered without complications.
This patient should be monitored closely through future pregnancies.
Autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery.
Background/Case Studies: A 15 year old Caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the Immunohematology Reference Laboratory (IRL) for antibody identification and RBC genotyping.
Initial serologic testing by the referring facility and the IRL demonstrated anti-D, anti-C and/or anti-G specificity with a positive auto control and IgG DAT.
Anti-G has an anti-D, -C specificity and is most frequently found in rr individuals exposed to r'r cells.
The G antigen is present on RBCs expressing either RhD and/or C and very rarely on D-C-G1 (r G r) cells.
Both RHCE*C and RHD genes encode Ser103 which determines G expression.
Rare RhD variant antigens lacking Ser103 are G-.
Study Design/Methods: Serologic evaluation included tube testing using Gamma LO-ION TM (Immucor, Inc., Norcross, GA) enhancement, elution studies (Gamma ELU-KITV R II (Immucor, Inc.)), EDTA glycine acid treatment (Gamma EGA TM Kit (Immucor, Inc.)), allogeneic adsorptions with papain treated intact RBCs, reagent and patient-derived RBCs and antisera.
Molecular testing was performed with BioArray Precise Type IVD HEA Assay (Immucor, Inc.).
Results/Findings: Molecular testing revealed an RHCE*cE genotype (with a C-E1c1e-predicted phenotype) and an otherwise unremarkable RBC typing report.
Serologically, the antibody(ies) demonstrated an anti-D, -C, -G specificity in the serum and eluate using R o r, R 2 R 2 , r'r, r G r and rr cells.
This patient is predicted to be R 2 R 2 (DcE/DcE) therefore, anti-C is possible but an allogeneic anti-D or -G is exceptionally unlikely.
Allogenic adsorptions using papain treated R o r and r'r cells excluded anti-C and anti-D, leaving anti-G as the only explanation of the initial findings.
Reactivity with the patient's EGA treated (DAT negative) cells against the "neat" serum, eluate and anti-G antisera confirmed auto anti-G.
Conclusion: Warm autoantibodies are common findings and often have an Rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used.
This anti-G had no reactivity with G-cells.
The differentiation of anti-G from anti-D and anti-C is generally academic as transfusion recommendations are the same: provide RhD-, C-units.
It is relevant and clinically important to determine the presence or absence of anti-D in RhD negative women of childbearing age who present with an anti-G specificity.
If anti-D is 135A TRANSFUSION 2017 Vol.
57 Supplement S3 excluded these women should receive RhIG as part of their prenatal care.
In this case differentiating anti-D, -C from an auto anti-G was necessary to provide transfusion recommendations.
Providing RhD-and C-units to give serologically compatible RBCs could result in formation of an allogeneic anti-e.
Automated Eluates: Comparison of Solid-Phase Red Cell Adherence and Gel Automated Eluate Testing Jayanna Slayten* 1 , Christa Voliva 1 , Kathy Fletcher 1 , Heather Vaught 2 and Tracie Ingle 1 .
1 Indiana University Health, 2 Indiana University Health (IU Health) Background/Case Studies: Acid Eluates (ELU Kit II.
Immucor.
Norcross, GA) are to be tested via tube IAT method in parallel with the recovered last wash per the manufacturer's package insert.
Finck et al (Immunohematology 2011; 27:1-5) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (ID-MTS.IgG Card.
Ortho Clinical Diagnostics.
Raritan, NJ) and automated solid-phase red cell adherence systems (Echo.
Immucor.
Norcross, GA).
Our study looked to compare the use of the automated gel microcolumn analyzer (VISION, Ortho Clinical Diagnostics.
Raritan, NJ) to the solid-phase red cell adherence analyzer (ECHO, Immucor.
Norcross, GA) for the testing of acid eluates in a regional Midwestern transfusion service.
Study Design/Methods: Twenty patient samples, less than 7 days from collection and drawn in EDTA, were used to prepare acid eluates (Elu-Kit II.
Immucor.
Norcross, GA) while retaining the last wash to be tested in parallel.
Two samples were >21 DAT positive, 2 were weakly DAT positive and 16 were DAT negative.
The prepared eluates were observed for color (bluegreen/BG, blue-brown/BB, blue-purple/BP), and the pH was documented for the prepared eluate (Whatman 6.0-8.1pH.
Whatman International.
Maidstone, England).
The prepared eluates and last washes were tested on the VISION and ECHO against an antibody screen.
If the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity.
Prior to the eluates being tested on the automated platforms, they were spun for 5 minutes twice to remove any RBC debris which could cause false positive reactions.
Results/Findings: The eluates prepared ranged in color: 5 BB, 14 BG and 1 BP.
The pH of all eluates ranged from 6.9-8.1 with the highest percentage of eluates at a pH of 8.1 (35%).
Sixteen of the 20 eluates tested yielded the same results in both automation platforms (concordance of 80%).
Four eluates with different results are summarized in Table 1 .
Conclusion: The study demonstrated that both analyzers may be used for eluate investigations.
Both methods yielded apparent false positive results on samples which were initially DAT negative.
The ECHO was more sensitive, yielding false positive results (3) when the VISION was negative, while the VISION was false positive with one eluate with ECHO negative.
There was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or pH of the eluate.
A larger study may be able to better elucidate the apparent false positive results noted in this study between ECHO and VISION eluate study.
Background/Case Studies: Blood agglutination observed by Landsteiner in 1900 led to the discovery of human blood groups.
In the ABO system >200 alleles have been described.
The glycosyltransferase encoded by most results in weakened expression of A or B or the null (Group O) phenotype.
As testing methods and reagents improve, donors may appear to change their ABO type.
Here we describe a frequent Group O blood donor (38 units over 17 years) who is actually A w .
Study Design/Methods: Donations were tested with the PK7300 instrument (Beckman Coulter Inc.).
Routine forward and reverse ABO testing was used to investigate the discrepancy.
Molecular studies were performed by DNA sequencing of ABO introns 1,2 and 4 and exons 6 and 7.
Specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by PCR.
The template used is genomic DNA extracted from whole blood collected in EDTA.
PCR-amplified exons are subjected to bidirectional DNA sequence analysis using standard Sanger dideoxy chemistry.
Seqscape software (ABI) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from NCBI.
Results/Findings: Serologic results are shown in Table 1 .
Tests with Anti-A, -A1, -B anti-A,B were negative as were the A2 cells in reverse testing.
The results of DNA sequencing of ABO introns/exons are shown in Table 2 .
The significant changes were found in exons 6 and 7.
In exon 6 there was a nucleotide (NT) deletion of 261G which resulted in a shortened transcript due to a stop codon, and another NT substitution lead to the amino acid change Gly117Ala.
Mutations in exon 7 included a NT substitution causing a Pro156-Leu change and a NT deletion 1061C resulting in shortened transcript.
Conclusion: Serologic testing of the donor plasma with A2 cells was nonreactive revealing the ABO discrepancy.
Molecular testing confirmed the donor genotype is heterozygote A/O [ABO*O.01.01/ABO*AW.02] which predicts A w phenotype.
Normally, donor RBCs are tested with Anti-A and -B and the reverse type confirmed by testing the with A1 and B cells.
This ABO discrepancy was caused by the presence of anti-A1 in the plasma causing the forward and reverse type to be interpreted as group O.
According to FDA guidelines, the donor is technically group A, and as such all donations need to be labeled as group A.
The donor was contacted and instructed to cease donating blood for transfusion.
If donations continue, the unit labeled group A would likely test as Group O at the transfusion facility resulting in an FDA reportable error.
There are numerous reports in the literature of the relative insusceptibility of A2 cells to destruction by anti-A, however, there is one hemolytic transfusion reaction to A x blood transfused to a patient with a potent anti-A titer >1:1000.
(Schmidt, Nacarrow et al.
1959) .
A review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion.
136A TRANSFUSION 2017 Vol.
57 Supplement S3 extraction of gDNA from EDTA-anticoagulated whole blood from pilot tubes derived from the unit.
DNA extraction from whole blood is performed on up to 96 blood tubes using the BioRobot Universal System (QIAGEN).
There is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature.
We set out to assess if blood up to 15 days post collection yielded suitable gDNA for downstream RBC genotyping.
Study Design/Method: 92 EDTA blood tubes collected from random blood donors were used to extract DNA from 200 microliters of whole blood on day 5, 12 and 15 days post collection.
Blood samples were stored at 2-8C before and after extraction.
Tubes were brought to room temperature and rocked before loading on the BioRobot.
Extraction was performed using the MDx Blood Minikit (QIAGEN).
Resulting DNA samples were assessed for gDNA yield and absorbance A260/A280 using a Nanodrop 2000 (Thermo Scientific).
The extracted gDNA was tested using PreciseType HEA Molecular BeadChip ("HEA", Immucor) and failure rates on both the BioRobot and the HEA were assessed.
Results/Finding: All three extractions were successful with no invalids (result50) on the BioRobot Universal Report.
No evidence of visible clots or splatter during extraction was noted by the technologist.
Out of the 92 samples, 20 samples were chosen at random and concentrations were measured using Nanodrop for each of the extracted plates.
DNA concentrations ranged from 10.8 to 62.6 ng/uL.
All readings with the exception of 1 (10.8ng/ uL) had concentrations >5 15ng/uL.
Interestingly, the one that was <15ng/ uL on day 5, yielded >515ng/uL on day 12 and 15 post collection.
Over the next 3 months, 67 sets of 92 samples were extracted and tested by HEA.
Eighty-three (1.3%) failed extraction and 82 (1.3%) failed HEA.
None of the samples that failed extraction were 12 or 15 days post collection; none of those that failed HEA were 15 days post collection; 3.7% were >10 <15 days post collection.
Conclusion: Based on these results it can be concluded that EDTA blood tubes up to 15 days post collection can be used as a source of gDNA for RBC genotyping without negatively effecting the concentration of the resulting DNA samples and the validity of the resulting genotyping.
Case Study: Investigation of Persistent Negative Antibody Screens on Patients Receiving Daratumumab Raeann Thomas 1 , Carlos Villa 1 , Rachel Davis-Rauser* 1 , Helen Carpenter 1 and Vrunda Patel 2 .
1 University of Pennsylvania, 2 Hospital of the University of Pennsylvania Background/Case Studies: Daratumumab is an anti-CD38 monoclonal antibody therapy that received FDA approval for treatment of multiple myeloma in 2015.
Communications suggest all patients receiving therapy would have a positive antibody screen because CD38 is a common antigen expressed on red blood cells.
Currently, 154 patients have been treated with daratumumab at a large academic medical center.
A wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens.
While there are several potential causes, neutralization of anti-CD38 antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity.
Study Design/Method: Samples received were drawn as a standard of care.
Indirect antiglobulin testing was performed using solid phase red cell adherence and gel.
Neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma.
A dilution control was made by adding saline to each positive patient's plasma.
Samples were incubated for 1 hour at room temperature and antibody screens were repeated.
Serial two-fold dilutions were also tested to determine if the neutralization could be titered.
Testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity.
Results/Finding: All control samples remained positive.
Positive/negative samples were negative in solid phase testing across all patient combinations at 1:1 dilutions.
Variable reactivity was observed in gel.
Serial dilutions showed that neutralization for 2 negative patients was observed up to a 1:4 dilution.
Conclusion: Results suggest that patients' plasma may have a substance that neutralizes the antibodies.
There is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab.
Additional studies are necessary to uncover how this correlates to patient outcomes.
Further studies using a standardized daratumumabspiked sample will be conducted.
Background/Case Studies: The MNS blood group is a red cell antigen system located on glycophorin A (GYPA) and glycophorin B (GYPB).
Individuals lacking GYPA or both GYPA and GYPB on their red blood cells may develop a rare antibody against the En (a) antigen.
The En (a) antigen is a highprevalence antigen, located on GYPA.
We present a case with a rare red cell phenotype and alloimmunization to the En (a) antigen.
A 28 y/o G1P0 at approximately 23 weeks gestation was discovered to have an anti-En (a) antibody in her plasma on a prenatal type and screen.
This was worrisome for both mother and fetus, as the En (a) antibody is of IgG isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN) [1, 2] .
Further testing with red cell antisera revealed that the patient lacked M, N, S, s, and U antigens.
A multiplex, allele-specific, PCR platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the M, N, S, s, and U antigens.
These findings were consistent with a null phenotype for both GYPA and GYPB antigens, i.e.
M (k) M (k) phenotype.
The patient's husband and father of her unborn baby demonstrated a M1N-S1s1 phenotype by the same serological and molecular means.
Given the exceedingly rare incidence of En (a-) individuals (positive frequency >99.9), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [1, 2] .
However, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).
The consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers.
If transfusions were required for the mother or fetus, our options were to either search for rare units lacking the En(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype.
At term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route.
The delivery was without event.
No transfusions were necessary antepartum or postpartum.
Study Design/Methods: N/A Results/Findings: N/A Conclusion: Anti-En(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman.
Providing this patient with rare En(a) negative red cells via national or international blood donor registries would have been an arduous task if needed.
This patient had many compatible family members available and willing to donate blood.
The M(k) null allele (s) within this family is likely due to a genetic recombination among the GYPA and GYPE genes rather than a mutation in both the GYPA and GYPB genes [3] .
This results in the absence of glycophorins A and B and the constitutive antigens of the MNS blood group system.
Our patient was exposed to the En(a) present on glycophorin A on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization.
In conclusion, this case report demonstrates a clinical approach in identifying a rare anti-En(a) antibody in a prenatal sample.
The clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it.
Background/Case Studies: Transfusions are essential for patients with SCD and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (RBC) alloantibodies and autoantibodies complicates transfusion therapy in such patients.
Routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and SCD.
Nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations.
The molecular understanding of blood groups has enabled the design of assays 137A TRANSFUSION 2017 Vol.
57 Supplement S3 that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units DNA typed.
Based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during 3 years for patients with SCD and thalassemia.
Study Design/Method: Blood group genotypes were determined in 67 DNA samples from chronically transfused patients with SCD, in 65 patients with thalassemia and in 3000 DNA samples from blood donors.
Laboratory developed tests (LDTs), HEA BeadChip TM , RHD BeadChip TM , RHCE Bead-Chip TM , and sequencing were used to determine the genotypes among patients and donors.
Molecular matching was performed in 3 levels: (1) RH and K matching; (2) extended matching and (3) extended matching including RH variants.
We considered the total of red blood cell units requested for each patient and a number of 2 donations per year for the compatible donors.
Results/Finding: According to the patients needs we performed molecular matching for 100% of our thalassemic and SCD patients at level 1, 90% for SCD patients and 70% for patients with thalassemia at level 2 and 30% for patients with SCD and 90% for patients with thalassemia at level 3.
The patients were transfused with a median of 36.4 RBC units.
After three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients.
Conclusion: Molecular matching has shown clinical benefits to the patients with SCD and thalassemia, contributing significantly to reduce the rates of alloimmunization to 5-10% with C E K matching and <1% with extended matching.
Improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their Hb levels and reduction in the % of HbS in SCD patients, better in vivo RBC survival and diminished frequency of transfusions.
Allahna Lilly Elahie* and Sandra Fazari.
Hamilton Regional Laboratory Medicine Program Background/Case Studies: The ideal manual backup method for an automated antibody detection system is an important choice.
Currently, our backup method is saline tube (6 drops plasma, 30 minutes incubation).
The change to either a low ionic strength solution (LISS) or polyethylene glycol (PEG) method would reduce incubation time to 10 minutes and specimen volume to 2 drops, both important laboratory considerations.
Objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of PEG and LISS, and to determine the most appropriate manual backup method for the existing automated solid phase system.
Study Design/Method: A total of 202 specimens were compared utilizing: automated solid phase red cell adherence assay (SPRCA) with manual tube PEG and LISS, some samples were not sufficient quantity to test in LISS.
Identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions.
Calculations were based upon comparison to SPRCA.
Results/Findings: A total of 164 clinically significant antibodies were detected using SPRCA technique, as well as 9 warm autoantibodies and 97 nonspecific reactions.
PEG demonstrated the highest sensitivity and lowest specificity while LISS was least sensitive and most specific for clinically significant antibodies.
For warm autoantibodies, LISS was more sensitive than PEG with both being 100% specific.
Both reduced the detection of nonspecific reactions.
While PEG had more nonspecific reactions (30 versus 13), it identified more clinically significant antibodies (129) than LISS (93).
(Table) Conclusion: Ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one.
PEG was selected as the backup manual method even though PEG has a higher sensitivity to nonspecific reactions.
This study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions.
Comparison of Thiol Reagents in Denaturing CD38 on RBCs Patricia A Arndt* 1 , Anthony Salazar 2 and Regina M. Leger 1 .
1 American Red Cross Blood Services, 2 Long Beach Memorial Medical Center Background/Case Studies: Monoclonal anti-CD38, e.g., daratumumab (DARA), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (IATs) due to expression of CD38 on red blood cells (RBCs).
This serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies.
One popular method is to denature the CD38 antigen by treatment of RBCs with thiol reagents, e.g., dithiothreitol (DTT) or 2aminoethylisothiouronium bromide (AET).
Chapuy et al described (2015) and validated (2016) 2), and 6% AET (pH 8.0) as per the AABB Technical Manual, 17th ed.
These treated and untreated RBCs were stored in Alsevers at 4C and tested on days 1, 2, 4, 5 and 8 by two methods: 1) polyethylene glycol (PEG) IAT using plasma from two myeloma patients who had received DARA (plasmas from 8 total DARA patients were tested with reactivity 5 1-31), and 2) flow cytometry using phycoerythrin (PE)labeled anti-CD38.
RBCs were also tested on days 1 and 5 or 8 with a serum containing anti-k by PEG IAT.
Results/Findings: The 0.2M DTT in pH 8.0 PBS had a final pH of 7.3 and the pH of the commercial 0.2M DTT was 6.5.
Results are in Table 1 ; flow cytometry results from days 2, 4 and 5 (data not shown) were similar to those from days 1 and 8.
RBCs treated with 0.2M DTT (both sources) or AET were nonreactive with anti-k and plasma from all DARA patients and gave very low results (% positive events) with PE anti-CD38 by flow cytometry for up to 8 days after treatment.
RBCs treated with 0.01M DTT reacted similarly to untreated RBCs with anti-k and DARA plasmas, and showed only some weakening (10-30%) of reactivity with PE anti-CD38.
Background/Case Studies: Clinically significant hemolytic disease of the fetus and the newborn (HDN) is often caused by feto-maternal RhD incompatibility.
With the discovery 1997 of cell-free fetal DNA (ccfDNA) in maternal plasma, it became possible to determine the RHD genotype of the fetus using non-invasive techniques.
However, the reliability of the non-invasive prenatal RHD test (NIP RHD) is dependent on sufficient amounts of cffDNA in the maternal plasma sample.
Recent studies show that the fraction of ccfDNA in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (BMI).
Thus, high maternal BMI, may impair the validity of NIP RHD.
The aim of this study was to examine the effect of maternal BMI on the correctness of NIP RHD and the correlation of maternal BMI with fraction of ccfDNA to total free DNA in the sample.
Study Design/Method: Measurements of body height and weight of pregnant RhD negative women in gestational week 12 were obtained from patient records and used for the calculation of maternal BMI.
Data on BMI were combined with the results from NIP RHD (real-time PCR targeting RHD exon 5 and 10) and sample fraction of ccfDNA (measured as threshold cycle [Ct] value of RHD) to total free DNA (measured as Ct of CCR5) in gestational week 25.
The correctness of NIP RHD was determined by correlation with postnatal serological RhD determination.
Results/Finding: A total of 1618 pregnant women were included.
NIP RHD was positive in 987/1618 (61%), negative in 582/1618 (36%) and inconclusive in 49/1618 (3.0%).
Compared to the postnatal RhD type, 9/987 (0.1%) of NIP RHD results were false positive (FP) and 4/582 (0.7%) were false negative.
In 5/49 (10%) of inconclusive NIP RHD, the postnatal RhD type was positive.
Mean BMI (n51618) at gestational week 12 was 25.3 (10-and 90-percentiles: 20.0 -32.4).
There was no difference in mean BMI between individuals who tested inconclusive or false negative by NIP RHD compared to the remainder (p50,71).
The fraction of ccfDNA was calculated for 150 randomly selected NIP RHD true positive cases.
Median ccfDNA ratio was 5.47 (the distribution had a highly positive skew, 10-and 90-percentiles: 0.64 -27.2).
There was no statistical correlation between BMI and fraction of ccfDNA to total free DNA (r2 50,012; p50.49).
Conclusion: Neither the correctness of NIP RHD test result nor the fraction of cffDNA to total free DNA appear to be correlated to maternal BMI with regard to maternal plasma samples drawn in the 25th gestational week.
Delayed Hemolytic Transfusion Reaction Due to Anti-Lan Antibody: A Case Report.
Adla DH Angelina*, Suneeti Sapatnekar and Suzanne Bakdash.
Cleveland Clinic Background/Case Studies: Lan is a high-prevalence antigen and the sole member of the LAN blood group system.
Anti-Lan is a very rare IgG antibody, with conflicting information regarding its clinical significance and potential for hemolysis.
We report a case of delayed hemolysis in a patient with anti-Lan antibody.
Study Design/Method: The patient's medical record and available literature were reviewed.
Results/Finding: An 83 year old man, O-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy.
The antibody screen and panel were panreactive by multiple test methods (gel, LISS, PEG) with negative autocontrols and DAT and a saline antibody titer of 1, suggestive of an antibody to a high-frequency antigen.
Anti-Lan antibody was identified by a reference laboratory.
Only 1 in 20,000 donors are Lan-, but two frozen RBC units were locally available and transfused postoperatively.
The patient's siblings were tested; one O-positive, Lan-sibling was identified.
Nine months later, the patient was admitted for surgical management of metastases.
At this time, the antibody screen was weakly reactive with 1 cell and new antibodies were ruled out.
Blood conservation measures were instituted, including limited blood draws and cell salvage for surgery.
Due to bleeding during and after surgery, 4 Lan-RBC units were transfused over 4 days, including rare donor units and units from the sibling donor.
Another surgical procedure was then performed; by post-operative day 2, the patient had symptomatic anemia with hemoglobin (Hb) 5.3 g/dL and serially increasing troponin.
No Lan-RBC units were available.
Four RBC units untested for Lan were transfused without adverse event; the units were presumed Lan1 but crossmatch compatible and phenotypically matched for the patient's other antigens.
A post-transfusion Hb of 10.2 g/dL was maintained for 4 days.
The antibody screen was negative on day 3 post-transfusion, but strongly panreactive on day 6, with a positive DAT (IgG 21, C3 11) and anti-Lan antibody identified in the plasma and eluate.
There was also evidence of extra-vascular hemolysis, including a progressive decrease in Hb from 10.9 g/dL on day 5 to 7.4 g/dL by day 8 with no bleeding identified, and increase in total bilirubin and LDH (peak 2.4 mg/dL and 304 U/L on day 7) with normal haptoglobin.
The patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection.
A Lan-RBC unit was transfused on day 8 with good response (Hb 8.1 g/dL).
The patient remained stable and was discharged to a skilled nursing facility 6 days later.
Conclusion: Transfusion of Lan1 RBCs caused a resurgence of anti-Lan antibody and a delayed hemolytic transfusion reaction 6 days after transfusion.
The rarity of Lan-units may require a patient with anti-Lan to be transfused with Lan1 units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion.
Delayed Serologic Transfusion Reaction Caused By Auto-Anti-f. Karen Yunker* 1 , Andrea Gerner 1 , Lynne Stewart 1 , Carol Sostok 1 , Mollie Bell 2 and Gregory R Halverson 2 .
1 St. Elizabeth Healthcare, 2 Hoxworth Blood Center Background/Case Studies: Anti-f was first described in 1953 by Rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused.
The f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the 6th antigen assigned to the Rh Blood Group System (ISBT RH6).
It is capable of causing significant transfusion reactions and mild HDFN.
We report in this case a 56 year old caucasian male, admitted for evaluation of suspected T-cell lymphoma, who appears to have had a Delayed Serologic Transfusion Reaction (DSTR) due to auto anti-f. ABSTRACT Study Design/Method: Antibody screen and compatibility testing was performed by automated solid phase (Echo and Neo, lmmucor, Inc).
Red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions.
Molecular genotyping was performed using the Bloodchip assay (Grifols, San Marcos TX).
Elution studies were performed using the ELU-Kit II (lmmucor, Inc.) Results/Finding: The initial antibody screen (AS) was negative and the patient was transfused 1 unit O-RBCs.
Two weeks later the patient received an additional O-RBC.
Within 4 days the Hgb had decreased from 8.3 to 7.1 g/dl, the AS and Direct Antiglobulin Test (DAT) were now positive, and Ounits were incompatible.
Anti-f was identified in the patient's plasma and eluate.
Three additional units were requested for transfusion.
Due to the rarity of O-f-RBCs, the patient was transfused 3 R 1 R 1 (DCe/DCe) RBCs with no reported complications.
The patient was discharged to follow up in clinic.
Molecular genotyping showed the patient was RhD deleted (RHO* del) and had normal RHCE (RHCE*ce/RHCE*ce) genes which predict a D-C-E-c1e1f1 phenotype.
The Rh phenotype and AS was repeated on a sample collected 18 days later.
The C typing was micro positive, mixed field only after 5 minute incubation.
The other Rh antigens were not mixed filed, and the AS was non reactive.
However, the DAT was weakly positive with anti-lgG.
No elution study was performed.
Conclusion: The expected post 24 hour Hgb increment from the receipt of a standard unit of blood should be near 1 g/dl (or 3% Hct.)
Throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation.
The first transfuion resulted in a 0.9 g/dl increase, and the second unit was only 0.5 g/dl.
The last transfusion of 3 units increased by only 1.2 g/dl.
Less than three weeks later, the RhC antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of 3 R 1 R 1 units was nearly complete.
In a case from 1989, Ohto and Kariyone (Transf.
1989; Vol29, No.
3) reported a 51 Cr Ásurvival study of f1 RBCs in a patient with anti-f.
They showed that the initital survival of f1 cells was fairly normal, however, after 18 days, there was a sudden increase of red cell destruction, and by day 27 all f1 cells were cleared from the circulation.
It is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood.
This patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity.
Background/Case Studies: Use of dithiothreitol (DTT) treated reagent red cells (RRBC) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by Daratumumab, an anti-CD38 drug for treatment of multiple myeloma.
Daily preparation of DTT-treated RRBC for testing of individual patients is burdensome for the laboratory and may delay patient care.
We evaluated the effectiveness of batch-prepared DTT-treated RRBC, stored up to 9 days after treatment, in antibody detection tests.
Study Design/Methods: In-date RRBC (Ortho Clinical Diagnostics, Raritan NJ) were selected based on phenotype to match the antisera to be tested.
RRBC were treated with 0.2M DTT (Sigma-Aldrich, St. Louis MO) and stored in reagent red cell diluent.
RRBC were tested with commercial antisera (Ortho Clinical Diagnostics, Raritan NJ and Immucor, Norcross GA) per the manufacturer's instructions for specificities from the Rh, Duffy, Kidd and MNS blood groups (see Table 1 ).
Patient source antibodies (anti-D, anti-c) were also tested.
Testing was performed before DTT treatment, on the day of DTT treatment and up to 9 days following the DTT treatment of RRBC.
Reactions were graded using standard serological grading of 0 (negative) to 41 (positive) reaction strength.
Stored DTT-treated RRBC were also observed for hemolysis during the storage period.
Results/Findings: See Table 1 for a summary of results.
Commercial monoclonal and human source antisera, and patient source antibody, were reactive with the DTT-treated RRBC throughout the storage period.
Reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested.
Mild to moderate hemolysis was noted in the DTTtreated RRBC's during the storage period.
Conclusion: DTT-treated RRBC showed adequate reactivity with various red cell antisera after storage for up to 9 days.
This suggests that DTTtreated reagent red cells can be stored for at least 9 days and used for the detection of alloantibodies with minimal effect on detection ability.
Batch preparation and storage of DTT-treated RRBC can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-CD38 therapy.
Interference: More Than Just Kell?
Marilyn Stewart*, Angela Treml and Geoffrey Wool.
University of Chicago Background/Case Studies: Daratumumab (DARA) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine Blood Bank antibody screening tests.
DARA is an IgG monoclonal antibody that binds CD38 that is present on the red cell surface.
At the University Of Chicago Blood Bank, we have seen many patients treated with DARA and were showing this interfering reactivity.
It has been well described that CD38 is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as DTT.
We performed a validation of DTT-treatment of reagent RBC to abrogate DARA interference.
Study Design/Methods: The validation was done to prove that DTT treated red cells could be used to screen patients receiving DARA and still detect clinically significant allo-antibodies.
Screening cells and panel cells selected for DTT treatment were those RBC homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma.
Several patients that had received the DARA drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not DARA treatment).
Reagent screening cells and panel cells were treated with 0.2M DTT prepared using the SOP from Judd's Methods in Immunohematology and the AABB Technical Manual.
The treated cells were preserved between testing episodes using Alsever's solution, stored at ABSTRACT 2-5C, and observed for hemolysis (none was seen) for up to 21 days.
All immunohematology testing using DTT-treated cells was performed using gel methodology.
Untreated and DTT treated cells were tested with anti k before any patient testing was done.
The untreated cells reacted 2-41 with the anti k, and the treated cells were negative.
These controls were run and tested each time DTT treatment was done.
Thirty eight patient samples, including six DARA patient samples were tested.
Results/Finding: Of the six patients who had DARA interference in their untreated antibody screens, all samples had negative reactions with the DTT treated cells except one patient, which had weak reactions in one cell.
This specimen was repeated three times and all repeats had weak positive reactions in the same cell.
This sample was sent to the ARC reference lab for DTT treatment and all clinically significant antibodies were ruled out.
Patients with allo-antibodies present in their plasma did react with the DTT treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-E antibodies in 4 patients.
Plasma from these four patients with a nascent anti -E all showed no reactivity with DTT treated cells.
Plasma from fourteen patients with a long history of anti-E (greater than 6 months) did react with the DTT treated cells.
Conclusion: DTT treatment eliminates DARA interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-E. Because of the negative testing with some of the alloanti E antibodies, DARA-treated patients at UCM will be given both Kell and E negative blood if they have immunohematology testing performed using DTT reagent cells.
Mahboubeh Rahmani* 1 , Monique Scott 2 , Garcia Curtis 2 , Ellice Wong 2 , Alexa J Siddon 1 and Christopher A Tormey 1 .
1 Yale-New Haven Hospital, 2 VA Connecticut Healthcare Background/Case Studies: Benign ethnic neutropenia (BEN) seen in approximately 25% to 50% of persons of African descent is characterized by neutrophil count of <1.5x10 9 /L with no obvious cause and no increased susceptibility to infection or any other adverse effect.
At present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis.
In this study, we investigated whether Duffy (Fy) blood group phenotyping would be a potentially useful modality to help identify patients with BEN; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting.
Study Design/Method: Cases included patients clinically diagnosed with BEN; and controls were chosen randomly from the pools of patients from whom a CBC and type and screen were checked for any other reason.
Cases and controls were tested for the RBC antigens Fy a and Fy b phenotype using serologic methods.
The Fy phenotype, absolute neutrophil count (ANC), white blood cell (WBC), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record.
Where appropriate, data were compared statistically using the Mann-Whitney U Test with significance set at P<0.05.
Results/Finding: Subjects who were clinically identified as having probable BEN included 7 patients (mean age 48.7; all self-identified as African-American; 6/7 were male) and controls included 50 patients (mean age 68.5; 10 self-identified as African American; (50/50 male).
All of the cases (100%) diagnosed with BEN had Fy(a-b-) phenotype.
Mean ANC (1.95x10 3 /uL) and WBC counts (4.04x10 3 /uL) were significantly lower in the cases with BEN and Fy(a-b-) phenotype (P50.0008 and 0.001, respectively) compared with controls (mean ANC 5 5.46x10 3 /uL ; mean WBC count 5 8.14x10 3 /uL).
There was no significant difference in mean platelet counts (161x10 3 /uL vs 213x10 3 /uL; P50.2301) or mean hemoglobin levels (12.4 g/dL vs 11.7 g/dL; P50.6031) between the two groups.
None of the patients with BEN had an accompanying marrow-suppressive hematologic disorder based on record review; however, 18 subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome.
Conclusion: Testing for Fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of BEN.
Further studies regarding Fy phenotyping comparing controls with neutropenia for any reason to our BEN population are in progress to better determine the positive predictive value.
These tests were compared to the ProVUE for concordance.
Additional samples tested with Anti-IgG,-C3d were correlated against tube testing for the DAT and antigen typing for: C, c, E, e, and K. Results/Findings: The IH-1000 had 100% concordance for all blood grouping assays.
For AHG assays, the IH-1000 detected an anti-Jka1E, anti-Fya 1 warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the ProVUE.
The IH-1000 identified one additional anti-E not identified on the ProVUE.
Discrepancies were also noted with the non-cord DAT results.
Five samples were positive on the IH-1000 with Anti-IgG,-C3d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube.
The table below summarizes the results.
Conclusion: This study demonstrated that the IH-1000 analyzer and associated IH-System TM Gel Cards are equivalent to the ORTHO ProVUE.
With random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the IH-1000 is an ideal immunohematology system for the hospital transfusion service environment.
Chris Elliott*, Susan Barnes, Fiona Lisle, Debra Smith and Whitehouse Natalie.
Background/Case Studies: The Erytra EflexisV R (Grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using DG GelV R technology.
Erytra EflexisV R analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large UK acute hospital transfusion laboratory.
Study Design/Methods: A comparison study was performed between the Erytra EflexisV R and Erytra (our routine system providing the reference platform).
A total of 2944 tests were performed on 1,214 adult patient samples and 208 donor red cell units.
ErytraV R Eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations.
Concordance between systems was assessed and discrepancies analyzed.
Time to first result (TTFR), overall turn-around time (TAT) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded.
For ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements.
Fault recognition and messaging was assessed by simulating failures e.g.
reagent absence.
Results/Findings: Blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested.
Concordant results between the Erytra EflexisV R Analyzer and reference method were obtained in 99.9% of samples tested.
There were 4 discrepancies, all antibody screening (2 false positives, 1 failure to detect a very weak prophylactic anti D and 1 positive reaction not detected on the Erytra but panels on both systems suggested a genuine anti Cw).
TTFR and TAT depended significantly on a number of factors including; number and variety of tests requested and whether the STAT functions were activated.
The analyser seemed to prioritise antibody screening Prioritization of the group, especially for STAT samples, was considered preferable The laboratory team found the software easy to use with some improvements over existing Ertyra software.
Physical design of the analyser was considered good with easy access to almost all areas.
Probe changing was quick and simple.
While the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased.
Conclusion: Results showed the Erytra EflexisV R offered a robust automated solution for routine transfusion testing.
The device could comfortably deal with a medium laboratory (processing 80-100 group and screens per day).
It is very flexible being able to deliver grouping, antibody screening and identification, DAT, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features.
This allows a compact design with maximum flexibility without compromising on turnaround times CP201 Evaluation of Two Monoclonal Anti-e As Reagents for the Detection of the Rh e Antigen and Its Variants Gregory A. Denomme* 1 , Kathleen Bensing 1 , Michael Schanen 1 , Cindy Piefer 1 , Randall W. Velliquette 2 , Christine Lomas-Francis 2 and Connie M. Westhoff 2 .
1 Immunohematology Reference Laboratory, Versiti/BloodCenter of Wisconsin, 2 Immunohematology and Genomics Laboratory, New York Blood Center Background/Case Studies: Monoclonal antibodies are used as reagents for automated and manual phenotyping.
False negative phenotypings have implications for variant antigens; e.g.
altered C antigen mistyped as a Cblood unit stimulating anti-C in a C-recipient.
The development of new 7/0 7/0 7/0 ceCF 1/1 2/0 2/0 RHCE*ce or RHCE*Ce compound heterozygotes ce254G 1 ce733G or ce48C,733G or ceS or ceTI 6/0 6/0 6/0 ce733G 1 ce48C,712G or 48C,733G 4/0 4/0 4/0 ce733G 1 ceS or ceMO or ceEK or ceEK(var) or CeRN 8/0 8/0 8/0 ce48C,733G 1 ce48C,712G or ceMO or ceTI 2/0 2/0 2/0 ce48C,712G/ce254G/733G; ceS/ceTI; ceAR/ceEK; ceEK/ceJAL; ceMO/ceBI 7/0 7/0 7/0 Total 106/8 110/4 113/2 142A TRANSFUSION 2017 Vol.
57 Supplement S3 reagents should include an evaluation of antigen variants to confirm fidelity.
We evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed Rh e variants.
Study Design/Method: Two monoclonal anti-e clones, RD9/4 and RD12/2, and a licensed comparator anti-e (P3GD5121MS63), all from Diagast (Loos, France), were evaluated.
RBC samples were either recovered from frozen storage (N 5 42) or EDTA blood from donors (N 5 72) and were tested using a manual tube method or on a PK7300 automated platform.
A score 6 (11) or greater was deemed acceptable for manual tube and a positive call for automated testing.
Results were tabulated by complexity of RHCE*ce alleles (Table 1) .
Results/Finding: The specificity of the monoclonal anti-e were confirmed using common Rhce haplotypes: R 1 R 1 , R 2 R 2 , R 1 r, and rr.
Twenty-one different RHCE*ce alleles were included in the extensive panel: 40 were RHCE*ce that were in trans to RHCE*cE; 16 were various RHCE*ce plus RHCE*ce48C compound heterozygotes; 31 were RHCE*ce or RHCE*Ce homozygotes; 27 were various RHCE*ce and RHCE*Ce compound heterozygotes.
The comparator reagent was negative or unacceptably weak for 6 RHCE*ce alleles in trans to RHCE*cE (RHCE*ceAR, RHCE*ceMO, RHCE*-ceJAL, RHCE*ceHAR), with 1 RHCE*ceAR/RHCE*ce48C compound heterozygote, and with 1 RHCE*ceCF homozygote.
RHCE*ceAR, RHCE*ceMO, RHCE*ceJAL, and 1 of 2 RHCE*ceCF homozygotes were detected using the comparator reagent.
RD9/4 and RD12/2 failed with 4 and 1 e variants, respectively (Table 1) .
Failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the RD9/4 clone.
None of the reagents detected e antigen variant expressed on 1 example of RHCE*ceHAR/RHCE*cE.
Conclusion: RD9/4 and RD12/2 anti-e reacted with more e variants than the comparator reagent.
The e antigen encoded by RHCE*JAL and RHCE*AR is not always detected when in trans to RHCE*cE.
However, double-dose expression was detected suggesting that the 3 monoclonal reagents bind weakly to the respective altered e antigen epitopes.
The e antigen encoded by RHCE*ceHAR continues to be a challenge to detect.
Meihong Liu*, Teresita Mercado, Orieji Illoh, Maria Rios and Zhugong Liu.
OBRR, CBER, FDA Background/Case Studies: Extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (RBCs) that match those of the recipient.
This is especially important in the management of chronically-transfused patients and patients with RBC alloantibodies.
Several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes.
Targeted next-generation sequencing (NGS) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples.
We developed and evaluated targeted NGS assays using two different target enrichment platforms for extended blood group genotyping.
Study Design/Method: Two custom design platforms SureSelect and Halo-Plex were used independently for preparation of probes that target the entire genes of 19 blood group genes associated with the expression of 56 blood group antigens from 17 blood group systems.
We used the Illumina's HiSeq 2000/2500 system to perform next generation sequencing first on SureSelect-enriched genes from 16 DNA reference samples with average target design coverage of 97.5%, and then on HaloPlex-enriched genes from 32 DNA reference samples with average target design coverage above 97.0%.
Twelve samples were enriched and sequenced in both methods to allow a direct comparison.
All reference samples were previously characterized for 38 blood group genetic variants in these 19 genes using TaqMan SNP assay and Sanger Sequencing assay.
Serological data were also available for these samples.
The NGS data were analyzed by CLC Genomic Workbench.
Sequencing variants were detected and annotated using dbSNP database.
Blood group genotype calls by the two targeted NGS methods were compared with the reference results.
Results/Finding: For the two targeted NGS methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of NGS, sequencing coverage, and genotype concordance.
A higher percentage of the HaloPlex reads (80.54%) were mapped to the target regions relative to the SureSelect reads (29.23%).
The mean sequence coverage depth of the targeted bases was around 200x for SureSelect method and 300x for HaloPlex method.
Some exons, such as RHD exons 4 and 8, 10, RHCE exon 10, ERMAP exons 5 and 12, CD55 exons 10 and 11, CR1 gene (most exons) and GYPB exon 5, are consistently covered with less than 10x coverage by both SureSelect and HaloPlex targeted NGS methods.
Both methods detected RHD gene deletion in a few representative samples.
The genotype call concordance on 38 blood group genetic variants was assessed by comparing NGS results to TaqMan genotyping and Sanger sequencing results, and more than 90% concordance was obtained for both targeted NGS methods.
Incorrect calls were restricted to four complex blood group genes: MNS, RHD, RHCE and ABO, and involved mainly heterozygous variants and indels.
Conclusion: Using two targeted NGS methods, we have correctly detected more than 90% blood group genetic variants in 19 selected genes.
Evidence RHCE*ceHAR Does Not Encode for Rh34 (Hr B ) Antigen Debra J Bailey* 1 , Trina Horn 2 , Paul Mansfield 2 , Najmi Qazi 1 , Pamela Nickle 2 , Jessica Keller 2 , Margaret A Keller 2 and Jan R Hamilton 1 .
Background/Case Studies: The RHCE gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete.
New information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation.
The RHCE*ceHAR allele was first described in 1996 and has a phenotype of C2E2c1e1 w f1 w , G2, Hr 0 1 w , Hr2, hr S 2, Rh:33, Rh:50 with a partial D antigen expression.
We describe new information regarding an RH haplotype that includes an RHCE*ceHAR allele and its apparent Rh:234 (Hr B 2) expression.
Study Design/Method: RBC typing was performed by standard tube methods with polyclonal and monoclonal antisera.
Antibody identification studies were performed by standard tube hemagglutination methods by published techniques.
Molecular immunohematology testing was performed on genomic DNA extracted from whole blood and included HEA, RHD and RHCE BeadChips (Immucor) and PCR-RFLP analysis for RHCE c.254C>G and RHD c.1136C>T.
Results/Finding: A sample from an African American female with a history of an anti-E and anti-K was evaluated for unexpected antibodies.
Her red cell serologic Rh phenotype on an untransfused sample was D1C1E2c1e1.
Her plasma contained an alloanti-S and an antibody that reacted strongly with all random E2K2S2 reagent red cells except her own.
The unidentified reactivity persisted following ficin and DTT pretreatment of reagent red cells.
Only D22 and Dc2 red cells were non-reactive in initial tests.
Differential adsorption studies excluded antibodies to all other common antigens and hr B except E, S and K. When subsequent examples of E2S2K2 red cells homozygous for the RHD*DIIIa-CE(4-7)-D, RHCE*-ce48C,733G,1006T haplotype (i.e., r' S /r' S ) and RHD*DIIIa, RHCE*-ce48C,733G,1006T/ RHD*DIIIa-CE(4-7)-D, RHCE*ce48C,733G,1006T (i.e., Bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-Hr B .
The patient's red cell antigen genotype identified the following probable RH haplotypes: RHD*01, RHCE*ceHAR and RHD*DIIIa-CE(4-7)-D, RHCE*ce48C,733G,1006T.
Additional antigen typing of the patient's red cells with unlicensed antisera indicated an Hr1 (2 of 2 sources) and Hr B 2 (2 of 3 sources) phenotype.
Conclusion: The RHD*DIIIa-CE(4-7)-D, RHCE*ce48C,733G,1006T haplotype is one of the RH haplotypes expressed by the original Hr B 2 individual Bastiaan.
The Hr B antigen status of red cells of individuals with the RHCE*-ceHAR allele has not been described.
We report an individual with the probable RHD*01, RHCE*ceHAR and RHD*DIIIa-CE(4-7)-D, RHCE*ce48C,733G,1006T RH haplotypes and production of alloanti-Hr B .
The specificity of the alloantibody produced and the red cell Hr B serologic antigen type supports the conclusion the variant allele RHCE*ceHAR does not encode for the Hr B antigen.
Excluding Clinically Significant Alloantibodies in the Presence of Interfering Antibodies with High-Titer, Low-Avidity Characteristics.
Background/Case Studies: High-titer, low-avidity antibodies (HTLA) are a group of clinically insignificant antibodies (AB) directed against highprevalence red cell antigens.
They interfere with the exclusion of clinically significant red cell AB and crossmatch testing, leading to long work-ups and potential transfusion delays.
We often use automated solid phase red cell adherence assay antibody panels (SP) when HTLA interference is seen by other methods, and undertook this study to determine its efficacy.
Study Design/Methods: A search of the laboratory information system database was conducted for patients with HTLA between 1/1/2006 and 3/ 31/2017.
All patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant AB (rule out).
Results/Findings: Over approximately 11 years, 81 patients had HTLA established at least once by titration studies.
Serological investigations on a total of 118 samples using a combination of gel, SP, and PEG and LISS tube methods, and occasional DTT and ficin panels, found that HTLA interference noted most frequently in gel (primary method) was, indeed, less often seen with SP.
However, the proportion of cases achieving rule out on SP was no greater than that with PEG testing (Table) .
For samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory.
Reference testing on 20 samples was successful in rule out in 60% of cases.
In an additional 12 patient samples, with negative antibody screens, HTLA were identified upon work-up for incompatible crossmatches.
Conclusion: SP is useful in avoiding interference from HTLA, but this conclusion is limited because SP was performed in only 40% of samples, and the inability to use select cell panels with SP made it difficult to complete rule out on samples containing multiple AB.
PEG testing was available for 71% of samples, and was at least as effective.
Further, manual testing allowed flexibility in selecting test cells when other AB were present.
Both SP and PEG testing may be used alone or in combination to avoid interference due to HTLA, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations.
Background/Case Studies: The DAU family of RHD alleles is characterized by c.1136C>T (p.Thr379Met).
The DAU0 allele harbors only this change, is not associated with depressed or altered D antigen expression, and is the ancestral allele from which other DAU alleles are purported to have evolved.
Srivastava et al (Transfusion 2016, 56:2520) recently summarized serologic characteristics and associated anti-D alloimmunization for 18 DAU family alleles.
We investigated two samples with the c.1136C>T change referred with weak D antigen expression.
Study Design/Method: Serologic testing was performed by standard tube methods using licensed anti-D reagents and the ALBAclone partial RhD typing kit.
Genomic DNA was isolated from WBCs and used in manual and array assays and for amplification and sequencing RHD.
Results/Finding: Sample 1 was from a 17 yo multiracial female.
Her RBCs reacted 11 S at immediate spin (IS) and 31 in IAT with Immucor Gammaclone and Series 4 and 5, and mi1 at IS and 41 in IAT with Ortho BioClone anti-D. RBCs did not react with 2 of 12 (LHM 174/102 & 57/17) anti-D in the partial D typing kit.
This pattern did not match any of the defined partial D epitope patterns.
RHD BeadChip found no changes but RFLP detected c.1136C>T characteristic of DAU.
RHD sequencing confirmed c.1136C>T and identified two adjacent changes, c.787G>T and c.788G>T (c.787_788delinsTT), in exon 5 encoding p.Gly263Leu.
Sample 2 RBCs reacted 1w at IAT with both Ortho BioClone and Quotient ALBAclone Delta, but were non-reactive with Immucor Gamma-clone, Series 4 and 5, and Quotient ALBAclone blend and alpha anti-D. Papain treated RBCs were 11s in IAT with Ortho BioClone.
These results suggested a D el like phenotype.
RHD BeadChip found no changes but RFLP detected c.1136C>T.
Sequencing confirmed c.1136C>T and found a new c.761C>T change (p.Ser254-Leu) in exon 5.
The c.761T has not been reported, but c.761G encodes a stop codon (p.Ser254Stop) in Japanese (Vox Sang 2015, 109:359).
Conclusion: We report two new alleles: RHD with c.787_788delinsTT (p.Gly263Leu) and RHD with c.761C>T (p.Ser254Leu), both also carrying the c.1136C>T (p.Thr379Met) characteristic of the African DAU cluster.
D antigen associated with p.263Leu is a partial D antigen with a novel epitope pattern.
The p.254Leu change is associated with a Del-like phenotype, the first observed to our knowledge for a DAU allele, and D antigen on the RBCs is not detected in IAT by 5/7 commercial anti-D.
The RHD nucleotide changes reported here are not in dbSNP database.
This study brings the DAU family of alleles to 21.
The number and diversity of alleles in the DAU cluster supports that the c.1136C>T change is a major ancestral African background allele (Wagner et al, Blood 2002,100:306).
Tae Eun Kim*.
KRC BTRI Background/Case Studies: There have been the cases of anti-D alloimmunization caused by the transfusion of serologically D negative blood component.
By analysis of genotype of the blood component, all of them were confirmed as Asian type DEL.
For that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay.
We established the algorithm for the genetic analysis of RHDin blood donors.
In this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test.
Study Design/Method: From September 2016 to present day we got 130 samples of repeated blood donors who are known to be D negative, C positive and/or E negative from 15 blood centers.
We obtained the consent for the test from all of the donors who provided samples.
As a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (PCR-SSP) for the region of promoter, exon 4, exon 7 and exon 10 in RHD gene.
Based on the results of PCR-SSP, we discriminated the results into total RHD deletion, RHD-CE-D hybrid and RHD variant.
When the results were discriminated to be RHD variant, we additionally analyzed the sequence of exon 9 to confirm the existence of c.1227G>A and c.1222T>A variations.
For the sample with indeterminate results, we performed sequencing for the full region of exon.
When the result was confirmed to be RHD deletion or RHD-CE-D hybrid, the blood components were regarded as RhD negative.
When the result was confirmed to be RHDvariant, the blood components were regarded as RhD positive.
Blood components were not supplied until the final results were obtained.
Results/Finding: For the 130 sample, we identified 71 cases (54.6%) of total RHD deletion, 18 cases (13.8%) of RHD-CE-D hybrid, and 41 cases (31.5%) of RHD variant.
39 of RHD variant were determined to be Asian type DEL with c.1227G>A variation.
2 cases of RHDvariant were regarded to be unknown variation.
Conclusion: The frequency of RHD variant in this study was 10 % higher than that of the general D negative donors not considering RhCE phenotype in a previous study.
For that reason, we considered that the genetic analysis of RHD targeting the donors of D negative, C positive and/or E negative is more efficient approach to identify RHD variant and better way to improve blood safety in the transfusion medicine related with RhD negative blood donors.
Lei Fang Tsai*, Ping Chun Wu, Shu Hui Feng, Yi Wen Tsai, Ming Hung Chen and Shun Chung Pai.
Taipei Blood Center, Taiwan Blood Services Foundation Background/Case Studies: Certain ABO subgroups or physiologic conditions may lead to mixed-field agglutination on ABO typing among blood donors.
The B 3 phenotype was found to be the most common subgroup in Taiwanese.
However, it is hard to distinguish the B 3 phenotype from other B subtypes also with mixed-field agglutination using routine serology without the genotype.
This study aimed to evaluate if flow cytometric method could alternatively differentiate different B subtypes with mixed-field agglutination rather than using molecular genotyping.
Study Design/Method: Blood samples from 30 Taiwanese blood donors exhibiting known common ABO phenotypes were included to establish normal flow cytometric patterns and genotyped.
Blood samples (n552) from B subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping.
Flow cytometric method was performed by FACSCalibur flow cytometry using the Gamma-clone anti-A and -B.
For genotyping, exon 6 and exon 7 of the ABO gene were amplified and sequenced.
The ABO*B3.03 allele was confirmed by PCR-RFLP analysis.
Results/Finding: Among 52 subjects with B 3 or AB 3 phenotypes, 47 were genotyped as ABO*B3.03.
The ABO*B3.03 group performed similar characteristic flow cytometric pattern and the profile was reproducible over time.
The pattern showed the main population of cells expressed no B antigen, while a percentage (37.81 6 6.62) of the RBCs exhibited B antigen levels diminishing with increasing of fluorescence.
Other 5 subjects with B 3 or AB 3 subjects, genotyped as ABO*B3.06(n51), ABO*BW.03(n51), ABO*BW.11(n51), ABO*BW.12(n51) and ABO*BW.29(n51), displayed flow patterns differed from the ABO*B3.03 group.
The ABO*BW.03, ABO*BW.11 and ABO*B3.06 subjects also showed a main population of cells expressed no B antigen and, however, less percentage of RBCs exhibited B antigen levels (<10% in ABO*BW.03 and ABO*BW.11 subjects and <20% in ABO*B3.06 subject).
Both ABO*BW.12 and ABO*BW.29 displayed a wedge-shaped pattern.
Conclusion: The flow cytometric method for the detection of B antigens on RBC might be useful in discriminating between B subtypes with mixed-field agglutination, especially ABO*B3.03 genotype.
This approach could assist the serological ABO subgrouping in clinical reference laboratory.
Frequencies and Specificities of "Solid-Phase Only" Detected Erythrocyte Antibodies: Is Solid Phase Testing Worth the Headache?
Karen Finegan*, Karen Gray, Jill Adamski, Theresa Kinard and Qun Lu.
Background/Case Studies: An effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated Gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system.
The data available from parallel testing on solid-phase, Gel, and PEG performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care.
Study Design/Methods: Throughout 2016, the transfusion service used automated solid-phase red blood cell (RBC) adherence as the primary method for antibody screening and identification.
All solid-phase antibody screen positive samples were re-tested using both Gel column agglutination and PEG method manually in order to determine which method should be used for antiglobulin phase crossmatch of RBC products.
All antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed.
Results/Findings: A total of 398 patients were positive on solid-phase antibody screen and re-tested on Gel and PEG antibody screen.
In 12% (n549) patients antibody reactivity observed in solid phase only and the concurrent Gel and PEG testing were completely negative.
Of them clinically significant RBC alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in 22% (n511), 4% (n52), and 74% (n536) of the cases, respectively.
RBC alloantibodies identified in solid-phase only included anti-E (n54), anti-Jka (n53), anti-K (n52), anti-Jkb (n51), both anti-E and Anti-C (n51) (see Table 1 ).
Conclusion: Solid-phase only RBC antibodies are clinically important in a significant portion of cases (roughly 1 in 4 cases).
Workup for solid-phase only antibodies is not "unnecessary" workload.
Transfusion of corresponding antigen negative RBCs to these patients prevented possible hemolytic transfusion reactions.
Full-Length Nucleotide Sequence of ACKR1 Alleles Encoding Duffy (FY) Antigens in Africans of Ethiopia Qinan Yin*, Kshitij Srivastava, Addisalem Taye-Makuria and Willy A Flegel.
Background/Case Studies: The human ACKR1 gene (previously known as DARC), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the Duffy blood group antigens (FY).
The Duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites Plasmodium vivax and P. knowlesi.
The study of FY variants in the low altitude and tropical Gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time.
In the present study, we determined the full length nucleotide sequence of the ACKR1 gene encoding FY antigens in donors from Ethiopia's southwestern Gambela region.
Study Design/Method: EDTA-anticoagulated whole blood was collected from study 60 volunteers in the Gambela region (NCT01282021).
The whole ACKR1 gene was amplified in one reaction covering 12,125 base pairs (bp).
This primary amplicon was re-amplified using nested primers covering 5782 nucleotides.
Nucleotide sequence was obtained by 14 sequencing reactions and manually annotated using NCBI RefSeq NG_011626.2.
The sequencing covered 1008 bp of both exons, 480 bp of intron, 2101 bp of 5'-flanking region, 947 bp of 5'-UTR, 53 bp of 3'-UTR and 1092 bp of 3'-flanking region and encompassed all the 470 variations present in dbSNP and NHLBI ESP databases.
Results/Finding: Among the 60 samples, a total of 15 SNPs, including one novel SNP in 5'-UTR were observed.
4 SNPs occurred in the exons, 5 in 5'and 3'flanking region, 4 in 5'-UTR and 2 in the intron.
All 60 individuals carried the SNP indicative of the common FY:2 phenotype; while 58 individuals were homozygous and 1 was heterozygous for the GATA box mutation.
No splice site mutation was detected.
As 46 individuals were observed as being homozygous or heterozygous for 1 SNP, we could unambiguously assign 8 distinct alleles.
In the remaining 14 individuals with 2 or more heterozygous SNPs, allele specific PCR is required to identify the alleles.
Conclusion: We sequenced more than 5.5 kb of the ACKR1 gene and identified at least 8 different alleles.
The present study found that the vast majority of alleles (117/120) in the Gambela population as defined by 15 SNPs, were similar to the clinically relevant FY*02N.01 allele, which in turn is defined by only 2 SNPs at positions c.1-67T>C and c.125G>A.
Out of the remaining 3 alleles, 2 were similar to FY*02 with the Fy(b1) phenotype and 1 was similar to FY*02W.01 with the Fy x phenotype.
The high frequency of FY*02N.01 (95%) in this study is similar to other studies conducted in Western, central and south-eastern regions from Gambia to Mozambique (95%-100%).
A more detailed analysis, including other regions of Ethiopia, will be useful to support transfusion care in the US for Ethiopian-Americans, the majority of whom may be of mixed Ethiopian ethnical background.
Judith Aeschlimann*, Sunitha Vege, Christine Lomas-Francis and Connie M. Westhoff.
Immunohematology and Genomics Laboratory, New York Blood Center Background/Case Studies: The homology, proximity, and inverted orientation of RHD and RHCE on the chromosome favor gene conversion events.
Regions of RHD are transferred into RHCE and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens.
RHD*DIIIa-CE(4-7)-D is the most common hybrid and is found in African Blacks.
It arose by conversion of exons 4-7 of RHCE*ceS into RHD*DIIIa and no longer encodes D antigen, rather (somewhat confusingly) encodes partial C antigen from the RHD locus.
This hybrid allele is in cis to RHCE*-ceS, together known as the r'S haplotype.
We investigated atypical RH genotyping results in three samples; two associated with weak D typing and one patient with sickle cell disease (SCD).
Study Design/Method: Serologic testing was by standard methods.
Genomic DNA was isolated from WBCs.
All samples were investigated by HEA PreciseType, RHD and RHCE BeadChip, RFLP, and Rh-cDNA sequencing.
SNP-specific sequencing was used to establish linkage/phasing.
Results/Finding: Sample 1 (male) and sample 2 (multiracial female), both C1c2E2e1 (presumed R1R1), presented with weaker than expected D typing; 11 IS and 31/41 at IAT.
RHD BeadChip identified the common African RHD*DIIIa-CE(4-7)-D hybrid encoding partial C antigen with apparent conventional RHD in trans.
These results did not provide an explanation for weak D antigen.
HEA indicated RHCE*Ce /Ce, concordant with the Rh phenotype, but c.733C>G and c.1006G>T (heterozygous) was also detected.
As RHCE*Ce with 733G and 1006T has not been reported, Rh-cDNA analysis was done.
Transcripts from the RHCE locus included one conventional RHCE*Ce in trans to RHCE*ceS with exons 2 and 3 replaced with RHD*DIIIa, and from the RHD locus, one conventional RHD and the hybrid RHD* DIIIa-CE(4-7)-D were found in both samples.
Sample 3 (SCD male), D1C2E2c1e1, by RH beadchip had one conventional RHD and RHD*DIII type 8, and RHCE*ce733G/ceS.
As RHD*DIIIa type 8 has never been found with either of these RHCE alleles, Rh-cDNA analysis was performed.
Transcripts representing a unique conversion event at the RHD locus, specifically RHCE*ce(48C) exons 1 and 2 had replaced those exons in the common hybrid RHD*DIIIa-CE(4-7)-D and expression of partial C antigen was lost These unique hybrid alleles have been deposited as GenBank#: KY926711and KY926710.
We report two different and novel complex RH rearrangements: two samples thought to be R1R1 had a unique RHCE locus representing a gene conversion into RHCE*ceS, designated RHCE*ceS-DIIIa(2-3)-ce.
In kind, a sample genotyped as DIII type 8 rather had a novel RHD locus representing a gene conversion into the common hybrid, designated as RHD*ce48C(1-2)-DIIIa(3)-ceS(4-7)-D .
These represent novel events on the r'S haplotype that can confound RH genotyping interpretations.
Interestingly, samples 2 and 3 have weaker than expected D antigen typing, despite the presence of a conventional RHD with RHCE*Ce [R1 haplotype (DCe)].
It is important to further investigate samples with unconventional results when interpreting RH genotypes.
High-Frequency Antibodies Anti-Lu(b-) and Anti-Yt(a-) in a Multi-Transfused Patient: A Case Study Nadia Baillargeon*, Carole Ethier, Cynthia Parent, Jessica Constanzo-Yanez, Maryse St-Louis, Marie-Claire Chevrier and Andre Lebrun.
Hema-Quebec Background/Case Studies: A 81-year-old Caucasian female was referred to our Immunohematology Reference Laboratory (IRL) for serological investigation.
She was diagnosed with anemia, renal failure and cardiac history.
Her hemoglobin level was recorded at 81g/L.
Her pregnancy history was not provided.
She had received 5 units of packed red blood cells (RBCs) in the past including 1 unit within the last 3 months.
None of the transfused unit was phenotypically-matched.
The referring hospital obtained panreactivity in gel with LISS-suspended RBCs and ficin-treated RBCs and negative direct antiglobulin test (DAT) and autocontrol (AT).
Study Design/Methods: ABO/Rh, DAT and antibody identification were performed by H ema-Qu ebec's IRL according to approved techniques.
In addition to LISS-suspended RBCs and papain-treated RBCs, trypsin-treated and chemical-treated reagent RBCs such as dithiothreitol (DTT) were tested.
Alloadsorption were done using papain-treated allogeneic RBCs (R 1 R 1 , R 2 R 2 , rr).
ID CORE XT platform (Progenika Biopharm / Grifols, Vizcaya, Spain) was used to analyse 29 polymorphisms which determine 37 antigens including Carthright and Lutheran blood groups.
PCR-SSP (Sequence Specific Primer) and PCR-RFLP (Restriction Fragment Length Polymorphism) were also performed to verify the absence of the high frequency antigens Yt a and Lu b .
Sibling samples were also requested to conduct a family study.
Results/Findings: Initial serologic testing showed strongly reactive panels in gel with LISS suspended RBCs, papain-treated RBCs as well as trypsintreated RBCs and DTT-treated RBCs but negative AT in each media leading to a probable antibody directed against high-frequency antigen.
Alloadsorption procedure allowed the identification of an anti-Jk a .
A select panel of high frequency antigens absent in Caucasian population was tested.
The patient's sera react weakly with one Jk(a-), Lu(a-b-) reagent cell.
In the meantime, genotyping results confirm the probable phenotype of the patient as Jk(a-) Lu(b-) Yt(a-).
Additional testing in gel using Trypsin and DTT differential effects on antigens Lu(b) and Yt(a) were performed to confirm antibody specificities.
No RBCs unit Jk(a-) Lu(b-) Yt(a-) were available for transfusion.
Selected units were Jk(a-) and Lu(b-) as alloanti-Yt a are known to cause none to moderate transfusion reactions.
Her daughter' sample were types as Yt(a1) and Lu(b1).
Conclusion: Serological study showed the presence of an anti-Jk a in addition to two antibodies directed against high prevalence antigen namely anti-Lu b and anti-Yt a .
The association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination.
Background/Case Studies: The KEL blood group system, consisting of 36 antigens encoded by the KEL gene, is organized into 19 exons.
There are approximately 30 KEL alleles associated with a Kell null phenotype (K 0 ) in which no Kell antigens are expressed, and 12 alleles associated with a Kell mod phenotype (K mod ).
Individuals with the K mod phenotype express very weak amounts of antigen on the surface of the RBC, and expression levels vary based on the allele present.
Here we describe the molecular and serologic testing that was performed in the case of a 53 year-old Hispanic male blood donor whose RBCs phenotyped K-k-Js(b-) Kp(b-).
Study Design/Method: The blood donor was phenotyped for K, k, Kp b and Js b antigens using standard tube agglutination methods.
Adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (American National Red Cross).
Genomic DNA (gDNA) was isolated from an EDTA blood tube using standard techniques.
DNA was genotyped for human erythrocyte antigens using the PreciseType TM HEA Molecular BeadChip (Immucor).
Exons 10, 11, 12,13 and 14 and flanking intron sequences were amplified and sequenced.
Total RNA was extracted using RNeasy Lipid Tissue Mini kit (QIAGEN) and KEL cDNA was amplified and the resulting PCR product was subjected to Sanger sequence analysis and aligned using Sequencher (GeneCodes).
Results/Finding: PreciseType TM HEA Molecular BeadChip testing predicted the sample to be K-k1 Kp(a-b1) Js(a-b1).
KEL-cDNA sequence analysis was performed and detected a single transcript species with c.578C, c.841C, 1790T, and missing the sequences corresponding to exons 11, 12 and 13.
Amplification of the exons from gDNA did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact.
cDNA analysis was repeated and the same aberrant transcript was detected.
Adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after 37C incubation at the PEG-IgG-AGT phase.
Conclusion: Here we describe a donor homozygous for a novel KEL*02 allele.
This donor was presumed to have a K 0 phenotype based on serology, but after molecular testing, has been reclassified as a K mod phenotype with extremely weak expression of k. The discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells.
The 11 variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations.
In contrast, the allele reported here is associated aberrant mRNA transcript.
We propose that this allele be named KEL*02M.12.
Here we report a case of a possible novel B subgroup observed in a pregnant black female.
The patient specimen was referred to our reference laboratory to investigate a possible ABO discrepancy.
The referring facility reported the patient's red blood cells were nonreactive with reagent anti-A and anti-B and the patient's plasma was reactive with A cells, but nonreactive with B cells using automated gel methodology.
Study Design/Methods: Serological testing of the patient's red blood cells was performed using routine and enhancement methods.
Molecular testing by PCR-RFLP was performed to determine the patient's genetic ABO typing and predicted ABO phenotype.
Results/Findings: Serological testing of the patient's red blood cells is summarized in Table 1 ; similar results were obtained with multiple sources of antisera.
Enzyme treatment failed to enhance reactivity.
Patient sera strongly agglutinated A1 and A2 cells, but failed to agglutinate multiple sources of B cells at all phases of testing.
Molecular testing by PCR-RFLP resulted in an uncommon banding pattern and indicated the presence of c.261 deleted G, characteristic of O alleles, c.467T, characteristic of A2 and some uncommon O alleles, and c.703A and c.1096A, characteristic of B alleles.
Genomic sequencing of exons 6 and 7 confirmed the presence of an O allele, ABO*O09 261del G, 318T, 467T), and the presence of a B allele (297G, 526G, 657T, 703A, 796A, 803C, and 930A), but did not reveal any changes associated with previously reported weak subgroups of B.
Conclusion: While serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone.
Additional ABO gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression.
Carine Arnoni* 1 , Tatiane Vendrame 2 , Janaína Muniz 3 , Diana Gazito 2 , Afonso Cortez 2 , Lilian Castilho 4 and Flavia Latini 2 .
1 Associa, 2 Associac¸ão Beneficente de Coleta de Sangue, 3 Hemocentro de São Jos e do Rio Preto, 4 Hemocentro Unicamp Background/Case Studies: After the elucidation of the molecular basis of VEL, molecular tools have been used to explain the reduced expression of Vel antigen in different populations.
Negative or weak reactions are generally related to the 17-bp deletion in SMIM1 in homozygous or heterozygous status.
However, other nucleotide changes have been described to reduce the Vel expression, as for example, the major A allele of the SNP rs1175550 located in the second intron of the gene, a regulatory region in erythroblasts.
This study aimed to characterize the genetic changes related to atypical Vel expression in a Brazilian population.
Study Design/Method: A total of 400 blood donor samples from the Southeast region of Brazil were typed for Vel with an anti-Vel serum from our inventory in GEL-IAT.
Samples typed as Vel-negative were further analyzed by adsorption-elution.
Molecular study was performed in samples with negative results, in samples reacting 21 and in samples with reactivity of 31.
DNA was isolated from peripheral blood and SMIM1 was sequenced.
Results/Finding: From 400 donor samples studied, 4 were serologically Vel negative by GEL-IAT but positive by adsorption-elution, 158 presented a 21 reaction and the remained samples showed a reactivity of 31.
Genotyping results showed that the 4 samples with negative results and 5 of 26 samples that presented 21 reaction were heterozygous for the 17 bp deletion and presented the A allele rs1175550 in homozygous status.
From the 21 of 26 remaining samples with reactivity of 21, 19 (90%) had the A allele of rs1175550 and 14 (66.7%) had the A allele of rs6673829.
In contrast, in the 16 samples with stronger reactions we found the A allele of rs1175550 in 5 (31.25%) samples and the A allele of rs6673829 in 3 (18.75%) samples.
Conclusion: The molecular changes rs6673829 and rs1175550 are located in intron 2 distancing 38 nucleotides.
This study reinforces the association of the A allele of rs1175550 with reduction of Vel expression and suggests the involvement of a new rs6673829 change in Vel expression.
In conclusion, the several patterns of Vel expression found in different populations can be influenced by different molecular changes.
Background/Case Studies: The D antigen is the most immunogenic antigen after ABO.
Consequences of misclassification of the D-antigen in patients or donors can be severe.
Some persons inherit mutations resulting in quantitative reductions of D antigen on the cell surface (weak D), some inherit Rh haplotypes that result in biochemical effects that reduce the availability of the D antigens to reagent anti-sera (Ceppellini effect), and others inherit D genes which are qualitatively different than wild type D. These latter individuals are often not identified until after they have formed anti-D. We hypothesize that some of these persons at risk of forming anti-D might be uncovered if they have weak and/or disparate D typing results with reagents that recognize different epitopes of the D antigen.
Study Design/Methods: All testing was performed using microtiter-well direct agglutination on the GalileoNEO or GalileoEcho (Immucor, Norcross,GA).
Any specimen that did not react as 0 (Rh Negative), or !31 on the NEO or !21 on the Echo (Rh Positive) for both series 4 and series 5 anti-D antisera were included.
Patients with discrepant historical types also were evaluated.
Any specimens meeting the inclusion criteria were tested on the NEO, Echo, and by saline tube method using series 4 and series 5 anti-D antisera.
Genotyping was performed from whole blood samples sent to Immucor genotyping laboratory in Warren, NJ using an algorithm of: RHD BeadChip, RHDxp (prototype assay), RHD zygosity, and RHCE BeadChip.
Results/Findings: 80 patients met inclusion criteria for molecular testing for the D antigen.
Weak or RhD variants were identified in 51 of 80 (63.7%) of the samples.
Ceppellini effect (i.e.
C in trans to RHD) resulting in weak D reactivity was seen in 16 of 80 (20%) of samples.
67 of 80 (83.8%) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm.
40 of 80 (50%) of tested samples had results indicating weak/variant D proteins with the potential to cause alloimmunization to the D antigen.
The remaining 13 of 80 (17.3%) samples did not have identified genetic cause for the weak and/or discrepant D test results and were presumptively classified as wild type D. Conclusion: Transfusion services that use the GalileoNEO or GalileoEcho to perform Rh typing should consider molecular testing of patients whose Rh typing results are discrepant, or positive but <31 on the GalileoNEO or positive but <21 on the GalileoEcho, as about half of these patients can develop anti-D.
This is particularly relevant for females of child-bearing potential where avoidance of D-positive transfusions and administration of RhIg during pregnancy is prudent until their D typing can be confirmed by molecular testing.
Carine Arnoni* 1 , Tatiane Vendrame 2 , Janaína Muniz 3 , Rosangela Person 2 , Lilian Castilho 4 , Afonso Cortez 2 and Flavia Latini 2 .
1 Associa, 2 Associac¸ão Beneficente de Coleta de Sangue, 3 Hemocentro de São Jos e do Rio Preto, 4 Hemocentro Unicamp Background/Case Studies: RhD and RhCE, are major protein constituents of red blood cell membrane, composing a complex together with RhAG.
Many variant Rh proteins have been described and most of them affect the integration of Rh proteins in the membrane.
D antigen expression can be affected by several molecular changes and also by the RHCEhaplotypes.
The present study investigated the score of reactivity of samples presenting a strong reduction in D expression.
Study Design/Method: A total of 108 samples were included in the study, being 69 previously genotyped as RHD*DAR1.2, 37 RHD*DAR3.1 and 2 RHD*DAU6.
The samples were phenotyped in NeoV R (Immucor) to D, C/c and E/e antigens by direct agglutinationin microplate.
Results obtained in NeoV R were expressed in a score from 0 -99 corresponding to the reaction intensity.
Zygosity assay was performed by a multiplex real-time quantitative PCR using a set of RHD-specific primers in RHD exon 10.
RHCE genotyping was performed by PCR-RFLP and SSP-PCR.
The presence of a D-CEhybridexon 3 was identified by amultiplex PCR.
Sequencing and identification of RHCE variants were also performed when necessary.
Results/Finding: Zygosity results showed that 10 of 108 samples (5 DAR1.2, 4 DAR3.1 and 1 DAU6) had 2 RHD genes, were phenotyped as C1E-c1e1 and genotyped as RHCE*ce/RHCEce.
RHD and RHCE genotyping in these 10 samples showed the presence of the D-CE-D S hybrid gene.
RHCE variants investigated in 2 DAR1.2 samples showed the RHCE*-ceAR/ce S genotype, in 2 DAR3.1 samples the RHCE*ceVS.02/ce S genotype and in the DAU6 sample the RHCE*ce S /ce genotype.
Table 1 describes the differences found in the reactivity of D among the samples carrying the (C)ce s allele and in the samples homozygous for RHCE*ce.
The results showed that the presence of RHCE*(C)ce S significantly reduces the expression of D antigen, probably due to the expression of the partial C partial antigen in trans to RhD.
Additionally, the samples with reduction on D expression carrying RHCE variant alelles phenotype can be useful to provide compatible blood to some patients with rareRH variant alleles.
Background/Case Studies: Drugs are known to interfere with routine blood bank testing.
A novel monoclonal humanized 5F9 antibody (Hu5F9-G4) that binds human CD47 has been entered into clinical trials for patients with acute myeloid leukemia, non-Hodgkin lymphoma and solid tumors.
We describe two cases of patients treated with Hu5F9-G4 (anti-CD47) who had ABO discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma.
Study Design/Method: This is a retrospective review of two cases with immunohematology work-up showing ABO discrepancy and plasma panagglutinin.
The first case is of a 69 year old female with progressive follicular lymphoma who was enrolled in Phase 1b/2 Trial of Hu5F9 G4 in combination with rituximab designed for patients with relapsed/refractory B cell NHL.
She had no prior transfusion history and her historical blood type was not known.
Two RBC units were requested in anticipation for a surgical procedure.
The second case is of a 48 year old male with refractory diffuse large B cell lymphoma enrolled in Hu5F9-G4 clinical trial.
His historical blood type was A Rh D positive with a negative antibody screen.
He received three RBC units within the past month prior to testing and receiving the anti-CD47 therapy.
Results/Finding: The ABO typing in the first case showed a discrepancy between the forward typing (41 with anti-A, non-reactive with anti-B) and the reverse typing (31 with both A 1 cells and B cells).
RhD typing was positive.
The extended reagent RBC panel tested with the patient's serum reacted with all cells tested at the immediate spin (IS) phase (11 to 41), at LISS-37C (11 to 31), at LISS-polyspecific AHG (m1), and at PEG-anti-IgG (m1 to 11).
Plasma reactivity at IS persisted with DTT or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or RESt adsorption.
Repeat testing, which avoided the IS and 37C readings, was non-reactive in the antihuman globulin (AHG) phase using both LISS and PEG enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens.
The Direct Antiglobulin Test (DAT) and autocontrol were negative.
The RBC units issued to the patient were crossmatch compatible at 37 o C AHG phase.
The ABO typing of the second case performed after anti-CD47 administration showed a discrepancy between the forward (41 with Anti-A) and the reverse (41 with both A 1 and B cells).
RhD typing was positive.
The antibody screen performed in solid phase technology was positive with all reagent red cells.
His plasma reacted with all reagent red cells at IS (21), at 37C in LISS (21), and LISS-polyspecific AHG (m1).
The DAT and autocontrol were negative.
His genotype was determined to be A1/O and full RBC phenotype by DNA analysis was obtained.
Repeat testing which avoided the IS phase did not show reactivity at PEG-AHG excluding all alloantibodies directed toward common red blood cell antigens.
Conclusion: Anti-CD47 therapy interferes with Blood Bank testing by causing ABO discrepancies and panagglutinin reactivity in the plasma at IS, 37C LISS, but not at AHG phase using Gamma-clone Anti-IgG, unlike the anti-CD38 interference.
Knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood.
Background/Case Studies: A middle-aged male with discrepant ABO typing results was investigated.
Initial forward typing was group O but no anti-B was seen in the reverse typing.
An unexpected 31 reaction was noted with an anti-A,B reagent.
Genotyping surprisingly showed ABO*O.01.01/ O.01.01, consistent with group O.
After initial testing at the referring center, samples were sent for extended analysis.
Study Design/Method: Standard serological methods and flow cytometry were used.
A panel of ABO reagents (n523) and lectins were tested with both native and papain-treated red blood cells (RBCs).
Lewis phenotyping was performed, as was genetic testing for ABO, GBGT1 and A4GALT.
Papain-treated patient RBCs were used to screen donor plasmas (n578) and two reactive plasmas were DTT-treated.
Results/Finding: Positive reactions were obtained with 3 polyclonal anti-A,B and a monoclonal anti-B (clone G1/2) when tested with the patient's papain-treated RBCs.
A panel of lectins gave negative results.
Genetic testing confirmed the predicted group O and ruled out the presence of FORS1 or NOR antigens.
The patient was Le(a-b1) and thus a secretor.
A positive crossmatch was seen with 47% of group O plasmas, while no reactivity was obtained with A or B plasmas.
DTT treatment of crossmatchpositive plasmas indicated the antibody to be mainly of IgG type.
This was confirmed by positive flow cytometry cross match using anti-human IgG secondary antibodies.
Reactivity remained after B-zyme treatment, thus excluding the normal (type 1 or 2) B antigen to be the underlying reason.
Inhibition with Lewis substance significantly decreased reactivity.
Enzyme activity assay showed the patient's plasma to contain a fully functional B glycosyltransferase.
On the suspicion that the patient had non-erythroid cells producing Breactive type 1 chains, a sample from a hematopoietic stem cell transplant (HSCT) patient (group B secretor receiving group O donor cells) was included as a control and gave the same type of reactions.
Conclusion: The medical history of the patient was queried and he had indeed undergone an HSCT $15 years earlier.
The reactions are likely due to uptake of recipient-derived BLe b (type 1) antigen (ISBT no.
007006), which is the dominant Lewis antigen in the recipient's original blood group, B Le(a-b1).
Interestingly, B-zyme did not affect BLe b .
Anti-BLe b is not simply anti-B plus anti-Le b but an inseparable and rarely reported specificity, which appears to be common among group O donors.
The phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups.
Background/Case Studies: The ProVUEV R and VisionV R (Ortho Scientific, Raritan New Jersey) automated analyzer use MTS-Gel TM card technology to perform immunohematology testing.
Benefits of automated testing include improved efficiency and enhanced reliability.
After eight years of using the ProVUEV R our Transfusion Medicine service switched to the ORTHO VisionV R analyzer in January of 2017.
Shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?")
results.
Additionally, some test columns were noted to be visually negative but called positive (11) by the analyzer.
The objective of this study was to investigate the cause of "?"
and apparent false positive results on VisionV R three-cell antibody screens.
Study Design/Methods: With assistance from Ortho Diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?"
results, and the gel card lot numbers used for testing from 1/29/2017 to 3/31/2017.
Reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results.
Investigation also included review of daily QC records, instrument maintenance, instrument diagnostics, and camera calibration.
Results/Findings: Of 19,647 columns run as part of antibody screens, 1,633 (8.3%) columns generated "?"
results.
Assuming 30 seconds of technologist time per "?
", we estimate that 13.6 hours were needed to resolve and update these results.
Among all potential causes investigated, only the gel card lot number was associated with the number of "?"
generated (table 1) .
In 26 cases, all three columns were visually negative but the analyzer reported 11 reactivity with 1 of 3 cells.
All cases had MTS-Gel TM antibody identification panels performed, 25 of 26 also had a MTS-Gel TM ficin panel.
The yield for the 51 panels performed was two routine panels with weak reactivity against HLA1 cells, and four ficin panels with weak reactivity with no apparent specificity.
Fourteen patients coincidentally had a subsequent Type and Screen; 13 were negative.
One patient newly demonstrated anti-Jka.
Fifty percent (13/26) of visually negative but analyzer positive samples were tested with gel card lot number 3, 38% (10/26) with lot 1, and 12% (3/ 26) with lot 2.
Conclusion: The incidence of "?"
and visually negative analyzer positive results is dependent on the specific lot of MTS-Gel TM cards used.
The difference between the lots is being investigated by Ortho Diagnostics, and remains to be explained.
To avoid unnecessary waste of technologist time and other resources, with assistance from Ortho Diagnostics, we have Results/Finding: Of the methods evaluated, the DTT method proved the most useful for mitigating DARA interference.
Cord cells were effective but in limited supply and alloadsorption was ineffective.
Of the three different DTT methods evaluated, the tube method initially failed which led to re-evaluation with the addition of LISS (passed).
The GC method was the most sensitive method.
Following release of DARA, samples from 28 patients (137 cross-match samples, 301 units issued) were tested using both LISS tube and GC IAT methods.
Despite DTT treatment, the GC method remained positive by IAT in 16/28 patients.
Further testing was performed in 9/16.
Eight were tested for the presence of antibodies at 188C and confirmed in 8/8.
Rouleaux formation was observed in 5/9 patients, 4/5 had reactivity detectable at 188C.
No transfusion reactions have been reported to date nor has alloantibody formation been observed to date.
Conclusion: As previously reported, the DTT method was the most useful for mitigating DARA interference.
The observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the LISS tube IAT.
This may be due to the washing phase in this technique which dissipates rouleaux formation.
Reactivity due to cold reactive antibodies can be eliminated by performance at strict 378C.
Our practice is now to use both DTT IAT methods on initial patient referral, if residual reactivity in GC is observed use LISS tube in preference thereafter in these patients.
A further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude DARA use if suspected.
Wendy Beres* 1 , Sandra Nance 2 , David Moolten 3 and P. Dayand Borge 3 .
(2):47-53).
Our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels.
This 2.5 year retrospective study was performed to assess levels of RBC-bound IgG, IgA and IgM in normal donors.
Study Design/Method: Residual EDTA-anticoagulated aliquots from 150 random allogeneic and 20 autologous blood donors were sequestered and tested per Institutional Review Board approved protocol.
The samples were tested by FC with fluorescein isothiocyanate (FITC)-labeled anti-human IgG and IgA (Jackson ImmunoResearch Lab, West Grove, PA) or FITC-labeled anti-human IgM (Life Technologies, Carlsbad, CA) at optimized dilutions in Dulbecco's PBS containing 0.6% BSA.
The Becton Dickinson FACSCalibur TM or FACScan TM (San Jose, CA) FC analyzed 50k RBCs from each sample.
EDTA-anticoagulated samples and Ig coated control RBCs were tested to determine FC settings and control for validity and cross-reactivity.
Controls reacted as expected.
There were less autologous donors tested and with a mean age of 62 these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured.
Despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated RBC-bound IgA, IgG, and IgM levels.
This emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing.
Long Range PCR Reveals the Genetic Basis of an Antibody in Pregnancy to a High Prevalence MNS Antigen Judith Aeschlimann* 1 , Anna Burgos 1 , Virginia Lew 2 , Sunitha Vege 1 , Susan Veneman 3 , Christopher J Gresens 3 , Jonathan Hughes 3 and Connie M. Westhoff 1 .
1 Immunohematology and Genomics Laboratory, New York Blood Center, 2 Blood Centers of the Pacific, 3 BloodSource Background/Case Studies: Recombination events have generated many GYPA and GYPB hybrids giving rise to glycophorin (GP) variants that express low-prevalence antigens (e.g.
Mia, MINY, Mur).
In rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g.
ENKT, ENEH, ENAV).
Complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes.
We investigated samples from a pregnant Asian (Hmong) woman with an antibody to an unidentified highprevalence MNS antigen, and samples from her sister.
Study Design/Method: Standard methods were used for RBC typing with licensed and unlicensed reagents and for antibody identification.
DNA was isolated from WBCs, and HEA PreciseType, exon-specific amplification and sequencing GYPB exons 1-6, and long range sequencing of exon 2-6 (5.4kb amplicon) were performed.
SNP-specific primers were used to associate changes (phasing) to specific alleles.
Results/Finding: RBCs of the pregnant proband typed S-s-(Gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high Background/Case Studies: The RhD antigen is clinically significant and immunogenic and therefore individuals who develop anti-D are at risk of haemolytic transfusion reaction.
RHD polymorphism shows substantial ethnic variability and at least 100 RHD variants associated with weak D alleles have been reported.
In this study, we report two new RHD alleles in Brazilian blood donors associated with weak D antigen expression.
Study Design/Method: The D status was evaluated with 4 commercially available monoclonal anti-D reagents: 1 blended IgM/IgG (clones TH-28/ MS-26), 2 IgM (clones MS201 and P3x61) and 1 IgG (MS26) in tube and on gel cards.
C, c, E and e phenotyping were performed in gel.
Most common weak D and partial D alleles were investigated by allele specific (AS) PCR and with the RHD BeadChip platform from Immucor.
Direct automated sequencing of the 10 RHD exons and flanking intron regions was performed by the Sanger dideoxy method.
In order to determine RHD allelic combinations, we also performed Rh-cDNA cloning and sequencing.
Background/Case Studies: Donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (RBC) genotyping panel.
When serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely.
Investigation of the discrepancy often leads to identification of variant antigens.
It is known that the set of GYP variants associated with expression of the St a antigen can also be associated with N typing discrepancies in M1N-individuals (Meyer et al.
Br J Haematol.
2016; 174:624-36) .
The St a allele, also described as GYP*401, is a hybrid GYPB-GYPAtranscript with the crossover in intron 3.
We sought to investigate five N typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel.
Background/Case Studies: A sample from a 24 years old pregnant, African American female G1P0 was sent to the blood bank for ABO/Rh and antibody screen.
The sample was analyzed using the Provue analyzer (Ortho Diagnostics).
The patient was typed as O pos with no reverse type discrepancy.
A retype of the same sample was performed using tube method with Biorad reagents per Hospital policy due to no previous ABO/Rh history on file.
The retype showed that the patient was A subgroup with Anti-A 1 antibody present in the plasma.
The sample was referred for genotyping, with the suspicion of A 3 like phenotype.
Genetic testing did not support the serological findings of A 3 subgroup and a new ABO allele, ABO*784C that has never been reported in correlation with an A 3 like subgroup was detected Study Design/Method: The patient RBCs were typed with anti-A 1 (Immucor) and anti-A,B (Biorad and Grifols DG Gel).
An Anti-A 1 antibody work up was performed using three different lots of A 1 cells and three lots of A 2 cells, as well as a type O screening cell and auto control .
The tubes were read at IS and also incubated at RT and 48C for 30min.
The patient 's initial antibody screen using Ortho gel was negative.
Conclusion: The patient delivered a healthy baby boy at 35 weeks of gestation.
The baby cord was sent to the laboratory.
The baby serological type showed an A 3 B phenotype and it was referred for genetic testing.
The baby RBCs showed the same ABO*784C found in the mother.
The previously reported ABO*784A allele encoded an Aspartic Acid to Asparagine change at position p.256 consistent with an A weak phenotype.
Also, at least five other alleles encoding an A 3 phenoytpe consisted of polymorphisms at positions c.745 through c.871, giving special characteristics to this new and unreported ABO allele.
From the data collected, it can be concluded that this A3/ Aweak phenotype is encoded by the variant allele ABO*784C.
This highlights the clinical relevance of confirming the serology of ABO subgroups by molecular methods.
Philip Berardi*, Jacqueline Cote, Gwen Clarke, Vito Scalia, Robert Liwski and Mindy Goldman.
Canadian Blood Services Background/Case Studies: Elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens.
The commonly used single nucleotide polymorphism (SNP) arrays require nucleic acid isolation which is typically achieved by extracting genomic DNA from whole blood.
This method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location.
DNA extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction.
Canadian Blood Services (CBS) has performed large scale DNA extraction and HLA genotyping for the OneMatch Stem Cell and Marrow Registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the US NMPD.
We sought to assess the accuracy and reliability of using DNA extracted from buccal swabs in predicting blood group antigen expression.
Study Design/Method: We performed parallel red cell genotyping on an automated typing platform, the Progenika/Grifols IDCoreXT assay (Progenika Biopharma-Grifols, Bizkaia, Spain) using DNA extracted from blood and buccal tissue from volunteers.
For antigen systems with available serologic reagents, we also compared results with serologic typing.
We evaluated three different methods of DNA extraction and performed testing regardless of DNA yield or purity.
Two buccal swabs (Puritan Medical Products, Guilford, Maine) were used for each test.
Swabs were stored at room temperature, and DNA extraction was performed within six days of collection.
In the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated Biorobot M48 Robot using the MagAttract DNA Mini M48 extraction method (Qiagen, Venlo, the Netherlands).
Extracted DNA had a mean concentration and purity of 38.1ng/ml and 1.83 respectively.
In the second phase of the study (n 5 39), DNA extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the QIAamp DNA mini kit, using either manual or an automated QiaCube robotic workstation (Qiagen, Venlo, the Netherlands).
Results/Finding: The manufacturer's recommended analytical range for DNA concentration was 20-80ng/ll and the recommended purity was an absorbance ratio of 1.63-2.1 (A260/280) for use of the ID CoreXT platform.
DNA extraction from buccal swab samples did not meet these specifications in several cases.
However, in most cases, a lower concentration of DNA was adequate for prediction of phenotype.
The Dombrock system was the most susceptible to failure of interpretation in the samples with a low DNA concentration, with "no call" results reported.
There was 100% concordance in genotyping results when source DNA was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results.
Conclusion: This study supports the use of genomic DNA extracted from buccal tissue on the ID CoreXT for predicting RBC phenotype with high accuracy.
Extraction methods may require optimization to achieve DNA yields within the recommended analytical range of the assay.
Performance Evaluation of ID RHD XT, a Genotyping Assay for the Detection of High-Prevalence RHD Negative and Weak D Types Araitz Molano 1 , Izaskun Apraiz 1 , María Azcarate 2 , Miguel Angel Vesga 2 , Montserrat Rubia 2 , Mercedes Piedrabuena 2 , Fernando Puente 3 , Barbera Veldhuisen 4 , Ellen Van Der Schoot 4 and M onica L opez* 1 .
1 Progenika Biopharma, a Grifols Company, 2 Centro Vasco de Transfusi on y Tejidos Humanos, 3 Banco de Sangre y Tejidos de Arag on, 4 Sanquin Blood Supply Research Background/Case Studies: It is well established that weak D 1, 2 and 3 phenotypes are not at risk for forming allo-anti-D, whereas a few weak D and all partial D and negative phenotypes are.
Routine serologic D typing does not distinguish among them, consequently RHD genotyping is recommended, especially in patients.
ID RHD XT (Progenika, Grifols) is a qualitative, PCR/Luminex V R xMAP hybridization-based genotyping test for the identification of the following RHD gene allelic variants: RHD*weak D type 1, RHD*weak D type 2, RHD*weak D type 3, RHD deletion, RHD*Pseudogene and RHD*DIIIa-CE(3-7)-D and ITGB3 gene: HPA1a and HPA1b, in genomic DNA extracted from whole blood specimens.
In this study the performance of ID RHD XT genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for Rh and HPA-1 blood group typing.
Study Design/Method: A cohort of 1000 previously serotyped samples for D antigen obtained from three European blood centers were analyzed with ID RHD XT at Progenika.
Samples were distributed as recommended by the Annex of the Common Technical Specifications 2009/108/CE for a IVD product of list A (!10% Clinical samples, >2% Neonatal Specimens and !2% Weak D donors).
For the intended use of the product, Weak D serotyped donors were enriched (n5160, 16%).
Commercial serology tests for D antigen predicted phenotype and Bi-Directional-Sequencing (BDS) for weak D type confirmation and HPA-1 predicted phenotype were used for comparison.
TRANSFUSION Results/Finding: No system failure, 100% call rate and no inconclusive results were obtained.
Discrepancies were found for D antigen between serology and ID RHD XT predicted phenotype results, although a 100% concordance was obtained when analyzed by BDS, considering ID RHD XT result correct.
Concordance between ID RHD XT and BDS results for the Weak D type was 100%.
The following ID RHD XT predicted phenotype results were obtained: D negative (n5361), No amplification variant (n515), Weak D Type 1 (n522), Weak D Type 1 heterozygous (n51), Weak D Type 2 (n532), Weak D Type 2 heterozygous (n51), Weak D Type 3 (n534), Weak D Type 3 heterozygous (n51), Weak D Types 1, 2 or 3 not detected (n5533).
Regarding HPA-1 blood group, the predicted phenotype results obtained by ID RHD XT were 100% concordant with BDS results: HPA-1a positive (n5157) and HPA-1a negative (n56), HPA-1b positive (n546) and HPA-1b negative (n5117).
Conclusion: ID RHD XT genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence RHD negative and Weak D types (100% specificity and 100% sensitivity for D antigen, HPA-1a and HPA-1b antigens).
That makes it a useful tool for the implementation of the RHDgenotyping recommendation on patient blood transfusion and anti-D prophylaxis.
Background/Case Studies: SCD patients form red blood cell (RBC) antibodies at higher rates than other transfused populations.
Multiple predictors of alloimmunization have been reported but not well replicated in large SCD cohorts.
We investigated the clinical, laboratory and genetic predictors of alloimmunization.
Study Design/Method: A large SCD cohort was established in Brazil to investigate disease outcomes.
At participating sites, patients are currently transfused with ABO/D/Cc/Ee/Kell matched RBCs prophylactically and extended phenotypically matched RBC after first antibody forms.
Policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study.
Transfused subjects with 11 RBC alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and T-test or Wilcoxon rank-sum tests as appropriate to compare continuous variables.
Backward elimination multivariable logistic modeling was used to generate odds ratios (OR) and identify independent predictors of alloimmunization using results of univariate analyses.
All subjects had peripheral blood whole genome SNP typing performed using an Affymetrix array, which included enhanced content for blood related SNPs.
Genome wide association (GWA) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization.
A p value <50.05 (clinical analysis) or <5x10 -8 (GWA) was considered statistically significant.
Results/Finding: Of the 2795 cohort patients, 2272 (81.3%) transfused subjects were included with 129 alloimmunized children <18 years (11.0% of 1172) and 224 alloimmunized adults (20.4% of 1100).
In multivariable logistic regression models, age (OR 4.2, p50.009, for age 501 compared to 0-4), gender (OR 1.3, p50.04, for female compared to male), transfusion history (OR 3.5, p<0.0001, for 811 transfusions compared to 1-5), site, hemolysis (OR 1.3, p50.05, for log transformed lactate dehydrogenase) and presence of autoimmune disorders (OR 4.5, p<0.0001) were independent predictors of alloimmunization.
GWA identified a single SNP of unclear biologic significance associated with alloimmunization (EEFSEC gene responsible for incorporation of selenocysteine into proteins).
Conclusion: RBC alloimmunization is primarily driven by transfusion burden in this SCD cohort.
Hemolysis remained significantly associated with alloimmunization after controlling for transfusions.
Presence of an autoimmune disease was also associated with RBC alloimmunization, indicating more systemic immune dysregulation may be present in SCD patients who develop RBC alloantibodies.
However, the GWA did not identify SNPs in immunoregulatory genes significantly associated with RBC antibody formation in the study population.
Background/Case Studies: Physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests.
To reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing.
Mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage.
There has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice.
Study Design/Methods: Transfusion Service tested sample pairs from 32 newborns less than 2,000 gr birth weight.
One of the samples was collected from the newborn as a heel stick sample, the other from the placenta.
The following tests were performed on the sample pairs: ABO, Rh, antibody screen and Direct Antiglobulin Test with IgG (DAT).
Results/Findings: ABO, Rh and DAT tests were performed on 32 sample pairs.
DAT test was negative on 30 sample pairs and two were positive.
There was 100% concordance with the ABO, Rh and DAT tests performed on these sample pairs.
Antibody screen was performed on 32 placental blood samples and 29 heel stick samples.
Twenty eight sample pairs were negative with the antibody screening test.
There was one positive heel stick sample, which was also positive using the placental sample.
One heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample.
This antibody which was detected only in the placental sample was a passive anti-D mother received during pregnancy.
This discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody.
Conclusion: This study shows that placental blood sample is not inferior to heel stick sample regarding ABO, Rh and DAT testing.
Based on this comparison study placental blood can be used for pre-transfusion testing for < 2,000 g birth weight newborns.
O-(7.4%), AB1(6.8%), B-(2.7%), and A-(0.6%).
Among the tested donors, 89.2% were D positive with R1r being the most common Rh phenotype.
In the Kell blood group system, 4.5% of the donors were K positive, while the k antigen was found to be 99.4%.
The most common phenotype in the Duffy blood group system was Fy(a-b-), while the Fy(a1b1) was found at a higher frequency compared to what has been reported in the black population.
(Table) The commonest phenotypes for the Kidd and MNS blood group systems were Jk(a1b1) and M1N-S1s1 at 47% and 22.6% respectively.
The Le a 1 and Le b 1 alleles were seen in 21.7% and 67.3% of donors respectively, while Lu b -phenotype was found in 3.3% of the donors.
The frequencies of the rare phenotypes Jk(a-b-), Le(a1b1) and Lu(a-b-) were 0.3% , 0.3% and 2.7% respectively, while the M1N-S-s-and M-N1S-s-phenotypes were not found.
The frequency of the P1 antigen was found to be at 78.9% similar to what has been reported in Caucasians.
Conclusion: This is the first study to examine the frequencies of RBC blood group phenotypes among the Omani blood donors.
The results show higher frequencies of the rare null phenotypes Fy(a-b-), Jk(a-b-) and Lu(a-b-) compared to what has been reported in Caucasians.
The frequencies of the Duffy blood group system resemble what has been reported in the black population.
This data is helpful in understanding the influence of the Arab ethnic background on the RBC blood group systems and warrants large genotype-phenotype studies in the region.
Quantitation of Anti-D in Serum Using Flow Cytometry Amanda Whitelonis*, Izekial Butler, Karen Leighton, Scott Jones and Anand Srinivasan.
QualTex Laboratories Background/Case Studies: Rh(D) antibodies (anti-D) are developed in Rhnegative individuals when exposed to D antigens.
This scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients.
Quantitation of anti-D in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn.
Quantitation of anti-D is also performed in quality control operations of organ procurement organizations and plasma fractionators.
It is a common practice to report the strength of anti-D in serum as antibody titer values but quality control operations require a quantitative value.
We have developed a screening assay using flow cytometry to quantitate anti-D in serum.
Study Design/Method: We have developed a method to quantitate anti-D in serum using flow cytometry, by modifying the protocols of Christensson et al., and Hilden et al.. Red blood cells from Rh-positive blood samples were washed three times in phosphate-buffered saline (PBS) at pH 7.2 and the supernatant was discharged.
A dilution buffer containing 2% human serum albumin (v/v) in phosphate buffered saline was prepared.
Serum samples or WHO anti-D standards, suspended in dilution buffer were mixed with 10ll of washed red cells.
The cell suspensions were incubated for 30 min at 378C.
Following incubation, FITC-labeled anti-IgG diluted in buffer was added and the mixture was incubated for an additional 15 min at 228C.
The samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals.
Green fluorescence was collected using a band-pass filter set for 515-548nm.
Events were recorded at a frequency of 1000 cells.
Results/Finding: Multiple dilutions of WHO anti-D reference standard were tested against Rh-positive red blood cells from five different donors.
The reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition.
After optimizing these factors, a linear regression was calculated to establish the standard curve.
The fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of Rh(D) antigens in reference standard.
Serum from thirty Rh(D)-immunized volunteers were analyzed for concentration of anti-D and the results were benchmarked with antibody titer values.
Conclusion: Based on our study, we conclude that the quantitation of Rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-D antibodies in serum.
The method is reproducible and advantageous over reporting antibody titer values.
The operations of this platform can be translated to a well-plate based high-throughput flow cytometry.
Sarah K Harm* 1 , Mark Yazer 2 , Nancy M. Dunbar 3 and Biomedical Excellence for Safer Transfusion (BEST) Collaborative 1 .
1 University of Vermont Medical Center, 2 University of Pittsburgh, 3 Dartmouth-Hitchcock Medical Center Background/Case Studies: The use of emergency issued group A plasma and uncrossmatched group O whole blood (WB) in patients without a valid ABO group is becoming increasingly common in the USA.
It is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established.
This study was designed to determine the rate of high titer donors using a titer threshold of 50.
Study Design/Methods: Three academic hospitals that routinely issue group A plasma units for emergency issue participated in this study.
Before issuing this plasma to patients, a 1:50 dilution of the donor's plasma in saline was produced and added to group B reagent red blood cells (RBC).
If any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group A or O recipients.
At these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added.
At one center samples were taken from the plasma of group O WB units and the same procedure was followed using A1 and B reagent RBCs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the WB unit was considered high titer and it was then centrifuged into an RBC unit for transfusion while the plasma and platelet components were discarded.
Two centers provided plasma testing data for a 4-year and 5-year period, respectively.
One center provided plasma and WB testing data for a 1-year period.
Results/Findings: In total there were 7106 group A plasma units tested and 654 (9.2%) had a high titer anti-B.
The range of high titer group A plasma units between these three centers was 7.4%-12.5%.
Of the 1778 WB units tested, 388 (21.8%) units had a high titer; 221/1778 (12.4%) of the units had a high titer anti-A, 61/1778 (3.4%) had a high titer anti-B, and 106/1778 (6%) had high titers of both anti-A and anti-B.
Background/Case Studies: Dithiothreiol (DTT) is a sulfhydryl reagent that denatures selective blood group antigens.
Reagent red blood cells (RBCs) treated with 0.2M DTT is used as a tool in identifying antibodies to high frequency antigens.
Recently, DTT has become widely used in destroying CD38 on reagent RBCs and render them free from plasma anti-CD38 drug interference.
Procedures for the preparation of 0.2M DTT has been published advocating the use of buffered saline at different pH levels.
In this study, an effect of pH on 0.2M DTT treatment time is investigated.
Study Design/Methods: Non-buffered saline (NBS, Thermo Fischer Scientific Inc, Middletown, VA), used in the preparation of 0.2M DTT, was adjusted to pH7.16, pH 7.56, pH 7.96 using sodium phosphate dibasis (Sigma Aldrich, Saint Louis, MO).
Reagent RBCs (Immucor, Norcross, GA)(n53) were treated with the 0.2M DTT solutions in parallel by mixing 1:4 ratio of packed RBCs to 0.2M DTT solution followed by incubation at 378C.
For up to 45 minutes during treatment, the expression of k antigens was measured every 5 minutes by tube method using 2 different sources of anti-k. To assure uniformity, all reactions were graded by the same investigator.
Each reaction grade (in each RBC and each antiserum) is converted into a semiquantitive score and an average score was calculated every 5 minutes for each pH level.
The reduction in average scores between different pHs were also calculated at every 5 minutes to measure the impact of 0.2M DTT reagent pH on the rate of k antigen destruction.
Results/Findings: The expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by !11) after 15 minutes of DTT treatment at pH 7.96; 15 minutes at pH7.56 and 20 minutes at pH7.16.
Complete loss of k expression was seen after 25 minutes of DTT treatment at pH7.96; 35 minutes at pH7.56 and 35 minutes at pH 7.16.
The reactivity patterns of k antigen tested with 2 sources of anti-k correlate with each other.
The reductions in average scores were seen between 15 to 30 minutes range of DTT treatment time when pH 7.16 was raised to pH7.56; 15 to 30minutes range when pH7.56 was raised to pH7.96; and 15 to 30 minutes range when pH7.16 was raised to pH7.96.
Conclusion: The use of higher pH buffered saline may shorten the treatment time it takes to weaken or destroy k antigen.
Based on the comparison of reaction scores between different pH levels, the pH levels did not have an impact on DTT treatment up to 15 minutes and/or beyond 35 minutes of incubation.
The pH of the 0.2M DTT reagent relative to the treatment time is a factor to consider during the validation of DTT-treatment process and qualification of 0.2M DTT reagent in a laboratory.
Background/Case Studies: Data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the United States.
The aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental United States.
Study Design/ Method: The aim of this retrospective study was to evaluate a cohort of prenatal patients (n 5 756,221) drawn between July 1, 2013 and June 30, 2014.
These patients were divided into United States Census Bureau regional and divisional categories according to their place of residence.
Prenatal blood work was collected which included an ABO, Rh(D) and a screen for unexpected alloantibodies.
Samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification.
Results/ Finding: In total, 11,647 patients were found to possess clinically significant red cell antibodies for an overall incidence of 1.5 percent.
The three most commonly encountered antibodies were anti-D (n 5 7639) 63.1%, anti-M (n 5 1288) 0.6%, and anti-E (n 5 1227) with a frequency of 10.1%.
A total of 455 (3.9%) prenatal women were found to possess two or more antibodies.
In general, the combination of anti-D and anti-C proved to be the most common, with 182 instances (40.0%) followed by anti-E and anti-c with 79 (17.4%), and anti-C, anti-e with 26 (5.7%).
Of the multiple antibodies identified, 435 (95.6%) included at least one antibody from the Rh blood group.
The South region had the largest number of antibodies identified with 7474 or 61.8% of the total.
The West had 2111 (17.4%), the Midwest 1538 (12.7%) and the Northeast with 979 (8.1%).
A contingency table, using the two-sided Fisher's exact test, was performed comparing the Northeast, South, Midwest and West regions.
The p value of anti-D was calculated to determine nonrandom associations and values of 0.05 and below was deemed significant from a region-to-region perspective.
With regard to anti-D, the Pacific division comprised of California, Oregon, Washington, and Alaska, had p values below the 0.05 thresholds when compared against seven of the eight other divisions.
The West South Central division (Texas, Oklahoma, Arkansas, and Louisiana) did not show statistically significant results when compared against the Pacific division (p 5 0.17).
Conclusion: Depending upon the antibody, statistically significant variations between geographical regions and divisions within the United States were observed.
This relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations.
Reduction in Repeat Testing Using Gel Technology Amy Mata* 1 , Lindsy Rich 1 , Sherry Stern 1 , Sharon Wangen 1 and Camille van Buskirk 2 .
1 Mayo Clinic, 2 Mayo Clinic Rochester Background/Case Studies: Our institution currently uses the IMMUCOR NEO (Immucor, Inc., Norcross, Georgia) to perform ABO/Rh and antibody screen (ABSC) testing utilizing solid phase technology.
When results are unable to be obtained from the IMMUCOR NEO, testing is repeated on the manual testing bench using tube agglutination.
This repeat testing can lead to significant expenses including reagents, supplies, and technologist time.
It was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard.
A side-by-side evaluation was performed between the IMMUCOR NEO and the ORTHO VISION (Ortho Clinical Diagnostics, Rochester NY) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed.
The evaluation looked at ABO/Rh and ABSC testing as those are the only tests that are currently automated in our laboratory.
Study Design/Method: Thirty specimens that were processed on the IMMUCOR NEO and resulted in No Type Determined (NTD) for ABO/Rh testing were selected to be tested on the ORTHO VISION.
Twenty-three specimens that were processed on the IMMUCOR NEO and produced positive results for ABSC testing were selected to be tested on the ORTHO VISION.
All specimens were EDTA tubes and were collected within the previous 4 days.
The timeframe between when the specimen was tested on the IMMUCOR NEO and the ORTHO VISION was 1 to 2 days.
Results/Finding: Of the 30 NTD specimens from the IMMUCOR NEO, 8 resulted in valid ABO/Rh typings on the ORTHO VISION.
Three results were flagged indicating possible extra reactivity.
Upon performing a visual review of all 3 results, it was determined that there was no reactivity and a valid result was present.
The other 19 samples required manual tube testing to interpret the ABO/Rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis.
Of the 23 ABSC specimens that were resulted out as positive on the IMMUCOR NEO, 11 specimens produced a negative result on the ORTHO VISION and were confirmed to be negative with manual tube testing using PEG as the enhancement media.
One specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative.
Nine specimens that were positive on the IMMUCOR NEO were also positive on the ORTHO VISION.
One specimen proved to be an anti-M that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out.
All showed discrepant results with monoclonal anti-C reagents, with a similar pattern of reactivity: 3-41 with MS24 (n515), 1-31 S with MS23 (n59), no reaction with MS273, DGC02, P3x255 (n514).
14 samples tested with a polyclonal anti-C showed a 1-31 reactivity.
3 D1C1E1c1e1 cases tested with a polyclonal and monoclonal anti-e (MS16, MS21, MS62, MS63) showed no weakened reactivity.
RHCE sequencing (genomic DNA or cDNA) showed a c.286G>A mutation in exon 2, predicted to encode the p.Gly96Ser substitution.
For 2 apparent R 1 R 2 donors, a f-negative type allowed the prediction of a RHCE*Ce286A/RHCE*cE genotype.
Altogether, our results are consistent with the presence of a very likely RHCE*Ce286A allele (C and e in cis) in all samples.
3 D1C1E1c1e1 individuals were reactive 11 S with the original source of anti-Rh55, slightly weaker when compared to RHCE*ce286A/RHCE*Ce RBC samples available from our cryobank (21).
Conclusion: Our results confirm that the c.286G>A mutation alters the conformational properties of the RHCE protein, either on a ce or Ce background, and encodes the low-prevalence LOCR antigen (RH55).
The LOCR reactivity appears to be rather similar when coded by RHCE*Ce286A or RHCE*ce286A alleles.
This was quite an unexpected finding, since the p.Gly96Ser substitution is close to the critical amino-acid for C/c expression (p.Pro103Ser).
None of our 15 cases made anti-C and/or anti-e but few were subject to a potential alloimmunization background.
However, as RHCE*-ce286A was reported to code for a partial c (Rh:-26), we consider that RHCE*Ce286A likely encodes partial C and e, this being also supported by the predicted localization of the p.Gly96Ser change on the second extracellular loop of the RhCe protein.
Background/Case Studies: Weak D genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a RhD typing discrepancy, or a serological weak D phenotype, to determine if they carried the weak D genotypes 1, 2 or 3.
The purpose of this study was to analyze the underlying RHD genotypes of the patient samples received for weak D genotyping since published recommendations, in particular those found to not carry the weak D 1, 2, or 3 genotypes.
Study Design/Methods: Between 9/2015 and 2/2017 50 samples were received for weak D genotyping.
Testing was performed using PCR-RFLP targeting the sequence variants in the RHD gene that have been previously defined.
Samples that did not have weak D types 1, 2, or 3 genotypes, but 156A TRANSFUSION 2017 Vol.
57 Supplement S3 had evidence of RHD genetic sequences in exon 7 and/or intron 4 in preliminary testing were evaluated by Sanger sequencing for RHD and RHCE exons 1-10 to determine the underlying RH genotype.
When provided, the patient's ethnicity and presence of anti-D was recorded.
Results/Findings: The majority of the samples were from obstetrical patients (62%) followed by transfusion patients (28%); 10% had no clinical indication provided.
34 samples (68%) were found to be weak D type 1, 2, or 3 (24, 6, and 4 samples, respectively).
5 samples (10%) appear to be genetically RHD negative.
Genetic sequencing was performed on 11 samples; 9 had RHD genetic variants that were not weak D types 1, 2, or 3 (Table) .
All of these variant RHD samples also showed some variation in the RHCE gene.
Two samples (4%) had wild type RHD alleles; further evaluation is ongoing.
Conclusion: Most samples tested by weak D genotyping were found to be weak D types 1-3.
Of the 11 samples that had evidence of an RHD gene and did not carry the known weak D types 1-3 polymorphisms, 9 (82%) of were found to have other RHD variants, and 2 (18%) did not have underlying genetic variation detected in the RHD gene.
The majority of the non weak D types 1-3 variants were DAR alleles, which are often associated with anti-D production.
Background/Case Studies: RHD genotyping has been recommended to guide transfusion of D-negative RBCs and administration of Rh immunoglobulin to patients with discordant or weaker than expected D typing, particularly for females and OB patients .
The recommendation is based on observational evidence, primarily from Europe (Flegel 2006, Curr Opin Hematol13:476) , that individuals with weak D types 1, 2, and 3 are not at risk for clinically significant anti-D.
The implications and utility of this approach for the diverse U.S. population are not yet clear.
Here we report 15 months experience with RHD genotyping on 352 samples referred with discrepant or weak D typing investigated from January 2016 to April 2017.
Study Design/Method: Serologic testing was performed by standard tube agglutination with licensed reagents.
DNA isolated from WBCs was used in manual RFLP and RHD BeadChip assays and RHD sequencing for some.
Ethnicity was known for 153 samples (53.3% Caucasian, 32.2% African American/African, 6.6% multiracial, 4.6% Hispanic, 2% Asian, and 1.3% other).
Results/Finding: RHD genotyping identified weak D types 1, 2, and 3 in 155/352 (44%) and alleles known to encode partial D phenotypes in 168/ 352 (47.7%) (Table) .
Uncommon or rare weak D alleles including types 6, 15, 40, 42, 45, 51, 57 (n52), 59, 61, 78, 91 , and 119 were found in 13 (3.7%).
The partial D alleles found were diverse, but the largest number included partial RHD*D 4.0 (n562) and *DAR ( Conclusion: In a multiracial cohort of 352 individuals with weaker than expected D typing 44% were due to weak D types 1, 2, or 3 and would not be considered at risk of clinical significant anti-D, but for 56% there is potential or unknown risk.
These studies are important to gain insight into the prevalence of specific alleles in the U.S. multiethnic population and to continue to evaluate and refine RHD genotyping for clinical practice.
CP242 RHD*07.02 Allele Causes Discrepant Genotyping Results for RHCE SMALL C Sabine Scholz* 1 , Sandra Schneider 1 , Sabrina K€ onig 1 , Susanne Helmig 1 and Vicky van Sandt 2 .
1 inno-train Diagnostik GmbH, 2 Rode Kruis Vlaanderen Background/Case Studies: In the human Rh blood group system the c, C, E, e and D antigens are expressed by the two highly homologous genes RHCE and RHD.
After D, c is the most immunogenic Rh antigen.
The difference between c (307C) and C (307T) is caused by the SNP on position 307 on the RHCE gene.
The RHD*07.02 allele (also known as RHD cat VII type 2) carries the SNP 307T>C on the RHD gene and additionally the SNP 329T>C.
This RHD*07.02 allele has been described to partially express RHc on the D polypeptide (Faas, Transfusion, 2001) .
Aims: Genotyping was performed to clarify the cause of the weak c expression.
Serology of a patient sample (Male, 81938) indicated a partial c phenotype with a CDe.
Study Design/Method: RhD and RhCE phenotyping was done by accredited routine protocols (monoclonal AB ID card: Diaclon Rh subgroups, seraclone anti-c).
Genotyping was performed with a TaqMan Probe assay (RBC-FluoGene vERYfy, inno-train Diagnostik GmbH), SSO (RBC-Lifecodes, Gen-Probe Inc.), in-house SSP-PCR (HILA, Rode Kruis-Vlaanderen) and commercial SSP-PCR (RBC-Ready Gene CDE, inno-train Diagnostik GmbH).
Sanger sequencing of the RHD gene was performed using an inhouse method (inno-train Diagnostik GmbH).
Results/Finding: Discrepant genotyping results were generated by different test systems: the TaqMan Probe based assay showed in repetition a CCee genotype, while the SSO system RBC-Lifecodes predicted in repetition a Ccee phenotype.
In SSP-PCR the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit.
The parallel analysis of the RHD gene with RBC-Ready Gene CDE test system revealed a variant D cat VII RHD allele.
Sequencing of the DNA sample identified two SNPs on one of the RHD alleles (307T>C, 329T>C) confirming a RHD*07.02 and one RHD*01 allele.
Hispanic female in preparation for surgery resulted in variable reactivity and weakly positive D reactions when using microtiter-well agglutination versus tube testing.
Determination of whether the D antigen expression represented a weak D or a variant D could not be resolved by serologic testing alone.
Here we report the characterization of a novel RHD gene mutation identified by RHD gene sequencing.
Study Design/Method: Serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms Galileo NEO and Galileo Echo (Immucor, Norcross,GA) and by standard tube testing using the Immucor Series 4 and 5 anti-D reagents.
Further immunohematologic evaluation was performed by standard tube testing (immediate spin -IS, and indirect antiglobulin -IAT) using OrthoBioclone, Immucor Gammaclone, Immucor Series 4 and Series 5, and ALBAclone anti-D reagents.
DNA isolated from WBCs was used in manual RFLP and RHD BeadChip assay (Immucor, BioArray) and RHD sequencing.
Results/Finding: RBC reactivity is summarized in the Table.
DNA testing detected a hybrid rhesus box associated with the RHD gene deletion, indicating the patient was hemizygous for RHD.
RFLP assay and RHD BeadChip did not identify any changes.
RHD gene sequencing identified a new c.463A>G change in exon 3 encoding an amino acid change p.Met155Val.
The predicted location of this change is within the fourth transmembrane segment of the RhD protein.
Conclusion: We identified a novel RHD allele with c.463A>G (p.Met155Val) change in exon 3.
Several SNPs, deletions, and insertions have been reported with changes in Exon 3.
Phenotypes of these genetic variations result in Rh negative, weak D types, and variant D. Since this change has not been previously identified, we are unable to determine if this confers a risk of anti-D alloimmunization, but the RHD c.463A>G SNP results in serologically weak phenotypic expression of D antigen when tested by microtiterwell agglutination on the NEO/Echo platforms.
In this case the combination of microtiter-well agglutination and DNA sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays.
Serologic and Molecular Detection of an Antibody to a High Incidence Antigen in Patient with History of Chronic Transfusions Georgia Spanos* 1 , Juan Merayo-Rodriguez 2 , Christopher Lough 1 and Nancy Eckert 3 .
1 LifeSouth Community Blood Centers, 2 Life South Community Blood Centers, 3 LifeSouth Community Blood Center-Headquarters Background/Case Studies: The Jo a antigen is one of three high incidence antigens in the Dombrock system.
The prevalence of this antigen is 100% in most populations and greater than 99% in the black population.
The Jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with Dithiothreitol (DTT), W.A.R.M.
TM (Immucor) and ZZAP treatment.
Anti-Jo a is an IgG antibody that demonstrates at AHG phase.
Hemolytic transfusion reactions to the Jo a antigen vary from none to moderate/severe.
Hemolytic Disease of the Fetus and Newborn (HDFN) has not been observed with any antibody associated in the Dombrock system.
There are two common phenotypes present in the black population:Hy negative/ Jo a negative and Hy weakly expressed/Jo a negative.
Study Design/Methods: An antibody identification and red blood cell (RBC) units were requested for an O positive, 57 year old, African-American female with a history of sickle cell disease and no history of pregnancy.
The patient was not recently transfused, however, had a history of chronic transfusions.
Last reported transfusion was three years prior to the current specimen.
There were no known RBC antibodies at the time of the request.
Facility reports that the patient's hemoglobin(g/dL)/hematocrit(%) (Hgb/Hct) is 6.4/ 18.8 and does not appear to be in sickle cell crisis.
A request for phenotypically matched units, as per hospital policy, for C, E, K and S was received by our Immunohematology Reference Laboratory (IRL).
Results/Findings: Anti-Jo a was detected in patient plasma reacting with LISS and PeG (tube method) and manual Gel-IAT.
The antibody was resistant when tested with DTT treated red cells.
In-house frozen reagent RBCs negative for the Jo a antigen (positive for Hy) were used to serologically prove the presence of the antibody to this high incidence antigen.
An allogenic PeG adsorption was performed to rule out other common clinically significant antibodies.
Anti-Kp a was identified using this adsorbed plasma.
Further testing with molecular genotyping (Grifols IdCore XT ) confirmed the patient's genotyping as antigen negative for the Jo a , Kp a and positive for Hy.
Conclusion: Molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout Florida, Georgia and Alabama.
Staff is able to search our database for any combination of antigen negative phenotypes using the internal 510(k) Blood Establishment Computer Software (BECS) Integrated Blood Bank Information System (IBBIS).
This enabled us to locate one refrigerated and three cryogenically preserved Jo a negative RBC units.
We found 251 eligible blood donors that could be recruited via an automatically generated call list.
The request for RBCs was cancelled.
Patient's clinical symptoms improved without transfusion and repeat Hgb/Hct increased to 7.1/21.
The patient's sibling is historically negative for the Jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf.
In order to continue having blood components available to meet all our patient's needs, IRL staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion.
RBCs of two females whose samples were referred for RHD genotyping with previously reported alleles for which serologic reactivity had never been investigated.
Study Design/Method: Serologic testing was performed by automated analyzer, Galileo Echo and NEO (Immucor, Norcross, GA), and by standard tube testing with licensed anti-D reagents and the ALBAclone advanced partial RhD typing kit.
Genomic DNA isolated from WBCs was used for Immucor RHD BeadChip assay, PCR-RFLP, and RHD sequencing.
Results/Finding: Patient 1 was a 29 yo female, C2E2c1e1, whose RBCs reacted 11 by ECHO and 31 by NEO with anti-D4, and '?'
with anti-D5.
Testing with D4 and D5 by tube gave 21 and 11 w on initial spin (IS) respectively and 41 by indirect antiglobulin test (IAT).
RBCs were non-reactive at IS with Ortho BioClone and BioRad Seraclone, and 1 w with Immucor Gammaclone anti-D, and all were 21 at IAT.
RBCs did not react with two (LHM 174/102 & 57/17) of 12 anti-D in the Alba partial D kit.
This pattern did not match any partial D identified by these clones.
RHD BeadChip detected an inactive RHD pseudogene in trans to RHD.
Gene sequencing confirmed the presence of the pseudogene, but RHD had a c.780C>A change encoding p.His260Gln.
Patient 2 was a 20 yo pregnant female, C2E2c1e1, whose RBC were 1 w at IS and 31 at IAT with Immucor Series 4 and 5 and Gammaclone, and moderately reactive, 21 IS and 41 IAT, with ALBA alpha, ALBA blend and delta anti-D. RBCs did not react with two (LHM 174/102 & 170/45) anti-D in the partial D kit with no known partial D pattern.
DNA testing predicted she was RHD hemizygous and RHD BeadChip detected markers for RHD*DAR but exon 2 gave low signal (LS).
Sequencing found a hybrid DAR with CE-specific nucleotides in exon 2 from c.150 to c.203 encoding amino acid changes p.Ile60Leu and Ser68Asn.
Conclusion: We found two previously reported rare alleles: RHD with a c.780C>A (p.His260Gln), previously found in France (LeFloch et al.
GenBank KU363612), and RHD*DAR with part of exon 2 replaced by RHCE, reported in sub-Sahara Africa (Granier et al.
Transfusion 53:3009) designated RHD*DAR(CE2:V505V-S68N) with an allele frequency of 0.002 to 0.016.
Blood samples were not available to test for alterations in D expression for either allele.
We provide serologic evidence that these alleles, found in two females evaluated by RHD genotyping, inform transfusion and Rh immune globulin prophylaxis, as they encode partial D phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-D. Background/Case Studies: Hu5F9-G4 is a human monoclonal IgG4 antibody recognizing CD47 that is in clinical trials to treat hematologic or solid malignancies.
CD47 is a transmembrane glycoprotein that binds to signalregulatory protein a (SIRPa) on macrophages and functions to regulate phagocytosis.
Blocking CD47 is thought to enhance phagocytosis and promote anti-tumor responses.
CD47 is also highly expressed on RBCs, and the purpose of this study was to evaluate anti-CD47 drug interference in blood bank testing.
Study Design/Method: Serologic testing was performed by standard methods.
Serial samples (n57) from 2 patients were tested over the course of 1 month treatment.
Plasma was tested at immediate spin (IS) and by IAT with R2R2, rr, D--, Rh mod and Rh null RBCs, as CD47 expression levels vary depending on Rh phenotype.
DTT and enzyme treated RBCs were also tested.
Both Immucor Gamma-clone anti-IgG (does not detect IgG4) and Ortho BioClone anti-IgG (total IgG) were used.
For titration plasma was diluted in PBS.
Allo-adsorptions were performed with papain treated rr RBCs and eluates were made using Gamma ELU-KIT II.
Results/Finding: Anti-CD47 was observed in plasma as soon as 1 hour post infusion.
Plasma reacted 31 to 41 at IS and 41 with all panel cells in PEG IAT using Ortho anti-IgG.
D--, Rh mod and Rh null RBCs were nonreactive at IS and weaker (31 and 21) in PEG IAT with Ortho reagent.
Reactivity with all panel cells by Ortho IgG gel card was 31.
In contrast, IAT reactivity using Gamma-clone anti-IgG was only 1 w to 11, and this reactivity was confirmed to be carry-over agglutination.
D--, Rh mod and Rh null were non-reactive in PEG IAT using Gamma-clone anti-IgG .
The anti-CD47 titer was 1 at IS and PEG IAT with Gamma-clone anti-IgG, but was !
256 with Ortho anti-IgG.
Plasma reacted with DTT, trypsin, papain, a-chymotrypsin or W.A.R.M.
treated RBCs.
Somewhat unexpected, autocontrols were negative and DATs were non-reactive or microscopic only.
Acid eluates (n54) were 31 reactive with Ortho, and non-reactive with Gamma-clone anti-IgG.
Plasma reactivity was removed after 4X allo-adsorption with papain treated rr cells, but in some samples low level (micro-11) reactivity remained.
PEG adsorption was invalid due to precipitation/complexing of antibody.
Robust plasma reactivity interferring in ABO reverse typing was observed, and weak spontaneous agglutination of the RBCs in the ABO forward and Rh typing.
Conclusion: Hu5F9-G4 anti-CD47 therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but ABO and Rh typing.
High levels of CD47 expression on RBCs results in plasma agglutination at IS, mimicking reactivity observed with IgM antibodies although Hu5F9-G4 is IgG4.
Reactivity was observed in all phases and with all test methods.
CD47 is not cleaved from RBCs by DTT, trypsin, papain/ ficin, DTT with ficin (W.A.R.M.)
or a-chymotrypsin, and treatment of RBCs with these does not mitigate interference.
Numerous adsorptions with papain treated rr RBCs were required to remove anti-CD47 reactivity from plasma.
Use of Immucor Gamma-clone anti-IgG, which does not detect IgG4, can mitigate interference in IAT although carryover reactivity may be observed.
Due to blocking by anti-CD47 on the patient RBCs, DAT and autocontrols were weak or non-reactive; however eluates prepared from the DAT1 RBCs were strong and pan-reactive using Ortho Anti-IgG.
Background/Case Studies: A Caucasian woman with history of a Caesarean section and a RBC TX in 1985.
In August 2016, she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions.
Five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation.
Ab screening was now positive, with an antibody reacting with all panel cells detected.
Because of the urgent need for RBC TX, two weakly cross-match positive Rh1K matched units were transfused with a warning of possible alloantibodies.
The patient got acute hemolysis.
Study Design/Method: A Gel technique was used in the Hospital Transfusion Laboratory.
In addition, various antibody identification panels and special serological and genotyping methods were used in the Reference Laboratory.
KEL sequencing was done by the International Immunohematology Center.
Results/Finding: The Hospital Transfusion Laboratory results were O RhD neg, DAT neg, and the ab identification was 21 with untreated and 31 with enzyme-treated cells, with weakly positive autocontrols.
A sample was submitted to the reference laboratory for additional investigation.
DAT was weakly positive, while ab identification results were similar to the hospital results.
Different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups.
After Pk, Vel neg, Jk:-3 etc.
had been excluded, k-phenotyping revealed a K 0 -phenotype.
A total of 38 silencing mutations are known for the KEL gene and the genotyping kits used did not recognize these.
The Anti-Ku antibody reacts with all cells apart from the K 0 -phenotype.
The presence of DTT-sensitive anti-Ku was confirmed with DTT-treated panel cells.
Anti-Ku may cause immediate and delayed hemolytic transfusion reactions.
Samples were taken from the patient's two siblings and daughter.
KEL sequencing revealed KEL*02N.19 with c.2023T encoding p.675Ter (reported in an individual from Austria in 2007).
There are two known K 0 -patients in our country, both homozygous for c.2023T.
The daughter was a c.2023T heterozygote, while the siblings did not have this variant.
A new operation is necessary but no K 0 -donors are available in our country.
With the help of the ISBT Rare Donor Working Party, a K 0 O RhD neg donor was found in Japan and one unit was delivered to us for use in the next operation.
Conclusion: An alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct Coombs is positive.
A combined serological and genotyping approach offers the best solution for problematic antibody cases.
Compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor.
Transfusion Strategy for the Serologic Weak D Phenotype in Tunisia Based on RHD alleles and RH haplotypes Mouna Ouchari* 1 , Kshitij Srivastava 2 , Houda Romdhane 3 , Saloua Jemni Yacoub 4 and Willy Albert Flegel 1 .
1 NIH, 2 DTM/CC/NIH, 3 Regional Blood Transfusion Center Sousse, 4 Regional Blood Transfusion Center Sousse, Tunisia Background/Case Studies: D antigen variants have been studied molecularly in many Arab populations, including Gaza, Tunisia, Egypt and Libya, 159A TRANSFUSION 2017 Vol.
57 Supplement S3 since 2009.
The Tunisian population has the largest known prevalence of weak D type 4.0 alleles, occurring in 1 of 105 RH haplotypes, compared to 1 in 6,060 or less in Europe.
A systematic study was missing for samples with the serologic weak D phenotype routinely found in blood donor and patient testing in Tunisia.
The study was designed to obtain data on weak D type 4.0 in a population known to harbor the greatest prevalence of such allele worldwide.
Study Design/Methods: A total of 13,431 random blood donors were serologically screened for the D antigen using 3 routine techniques.
Samples with weak reactivity were tested with a panel of 6 monoclonal anti-D (Partial RhD-Typing Set) to identify partial D phenotypes.
The RHD gene was sequenced in all samples with serologic weak D phenotype.
The RHCE gene was also tested molecularly by either direct sequencing or using the RHCE BeadChip kit to ascertain the RHCE allele linked to the RHD allele.
Results/Findings: A total of 67 discrepant samples (0.5%) were observed and expressed the serologic weak D phenotype.
Among them, 60 carried an allele of the weak D type 4 cluster (89.6%), of which 53 samples (88.3%) showed the weak D type 4.0 allele.
Only 1 sample each was found for the weak D types 1, 3 and 100 and the DVII, while 3 samples showed the consensus RHD sequence.
No mutation in any of the 10 RHD exons was detected in another 3 samples.
The molecular analysis of the RHCE gene showed that 59 out of 67 samples with serologic weak D phenotype (88.06%) had a variant RHCE allele and the most common associations were: weak D type 4.0 linked to RHCE*ceVS.04.01; weak D type 4.2.2 with ceAR; and weak D type 4.1 to RHCE*ceVS.02, while the other RHD alleles were linked to one of the common RHCE alleles.
Conclusion: Almost 90% of the weak D phenotypes in Tunisia were caused by alleles of the weak D type 4 cluster, of which 88% represented the weak D type 4.0 allele.
Based on established RH haplotypes for variant RHD and RHCE alleles and the lack of adverse clinical reports in Tunisia, we recommend D positive transfusions for patients and no RhIG administration for pregnant women with weak D type 4.0 in Tunisia.
We propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-D immunization would occur in Tunisia associated with weak D type 4.0 phenotype.
There is a possibility that the RHCE*ceVS.04.01 allele, typically associated in Tunisian individuals, may protect from allo-anti-D immunizaton and other RHCE alleles, such as RHCE*ce more often associated in individuals of other ethnic groups, may not.
However, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations.
Martha Rae Combs* 1 , Heather Simmons 1 , Christine Lomas-Francis 2 , Gayane Shakarian 2 , Sunitha Vege 2 , Lauren Hutelmyer 3 , Sandra Nance 4 , Jessica Poisson 1 , Nicholas Bandarenko 1 and Connie M. Westhoff 2 .
1 Duke University Hospital, 2 Immunohematology and Genomics Laboratory, New York Blood Center, 3 ARC PennJersey, 4 American Red Cross, Immunohematology Reference Laboratory, Biomedical Services Background/Case Studies: Plasma from a transfused, A1, 2 year old white female, post liver transplant with RBC aplasia, reacted at RT and in PEG IAT with all RBC samples tested except her own.
Study Design/Method: Standard hemagglutination methods were used for antibody ID and antigen typing.
Acid eluates were prepared using Gamma ELU-KIT II (Immucor).
Genomic DNA was isolated from WBCs and used for HEA PreciseType array and KEL and SC gene sequencing.
Samples from the proband and her mother were tested, as applicable.
Results/Finding: The patient's DAT was negative.
Her plasma reacted with 0.2M DTT-treated and papain-treated RBCs, all available RBC samples lacking high-prevalence antigens, and with phenotypically similar RBC samples [C2, K2, Fy(a2),S2].
Reactivity was detected to a titer of 64; it was not removed by prewarm technique or by 4x PEG alloadsorption.
The adsorbed plasma reacted with 0.2M DTT-treated RBCs.
Extensive RBC phenotype results were unremarkable except for the following: K2, k2, Js(b2), Kp(a2b2) and Sc:21,23.
Her plasma reacted with K o , McLeod, Sc:21,22 RBC samples and DTT-treated Sc:21 RBCs at RT and PEG IAT but her diluted plasma and pretransfusion eluate showed relative Kp b specificity.
The patient was transfused 4 aliquots of crossmatch incompatible Kp(b2), S2 RBCs.
Her post-transfusion DAT was 21 with anti-IgG, 11 with anti-C3d.
The eluate reacted with all RBC samples except 1 Kp(b2) sample.
She tolerated additional aliquots from 4 phenotypically similar RBCs untested for high-prevalence Kell or Scianna antigens.
The HEA Precise-Type predicted K2, k1, Kp(a2b1), Js(a2b1) and Sc:1,22, discordant with her RBC phenotype.
KEL gene sequencing identified a homozygous change, c.1481A>T (p.Glu494Val) (KEL*02.10) encoding the low prevalence antigen, Ul a , but no changes associated with lack of Kell system antigens; however, her RBCs typed Ul(a2).
SC sequencing found heterozygosity for a 5'-2g>a change (rs12124733, 24 to 30% prevalence) and conventional SC*01, predicting Sc:1,22,3.
KEL and SC results on the mother were KEL*02/KEL*02.10, heterozygous for the SC change 5'-2g>a, and her RBCs typed K2k1 Kp(a2b1), Sc31, Ula1, consistent with DNA predictions.
Plasma collected 7 months later was nonreactive at RT and in PEG IAT.
Her RBCs were DAT2 and now typed k1, Kp(a2b1), Ul(a1) Sc11 and Sc31,concordant with predicted Kell and Sc phenotypes.
Conclusion: We report an example of Kell and Scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the KEL system.
To our knowledge, this is the first report of a Ul a KEL*02.10 homozygote.
The RBCs may lack a high-prevalence antigen antithetical to Ul a .
Without DNA testing and gene sequencing, the patient would be presumed to have Kell null and Sc null phenotypes, a search for K o , and/or Sc:21,23 RBC units would be performed and we would not have been prompted to re-type her RBCs when the DAT was negative.
Background/Case Studies: Anti-Jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction.
In nearly all cases, this antibody is identified in the context of a phenotypically homozygous Jkb patient, Jk(a-b1).
Other scenarios are quite rare.
We present two cases of anti-Jka in which this phenotype was not observed.
Study Design/Method: Patient A is a 55-year-old multiparous female with no known transfusion history.
Her blood typed as O positive with a positive antibody screen, negative DAT, and a clearly identified anti-Jka in plasma.
The patient phenotyped as Jk(a-b-).
Genotyping revealed the presence of the JK*B allele, but not the JK*A allele.
Complete sequencing of the JK gene showed an intron 5 polymorphism in homozygosity.
Specifically, the patient showed a JK*B(IVS5-1A)genotype, associated with a Jkb null phenotype.
Anti Jk3 was not identified.
The conclusion was an allo-anti-Jka in a Jk null patient.
The patient did not receive any transfusions.
Patient B is a multiply transfused 64 old female.
Her blood typed as A positive with a positive DAT and antibody screen.
Both the plasma and eluate revealed an anti-Jka.
Despite the recent transfusion, the patient phenotyped as Jk(a1) 41 and Jk(b1) 21.
Genotyping showed the presence of both JK*A and JK*Balleles.
Whole gene sequencing was not performed.
There was no hematologic or biochemical evidence of hemolysis.
The patient was considered to have an auto-anti-Jka and Jka negative cells used for transfusion.
Results/Findings: Patients A and B both developed anti-Jka while having uncommon phenotypes/genotypes.
Conclusion: It is common for Jk null patients to develop anti-Jk3.
However, we speculate that expression of the Kidd glycoprotein with the Jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-Jk3 or anti-Jkb.
Auto-anti-Jka is usually reported in the context of an active hemolytic process, but Patient B illustrates an auto-anti-Jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for Rh epitopes.
These rare cases of anti-Jka require phenotypic and genetic analysis for the Jkb epitope and JK*B allele respectively, and in more complex cases whole gene sequencing.
Background/Case Studies: Donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories.
Package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification.
These outcomes may go unrecognized unless serological results are available for comparison.
Study Design/Method: A routine blood donor, self-identified as African American, was selected for red blood cell genotyping.
DNA was extracted and genotyping was performed using two commercial platforms 160A TRANSFUSION 2017 Vol.
57 Supplement S3 (PreciseType, BioArray, Warren NJ; IDCore XT , Grifols, Emeryville, CA).
Genotype results were compared to historical serological results.
Discrepancies were resolved by Sanger sequencing (Grifols IH, San Marcos, TX).
Results/Finding: Genotyping results showed variants in both the Duffy (FY) and Kell (KEL) blood group systems.
The donor's genotype was concordant on both platforms, FY*A/FY*B_GATA, KP*A/KP*A, for a predicted phenotype: Fy(a1b-); Kp(a1b-).
When genotype results were compared to historical serology, it was noted that the donor previously typed Fy(a-) on 3 separate donations.
No previous Kpa or Kpb serotyping was available.
Sequencing of FY exon 2 revealed a 287G>A mutation, FY01*N.04, known to silence Fya.
Sequencing of KEL exons 1-19 exposed a silent polymorphism in exon 8, 846G>C.
This polymorphism causes a dropout artifact yielding a false negative Kpb interpretation.
Conclusion: The discrepant FY*A result, as well as the unlikely Kp(b-) type prompted the request for sequencing.
The rare FY01*N.04 mutation has been reported in people of Caucasian descent.
This is the first example of this FY mutation identified in this regional population.
The Kpb antigen is present in nearly 100% of all populations.
However, Kp(b-) is most frequently seen in people of Caucasian descent.
To date, 64 self-identified African American donors have been genotyped as KP*A/KP*B at this blood center.
Given the diversity of regional heterogeneity, it is feasible to identify a Kp(b-) donor, self-reporting as African American.
Red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization.
Donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types.
In this case, a donor selected for genotyping based on ethnicity was initially thought to have 2 genetic variants not previously reported in those of African descent.
Only 1 was proven to be present.
This case acts as a reminder that genotype limitations must be considered even when using licensed methodologies.
This case report presents two group O pediatric patients who had been on enteral feeds and had absent/weak anti-B that became strong over time in patient 1.
Study Design/Methods: Patient 1 was a 7 year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at 3 years of age.
Anti-B changed from undetectable/weak to strong at the age of 7 years.
Patient 2 was a 17 month-old female with a metabolic urea cycle disorder who underwent a liver transplant.
Anti-B was 0/11.
Both patients were on total parenteral nutrition (TPN) since birth and had strong anti-A and normal immunoglobulin testing.
ABO typing with enhancing techniques is presented in Table 1 .
Results/Findings: Both patients typed as group O on forward typing.
Anti-A was strong in both patients.
Anti-B varied in strength in patient 1 with 0-11 reactions up to 7 years of age.
Thereafter, ABO typing showed mainly strong anti-B.
Patient 2 had 0/11 anti-B.
Conclusion: Intestinal bacteria stimulates production of anti-A and -B.
Unexpected changes in anti-B that caused ABO discrepancies are reported here for 2 children on long-term TPN.
Patient 1 had absent/weak anti-B since birth up to 7 years of age, then developed strong anti-B with no change in feeding regiment and medications.
Patient 2 had consistently strong anti-A and absent/weak anti-B.
These findings support the notion that normal colonization of the gut is important in the development of anti-A and -B and suggests that microflora of the gut in patients on prolonged TPN is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet.
Difference in strength of anti-A and-B could be due to stronger A than B antigen expression on gut bacteria.
Results/Finding: A daratumumab protocol was established that incorporated use of the cord panel.
Multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, DAT and genotype.
After daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication.
Repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication.
The cord panel ruled out underlying alloantibodies.
Selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity.
Conclusion: The cord panel was used 28 times over a five month period to rule out underlying alloantibodies.
Tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution.
The daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner.
Teresa Gorey* 1 and Elizabeth Hart 2 .
1 Brigham and Women's Faulkner Hospital, 2 University of Massachusetts-Dartmouth Background/Case Studies: The purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies.
Several antibody detection methods (Polyethylene glycol (PEG), LISS, and albumin) are routinely used in small transfusion services.
The utility of PEG is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure.
The Code of Federal Regulations, Title 42, CFR part 493.1271(a), states the manufacturer's instructions are followed when testing for unexpected antibodies.
The package insert for Gamma PeG TM (Immucor Inc., Norcross, GA), states that negative reactions may be examined with an optical aid.
Based on these directions, our institutional policy is to confirm all negative reactions using the microscope.
Study Design/Method: A one-year retrospective document review was performed on all patient samples in which a positive antibody screen (ABSC) triggered the antibody identification (ABID) to be performed in 2016.
A total of 232 samples were evaluated.
Each ABID was subcategorized; (1) as being a new antibody for our facility or in the patient's shared electronic health record within the PartnersV R Healthcare system and (2) whether a microscopic ABSC result triggered the ABID.
Also, patients with known antibodies were grouped according to a microscopic ABSC result.
A comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant.
Results/Finding: A total of 83 ABIDs were performed on new patient samples.
Of the new ABID samples, 29 (35%) had microscopic ABSC results.
For the previously known antibody patients, there were 35 which accounted for 15% of the total ABIDs performed.
When reviewing the total ABID workups, a total of 64 (28%) of the ABSCs had microscopic results which resulted in an ABID being performed.
The antibodies identified in the 29 new antibody samples were: Conclusion: A total of 86% of the new antibodies identified based on a microscopic ABSC were clinically insignificant.
The manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results.
Due to the results of this study, a decision will be made to: (1) discontinue the use of the microscope, (2) switch to a PEG manufacturer whose directions indicate to observe macroscopically for agglutination, or (3) define the use of the agglutination viewer as the optical aid.
Decreasing the number of ABIDs will save time and money while providing potential RBCs for transfusion in a timely and efficient manner.
Anton") has a prevalence greater than 99% in all populations.
Hereditary absence of AnWj has only been described once (in a single family).
However, red cell expression of AnWj may be markedly decreased to near undetectable levels in blood donors of the In(Lu) (or "Dominant Lutheran Inhibitor") phenotype.
Similarly, anti-AnWj antibody formation is rare, with only 10 cases reported in the literature.
The antibody developed in the context of hereditary absence of AnWj (i.e., a true alloantibody) in only one of the cases.
In the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient AnWj antigen suppression.
Most of the reported cases lacked clinical or laboratory evidence of hemolysis.
However, in the most recently reported case, involving a 56-yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after RBC transfusions, necessitating transfusion support with AnWj-negative and In(Lu) RBCs.
The case was also unique in that the anti-AnWj resulted in a direct antiglobulin test (DAT) that was positive for complement only, rather than IgG like all previous cases in which the DAT was performed and was positive.
Study Design/Method: A 59-year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (AHTR) with development of a panagglutinin on indirect antiglobulin test (IAT) screens.
Prior to identifying the specificity of the panreactive antibody, the patient received 10 RBC transfusions and showed signs of hemolysis with six of them.
The first three transfusions were prior to her positive IAT and were electronically crossmatched.
The next seven transfusions were incompatible by antihuman globulin (AHG) phase crossmatch, but were extended phenomatched for clinically significant antigens.
The patient's AHTR signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and LDH.
The DAT, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for IgG only (1-21), and negative with anti-C3b, C3d reagent.
The antibody showed a peak gel-IgG IAT titer of 32.
Results/Finding: The antibody was identified as having AnWj specificity.
The patient's pre-transfusion sample showed weak AnWj expression (w1), altogether suggesting an auto-anti-AnWj.
Monocyte monolayer assay testing using the patient's plasma and RBCs from the AHTR-implicated units yielded monocyte indices ranging from 33 to 83%, consistent with the clinical hemolysis observed.
Given the patient's group O, Rh D negative blood type and continuing transfusion dependence, in order to avoid further AHTRs, international collaboration was necessary in order to procure and provision group O, Rh D negative RBCs that were also serologically negative for AnWj.
The patient was successfully transfused three such units without further incident.
Conclusion: This is the second documented case of anti-AnWj in a patient with aplastic anemia and, overall, the third anti-AnWj case associated with AHTR.
This case also underscores the importance of international collaboration.
Cold auto-antibody 12 Anti-P 1 4 Anti-M 2 Anti-Sd a 6 Anti-Le b 1 Anti-Jk a 1 Anti-K 1 Anti-E 1 Anti-C 1 Results/Finding: Three hundred and ninety weak D genotypes have been determined to this day with frequencies of 21% (type 1), 5% (type 2), 9% (type 3), 25% (type 42) and 40% other than 1, 2, 3 or 42.
Further investigation was conducted to determine the molecular identity of the «others».
Out of 157 samples, 119 (75%) were confirmed to be legitimate serological weak or partial D, mainly deletions of exon 5 or both exons 4 and 5.
A surprising amount of 38 samples were discovered to be normal RHD.
Conclusion: Along with Sandler et al.
(2015) data, our findings highlight the difficulties hospitals face in interpreting serological weak D. Trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point.
Altogether, our findings allow to share the frequency of weak D types 1, 2, 3 and 42 obtained in serological weak D, 45 years old Quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical D typing.
were classified as FNHTR.
TACO incidence was 0,6%.
No TRALI happened in the period.
Prophylaxis were used in 98% of patients.
Conclusion: FNHTR is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions.
FNHTR occurred 3 times less than allergic reactions.
This might be explained by universal leukoreduction and universal prophylaxis adopted at our institution.
Further studies are necessary to evaluated the benefit of this approach.
that cannot be associated with a specific RBC unit or were deemed unrelated to transfusion, 358 RBC transfusion AEs were analyzed.
Chi-square test and logistic regression were used to compare the AE incidences among transfusion groups.
Results/Finding: Univariate and multivariate logistic analyses showed that irradiated RBCs were associated with a significantly increased incidence of transfusion-related AEs (p <0.05).
There was a significant difference in febrile non-hemolytic transfusion reaction (FNHTR) (0.27% vs 0.11%, p <0.001) or AEs with a non-allergic type inflammation etiology (0.30% vs 0.14%, p <0.001) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated RBCs and non-irradiated RBCs.
In contrast, the incidences of allergic AEs (0.028% vs 0.024%, p 5 0.614) were similar between these two groups.
The incidences of inflammation AEs after transfusion of irradiated RBCs that were stored for 1, 2, 3, and 4 weeks were 0.25%, 0.32%, 0.39% and 0.41%, respectively (p 5 0.084, logistic regression) but there was a significant difference in the incidence of inflammation AEs caused by irradiated RBCs stored for a week (0.25%) and longer than a week (0.35%) (p < 0.05).
Conclusion: Irradiated RBCs associated with a higher incidence of transfusion inflammation AEs compared to non-irradiated RBCs and this risk increased when RBCs were stored longer than 1 week after irradiation.
While it is likely the patient population is a factor in AE caused by irradiated RBCs, it is also possible that RBC radiation damage, as shown in previous studies, contributed to this increased AE incidence.
A list of patients with one of these icd10 codes was generated.
The EMR was searched to find the clinical scenario in which TRALI was mentioned.
These patients' records were then searched within our laboratory information system (CoPath), to determine if they had a transfusion reaction reported to our transfusion medicine service.
Results/Finding: The search of our electronic medical record found 11 patients from 2011-2016, who had TRALI mentioned in their chart as a diagnosis or possible/likely diagnosis.
One patient was excluded from our study because TRALI was mentioned as a past medical history from an outside hospital.
Only the patients who had TRALI listed as a diagnosis or possible diagnosis were included in this study.
These 10 patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress.
The clinical teams caring for these patients were either giving a diagnosis of TRALI or considering TRALI as a possible diagnosis.
Of these 10 cases, only 2 of them were reported to our transfusion medicine service as transfusion reactions.
Of the reported cases, one was determined to be TRALI and the other one was consistent with TACO.
Eight out of those 10 cases were never reported.
Background/Case Studies: Despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (DHTR).
This transfusion reaction is seen in as many as 1 out of 100 transfused products.
Therapeutic plasma exchange (TPE) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe TPE for clinical management after profound hemolysis.
Study Design/Method: Case review of a patient was performed after diagnosis and treatment of severe DHTR.
Results/Finding: A man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria".
He had a known history of anti-D and anti-C, and was transfused two units of crossmatch compatible RBCs seven days prior during a previous admission.
Readmission hemoglobin (Hb) was 8.7 g/dL but declined to 7.3 g/dL the next day.
An antibody screen was consistent with anti-D, anti-C, and direct antiglobulin test (DAT) was negative.
He received three units of crossmatch compatible RBCs over days 2 and 3 with poor responses.
On day 4, routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from 1.0 mg/dL to 1.8 mg/dL (reference 0.8-1.3 mg/dL), and lactate dehydrogenase was above reportable linearity, >2500 u/L (reference 122-222 u/L).
Testing revealed additional anti-E, anti-Jkb, DAT C31, plasma free Hb 64.4 mg/dL (reference 1-15.2 mg/dL), and hemoglobinuria.
Four of five transfused RBC units were Jk(b1), one of which was also E1.
One volume TPE was performed to remove free Hb on days 5, 6, and 7 using fresh frozen plasma as replacement fluid for haptoglobin supplementation.
Creatinine peaked at 3.7 mg/dL on day 13, decreased to 2.3 mg/dL before discharge on day Results/Findings: Twenty three cases were identified, of which 20 had medical records available for analysis.
Ten (50%) patients were male, the mean age was 50.4 years (range 24-76 years), 15 (75%) had an underlying hematologic malignancy or bone marrow disorder, and 3 (15%) had a history of coronary artery disease (CAD).
The implicated units included 14 (70%) red blood cells and 6 (30%) platelets; 17 (85%) patients received a single unit, and 3 (15%) received two or more within the previous 6 hours; the mean volume transfused was 153.3 mL (range 20-280 ml).
The mean time to onset of chest pain was 92.15 minutes (SD 85 minutes), with 90% of patients presenting within 2.5 hours and 100% within 6 hours of starting the transfusion.
Chest pain was present as the only symptom in 35% of the cases, and for the other cases the accompanying symptoms included dyspnea (30%), fever (25%), back pain (20%), and hypo-and hypertension (10%).
A post-transfusion chest X-ray was performed in 65% of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (TACO).
Electrocardiogram was performed in 70% of cases and showed no findings to suggest acute ischemia.
Three (15%) patients had a minimal increase in their troponin levels, although 1 had a history of chronically elevated troponin due to stress cardiomyopathy.
Fourteen (70%) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than 10 minutes in the majority of patients (90%).
No cases resulted in new admission to the ICU or procedure cancelation.
Conclusion: Chest pain associated with transfusion was infrequent, but several such cases were identified during the review period.
This symptom is not a diagnostic criterion for any of the other Hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain.
Larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies.
Background/Case Studies: Thrombotic microangiopathy (TMA) in children is most commonly seen in the form of hemolytic uremic syndrome (HUS).
However, TMA may be seen in the presence of Streptococcus pneumoniae (SPN).
The action of bacterial neuraminidase of SPN results in exposure of the normally "hidden" Thomsen-Freidenreich antigen (T-antigen) found on erythrocytes and other tissues.
Ultimately, this may lead to SPN induced hemolytic uremic syndrome (pHUS) with subsequent hemolysis and end organ damage by naturally occurring anti-T antibodies against the exposed T antigen.
Specific lectins or anti-sera can confirm exposure of the T antigens in pHUS.
Alternatively, pHUS can be identified by minor crossmatch incompatibility resulting from agglutination of exposed T antigens on recipient's erythrocytes to anti-T antibodies in the plasma portion of blood products.
We present a case of suspected pHUS that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical HUS (aHUS).
Study Design/Method: A 6 months old boy presented with respiratory failure.
He was found to have blood cultures positive for SPN as well as hemolytic anemia, thrombocytopenia, and acute renal failure.
He was Shiga toxin negative and had normal levels of ADAMTS 13.
Based on the findings, the clinical team was concerned for pHUS.
Therefore, he received washed erythrocytes.
For his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product.
As a result, a minor crossmatching was suggested and performed to determine if T activation was present.
Results/Finding: Minor crossmatch was performed with patient's erythrocytes and plasma of ABO-identical platelets to be transfused.
No agglutination was seen at immediate spin, 37 degree, or anti-human globulin phase.
Check cells were found to be 21.
These findings were conveyed to the clinical team and platelets were issued without washing.
Due to the lack of identification of T activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with aHUS.
The patient was then treated with Eculizumab with clinical and laboratory improvement.
We present a case clinically consistent with pHUS.
Confirmation of this diagnosis is done with lectins or anti-sera that are not readily available.
An alternative means of identifying pHUS is by minor crossmatch incompatibility.
By demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of aHUS with appropriate management.
Background/Case Studies: Orthotopic liver transplantation (OLT) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding.
We report a case of massive transfusion in an OLT patient necessitating an ABO blood group switch (from O1 to A1) to sustain transfusion support and minimum group O RBC inventories.
Study Design/Methods: Type & screen (TS, gel) and anti-A titers (tube) were performed using routine methods.
A chart review was performed for pertinent medical and laboratory findings.
Results/Findings: The patient was a 63-year-old O1 man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha-1 antitrypsin deficiency who presented for OLT (donor O1).
During OLT, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy.
He required rapid high volume RBC and plasma support, which strained hospital inventories.
After receiving 46 units of O1 RBCs and 26 units of O1 plasma with ongoing severe hemorrhage, he was switched to group A products.
Ten units of A1 plasma were transfused to wash out anti-A antibody prior to transfusion of A1 RBCs.
Due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for 7 hours post-transplant.
The patient's total estimated blood loss was >20L.
He received a total of 71 units of RBCs (including 23 A1), 63 units of plasma (including 37 A1), 6 units of cryoprecipitate, and 9 units of platelets.
Towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two RBC units he received were O1.
On postoperative day (POD) 1, a TS showed predominantly A1 RBCs with trace O1 RBCs, as well as very low anti-A IgM and IgG titers (Table 1) .
He received two additional O1 RBC units (1 each on POD 4 and POD 12) with increasing O1 RBCs on TS and rising anti-A titers.
His blood type was unequivocally O1 by POD 13.
The patient showed recovery of liver synthetic function on POD 1 (Factor 5 activity 5 58%) complicated by cholestasis.
Conclusion: This study shows successful switching of a group O patient to group A in the setting of rapid hemorrhage and massive transfusion.
By POD 13, the patient had reverted to O1 with recovery of anti-A titers.
At 3 months post-OLT, the patient is alive with signs of improving biliary graft function.
A New RFID Transfusion Safety System Anna Millan* 1 , Alfred Mingo 1 , Maria Isabel Gonzalez 1 , Antoni Mena 2 and Juan Pedro Benitez 2 .
1 BST, 2 AT-Biotech Background/Case Studies: A new Transfusion Safety System (TSS), based on processes and technologies, especially, identification by radio frequency (RFID), is currently implemented in two hospitals, a general one (H1) and an Oncology center (H2).
The TSS is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (NM) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (PSE) and the blood components administration (BCA) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (TIS).
The TSS allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of NM.
Study Design/Method: Retrospective analysis of 2016 transfusion activity in both H1 and H2 shows 7970 PSE and 12572 BCA, out of 13163 and 20676 respectively, since the TSS deployment in 2015.
Retrospective analysis and classification of 6700 security events has been done.
Results/Finding: Activity results for both hospitals are shown in the table below.
The safety events have been classified in pretransfusion sample extraction (PSE), blood component assignment (BCAS) in the transfusion service and blood component administration (BCA) near misses (NM).
For H1, NM related to PSE accounted for 42.39% of all, being the mistake in concordance between patient identification and prescription order the most frequent (52.03%).
The NM detected in BCAS were 12.1% of all and mostly (74.52%) occur when the patient information in the TIS does not match the one registered in the TSS.
The NM detected in BCA are 45.51% of all and mostly (36%) the systems detects a not assigned bracelet.
For H2, NM related to PSE accounted for the 47.49% of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent (65.84%).
The NM detected in BCAS accounts for 24.48% of all and in 69.08% occurs when the patient information in the TIS does not match with the one registered in the TSS.
The NM detected in BCA are 28.02% of all and in 65.61% of them the blood components were assigned to another patient.
(1, 3, 4, 5, 8, 9, 12, 14, 19, 23, 26, 51, 56, 68) were analyzed via a commercially available ELISA.
Comparison of adequate response to PPV23, defined as !
2 mcg/mL for >7 serotypes, was perform based on alloimmunization status.
Statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests.
Results/Findings: Pre-vaccine SP titers were available in 72 patients (alloimmunized, 15); pre-and post-vaccine titers were available for 19 patients (alloimmunized, 6).
Of the 72 patients, 25 were on chronic transfusions, 24 were on hydroxyurea, 11 were surgical splenectomized, 58 patients had no history of surgical splenectomy or status was unknown.
Forty-four patients had a previous history of PPV23 in the previous 10 years; 9/44 also reported previous history 13-valent SP conjugate vaccine within the last 5 years.
Baseline pre-vaccination titers (N572) showed no difference between alloimmunized and non-alloimmunized patients (all p-values >0.13).
In the group with pre-and post-vaccination (N519) titers available, 11 out of 13 (85%) non alloimmunized patients had an adequate response versus 4 out of 6 in the alloimmunized group (67%, p 5NS Background/Case Studies: Blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance.
To safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (TREs) as they occur from "vein-to-vein", i.e.
from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time.
Multiple over-lapping error documentation processes are needed to capture and report TREs from within and outside of Blood Bank (BB).
We present a comprehensive error management program along with data on five years of benchmarking TREs at a large academic medical center.
Study Design/Method: TREs were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations.
In addition, TREs as observed and reported by providers and clinical staff (i.e.
blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the BB and hospital quality through the Veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm.
All serious errors were reviewed daily and summation of TREs was discussed on a monthly basis.
Mapping TREs within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from 2012 to 2016).
Patient harm events recorded within the Veritas system from January to July 2016 were investigated in depth.
Transfusion reactions were excluded in this analysis.
Results/Finding: An average of 114 TREs per month and 1300 per year were found over five years.
81% of TREs are associated with pre-BB activities, 10% occur within BB, and 9% are post-BB events.
Sample collection and handling represent 80% of total TREs.
Most TREs (96%) were reported by BB staff, 4% were reported by non-BB staff.
Patient harm analysis revealed an average of four Level 0 (near miss), three Level 1 (no known harm), and 0.3 Level 2 (patient harm) per month.
No deaths related to TRE were detected over the seven month January to July 2016 period.
Patient harm was associated with TREs occurring in the BB (17%) and post-BB (83%).
These events were reported externally (78%) and by BB staff (22%).
Conclusion: Although most TREs were detected in the pre-BB phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm.
The TREs causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-BB and BB phases.
These results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-BB phase aimed at reduction of waste associated with sample collection and handling, and the post-BB and BB phases aimed at improving TRE detection and decreasing patient harm.
Background/Case Studies: Uncrossmatched red blood cells (RBC) and emergency issued platelets (PLT), plasma and cryoprecipitate (CRYO) are lifesaving in a bleeding patient without a valid type and screen.
Collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed.
This study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued.
Study Design/Methods: A list of patients on whom blood products had been emergently issued between January 1, 2013 and March 28, 2017 was obtained from the blood bank at a regional maternity care hospital.
Patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded.
The total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within 6 hours of the first emergently issued products were enumerated.
Apheresis PLT units were multiplied by 5 and added to the number of individual whole blood PLTs; apheresis plasma units were multiplied by 2 and added to the number of whole blood plasma units.
Results/Findings: Seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified.
Average age was 31.
The majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted.
That PLT wastage was higher than the other products was likely due to the 4-hour post-pooling room temperature shelf life.
Keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank.
.
Patient blood platelets were higher before prophylactic than therapeutic transfusions (18[10 9 /l vs. 14[10 9 /l, p50.029).
There were no significant differences in the frequency of effective therapeutic (55% vs. 72%, p50.1) and prophylactic (63% vs. 54%, p50.09) transfusions between the PRCs and 25GyPCs.
We did not find significant differences between PRCs and 25GyPCs in CCI1 after prophylactic (16.0 6 7.1 vs. 19.2 6 8.7) and therapeutic (11.3 6 9.0 vs. 11.8 6 5.8) transfusions, in CC24 after prophylactic (20.0 6 9.2 vs. 22.5 6 12.8) and therapeutic (13.3 6 8.9 vs. 13.9 6 8.)
transfusions.
There were no significant differences between PRCs and 25GyPCs also in MA1 after prophylactic (62.2 6 8.5 vs. 60 6 8.5, p50.7) and therapeutic (61.3 6 9.9 vs. 60.9 6 12.7, p50.08) PC transfusions.
Reduction of the severity of bleeds was obtained in 78 (86%) of the 91 cases after PRPC transfusions and in 51 (84%) of 61 cases after 25GyPC transfusions.
There were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, 3 and 2 cases).
Background/Case Studies: An 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in 2006.
It enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components.
An error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in 2014 under the category 'errors relating to information technology'.
This error was the incorrect use of the emergency transfusion process for non-emergency transfusions.
The standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag.
The emergency transfusion option is only intended for use with 'emergency group O RhD negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label.
The emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion.
It was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient.
Study Design/Method: This center worked with the software supplier to develop a solution which corrects the weakness in the process.
The revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion.
The use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not.
Results/Finding: There were 593 units administered using the emergency transfusion process in the 3 months before the change was implemented.
It was found that 51/593 (9%) units were non-emergency units administered incorrectly without a bedside compatibility check.
Following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month (2530 components administered), 109/2530 (4%) were emergency units which were administered correctly.
Users of the system reported the revised process was quicker, safer and unified with other functions on the device.
Conclusion: The improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check.
This report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety.
Background/Case Studies: Recent recommendations indicate one red blood cell (RBC) unit should be transfused at a time with reassessment after each transfusion to determine the need for more.
However, the practices of Canadian transfusion medicine (TM) experts and what constitutes a reassessment are unknown.
Therefore, we conducted a survey of TM experts across Canada to gather information on their practices and criteria for reassessment.
Study Design/Method: TM experts were identified and contact information obtained from the Canadian National Advisory Committee (NAC) and from contacting least one TM expert per province.
Each respondent was assigned a unique study ID after consenting to the survey, allowing for anonymity on analysis.
The survey contained demographics, general practice questions, and questions regarding transfusion in: 1) a stable anemic inpatient, 2) a stable anemic inpatient to be discharged, and 3) an asymptomatic post-operative inpatient.
Results/Finding: We identified 67 Canadian TM experts: 48 (71.6%) provided a response and most had a primary place of practice in a laboratory setting (38/48; 79.2%).
For a stable, non-bleeding, anemic inpatient, 87.5% of respondents recommended transfusing one RBC unit, then reassessing.
Recommendations were more variable in outpatient settings, with 31.2% generally recommending transfusing two RBC units then reassessing.
Recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period (1-2 hours), a repeat hemoglobin >18 hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings.
Lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds.
With an asymptomatic patient to be discharged, fewer respondents chose to transfuse (38.1%) compared to an inpatient potentially symptomatic due to anemia (72.1%).
None of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down.
Conclusion: TM experts generally recommend transfusing one unit at a time in stable inpatients.
Assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause.
"Top-up" transfusions were not recommended.
These recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices.
Background/Case Studies: Current evaluation of red blood cell (RBC) post transfusion recovery is based on ex vivo labeling of stored RBCs with radioactive chromium-51 ( 51 Cr).
This method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple RBC populations in the recipient.
RBC labeling with s-NHS-biotin (Bio-RBCs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused RBCs in vivo.
The purpose of this study was to scale up and optimize the biotinylation procedures to the current Good Manufacturing Practice (GMP) environment.
Study Design/Method: Packed RBC units (n514) were divided into two 150mL aliquots, which were labeled with selected concentrations of s-NHSbiotin (3 and 30 lg/mL) in a CGMP closed system (average Bio-RBCs hematocrit of 38.4 6 1.6%).
Optimization of labeling efficacy was determined by flow cytometric analysis of Bio-RBCs using fluorochrome-conjugated streptavidin (SA).
Approximately 2 million RBCs were measured in triplicate.
Quantum Simply Cellular Beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, MESF) and infer number of biotin molecules per RBC.
The lower limit of detection was determined for RBC labeled with varying amounts of biotin.
Product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after RBC biotin labeling.
Results/Finding: Investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled RBCs revealed that 561nm excitation of phycoerythrin (PE)-SA and high laser power (150mW) provided the best separation between the two Bio-RBC populations, and between labeled and unlabeled RBCs.
Labeling with 3lg/mL of biotin resulted in $50,000 MESF/RBC, and were detectable among unlabeled RBC at a lower limit of detection (LLD, 95% CI) of 1 in 380,000 (0.0003%).
The LLD95 for RBC labeled with biotin at 30lg/ mL was $ 1 in 1 million (0.0001%).
Biotinylation was not associated with increased levels of hemolysis (0.40 6 0.22% before labeling versus 0.34 6 0.12% after labeling; p50.09) or bacterial contamination.
Conclusion: The resulting manufacturing process produces large volumes (150mL/transfusion) of Bio-RBCs with low risk of contamination or hemolysis.
The flow cytometry assay can detect Bio-RBC in unlabeled blood at very low frequency.
We plan to use this technology to study the impact of donor characteristic on RBC storage stability and post-transfusion survival.
Background/Case Studies: Blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs.
Hemoglobin-based oxygen carriers (HBOCs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available.
Since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer-250 (HBOC-201) effects on coagulation parameters alongside freeze-dried plasma (FDP) in an in vitro model of hemorrhage/resuscitation.
Study Design/Method: Whole blood (WB) was collected from healthy donors under an approved institutional standard operating procedure.
In the first study (limited resuscitation), samples were: (1) WB, (2) WB110% HBOC volume (model of two units in an adult), (3) WB110% FDP, and (4) WB110% HBOC110% FDP.
Samples (5)-(8) simulated autoresuscitation by adding 25% plasmalyte to 1-4.
Susceptibility to lysis was tested with 75ng/ml tissue plasminogen activator (tPA).
Follow-up studies were performed with severe resuscitation simulations of 50%, 60%, 75%, and 100% volume replacement with HBOC and/or FDP, with or without prior 25% plasmalyte dilution.
Coagulation parameters were obtained with a coagulation analyzer and thromboelastography (TEG).
RBCs/hemoglobin were measured on a hematology analyzer.
Thrombin generation was quantified by thrombogram.
Platelet aggregation was measured in Multiplate and adhesion to collagen under shear in BioFlux.
Viscosity was evaluated by rheology.
Results/Finding: A limited resuscitation model with HBOC and/or FDP had no effects on fibrinogen, PT, aPTT, pH, Hct, or hemoglobin.
In TEG, WB, WB1HBOC, and WB1HBOC1FDP had reduced clot strength with dilution and tPA.
There was increased susceptibility to tPA-induced lysis between WB and WB1HBOC in autodilution simulation (mean lysis 4.79% vs. 16.36%; p<.05).
HBOC and FDP had no statistically significant impact on thrombin generation.
No effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow.
HBOC (10%) did not significantly change viscosity.
Severe resuscitation simulations had increased PT/PTT and reduced clot strength, particularly in HBOC-only resuscitation; however, even 75% HBOC volume replacement produced clots with acceptable TEG parameters.
Conclusion: In a limited resuscitation model with HBOC-201, there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting.
Considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of HBOC1plasma as initial resuscitation of hemorrhagic shock.
therapy in patients with acute major bleeding.
While published literature has largely focused on the efficacy and safety of PCC, actual usage practices are less characterized.
Our aim was to describe the PCC usage practices within a tertiary care center.
Study Design/Method: We conducted a retrospective review of the electronic medical records of patients who received PCC between its addition to our institution's formulary in 8/2013 and 2/2015.
We compiled information about the usage of PCC in these patients.
Descriptive statistics were generated with Microsoft Excel.
Results/Finding: Of 81 patients, 24 were on warfarin.
PCC was most frequently prescribed for hemorrhage due to surgery (43%).
PCC was given for warfarin reversal in 31% of cases.
A subset of patients received plasma within 2 hours prior to PCC (40%) or 24 hours after (47%).
PCC was most frequently ordered in the OR/perioperative service (25%).
Conclusion: The majority of PCC usage was "off-label" in terms of being prescribed for indications other than warfarin reversal.
The most frequent indication was hemorrhage due to surgery, and PCC was most often ordered in the OR/perioperative service.
Although guidelines recommend the use of PCC as a plasma alternative, plasma was administered within hours of PCC in a notable subset of patients.
Background/Case Studies: In Emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently.
However, in some cases, poor communication and lack of clear expectations between the Blood Bank and patient care areas can lead to frustration and delays in the timely provision of blood products.
An incident prompted an appraisal of our emergency release protocol (ERP), which revealed gaps in communications and expectations by both the Blood Bank and nursing personnel.
Thus, it is imperative that there is a standardized ER protocol with clear communications for both the Blood Bank and Nursing personnel.
Reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (TAT) for our ER protocol.
Study Design/Method: In 2016, several meetings were conducted with stakeholders (Critical care units (ICU), emergency department (ED), internal medicine, interventional radiology (IR) etc.)
in an effort to identify process gaps, improve communications, and expectations for ER episodes.
The goals was to design a process for emergent blood product request and release in life threatening situations that will; 1) Simplify and expedite the process; 2) improve communication and expectations to decrease TAT; 3) improve patient safety and meet compliance.
In order to achieve these goals, a series of activities were conducted.
These included meetings with all stakeholders to ensure process improvement meet the needs intended.
A series of training sessions with nurse educators in ICU, ED, IR and Surgery managers were conducted.
During the meetings, communication goals, and expectations were defined and agreed upon.
Training sessions included PowerPoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow.
The impact on the current process was analyzed and, as a result, led to the revision of the current SOP, addition of pre-labeled Emergency Pack blood (4 units of O Neg RBC's) and implementation of an Electronic Emergency Blood order set.
Results/Finding: In the ten months post implementation of our improved, standardized ER Pack Protocol, A total of 61 ER episodes were received.
The average TAT from order to delivery at the bedside was reduced by 50% (7.0 minutes compared to 14 minutes previously), while the compliance rate for ER orders and physician documentation was 100% (61/61), with no current wastage of blood products.
Conclusion: The implementation of the improved standardized ER protocol significantly improved communications and expectations, decreased TAT and delays in transfusions while ensuring patient safety and compliance to regulatory requirements.
Background/Case Studies: Massive transfusion (MT) in the trauma setting has been extensively studied.
Yet, the literature in non-trauma areas, especially oncology is rather sparse.
The following study was conducted to understand the background and outcomes of MT in cancer patients.
Study Design/Methods: This was a single center retrospective study performed at a large cancer center between February 2016 -February 2017.
MT was defined as the transfusion of !
10 RBC units in a 24-hour period.
The following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (MTP) was activated, and survival at 30 days.
Results/Findings: Thirty MTs occurred during a one year period.
A total of 192,441 blood products were transfused during that time period.
Gender distribution was 21/30 (70%) males, and the average age of all patients was 68 with a range of 21 to 70 years of age.
Surgical patients accounted for 26/30 (86.7%) MTs, and 4/30 (13.3%) were critical care patients.
Tumor categories included carcinomas (14/30), sarcomas (13/30), leukemias (2/30) and lymphoma (1/30).
Resection of tumor followed by complex reconstruction was the cause of the majority of MTs.
Metastatic renal cancer (6/30) was the most common disease seen followed by sacral chordoma (4/30).
MTPs were activated in only 8/30 (26.7%) cases.
Thirty-day survival was seen in 25/30 (83.3%) patients.
Only 1 of 5 mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by GI hemorrhage (3/ 5) or perisplenic hematoma (1/5).
The overall ratio of RBC:FFP in the entire Patients ( Background/Case Studies: Plasma is a straw-colored supernatant of blood that is used for type and screen (T&S) and crossmatch.
In the analytic phase of testing, plasma is examined prior to processing.
Plasma occasionally becomes discolored, interfering with crossmatch procedures.
Timely identification of the etiology allows for corrective actions and minimizes delay in transfusion.
Study Design/Method: During the analytic phase of blood bank testing, samples were evaluated for T&S and crossmatch; this identified three samples with discolored plasma.
We present a series of cases that illustrate the testing process.
Results/Finding: A 54-year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy.
A preoperative T&S specimen contained bright green plasma.
Review of her preoperative case revealed exposure to intravenous methylene blue.
This dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects.
Alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use.
Although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique.
A specimen drawn one week later contained clear plasma.
A 26-year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies.
Administration of a synthetic blood product resulted in dark maroon colored plasma.
The most common cause of a dark red color is hemolysis of the sample, which is usually discarded.
In this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples.
With this in mind, the sample was not discarded and testing was completed by tube method.
A 70-year-old woman admitted with acute stroke was treated with a thrombolytic.
Her T&S revealed cloudy white plasma that could not be used for the crossmatch procedure.
Common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol.
Although an etiology could not be identified a repeat specimen drawn several hours later was clear.
Conclusion: These cases highlight the importance of an appropriate evaluation of discolored plasma.
Once a discolored sample is identified, a repeat sample is required to confirm the change in color.
In the first two cases, the discoloration persisted, prompting further clinical investigation.
Once the etiology was identified, need for further testing and eligibility for further transfusion was determined.
Testing by tube method could be performed in two cases.
In the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay.
Decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists.
Caleb Wei-Shin Cheng* 1,2 , Rebecca Ross 2 , Christopher A Tormey 1,2,3 and Amit Gokhale 1,2 .
1 Yale University School of Medicine, 2 Yale-New Haven Hospital, 3 VA Connecticut Healthcare Background/Case Studies: Daratumumab (Dara) is a IgG1 monoclonal antibody therapy that specifically targets CD38, a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma.
Dara interferes with blood bank testing as it binds to CD38 expressed on red blood cells, causing pan reactivity.
The Dara interference can be overcome with the use of dithiothreitol (DTT) treated reagent red blood cells.
To minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a Dara protocol in our blood bank.
The purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during Dara treatment at our institution.
Study Design/Method: All Dara patients' antibody workups were completed using DTT pre-treated reagent red blood cells.
If the antibody screen was negative, K antigen negative RBC products are provided.
If an antibody is identified, K negative along with that particular antigen negative blood is provided.
Our electronic medical record (EMR) was searched for patients who received Dara over the past eight months.
Study subjects were examined to see if they had pre-existing alloantibodies before Dara treatment and whether they formed new alloantibodies during Dara treatment.
The age, gender, type and screen pre-Dara treatment, type and screen post-Dara, intervening blood transfusions, and the date of first Dara treatment was recorded.
Results/Finding: Overall, 54 subjects were identified for analysis.
Their mean age was 67.8 years, with 29 male and 25 female subjects; all were diagnosed with multiple myeloma.
We found an alloimmunization rate of 0% (0/54) prior to administration of Dara.
Of these patients, 22 were transfused with red blood cells (RBCs) after initiation of Dara therapy.
Following our testing/matching protocol, none of these (0%; 0/22) patients formed a confirmed, new alloantibody during Dara treatment; each of these patients underwent at least one follow-up screen after their first RBC unit.
We also found no complications in providing crossmatch compatible units to any of the 22 patients.
Conclusion: To our knowledge, this is the largest case series reporting on results of overcoming Dara interference with blood bank type and screen testing.
The protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving Dara therapy.
It is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted.
Background/Case Studies:A multi-facility transfusion service began stocking liquid plasma in September of 2015 for use in massive transfusion and trauma situations.
Due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use.
A policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented.
This study evaluated the effects of that policy on INR values of plasma recipients.
Study Design/Methods:A retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (FFP) and liquid plasma (LQP) in changing INR values of recipients.
All plasma units transfused within the facility from September 1, 2015 through April 7, 2017 were identified.
The following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion INR values, and whether or not vitamin K was administered.
Patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin K. The change in INR for each recipient was calculated, along with the average change in INR for each group.
Background/Case Studies: In gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation.
Transfusion could generally be avoided in those without haemodynamic instability.
The oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia.
Besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (Hb).
The present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in Hong Kong.
Study Design/Methods: Anonymous data of gynaeological patients age 60 was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion Hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge.
All transfusion episodes associated with surgical operations during same admission are excluded.
Results/Findings: In 2016, 2,523 unique women receiving a total of 5,889 units of red cells (RC) in 2,906 transfusion episodes were identified.
Their median age was 45 (range 11 -60).
The distribution of pre-and post-transfusion Hb and units of RC transfused were summarized below: In this cohort, pre-and post-transfusion Hb were absent in 46 (1.6%) and 283 (9.7%).
635 (21.9%) transfusion episodes were associated with the use of 3 units or more RC.
As a result, 1385 (47.7%) episodes resulted in a post transfusion Hb !
9g/dL.
Parenteral iron or tranexamic acid was uncommon during hospitalization and was given 2 (<0.1%) and 116 (4.6%) women respectively.
Upon discharge, 442 (15.0%), 84 (3.2%) and 1,994 (65.8%) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively.
However, neither were given to 386 (16.0%) women.
Conclusion: In the present study, it is observed that 49.7% transfusion episodes were given at Hb !
7g/dL.
A substantial number of episodes (71.7%) were transfused with multiple units and resulted in almost half having a post transfusion Hb level (!
9g/dL).
For iron replenishment and bleeding control, up to 16.0% transfused women were not given iron or tranexamic acid at discharge.
The results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved.
It is recommended that in-depth education and training should be provided for a better gynaecological patient blood management.
Background/Case Studies: Granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection.
However, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions.
We report a case of granulocyte transfusion therapy following chimeric antigen receptor T-cell (CAR-T) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed B-cell acute lymphoblastic leukemia (B-ALL).
Study Design/Method: Granulocytes (0.8-1.3x10 10 per unit) were collected from ABO-identical unstimulated donors at a regional blood center.
Each unit was irradiated with 25 Gy and transfused over 3-4 hours within 24 hours after the time of collection.
The patient's response and laboratory data were reviewed in the medical record.
Conclusion: This data suggests that a diagnosis of AML is associated with anti-HLA antibodies.
An increased frequency of blood group A in patients with AML has been reported, but here no statistically significant difference between ABO blood group frequencies was found in any category except the patient's with HLA antibodies.
Blood group B has a significant association with HLA alloimmunization in the studied patients.
It has been reported in a large study of female blood donors that no difference in HLA antibody frequency was observed based on ABO blood group at centers using the flow-based assay.
Although the reasons for the higher rate of group B blood type among patients with anti-HLA antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially AML.
Females with AML who are blood group B appear to be most likely to have HLA alloimmunization among patients with hematologic malignancies.
Implementation of Electronic Solution to Reduce Risk of Mistransfusion in a Regional Transfusion Service Debra Lane* 1 , Lee Grabner 1 , Brenda Herdman 2 , Robert Fallis 1 , Amin Kabani 3 and Charles Musuka 3 .
1 Canadian Blood Services, 2 Kenora Rainy-River Regional Laboratory Program, 3 Diagnostic Services Manitoba Background/Case Studies: Patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion.
The rate of mistransfusion has remained unchanged in over 50 years.
Attempts have been made to reduce mistransfusion including barrier devices, barcoding and RFID.
Within a Regional Background/Case Studies: The role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (RBC) units is receiving increased attention.
However, the impact of donor characteristics on efficacy of RBC transfusion has not been studied in largescale donor-recipient outcomes databases.
Study Design/Methods: We conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between 2008 and 2011 before patient blood management initiatives.
Linkage was performed between blood donor characteristics and hospitalized RBC transfusion recipients who received a single RBC unit.
Studied exposures for this analysis were blood donor sex and age in addition to RBC storage age.
The Wilcoxon test was used to examine changes in hemoglobin level following RBC transfusion, and , and 38% were male.
Recipients of RBC's from male and female donors had similar pre-transfusion hemoglobin levels (8.7 g/dL; p50.94); however, transfusion recipients of male donor RBC units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female RBC units (9.9 vs 9.8 g/dL; 1.2 vs. 1.1 g/dL; both p50.02).
Female recipients had a larger rise in hemoglobin per RBC unit compared to male recipients (1.3 g/dL vs. 1.0 g/dL; p<0.001).
Female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p50.01).
RBC storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p50.53 and p50.32, respectively.
Conclusion: RBC units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes.
This suggests that the dose of hemoglobin is lower in female than male RBC units.
This analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and RBC efficacy, hemolysis and other donor-component-recipient interactions.
Background/Case Studies: People who identify as Jehovah's Witnesses (JW) comprise less than 1% of the population of the United States.
However, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings.
The degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted.
However, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider.
As such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution.
Study Design/Method: The electronic medical record (EMR) utilized in this study in an institutionally modified version of EPIC EA Best Practices Alert (BPA) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (Jehovah's Witness) discrete data fields.
The alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override.
Data on the triggers are automatically collected through the EMR systems and generated into a report by informatics personnel.
Results/Finding: The available data covers triggers in the two month postimplementation of the BPA.
The BPA triggered 33 times in total, affecting 15 patients and 21 users.
Stratified by location, the majority of triggers occurred in the perioperative areas (18 times) and the liver ICU (6 times) with a minority occurring on the regular hospital floors and emergency department.
Nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected.
Orders that triggered the BPA included type & screens, Human albumin 25% IV solution, Human albumin 5% IV solution, Immune Globulin (Human) solution.
Conclusion: Despite the limited and very preliminary data, the user action findings seem to indicate that the BPA is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders.
Given the limited types of orders that the BPA is triggering on, the pattern suggests that the BPA is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many JW patients.
Despite these positive initial findings, this is an ongoing study to track the efficacy of the BPA and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal.
Intervention to Address Inappropriate Cryoprecipitate-AHF Orders at a Tertiary Medical Center Sirisha Kundrapu* 1,2 , Mahmut Akgul 1,2 , Hollie M Reeves 1,2 , Robert W Maitta 1,2 , Marcie Pokorny 2 , Anne Capetillo 2 and Katharine A Downes 1,2 .
Background/Case Studies: Although introduced for the management of hemophilia A, now cryoprecipitate is primarily indicated for low fibrinogen levels.
At our institution the transfusion medicine service (TMS) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders.
We aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders.
Study Design/Method: We conducted a 7-month retrospective study (January-July 2016) of adult cryoprecipitate order quality assurance forms.
The reference range for fibrinogen was 200-400 mg/dl with critical value of 50mg/dl.
Cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the TMS.
During the study period, TMS evaluated orders for appropriateness of dosing and agreement with estimated required doses.
Post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels.
Statistical analysis was performed using chi-square and t-tests.
Results/Finding: There were 301 adult (>18 years) orders reviewed by TMS out of which 299 were approved.
Of the 299 approved orders, 136 (45.5%) were in agreement with TMS's estimated dose.
Of 163 (54.5%) orders that were not in agreement with the TMS's estimate, 142 (47%) were underestimated and 21 (7%) were overestimated.
Seventeen of 299 orders had no post-transfusion fibrinogen levels.
Without intervention, there would have been a median deficit of 23.6 mg/dl (range 0.3 to 124 mg/dl) and a median excess of 13.3 mg/dl (range 0.6 to 155 mg/dl) of fibrinogen from the target.
Median difference between target and actual post-transfusion fibrinogen level was 12 mg/dl above target, which is significantly higher with intervention than without (which could have been 12 mg/dl below the target; p<0.0001).
Median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention (11 vs. 2.7 mg/dl, p50.07).
Median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention (15 vs. -23.5 mg/dl, p<0.0001).
Seven of 299 (17) 40 (24) orders were for critically low fibrinogen (<50 mg/dl) and 4 of these were under-estimated requests and reached target fibrinogen with TMS's estimate and approval of required units to be transfused.
Overall most frequent orders were 10 and 5 units (59.5% and 25%) i.e.
2 and 1 pools and the most frequent orders in the disagreement group were 10, 1, 5 and 2 units (33%, 20%, 17% and 16%).
There is a significant difference between agreement and disagreement groups based on clinical service ordering the units (Table) .
Conclusion: Intervention by TMS to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels.
Further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage.
Patient characteristics, medical records, vitamin K administration, and adverse events, were collected (Table) .
Results/Finding: The average pre-transfusion INR was 2.36 and posttransfusion was 1.91.
Only 22% of patients had their INR corrected to 1.5, while 28% had no change, or had increased INR.
(Table) .
The majority (67%) of patients received 2 units of plasma.
The mean plasma dose was 6 mL/kg.
There were 4 transfusion reactions reported, 1 non-hemolytic and 3 transfusion associated circulatory overload reactions in which 1 required admission to the ICU.
Two patients experienced bleeding during IR procedures (TIPS) and 1 developed a hematoma (tunneled central line).
The median of INR correction in this study was 1.9 with no relationship to the number of units of plasma transfused and/or if vitamin K was administered.
This study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when INR is 1.9.
Randomized trials are needed to assess whether the INR is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit.
3 of the 111 patients experienced bleeding complications indicating that INR of 1.9 may be considered safe in some lower risk procedures.
Current practices may provide little or no benefit, with substantial risk of life threatening complications.
Background/Case Studies: Group AB plasma, which lacks anti-A and anti-B antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available.
Approximately 4% of the population is group AB, which limits the available inventory of group AB plasma.
Of group AB population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (TRALI).
This makes type AB plasma a limited resource.
Our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known.
Group O individuals make up 45 % of the population and have no A or B antigens on their cells.
Group A is the second most prevalent blood group in the US population (40%) and has no B antigen on their cells.
So, group A plasma is compatible with both group O and A patients, approximately 85% of the patient population.
Before patient's blood type is known, type O red cell units are transfused with A plasma, which decreases the chance of hemolysis.
To conserve AB plasma, we instituted a policy effective July 1, 2014 as follows: 2 units of group A plasma and 3 units of group AB plasma is provided for the Massive transfusion protocol (MTP) along with 5 units of O negative RBC until patient blood type is known.
Study Design/Method: This prospective study is designed to monitor the use of group A plasma in MTPs at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma.
Direct antiglobulin test (DAT) is performed if patient received incompatible plasma.
If DAT is positive, lactate dehydrogenase (LDH), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction.
Results/Finding: We reviewed 235 MTPs at our institution between July 2014 and March 2017.
Twenty patients (8.5 %) were transfused with incompatible group A plasma (5 group AB and 15 group B patients).
Five patients died due to severe injury, and follow-up testing of these patients could not be performed.
The remaining 15 patients had negative DAT, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis.
None of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion.
Conclusion: Our study adds more evidence of the safety of group A plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known.
Based on this and other recently published studies, starting in April 2017, our institute will provide only group A plasma for emergency release and MTP cases before the patient blood type is known.
Average ( Background/Case Studies: In 2013, Bonfils Immunohematology Reference Lab (IRL) sent out approximately 245 special platelets for patients with HLA antibodies.
By 2016, HLA platelet orders increased dramatically and the IRL sent out over 650 special platelet products.
The purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time.
Study Design/Methods: Bonfils Blood Center has over 10,000 donors in the database with historical HLA typing.
However, only approximately 3500 of those donors actively donate.
In the Denver area, one of the most common HLA types is A1 A2 B7 B8.
Only 81 of the 10,000 donors have this type (0.81%).
Therefore, to fill an HLA platelet order request for a common HLA type, only 28 donors in the system would be a perfect HLA match.
With that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order.
After a donor is recruited and donates, it takes at least two days to fill an order.
For a less uncommon HLA type like A9 A11 B17 B35, there is only 1 out of 10,000 donors (0.01%) that match perfectly.
In those cases, there are no donors to recruit to fill such an order.
In some complicated cases, the IRL was provided with an HLA antibody list or Panel Reactive Antibody test (PRA).
In order to find product for these patients, lists of platelets in inventory with corresponding HLA types were printed.
If a patient had an antibody to A1 for example, all of the A1 positive platelets were crossed off the list.
This cross-out process would continue manually until the only platelets on the list were the ones positive for HLA antigens to which the patient did not have antibodies.
These platelets are PRA matched to the patient.
In order to automate this process a report linked to the donor database was created to find both PRA platelets in inventory and donors for recruitment.
The blood center medical director began suggesting that hospital clients order a PRA for each patient with platelet refractoriness.
The PRA test is fast and it is a definitive method to discern HLA antibody mediated refractoriness from platelet refractoriness due to other causes.
Results/Findings: In all but the most complicated cases with rare HLA patient phenotypes, it was much easier to find a PRA patient matched platelet on the shelf than an HLA match donor.
In 2012, approximately 27% of these special order platelets were PRA matched and the remaining 73% were HLA matched by donor recruitment.
By 2017, approximately 59% of special platelets sent are PRA matched.
This change resulted in a 2.2 fold increase of finding product in inventory to fill orders quickly.
Conclusion: Developing a system to provide PRA matched platelets is a faster alternative to finding HLA matched platelets thus contributing to better patient care.
Background/Case Studies: In urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (MTP) is critical to the timely delivery of these products.
Each MTP pack at UCM contains 6 packed red blood cells (pRBCs), 4 fresh frozen plasma (FFP) units, and 1 plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in Labor and Delivery (L&D) or if one is requested.
At UCM, blood products are generally transported through the pneumatic tube system (PTS).
We undertook a review of our MTP issuing practices and efficiency patterns over the last three and half years.
Study Design/Method: The electronic archives of the Blood Bank laboratory information system and electronic medical record at our institution were queried for patients who had MTP activations.
The archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on.
Results/Finding: Between August 2013 to March 2017 MTPs were activated at UCM, of which 251 orders could be traced to the origin: 118 on inpatient floor (including ICUs), 58 in the operating rooms, 46 in the Emergency Department, 25 in Labor and Delivery, and 4 in other procedure rooms.
Of the 2207 pRBCs that were issued, 1406 were transfused (64% utilized); of the 1446 units of FFP that were issued, 901 were transfused (62% utilized); of the 359 platelet packs that were issued, 246 were transfused (69% utilized); of the 64 units of cryoprecipitate that were issued, 49 were transfused (77% utilized).
Since March 2016, the time of first product issue after the initiation of an MTP has also been tracked.
Of the 84 events that fall within this time period, 39 (46%), had the first product issued in 5 minutes or less.
Another 31 (37%) were issued between 5-10 minutes, resulting in over 80% of patients being issued their first blood product within the first 10 minutes.
Only 15 of 84 (17%) events had an initial time greater than 10 minutes and none were greater than 21 minutes.
Conclusion: The majority of our activations currently come from inpatient floors (primarily ICUs).
As our institution anticipates the introduction of an adult level 1 trauma center, we anticipate this balance will shift.
In addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during MTP activations ($60-70%).
Again, we anticipate utilization rate of issued MTP products to increase with the introduction of a new adult trauma center.
We have recently begun tracking time to last product issued during an MTP, but cannot report on that variable at this time.
Overall, our data show that our transfusion service is generally performing adequately to issue the first product within 10 minutes of MTP protocol activation.
This data only reflects time to issue in the PTS; patient care areas can experience additional minutes delay in PTS delivery and arrival of product at bedside.
We must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient MTP care.
Mehreen Yasin* 1 , Shailesh Macwan 1 , Arline Stein 1 , Jane Fischman 1 , Nancy Nikolis 1 , Matthew Bank 1 , Lennart Logdberg 1 , Alexander Indrikovs 2 , Sherry Shariatmadar 1 and Vishesh Chhibber 1 .
1 North Shore University Hospital, 2 Northwell Health Background/Case Studies: Massive bleeding is generally defined as any patient who requires 1 blood volume replacement within 24 hours and/or receives transfusion of greater than or equal to 4 units in one hour with 177A TRANSFUSION 2017 Vol.
57 Supplement S3 ongoing bleeding.
Our MTP was officially implemented in 2013 in preparation for an initial verification as a Level 1 Trauma Center by ACS.
Our MTP has the following packages: 1st pack has a ratio of 4:4:1 (RBCS, Plasma & Platelets) and subsequent packs a ratio of 6:6:1.
Our MTP also includes Prothrombin Time (PT), activated Partial Thromboplastin Time (aPTT) and Fibrinogen testing after each pack is transfused.
This data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies.
However, attempts to supplement MTP packs with cryoprecipitate (CRYO) and prothrombin complex concentrate (PCC) were challenging to accomplish in a timely manner.
Study Design/Methods: Due to challenges in timely supplementation of MTP packages with CRYO and PCC, the protocol was modified in March 2016 to add CRYO and PCC at a defined point in the MTP (CRYO is included in the 3rd pack and PCC in the 4th pack).
In order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received >20 RBC at our institution as these massively hemorrhaging patients would receive PCC based on our current protocol.
We reviewed the blood products received by these patients and their available laboratory data.
Results/Findings: We had 8 patients who received >20 RBC in 2015 and 2016.
MTP had been activated for all patients and all patients received between 0.5 to 1 unit of plasma for each RBC unit transfused.
Despite receiving these ratios of blood products, all patients had elevations of their PT >16 seconds and many had elevations of the aPTT and fibrinogen levels less than our institution's target of 200 mg/dL (Table 1) .
As anticipated, improvement in the coagulation parameters was noted with CRYO and PCC supplementation.
Conclusion: Our data on massively hemorrhaging patients supports a role for supplementation of our MTP with CRYO and PCC in patients who require transfusion of >20 RBC.
Our current protocol with the addition of CRYO and PCC at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients.
Background/Case Studies: Red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (TA-PLS).
However, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype.
A living donor liver segment transplant resulted in a case of TA-PLS with donor-derived anti-D that had the potential for causing a clinically significant hemolytic event.
The donor's plasma contained anti-D. Anti-D was absent in the recipient's pre-transplant plasma, but present in the recipient's 5-day and 114-day post-transplant plasma.
Although these findings established a diagnosis of TA-PLS, hemolysis did not occur because the recipient's blood group phenotype was D-.
The conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of PLS and limits capturing the true scope of the syndrome.
Study Design/Method: To determine the standard of practice for detecting and diagnosing TA-PLS, a retrospective 10-year PubMed search for peerreviewed English-language journal articles was conducted using key words "passenger lymphocyte syndrome."
Cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies.
Results/Finding: Of 63 published cases (31 reports) of TA-PLS, 8 (4 reports) were stem cell and 55 (27 reports) were organ transplants.
All 8(100%) stem cell transplants and 52 (95%) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying TA-PLS associated with hemolysis.
Of the 4 reports of stem cell TA-PLS, 3 actively screened for antibodies in the immediate post-transplant period, and of the 27 reports of organ TA-PLS, 1 actively screened for antibodies.
These screens detected 5 cases of stem cell TA-PLS before hemolysis became apparent and 2 cases of organ TA-PLS with antibodies without hemolysis.
It can be inferred that TA-PLS is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely.
Conclusion: A new category of "non-hemolytic TA-PLS" is recommended to capture otherwise undiagnosed cases where TA-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case.
To ensure including the full scope of TA-PLS, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated.
Occult Hemolytic Anemia Due to Anti-Mur in a Patient Receiving Blood from a Region with a Prominent Asian Donor Population Jean Oak* 1 , Rosario Mallari 2 , Marc de Asis 2 , Elaine Shu 3 , Jonathan Hughes 4 and Tho Pham 1,3 .
1 Stanford University, 2 Stanford Health Care, 3 Stanford Blood Center, 4 BloodSource Background/Case Studies: Mur antigen is present in 7-10% of individuals in Southeast Asia, Taiwan, and parts of southeastern China, but is rare elsewhere.
Antibodies against Mur antigens are clinically significant, hence many countries in Asia routinely screen for it while other countries, including the US, does not include Mur in the standard screen.
We describe a case of an occult anti-Mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large Asian donor population.
41 year old Hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia.
Initial antibody screen and DAT were negative, and the patient received 1-2 RBC units every 1-2 weeks to maintain a hemoglobin (Hb) level of 8 g/dL.
The patient remained stable for 5 months when his Hb level acutely dropped to 6.6 g/dL.
The antibody screen remained negative for an additional 2 months when it became positive for anti-Jka and anti-Mur.
Donor ethnicity data was available for 30 of the 33 RBC units he received.
3 units were from an Asian donor, and a unit transfused 13 days prior to the Hb drop was from a Caucasian/Chinese donor.
Study Design/Method: We reviewed the ethnicity data of 64,495 donors at a hospital-associated blood center located in a region where Asians comprise approximately 30% of the population.
Results/Finding: 6.6% of donors identified as Chinese, Vietnamese, Filipino, or other Southeastern Asian.
These donors account for 5245 of 37933 (13.8%) RBC collections.
Conclusion: Identification of anti-Mur in this patient was triggered by the presence of a concurrent anti-Jka alloantibody.
Since over 10% of the RBC supply in the local blood center was collected from Chinese or Southeast Asian donors, chronically transfused patients are at risk of developing anti-Mur-mediated hemolysis that could be missed on a standard screen.
This finding raises a possible need for blood banks located in regions with a prominent Asian population to implement screening for anti-Mur.
Brian Adkins* 1 , Princess Maynie 1 , Carol Chandler 1 , Shelia Garret 1 and Pampee Young 2 .
1 Vanderbilt, 2 Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology Background/Case Studies: Antibody titration is a testing modality vital to both obstetric and transplant services.
Manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive.
In fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method.
The OrthoVision automated analyzers offers automated titering of patient plasma using gel technology.
Although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing.
The higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation.
Moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision.
[3] [4] [5] There is minimal information on the comparisons of tube titers to OrthoVIsion automated titers or assessment of the reproducibility of this automated method.
Study Design/Method: Rh and non Rh minor RBC antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ORTHO VISION analyzers to assess precision and inter-instrument reproducibility.
Results/Finding: A total of 26 samples have been analyzed (Table) , 17 Rh and 9 non-Rh antigens.
Titers via automated testing on OrthoVision resulted in a mean titration being 2.77 (range 1-7) times higher.
The average fold change for RhD/C/E antibody titers were 3.2, whereas the average fold change for non Rh titers was 1.03 (range 1-2).
The range for anti D titers was particularly variable, 2-7, whereas for C/E, it was 1-3.
The overall reproducibility/precision of the automated analyzer was $90%.
To correlate the 178A TRANSFUSION 2017 Vol.
57 Supplement S3 increased titers observed with some classes of antibodies, particularly anti D, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values.
Conclusion: Automated titration of antibodies using the OrthoVision Analyzers resulted in highly reproducible results between different instruments using the same sample.
However, the automated analyzers consistently yielded higher values, particularly with Rh D, with results $3 times higher than in manual tube testing.
Interestingly, the difference in titers of non Rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small.
In order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus.
Platelet Additive Solution Reduces the Isoagglutinin Titer in Apheresis Platelet Units Maxim Tynuv*, Elizabeth J Furlong and Willy A Flegel.
DTM/CC/NIH Background/Case Studies: Isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-A and/or anti-B may cause a hemolytic transfusion reaction (HTR) in a recipient with cognate antigen.
Apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of HTR due to plasma incompatibility if given based on short outdate and not ABO type.
At our facility testing is performed on all apheresis platelets with a cutoff titer of 250.
Units above the cutoff are marked as "high titer" and only given to ABO plasma-compatible recipients or washed with saline to reduce plasma.
However, washing platelets is a time consuming process that results in a loss of up to 33% of the platelets.
Platelet additive solution (PAS) is used as an alternative collection and storage solution, replacing approximately 65% of donor plasma in the final product.
The goal of this study was to determine what affect PAS has on isoagglutinin titers and whether using PAS could lead to a revision of one facility's procedure for management out of group platelet transfusions.
Study Design/Method: Isoagglutinin titers of whole blood EDTA samples were compared to the final apheresis platelet unit collected in PAS (Intersol, Fresenius Kabi, Lake Zurich, IL).
Using two-fold dilution steps, plasma was tested with pooled red cells (equal mix 0.8% suspension of A1 and B cells, Ortho, Raritan, NJ) in a gel matrix test (MTS Buffered card, Ortho, Raritan, NJ) with 15 min incubation (room temperature) prior to centrifugation (MTS Ortho Workstation).
Fifty two donors were group O, 32 group A, and 16 group B. Results/Finding: Of the 100 whole blood EDTA samples tested, 26 (25 Group O and 1Group B) exceeded a high titer threshold of 250.
When the PAS samples of these 26 donors were tested, only one (Group O) exceeded the same threshold.
PAS specimens showed a consistent two-fold decrease in titer compared with whole blood specimens.
Nearly half of the group O donors exceeded a titer of 250 when whole blood specimens were tested.
Conclusion: Only one sample from apheresis platelets collected in PAS exceeded our clinically applied titer threshold of 250, a 96% decrease from the number of whole blood specimens exceeding the threshold.
Testing the platelet bag collected in PAS instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed.
Furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using PAS or not.
other components.
The majority of blood components in Israel are collected and distributed by Magen David Adom (MDA), from 2 main locations.
Several hospitals in Israel also collect platelets in-house.
As part of an effort to understand PLT utilization, a nationwide survey of PLT transfusion and expiration was conducted.
Study Design/Methods: Data on the disposition of all PLT units, acquired from MDA and collected in-house, during the calendar year 2016 was requested from all hospitals in Israel.
The number of PLT distributed to hospitals by MDA was also collected.
PLT wastage was defined as the sum of PLT that were returned and not reissued from the hospital blood banks and PLT that expired on blood bank shelves.
Results/Findings: Sixteen of the 27(59%) hospitals in Israel, along with MDA, participated in the survey, listed as A to P. The results are presented in the table along with each hospital's distance from the 2 MDA facilities.
For some hospitals, the sum of transfused and wasted PLT was slightly less than the number of PLT supplied by MDA; this is likely due to the small number of PLT that had not either been transfused or expired by the time the data collection period ended.
Three of the largest hospitals (C, B and A) collected PLT in-house in addition to acquiring units from MDA.
These 3 hospitals had a lower overall rate of wastage including their own donations than the other 13 hospitals that did not collect in-house PLT.
The other 13 hospitals had wastage rates ranging between 9-54%.
No correlation was apparent between the hospital's distance from the MDA facility or its number of beds and the PLT wastage rate.
Conclusion: There is considerable platelet wastage in Israel.
Large hospitals in Israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals.
Factors known to affect PLT utilization and wastage such as patient diagnosis mix, policies about how PLT are issued and accepted back into hospital inventory, PLT inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates.
Possible Immune-Mediated Hemolysis Due to Platelet Transfusion Masked By Underlying Hemolysis in a Patient with Blast Crisis Sirisha Kundrapu* 1,2 , Christopher J Gresens 3 , Anne Capetillo 2 , Hollie M Reeves 1,2 and Katharine A Downes 1,2 .
1 Case Western Reserve University School of Medicine, 2 University Hospitals Cleveland Medical Center, 3 BloodSource Background/Case Studies: Transfusion-related hemolysis with ABOmismatched platelets is rare with a reported incidence of <0.1%.
Most commonly in such cases Group O platelets having high titer anti-A result in clinically significant hemolysis when transfused to a Group A or AB recipient.
We present a patient with a possible hemolytic reaction following transfusion of ABO mismatched platelets presenting in the setting of underlying disease associated hemolysis.
Study Design/Method: A 58-year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (SDP).
Two hours post transfusion he developed chills, rigors, and increased blood pressure (117/65 mm Hg to 205/89 mm Hg) followed by hematuria (500 mL).
Chills and rigors resolved; blood pressure stabilized after 15 min with diphenhydramine, solumedrol, and acetaminophen.
negative.
Patient ABO group, Rh (D) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies.
Laboratory indicators of hemolysis are summarized in Table.
Notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels.
Despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-A.
This revealed donor anti-A titer results of 256 (IgM) and 1,024 (IgG); donor was deferred from future platelet donations.
Conclusion: While the post-transfusion sample had no visible hemolysis and a negative DAT, increased total/ indirect bilirubin after transfusion and high titer donor anti-A are supportive of immune mediated hemolytic transfusion reaction.
The key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-A.
Ana Paula Hitomi Yokoyama* 1 , Leila Patricia de Sousa Fontenele 1 , Isabel Nagle Reis 1 , Carolina Bonet Bub 1 , Araci Sakashita 1 , Raffael Zamper 1 , Cristiane Nakazawa 1 , Tatiane Almeida Omura Paula 1 , Patricia Silva Batista 1 , Marcio Dias Almeida 1 , Fernanda Loureiro de Andrade Orsi 2 and Jose Mauro Kutner 1 .
1 Hospital Israelita Albert Einstein, 2 Hemocentro Unicamp-Universidade Estadual de Campinas Background/Case Studies: Orthotopic liver transplantation (OLT) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease.
Despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements.
Peri and intraoperative transfusion of red blood cells (RBC) have been previously reported as major predictors of post -operative mortality .
Identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in OLT.
We conducted a single center retrospective analysis of 671 cases of OLT performed between 2011 and 2015 in Brazil in order to identify predictive factors for red blood cell transfusion Study Design/Method: A retrospective analysis in a single institution was performed, and charts of 671 consecutive patients submitted to liver transplantation between 2011 and 2016 were reviewed.
The following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (MELD), duration of warm and cold ischemia.
Categorical variables were analysed using Pearson chi-square test.
Continuous variables were analysed using t-Student test.
A forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant.
Results/Finding: In univariate analysis, female patients, absence of hepatocellular carcinoma (HCC), primary diagnosis, corrected MELD and warm ischemia time were significantly associated with consumption of RBC use in the intraoperative period.
Multivariate logistic regression of these factors showed that female patients (OR 1,726 -95% CI: 1,147-2,597, p:0,009), absence of HCC (OR 0,295 -95% CI:0,199-0,437, p:0,0), cirrhosis of any cause (OR 4,161 -95% CI 1,816-9,534 -p:0,001), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (OR 5,236 95%IC 2,212-12,394) and retransplantation due to primary non function of the graft (OR 5,791 95%CI 1,33-4,25,206, p: 0,019) were independently associated with RBC transfusion requirements.
Conclusion: In this study, female patients, absence of HCC, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with RBC consumption in intraoperative period.
Determination of RBC transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies.
Prevalence of High-Titer Anti-A1/B in Group O Platelet Products.
Charles K. Childers* 1 , Mark Destree 2 , Ashley Rose 2 and Theresa Nester 3,4 .
1 Madigan Army Medical Center, 2 Bloodworks Northwest, 3 Bloodworks NW, 4 Dept of Laboratory Medicine, University of Washington Background/Case Studies: With platelet substitution policies, minor ABOincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply.
However, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high Anti-A1 or anti-B titers, typically in a group O donor.
One method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention.
The percentage of high titer Anti-A1/B in group O platelet products is presented from a large regional blood center collected over 10-12 months.
Data from both pre-storage pooled platelet units (PSPP) and apheresis derived platelet units (APLT) is shown.
Study Design/Method: Platelet component samples were collected in 2 mL EDTA sample tubes.
A single 1:150 dilution of plasma was prepared using a Hamilton MicroLAB 600 series dilutor using 2235.0 mL saline diluent and 15.0 mL platelet component sample.
Using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of A1 or B red blood cell reagent.
Reaction tubes were centrifuged immediately in a serological centrifuge at 3175 rpm for 20 seconds.
Reactions were read using a lighted agglutination reader.
The presence of macroscopic agglutination (weak or greater) with either the A1 cells or B cells was recorded as a positive reaction, indicative of a high titer Anti-A1 or Anti-B.
Retesting of samples was performed to confirm high titers.
Results/Finding: The above results indicate that, when a titer cut-off of 150 is used, approximately 3% of group O apheresis platelets will have a high titer, most commonly with anti-A1.
Less than half of a percent of PSPP units will have a high titer.
Testing units for the titer can help to change ABO out-of-group platelet substitution policies.
In our example, the Bloodworks transfusion service was able to change from a policy of volume reducing any group O apheresis platelets being issued to a group A or AB patient, to giving high titer products to only group O patients.
The subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their 5 day outdate.
After 10 months of testing PSPP units and verifying that the products rarely had a high titer (0.28%), the blood center stopped performing this testing for PSPP units.
Rh1) ] started complaining of worsening back pain two and half hours after receiving one unit of RBC for a drop in hematocrit to 19% (from 24% on the previous day).
His hematocrit did not increase (18%), and over the ensuing 12 hours, he became anuric and jaundiced.
Clerical checks confirmed that his forward type was A positive, which was also the type of the RBC unit transfused, but revealed anti-A at a titer of 8 in his plasma.
Furthermore, the direct antiglobulin tests (DAT) were positive for C3 in the pre-and post-transfusion blood samples.
Anti-A was not detected in his plasma collected three days earlier, however.
Although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (LDH) and total and indirect bilirubin Results/Finding: The positive DAT in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the A RBC unit.
In the setting of recent ABO-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-A by proliferation of donor lymphocytes, or PLS.
We performed an emergent RBC exchange using O RBCs with a goal hematocrit of 24% while reducing the number of A RBCs in his circulation by approximately 70%.
His pain improved rapidly thereafter, and he had complete recovery of renal function.
Conclusion: PLS should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in ABO-mismatched HPC transplant recipients, especially when the HPC source is from peripheral blood.
As in our patient, it usually takes 7-14 days for antibodies to develop and they are short-lived (3-5 weeks).
Due to the severity of his manifestations, we performed an emergent RBC exchange successfully.
Furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for ABO-mismatched transplant recipients, which has since been remediated electronically Background/Case Studies: Group O RhD negative (ONEG) red blood cells (RBCs) are a precious resource.
To conserve the ONEG inventory while minimizing the risk of RhD alloimmunization in ONEG females of childbearing age, transfusion services may automatically provide group O RhD positive (OPOS) RBCs to RhD negative males and/or RhD negative postmenopausal females during bleeding emergencies.
Despite these conservation strategies, shortages of ONEG RBCs occur.
The goal of this study was to determine how the utilization of ONEG RBCs can be optimized using agebased OPOS switching for routine transfusions in ONEG patients.
Study Design/Methods: Recipient age and ABO/RhD group were obtained for all allogeneic RBC transfusions during the 2016 calendar year from 9 hospitals.
An additional hospital* provided data for August-December 2016.
RBC transfusions in patients <1 year of age, and in patients whose age and/ or ABO group were unknown, were excluded from analysis.
The ABO/RhD group of each RBC unit was compared to that of the recipient to determine the number of ONEG RBCs transfused to all patients, the number of RBCs transfused to ONEG patients and the number of ONEG RBCs transfused to ONEG patients.
The number of ONEG RBCs transfused specifically to ONEG patients >/5 70 years was also determined.
Results/Findings: See table 1.
The fraction of all transfused RBCs that were ONEG ranged from 5-14% (row F).
The percentage of ONEG RBCs transfused to ONEG patients ranged from 37-89% (row G); thus, NON-ONEG patients received 11-63% of the ONEG units transfused (row H).
Hospitals differed widely in the practice of issuing ONEG RBCs to ONEG patients (68%-100%; row I).
Overall use of ONEG RBCs could have been reduced by 10%-39% if OPOS units had been given to all ONEG patients >/ 5 70 years old (row J).
Conclusion: During times of ONEG shortage, age based OPOS switching rules may be applied for routine transfusions.
This would help to ensure the availability of ONEG RBC units for ONEG females of childbearing age.
Rasha Eldeeb Mohammed* 1 , Nehad Mohammed 2 , Marwa Aly 2 and Nashwa Fahmy 2 .
1 National Blood Transfusion Services, 2 NBTS Background/Case Studies: Sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients.
Splenectomy has been shown to increase human leucocyte antigen immunization.
The aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans.
Study Design/Method: This study was conducted on 206 multitransfused patients who received blood transfusion chronically at our central blood center.
They were 129 thalassemia patients (128 bthalassemia patients, one patients with a thalassemia), 10 sickle cell anemia patients and 6 immune hemolytic anemia patients (4 auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients).
29 Oncology patients, 32 Chronic diseases patients.
History and demographic data were documented.
All the patients who received blood are examined for the presence of the spleen.Our patients were subjected to Direct & Reverse Blood grouping (ABO& Rh) tests, alloantibody screening and detection.
Results/Finding: Statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients.
The study revealed that: 32 out of 48 (67%) alloimmunized patients and 16 out of48(33%) non alloimunized patients(p<0.001) .
statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form Conclusion: Patients who had splenectomy had a higher alloimmunization rate Removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components.
restrictive transfusion triggers rather than specific evidence.
Therefore, two systematic reviews of A) RBC transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and B) to identify studies comparing strategies were performed.
Study Design/Method: Methods MEDLINE, EMBASE, CINAHL, Web of Science, National Guideline Clearinghouse, and the Trip Database were searched from inception to June 2016.
Screening and data abstraction were done independently by two assessors.
For review A, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles.
For review B, the primary outcome was RBC utilization.
Secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality.
Meta-analysis was done using the Mantel Haenszel random effects model.
Results/Finding: Review A identified 136 articles for data abstraction, where 48 articles were transfusion guidelines.
There were 12 guidelines (25%) that made a recommendation, 11 for a single unit and 1 for multiple unit transfusion strategy (Table 1) .
Review B identified 3 retrospective cohort studies that were eligible and data abstraction was performed.
All utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period.
Meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (OR 9.4, 95% CI 5.02-17.60), although heterogeneity was high (I 2 597%).
Conclusion: Our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of RBCs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies.
Additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used.
Guidelines groups should review research in this area to determine if a recommendation can be made.
Background/Case Studies: Platelets made with platelet additive solution C (PAS C) and treated for Pathogen Reduction (PR) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma.
With the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use.
The literature from Europe has shown that platelet and red cell use does not increase when PR and PAS products are used.
Evaluation of RBC use at our institution has shown no change in the number of products transfused per patient per month.
We are evaluating whether the mixed inventory has led to more platelet transfusions.
Study Design/Method: We looked at occasions when patients received all of their platelet transfusions on a single day.
By doing this we were able to exclude refractory patients from the analysis.
The information obtained from routine Quality Management audits of transfusions between December 2016 and February 2017 was used for this analysis.
The information included the ordering service, product release time, product code, pre and post counts.
Statistical analysis was performed using Minitab.
Results/Finding: During the 3 months, 1723 units of platelets were transfused to 238 recipients.
Over the 3 months, a median of 4 units was given to each patient with a range of 1 to 69.
The overall distribution of products used was 58% plasma, 24% PR, 7% PAS F and 11% PAS C. Thirty percent of patients (N572) received all of their products on a single day.
Single units were given to 54 patients while 14, 3 and 1 received 2, 3, and 4 units respectively.
The distribution by product type was 56% plasma, 25% PR, 13% PAS C and 4% PAS F. This same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the 3 month period (P5 1.00).
The distribution by service was different for the groups receiving multiple units.
For single units the distribution was 44% hematologic malignancy, 22% infusion clinic (NOS), 13% solid tumor medicine, 11% surgery, and 9% pediatrics.
For those receiving multiple units the distribution was 50% surgery and 16% each for solid tumor, hematology and infusion (NOS).
The chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a P value of 0.022.
Conclusion: The distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the 3 month period.
The patient's clinical service was a better predictor of the use of multiple products than the type of product given.
This suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario.
The Effect of Red Blood Cell Transfusion on Iron Metabolism in Critically Ill Patients Margit Boshuizen* 1,2 , Yvemarie B.O.
Somsen 2 , Maike E. van Hezel 2 , Marleen Straat 2 , Robin van Bruggen 1 and Nicole P Juffermans 2 .
1 Sanquin Research and Landsteiner Laboratory, 2 Academic Medical Center Background/Case Studies: Anemia of Inflammation (AI) has a high prevalence in critically ill patients.
In AI, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis.
AI is treated by red blood cell (RBC) transfusions.
It is known that RBC transfusions increase iron level in neonates and thalassemia patients, but the effect of RBC transfusion on iron metabolism during inflammatory processes is unknown.
Since one unit of RBCs contains 220 mg of iron and 25% of the RBCs are cleared by macrophages within 1 hour following transfusion, RBC transfusion could increase iron levels and iron availability for erythropoiesis.
We investigated the effect of RBC transfusion on iron metabolism in ICU patients, and additionally compared the effect in septic patients to non-septic patients.
Study Design/Method: In a prospective cohort study in 52 ICU patients who received one RBC transfusion, different iron parameters were measured before and 24 hours after transfusion, to determine the effect of a RBC transfusion over a period of time.
Next, the impact of a RBC transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed.
Plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and IL-6 levels were determined.
Results/Finding: In this cohort, serum iron levels were low and did not change following transfusion (4.1 vs. 4.3 mmol/L, p50.69).
Also, the transfusion had no effect on transferrin saturation (12 vs. 13 %, p50.13), ferritin (531.0 vs. 599.0 mg/L, p50.74) and IL-6 levels (35.0 vs. 25.5 pg/ml, p50.09).
Hepcidin levels increased in these ICU patients after RBC transfusion (223 vs 332 ng/ml, p50.01).
In septic patients, RBC transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (-2.7 vs. 3.7 % change, p50.05).
Other iron parameters did not differ between septic and non-septic patients.
Conclusion: Transfusion of one unit of RBCs does not increase iron levels in ICU patients.
The increase of hepcidin suggests RBC transfusion induced upregulation of hepcidin, despite the absence of a significant increase in IL-6 or plasma iron levels.
This increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis.
In sepsis, RBC transfusion decreases haptoglobin levels, suggestive of hemolysis.
In conclusion, RBC transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion.
The Effects of PAS and PR on Platelet Use Barbara Mendez, Judith DelMonte, Elizabeth McCabe and Joanne Becker*.
Roswell Park Cancer Institute Background/Case Studies: With the anticipated release of the FDA guidance: Bacterial Risk Control Strategies for Blood Collection Establishments and Transfusion Services to Enhance the Safety and Availability of Platelets for Transfusion, the use of pathogen reduced platelets (PR) which are often produced from products made with platelet additive solution (PAS) may become more common.
Our institution has been transfusing platelets made with additive solutions since 2011 and pathogen reduced platelets have been available since 2016.
In our data validating PAS and PR, the post counts from transfusion of PAS-C and PR products have been statistically lower than platelets in all plasma (PP) or PAS F products.
Our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused.
Study Design/Method: The data was obtained from the routine quality reports produced for the blood utilization committee at our facility between 2012 and 2016.
During this time PAS C, PAS F and PR went from 13% to 40% of all platelet products given.
All recipients had an oncology diagnosis.
The data collected included the service, unit number and product code.
The number of unique recipients was determined monthly.
The data was converted to PLT/month/recipient for analysis.
Statistical analysis was performed using the two sample T-test Results/Finding: The data was normalized to PLT/recipient/month.
In 2011 patients received an average of 5.41 units/recipient/month and in 2016 the average was 5.39 units/recipient/month.
The intervening data points for 2013, 2014, and 2015 were 5.92, 5.66, and 5.92 respectively.
The 5 year average was 5.66.
The slope of the graph for all 5 points was y5 -0.004 15.672.
The two sample T-test showed that the PLT/recipient/month from 2012 to 2016 was not statistically different with a P value of 0.81.
Conclusion: The implementation of PAS and PR platelets in the oncology environment has not increased in the number of platelet transfusions given.
In additional analysis, the red cell use has decreased (data not shown).
This can be interpreted as indicating that patients have not had increased episodes of bleeding.
Although the post platelet count from PAS/PR platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given.
Background/Case Studies: It is reported that the incidence of alloimmunization in AML patients is unrelated to the number of transfusions the patient receives and most patients who have HLA antibodies do not exhibit platelet refractoriness.
Many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests.
Recent data in leukemia and hematopoietic stem cell (HSCT) recipients transfused exclusively with leukoreduced products show that 4% to 8% develop Alloimmune platelet refractoriness.
Objective: To determine an improvement in platelet count with the match grade and/or the ABO blood group of the HLA matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction.
Study Design/Method: Clinically documented platelet refractory patients, who received HLA matched irradiated SDA platelets with their HLA typings for HLA-A/-B and HLA Antibody Identification were reviewed.
There were two strategies utilized, the HLA strategy (matching recipient and donor HLA-A/ -B types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those HLAs to which the patient had antibodies) strategy.
Statistical Analysis: A One Sample t-test using Minitab 17 Statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least 5 K/ul.
The analysis revealed that the mean of 9.35 K/ul (n584) had a 95 percent lower bound confidence interval platelet increment of 7 K/ul (p<50.001) Results/Findings: 123 (median 4 range [1-43]) HLA matched leucoreduced irradiated SDA platelets were transfused to 17 (6M/11F) patients, median age 60 years (range 27-83).
15/17 (88%) patients showing broad alloimmunization to HLA Class I/Class II antigens.
2/17(12%) patients had anti-HPA antibodies (GP IIb/IIIa and GP IIb/IIIa and GP Ia/IIa).
The majority 16/17 (94%) had a diagnosis of hematologic malignancy (AML/MDS/MPN/ CMML/MM); 9/11 (81%) female patients had prior exposure via pregnancy and 4/11 (24%) had a history of HSCT.
63 (51%) platelets were ABO identical-platelet increment median 7 K/uL (range -14 to 61), 53 (43%) were ABO compatible -platelet increment median of 2K/uL (range -10 to 46) and 7(6%) were ABO incompatible with platelet increments median 8K/uL ( range -10 to 32).
Platelet counts were performed within 24 hours in 73 (57%) transfusions.
The HLA match grade of the transfused platelets were as follows: The Use of Massive Transfusion Protocol (MTP) in a Community Hospital Rohini Patel* 1 , Renee LeBlanc 2 , Dongfu Xie 2 , Alice Cabe 1 and Yanyun Wu 2 .
1 Overlake Hospital, 2 Bloodworks Northwest The Use of Massive Transfusion Protocol (MTP) in a Community Hospital Background/Case Studies: The establishment and use of massive transfusion protocol (MTP) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding.
However, it is not well established if the use of MTP also has value in small hospitals and community hospitals, and how MTP is used in Fig.
1 these settings, such as indication for MTP, blood products used, and the outcomes of these patients.
Study Design/Method: Retrospective review of transfusion data from a community hospital with a bed size of about 350 for 3 years (from 2014 to 2016) was performed.
Patients with MTP requested are included in this study.
Results/Finding: Please see the table below for the summary of data.
Notably, patients with GI bleed and OB bleed are the two most common indications for MTP, and 68 % of patients survived with the support of MTP.
In one case, no blood product was used.
The establishment and readiness of MTP can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals.
In these settings, MTP is most commonly used for patients with massive GI bleed and Ob bleed.
If the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible.
Included in the MNS system, and residing on Glycophorin B (GPB), the U antigen is absent in less than 0.25% of the black population.
Those with altered forms of GPB, known as U variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).
This case illustrates the balance between the need to transfuse and avoiding complications thereof.
A 32-year-old Ghanaian woman with SCD and history of chronic transfusion presented with diffuse pain and a hemoglobin value of 6.4 g/dL (baseline 9-10 g/dL).
She is known to be E, C, K, Fya, Jkb, S, s negative, U variant, and has anti-E, C and U antibodies.
There were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible U variant units.
The decision to transfuse was made.
The patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was 5.9 g/dL.
A transfusion reaction work-up was ordered.
Post-transfusion serum sample was negative for hemolysis and no new antibodies were identified.
The post-transfusion DAT was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin (8.8 to 6.2 mg/dL).
Two additional U variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to 6.2 g/dL.
The patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline.
Study Design/Methods: Molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services.
Conventional methods were used to monitor the patient's condition pre and posttransfusion.
Results/Findings: Each donor unit came from a different donor but all were the same GPB genotype as the patient.
The patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case.
Conclusion: Transfusion of U variant red cells to a U variant patient should be undertaken with great caution due to epitope and antibody heterogeneity.
This case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, SCD patient with anti-U.
Sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff.
Background/Case Studies: Patients with decompensated WAIHA may require transfusion with red blood cell (RBC) products that are cross-match incompatible due free autoantibodies.
The feasibility of blood transfusions in WAIHA patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused RBCs may be destroyed more rapidly in patients with active hemolysis.
To study the actual vs. theoretical risk of increased hemolysis in WAIHA patients, we investigated the post-transfusion (post-tfn) hematocrit (Hct) change in WAIHA patients who were transfused compatible RBCs compared to those who received LI blood.
We further hypothesized that a post-tfn Hct would be inversely related to the degree of AHG-phase incompatibility.
Study Design/Method: We reviewed all transfusions to patients in our quaternary-care hospital with a history of WAIHA from October 2015 to March 2017.
Patient Hcts were ordered by prescribing physicians for clinical purposes.
A transfusion episode was defined as all units released in the interval before a post-tfn CBC.
AHG-phase crossmatch was tube tested in saline per clinical procedure.
Transfusion medicine physicians determined the release of least-incompatible units.
Statistical tests were performed with STATCALC (EpiInfo, CDC) and www.socscistatistics.com.
Results/Finding: There were 139 RBC products transfused to 40 WAIHA patients.
Twenty-three (57.5%) patients received at least 1 incompatible unit.
The mean age was 51.4 years (range 4-93 yrs) with 50% women.
Ethnic composition was 55% African-American, 40% Caucasian, and 5% patients of mixed/other ethnicity.
One hundred fourteen (82%) of these products were released as LI products and 25 (18%) were compatible.
Ninetythree (81.6%) of the LI product transfusions had a post-tfn Hct change of <3% whereas only 14 (56%) of the compatible product transfusions resulted in a post-tfn Hct change of <3% (p50.0092, v 2 (1), exact methods).
The mean Hct increase in the compatible group was 1.83% per unit vs. a slightly lesser per-unit increase of 1.71% in the LI group (p50.82, t-test, 2-tailed) Within the LI group, there was no difference in the per-unit Hct change according to strength of incompatibility (Table) .
Strength of AHG incompatibility was not available for 12 units.
Units that were 31 incompatible had a lower mean post-tfn Hct rise compared to all other LI units (1.49% vs. 2.15%); however, this difference was not statistically significant (p50.38).
Conclusion: The post-tfn Hct change for transfusions of LI units to patients with WAIHA was less than the expected 3% per unit more frequently than it was for WAIHA patients who received compatible products (81.6% vs. 56%).
However, likely due to our small sample size, the mean differences were not statistically significant.
Interestingly, there was no difference in the per-unit post-tfn Hct according to differing strengths of incompatibility in our sample, although the mean increase for the 31 LI products was less than all other LI products combined.
The increase was unexpectedly low for weaklyincompatible units, which we are further studying.
Future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications.
transfusion management was summarised by individual hospital, type and total cases.
In-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of RBCs in the first 4-hours from MT onset) was calculated with 95% and 99.8% control limits to indicate potential outliers.
Data were analyzed using statistical software (Stata).
Results/Finding: There were 5482 MT cases from 25 hospitals (17 tertiarylevel, 6 smaller/medium sized acute-care and 2 specialist women's).
Number of MT cases per hospital ranged from 5 to 721.
Patient median age was 65 years (IQR 49, 76), 62% were male and 73% required admission to intensive care.
The most common clinical groups were cardiac surgery (21% cases), trauma (20%) and gastrointestinal hemorrhage (13%); however there was marked variation between hospitals.
Ratios of transfused products, analyzed according to bleeding context, varied between hospital types.
The pooled average adjusted in-hospital mortality for the 17 tertiary-level hospitals was 21% (range 13% to 33%) and 16/17 (94%) were within the 95% control limit.
CB that required !10 RBCs within 24-hours of MT onset occurred in 40% of cases.
Comparison of transfusion management for this subset of MT cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals (67% versus 78%; p50.03).
Conclusion: Patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable.
Results are made available to participating hospitals in the ANZ-MTR to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation.
Data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research.
Background/Case Studies: In hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer.
With this, infusion fluids, including blood products, are administered under pressure.
This is done because veins of trauma patients are often not suitable for infusion of fluids.
Suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes.
There is no information available concerning transfusion of platelets under pressure via a bone needle.
The aim of the study was to investigate the effects of warming and administration of a platelet concentrate (PC) under pressure via a bone needle on the in vitro quality of platelets.
Study Design/Method: Pools of 5 BCs and 280 mL of platelet additive solution III (PASIII) were used to produce PCs (n55).
PCs were stored on a flatbed agitator (60 cycles/min) in a temperature-controlled cabinet at 22 6 28C for 4-7 days.
To mimic hospital conditions, PCs were warmed using a blood warmer and transfused via a bone needle to a transfer bag.
On the PCs a pressure of 300 mm Hg was applied.
Using clamps, a flow velocity of 90-120 mL/minute was realized.
Platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters.
Results/Finding: Due to priming of the transfusion disposable with saline, the PCs were diluted 10-30%, resulting in a significantly increased PC volume and decreased platelet concentration after simulated transfusion.
Because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion.
After simulated transfusion, the PCs still fulfilled the requirements for platelet concentration (0.8-1.6x10 11 /L) and number (>250x10 9 /unit).
Simulated transfusion had no effect on the percentages of CD62P and Annexin V positive cells, indicating no activation or induction of apoptosis.
pH was not influenced by simulated transfusion.
Due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion.
Conclusion: Warming and simulated transfusion of PCs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets.
Transfusion of warmed PCs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets.
It is recommended to study the in vivo effects in a limited clinical study.
Alesia Kaplan* 1,2 , Joan Sevcik 2 and Joseph E. Kiss 1,2 .
1 University of Pittsburgh, 2 Blood Systems Inc. Background/Case Studies: Low titer A plasma has been safely used as a substitute for AB plasma in trauma patients.
Low inventories of AB plasma can cause a delay in life saving therapeutic plasma exchange (TPE) procedures in AB patients needing plasma replacement.
Here, 2 AB non-bleeding patients are presented who safely received AB and low titer A plasma for TPE.
One AB patient who received AB plasma only was used as control to compare hemolysis laboratory data over TPE course.
Study Design/Method: A retrospective review of TPE procedures for 3 patients was conducted from medical records.
Number of procedures, volume replaced, total number of plasma units, number of A plasma units, quantity of A plasma and hemolysis laboratory data were recorded.
Average quantity (ml) for A plasma and % of A plasma out of total volume of plasma used were calculated.
All A plasma units were low anti-B titer units.
In the laboratory, plasma dilution 1:50 is prepared and tested with reagent B cells.
If agglutination is not observed, the unit is labeled as "low titer anti-B".
Hemolysis laboratory data was traced with linear graphs and trends were compared between patient 1 and 2 and 3 (control).
Results/Finding: All 3 patients were AB blood type.
Patient 1, a 57 year old female with recurrent ADAMTS13 deficient TTP, received 2 courses of TPE (total 12 TPE procedures) for relapse and exacerbation.
Ten out of 12 procedures were performed with AB and A plasma (average 916 ml of A plasma or 24% of total plasma volume for 10 TPE procedures).
Patient 2, a 27 year old female with thrombocytopenia, schistocytes and presumed TTP, received a total of 12 TPE procedures.
Four out of 12 procedures were performed with AB and A plasma (average 1210.5 ml of A plasma or 48% of total plasma volume for 4 TPE procedures).
Patient 3, a 33 year old female with ADAMTS13 deficient TTP who served as a control, received a total of 10 procedures with AB plasma only.
Haptoglobin, LDH, hemoglobin and total bilirubin were graphed and compared between 3 patients.
The trends of hemolysis laboratory data for patient 1 and 2 were comparable with patient 3.
All 3 patients had negative DAT.
Only patient 3 received 2 RBC transfusions.
All 3 patients had a favorable clinical outcome with TPE treatments and adequate platelet recovery.
Conclusion: In this study, TPE was effectively performed without evidence of increased hemolysis using up to 48% of low titer A plasma.
This approach can reduce strains on limited supplies of AB plasma while providing a vital treatment alternative for AB patients undergoing TPE who require plasma replacement.
When CD36 Negative Platelet Unit Is Not Available for a Patient with Anti-CD36 Antibodies Sameer Khatri* 1 , Charles Harmon 1 , Brian R Curtis 2 and Chisa Yamada 1 .
Background/Case Studies: Refractoriness to platelet (PLT) transfusion can be caused by antibodies (Abs) against Human Leukocyte Antigen (HLA) class I antigens (Ags) or less frequently against PLT specific Ags (PSAs).
Glycoprotein IV (CD36) is one of the identified PLT surface Ags and deficiency is rare, but found in Asians (3-11%), sub-Saharan Africans (7-8%) and also in some people from Mediterranean descent.
Two types of CD36 deficiency have been described.
Type 1 deficiency is the complete lack of CD36 on both PLTs and monocyte-macrophages whereas type 2 deficiency lacks CD36 on PLTs with variable expression (12-99%) on monocytemacrophages.
Transfusing PLTs in a patient with CD36 deficiency is challenging given the rarity of CD36 negative phenotype and risk of further immunization when giving Ag non-matched platelets.
Study Design/Method: A patient with CD36 negative phenotype who received multiple PLT units was reviewed in the electronic medical record.
Results/Finding: A 21 year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple PLT and RBC transfusions.
He received more than 20 units of apheresis PLT units over a 2 week period without any significant increase in PLT count.
Cross-match compatible PLT unit found in 1 of 32 units and HLA matched units were tried without success.
At that point, a CD36 Ab was identified in the serum and the patient's type 1 CD36 deficiency was confirmed by flow cytometry.
His HLA class I Panel Reactive Ab (PRA) was 95% due to multiple PLT transfusions, although all Abs were low levels.
The patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in PLT increase.
Following three doses of IVIG, he received a CD-36 negative (but blood type different and HLA 187A TRANSFUSION 2017 Vol.
57 Supplement S3 unmatched) PLT unit from his relative with only a slight increase in PLT count.
However, he started to respond to CD36 non-tested apheresis PLTs after receiving a fourth IVIG and two rituximab infusions.
Since then, he has received IVIG every 2 weeks.
Other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia.
The mean corrected count increments (CCI) when post-transfusion PLT count was available are shown in Table.
With desensitization therapy, his CD36 antibody positive reactivity in serial dilutions has reduced from 1:32 to 1:2 dilutions and his HLA Class I PRA has decreased to 37%.
He is currently receiving 2 apheresis PLT units twice a week and RBC units periodically.
His bone marrow (BM) has been slowly recovering evidenced by increased WBC count from zero to up to 1.0 K/mL and slow increase of reticulocyte counts.
Current plan is RBC/PLT transfusion support until BM recovers or a haplo-identical transplant if BM recovery fails.
Conclusion: We report a case with anti-CD36 Abs that received multiple PLT transfusions.
This case demonstrates that decreasing Ab level with immunomodulation can be an alternative option for successful PLT transfusion when compatible PLTs are not available for patients with rare or multiple Abs to PLTs.
Table: Mean available CCI for PLT Transfusions A Blood Center's Experience Screening Donations for Babesia Microti Using Enzyme-Linked Immunoassay Methodology Nancy Van Buren*, Jed Gorlin, Vanessa Reynolds and Deborah Anderson.
Background/Case Studies: Our blood center, located in an area considered to be moderately endemic for Babesia microti, implemented universal screening of red cell collections from Minnesota and Wisconsin under an investigation new drug (IND) study in Oct 2015 utilizing the Immunetics investigational enzyme-linked immunoassay (ELISA) performed by Creative Testing Solutions (CTS).
This test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational IFA/PCR test combination.
Study Design/Methods: We performed a retrospective analysis of our screening test results and deferral rates for 2016 to evaluate for seasonality, donor ABO bias, deferral rates, and outcomes of lookback investigations.
Since an opt-out of this research test was originally offered, we report donor opt-out rates.
Results/Findings: From Jan through Dec 2016, 101,854 blood donations were screened for B microti by Immunetics ELISA.
Of those, 267 (0.26%) were positive.
The percent of positive donations was evaluated monthly revealing a variable reaction rate between 0.08% and 0.42%.
No patient Babesia transmission has been reported since implementing this test, but we only had 4 documented Babesia TTD cases from 2007-2017.
Donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in Minnesota and Wisconsin.
Test performance characteristics were analyzed by ABO group with no demonstrable differences in positive rates.
The opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test.
Of 267 positive test results, 160 lookback investigations were initiated representing 59% of positive donations.
Lookbacks were only performed when there was a donation within 12 months of the new positive screening test, according to IND protocol.
No confirmatory testing was performed per IND protocol or for donor counseling, so the true positive rate is unknown.
In the prior IND trial, up to 80% were unlikely to transmit infection in our region, i.e.
were PCR and blood smear negative.
Although a small number of antibody positive, PCR negative donors may be actively infected, no transfusion-transmitted Babesia infections were identified by lookback investigations.
Notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup.
Overall, donor deferral represented 0.25% loss of eligible donors during this follow-up period.
Deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility.
Conclusion: Testing for B microti may help improve blood safety, particularly in endemic regions.
Although only 0.25% of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection.
A direct test capable of detecting Babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal.
A reinstatement protocol for donors who test positive should also be considered.
Nonetheless, the current method of screening is inexpensive compared to PCR-based methods.
Background/Case Studies: Human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens.
They establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter.
Some studies suggest they are ubiquitous, present in >90% of the human population, and that immune surveillance is required to control the level of the virus load.
Study Design/Methods: We have developed a quantitative DNA PCR assay for the most conserved region of the Anellovirus genome that detects all known genotypes of the virus.
We used this assay to examine viral loads in the plasma of US blood donors and transplant recipients pre-transplant and three months post-transplant.
Results/Findings: For 53 blood donors, 51 were positive with an average load of 1.49x10 2 copies/mL of plasma, a median value of 80.5 copies/mL of plasma, ranging from 0 to 1.87x10 3 copies/mL.
Pre-transplant viral loads were similar.
For 41 transplant candidates, 40 were positive with an average of 3.70x10 2 copies/mL of plasma, a median value of 88 copies/mL of plasma, ranging from 0 to 1.18x10 5 copies/mL.
Post-transplant viral loads were remarkably different.
For 94 transplant recipients, all were positive with an average of 3.14x10 5 copies/mL of plasma, a median value of 1.25x10 5 copies/mL of plasma, ranging from 0 to 4.6x10 7 copies/mL.
Conclusion: These results validate the PCR assay that was developed and confirm that detectable viral loads of around 100-200 copies were present in >90% of the blood donors surveyed.
In addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least 2 orders of magnitude above that of non-immunosuppressed individuals.
Background/Case Studies: The screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens.
Furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance.
The development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Yellow Fever virus (YFV), Usutu virus (USUV) and Chikungunya virus (CHIKV).
Study Design/Method: An innovative diagnostic approach combining generic RT-PCR amplification and identification on low cost microarrays has been developed.
We have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific Mean CCI Pre-IVIG: All PLT (17) 0.2 Post-IVIG: All PLT (41) 5.5 Post-IVIG: CD36-negative PLT from relative (1) 0.8 Post-IVIG: Single Donor Apheresis (23) 4.3 Post-IVIG: Cross-match Compatible (15) 6.1 Post-IVIG: Flow Cross-match Compatible PLT (2) 12.6 188A TRANSFUSION 2017 Vol.
57 Supplement S3 detection of the viral genomes.
Analytical performances of the test were evaluated on viral standards and on clinical samples: DENV (1/2/3/4), WNV, ZIKV and CHIKV.
Forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls.
We have designed two sets of degenerated primers for the generic RT-PCR amplification of all flaviviruses and for CHIKV.
Biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate.
After addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader.
Results/Finding: One original generic probe for DENV and specific probes designed for each of the four DENV serotype, WNV, the two ZIKV lineages and for CHIKV, were validated.
The use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows 100% of specificity, with no false positive results on the 40 control samples, and no cross reactions.
Using viral reference standards, we have observed sensitivities of 1 TCID 50 /mL for DENV-1, DENV-3 and CHIKV and of 10 TCID 50 /mL for DENV-2, DENV-4 and ZIKV.
Finally, the first results obtained on 110 DENV(1), 69 ZIKV(1) and 50 CHIKV(1) clinical samples show 85%, 87% and 96% correlation respectively between our approach and commercial or in house real time RT-PCR methods.
Conclusion: This innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses.
This methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections.
Babesia Microti Serological Testing with Pooled Samples: A Feasibility Study Laura Tonnetti*, Aaron Thorp, Letitia Dixon and Susan L Stramer.
Background/Case Studies: Blood donation screening for Babesia microti, a tick-borne intraerythrocytic parasite endemic in the Northeast and Upper Midwest US, is performed under an investigational study using nucleic acid and immunofluorescence assays (IFA).
However, IFA is a time consuming and labor intensive procedure.
With the possibility of an FDA licensed screening assay(s) in the near future, we investigated if B. microti testing by IFA in pools of plasma or serum could be a feasible screening approach.
Study Design/Method: To test if the increased amount of plasma or serum interferes with background fluorescence, pools of 4, 8, 16 and 32 were prepared from 192 plasma or 192 serum samples determined to B. microti-negative by individual IFA screening.
The pools were tested by IFA with or without a blocking step using bovine serum albumin (BSA) and goat serum to minimize background fluorescence.
Potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from 1:128 to 1:1024 (2-fold dilutions) in the pools.
Results/Finding: Non-specific fluorescence was visible in pools of 16 or higher and was not eliminated by the addition of a blocking step.
Pools of 4 or 8 samples did not show significant increased background.
There was no difference between testing of pooled serum or plasma samples.
When one single positive sample was included in the pools of 4 or 8 samples, the pool tested positive and the final titer was the same as the positive sample tested individually.
When two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer.
Conclusion: This study represents a proof of concept that serological testing for B. microti by IFA in pools of up to 8 plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test.
Background/Case Studies: The rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components.
The BacT/ALERT VIRTUO* is an advanced, next generation system with improved automation, connectivity and with data management systems.
The VIRTUO's new algorithm significantly reduces the time to detection (TTD) of microorganisms during quality control testing of platelet preparations using BacT/ALERT (BTA) BPA (aerobic) and BPN (anaerobic) bottles.
As plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for VIRTUO studies.
Study Design/Method: Human plasma (thawed and pooled) and saline controls were seeded with $100 CFU/mL of 12 organisms associated with platelet contamination and incubated at room temperature for 18-24 hours.
Colony counts were performed initially and after incubation.
Plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher (1 1 Log) than the colony count of the seeded saline after incubation.
Results/Finding: The serially diluted strains and all BioBall TM strains except P. aeruginosa, NCTC 12924, were determined to be plasma resistant.
The BioBall TM P. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into 4 mL of Leukocyte Reduced Apheresis Platelets (LRAP) and inoculated into BTA BPA bottles and loaded into the BTA 3D and VIRTUO the organism was recovered 100% .
Conclusion: Results confirm that previously tested organisms and additional strains are plasma resistant with the exception of P. aeruginosa, NCTC 12924.
However, the BPA bottles still recover P. aeruginosa in the presence of LRAP.
BPA/BPN bottles inoculated with select organisms from this panel in the presence of 4mL LRAP demonstrated 100% recovery when loaded onto the VIRTUO and 3D ( Table 1) .
Further studies may be required to determine if higher test volumes of LRAP could affect the recovery of plasma sensitive strains.
* VIRTUO is not FDA cleared for platelet testing A. notoscriptus, identified as a major urban vector of RRV, is also capable of transmitting Dengue virus 1-4, and has recently been found in Los Angeles, illustrating an expansion in range.
With the growing geographical distribution of Aedes species mosquitoes, the potential for RRV to enter local transmission cycles outside of Australia is significant.
In 2014, a probable transfusiontransmission (TT) was confirmed as the cause for an RRV infection in Australia, validating the reality that RRV TT can occur.
RRV morbidity leads to clinical manifestations that are similar to CHIKV infection, with varying degrees of arthralgia, which can become debilitating.
Various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional TT in endemic areas and could mask the spread of the disease globally.
Study Design/Method: Platelet concentrates (PC) prepared in PAS were inoculated with RRV, amotosalen was added to final concentration of 150 mM and the units were treated with UVA light.
Pre-and post-treatment illumination samples were collected for titration.
AS-5 RBC units were contaminated with RRV, mixed with processing solution/glutathione (GSH) and treated with amustaline at a final 200 mM concentration.
Pre-and post-treatment samples were removed prior to amustaline treatment and 3hrs after amustaline addition, respectively, for titration by plaque assay on Vero76 cells.
Log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples.
Results/Finding: Inactivation of RRV was achieved to the limit of detection in PC and RBC.
In PC, >5.1 log 10 or log 10 /mL of RRV was achieved, with >5.5 log 10 or >5.2 log 10 /mL of RRV inactivated in RBC.
Conclusion: These studies illustrate that amotosalen/UVA and amustaline/ GSH treatments are effective at inactivating RRV in PC and RBC, respectively.
These data corroborate previous results achieved with other alphaviruses, including CHIKV and Mayaro virus which are inactivated at high titers in PC and RBC, demonstrating the ability for these systems to mitigate TT potential and maintain safe blood component availability in endemic areas.
(Data have not been submitted for FDA review and INTERCEPT for red blood cell is not approved for commercial use).
Background/Case Studies: The INTERCEPTV R Blood System for platelets is designed to inactivate pathogens and contaminating leukocytes.
This photochemical treatment process utilizes amotosalen and low energy ultraviolet A (UVA) light.
The current available sets include Small volume (SV; 255-325 mL), Large Volume (LV; 300-390 mL) and Dual Storage containers (DS; 300-420 mL) designed to treat platelet doses between 2.9 and 8.0x10 11 .
The new Triple Storage (TS) set was designed to expand the dose range to 12.0x10 11 and the maximum volume to 650 mL, generating either 2 or 3 doses of pathogen reduced platelet components (PC).
The objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in PAS, or 100% plasma using TS Set.
Study Design/Methods: For each experiment, a platelet pool was prepared either in 47% plasma/53% PAS or 100% plasma with a final volume of $650 mL and a dose of 9-12 3 10 11 platelets.
These conditions represent inactivation using the lowest amotosalen concentration (135 mM) and highest concentration of platelets.
Platelet units were inoculated with high titers of viruses, or bacteria and treated.
Control (Pre-UVA) and Test (Post-UVA) samples were serially diluted and cultured.
Plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays.
Log reduction was calculated as the difference between the log 10 titers in Control (pre-UVA) and Test (post-UVA) samples.
Conclusion Dromedary camels were identified to be the reservoir of MERS CoV, transmission to humans occurs through direct and indirect contact.
MERS CoV has been detected with high genomic titers of 6-10 logs in respiratory secretions of MERS patients, and with lower genomic titers of 4-5 logs in blood.
The presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas.
The high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude MERS CoV contamination of blood products.
Pathogen Reduction with Amotosalen/UVA technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of Amotosalen/UVA treated blood products.
The aim of the study is the assessment of the MERS CoV inactivation efficacy in human plasma with Amotosalen/UVA pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units.
Pre-UVA Titer Post-UVA Titer Log 10 Reduction/mL (log 10 /mL) 47%Plasma/ 53% PAS E .coli 6.0 <-1.0 >6.0 E. cloacae 6.4 <-1.0 >6.4 K. pneumoniae 6.6 <20.1 >6.5 S. aureus 6.7 <-1.0 >6.7 Blue Tongue Virus 4.9 <-1.0 >4.9 Bovine Viral Diarrhea Virus 4.6 <-1.0 >4.6 Adenovirus-5 1 3.9 <-0.6 >3.9 100%Plasma K. pneumoniae 1 6.5 -0.5 >6.5 S. aureus 1 6.2 <-0.7 >6.2 Adenovirus-5 1 4.5 <-0.6 >4.5 1 N53 190A TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT Study Design/Method: Four therapeutic human plasma units were spiked with a fully characterized MERS CoV clinical isolate followed by pathogen inactivation with Amotosalen/UVA (INTERCEPT Blood System, Cerus Corporation) at four different days.
Pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-PCR.
Samples post pathogen reduction have been passaged 3 times up to 9 days, assessing the infectious titer and genomic titer every 3 rd day to exclude the presence of low-titer infectious particles.
Results/Finding: All viral particles in the plasma units were completely inactivated with an average efficacy of !5.8 log infectious titer.
No viral replication was observed after 9 days of passaging post inactivation.
The genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer.
Conclusion: Amotosalen alone had a slight effect on the infectious titer while Amotosalen/UVA effectively inactivated all infectious MERS CoV viral particles in the plasma units with an inactivation efficacy above 5 logs infectious titer, giving evidence for improved blood safety of Amotosalen/UVA treated plasma in MERS CoV endemic regions.
Estimating the Prevalence and Incidence in a National Blood Service in Taiwan for HCV Eradication Program Yun-Yuan Chen* 1 , Jen-Wei Chen 1 , Chi-Ling Chen 2 , Sheng-Nan Lu 3 and Pei-Jer Chen 2 .
1 Department of Research, Head Office, Taiwan Blood Services Foundation, 2 Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, 3 Division of Hepatogastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital Background/Case Studies: World Health Organization (WHO) has set a goal to eliminate HCV by 2030, and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness.
This study aimed to evaluate the prevalence and incidence of HCV infection in Taiwan.
Study Design/Method: In Taiwan, anti-HCV (since 1992) and 8-sample mini-pools triplex nucleic acid test of HCV, HBV and HIV (since 2013) have been used in the routine blood screening.
Prevalence of anti-HCV and HCV RNA were estimated in the first-time donors during 1999-2016 and 2013-2016, respectively.
Age-standardized prevalence and its 95% confidence interval (95% CI) were calculated with adjustment of WHO world standard population 2000-2025.
For the incidence study, donors who have donated blood two or more times during 2013-2016 and who were without a history of anti-HCV positive before the follow-up period were included.
The incidence and its 95% confidence interval was estimated from the number of new HCV RNA positive cases divided by the person-years of follow-up.
Results/Finding: The crude prevalence of anti-HCV in the first-time donors was dramatically decreased from 15.2 per 1,000 donors (95% CI: 14.8-15.7) to 4.0 per 1,000 (95% CI: 3.7-4.3) during 1999-2016, and the agestandardized prevalence was also decreased from 27.0 per 1,000 donors (95% CI: 25.6-28.4) to 7.7 per 1,000 (95% CI: 6.9-8.5).
The agestandardized prevalence of anti-HCV was generally higher in female donors before 2015, but it was significantly higher in male donors at 2016 (p-value50.03).
A total of 1,036 HCV RNA positive cases, 1.9% of them were anti-HCV negative, identified from 579,286 first-time donors during 2013-2016, and the crude and age-standardized prevalence of HCV RNA was 1.8 per 1,000 (95% CI: 1.7-1.9) and 5.0 per 1,000 (95% CI: 4.3-5.7), respectively.
Crude prevalence of HCV RNA was significantly higher in female donors (p value <0.0001), but no significant difference was found after age standardization (p value50.93).
Both the prevalence of anti-HCV and HCV RNA were increased with age (p for trend<0.0001).
In the incidence study, a total of 68 new HCV RNA positive cases, 23.5% of them were anti-HCV negative, found from 1,202,165 donors followed for 2,415,668 person-years.
The incidence of HCV RNA was 2.8 per 100,000 person-years (95% CI: 2.2-3.5), and no significant difference was observed between both genders (p-value50.41) and between age groups (p for trend 0.37).
Conclusion: The prevalence of HCV infection has been dramatically decreased by 71.5% during 1999-2013.
It becomes significantly higher in male donors and that needs to monitor in the future.
Incidence of HCV RNA is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics.
Dalia Ashour* 1 , Sahar Muhmmad 2 and Dalia El Dewi 2 .
1 National Blood Transfusion Services, 2 Azhar University Background/Case Studies: Blood safety is a challenge in Egypt because of the high prevalence of HCV and HBV.
Nucleic acid amplification test (NAT) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion.
The primary benefit of NAT is the ability to reduce residual risk of infectious WP donations.
The estimated reduction of the WP utilizing NAT for HCV is 70-12 days, HIV from 22 to 11 days, and HBV from 25-30 days.
Study Design/Method: This cross sectional study was conducted in National blood Transfusion Center (Giza, Egypt) from 2012 to 2015, The total number of donor samples to be screened is 178685, The age of the donors ranged from 18 to 50 years, and they were of both sexes (M: F 5 3:1).Screening by NAT Ulterio assay (Grifols Diagnostics; formerly Novartis Diagnostics) was done in parallel with EIA testing for HBsAg, HCV-Ab and HIV Ag/ Ab.
using individual donation NAT (ID-NAT).
Multiplex NAT yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample.
Statistical analysis Chi-square (v2) test was used to measure the association between two qualitative variables.
Results/Finding: NAT screening detected a total of 75 NAT yield donations among 178685 (0.04%) seronegative donors.
Among these 75 NAT yields cases, 53 (0.03%) were reactive for HBV, 20 (0.011%) were reactive for HCV and 2 (0.001%) were reactive for HIV-1.
We stratified the age of the donors into 3 groups; group A (18 -28 years), group B (29 -39 years) and group C (40 -50 years).
The prevalence of NAT yield to the three viruses was significantly higher in either group B or C, compared to group A (p 5 0.0089; with 95% confidence interval (CI) 5 0.0085 -0.0520 & p 5 0.0247; with 95% CI 5 0.0025 -0.0534 respectively).
Prevalence of NAT-HBV; was significantly higher in age group B, as compared with group A (p 5 0.0224; with 95% CI 5 0.0032 -0.0413).
On the other hand, there was no statistically significant difference between groups C and A and between groups B and C. Comparing groups B and C combined with group A found a significantly higher prevalence of HBV in the former (p 5 0.0335; with 95% CI 5 0.0015 -0.0352).
NAT-HCV; did not differ significantly between the three groups (p 5 0.3222; with 95% CI 5-0.0089 to 0.0161 between groups A and B & p 5 0.1340; with 95% CI 5 -0.0055 to 0.0270 between groups A and C & p 5 0.4277; with 95% CI 5 -0.0080 to 0.0215 between groups B and C).
NAT-HIV; did not also differ significantly between the three groups (p 5 0.3801; with 95% CI 5-0.0077 to 0.0077 between groups A and B & p 5 0.3172; with 95% CI 5 -0.0056 to 0.0077 between groups B and C).
In either group A and C, no NAT-HIV detected.
NAT yield to the three viruses was significantly higher in males than in females (p 5 0.0013; with 95% CI 5 0.0136 to 0.0507).
NAT HBV was significantly higher in males (p 5 0.002; with 95% CI5 0.0103 -0.0413), but the prevalence of either HCV or HIV did not differ significantly between males and females (p 5 0.3835; with 95% CI 5 -0.0077 -0.0145 & p 5 0.2751; with 95% CI 5 -0.0044 -0.0066; respectively).
Conclusion: In this study The NAT yield of 75 in 178685 assumes more significance when one considers the fact that single donation is used for generating 3 components that can be used by 3 recipients.
Hence, in effect the NAT yield becomes 3 times that is, 225 in 178685.
Saving 225 recipients from TTI out of 178685 (0.13%) is indeed very significant.
Results/Finding: Of the 303,569 donors who were tested by our donor center, 1,386 (0.460%) were repeat reactive.
A seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring.
There was a single case of transfusion transmitted babesiosis reported from our center during this period.
A patient who was transfused with two units of packed red blood cells (RBCs) from two donors in the beginning of July presented in August for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis.
The donors were called back, however one of them could not be tracked.
Samples were sent to the state for further testing: An Immunofluoresence assay was performed (combination of IgG, IgM and IgA).
The test was positive at 1:128 titer.
The screening ELIA S/CO of this donor was 0.2782.
Both donors were indefinitely deferred as blood donors.
Conclusion: Our data confirm a decreased risk in transfusion transmission with the use of a screening assay.
Prior to implementation of the screening there were 6-10 transfusion transmitted babesia cases per year from 2008-2015 (Table 2 ).
In the 11 months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed (1 in 303,569 donors tested).
Thus the babesia EIA screening test effectively prevents TTB.
However, there was a substantial loss of donors due to being screen positive.
Four Years of Experience with ID-NAT at a Tertiary Care Centre in North India: Implications for Transfusion Transmission and Donor Screening.
Jasmeet Singh*, Amarjit Kaur, Gurpreet Kaur, Rajesh Kumar and Sonia Gupta.
Dayanand Medical College and Hospital Background/Case Studies: Transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe.
Blood safety is a formidable task especially in a high population country like India.
Newer technologies like ID-NAT equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods.
This study aims at examining the effect of ID-NAT as an additional test on the safety of blood supply.
Study Design/Method: A retrospective observational study was conducted to analyze the data of 4 years of additional NAT testing at Blood Bank, DMCH, Ludhiana from September 2012 to December 2016.
Results/Finding: Results 1.73% (2041 of 118021) units were initially NAT reactive.
These units were further tested, of which 90.98% were discriminated (70 HIV, 1051 HCV, 726 HBV and 10 co-infections).
The remaining 6.71% (137) were repeat non-reactive and 1.91% (39) could not be discriminated.
Overall, NAT yield rate was one in 837, whereas virus-specific NAT yield rates were one in 59,010 for HIV, one in 1873 for HCV, one in 1639 for HBV and one in 29,505 for HBV/HCV Coinfections, respectively.
Conclusion: ID-NAT screening of all blood donations at our institution over past 4 years has increased the screening sensitivities to check viral load and prevented transmission of 141 probable transfusion transmitted viral infections.
Assuming 100 % component preparation it saved 423 transfusion recipients from harm.
Implementation of NAT along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers.
Min Xu 1,2 , Wei Mao 3 , Tao He 3 , Yashan Yang 1,2 , Zhan Gao 1,2 , Chunhong Zhang 3 , Hongmei Liao 3 , Jingxing Wang 1,2 and Miao He* 1,2 .
1 Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, 2 Sichuan Blood Safety and Blood Substitute International Science and Technology Cooperation Base, 3 Chongqing Blood Center Background/Case Studies: Many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients.
With more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations.
Therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety.
Study Design/Method: Pooled plasma sample were collected from 5,000 voluntary blood donors from Chongqing, China.
Total DNA and RNA were extracted and amplified with random primers PCR respectively in order to construct a 250PE library to peform deep sequencing by Illumina Miseq.
All reads were trimmed to remove low quality bases and adapter sequences.
The fully overlapping paired-end reads passing the quality filter were concatenated using PEAR.
We classified the final reads using Kraken and a Kraken database made from complete RefSeq bacterial, archaeal and viral genomes, along with the GRCh37 human genome.
The unclassified reads by Kraken were aligned to NCBI nt database using BLASTn with cut-off Evalue as 1E -5 .
The best alignment hits were used to classify the reads.
Krona was used to generate all taxonomic distribution plots.
Finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience.
ABSTRACT Results/Finding: 1.23 GB raw data with 2,450,046 reads were generated in the DNA library.
Meanwhile, 1.98GB raw data with 3,967,242 reads were generated in the RNA library.
After cleaning the human background, 211 reads from bacteria, 98 reads from viruses, and 341 reads from parasites were identified (Table 1) .
No hazardous viruses were identified as potential threats to blood safety.
Except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as Plasmodium sp.
and Leishmania infantum (Table 1) .
Conclusion: The investigation has revealed the metagenomics of the qualified blood donations in Chongqing, China.
The results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety.
The displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety.
However, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens.
The validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens.
Background/Case Studies: The Caribbean has become an endemic region for several emerging viruses in the last decade.
After a Chikungunya outbreak in 2015 most recently Zika was shown to be endemic on the Caribbean island of Curacao.
To effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply.
Pathogen reduction (PR) is considered an important new approach with potential benefits.
The introduction and experience of use of PR platelets in the Dutch Caribbean over a period of one year is presented.
Study Design/Method: Pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (Mirasol PRT, Terumo, Belgium) was introduced.
All thrombocyte concentrates provided to the general hospitals on the Dutch Caribbean islands of Curaçao, Bonaire and Sint Maarten were PR and data collected over the period of 1 February 2016 to 1 February 2017.
Thrombocyte concentrates are prepared out of 4 single donation units by the buffycoat method.
Results/Finding: Over the period 260 platelet concentrates were provided to adult and pediatric patients.
These included patients on the intensive care and neonatal intensive care departments.
No adverse events were reported and the CCI for each transfusion was within the expected outcome.
Introduction of PR had minimal impact on the logistics of thrombocyte concentrate preparation and availability.
Furthermore no transfusion related bacterial contaminations were reported.
Conclusion: PR of platelet concentrates seems viable and safe for use in a small scale Caribbean setting with endemicity for emerging viruses like Chikungunya and Zika.
It offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability.
Michael Phillips* 1 , Germ an Leparc 2 , Phillip C Williamson 1 , Lani Palmer 1 , Ben Reynolds 1 , Maria Noedel 1 and Lindsey Houghton 1 .
1 Creative Testing Solutions, 2 OneBlood Background/Case Studies: Due to the risk of travel and sexually transmitted Zika infections, the Food and Drug Administration issued a guidance document on February 16, 2016 recommending that blood centers in Puerto Rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by March 1, 2016.
With the high incidence of Zika virus (ZIKV) in Puerto Rico and uncertainty of the impact to the continental U.S. blood supply, there was intense pressure to implement a donor screening test for ZIKV.
The project was initiated on February 16, 2016 and included clinical trial requirements, client onboarding and laboratory operations.
Stakeholders consisted of clients, the manufacturer, Institutional Review Boards (IRB), Informational Technology (client and lab based), the Food and Drug Administration (FDA), the Centers for Disease Control CDC, and the Florida Department of Health.
Clinical trial requirements included development of instrument and assay validations, SOP creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling.
Client onboarding began with confidentiality agreements between the client and the sponsor.
A Zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities.
The complexity of the project increased when mosquito borne Zika transmission was identified in two counties in Florida.
This required ZIKV testing to be performed on collections in both Florida and Puerto Rico.
The ZIKV-NAT is performed in singlet, unlike the MPX and WNV assays which are run in minipools.
This had a significant impact on instrument capacity.
Despite these obstacles and the changing regulatory requirements, the ZIKV screening test was implemented within six weeks.
Study Design/Method: One metric used to measure client service levels is our ability to meet established upload time goals for individual clients.
The percentage of samples released on time is evaluated daily with a running monthly total.
Our upload time goals were negatively impacted from July through September due to the unexpected increase in ZIKV testing, the requirement to perform testing in singlets and the resulting instrument capacity issues.
Additional instruments were sourced in October and operations stabilized.
Conclusion: On February 16, 2016, the project to implement a ZIKV IND test was initiated.
Six weeks later, testing was performed on the first batch of samples.
Despite the changing regulatory requirements over time, the implementation was extremely successful.
Initiating a new IND testing within 6 weeks is unprecedented and required exceptional collaboration between all participants and stakeholders.
Background/Case Studies: Plasmodium falciparum (Pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in Africa.
In 2015, WHO reported $212 million new cases worldwide, resulting in >400,000 deaths.
Malaria prevalence is highest in sub-Saharan Africa, home to 90% of all infections accounting for 92% of mortalities.
Both the incidence and prevalence of malaria in Africa significantly increase the potential for transfusion-transmission (TT), with little to no screening of products in developing countries.
The objective of this study was to evaluate the inactivation of Pf in Whole Blood (WB) using a system specifically developed for the realities of the developing world and in support of the Swiss Red Cross Humanitarian Foundation for Whole Blood Pathogen Inactivation for Africa.
The inability to consistently supply blood components leads to routine WB transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust WB pathogen inactivation system is desirable.
The approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication.
The process includes addition of 0.2 mM amustaline and 2 mM glutathione (GSH) and a 24h at room temperature (RT) incubation after which the treated WB unit is suitable for storage up to 7 days at RT.
Study Design/Method: For each experiment, a WB unit was spiked with ring-stage Pf-infected red blood cells (iRBC).
A pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed 24h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation.
These samples were serially diluted in flasks containing medium with 5% fresh RBCs.
The diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting iRBC in blood smears and by flow cytometry.
Pretreatment cultures were terminated after reaching >1% parasitemia, while no residual Pf was detected in post-treatment cultures.
Log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples.
Results/Finding: Robust inactivation of Pf in WB was achieved to the limit of detection, at >7.5 log 10 or >6.0 log 10 /mL.
Conclusion: Pf was inactivated to the limit of detection in WB after treatment with amustaline/GSH, illustrating that the system has potential to mitigate the risk for Pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on WB transfusion.
(This system for WB is not approved for commercial use).
Increased Patient Safety and Improved Inventory Management with 7 Day Apheresis Platelets Nancy M. Dunbar* and Zbigniew M. Szczepiorkowski.
Background/Case Studies: A pathway currently exists for apheresis platelet (AP) outdate extension from 5 to 7 days using an FDA cleared rapid test (RT).
In February 2016, our hospital based transfusion service implemented the use of RT on day 5, 6 and 7 to routinely extend AP shelf life to 7 days.
Prior to this, we tested APs by RT on day 4 and transfused day 6 or day 7 units with physician approval when deemed medically necessary.
This report describes changes observed in transfusion practice and platelet inventory management one year following routine use of 7 day platelets.
Study Design/Methods: Data were obtained for two 12-month study periods: October 2014-September 2015 (pre-implementation) and February 2016-January 2017 (post-implementation).
The interval transition period was intentionally excluded.
For each study period, we determined the total number of APs transfused, RT status on the day of transfusion, total number of RTs performed, expired AP units, and APs obtained from suppliers using ad-hoc ordering.
We also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index.
Results/Findings: Data are shown in Table 1 .
The number of AP transfusions increased by 7% post-implementation, comparable to a 4% increase in inpatient admissions and an 11% increase in surgical volumes.
The hospital length of stay and case mix index were similar for both periods.
The average number of platelet transfusions per patient was not statistically different (3.16 pre; 3.12 post, p50.91).
The number of RTs performed increased by 130%.
The percentage of transfused units tested at least once by RT prior to transfusion increased by 21% (p<0.0001).
The outdate rate decreased from 5% to 2% (p<0.0001).
Ad-hoc ordering decreased from 21% to 9% (p<0.0001).
Conclusion: Use of an approved RT for routine AP outdate extension to day 7 was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion.
Increased cost of RT was offset by reduced AP waste and less frequent need for ad-hoc ordering.
Sheila O'Brien* 1 , Vito Scalia 1 , Carla Osiowy 2 , Michael Carpenter 2 , Anton Andonov 2 and Margaret Fearon 1 .
1 Canadian Blood Services, 2 Public Health Agency of Canada Background/Case Studies: The rates of hepatitis B (HBV) and hepatitis C virus (HCV) positive donations are low (6.6 and 4.9 per 100,000 donations, respectively) and most are among first time donors.
We aimed to determine the frequency of various genotypes of HBV and HCV in Canadian blood donors confirmed positive for HBV and HCV.
Study Design/Methods: In 2011 the Roche multiplex assay (HCV/HIV/ HBV) was implemented in minipools of 6 units.
HCV NAT was in place since 1999 (using minipools of 24) but this is the first time donors have been screened by HBV NAT.
HBsAg, anti-HBc and anti-HCV were tested using the Abbott PRISM assay.
Confirmatory testing for HBsAg was by the PRISM neutralization assay.
Anti-HCV repeat reactivity was confirmed by the Inno-Lia HCV Score Line Immunoassay.
Since March 2016 all samples testing HBV NAT positive, or confirmed positive for HBsAg and all HCV NAT positive or anti-HCV confirmed positive samples were considered positive and samples were sent to PHAC for sequencing.
A sample from each positive donation was aliquoted and frozen at -20 o C. Genotyping was carried out by sequence and phylogenetic analysis of the HBV surface antigen coding region.
HCV viral RNA was extracted and subjected to reverse transcription and PCR amplification in the 5' NTR-E1 and NS5B regions.
Sanger sequencing of these regions represents approximately 15% of the genome.
Results/Findings: All confirmed positive donations were whole blood donations.
There were 42 HBV positive donations.
Of these, 37 had tested HBV NAT positive.
Genotypes were 8 type A, 6 B, 4 C, 17 D and 2 E. There were 5 samples HBV NAT negative but HBsAg positive (2 were anti-HBc reactive).
Of these, 4 could not be sequenced and one was genotype A (also anti-HBc reactive).
There were 30 samples considered HCV positive.
Of these, 17 samples were HCV NAT positive.
Genotypes were 5 type 1a, 3 1b, 3 2c, 2 2b and 4 3a.
There were also 13 samples HCV NAT negative but anti-HCV positive.
None of these could be sequenced.
Conclusion: The first 8 months of molecular surveillance show a range of genotypes for HBV and HCV for samples identified as NAT positive.
To date no samples that were NAT negative anti-HCV reactive could be sequenced, however one NAT negative sample that was positive for HBsAg and anti-HBc reactive was HBV genotype A. Surveillance over a longer period is Background/Case Studies: The BacT/ALERT 3D Microbial Detection System (BTA 3D) is currently FDA cleared for the quality control testing of Leukocyte Reduced Apheresis Platelets (LRAPs).
The BacT/ALERT VIRTUO Microbial Detection System (VIRTUO) (bioM erieux, St. Louis, MO) is a new generation of BacT/ALERT instrumentation.
The underlying colorimetric technology used in previous generations of BacT/ALERT is used in the VIR-TUO and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection.
The objective of this study was to compare the performance of the VIRTUO and BacT/ALERT 3D (BTA 3D) instruments, using BacT/ALERT BPA (aerobic) and BacT/ALERT BPN (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (LRAPs).
* Study Design/Method: The study was performed at two institutions, one in the US and the other in the UK.
Aliquots of LRAPs were seeded with low levels (1-20 cfu/mL) of 11 bacterial species commonly associated with platelet contamination, and 20 replicates (10 per instrument) of 4 mL aliquots per bottle were inoculated into BPA and BPN bottles.
One set of bottles was loaded into BTA 3D and the other into VIRTUO and incubated until signaled positive by the instruments or for up to 7 days.
Overall detection rates and time to detection of bacterial contaminants between instruments were compared.
Additionally 98 bottles were tested in each instrument (LRAPs only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations.
Background/Case Studies: The implementation of Nucleic Acid Testing (NAT) blood screening is still a challenge in resource-limited countries.
At the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited.
A higher prevalence of infections is observed in relation to developed countries.
As a consequence, more incident cases of infections can be expected.
In our country, some hospital blood banks could not afford NAT due to high costs, but belong to a net that centralizes NAT in a reference Blood Center.
The process to consolidate small blood banks in Regional Blood Centers, which will be able to implement NAT, is not yet complete.
Although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved.
The aims were to compare the prevalence of HIV, HCV and HBV by NAT screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the NAT yield rates for HIV, HCV and HBV in a period of three and a half year experience.
Study Design/Method: A Regional Blood Donor Center (RBDC) has centralized NAT screening from centers in different regions of the country due to since August 2013.
This process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results.
When a window period was suspected, the NAT screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm NAT results.
This RBDC have also been developed a 100% Voluntary Donor Program since 2011 and is the only center in the country that has achieved this goal.
Results/Findings: A total of 264,343blood donations were studied from August 2013 to December 2016.
In the RBDC, where only voluntary blood donations are recruited, the prevalence was 18 per 100,000 donations for HIV (IC95% 8-34:100,000); 14 per 100,000 for HBV (IC95% 7-29:100,000) and 18 per 100,000 for HCV (IC95% 8-34:100,000).
In all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was 89 per 100,000 donations for HIV (IC95% 77-103:100,000); 70 per 100,000 for HBV (IC95% 59-83:100,000) and 78 per 100,000 for HCV (IC95% 66-91:100,000).
Window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving NAT yield rates of 1: 66,086 for HBV; 1: 132,172 for HIV and 1: 264,343 for HCV.
Conclusion: The HIV, HBV and HCV prevalence was lower in a center where the tasks to sustain a Voluntary Blood Donor program were developed.
NAT yield rates could be reduced in the region if this program could completely be applied in all centers.
Mechanisms leading to OBI include various factors such as imperfect host's immune response and viral variation factors.
This study was to determine the viral loads of OBI under currently recruitment and screening among blood donors in five Blood services of Zhejiang Province, China.
Study Design/Method: Before donation, the donors were screened and precluded with HBsAg preliminary test positive and ALT level abnormal.
Following, the samples were detected for HBsAg twice using different ELISA reagents and HBV DNA using TMA or QT-PCR techniques.
Then, the samples with HBV DNA positive and ELISA negative were tested for the viral loads using TaqMan technique in COBAS S201 system.
HBV S region was also sequenced.
Results/Finding: 234 OBI were found in the 230,000 donations.
In the viral loads assay, 43 samples were negative and 104 samples' viral loads were lower 20IU/ml.
The mean viral loads was 1.85 6 0.41 (log10) IU/ml in other 87 samples,while the mean viral loads with HBsAg1/HBV DNA1 samples was 2.38 6 0.83 (log10) IU/ml.
60 samples of OBIs have analyzed the HBV genotype, which B was the most prevalent subtype (69.0%) and the other was HBV C genotype(31.0%).
Compared the samples with HBsAg1/HBV DNA1 ,We found two OBI samples carrying with 318T>C mutation, which could cause an amino acid S55F.
Conclusion: In this study, the viral loads of OBI infection in donors was much low than HBsAg1/HBV DNA1, and some unique variation was identified in the OBI individuals.
195A TRANSFUSION in general population.
Screening of blood donors for HBV in India is primarily based upon detection of Hepatitis B surface antigen (HBsAg) in donor's sera.
The current study was undertaken to determine the prevalence of occult HBV infection (OBI) in voluntary blood donors and to analyze the burden of HBV window period donations.
Study Design/Method: This is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (NABH) accredited apex blood bank, located in Maharashtra state, India.
Monolisa HBsAg ULTRA (Bio-Rad, France)sandwich type ELISA using monoclonal and polyclonal antibodies was used for HBsAg detection in donor's sera.
All the ELISA non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (MPX-PCR) by cobasV R TaqScreen MPX test.
The donors which were found to be positive for HBV DNA were followed upat 15 th days, 1month, 3 months& 6 months by Monolisa HBsAg ULTRA (Bio-Rad, France) to analyze interval of window period and to delineate the window period donations (WPD) & true OBI.
Background/Case Studies: Occult hepatitis B infection (OBI) is characterized by hepatitis B virus (HBV) DNA-positive, but HBV surface antigen (HBsAg) -negative.
Since May 2015, we have been testing apheresis donors for HBV nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection.
The number of apheresis collections increased significantly year by year, however, data on hepatitis B virus marker rates among these donors continue to be lacking.
The aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of OBI among apheresis donors in a region of central China.
Study Design/Method: Apheresis donors' data from May 2015 to Dec 2016 was retrospectively analyzed.
All samples were tested for HBsAg, HBV DNA, and other markers.
Nucleic acids testing (NAT) was performed on the Roche cobas s201 platform using pools of 6 serologically negative samples and any pools positive would undergo NAT again individually.
HBsAg negative, but HBV DNA positive were further tested for HBV DNA quantitative PCR, antibody to hepatitis B surface antigen (HBsAb), antibody to hepatitis B core antigen (HBcAb), hepatitis B e antigen (HBeAg) and antibody to hepatitis B e (HBeAb).
Results/Finding: In the evaluation, 68547 seronegative donations were screened by NAT and a total of 20 HBV DNA-reactive/HBsAg-negative donors were detected.
No HIV RNA -reactive or HCV RNA -reactive sample was detected.
Complete serologic screening of the index donations indicated that the majority of these donors had an occult HBV infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations.
Age distribution of the age group 31-55 years old showed a large proportion, who accounted for 80% of reported infections.
Most of the HBV DNA cases (about 80.0%) reached senior high school education.
The average HBsAg DNA positive rate was 0.029% (20/ 68547).
Incidence among apheresis donors in this period for HBsAg DNA were 2.91/10000.
These estimates were comparable to those among repeat whole blood donors.
We developed pathogen reduced (PR) cryo derived from FFP and PF24 with 5 day stability at 228C.
Study Design/Method: Six replicates of type-matched pools of whole blood derived (WBD) and Apheresis (Aph) plasma were split to produce conventional control (225 610 mL) and test components (625 mL 625 mL).
Test components were PR with amotosalen and UVA light.
Aph and WBD FFP were produced by freezing plasma within 8 hr and WBD PF24 within 24 hr.
Cryo was manufactured according to site SOPs and frozen at -308C (Test 62 6 2 mL, Control 22 6 2 mL ).
Test and Control Cryos were thawed at 378C, and characterized immediately post thaw (t50), and after 5 d storage at 228C and tested for FB and FVIII function, thromboelastography (ROTEM) and thrombin generation (CAT).
Results/Finding: PR cryo retained sufficient FB and FVIII activity post thaw and over 5d at 228C (Table) for hemostatic capacity.
ROTEM (EXTEM) showed retention of fibrin formation (a angle) and clot quality (MCF) (Table) .
Thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (ETP), and time to peak (tt) despite lower FVIII levels.
These parameters were maintained through 5d storage at 228C.
Conclusion: PR cryo can be processed from 3 plasma sources, including PF24, and stored at RT for 5 days.
PR plasma provides adequate levels of FB with hemostatic capacity equivalent to control as demonstrated by ROTEM and CAT.
Use of PF24 with stability over 5 days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection.
Cryo produced with psoralen-treated (PR) plasma is not approved for use in the US.
Performance of a New Automated Alinity s Assay for Antibodies to T. Cruzi Darwin Smith* 1 , Ed Bakker 2 , Anton van Weert 2 , Jane Bryant 1 , Mark Paradowski 1 , Lynne Fleischmann 1 , Mirjana Sarac 3 , George Chen 1 , George Schlauder 1 and Gregg Williams 1 .
1 Abbott Diagnostics, 2 Sanquin Diagnostics, 3 Abbott GmbH & Co. KG Background/Case Studies: The parasite, Trypanosoma cruzi (T. cruzi), is the cause of Chagas disease which is endemic to the Americas and infects 6-8 million people.
In order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-T. cruzi assays with good specificity and sensitivity.
In nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
Study Design/Method: The performance of the new automated chemiluminescence immunoassay for the detection of antibodies to T. cruzi was evaluated on the Alinity s automated platform and compared to another onmarket chemiluminescent immunoassay.
Precision was assessed over 20 days using a panel of positive and negative samples.
Sensitivity was evaluated on 407 presumed antibody positive specimens and specificity was evaluated on 7621 random blood donor samples.
Results/Finding: Precision was 7% CV or less for positive samples over 20 days.
The overall specificity in a blood donor population was 99.99% (7620/ 7621).
Sensitivity was 100.00% for 407 presumed antibody positive samples.
Conclusion: These results indicated that the new automated Alinity s Chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-T. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries.
Performance of a New Automated Alinity s Assay for Hepatitis B Surface Antigen and Hepatitis B Surface Antigen Confirmatory Randal Makela* 1 , Anton vanWeert 2 , Ed Bakker 2 , Jane Bryant 1 , Mark Paradowski 1 , Lynn Martin 1 , Daniela Kaleve 3 , George Chen 1 , Gregg Williams 1 and George Schlauder 4 .
1 Abbott Laboratories, 2 Sanguin Diagnostics, 3 Abbott GmbH & Co. KG, 4 Abbott Diagnostics Background/Case Studies: Despite the development of sensitive NAT methods, blood transfusion in many parts of the world relies on serologic screening for Hepatitis B surface antigen (HBsAg) to prevent transfusion transmitted HBV infection.
Sensitive HBsAg assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
Study Design/Method: The performance of a new automated chemiluminescence immunoassay for the detection and confirmation of HBsAg was evaluated on a next generation automated platform, Abbott Alinity s. Precision was assessed over 20 days.
Sensitivity was evaluated using 511 known positive samples, 30 commercially available seroconversion panels, the WHO standard, 23 HBsAg mutants, and 94 HBsAg genotyped specimens (A through H).
Specificity was evaluated on random blood and plasmapheresis donors.
Results/Finding: Precision was less than 8% CV for positive samples over 20 days.
The blood donor specificity was 99.98% (7998/8000).
Sensitivity was 100% for 511 presumed positive samples.
Sensitivity was 100% for all genotypes.
100% of the mutants were detected vs 83% for the comparator assay.
Seroconversion detection was equivalent to the comparator assay with 157 reactive samples detected with the Alinity s assay and 154 reactive samples detected by the comparator assay.
Analytical sensitivity ranged from 0.015 to 0.016 IU/ml.
The Alinity s HBsAg Confirmatory Assay confirmed all known positive HBsAg specimens, including 3 HBsAg mutant samples that were not confirmed by the comparator HBsAg Confirmatory Assay.
Conclusion: The new automated Alinity s HBsAg assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay.
However, the Alinity s HBsAg assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants.
Performance of a New Automated Alinity s Immunoassay Assay for HIV Darwin Smith* 1 , Ed Bakker 2 , Anton van Weert 2 , Jane Bryant 1 , Mark Paradowski 1 , Kevin Callear 1 , Susan Sullivan 1 , George Chen 1 , George Schlauder 1 and Gregg Williams 1 .
1 Abbott Diagnostics, 2 Sanquin Diagnostics Background/Case Studies: Blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus Types 1 and 2 (anti-HIV-1/2).
Blood centers require very high throughput anti-HIV-1/2 assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
In the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-HIV-1/2 antibodies and HIV-1 p24 antigen.
Study Design/Method: The performance of the new chemiluminescence combination immunoassay for the detection of anti-HIV-1/2 antibodies and HIV-1 p24 antigen was evaluated on the Abbott Alinity s System.
Precision was assessed over 20 days evaluating positive samples.
Specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors.
Sensitivity was evaluated using presumed positive samples for HIV-1, HIV-2 and HIV Group O antibodies and HIV-1 p24 antigen.
Seroconversion sensitivity was evaluated with 41 commercial seroconversion panels.
Results/Finding: Precision was less than 8% CV for positive samples over 20 days.
The blood donor specificity was 99.96% (8082/8085).
Sensitivity was 100% for 813 presumed antibody positive samples comprised of HIV-1, HIV-2 and HIV-1 Groups O, N, P, CRF and URF samples.
Also, sensitivity was 100% for 102 antigen positive viral isolate samples comprised of HIV-1, HIV-2 and HIV-1 Groups O, N, P, CRF and URF samples.
Seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the Alinity s assay and 135 reactive samples detected by the comparator assay.
Conclusion: These results indicate that the new automated Alinity s HIV Ag/Ab Combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay.
Performance of a New Automated Alinity s Immunoassay for the Detection of Anti-HBc Antibodies Randal Makela* 1 , Anton vanWeert 2 , Ed Bakker 2 , Jane Bryant 1 , Mark Paradowski 1 , Joyce Siregar 1 , Angela Vockel 3 , George Chen 1 , Gregg Williams 1 and George Schlauder 4 .
1 Abbott Laboratories, 2 Sanguin Diagnostics, 3 Abbott GmbH & Co. KG, 4 Abbott Diagnostics Background/Case Studies: In countries with a low prevalence of Hepatitis B, blood donations are commonly screened to detect the presence of antibodies to hepatitis B core antigen (Anti-HBc) alongside HBsAg and HBV NAT to detect donors with occult Hepatitis B infections (OBI).
Blood centers require anti-HBc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
In the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-HBc on the Alinity s System.
Study Design/Method: The performance of a new chemiluminescence anti-HBc assay for the detection of anti-HBc antibodies was evaluated on the next generation automated Abbott Alinity s System.
Precision was assessed over 20 days evaluating positive samples.
Specificity was evaluated on samples obtained from random blood donors.
Sensitivity was evaluated using specimens characterized as anti-HBc positive by means of serologic methods.
Analytical sensitivity was assessed using the WHO 1st International standard.
Seroconversion sensitivity was evaluated using 10 commercial seroconversion panels.
Results/Finding: Precision was less than 6% CV for positive samples over 20 days.
The blood donor specificity was 99.93% (6946/6951).
Sensitivity was 100% for 500 samples presumed to be anti-HBc positive.
Analytical sensitivity results on the Alinity s Anti-HBc assay ranged from 0.57 to 0.62 IU/mL.
Seroconversion detection was equivalent to the comparator assay with 136 reactive samples detected with the Alinity s assay and 134 reactive samples detected by the comparator assay.
Conclusion: These results indicate that the new automated Alinity s Anti-HBc assay provided good performance in specificity, sensitivity and precision versus the comparator assay.
Performance of a New Automated Alinity s Immunoassay for the Detection of HTLV I and HTLV II Antibodies Melanie Anderson* 1 , Anton vanWeert 2 , Ed Bakker 2 , Mark Paradowski 1 , Jane Bryant 1 , Tuan Bui 1 , Joyce Siregar 1 , George Chen 1 , George Schlauder 3 and Gregg Williams 1 .
1 Abbott Laboratories, 2 Sanguin Diagnostics, 3 Abbott Diagnostics Background/Case Studies: In endemic countries, universal blood screening is necessary to prevent transfusion transmitted HTLV infections (anti-HTLV I/HTLV II).
In non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high HTLV prevalence.
Blood centers require high throughput anti-HTLV I/HTLV II assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
In response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against HTLV-I/II antibodies for the Alinity s System.
Study Design/Method: Precision was assessed over 20 days using HTLV I and HTLV II positive samples.
Specificity was evaluated using 8,001 blood donor specimens from Europe and 200 diagnostic samples obtained from the United States.
Sensitivity was evaluated using 500 preselected HTLV I and HTLV II positive samples.
Sensitivity and specificity samples were split across 3 reagent lots during testing.
Confirmation of repeatedly reactive samples was done using the MP Diagnostic HTLV Blot 2.4.
Results/Finding: Imprecision was less than 7.0% for positive samples over 20 days.
Clinical sensitivity was 100.00% (500/500) on preselected HTLV I and HTLV II positive samples.
The specificity was 99.98% (7,999/8,001) on a blood donor population and 100.00% (200/200) on diagnostic samples.
Conclusion: These results indicate that the new Alinity s automated HTLV I/ II assay provided very good performance in specificity, sensitivity, and precision.
Sensitivity and specificity were comparable to the comparator assay.
Claudia Ramirez 1 , Michel Garcia* 2 , Fernando Palomino 3 and Guillermo Orjuela-Falla 1 .
1 National Blood Bank Colombian Red Cross, 2 Universidad Del Rosario, 3 FUATS Background/Case Studies: Current hepatitis C virus (HCV) supplemental testing algorithm for blood donations in Colombia, requires that an immunoblot assay be performed on every HCV enzyme immunoassay (EIA) repeatreactive sample.
A higher proportion of indeterminate (IND) results by immunoblot assays has been documented for non-US donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors.
This work aimed to establish the distribution of immunoblot results in Colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots.
Study Design/Method: In total, 387 anti-HCV-reactive donor samples (signal-to-cutoff (S/CO) ratio greater than 1.0; Abbott Architect i2000SR) underwent supplemental testing by immunoblot (either Chiron RIBA HCV 3.0 SIA or HCV Blot 3.0 test, MP Diagnostics).
Negative (NEG), indeterminate (IND) and positive (POS) blot results were grouped by S/CO ranges as follows: 1-4.99, 5-9.99, >10.
Band detection and intensity were independently analyzed for indeterminate and positive results.
Results/Finding: Immunoblot results were negative in 57.9% (224/387) of samples, indeterminate in 30.7% (119/387) and were positive in 11.4% (44/ 387).
A direct relationship was observed between positive immunoblot and increased S/CO.
The proportion of IND results were higher in the S/CO group 5-9.99 (63.2%) compared with the 1-4.99 (29.9%).
In samples with indeterminate results, NS3_2 was the most frequent band detected (52,9%).
In contrast, the most frequent band in the group of positive results was CORE (93,2%).
Only one sample from the indeterminate group (0.8%) had a strong band intensity (31), compared with 10 samples from the positive group (22.7%).
Conclusion: The proportion of indeterminate immunoblot results in this sample of Colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of US donors.
The high proportion of IND results found in the S/CO group (5-9.99) suggests that the optimal S/CO ratio for predicting a confirmed anti-HCV result in this population should be higher than the one recommended by the CDC for US population (>5).
Overall, these results suggest that the supplemental testing algorithm for blood donations in Colombia could be improved not only by using high S/CO ratios as an alternative to immunoblot, but also by introducing HCV genomic assays instead of immunoblots, at least for samples with intermediate S/CO ratios.
NS3_1 and NS3_2 cross-reactivity in Colombian population warrants further investigation.
Performance of the Alinity s Immunoassay for the Detection of Syphilis Antibodies Melanie Anderson* 1 , Ivanka Mihaljevic 2 , Manuela Miletic 2 , Miljana Stojic Vidovic 2 , Irena Jukic 2 , Jane Bryant 1 , Mark Paradowski 1 , Angela Vockel 3 , George Chen 1 , Gregg Williams 1 and George Schlauder 4 .
1 Abbott Laboratories, 2 Croatian Institute of Transfusion Medicine, 3 Abbott GmbH & Co. KG, 4 Abbott Diagnostics Background/Case Studies: Blood donations are commonly screened for Syphilis in order to detect the presence of antibodies to the bacterium Treponema pallidum.
In addition, continued pressures on laboratory operations demand that the full panel of TTID assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
In response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to T. pallidum.
Study Design/Method: Performance of the new automated chemiluminescence immunoassay for the detection of antibodies to Treponema pallidum was evaluated on the Alinity s System.
Precision was assessed over 20 days using positive samples.
Specificity was evaluated on samples obtained from 9,101 blood and plasmapheresis donors from the United States and Europe and 200 diagnostic samples obtained from the United States.
Sensitivity was evaluated using 514 preselected positive samples.
Sensitivity and specificity samples were split across 3 reagent lots during testing.
Confirmation of repeatedly reactive samples was done using a testing algorithm with 3 confirmatory assays, INNO-LIA TM Syphilis Score, and Mikrogen recomLine Treponema IgG and IgM blots.
Results/Finding: Imprecision was less than 6.0% CV for positive samples over 20 days.
Clinical sensitivity was 100.00% (514/514) on preselected Syphilis positive samples.
The specificity was 99.97% (9,063/9,066) for blood donor specimens and 100.00% (200/200) on diagnostic samples.
Conclusion: These results indicate that the new automated Alinity s Syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay.
Performance of the New Automated Alinity s Assay for Anti-HCV Melanie Anderson* 1 , Ed Bakker 2 , Anton vanWeert 2 , Jane Bryant 1 , Mark Paradowski 1 , Tuan Bui 1 , Lynn Martin 1 , George Chen 1 , Gregg Williams 1 and George Schlauder 3 .
1 Abbott Laboratories, 2 Sanguin Diagnostics, 3 Background/Case Studies: Serological screening for antibodies to Hepatitis C virus (HCV) often in conjunction with nucleic acid testing (NAT) is used worldwide to prevent transfusion transmitted HCV infections.
While NAT provides improved sensitivity and detection of HCV in the pre-seroconversion window, serological testing provides continued detection of HCV in infected individuals and individuals with resolved infections with no detectable HCV RNA.
Blood and plasma centers require very high throughput anti-HCV assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply.
In addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management.
Study Design/Method: The performance of a new automated chemiluminescence immunoassay for the detection of antibodies to HCV was evaluated on the Alinity s System.
Precision was assessed over 20 days evaluating positive samples.
Sensitivity was evaluated using 501 preselected positive samples and 30 seroconversion panels.
Specificity was evaluated on samples obtained from 8,113 blood and plasmapheresis donors from the United States and Europe and 200 diagnostic samples obtained from the United States.
Sensitivity and specificity samples were split across 3 reagent lots during testing.
Confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the INNO-LIA TM HCV Score and NAT/HCV Discriminatory NAT assays.
Results/Finding: Imprecision was less than 7.0% CV for positive samples over 20 days.
Overall clinical sensitivity was 100% on 501 preselected anti-HCV positive samples.
Seroconversion sensitivity was better than the comparator as evidenced by the new Anti-HCV assay identifying 5 more bleeds than the comparator assay.
The specificity was 99.99% (8,111/8,112) for blood donor specimens and 98.
98% (194/196) Background/Case Studies: ZIKA virus (ZIKV), which has been outbroken in South America and the United States since middle of 2015, was declared as the public health emergency of international concern by WHO in Feb 2016.
In addition to mosquito, ZIKV can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion.
The potential for transfusion-transmitted Zika virus was shown in French Polynesia where 2.8% of asymptomatic blood donors tested were positive for Zika virus RNA using nucleic acid test (NAT).
Several case reports have confirmed that ZIKV can be transmitted by transfusion.
It has been shown that among blood donors, 73.8% of the ZIKV infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in Micronesia was approximately 1:5 to 1:6.
Thus ZIKV has raised a great challenge to transfusion safety.
Measures should be taken to prevent transfusion-transmitted ZIKV, including temporary deferral of blood donors in epidemic locations, donor self-reporting of ZIKV symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, NAT of blood donations, and pathogen inactivation of blood products.
In this study, we evaluated ZIKV inactivation in plasma by using methylene blue photochemical treatment (MBPT).
Study Design/Methods: Plasma units from randomly selected healthy donors were collected and spiked with ZIKV.
Samples were added by MB at a final concentration of 1lM and assayed after illumination with visible light from both sides for 5, 15, and 30min.
Viral infectivity and ZIKV RNA loads (reverse transcription PCR) were measured in spiked plasma before and after MBPT and confirmed using repetitive passages in cell culture.
Control was ZIKV spiked plasma without photochemical treatment.
Results/Findings: ZIKV titer of control sample was 4.5 log 50% tissue culture infectious dose (TCID 50 )/mL.
No viral infectivity was detected after MB photochemical inactivation treatment for 5min, 15min or 30min and the losses of the infectivity were further demonstrated by 3 repetitive passages of cell culture.
Meanwhile, ZIKV RNA loads decreased significantly during the initial 5min of treatment whereby Ct-value jumped from 18.25 (control) to 25.50 (MBPT for 5min) (Table 1) .
Conclusion: It showed that MB photochemical treatment could effectively inactivate ZIKV in plasma.
RNA lesions were induced during MBPT process so that nucleic acid reverse transcription and amplification were inhibited.
MBPT is proved to be an efficient method to prevent plasma transfusiontransmitted ZIKV infections.
Gilles Delage* 1 , Margaret Fearon 2 , Susan L Stramer 3 , Megan L Nguyen 3 , France Bernier 1 , Sheila O'Brien 2 , Vito Scalia 2 , Sakina Smith 3 , Yves Gr egoire 1 and Boris Hogema 4 .
1 H ema-Qu ebec, 2 Canadian Blood Services, 3 American Red Cross, 4 Sanquin Background/Case Studies: Hepatitis E virus (HEV) is known to be transfusion-transmissible.
As part of the risk assessment for this infection, a study was carried out in 14,000 Canadian blood donors in 2013.
In a subset of 4,000 donor samples the seroprevalence was 5.9%.
However, no donor samples were positive for HEV by an in-house nucleic acid test (HEV-NAT).
Since that study suggested exposure to HEV in Canada but used an HEV-NAT with a limit of detection of 250 IU/mL, a larger study was performed using a more sensitive HEV-NAT assay.
Study Design/Method: Donors were informed about the study in the predonation reading materials.
Linked samples from approximately 50,000 Canadian whole blood donors including 30,000 from Canadian Blood Services (CBS) and 20,000 from H ema-Qu ebec (HQ) were collected.
Clinics were selected to ensure representative sampling of the donor population.
All 199A TRANSFUSION 2017 Vol.
57 Supplement S3 donations with available plasma samples were tested by individual donation NAT at the American Red Cross laboratory in Gaithersburg, MD, using the cobas V R HEV test (95% LOD 18.6 IU/mL, 95% CI 15.9-25.6) for use on the cobas V R 6800/8800 System.
This test is not currently approved in Canada or the USA, but is available as a CE marked test.
All NAT-reactive donors are questioned concerning risk factors for recent HEV infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate NAT, viral load, genotyping and IgM/IgG serology), are notified by letter, and deferred from donating for 6 months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous 6 months are destroyed.
Recipients will be traced in the event of any products transfused in the previous 6 months.
Results/Finding: As of April 10, 2017, 9 of 39,834 (19,395 CBS, 20,439 HQ) tested samples with valid results have been found HEV-NAT reactive: 8 donors have been confirmed by further testing to date.
Confirmation is pending in 1 donor.
Of the 9 donors, 7 were from Quebec, and one each from Nova Scotia and Alberta (7 male, 2 female).
Ages ranged from 21 to 70 years.
Only two donors reported non-specific symptoms (fatigue).
In terms of risk factors: 6 ate pork (including 3 who ate pork liver), 4 ate shellfish, 2 ate venison, and 3 drank well water.
One donor had no identifiable risk factor.
Viral loads ranged from 3 to 151 IU/mL, of which 2 were <10, 3 were 10-50, and 3 were >50 IU/mL; 2 were anti-HEV IgM positive and 4 anti-HEV IgG positive at index (Wantai assay).
Conclusion: The prevalence rate of acute HEV infection in this donor population appears to be around 1/4400.
The data from this study will contribute to the ongoing risk assessment of transfusion-transmitted HEV infection in Canada.
Prevalence of Malaria Parasite in Donated Blood at Nakasero Blood BANK, Uganda Gerald Nsubuga* and Musiisi Ezra.
UGANDA BLOOD TRANSFUSION SERVICE Background/Case Studies: Introduction Infectivity of donated blood with malaria is a significant health problem facing humanity.
In Uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current Uganda National Blood Transfusion Service (UBTS) guidelines by the Ministry of Health.
As a result, the proportion of donated blood that is infected with malaria is largely unknown.
Malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about 300 to 500 million cases and 2 to 3 million death occur per year.
However the study aimed at determining the prevalence of malaria parasites in donated blood at Nakasero Blood bank, Kampala, Uganda Study Design/Method: A cross sectional study was carried out in Nakasero blood bank, Kampala, Uganda in four hundred and seventy randomly selected donor samples at the blood bank between June and August 2014.
Both thin and thick glass stained blood smears of 417 blood samples with Giemsa was examined using microscope.
Results/Finding: Of the 417 donated blood samples, 17 (4.1%) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of Plasmodium in relation to sex, age and blood group (P>0.05), majority of the blood donors that tested positive belonged to blood group O (64.71%).
The prevalence of malaria parasite in the study was 4.1%.
Regardless of the prevalence, the presence of malaria parasite (Plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in Uganda.
The Ministry of Health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia.
Using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities.
components.
The BacT/ALERT VIRTUO* (VIRTUO) is an advanced, next generation system with improved automation, connectivity, and with data management systems.
Most importantly, the VIRTUO's new algorithm significantly reduces the time to detection (TTD) of microorganisms during quality control testing of platelet preparations using BacT/ALERT BPA (aerobic) and BPN (anaerobic) bottles.
BPA and BPN bottles were tested on VIRTUO and BacT/ ALERT 3D (BTA 3D) to evaluate repeatability to detect growth in seeded Leukocyte Reduced Apheresis Platelets (LRAP) without Platelet additive Solution (PAS), throughout platelet shelf life (3, 4 and 5 days after collection).
Study Design/Method: Pooled LRAP were seeded with low levels of 6 organisms commonly associated with platelet contamination at 3, 4 and 5 days post collection.
The seeded LRAP were inoculated into BPA and BPN bottles on 10 different days (not consecutive) alternating between 2 teams of 2 people each.
Seeded bottles were loaded into a VIRTUO and a BTA 3D and incubated until declared positive or negative (up to 7 days).
Additionally, BPA and BPN bottles inoculated with 4 mL of unseeded LRAP were tested on the VIRTUO and the BTA 3D (120 and 40 bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by LRAP Results/Finding: The repeatability of the VIRTUO to detect organisms in LRAP was demonstrated by a recovery rate of seeded bottles of 99.9% for the VIRTUO and 99.5% for the BTA 3D.
The VIRTUO demonstrated an average improved TTD of 3.2 hours, when compared to the BTA 3D in the presence of 4 mL LRAP platelets.
The LRAP did not cause false positives.
Additionally, the age of the LRAP units (within 5 day expiry),did not impact the TTD when seeded with organism Background/Case Studies: Zika Virus (ZIKV) is an emerging flavivirus that is transmitted by the Aedes aegypti mosquito and sometimes A. albopictus mosquito.
Most infections are asymptomatic.
ZIKV Nucleic Acid Testing (NAT) became a required test for blood donors per the FDA Guidance entitled, "Revised Recommendations for Reducing the Risk of Zika Virus Transmission by Blood and Blood Components".
Based on our geographical location, implementation of this testing began 12 weeks after this guidance was issued.
We performed ZIKV NAT for donors of whole blood and blood components under an Investigational New Drug (IND) Study (sponsored by Hologic, Inc.).
We performed a retrospective analysis on all NAT results as there is a potential to defer donation due to false positive screening results.
Study Design/Method: Donors that consented to donate blood and be tested for the ZIKV were obtained from three Blood Banks in Colorado and Nebraska.
NAT was performed using the Procleix Virus assay which is a qualitative in vitro nucleic acid assay system that detects ZIKV RNA in plasma specimens.
The assay was performed on the automated Procleix Panther system.
All testing was performed according to the manufacturer package insert.
Results/Findings: In the event of a Reactive result, donors would be retested by NAT in addition to other testing (IgM antibody testing, neutralization test).
Donors are deferred for 120 days barring continued ZIKV testing and NonReactive results.
A total of 2,485 donors were screened for ZIKV.
All donors screened for ZIKV were NonReactive by NAT.
No invalid test results were obtained.
In addition the number of failed test runs due to instrument or assay issues were experienced were quite low (1.0%).
This data indicates that both the assay and instrument are robust.
There was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our Blood Bank customers.
Conclusion: The reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results.
This screening is important to continue to ensure blood safety in the United States.
Robust Inactivation of the Yellow Fever Virus 17D Strain Can be Achieved Using Amotosalen and UVA Light for Pathogen Reduction Treatment (PRT) of Platelet Components Andrew Laughhunn 1 , Felicia Santa Maria 1 , Yvette Girard 1 , Peter Bringmann 1 , Marion Lanteri* 2 and Adonis Stassinopoulos 2 .
1 Microbiology Department, Cerus Corporation, 2 Scientific Affairs Department, Cerus Corporation Background/Case Studies: Yellow fever virus (YFV) is known to cause explosive outbreaks, such as the one in Angola in 2015.
The rapidly increasing number of infections in Brazil, with hundreds of fatalities since December 2016, is of concern.
YFV is a Flavivirus transmitted by Aedes mosquitoes and could spread, like Zika virus, to other parts of the Americas where the vector is endemic.
With no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the 17D-YFV strain.
YFV vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects.
In addition, transfusion transmission (TT) of live attenuated YFV has been reported with severe clinical outcomes, especially in immunosuppressed patients.
In order to prevent TT by YFV vaccine strain, the AABB recommends a 2 weekperiod deferral after YFV vaccination.
YFV outbreaks and vaccination campaigns may therefore reduce blood availability.
This pilot study evaluated the ability to inactivate 17D-YFV using amotosalen (S-59) and UVA light PRT of platelet components (PC).
Study Design/Method: PC in 65%PAS (n53) or 100% plasma (n51) were spiked with high titers of 17D-YFV and treated with S-59/UVA PRT.
Samples were taken pre-and post-UVA illumination and infectious titers were determined, by plaque assay using Vero76 cells.
The extent of inactivation was quantified by comparing titers before and after inactivation.
Results/Finding: Pre-PRT infectious titers were 4.71 6 0.7 Log 10 PFU/mL for PC in 65% plasma and 5.19 Log 10 PFU/mL for PC in 100% plasma while titers in post-PRT samples were <-0.7 6 0.0 Log 10 PFU/mL for PC in 65% plasma and <-0.7 Log 10 PFU/mL for PC in 100% plasma.
Inactivation to the limit of detection of >5.41 6 0.7 Log 10 or inactivation of >4.71 6 0.7 Log 10 PFU/mL was achieved for PC in 65% plasma.
Inactivation to the limit of Background/Case Studies: The use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients.
This high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology.
Although Abbott's Alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the Alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels.
Study Design/Methods: The purpose of this study was to determine if the eight developed Abbott Alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin.
For each of the Alinity s assays evaluated (HIV Ag/Ab Combo, HTLV I/II, Anti-HCV, Chagas, HBsAg, Anti-HBc, Syphilis, and CMV IgG), samples spiked with a concentration of biotin at approximately 1000 ng/mL were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples.
Two samples, one negative and one positive, were tested with all assays, except the HIV and HTLV assays, which each tested two positive samples (1 HIV-1 antibody and 1 HIV-1 p24 antigen, and 1 HTLV-I antibody and 1 HTLV-II antibody, respectively).
Results/Findings: For the negative samples, the sample to cutoff (S/CO) differences between the biotin spiked and control were 0.00 for HCV, HBc, Syphilis, CMV IgG, and Chagas, 0.01 for HIV Ag/Ab and HTLV I/II, and 0.03 for HBsAg.
For the positive samples, the mean S/CO % differences between the biotin spiked and control were 0.00 % (antibody sample) and 0.36% (antigen sample) for HIV Ag/Ab Combo; 0.90% (HTLV I antibody sample) and 0.32% (HTLV II antibody sample); -1.43% for Anti-HCV, -2.52% for Chagas, -0.71% for HBsAg, -0.37% for Anti-HBc, -1.62% for Syphilis, and -0.59% for CMV IgG.
Conclusion: Eight Abbott Alinity s assays were evaluated to determine if they were susceptible to biotin interference.
These results indicate that the eight Alinity s assays do not show susceptibility to biotin interference at an approximate concentration of 1000 ng/mL.
Robustness of the Abbott PRISM Methods to Biotin Interference C Fischer 1 , R Schneider 2 , W Leonard 2 , M Cobb 3 , G Schlauder 3 , G Williams 3 , M Zuske 2 M Janulis* 2 .
1 Transfusion Medicine, Abbott Diagnostics, Wiesbaden, Germany, 2 ADD Diagnostics, 3 Transfusion Medicine, Abbott Laboratories, Chicago, United States Background/Case Studies: The use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses.
The increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology.
The purpose of this study was to identify any Abbott PRISM assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin.
After a comprehensive review of Abbott's current on market PRISM assays, no assays were identified that utilize biotin-streptavidin capture; however, 3 assays were identified for subsequent testing as they contain biotin in their assay design.
Background/Case Studies: Bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies.
While bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth.
Based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from 10 to 100 colony forming units (CFUs)/bag (i.e.
0.03 to 0.3 CFU/mL).
One major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of 10 8 -10 9 CFU/mL over the 5 days product shelf-life.
Moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test.
The aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates.
The adaptability of the process with the blood transfusion services requirements was of major concern.
Hence, attention was focused on an easy to automate technique able to deliver results on Day 2 after collection.
Study Design/Method: A large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps.
An original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay.
As recommended, 24 hours (Day 1) after collection a sampling volume of spiked platelets (0.1-1 CFU/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format.
An immunoassay was performed for the detection of the captured bacteria.
Results/Finding: This approach was tested on a panel of 25 bacterial strains involved in transfusion reactions.
The pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection.
The full test developed in this study combining a pre-analytical culture step followed by an There are many stakeholders are involved in HCV eradication program, including government authority such as Centers for Disease Control and Prevention, National Health Insurance and Health Promotion Administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, NPOs and academia.
Results/Finding: TBSF is a private nationwide single blood services program in Taiwan, and performs Anti-HCV screening test and NAT confirmatory test on every collected blood, which is a large-scale population screening of HCV in Taiwan because of its high blood donation rate (7.5%).
TBSF confirmed positive test result of repeated blood donors, and can identify HCV RNA seroconversion cases as recently-infected hepatitis patients.
Those infected patients would be referred to physician for further medical care and deferred permanently by TBSF to secure blood safety.
By interviewing the newly-infected cases, the risk factors of HCV patients can be studied and then help identifying and eliminating sources of HCV infection.
TBSF also contribute to health education by teaching our donors being aware of potential risks of HCV infection and keep monitoring every parameters of HCV epidemiology to evaluate the efficacy of HCV eradication program.
Conclusion: In HCV eradication program, TBSF can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation.
Thus, among all stakeholders, TBSF is particularly important and can play a pivotal role in eradicating HCV by 2030 in Taiwan.
The Theraflex UV-Platelets Technology Efficiently Inactivates Transfusion-Relevant Bacteria Species in Contaminated Platelet Concentrates Ute Gravemann 1 , Frank Tolksdorf 2 , Wiebke Handke 1 , Thomas H. M€ uller 1 and Axel Seltsam* 1 .
1 German Red Cross Blood Service NSTOB, 2 Maco Pharma International, GMBH Background/Case Studies: The THERAFLEX UV-Platelets system (Macopharma) is a UVC-based pathogen inactivation system for platelet concentrates (PCs).
Inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans.
Previous studies with the first set of bacteria species of the WHO International Repository of Platelet Transfusion Relevant Bacterial Reference Strains revealed a high inactivation capacity for clinically relevant bacteria.
Aim of the current study was to investigate the bacteria inactivation efficacy of the THERAFLEX UV-Platelets system for Enterobacter cloacae, Pseudomonas fluorescens, Staphylococcus aureus and Streptococcus bovis which have recently been added to the WHO International Repository.
Study Design/Method: PCs were produced from 5 buffy coats using the additive solution SSP1 (MacoPharma) with a residual plasma content of 35%.
For inactivation kinetics, PCs (n53) were spiked with bacteria to a final concentration of approx.10 6 colony forming units (CFU)/mL and irradiated with increasing doses until the full UVC dose was achieved.
Samples were taken for the bacterial titer determination after each irradiation step.
For sterilization studies, two PCs were pooled and inoculated with bacteria to a final concentration of approximately 0.3 CFU/mL.
Bacteria were allowed to grow for 6 h in the PCs at 22 6 28C under agitation.
After splitting, one PC remained untreated (growth control) while the other one was UVC-treated.
After storage for seven days, samples were taken from both bags for sterility testing by BacTALERT (Biomerieux) and for determination of the bacterial titer in the untreated control units.
Results/Finding: Bacteria in PCs were inactivated in a dose-dependent manner by treatment using the THERAFLEX UV-Platelets system.
Mean log 10 reduction factors ranged from 6 to 7 for Enterobacter cloacae (6.3 6 0.6, PEI-B-P-43), Pseudomonas fluorescens (7.1 6 0.4, PEI-B-P-77), Staphylococcus aureus (6.6 6 0.4, PEI-B-P-63), and Streptococcus bovis (7.0 6 0.3, PEI-B-P-61).
PCs (n512 for each species) spiked with these different bacteria species were efficiently sterilized (12 out of 12).
Treated PCs remained sterile during storage for 7 days, while bacteria in non-treated PCs grew to high titers of 10 6 -10 8 CFU/mL.
The THERAFLEX UV-Platelets system efficiently inactivates a broad range of different bacteria species, including the WHO reference strains.
Sterility is maintained over a storage period of 7 days.
These results suggest that the UVC-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions.
Transfusion Transmissible Infections Among Blood Donors and Strategy on Direct Laboratory Testing Cost of Blood Screening at National Blood Bank Center, Addis Ababa, Ethiopia Abraham Zewoldie*.
National Blood Bank Service Background/Case Studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (TTIs).
Hepatitis B virus (HBV), Hepatitis C virus (HCV), human immunodeficiency virus (HIV) and syphilis are the most serious infections transmitted during blood transfusion.
Serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of TTIs.
Knowing the current prevalence of TTIs among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup.
Study Design/Method: A retrospective analysis of blood donors' record covering the period from July 1, 2008 to July 30, 2013 was conducted.
The data was collected from the Nation al Blood Bank (NBB) center Donor Data base.
In addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs.
Data was first exported to SPSS version 16 software for analysis.
Data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables.
P values less than 0.05 were considered significant.
Results/Finding: A total of 173, 207 consecutive blood donors were screened between 2008 and 2013.
The overall seroprevalence rate of HBV, HIV, HCV and syphilis of blood donors was 5.0%, 1.6%, 1.4% and 0.1% respectively.
The HIV-HBV co-infection was higher among blood donors 135(41.79%) followed by HBV-HCV co-infection whish accounts about 103(31.89%).
Significantly increased sero-prevalence of TTIs was observed in among Family replacement donors, factory workers, daily labors and the age group of 26-35.
In this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was 746,773.9 Ethiopian Birr.
Conclusion: A significant percentage of the blood donors harbor TTIs.
The NBB center should work on voluntary blood donor mobilization and develop culture of voluntarism.
The direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm.
Thus, the new strategy can be implemented to make screening of TTIs cost effective in NBB center.
Transfusion Transmitted Malaria in a 14 Month Old Infant Patricia Davenport* 1 , Geeta Paranjape 1 and Laurie Sutor 1,2 .
1 Carter BloodCare, 2 UT Southwestern Medical Center Background/Case Studies: In 2017 at a large pediatric hospital, a 14 month old infant was supported for 31 days by extracorporeal membrane oxygenation (ECMO).
Over this time 113 blood products were transfused.
About 10 days after end of ECMO support, a routine blood smear examination revealed inclusions in some of the patient's red cells.
The patient had also been having intermittent fever.
Malaria was confirmed by PCR as Plasmodium ovale (P. ovale).
Because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products.
Study Design/Method: The investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote.
We identified 27 donors of red cell products.
Each donor was contacted and was asked four questions.
Additional questions were asked for clarification if needed.
Based on donor response, risk for active malaria infection was assessed.
We also considered areas where P. ovale is, or is not found.
Donors identified as having possible risk were tested for antibodies and parasitic DNA.
Results/Finding: The five donors who had been ill all had common cold or bronchitis like symptoms.
Donors who traveled went only to non-risk areas.
Three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the U.S. as adults.
It was discovered that one of these three did not meet all donor criteria.
The donor had failed to disclose that he had not completed 3 years stay in the U.S. after emigrating from Cameroon, an area endemic for P. ovale.
He had not travelled anywhere after coming to the United States in October 2014 and answered "No" to travel.
Antibody tests on this donor were positive for P. ovale and P. falciparum, but PCR tests were negative.
Another possible at-risk donor, a former resident of Iran was tested and was PCR and antibody negative.
The third donor has not yet been tested but the country of residence does not have P. ovale malaria.
Conclusion: While it could not be definitively proven that the donor with antibodies to P. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment.
Transfusion-Transmitted Babesiosis Outside an Endemic Area: A Case Report German Felix Leparc*.
OneBlood Background/Case Studies: An 81 y.o.
male patient was admitted to the Emergency Room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically.
During this admission, he received a total of 13 units of Red Blood Cells.
Approximately 4 weeks later, he was re-admitted due to another episode of GI bleed manifested by melena.
As part of his routine evaluation, a CBC was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with Babesia sp.
Study Design/Method: Upon notification of a suspected case of Transfusion-Transmitted Babesiosis, lookback of all donors involved in prior transfusion event was initiated.
Results/Finding: To confirm the presumptive diagnosis of babesiosis, PCR was performed and Babesia microti DNA was detected.
An evaluation of the patient's risk factors revealed that prior to the GI bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery.
No travel history to the US Midwest, and while he travelled to New England two years ago he did not spend time outdoors.
He was splenectomized in his mid 20's.
Donor lookback identified a donor who lived in New London County, Connecticut but spent the winter season in Central Florida, where the blood donation (double RBC collected by apheresis) took place.
He had never been diagnosed with babesiosis, but participated regularly in outdoor activities in Connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it).
Upon testing, he was found to be negative for B. microti on PCR as well as IgM antibodies, but had IgG antibody titers of 1:256.
The recipient of the other RBC unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed.
During phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative.
Conclusion: While transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case.
Once licensed assays for Babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration.
Transfusion-Transmitted Stenotrophomonas Maltophilia from a Red Cell Unit: A Case Report Ashley C Gamayo* 1 , Andrea J Linscott 2 and Donny Dumani 3 .
Background/Case Studies: Transfusion-transmitted bacterial infections (TTBI) are rare, but serious complications of blood product transfusions.
From 2011-2015, 8% of 173 transfusion-associated fatalities reported to the FDA were attributed to bacterial contamination.
Red cell units are rarely implicated in severe and fatal TTBI.
When present, contaminants are often gram-negative rod (GNR) bacteria with psychrophilic properties.
We present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with Stenotrophomonas maltophilia (S. maltophilia).
Study Design/Methods: A 27-year-old female with sickle cell disease was admitted to the hospital for possible pain crises.
Pre-transfusion blood and urine cultures collected on Day 1 of hospitalization showed no growth after five days.
On Day 3, the patient required a blood transfusion for which she was issued a CMV-safe, irradiated, HbS-negative, crossmatched, O-negative red cell unit.
The 318 mL unit had been aliquoted via sterile connecting device 12 days prior for a pediatric patient.
All 26 mL of the pediatric aliquot were transfused without adverse effects.
The patient's pretransfusion temperature was 37.18C.
Within 45 minutes of starting the transfusion, the patient's temperature increased to 39.38C and subsequently reached a maximum of 39.58C.
The transfusion was stopped and the blood bank notified immediately.
Gram stain of the remainder of the transfused component revealed GNR bacteria.
Blood was collected from the patient for culture and antibiotic treatment initiated.
Results/Findings: Initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test.
Blood cultures from both the patient post-transfusion and the implicated red cell unit grew GNR bacteria identified as S. maltophilia.
Further microbial testing revealed the cultured pathogen was able to proliferate at 4-68C; a finding not characteristically observed in S. maltophilia.
Conclusion: This is the first definitive case of TTBI with S. maltophilia.
This bacterium is a globally emerging GNR that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients.
Contamination was unlikely due to an asymptomatic donor.
There was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component.
The patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified.
The patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization.
The transfusion reaction was classified as definitive, severe TTI of definite imputability.
Validation of Commercial Immunoassays for Detecting HBsAg and HIV Antibodies in Production Pools Karen Leighton, Izekial Butler and Scott Jones*.
QualTex Laboratories Background/Case Studies: Plasma fractionators test plasma production pools for HBsAg and HIV antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling.
The European Medicines agency (EMA) has published guidelines for the validation of immunoassays for the detection of HBsAg and HIV antibodies in production pools.
The aim was to validate commercial immunoassays for the testing of production pools for HBsAg and HIV antibodies utilizing the EMA guidelines.
Study Design/Method: A lower calculated cutoff value for the ABBOTT PRISM HBsAg and HIV O Plus assays was determined by calculating the mean signal-to-cutoff ratio (S/CO) plus 3 standard deviations of four different types of plasma production pool samples.
The calculated cutoff values were utilized for the rest of the validation.
The detection limit was determined by testing in triplicate, serial dilutions of WHO HBsAg and HIV antibody standards diluted in plasma.
A normalized detection limit was calculated for the HBsAg assay using production pools containing low, typical and high anti-HBsAg titers.
Intra-assay variability was determined by testing a minimum of 6 determinations of a low positive control in 1 run.
Inter-assay variability was determined by testing at least 3 representative negative production pool samples, at least 1 low positive sample (about 3 S/CO) and a titration series of WHO standard spiked into plasma production samples.
Runs were performed on six separate days using two different instruments and two different lots of assay reagents.
Results/Finding: The lower calculated cutoff values for the HBsAg and anti-HIV assays were both below the manufacturer cutoffs of 1.00 and were 0.72 and 0.48 respectively.
The HBsAg assay detection limit was 0.065 IU/ mL for source plasma and 0.120 IU/mL for recovered plasma samples.
The normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at 15-25 0 C where all samples were still reactive for HBsAg.
The anti-HIV lowest positive dilution for all replicates varied between 1:10,000 to 1: 1,250,000 depending on subtype and group.
The % CV of the S/CO values of the replicates of the intra-assay variability validation were less than 5% for both assays.
The %CV of the S/CO values of the panel of samples of the inter-assay variability validation were less than 14%.
Conclusion: A lower calculated cutoff value could be determined for commercially available immunoassays for HBsAg and anti-HIV.
These immunoassays could meet all of the recommendations in EMA validation guidelines.
The ABBOTT PRISM HBsAg and HIV O Plus assays can be utilized to test production pool samples.
was performed on 6 donors (3-17 days after the index donation) -3 donors in the follow up study and 3 tested by the DOH.
No donors tested by the DOH participated in the follow up study.
Follow up testing was negative for all 6 donors.
DENV antibodies were negative in 9 donations and equivocal in 1.
Our initial reactive rate is higher than that reported to date for the Procleix ZIKV TMA of 1 per 23, 342 [P. Williamson, et al Transfusion, in press] .
Conclusion: Universal testing under IND was successfully implemented and incorporated into blood center operations.
We have noted an initial reactive Other demographics that should be analyzed for their potential to be used to predict CMV seroconversion rate include gender, age, race, ethnicity or a combination of these.
Background/Case Studies : Growing the geographic footprint has been a priority for the organization since 2014.
Over a four year period, the organization doubled the number of blood centers, with continued growth expected.
With the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk.
The internal audit function was centralized in late 2011, for which the program consisted of 2 types of audits, an operational compliance audit and a support systems compliance audit.
Each type was performed twice per year at each main center.
This model was no longer serving the changing organization.
Study Design/Methods: Lean Six Sigma concepts were applied to this project.
Survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program.
This information was the primary input to the SWOT Analysis (Strengths, Weaknesses, Opportunities, and Threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design.
Potential solutions were placed into a Pugh Matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection.
Each potential solution was compared to criteria for evaluation and selection of the best solution.
Results/Findings: The program was re-designed to perform internal audits annually as a single, team-based comprehensive audit.
Remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings.
A formula was created to determine on-site audit time that included adjustable risk factors.
The audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs.
The team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns.
Conclusion: The comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing Quality or increasing compliance risk.
External inspection performance has achieved record performance levels the past year.
Diversity of auditor skills led to a stronger skill presence, which was consistently applied across system.
Auditing is more efficient and effective.
Stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system.
Auditors and auditees have increased in knowledge, and the internal quality audit program has improved.
Background/Case Studies: In many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma).
Banc de Sang i Teixits (BST), adopted the fully automated Reveos system (Terumo BCT Inc, Lakewood, CO) few years ago to manufacture blood components.
In June 2016, BST started a validation of new blood bags manufactured by Terumo BCT with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter.
Study Design/Method: To perform this validation, 300 blood donations were used under different conditions (see Table below ).
The current filter evaluated for the platelet pool (LRF-XL, Haemonetics Corporation) was compared to a new filter (Terumo BCT Inc.).
The new blood bags were manufactured using a new vinyl supplier.
A portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (QC) tests (routine QC performed at BST following European Directorate for Quality of Medicines & Healthcare; cytokine analysis, such as P-Selectin and platelets recovery through the filter).
Results/Finding: The results are very similar between both bags, current and new one, as well as filters.
All the analysis done to evaluate the quality of the blood components were similar in all conditions.
Also, it was shown a better performance on platelets pools, when they came from bags centrifuged with 60 ml of plasma, vs. 30 ml of plasma and additive solution.
Conclusion: These new bags and filter have shown a similar behavior when using them for manufacturing blood donations with Reveos system in our blood bank.
Regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools.
No issues should be found if they are implemented in routine use.
It's planned to start this implementation during this year, 2017; so then there will be larger results in order to have a proper procedure qualification.
Conclusion: Patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood.
It is clear that some requests for exquisitely rare types are not able to be filled with current donors.
Molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years.
It is recognized that commercial platforms do not include many of these making these rare types even more challenging to find.
Consideration should be given to testing more donors of all ethnicities to identify more rare donors.
Recommendation #1: Updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation.
Updating our educational materials will likely have a minor impact.
Recommendation #2: Implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency.
Initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over 120, 000 donors.
The recommendation to limit the number of donations would have a substantial impact.
For this analysis, the focus was on 16-18 year olds and premenopausal women (ages 19-55) donors.
On average, 16-18 year olds donate 1.3 times a year and premenopausal women donate 1.49 times a year.
If both of these groups were limited to donating once a year, a total of 4,845 donations from 16-18 year olds and 9,272 donations from premenopausal donors would not be collected.
Conclusion: After analyzing the impact of the AABB Association Bulletin #17-02, the bulletin will have a significant impact on both donors and our local blood supply.
More than half of donors would receive either ferritin testing or iron supplementation.
If the only measure employed is limiting the number of times a donor could donate for 16-18 year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals.
Background/Case Studies: Transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians.
These deficiencies may be due to the frequency and type of education.
The majority of medical students in the United States receive four or fewer hours of transfusion medicine education.
The Transfusion Medicine Academic Award Group published educational content guidelines for medical school, residency and fellowships.
However, the frequency and educational methods remain poorly evaluated and with little guidance.
We investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners.
Study Design/Method: Three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications.
The simulation group received all educational activities within a 2.5 hour inperson, high-fidelity live session.
The hybrid group received some educational component online and also attended an in-person high-fidelity simulation session.
The online only group received all educational materials online, including a pre-recorded-video simulation session.
The learners were second year medical students enrolled at one institution.
The same faculty members taught all live sessions and developed all online materials ensuring the content was the same.
A pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications.
The educational session was evaluated by the Likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding).
Results/Finding: 97% (101/104) of the simulation group students improved their post-test scores and had an average Likert scale rating of 4.1 (very good).
89% (63/71) of hybrid group students improved their post-test scores and had an average Likert scale rating of 4.2 (very good).
89% (90/101) of online only students improved their post-test scores and had an average Likert scale rating of 3.0 (good).
The average changes in scores were statistically significant within all training groups (p value < 0.0001).
Additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p<0.0001) and the hybrid group (p<0.0001).
Conclusion: Our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum.
The high-fidelity simulation curriculum is also preferred over the online only education as indicated by the Likert survey results.
Aaron J Wyble*, Yeon Mi Kim and Barbara J Bryant.
University of Texas Medical Branch Background/Case Studies: Diagnostic management teams (DMTs) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care.
DMTs employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations.
The interpretations must be of moderate to high complexity in order to be clinically valuable.
Recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care.
The timeliness of the DMT reporting is vital to patient management.
The inherent design of a DMT also provides an educational opportunity for trainees at academic centers.
Study Design/Method: The transfusion medicine service at a large university-based academic medical center implemented a DMT in 2016.
All cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from July 2016 through January 2017 were evaluated by transfusion medicine residents.
The electronic medical record (EMR) of each patient was also reviewed to determine relevant clinical history.
All significant findings were presented at the Transfusion Medicine DMT conferences.
The DMT was comprised of physicians from Transfusion Medicine, Hematology/Oncology, Anesthesiology, Transfusion Service technical staff as well as visiting clinical staff from Surgery, Obstetrics and Gynecology, Transplant Services, and Pediatrics.
The DMT integrated the clinical and laboratory data to formulate relevant interpretations and recommendations.
The final DMT reports were placed into the EMR for access by health care providers.
Financial benefits of a transfusion medicine DMT were also evaluated.
Results/Finding: In a 7-month period, 504 cases of complex antibody identification workups (65%), transfusion reactions (2%), consultations for blood component utilization (6%), and deviations from standard operating procedures and massive transfusion protocols (27%) were presented at the Transfusion Medicine DMT conferences.
The placement of DMT narratives in the EMR as progress notes and laboratory reports provided informative and timely communications.
Residents participating in DMTs demonstrated improved clinical and laboratory correlation skills.
As a result, resident competency in transfusion medicine was enhanced.
Over $68,000 of revenue was generated utilizing the standard professional component CPT codes.
Conclusion: DMTs encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services.
Additional benefits of a DMT program include resident, clinician, and technical staff education and the generation of revenue for the institution.
Streamlining a Blood Center and Hospital Transfusion Service Supply-Chain with an Informatics Vendor-Managed Inventory Solution Hamilton C. Tsang* 1 , David Lancaster 2 , Dianne Geary 2 , Robert Scott 1 , Anh Thu Nguyen 1 , Adam Garcia 2 , Raina Shankar 1 , Leslie Buchanan 1 and Tho Pham 2 .
1 Stanford Health Care, 2 Stanford Blood Center Background/Case Studies: Inventory management is both a major challenge and an integral part of hospital transfusion service (HTS) and blood centers (BC) operations.
The current process at our institution involves twice-per-day shipments from the BC to the HTS, with each shipment predicated upon current stock levels at HTS.
Manually obtaining inventory levels for each product is time-consuming.
The manual determination is also errorprone.
We aim to enhance inventory management operations by developing an informatics solution to (1) streamline the ordering process to accurately reflect inventory status and transfusion practices and (2) re-allocate valuable HTS tech time.
Study Design/Method: At our HTS, the general inventory accounts for over 50 product categories broken down by component, blood type, irradiated status, and CMV-serology status.
We therefore sought to establish an electronic method to reliably infer the general inventory level.
Since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g.
special antigen units, cross-matched units), over a 5-month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory.
Once we had a reliable electronic method to determine inventory levels, we implemented a 3-month pilot period.
We analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: (1) the ratio of units transfused per week to the number stocked (T:S), (2) the number of products ordered as STAT, and (3) the number of expired products.
We created in-house programs on Visual Basic for Applications (Microsoft, Redmond, WA) for both the calibration and pilot periods.
2486 lines of code were written for both programs, including 2 class modules and 34 distinct subroutines.
Results/Finding: During the pilot period, we investigated our system's noninferiority.
The average weekly T:S ratio for cryoprecipitate, plasma, and RBC, respectively, were 1.03, 1.21, and 1.48 before the pilot period compared with 0.88, 1.17, and 1.40 during the pilot period.
These differences did not reach statistical significance (p50.28).
We also monitored the number of stat ordered products before and during the pilot period, which were 27 and 31 stat units per week, respectively (p50.86).
Lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were 226 and 196 units, respectively (p 5 0.28).
An estimated 7 hours per week of technologist time was reallocated to other tasks once the electronic census was adopted.
This translates to 0.175 FTE and $18,200 per year saved from labor costs per year if permanently adopted.
Conclusion: We created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering.
Our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same T:S ratio, and not increasing the number of expired products.
This is achieved while freeing up over 360 hours of staff time per year.
Future directions include full automation with involvement from HTS informatics department.
Transfusion Practice Improvement: Gaining Traction through the Use of a Provincial Transfusion Quality Improvement Plan Denise Evanovitch* 1 , Yulia Lin 2 , Troy Thompson 1 , Allison Collins 1 and Sheena Scheuermann 1 .
1 Ontario Regional Blood Coordinating Network, 2 Sunnybrook Health Sciences Centre Background/Case Studies: A provincial regional blood coordinating network (PRBCN) held a "Quality Focus Day" (QFD) in 2014 to explore transfusion quality indicators to be included in a province wide quality improvement plan (QIP).
The plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use.
The following recommendations were made: Select a blood component that most hospitals could monitor Display progress in a public forum so that hospitals could compare themselves to peers Strike a province-wide transfusion QIP committee to guide the development of the plan, supporting resources and ongoing improvement initiatives Study Design/Method: A provincial transfusion quality improvement plan (PTQIP) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient.
There was further collaboration with other organizations such as the provincial health quality division, Choosing Wisely After the launch, an informal survey indicated that 74 of the province's 158 hospitals were interested or had already adopted portions of the PTQIP.
To further assist hospitals in advancing their QIPs, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success.
Both hospital and provincial reports can be generated from the tracking tool.
A more formal survey conducted in 2017 indicated that 93% plan to implement or already have implemented the PTQIP and 43% of the respondents already have put prospective order screening by technologists in place.
Conclusion: Helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives.
Taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses.
Background/Case Studies: Military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as Pre-Hospital Transfusion (PHT).
Helicopter emergency medical services (HEMS) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications.
There is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a PHT program.
Herein, we report our experience as a large hospital system embarking on the development of a multi-state PHT service.
Study Design/Method: In October 2016 a work group was formed to establish PHT services for the HEMS providing care to over thirty regional hospitals.
Composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: Federal/State Regulations; Inventory Structure/Management; Product Storage/Testing; Tracking/Traceability; Emergency Release Protocol; and Staff Training.
While there are no specific regulations governing PHT, the regulations pertaining to blood product storage, validation, and monitoring apply.
The FDA, AABB, and state agencies were each consulted to ensure compliance with all directives.
Results/Finding: The largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state HEMS.
Similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites.
The system's FDA licensed blood supplier was deemed responsible for product consignment and transport between the four HEMS sites.
The blood inventory at each site was designed to contain: group O positive RBCs, group A low anti-B titer liquid plasma, and four-factor prothrombin complex concentrate.
A military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed.
Validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan.
Staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the HEMS medical director with additional oversight provided by transfusion medicine physicians.
Conclusion: Our work group successfully identified the challenges associated with a multi-state PHT helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training.
Our PHT service will go live in 2017.
Publishing this experience may benefit future sites as they launch similar PHT initiatives.
Blood Transfusion during Humanitarian Emergencies Yetmgeta E. Abdella* 1 , Rana Hajjeh 1 and Cees Th.
Smit Sibinga 2 .
1 World Health Organization Regional Office for the Eastern Mediterranean, 2 International Quality Management (IQM) Consulting Background/Case Studies: More than 76 million people are affected by humanitarian emergencies in the Eastern Mediterranean Region of the World Health Organization (WHO), where some of the most affected countries in the world are located.
In these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances.
Humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex.
Despite these obvious needs, across the Region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies.
Study Design/Method: We searched PubMed and Index Medicus for the WHO Eastern Mediterranean Region for data on availability and safety of blood transfusion in humanitarian emergencies.
We conducted a structured survey of blood transfusion services (BTS) in all countries in the Region to identify the following: type of humanitarian emergencies between 2006 and 2016; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges.
Additional information was collected during a regional consultation (Eastern Mediterranean Region) held in May 2016 in Tunisia.
Results/Finding: We found 24 publications on disaster from five countries in the Region and 16 publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the Region.
However, none dealt with the questions of availability and safety of blood transfusion during emergencies.
Twelve countries (54.5%) responded to the survey.
Armed conflicts and terrorism are the commonest types of emergencies with estimated 10-85% of the injured requiring blood transfusion.
Nine countries have emergency preparedness and response plans for BTS.
Potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors.
Seven of the responding countries keep an emergency blood stock.
Collaboration between the different stakeholders exists in seven countries.
Lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified.
Conclusion: There is a need to integrate BTS in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise.
(63, 85, and 108 for 2014-2016) .
The number of collections per registered TRT donor varied significantly, ranging from 0 to 12 therapeutic draws/donor per year.
Excluding those that didn't present for a therapeutic blood collection, the average number of TRT collections/donor per year decreased from 3.8 to 2.8 between 2014 and 2016.
Conclusion: Our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on TRT referred for therapeutic phlebotomy due to elevated hemoglobin values from 2014 through 2016.
It is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on TRT, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per TRT donor decreased during this timeframe.
The percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral.
Our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population.
As it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than 56 days, is sufficient to mitigate the risks of testosterone therapy.
Hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements.
Approaches Involving the Use of a Vein Illumination Device in a Blood Donor Center Sara Matheson*, Kimberly J Duffy, Audrey E Traun, Mary M Benike, James R Stubbs and Justin D Kreuter.
Mayo Clinic Background/Case Studies: Venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge.
Unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration.
In a blood donor center, the primary selection of a vein is done by palpation within the antecubital area.
Prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed.
Vein illuminator (VI) devices are available to aid in visual display of potentially suitable veins.
Such a device was made available to staff in March of 2010.
After an initial testing and instructional period, the VI has since not been used by staff.
The objective of this study is to discover reasons why staff does not use the VI to identify potentially suitable veins.
Study Design/Method: A staff survey was developed and distributed to staff in March 2017 to inquire about usage of the VI and obtain feedback about the device.
At the time that the survey was sent, the device had been available for several years.
The survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback.
Results/Finding: The survey had a 77% response rate (n533).
Of these, 78.8% have never or very rarely utilized the VI.
Self-reported reasons for low utilization focused on two dominant themes.
First, that the device is not needed and second that it doesn't accurately show veins.
87.9% of respondents are aware of where the VI is stored and a more accessible location to share the device was not identified.
Although 93.9% of respondents have been provided training on using the VI, the group was mixed regarding their comfort level in using the device independently.
Only 48% of the group was willing to try VI.
Conclusion: Infiltration and incomplete collection account for approximate 3% (770 units/year) of qualified blood donors, yet VI does not appear to be a viable solution for our blood donor program.
There seems to be both an opportunity and challenge with VI implementation.
The opportunity is to create critical awareness of problems with vein cannulation.
The challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation.
Benefits of Converting From MCS1 To Alyx Penny Schroeder* and Elizabeth Parker.
Indiana Blood Center Background/Case Studies: In 2015, apheresis red cells (aRC) represented 4.7 % of total red cell collections at our center.
HAE MCS1 LN8150 was utilized to collect aRC.
Due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies.
Study Design/Method: Fresenius Kabi demonstrated the Fenwal Alyx technology as well as the business case to the primary stakeholders.
All implicated departments were involved in the initial impact assessment.
A multidepartment kick off meeting was held and project team formed.
Due to product demands, the decision was made to validate aRC and plasma apheresis.
The primary departments affected were Blood Collection and Production.
Fresenius Kabi provided sample validation plans, SOPs, training and training materials for use.
Four mobile-carts were purchased for easy transportation of Alyx and quick-connect feet for installation on mobile buses.
The Lead Trainer and the BC Technical Administrator traveled to an affiliate blood center to observe their Alyx program and identify best practices.
A team of Blood Collection trainers and preceptors were the initial group trained and validation performed.
This team also served as the subject matter experts and field preceptors.
Fresenius Kabi returned for Advanced Alyx Operator training.
The training plan targeted previous MCS1 operators first and then operators new to apheresis with a training goal of 30% of mobile staff.
Validation of the 12 Alyx began 06/01/16 and took approximately 45 days to complete.
During this time, Fresenius Kabi conducted Alyx education and apheresis recruitment training to all Collection and Recruitment staff.
The MCS1 machines were removed from service 07/08/16.
Alyx Go-Live occurred 07/13/16.
Additional operator training continued through September 2016.
Results/Finding: Due to ease of mobility and use of Alyx, reduced procedure time compared to MCS1 and donor conversion training we increase components collected.
Alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles.
This decreased kit cost by $19.21 each providing an estimated annual savings of $239,000.
Conclusion: With the multiple Alyx donation types we were able to increase our collection of right type procedures by approximately 2.5% and decrease our kit costs by 22%.
With Alyx the collection plasma on mobile blood drives is now possible.
Due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from October 2016-March 2017.
Background/Case Studies: High frequency of donation is a risk factor for iron deficiency.
Because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk.
Minimum hemoglobin (Hb) has long been the same for males and females at 125g/L, but for males this falls below the normal limit.
As a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum Hb increased to !
130 g/L) and for females (minimum interdonation interval increased from 56 to 84 days).
The longer interdonation interval in females was gradually implemented, starting with donor messaging in October 2016, changes in rebooking of donation appointments in December 2016 and culminating with eProgesa criteria changes on March 5, 2017.
Both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in Hb deferral rates in female donors.
We aimed to assess the impact of these changes on Hb deferral rates.
Study Design/Method: Percentages of Hb deferrals were calculated as the number of donation attempts that resulted in Hb deferral divided by the number of successful donations plus Hb deferrals multiplied by 100.
Percentages were calculated for male and female donors before and after changes were made.
Results/Finding: The percentage of Hb deferrals increased in male donors from 0.89% in the 3 weeks pre-implementation to 2.16% in the 3 weeks post-implementation of the change in the Hb criterion.
Hb deferral rates for female donors were 12.6% in September, 12.0 % in October/November, and 9.9 % from December to March, 2017.
Conclusion: Hb deferral was more frequent in male donors after the minimum Hb was increased to 130 g/L.
The gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased Hb deferrals.
A longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention.
In the past 8 years, 59,223 blood products, derived from 10,509 procedures, were distributed to 185 different investigators in over 200 laboratories.
Whole blood was the most common product (45.2%), followed by unmanipulated mononuclear cell collections (28.6%), and elutriated monocytes or lymphocytes (19.8%).
Less common requests included platelets (2.5%), plasma (2.5%) and granulocytes (0.8%).
Adverse donor reactions were infrequent (0.33% of procedures).
Conclusion: We report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank.
Research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion.
Hospital-Based Blood Donor Center's Experience with Implementing Platelet Pathogen Reduction System Kimberly J Duffy*, Mary M Benike, James R Stubbs and Justin D Kreuter.
Background/Case Studies: The safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats.
The recent FDA approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers.
In order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications.
The objective of this study was to determine the optimal collection settings to enable 100% collection of pathogen reduced platelets while limiting the loss of products.
Study Design/Methods: The collection instrument evaluated for this study has FDA approval for platelets suspended in 100% plasma.
The corresponding pathogen reduction system used for the study has 3 kits with 3 different collection specifications.
All apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer.
The yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device.
In order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections.
Staff was instructed to collect the highest available yield per donor.
After collection, volume, platelet yield, and concentration data was obtained.
This data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than 6.8 x10 11 , thus meeting the criteria for a double product.
Results/Findings: A higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than 1800 x 10 3 /mL was more likely to be outside the specification of the pathogen reduction kit.
The platelet concentration target of 1867 x 10 3 / mL results in discarding products and was quickly removed from instrument settings.
Collections with a platelet yield as low as of 3.5 x10 11 and platelet concentration of 1167 x10 3 /mL were more likely to produce a product that was not within the specification of the pathogen reduction kit.
ABSTRACT Conclusion: The loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise.
The goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications.
Final collection instrument configurations are platelet yield of 7.0 x10 11 and 6.8 x10 11 at the volume of 400 mls and platelet yield of 4.2 x10 11 , 4.0 x10 11 , and 3.5 x10 11 at the volume of 300 mls.
Moving from Subjective to Objective Donor Eligibility Screening Platforms: A Blood Center's Journey Angela Dirr* 1 and Steve Cihura 2 .
1 Bonfils Blood Center, 2 BBC / BSI Background/Case Studies: In 2012, the device used by Bonfils Blood Center to determine donor hemoglobin and donor eligibility was reaching its end of life, and BBC needed to define a path forward for a reliable replacement device.
Study Design/Method: BBC evaluated 3 devices with the following criteria in mind: 1) device disposable costs, 2) reagents/controls/quality control, 3) objective Hgb/Hct measurement, 4) portability and durability for a mobile environment, 5) ease of use, 6) donor experience, 7) battery life, 8) validation requirements plans, 9) blood center suitability, and 10) ability to link to BECS.
Multiple departments including Donor Care, Equipment Management and Validation, Quality, and Regulatory Affairs were involved in the evaluation and product selection.
BBC tested 50 donors per each device at both a fixed and a mobile site.
BBC also considered donor feedback for the choice of replacement technology.
The project started February 2013 with a targeted implementation date of July 2013.
After creating necessary SOPs and adopting existing SOPs, BBC successfully completed the validation of the devices, and chose the CompoLab technology from Fresenius Kabi as the new device for BBC blood bank.
Results/Finding: The CompoLab was selected as it met project scope and selection criteria.
It was important for BBC to reduce paperwork and daily tasks.
The CompoLab eliminates daily QC reducing paperwork, time and improves error management.
After converting to the new technology, BBC donor deferral rates increased by approximately 15%.
As a consequence to this increase, BBC conducted reminder training with BBC staff to ensure proper sampling technique and higher sample quality.
Over time, BBC deferral rates stabilized to 4.59% in 2014 and 4.29% in 2015.
During this time period, BBC also successfully recruited new blood donors to BBC program, which may have contributed to an increase in deferral rates.
In 2016, the deferral rate increased again, probably due in part to the FDA Final Rule "Requirements for Blood and Blood Components Intended for Transfusion or for Further Manufacturing Use", which went into effect in May 2016.
Conclusion: During the evaluation for new equipment, BBC learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business.
After comprehensive evaluation of multiple donor eligibility screening platforms, the CompoLab device was selected at BBC facility.
It met the majority of all aspects of the project scope and qualifying criteria.
BBC also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates.
Flowmetry on Platelet Apheresis.
Tetsu Yamamoto* 1 , Ayumi Araki 1 , Hiromi Sanyoshi 1 , Hiromi Kanai 1 , Hiroya Kikuchi 2 , Katsushi Tsukada 2 and Kazuhide Mure 2 .
1 Hokkaido Red Cross Blood Center, 2 Japanese Red Cross Hokkaido Blood Center Background/Case Studies: Vasovagal reaction (VVR) is known to be the most common adverse reaction to blood collection, but effective measures for preventing VVR have not yet been developed.
Effective timing of interventions during apheresis donations in particular should hold the key to predicting VVR, but no research has been done on the topic.
Study Design/Methods: This study investigated the potential to predict VVR from fluctuations in peripheral blood flow measured by laser Doppler flowmetry in platelet apheresis donors, a population highly likely to experience VVR.
Data were collected from 354 individuals who donated platelets during the 6-month period between February and August 2015, and data from the 30 donors who experienced VVR were analyzed.
To calculate the level for issuing VVR alert, the percent decrease in blood flow (DBF) and the percent decrease in heart rate (DHR) were calculated, the time from alert to VVR was estimated for three DBF levels, and the detection performance of each alert level was calculated.
Results/Findings: Eight of the 156 men (5.1%) and 22 of the 198 women (11.1%) experienced VVR.
One donor did not experience VVR during blood collection, but had a delayed reaction while resting afterward.
Mean maximum DBF in the 30 donors in the VVR group was 64.7 6 13.7%, which was significantly higher than the 25.6 6 11.7% in the non-VVR group.
At a maximum DBF threshold of 45%, sensitivity for discriminating between VVR and non-VVR donors was 93.3% and accuracy was 94.4%.
When 45% DBF was used as the alert level, alerts were issued for 44 donors, including 25 in the VVR group.
Therefore sensitivity for predicting VVR was 83.3% and specificity was 94.1%.
Mean time from alert to diagnosis in the VVR group was 4.03 6 4.35 minutes, and accuracy of the alert was 56.8%.
Some of the VVR could not be predicted even the value of maximum DBF exceeded 45%.
The reason was supposed to be the difference of donor susceptibility on DBF.
Conclusion: We investigated whether VVR in platelet apheresis donors can be prevented by prediction and found that it is possible to predict VVR early enough before onset to intervene by monitoring DBF in real time during blood collection using laser Doppler flowmetry.
Future research must also investigate whether the incidence of VVR can actually be reduced by interventions such as adjusting extracorporeal circulation.
The Risks of Alloimmunization in Sickle Cell Patients Using C, E, K Negative Blood: Experience of a Hospital Apheresis and Transfusion Service Grace Banez Sese* 1,2 , Salam Abdus 3 and Shabrina Shah 3 .
1 Inova Blood Donor Services, 2 Inova Fairfax Medical Campus, 3 Inova Fairfax Medical Campus Transfusion Services Background/Case Studies: Red blood cell (RBC) transfusion is often a lifesaving measure for patients with sickle cell disease (SCD).
It is critical in the management of SCD complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome.
A wellrecognized complication of chronic transfusion in SCD patients is alloimmunization to RBC antigens.
To prevent alloimmunization, transfusion with RBCs negative for C, E, and K antigens has been advocated.
This has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions.
We report a summary of our three year experience with the prophylactic transfusion of RBC units negative for C, E, K antigens for SCD patients during red blood cell exchange transfusions (RBCx).
Study Design/Method: Retrospective review of 10 SCD patients with a history of stroke, refractory sickle pain crisis and priapism was done.
RBCx was performed every 4 to 8 weeks from December 2013 to March 2017.
Blood bank work-up used the MTS gel method for antibody screen and identification.
Our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner.
Results/Finding: A total of 10 patients, 3 females and 7 males, who underwent a total of 178 RBCx from October 2013 to March 2017, using an average number of 7 RBC units per RBCx.
RBC units negative for C, E, and K antigens were used during RBCx for 8 patients.
Two patients positive for C antigen underwent RBCx, using E and K antigen negative RBC units.
Review of the antibody screen test results performed prior to each of the 178 RCE showed that no new clinically significant alloantibodies were formed after exposure to multiple RBC units.
Conclusion: Although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for SCD patients, studies suggest that the standard of care for transfusion of all patients with SCD is to provide RBC negative for C, E, and K antigens.
This ability to find these rare units is also affected by the characteristics of one's institution and blood supplier.
It is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units.
The approach by our institution to transfuse RBC units negative for C, E, K or Study Design/Method: Venous blood specimens of 168 healthy volunteers were collected before blood donation and after blood donation immediately, 1 day, 1 week, 4 weeks, and 12 weeks among men and 16 weeks among women.
Immunoglobulin G (IgG), immunoglobulin M ( IgM) , immunoglobulin A ( IgA)and complement component 3 ( C3) , red blood cell (RBC), white blood cell count ( WBC) , hemoglobin (Hb), hematocrit (HCT), and serum iron (Fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality.
Results/Finding: the level of IgG slightly decreased after blood donated immediately, IgA and C3 decreased significantly but still within their normal ranges, IgM did not change after blood donation.
The level of IgA significantly decreased at 12 weeks among men and 16 weeks among women, while C3 significantly increased at the same time period.
IgG, RBC, Hb, HCT and Fe started to recover 1 week after blood donated and reached their levels before blood donated within 12 weeks among men and 16 weeks among women.
Conclusion: the biomarkers mutually changed over the course of 12 weeks among men and 16 weeks among women.
Donating 400 ml blood will not significantly affect overall blood quality.
Utilizing Amicus Dxt Relay Data Managment Solution to Increase Platelet Split Rate and Improve Amicus Productivity Janelle Wilhelm* and Jennifer Kaluza.
Memorial Blood Centers Background/Case Studies: With the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base.
We also faced the challenge of managing multiple collection sites in multiple states.
The decision was made to implement Amicus DXT Relay Data Management Solution to provide us insight into procedure details to make data driven decisions.
Day to day variability previously dipped as low 40% split forcing reactive planning.
Study Design/Method: Incorporate DXT to strategically plan our day to day operations.
DXT reports were monitored by management and with the Fresenius Kabi team for productivity by site, phlebotomist and device.
Reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis.
This allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction.
Reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate.
Results/Finding: The monthly DXT report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate 15 percent and increased concurrent plasma collections by 24 percent.
With utilization of the DXT reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than 62 percent split.
Phlebotomist QNS rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall QNS rate to consistently below 3 percent.
Conclusion: DXT was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers.
DXT provides invaluable tools for the Operational Supervisors to monitor their staff and improve productivity at their multiple sites.
Next step is to develop the plan for implementation of paperless documentation with DXT and Healthcare-ID.
The ability to immediately review data directly from Amicus was key in the productivity improvements realized.
Evaluating the Impact of a Background/Case Studies: As blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety.
It is a prerequisite for all Registered Nurses (RNs) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice.
Aim: The aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program.
The results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme.
Study Design/Method: All RNs from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in October 2015.
After which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions.
The same questionnaire was administered to the RNs one year later in September 2016 for post-training programme evaluation.
Individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation.
Results/Finding: In 2015 and 2016, a total number 1,097 RNs and 965 RNs completed the questionnaires, giving a response rate of 78.5% and 67.4% respectively.
The overall mean score in 2015 was 6.24 points (range 0 to 8).
The mean score in 2016 was 6.57 points (range 2 to 8).
The percentage of RNs having perfect scores of 8 increased from 8.8% in 2015 to 20.5% in 2016.
Table I below shows the results for each question item.
The implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst RNs.
Further training may be needed in the preparation of blood sets and management of fever.
Background/Case Studies: Clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain.
This global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses.
Study Design/Methods: A descriptive 'ex-post facto' survey design was used; 264 purposively selected blood prescribing clinicians and nurses from 60 hospitals in 13 countries of the 4 Human Development Index (HDI) groups (Low, Medium, High, and Very High) participated.
Three research questions were answered, while seven null hypotheses were tested at .05 level of significance.
Descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -Pearson Product-Moment Correlation Coefficient (PPMC), Analysis of Variance (ANOVA), were used to analyse the hypotheses.
Results/Findings: Quality of clinical use of blood was positively and significantly correlated with levels of awareness (r5 .137; p5.03; df5262) and accessibility (r5.184; p5.01; df5262) to e-continuous learning among blood prescribing clinicians/nurses.
There was significant difference in levels of awareness [F(3,260) Conclusion: Today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood.
The higher the HDI level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs.
There is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion.
Accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the Low and Medium HDI countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors.
The results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes.
Background/Case Studies: Transfusion medicine (TM) didactic teaching materials for pathology residents are not widely available to share among residency training programs.
The Advancing Blood KnOwledge (ABO) Leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (CoP).
Educational theorist Etienne Wenger defined CoPs as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly.
Study Design/Method: As a pilot project, 7 junior faculty co-investigators from 5 west coast institutions each had 2 months to create a 30 minute PowerPoint presentation on a fundamental TM topic, after which 2 other members had 2 months to review and edit.
Therefore, each member created 1 and reviewed 2 presentations (three total steps).
During each step, members wrote 2 multiple-choice questions for those particular topics.
In the end, each topic would have 6 quiz questions to assess learning.
At completion, 7 evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents.
Three methods were planned to measure effectiveness of these materials: 1) Pre and postlecture ABO Leaders exam using the questions made for each topic to assess learning; 2) Pre and post-lecture 20 question validated examination (BEST Collaborative) to assess learning; 3) Resident In-service examination trends specific to TM.
Results/Finding: Six presentations were developed as 6 of the 7 ABO Leaders members continue to participate in this CoP for TM education.
ABO Leaders and BEST pre-test results are shown in tables 1 and 2.
ABO Leaders pre-test data could not be obtained for institution B, and 3 trainees declined to participate in the examinations at institution A.
Challenges experienced by the CoP have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all 6 presentations during the defined institution-specific teaching period.
Post-test results will be included when assessments are complete.
Conclusion: Despite logistical and organizational challenges, it is feasible to create a multicenter CoP for TM education.
The impact of such a group on resident learning will be assessed and plans for growth will be evaluated.
Background/Case Studies: The traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases.
The use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine.
Our goal is to use and evaluate the relevance of this approach in resident education.
Study Design/Method: A clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation.
During a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics.
Each resident completed a 10 question preand post-test on topics related to the vignette.
Several meetings for review and adaptation of the script, topic discussion, and rehearsals were held.
There were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance.
During the skit, each resident presented at least one major transfusion management teaching point.
Results/Finding: The educational activity, including the 40 minute vignette performance and the 20 minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis.
All performers significantly improved their scores in the post-test (mean 95 1 4%) when compared to the pre-test scores (mean 67 1 26%) ttest p<0.017.
During the vignette discussion, residents together identified all the intended non-conformances and answered related questions.
Residents in the audience actively participated in the post skit discussion and 90% reported a satisfactory learning experience.
Conclusion: Dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents.
With specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember.
The skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care.
Hannele Sareneva*, Susanna Sainio, Inna Sareneva, Tiia Kivipuro and Taru Jaske.
Finnish Red Cross Blood Service Background/Case Studies: The Finnish Red Cross Blood Service (FRCBS) is the nationwide blood service provider in Finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers.
The FRCBS serves as the National Blood Group Reference Laboratory and provides a wide range of other laboratory services e.g.
tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation.
FRCBS also performs antenatal blood group and RBC antibody tests covering whole country.
As a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services.
Study Design/Methods: We have performed customer surveys to healthcare professionals to assemble the needs for education.
Based on these results and continuous feedback FRCBS provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * Handbook for Blood Products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports Regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians.
For every education we collect numerical feedback as following: "How did the education responded Your expectations" and "Can You utilize the knowledge in practice".
We also inquire "How likely You would recommend the training for Your colleges" indicating net promoter score (NPS).
Results/Findings: More than 350 healthcare professionals participate training days at FRCBS annually.
In addition our experts give tens of lectures at hospitals across Finland.
Feedback from educations has been very good, varying between 8.3 to 9.4 (in the range of 4-10).
NPS varies between 83 and 98.
According to customer surveys FRCBS provides appropriate education to healthcare professionals.
This score has increased 2011-2016 from 8.4 to 9.0.
Conclusion: Feedback, NPS scores and surveys ensure that education and training program of FRCBS responses to customer needs.
Hospitals can utilize annual courses of FRCBS in their own initiation programs.
Together with clinical contact persons in hospitals our aim is to ensure Patient Blood Management (PBM) and to optimize use of blood products.
We also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students.
Educational Outreach and Effect on Reporting Septic Transfusion Reactions Kathleen M Grima* 1 , Anne Eder 2 , Beth A. Dy 1 and Mary O'Neill 1 .
1 American Red Cross, 2 Georgetown University Background/Case Studies: Hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center.
About 1 in 100,000 apheresis platelet donations are implicated in septic transfusion reactions (STRs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions.
A large blood center designed an educational outreach program to increase awareness of STRs and assessed its effect on the rate of STR reporting to its national hemovigilance program.
Study Design/Method: In Dec. 2015, a large blood center developed a web based course on STRs for CME/CEU credit.
Letters were sent to 2,300 hospital customers about recognizing and reporting STRs, and alerting them to the availability of the course.
Blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting STRs, using the online educational content.
The physicians tracked their interactions.
The blood center's national hemovigilance program compared the number of STRs reported in the 12 months before and after launching the educational outreach.
Results/Finding: The web based course was completed by more than 700 participants; 117 were physicians.
Based on a review of the evaluations, the course was highly valued with 93% of participants rating it excellent or very good.
The blood center physicians gave over 200 presentations to hospital customers.
Reporting of suspected STRs in 2016 increased by 23% compared to the prior year.
The increased reporting came from 2 specific regions.
The total number of STRs that met the hemovigilance definitions for definite (culture-confirmed) and probable STRs in the nationwide system increased but did not change significantly compared to the previous years.
The educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of STRs.
While the number of reports of suspected STRs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed STRs across the national blood system.
This finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside.
More targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine.
Implementation of Subscription-Based cGMP e-Learning Laurie McGraw*, Courtney Saphier, Helene Belton, Sallie Bittner and Ward Scott.
Gulf Coast Regional Blood Center Background/Case Studies: Previous cGMP e-learning courses we developed required 30-45 minutes for learners to complete.
While feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules.
At the same time, a shift in design trends suggest that subscription-based learning is more effective (Thalheimer, 2014.)
Subscription-based e-learning utilizes 5-7 minute modules, delivered at regular intervals.
This changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind.
Study Design/Method: We began developing cGMP subscription-based elearning in 2016 by selecting our first five series topics: Equipment, Personnel, Labeling, SOPs, and Records.
The first topic, Equipment, was divided into modules on Selection, Validation, Calibration, Quality Control, and Maintenance.
These modules, and pre-and post-quizzes for the Equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system.
The pre-quiz was assigned to employees in June 2016, with a new Equipment module assigned each month for the following five months.
The series concluded in December 2016 with the post-quiz.
Results/Finding: Using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four Kirkpatrick levels.
While our previous cGMP courses received good ratings from learners, the Equipment series received the highest rating of 3.5 on a 4-point scale.
Of employees who completed all versions of our cGMP courses, the majority preferred the Equipment series over all previous courses combined.
Comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with 21 positive and 1 negative comment.
Level 2: Learning The average score of users increased 13% from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees.
A two-sample t-Test determined the result to be statistically significant with a t-Critical value of 1.647 and a t-Stat value of 5.641.
Level 4: Results While equipment-related errors decreased by 20% after training, there is not enough data to demonstrate a statistical significance.
Conclusion: Our Level 1 and 2 evaluation data validated that the subscription approach was effective.
Knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements.
As a result, we are continuing development of the remaining series.
Background/Case Studies: The interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams.
We hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in October 2013, a multidisciplinary educational meeting called Friday Blood Conference (FBC) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice.
During these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion.
Study Design/Method: A survey was sent to FBC participants (n5151) to retrospectively capture the effect of FBC on interdepartmental collaboration.
The survey was structured to obtain formative feedback using the published Interprofessional Collaborative Practice Competencies (ICPC) as a guide.
These core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care.
Results/Finding: Our survey response rate was 35%.
Of those, 96% endorse that FBC creates a climate of respect within our transfusion practice, 94% believe it has improved communication between work units, and 98% feel that FBC leads to increased understanding of interdepartmental processes.
Notably, laboratory scientists and transfusion nurses have the highest attendance rate.
Furthermore, those attending via the online broadcast report the lowest satisfaction, with only 56% responding positively.
The main reasons individuals attend FBC are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care.
Suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of FBC for later viewing.
Conclusion: The application of ICPC in transfusion medicine was an effective lens to assess the value of interprofessional collaboration.
Although there is room for improvement, the results support that FBC has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice.
Novel Approach to Curriculum Development: Demystifying Transfusion Medicine Ritcha Saxena* and Ananya Saxena.
All Saints University School of Medicine Background/Case Studies: Transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel.
Transfusion carries considerable advantages as well as risks.
Consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps.
And the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine.
Study Design/Methods: 41 students of undergraduate Semester 3 and 59 students of Semester 4 participated in the study.
Self-directed learning resources combined with modules of interactive instruction were implemented in a TBL course design.
Five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students.
The students' reaction to TBL in Transfusion Medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity.
The participants were first assessed with Readiness Assurance Testing (RAT) to guarantee that they understood the concepts and their application followed by case study based test questions.
Results/Findings: Students' reaction to TBL was primarily positive, with 86% of students giving a positive feedback.
Evaluation through Readiness Assurance Testing (RAT) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study.
Students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications.
Anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation.
Conclusion: Our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians.
TBL is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students.
Results/Finding: Open house attendees were given tours of the BB, led by a BB attending, BB residents, BB supervisor, or BB Quality Coordinator.
The Patient Blood Management nurse was also in attendance to answer attendee questions and educate about Patient Blood Management.
Light refreshments were offered to the attendees in the BB break room.
The first BB open house was held on Wednesday, 12/7/16 from 9-11am.
There were 17 attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in Facilities Management and the University Office of Admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the University Office of Admissions.
The second BB open house was held on Thursday, 4/20/17 from 7-11am.
There were 14 attendees, including 2 regular blood donors (who were also employees in the Office of International Affairs and Supply Chain, respectively), a hematology/oncology fellow, and 11 surgical residents.
Background/Case Studies: Simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (SCD) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells.
It is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure.
Given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks.
Study Design/Method: A web application was generated (www.Phamcalcs.com).
The performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data.
The performance of the automated and depletion RBCx calculators was validated using the Terumo BCT (Lakewood, CO) calculator up to a fraction cells remaining (FCR) 50% as patients with FCR 50% may benefit from delaying the procedure for performance in the future.
Validation process included (1) a Deming regression to globally assess the predicted vs. actual results and (2) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |D| 15%.
Validation was performed for hematocrit (Hct) and hemoglobin S (HgbS) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion RBC exchange.
Results/Finding: See Table 1 Background/Case Studies: With the focus on new technologies the modern medicine requires more expenses.
Despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment.
The issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency.
In Russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs.
As a multidisciplinary approach, PBM optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution.
However, the prosperous implementation of PBM also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist.
Study Design/Method: At the Medical Simulation Centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion.
The main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods.
During 2 days (18 academic hours) the trainees can attend 6 lectures, discuss the methodical materials, participate in 3 seminars, 2 interactive clinical discussions, a Master Class and a game that presents the modelling of working processes.
Since initiating the project in June, 2016 with the group capacity 220A TRANSFUSION 2017 Vol.
57 Supplement S3 of up to 35 people the number of medical specialists who have attended the training is nearly 450.
Results/Finding: The medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards Conclusion: The launch of the program "Guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of Patient Blood Management and reach the Compliance in practice.
The Effect of Emergent Situation Drills on Technologist Teamwork and Comfort Levels Abigail Neils*, RaeAnne Stensgard, Rebecca Wren, Elisabeth Greer, Amy Mata and Camille van Buskirk.
Mayo Clinic Rochester Background/Case Studies: Teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously.
In an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (ESD).
The ESD were based on common emergent situations encountered in the lab and were run once per month per shift.
The main goal of ESD was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (SOP) deviations.
Study Design/Method: Prior to ESD implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations.
One year post ESD implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations.
The surveys asked employees to rate satisfaction and comfort level on a grading scale of 1-100; 1 being least satisfied/comfortable and 100 being most satisfied/comfortable.
The pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey.
In addition, unplanned SOP deviations related to emergent situations were counted for one year before and one year after ESD implementation.
Results/Findings: Out of 35 total technologists, 31 technologists took the pre ESD survey and 25 technologists took the one year post ESD implementation survey.
Table 1 shows the lab averages from the pre and post surveys as well as the percent difference.
Out of the employees who responded to the post survey, 19 (76.0%) answered "true" to the statement "ESD have improved my comfort level with emergent situations."
In the year prior to ESD implementation there were 14 unplanned SOP deviations; in the year after ESD implementation there were only 5 deviations.
Conclusion: All but one area increased in comfort level post ESD implementation.
Also most technologists agreed that the ESD helped improve their overall comfort level with emergent situations.
The goal of implementing ESD has been met based on the unplanned SOP deviation decrease and technologist satisfaction increase; therefore ESD were deemed effective.
Monthly ESD will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels.
Therapeutic Background/Case Studies: Category I indications for red blood cell exchange (RBC exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention.
As described in the first installment of this series about therapeutic plasma exchange (TPE), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population.
RBC exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood.
Nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care.
Providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery.
A previous project explaining TPE to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including TPE and RBC exchange.
Study Design/Method: In collaboration with a Child Life Specialist, an ageappropriate story-driven explanation of the RBC exchange procedure was adapted from a previously implemented project related to TPE.
Artwork was produced with the aid of a medical illustrator to complement the story-line.
Results/Finding: The story board addresses why RBC exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure.
The idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy.
The booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use.
Conclusion: Using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety.
This second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies.
Transfusion Safety Officer Resource Manual Leonor De Biasio*.
It is also intended to be utilized by hospitals that do not have a formal TSO position but which have delegated the responsibilities to other staff.
The resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood.
The resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the TSO role.
Turning on Pathogen Reduction: A Case of Flipping the Switch Kassandra Poffenberger*, Darla Wendt, Jennifer Vrieze and James R Stubbs.
Mayo Clinic Background/Case Studies: A critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care.
With the implementation of Pathogen Reduction Technology (PRT) using INTERCEPTV R Blood System for Platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections.
Our institution collects the majority of its blood products and supplements inventory from a major blood collection center.
It was crucial for the Component Laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors.
Our approach in introducing PRT for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory.
Study Design/Method: It was essential to prioritize who would be trained first.
Collections occur Monday through Friday from 0600 to 1600.
The first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by Cerus deployment team.
The second group was those who would process platelets on weekends and evening hours without direct management support.
The last group was the technologists who would be working during normal hours with direct management support.
PRT processing for platelets in 100% plasma is broken up in to two days.
On Day 0 platelets are treated with amotosalen and placed in a Compound Adsorption Device to remove residual amotosalen for 12-24 hours.
On Day 1 products are removed from the CAD and modified into final product codes and labeled.
Each technologist was trained one on one, over a one week period.
The trainers alternated training processing days for Day 0 and Day 1.
In the weeks following training it was important that each technologist rotated back thru PRT processing to maintain proficiency.
Results/Finding: 13 of 18 employees were trained in a two month time period.
Prior to "flipping the switch" the daily average of products collected was 21.
For the two month training period the daily average rose to 23.
Conclusion: Our "flip the switch" training plan for implementing PRT platelets in 100% plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process.
It was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts.
Technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory.
accepted depending on each individual's conscience.
Due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care.
We describe the process of identifying JW in our hospital and communicating treatment needs to staff.
Study Design/Methods: Proper treatment of JW requires the ability to identify the patient and his/her needs.
When a JW is admitted to our hospital, our electronic medical record (EMR) triggers several processes based on the patient's listed religion.
One process creates an order that reminds caretakers to complete the Declining Blood Consent (DBC) with the patient.
The DBC contains language declining MABF and reviews the MIBF with the patient to identify any that would be accepted.
The EMR order regarding the DBC provides educational links that include a bloodless policy, step-by-step instructions on obtaining the DBC, and information on alternatives to transfusion.
A second EMR process triggers a stop-gate to prevent the completion of any MABF order or MIBF order for a product that the patient has declined.
A third enrolls patients in the Minimal Blood Volume Labs Protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws.
Additionally, at Registration, a Bloodless Packet is added to the patient's paper chart.
This Packet contains the DBC, a glossary of DBC terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart.
These steps remind the caretakers of the patient's special requests.
Finally, the Patient Blood Management (PBM) Department receives EMR developed reports which identify JW presenting to the hospital.
These patients are followed by the PBM nurses and medical director during the duration of care.
Treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed.
Results/Findings: Nearly 100% of JW that enter our hospital have a DBC completed.
This has resulted in increased education of the medical staff.
In addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients.
Conclusion: Our hospital has found success by using an education-based team-oriented approach involving EMR, PBM, and caretakers when caring for the JW patient.
This approach has set up a foundation for treating other bloodless medicine patients.
Background/Case Studies: Transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers.
This process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes.
Cost management has a great impact on technical processes guiding decisions upon supplies and technical staff.
Activity-based costing (ABC) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions.
We demonstrate how an effective ABC approach can result in financial savings without compromising process quality in a mid-size transfusion service.
Study Design/Method: Materials costs can represent as far as 90% of an activity.
In 2015 we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock.
Purchases were performed on demand.
At the end of 2015 we performed a supply inventory on all departments to plan future purchases and control residual stocks.
In 2016, we implemented annual purchases and satellite storages were supplied only to replenish programmed stock.
Cost drivers were defined upon activities on standard operational procedures (SOPs) resulting in a 2016 cost estimate.
Technical staff was involved in cost driver calculations to indicate possible changes to SOPs, supplier and deliveries.
To minimize seasonal fluctuations we compared last quarter 2015 (Q4/15) with last quarter 2016 (Q4/16).
In this work we present activity data from blood collections to illustrate ABC method.
Results/Finding: In Q4/15 1756 blood bags were used compared to 1998 in Q4/16, demonstrating an activity "13.78%.
Price negotiation resulted in 12.58% readjustment.
Both indicated an estimated cost "28.10% with a possible impact of over US$ 35,000.
We have identified a real cost #2.31% in Q4/16, representing an overall #14.89% and US$ 3,716.72 (R$12,235.16) savings.
Conclusion: Economy had deteriorated in our country in 2016 with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical.
Even with adverse economy, ABC showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes.
Cost drivers calculations demanded review of SOPs and suppliers by technical staff resulting in optimization of activities.
Also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly.
Automated Verification of Immunohematology Results and the Impact to Donor Testing Barbara J Bachman* 1 , Candace Williams 1 , Carmen Meyer 2 , Paul Lamonby 1 , Anne Cleverley 1 and Silke Milbradt-Pohan 1 .
1 Bio-Rad Laboratories, 2 Diamed Gmbh Title: Automated Verification of Immunohematology Results and the Impact to Donor Testing Background/Case Studies: Staffing challenges in today's blood banks require instrumentation with minimal operator intervention.
Technology advances have developed where every immunohematology result does not necessarily require operator visual review.
This study evaluates the impact of automated result verification on the Bio-Rad IH-1000 TM Immunohematology System through the IH-Com TM Data Management System (DMS) for donor processing laboratories.
Study Design/Method: A multi-center study was performed on donor samples as shown in Table A evaluating two of the most commonly used IH-System Gel Cards available in the US.
Workflow data was analyzed using Process Modellar APP (iPad).
This study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to LIS data transfer).
Operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined.
Speed metrics were analyzed using Minitab v17, statistical The transfusion team collaborated with multiple user groups to educate them regarding the new processes.
A gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options.
The use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus.
Results/Finding: User groups requested additional training sessions as questions arose regarding use of the eHR for blood ordering.
Because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood.
Department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order Rh immune globulin, a cord blood workup, etc.
Conclusion: Leadership was challenged to provide a stable and positive environment during a complex set of changes.
The simultaneous hospital move/merger and implementation of a new eHR constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance.
Training is essential to the success for a scope of change this big and should not be minimized.
While training was thorough prior to the move, gaps were nonetheless discovered following the move.
ABSTRACT Conclusion: strategic development partners funding and support based on newly developed government strategy on Blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in Ethiopia.
Even though the identified positive impacts mentioned are achieved, the BTS remains with multiple challenges and needs continuity of funding and more partner support and government commitment.
Pilot Implementation of a Comprehensive Hybrid Performance Management System at National Blood Service Zimbabwe Blessing Mukwada*, Judith J Parirewa and Tonderai Mapako.
National Blood Service Zimbabwe Background/Case Studies: The National Blood Service Zimbabwe (NBSZ) introduced its first Performance Management System (PMS) in 2006.
In the 2015-2018 NBSZ strategic plan it was noted that the current PMS lacked objectivitety and there was no relationship between perfomance and remuneration.
In order to revise the PMS, the NBSZ set up a three membered committee at the Executive Management level to spearhead the revamp of the NBSZ PMS.
The aim of the new PMS was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan.
In this paper, we share how NBSZ revamped and implemented its new hybrid PMS that derived its inputs from established PMSs and NBSZ monitoring and evaluation (M&E) process that have been linked together.
One-selected departmental results for one quarter are shared to demonstrate how the system works.
Study Design/Method: PMS Committee developed and shared with Executive Management a PMS conceptual and implementation framework.
Consultations including field visits were done on three established PMS to assess suitability for NBSZ adoption.
A hybrid PMS was adopted for NBSZ and a pilot application for one quarter on selected department was done.
Review of policy, procedures to including hybrid PMS templates and forms were done.
PMS Committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new PMS, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools.
Throughout the process risk assessment were done.
Results/Finding: The NBSZ Hybrid PMS is based on five levels of planning namely Strategic, departmental, branch, sectional and individual.
The fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels.
The levels of accountability were properly defined for each level of planning.
A weighted overall integrated individual scorecard (IIS) is determined based on 60% individual and 40% for the other four levels (10% for each).
The bonus (%) is calculated based on the IIS as follows; Category A: 100% (IIS >575%), Category B: 75% (IIS: 50 -<75%), Category C: 50% (IIS: 25-<50%) and Category D: 0% (IIS < 25%).
On the pilot implementation, the individual scores for 12 staff ranged from 71% to 100%.
The IIS were 76% to 81%.
The number of staff in each bonus categories were 11, 92% (Category A) and 1, 8% (Category B).
Conclusion: The new hybrid PMS was generally accepted by all staff and it was easily implemented at various staff levels.
This provides a basis for the full implementation of the new PMS and this simplified PMS can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision.
Rare Donor Engagement with American Rare Donor Program (ARDP) Margaret C Manigly* 1 , Deborah R Fludd 1 and Sandra J Nance 2 .
Background/Case Studies: Rare donors are defined as a blood type occurring in less than 1 in 1000 people in a given population.
These donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor.
Once found, if the facility is a member of the American Rare Donor Program (by being an AABB Accredited or American Red Cross accredited IRL), the donor is registered in the ARDP database as a rare donor.
In 2016, there were 65,801 active rare donors in the ARDP.
With the mobility of the population in the USA, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need.
In addition, when recruitment is needed for a patient need, correct contact information on the donor is required.
Study Design/Method: The ARDP procedure for ARDP members requires that donors be contacted every six months to ensure that ARDP (or the facility) has their latest contact information.
The timing is determined by the postal service time limit of six months to forward mail to a new address.
This contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate.
This contact is achieved by ARDP sending a contact card by postal mail twice yearly to all donors for whom the ARDP has address demographics.
Results/Finding: The ARDP reports on the information obtained from the contact cards returned in the ARDP Annual Activity Report to the ARDP Members at the AABB Annual meeting.
Of the 6398 (9.7% of total active donors) returned contact cards alerting ARDP of changes in calendar year 2016, 355 (5.5%) were donors moving from one ARDP facility to another, 1369 (21.4%) were donors no longer eligible to donate, and an additional 4324 (68.4%) were address changes.
Other changes were 115 (1.8%) reactivated donors and 235 (3.5%) donors who we were notified were deceased, or did not want to be listed in the ARDP.
In 2016, 5390 new rare donors were submitted to ARDP for registration.
The number of donors that could potentially be lost to follow-up in 2016 was 4709 (355 1 4324), which would be 87.4% of the new donors submitted.
Conclusion: With nearly a 10% response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ARDP contact card, and inform ARDP of address changes and changes in their health status that affects their ability to donate.
This is evidence of the importance of the card in ensuring correct donor contact information.
In 2016, 4709 donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move.
The ARDP contact card is effective in retaining the relationship with the ARDP registered donors and keeps the address information of rare donors current.
Workflow Comparison of Two Gel Analyzers in a Large Transfusion Service J Peter Pelletier* 1 , Barbara J Bachman 2 , Mike Leamy 2 , Susan Olson 2 and Candace Williams 2 .
1 University of Florida College of Medicine, 2 Bio-Rad Laboratories Background/Case Studies: Vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market.
The purpose of this study was to evaluate the ProVue (Ortho Clinical Diagnostics) against the IH-1000 (Bio-Rad Laboratories, Inc.) in a large volume transfusion service using LEAN process flow.
Study Design/Method: Twenty-two (22) runs of one to six (6) samples per run were observed for two Ortho ProVues alternating testing at a large Transfusion Service performing 153,000 Types, Screens, Type & Screens (T&S) annually.
The workflow patterns observed were then repeated on the IH-1000 and compared.
Each process was mapped in detail by direct observation using Process Modellar APP (iPAD).
The evaluation started at sample centrifugation completion and ended with results sent from analyzer to LIS (Lab Information System).
Each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured).
Time studies were analyzed using Minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant.
Regardless of quality or speed metrics evaluated, the IH-1000 demonstrated a significant reduction (improvement) in process steps and associated times when compared against the Ortho ProVue (p < 0.001).
IH-1000 process steps and time studies addressed in the table below did not account for the IH-1000 reagent storage capacity.
In reality, the improvements would be greater than what was displayed here in a real-life operation.
Evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the IH-1000 (77% reduction, a difference of 43 hours/year).
Conclusion: This study verified the IH-1000 provided significant efficiencies and cost avoidance over the Ortho ProVue for a large volume Transfusion Service.
Workflow Comparison of Two High Volume, High Throughput Analyzers Aaron Samson* 1 , Kimberly Monnin 1 , Barbara J Bachman 2 , Kyla Warren 2 , Susan Olson 2 and Candace Williams 2 .
1 Clinical Pathology Labs, 2 Bio-Rad Laboratories Background/Case Studies: Few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities.
The purpose of this study was to evaluate the Galileo V R Neo (Immucor) against the IH-1000 TM (Bio-Rad Laboratories, Inc.) using LEAN process flow.
Study Design/Method: A total of 12 separate test runs of 72 or 144 samples per run were observed over a three day period on the Galileo Neo at a reference laboratory annually performing approximately 211,500 Type & Screens (T&S).
The workflow patterns observed were then repeated on the IH-1000 and compared.
Each process was mapped in detail by direct observation using Process Modellar APP (iPAD).
The evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to LIS (Lab Information System).
Each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured).
Time studies were analyzed using Minitab v17, and statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant.
Results/Finding: Detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (Table, Part A) .
Time studies focused on operator time, analyzer time, and maintenance time (Table, Part B).
Regardless of quality or speed metrics evaluated, the IH-1000 demonstrated significant reduction (improvement) in process steps and associated times when compared against the Galileo Neo (p < 0.001).
Evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the IH-1000 (difference of 120 hours/year).
Conclusion: This study verified the IH-1000 provided significant efficiencies and cost avoidance over the Galileo Neo for high volume/high throughput testing facilities.
Workflow Impact of Automated Result Verification for Patient and Donor Blood Typing Barbara J Bachman* 1 , Candace Williams 1 , Carmen Meyer 2 , Paul Lamonby 1 , Anne Cleverley 1 and Silke Milbradt-Pohan 1 .
1 Bio-Rad Laboratories, 2 Diamed Gmbh Background/Case Studies: Immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety.
To alleviate these challenges, the IH-1000 TM instrument and complementary IH-Com TM Data Management System (DMS) were designed to provide lean automation to enhance blood testing facility workflow.
The purpose of this study was to focus on the lean functionality of automated result verification on the IH-1000 and IH-Com DMS and determine its impact on workflow.
Study Design/Methods: Internal and external studies using the IH-1000 with the IH-Com DMS were performed with patient and donor samples.
Assays included ABO/Rh blood grouping and antibody screening (ABS) as shown in Table A .
Workflow data was analyzed using Process Modellar APP (iPad).
The evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to LIS data transfer).
Operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined.
Speed metrics were analyzed using Minitab v17.
Statistical significance was assessed using the paired t-test, with p values of <0.05 considered significant.
Results/Findings: Using automated result verification, only 0.93% out of 6,339 samples evaluated for ABO/Rh testing would require visual verification, resulting in a 98% reduction in operator touchpoints (p < 0.001) and a labor saving of 444 minutes (7:01 hh:mm) for ABO/Rh testing.
For 8,750 antibody screens, automatic validation of results would result in 99.5% reduction in operator touchpoints (p < 0.001) and a labor savings of 378 minutes (6:18 hh:mm).
No false positive or false negative typing results or false negative screenings occurred with results auto-verification.
(RBC) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management.
Over the past 2 years a steady increase in demand for O Neg RBC compared to other blood types has been observed at our blood center.
Utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits.
Despite heightening awareness, percent O Neg RBC sales continued to rise by 1% annually and peaked at 16% in mid 2016.
To better understand this increased demand a survey was conducted to gather insight for improved utilization.
We speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of O Neg RBC sales.
Study Design/Methods: A tie tag was designed as a survey tool and attached to each O Neg RBC distributed to hospital customers for an 8week period in late 2016.
Hospital transfusion service staff were asked to record the final disposition of the O Neg RBC (transfused, wasted, returned) on the tie tag.
Information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers.
Completed tags were returned to the blood center.
Customers are allowed to return RBC units with greater than 10 day shelf life remaining.
Units with tie tags attached were in hospital inventories for up to 3 months due to the shelf life of RBC.
Return rates and percent of net sales (gross sales minus returns) by ABO/Rh type were tracked monthly before, during and after the survey.
Results/Findings: Participation was 100% of the 56 hospitals surveyed.
Mean percent O Neg RBC gross sales for a 3 month period before, during, and after the survey was 16.6%, 16.0% and 16.5%, respectively.
Mean percent O Neg net sales during the 3-month survey fell to 13.5% compared to an average of 15.4% in the 3 months prior.
During the 3-month survey period O Neg RBC monthly return rate increased to an average of 28.4% compared to an average of 23.0% in the 3 months prior.
For the 3 months after the survey the average O Neg RBC return rate further increased to 28.9% while mean percent O Neg RBC net sales trended slightly upward to 14.1%.
When customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported.
Conclusion: During and after the survey percent O Neg RBC gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged.
However, during the same period the increase in O Neg RBC return rate and corresponding decline in percent net sales suggests improved O Neg RBC utilization.
Increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent O Neg RBC net sales.
Tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving O Neg RBC utilization.
Acoustophoretic Separation of Platelets from Whole Blood: A Relevant and Practical Alternative to Centrifugation Pierre Bohec* 1 , Jeremie Gachelin 1 , Veronique Ollivier 2 , Thibaut Mutin 1 , Xavier Telot 1 , Benoit Ho Tin Noe 2 and Sandra Sanfilippo 1 .
1 Aenitis Technologies, 2 Hôpital Bichat, INSERM U1148 Background/Case Studies: Shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates.
Transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions.
Aims: Here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets.
Study Design/Method: Whole blood was obtained from 14 donors and fractionated using an acoustic-based device.
Platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples.
Quality of isolated platelets was evaluated using the surface expression of two activation markers (P-selectin, PAC1) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation.
Platelets isolated using a soft-spin protocol, were used as inactivated control.
Results/Finding: Fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than 80 % (p< 0.001) and a purity of platelets close to 91.0 %.
We did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol.
Conclusion: This acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings.
Automation in Blood Bank Processing: Where We Go?
Robert Fernandez, Lluis Puig, Pilar Ortiz, Joan Ovejo, Nuria Martinez, Elena Valdivia and Susana G Gomez*.
Banc de Sang i Teixits Background/Case Studies: Nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production.
At Banc de Sang I Teixits (BST), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over 250.000 blood donations for an area with more than 7 million people.
Study Design/Method: The automation of blood donations process, BST has done different changes on the equipment.
In 2005, Orbisac (Terumo BCT) was the equipment to obtain buffy coats and from this product, we got platelets concentrates.
It was in 2007, when we moved from this equipment to Atreus 2C (Terumo BCT), to get red blood cells, buffy coat and fresh frozen plasma.
Then we did some updated on Atreus; in 2011 we changed to Atreus 3C (Terumo BCT) and finally in 2013, we moved to Reveos system (Terumo BCT).
Since the changes in 2007, our blood components were red cell concentrate, plasma, platelets and a leukocyte residue.
While all these changes in processing equipment, we added also some automation in our registration (donation ID, weight and temperature) and labeling steps, implementing two homemade robots.
And finally, to get better results and more efficacy in our production, in 2009, BST incorporated an engineer to introduce lean manufacturing methods.
These methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure.
Results/Finding: Once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations.
This evaluation was done for processes during 2008 and 2016.
Conclusion: With these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way.
We encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary.
In a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank.
Background/Case Studies: The laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of Remote Blood Allocation Devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload.
As part of this initiative, BloodTrack HaemoBanks (HB) (Haemonetics, Braintree, MA) were installed and interfaced to the existing SafeTrace Tx (Haemonetics, Braintree, MA) Laboratory Information System.
One HB was installed in the Methodist Hospital (RMH) campus which includes a busy outpatient Infusion Therapy Center (ITC).
Study Design/Method: An assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff.
Frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times.
For the ITC, with a daily outpatient volume of 140-160 patients and an average, round-trip travel time of approximately 15-minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects.
ITC nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care.
About 30% of patients in the ITC have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow.
Often for patients in the ITC, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood.
These inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the ITC.
Results/Finding: HB devices allow nursing staff to access red blood cells (RBC) for the majority of their patients at the point of care.
Since implementing in November 2015, the HB has significantly improved the turnaround time of RBC issue -from 15-minutes to less than 60-seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window.
Prior to HB implementation, blood bank staff at RMH were issuing approximately 750 RBC per month out of the window for non-surgical patients.
This has been reduced to approximately 300 RBC per month, a 60% average monthly reduction.
Conclusion: Having the HB located in the ITC has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders.
The use of HB devices has not resulted in a reduction in blood bank FTE, but rather a shift in workload; from issuing products to monitoring inventory and restocking.
consists of registered paramedics that are pararescue specialists and helicopter personnel.
When in combat, the squadron conducts personnel recovery operations and rescues downed airmen.
When stationed in the US, they mitigate in state emergencies and perform aeromedical evacuations.
In 2015, they supported a civilian medical emergency and the patient needed a transfusion in the field.
They procured blood products from a distant air force base with adjacent medical facility.
At the debriefing, members of the 131 st Rescue Squadron (131 RQS) decided to find a local civilian blood supplier.
The Master Sergeant contacted our blood center and set up a contract for blood supply.
Study Design/Method: Blood center representatives met with the 131 RQS Master Sergeant in January 2016.
We asked what 131 RQS's order and delivery expectations were.
He said sporadic use and the blood order would be 2 RBCs.
We wrote a procedure for consignment and packaging, using standard blood transport boxes.
We developed a communication template for staff to anticipate the 131 RQS needs.
Staff was trained based on data from January 2016 meeting.
We contacted the 131 RQS in September 2016 to perform a trial run.
At that time, we learned the Master Sergeant was shipped out to military theater.
We invited his replacement to the blood center.
This pararescue Senior Airman had just returned from Syria and was assigned to civilian duty.
He had no prior knowledge of the 131 RQS association with a civilian blood center.
Based on his field experiences, he changed the blood order from 2 to 6 RBCs.
He introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions.
We rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers.
The blood center and 131 RQS performed a mock run on October 31, and we felt prepared for any future events.
Results/Finding: On November 11, 2016, the 131 RQS was deployed to a civilian aeromedical evacuation.
We anticipated a 6 RBC order.
The actual order was 7 RBCs and 4 FFP.
Staff was preparing frozen FFP to ship, as was their norm for filling hospital orders.
Realizing that they could not thaw plasma in flight, we contacted the 131 RQS and offered Liquid Plasma instead, which they accepted.
Product was consigned and picked up at 4:30am by the 131 RQS.
The patient was transfused in the field and then taken to a nearby hospital.
At our joint debriefing on November 28 th , we established a maximum blood order of 10 RBC and 4 Liquid Plasma, noting future orders may request fewer products, yet meet the preferred 2 RBC; 1 Plasma transfusion ratio.
Conclusion: Military personnel are adapted to instantly adjust to an ever changing environment.
Regulated blood centers are not as adaptable.
With clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the Air National Guard.
(Table 1 ).
The highest mean Fib concentration was 535 mg/donor unit; lowest mean Fib concentration was 264 mg/donor unit.
All sites had a mean Fib concentration at least 100 mg/donor unit above the FDA minimum requirement of 150 mg/donor unit.
Fifteen of 17 blood centers completed the manufacturing process survey.
One used a leukocyte reduction filter with AHF destined plasma.
All blood centers manufactured single donor Cryoprecipitate; 12 manufactured pooled donor Cryoprecipitate.
Most froze plasma in a -188C or colder blood bank freezer.
One froze plasma using dry ice, and one used a blast freezer.
Two blood centers method of thawing frozen plasma took longer than 10 hours.
Conclusion: Blood centers consistently met the overall Fib minimum requirement with a mean of 345 mg/donor unit, over double the FDA requirement.
However there is variability in Fib levels amongst blood centers.
In general, manufacturing processes were similar with a few exceptions.
Blood centers should inform their hospital customers of their average Fib level in Cryoprecipitate in order to most appropriately care for patients receiving this product.
Compliance & Productivity Improvement Via Engineered-Staffing/ Scheduling Calculator Application (APP) Mary Deck, Mark Angelelli and Kevin Lee*.
American Red Cross Background/Case Studies: The healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure.
Applications of basic Industrial Engineering tools, coupled with Lean-Six Sigma techniques such as time study analysis, bottleneck elimination & process standardization to TRANSFUSION reduce variation has been transformed into an Application (for short "APP"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking.
Study Design/Methods: A time study analysis offers valuable data about the process requirements.
Once this baseline has been established, translating the data into a user-friendly APP would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations.
Important concepts such as Lean-Pull Production System, bottleneck elimination, work-load balancing together with basic development of the APP using MS Excel software will be demonstrated.
Results/Findings: Successful rollouts and implementations of the Staffing/ Scheduling Calculator APP across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard.
The APP interactive-based approach, programmed via a commonly used software, MS Excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e.
match incoming volume/work content to staffing availability).
The Staffing/Scheduling Calculator APP has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking.
Conclusion: Besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the Staffing/ Scheduling Calculator APP will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization.
All coolers were prepared in a walk-in refrigerator.
Two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with RBC units on top of the ice.
A quality-controlled thermometer was placed on top of the RBC units.
A control thermometer was place at the interface between the ice and the RBC units in one large and one medium cooler.
The start temperature was recorded and then the temperature was recorded every 15 minutes for a 12 hour period or until the temperature exceeded 68C.
Results/Finding: The temperature recorded from the thermometer on top of the units in all five coolers reached >68C in 75 minutes as shown in Table 1 .
The control thermometer recorded temperatures maintained at 1-68C for the entire 12 hour observation period in both the large and medium cooler.
Conclusion: When units are placed on top of the ice in a cooler, the temperature is not reliably maintained at 1-68C for more than 45 minutes.
These data support a policy of wasting units that are returned to the blood bank with RBC units on top of the ice.
Background/Case Studies: An FDA draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (PC) via pathogen reduction (PR) or secondary rapid testing (RT).
Hospitals must understand the cost implications that may result.
Our objective was to create an interactive model to analyze the budget impact for different PC types across the range of existing US hospitals.
Study Design/Methods: An Excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to 27 US hospital transfusion service directors.
The model was reviewed and refined by a panel of seven transfusion medicine physicians.
The model allows base-case assumptions to be overwritten with values specific to the institution.
Three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (AE), shelflife, and reimbursement for a hospital that purchases all of its PCs: 100% conventional (C-PC), 100% PR-PC, and mix of 75% C-PC/25% PR-PC.
The model predicts a modest ($4%) cost increase for PR-PC compared to C-PC depending on the degree of PR conversion; this takes into account cost offsets such as elimination of BD and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement.
The effective PC shelf-life is potentially increased with PR due to elimination of BD, and is dependent on NAT turnaround time.
Benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses.
Future iterations of this model will also enable hospitals to consider scenarios in which RT is used.
This model can serve as an important tool for hospitals considering PR adoption.
in January 2011.
A report was created to identify donors previously classified as rare according to the American Rare Donor Program (ARDP) criteria.
Donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or IgA deficient.
The new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate.
A database was created to track the letters sent to rare donors.
In August 2013, inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (RBC) collected to minimize unit age at transfusion.
The inventory reduction occurred in phases and was completed by January 2015.
A study was performed to determine the impact of the inventory reduction on the number of rare donor donations.
Study Design/Methods: The total number of allogeneic RBC donations, rare donor donations, and number of rare donor letters sent was analyzed from 2010 to 2016 (see Table) .
The percentage of rare donor donations per year was calculated.
Background/Case Studies: Blood Centers (Clients) often carry low inventory of blood and blood components.
Laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products.
In order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to Clients.
In 2016, ZKV-NAT testing was implement for travel deferral donors (July), followed by universal individual donor screening in September and November in response to the FDA recommendations for "Reducing the Risk of ZKV Transmission by Blood and Blood Components".
Per the FDA guidance we implemented mandatory ZKV testing for Clients with proximity to areas with locally acquired mosquito-borne cases of ZKV within 4 weeks (Sept.
Phase 1) and nationwide within 12 weeks (Nov.
Phase 2).
ZKV testing is performed on individual samples, unlike all other NAT tests that are performed in minipools (16-donations).
Therefore ZKV testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study.
Study Design/Method: Within two regional testing labs, participating in the same clinical trial, Lab 1 had 86% and Lab 2 had 77% of Clients requiring universal ZKV testing.
We evaluated a 12-month test result upload performance period to determine the impact of ZKV test implementation.
Results/Finding: During 2016, Lab 1 upload time performance ranged from 92% to 94.2% from January to July; upload time performance fell between August through November, returning to 94.2% performance in December.
Lab 2 upload time performance ranged from 91.4% to 95.3% January to August.
Performance fell September through December 83.3% -88.5%.
Lab 1 experienced a low of 75% upload time performance during Phase 1 when there was a rapid implementation; 69% Clients required ZKV NAT.
Improved performance was observed during Phase 2, with a 16% increase in ZKV Clients.
For Lab 2: Phase 1 experienced a modest decline of upload performance ranging from 83.3% to 88.5% with 33.3% of Clients implementing ZKV NAT.
Performance was 87.1% in Phase 2, when an additional 43.3% of Clients implemented ZKV testing.
Conclusion: With an unprecedented rapid implementation of ZKV testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric.
Enhanced Platelet Bacterial Screening in an Eight-Hospital System Robin Larson* 1 and Colleen A. Aronson 2 .
1 Advocate Lutheran General Hospital, 2 ACL Laboratories/ Advocate Hospitals Background/Case Studies: In response to two platelet-related septic transfusion reactions and the draft FDA guidance released in March 2016 regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the Verax PGD enhanced platelet bacterial screening test in 6 of the 8 hospital transfusion services.
The 2 sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier.
The 6 sites which implemented the Verax PGD test perform testing on all day 4 and day 5 platelets to be issued for transfusion.
This abstract summarizes the data collected for the first 5 weeks of testing.
Study Design/Methods: Platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day 4, day 5, and those that were tested twice.
Inventory reports were reviewed for platelets issued on day 2 or day 3 that did not require testing, and for the total number of platelets issued over the time period studied.
Results/Findings: In the month of February (1 week of performing the test), 48.1% of all platelets issued by the 6 participating transfusion services were day 2 or day 3 platelets.
In March that number dropped to 29.9%.
It is expected that this number will level off at some percentage at or below 29.0% with further data collection.
In February 25.9% of platelets were tested twice prior to final issue from the transfusion services.
In March Conclusion: The percent of platelets issued fresh (day 2 or day 3) will likely level off at some number at or below 29.9% due to inventory management from both the blood supplier and the individual transfusion services.
Testing platelets twice is undesirable.
Ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing.
In addition, 5 of the 6 sites performing testing are Level 1 Trauma Centers and need to have tested platelets available at all times.
This will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed 100%.
As the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease.
Background/Case Studies: In order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(PCs) for providing a reliable source for clinical application.
To speed up the storage research of pooled PCs in China, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for PCs and the quality changes during storage in PVC-BTHC blood bags.
Study Design/Method: PCs were prepared from 400 mL virus free whole blood by platelet-rich plasma (PRP) method.
Five or six bags of ABOmatched PCs were pooled and filtered with leukocytes filter for PCs(n57).
The swirling phenomenon, pH, automatic blood count, platelet aggregation, hypotonic shock response (HSR), the extent of shape change(ESC), CD62p expression, ATP level in platelet, glucose and lactate concentration were detected before and after filtering, and on days1, 3, 5 and 7 of storage, respectively.
Results/Finding: The platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was (86.7 6 1.6)%, relative change rate of HSR was (3.87 6 12.75)%, the residual leukocytes were (0.15 6 0.15)310 6 .
The pH, HSR, and the CD62p expression of pooled platelet concentrates before and after filtering were (7.00 6 0.17) vs (7.06 6 0.16), (66.96 6 12.35)% vs (63.22 6 8.26)% and (28.94 6 14.25) % vs (31.60 6 16.77)%.
There is significant change for WBC after filtering (P<0.01).
During storage in PVC-BTHC blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table 1.
Conclusion: Storage in PVC-BTHC blood bags for five days, the quality of pooled PCs met the requirements of Chinese standards (GB 18469-2012) .
It can be a complementary source for apheresis platelets supplement in China.
Evaluation of SamplokV R Segment Sampler to Obtain and Measure Samples from Blood Component Tubing Segments Abbejane Blair*.
AJBlair Laboratory Consulting Background/Case Studies: Current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination.
ITL BioMedical has developed SampLokV R Segment Sampler (SS), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments.
SS was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure.
Study Design/Method: SS obtains fluid samples from sealed tubing segments into a needleless syringe.
It consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device.
A needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the SS chambers and, gently pushed onto the needles to pierce each end of the segment.
The sample from the segment is then withdrawn into the syringe.
The study was performed at Rhode Island Blood Center (Providence, RI) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback.
Two lengths of tubing segments were filled to contain sample volumes of 500mL and 1000mL.
Two users then evaluated the SS tubing segment types with 500mL or 1000mL samples for a total of 10 data points.
Samples were collected into the attached 1mL or 3mL syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube.
Results were tabulated as PASS or FAIL.
Results/Finding: A total of 10 SS were evaluated by two users.
All samples were successfully collected and transferred into tubes.
Insertion of the segment edge requires observation to ensure placement onto the needles.
Any air bubbles collected into the syringe could easily be moved to the top by Background/Case Studies: The management of platelet inventory is crucial due to a number of factors including the 5 day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g.
platelet collection kits, testing, product processing).
The use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste.
The objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (FTE) allocation.
Study Design/Method: In January 2015, a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day.
The tool uses daily historical transfusion data from the last five weeks.
Additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate.
The number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends.
The effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and FTE information for a one year period prior to the implementation of platelet inventory management to one year period following implementation.
Results/Finding: By implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs.
The staffing adjustments and targeted collections have lowered FTE and outdate cost by 22%.
The platelet outdate rates dropped after implementing the platelet inventory tool from 14% (1324 units) to 11% (874 units); a 21% decrease.
FTE was able to be monitored closely with the donor schedule and lowered from a yearly average of 7 FTE to 6.2 FTE, lowering FTE by 11%.
Conclusion: Considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections.
Staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets.
Given these positive results, we are beginning to develop a similar tool for our whole blood collections.
Identifying Opportunities to Right-Size Hospital Inventory Using Compotrace Radio Frequency ID Inventory Management System Nanci Fredrich* 1 , Jaclyn McKay 1 , Jennifer Curnes 1 and Rowena Punzalan 1,2 .
1 BloodCenter of Wisconsin, 2 Children's Hospital of Wisconsin Background/Case Studies: The ability to track inventory of blood components in real time is challenging for both hospital transfusion services (TS) and blood centers (BC) using current blood bank information systems (BBIS).
In addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain.
A pilot was designed to track and monitor all blood components from distribution at the BC to issue in a hospital TS using Fresenius Kabi CompoTrace Radio Frequency ID (RFID) enabled Inventory Management System.
The objectives were to determine feasibility of the CompoTrace system and analyze CompoTrace data for real-time usage and optimal inventory levels.
Study Design/Method: A 3 month pilot was conducted at a pediatric hospital and its BC using both BBIS and CompoTrace systems to track all adult-size blood components.
Staff were trained on use of CompoTrace system.
Upon receipt of order from pilot hospital, BC staff applied RFID tags to all component bags and scanned components into the CompoTrace system.
Components were transported and delivered to TS following established procedures.
Upon receipt at the hospital, components were scanned into inventory using both the TS BBIS and CompoTrace systems.
Dual scanning of components occurred upon issue to or return from floor, component modification or return to BC.
Products for emergency use or at time of high demand were not RFID-scanned.
A priori, the pilot would stop if the Compo-Trace system hampered current workflow, component issue was delayed or if TS errors increased.
No inventory changes were made during the pilot.
Results/Findings: Real-time data from CompoTrace system provided actual usage for all blood components including component disposal and shipment to and from BC.
Average daily RBC inventory levels and usage for selected blood types is shown in table.
Lessons learned related to equipment and workflow: (1) Use of smaller irradiation canister may damage RFID tag, which was resolved by relocating tag, (2) TS workflow and STAT orders challenged consistent use of dual processes to track component status.
However no increase in TS errors or delay in issue of components occurred.
Conclusion: Use of RFID to track blood components from BC to final disposition is feasible.
Real-time data from CompoTrace system identified optimal inventory levels for RBC at the pilot TS.
Use of real-time RFID to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory.
Background/Case Studies: Physicians expect blood to be available at all times.
Following a national appeal in July 2016 for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (TS) recognized a potentially dire situation given the institution's patient acuity.
Our hospital-based TS supports a full range of services: a Level I trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services.
A regional donor center supplies our blood products.
To insure appropriate response to patient needs, the TS created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages.
The approach is described herein.
Study Design/Method: At the direction of the Transfusion Committee (TC), TS directors presented the concern for impending shortages to the hospital Quality Directors (QD) committee.
The QD committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional Health Care Quality (HCQ) activities.
The QD recommended creation of a multidisciplinary team: "the blood shortage task force (BSTF)", analogous to an existing task force started for management of drug shortages.
Results/Finding: With HCQ and TC support, the TS created the BSTF and blood shortage management algorithm (BSMA).
Standing Members of the BSTF include TS Medical Director (Chair), Senior Vice President (SVP) of HCQ, SVPs of Clinical Services Director of Regulatory Affairs, Legal Counsel, and representatives from Ethics, Social Work, Pharmacy, Patient Referrals, and Communications.
Ad hoc members include those whose patients would be most impacted by the specific shortage.
The BSMA designed by the BSTF provides a framework for TS's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the BSTF.
Trigger criteria include: marked TS concern; essential product; high likelihood of inventory depletion; broad patient impact.
Once convened, the BSTF is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care.
Conclusion: Faced with the potential for limited blood supply, the TS reached beyond the laboratory and engaged the TC and members of HCQ to assemble a robust, multidisciplinary task force.
This resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients.
ABSTRACT Background/Case Studies: Cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants.
The collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within 8-hours after collection.
This tight 8hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products.
Study Design/Methods: The American Red Cross (ARC), in partnership with researchers from the Georgia Institute of Technology (GT), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate.
After reviewing blood collecting and processing schedules, collection locations, and other factors, ARC-Cryo subject matter experts together with GT researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model.
Results/Findings: To facilitate implementation, a Decision Support Tool (DST) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized.
The implementation of the DST led to an increase in the number of whole blood units satisfying the tight 8-hour completion time constraint for cryo production (capacity expansion).
In particular, during the 4 th -Quarter of 2016, a blood processing region was able to process about 1000 more cryo units/month (an increase of 20%) at a slightly lower collection cost (cost avoidance), resulting in an approximately 40% reduction in the per unit collection cost for cryo.
Conclusion: By utilizing Operations Research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved.
This interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a Finalist on the 2017-The Franz Edelman award, recognizing outstanding achievements and practices in Operations Research.
Inventory Management and Transfusion Practice before and after 7-Day Apheresis Platelets Sarah K Harm*.
University of Vermont Medical Center Background/Case Studies: The shelf life of apheresis platelet (AP) units stored in plasma may be extended from 5 to 7 days in the USA using an FDA cleared rapid test (RT).
In August 2016, our hospital based transfusion service began using a RT on day 6 and 7 to routinely extend AP shelf life to 7 days.
This report describes changes in platelet inventory management and transfusion practice six months following routine use of 7-Day AP.
Study Design/Methods: Data were obtained for two study periods: September 2015-February 2016 (pre-implementation) and September 2016-February 2017 (post-implementation).
The study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region.
The transition period from 5-Day to 7-Day AP inventory was excluded.
The following data was collected for each study period: the total number of AP transfusion recipients, AP units transfused, expired AP units, AP units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay.
Results/Findings: Data are shown in the Table.
The number of AP transfusions decreased by 3% post-implementation while inpatient admissions and surgical volume increased by 1% and 3%, respectively.
The hospital length of stay was similar for both periods.
AP inventory decreased by 36% post-implementation and the outdate rate decreased from 29% to 15% (p<0.0001).
Ad-hoc ordering was not statistically different between study periods (p50.10).
The average number of AP transfusions per patient between pre-and post-implementation periods was not statistically different (2.1 and 1.9, respectively, p50.65).
Furthermore, a new "rejection threshold" for lipaemic products will be implemented.
This threshold represents the TG concentration above which viral marker testing for donor screening will be affected.
In KCBB Abbott's Prism Assays are used for: HBsAg, anti-HCV Ab, anti-HIV Ab, anti-HTLVI/II .
Results/Finding: Using data management system and file records in KCBB as regard discarding blood components due to lipaemia during the last five years (2012) (2013) (2014) (2015) (2016) , it was demonstrated that number of discarded RBCs due to lipaemia during the whole period was 4892 units.
Number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was 8546, 24, and 2 units respectively.
The mean number of discarded RBC units of the five years of the study exceeds 50% of the tested ones.
Literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations.
By reviewing sample requirements for viral marker testing in KCBB, the accepted level for TG in blood samples is below 1000 mg/dL, and so the rejection threshold for lipaemia is level equal to or more than 1000 mg/dL.
Conclusion: Many blood product units are discarded needlessly in KCBB due to lipaemia in the last five years (including 4892 RBCs, 8546 plasma products and 24 apheresis platelet units).
In an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from 200 mg/dL to 1000 mg/dL which does not affect blood safety.
A follow up study is recommended after applying the new threshold to evaluate the new policy.
Logistical Management of the Incorporation of Pathogen Reduced Single Donor Platelets (PR-SDP) into Inventory at a U.S.
Tertiary Care Medical Center Eric Gehrie* 1,2 , Rebecca Ross 3 , Debra Mraz 3 , Anne Baker 3 , Zenna Neal 3 , Melanie Champion 3 and Edward L. Snyder 2,3 .
1 Johns Hopkins University School of Medicine, 2 Yale University, 3 Yale-New Haven Hospital Background/Case Studies: The approval of PR-SDP by the FDA provided an opportunity to improve the safety of our platelet inventory across all patient demographics.
We outline our approach and address issues we faced during the first 4 months of PR-SDP availability.
Study Design/Methods: Our nursing education team provided presentations to the nursing and clinical unit support staff.
A company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards.
Presentations to physicians were made by the blood bank medical staff.
Information Technology personnel created a new product type in the blood bank computer system, tested the ABO/Rh truth tables, and ensured that billing codes were in place.
The necessity for transiently supporting a dual inventory of PR-SDP and conventional platelets led to consultation with the ethics committee and risk management, to confirm that PR-SDP and conventional platelets (C-PLTs) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care.
We chose to not gamma irradiate any unit of PR-SDP, consistent with the package insert.
Results/Findings: The ethics committee and risk management confirmed that informed consent was not needed for transfusion of PR-SDP.
PR-SDP available from our blood supplier incremented monthly.
Over the first four months of PR-SDP availability, 777 PR-SDP were transfused at our hospital (out of a total of 3286 platelets transfused).
After 4 months of scale-up, PR-SDP were approximately 30% of inventory.
Questions received during the nursing and medical conferences related to: the risk of bacterial contamination with C-PLTs vs. PR-SDP; toxicology of the PR process; scanning PR-SDP labels into the electronic medical record; and the need to irradiate PR-SDP.
Our use of a "safety measure" addressed concern over bacterial contamination of C-PLTs.
Published PR-SDP toxicology data comparing the content of psoralens in food products such as grapefruit ($12 mg per 100g) to the content in PR-SDP (<1 ng per mL) addressed toxicology concerns.
Nursing/IT allayed concern over scanning issues with a simple demonstration.
Finally, we ensured that all parties were aware that FDA did not require irradiation of PR-SDP.
Presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization.
Company personnel did not present at medical or nursing conferences per institutional policy.
No Background/Case Studies: Ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations.
In September 2017, the apheresis collection process (ACP) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (DPD).
The process review has led to several changes, including the substitution of the pre-donation platelet (PLT) count measurement before donation type allocation, in favor of the use of the donor's past donation records.
Multiple processing steps were eliminated, and the evaluation of PLT concentration as a function of time, deduced from complete blood count (CBC) measurements, allowed the centralization of the analysis at the QC Department.
Finally, introducing the concept of non-optimal donations has led to an increase in the proportion of DPD.
Study Design/Method: At the donation centers, whole blood (WB) from 10 donors was collected in K 3 EDTA tubes.
PLT concentrations were determined at the QC Department using the Coulter AcT 5 diff hematology analyzer (Beckman Coulter).
Sample tubes were stored at 20-248C and measured at 24, 48 and 72 hours post-collection.
Single platelet donations (SPD) or DPD were collected using the Trima Accel.
Units were pooled and split in ELP (Extended Life Platelet, Terumo BCT) storage bags to mimic SPD (250 mL; n58) or DPD units (500 mL; n54).
PLT pools were stored at 20-248C under mild agitation for seven days except for DPD, which were split in two 250-mL bags after 18 6 1 h. Samples were taken on days 1 and 7. pH, pO 2 and pCO 2 , hypotonic shock response (HSR), extent of shape change (ESC), CD62p expression, ATP content, lactate and glucose concentrations were used as in vitroquality markers.
Results/Finding: PLT concentration as a function of time, determined from WB CBC measurements, showed no significant difference at 24h (247 6 32 PLTx10 9 /L), 48h (247 6 27 PLTx10 9 /L) and 72h (247 6 32 PLTx10 9 /L) postdonation.
DPD can be stored in the same collection bag for 24h after donation without any significant impact on PLT quality markers.
PLT concentrations were within the manufacturer's acceptable limits (1141-1526 PLTx10 9 / L) before splitting.
On day 1, lactate and pCO 2 concentrations increased, and pO 2 decreased in DPD.
However, these values normalize to those of control units at the expiration day.
Conclusion: This project was approved by Health Canada and implemented in our organization in March 2017.
There are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality.
Post-implantation efficiency data will be compared to the targeted 12% increase in the targeted number of PLT units per donation ratio.
Phased Implementation of Pathogen-Reduced Platelets in a Health System Elizabeth S. Allen* 1 , Colleen Vincent 2 and Patricia Kopko 1 .
1 University of California -San Diego, 2 American Red Cross Background/Case Studies: Pathogen reduced platelets (PRP) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex.
Early evidence shows only 40-45% of double platelet collections meet requirements for pathogen reduction treatment.
1 Blood centers need hospitals to implement PRP to start manufacturing, but hospitals may not wish to use PRP until they can provide the product to all patients.
Scaling up manufacturing at the blood center and phasing in PRP across patient populations meets both parties' needs.
We evaluated this strategy at our university health system (transfusion volume: 9,500 apheresis platelets annually), which includes two hospitals (750 inpatient beds) and an outpatient cancer center.
Study Design/Method: Before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained.
Live training was provided for nurses in the outpatient cancer center (week 0) and the bone marrow transplant (BMT) ward (week 6).
An e-mail communication explained the change to all physicians and nurses.
In Phase 1, we implemented PRP in the outpatient cancer center.
These patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur.
In Phase 2, we expanded usage to include the inpatient BMT ward.
In Phase 3, we lifted all restrictions so PRP could be used throughout the health system, with the goal to reach 100% PRP within 6 months.
Results/Finding: In Phase 1 (weeks 1-6), we requested 31 PRP products weekly, based on typical usage in the outpatient cancer center.
Our blood supplier provided an average of 23 PRP weekly (range 9-33), and PRP constituted 44% of platelet transfusions in the cancer center.
In week 2, excess PRP inventory required use of PRP in the inpatient BMT ward ahead of schedule, a practice which continued throughout Phase 1.
In Phase 2 (weeks 7-8), we formally expanded issuing of PRP to include the inpatient BMT ward and requested 91 PRP products weekly.
Our blood supplier provided an average of 57 PRP weekly (range 44-69), and PRP constituted 53% of platelet transfusions in the phased-in areas.
In Phase 3 (weeks 9-10), we began issuing PRP throughout the health system.
Our supplier provided an average of 70 PRP weekly (range 61-78), and PRP constituted 43% of all platelet transfusions.
Scaling-up is ongoing.
Conclusion: Phased implementation of PRP by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing.
Background/Case Studies: Maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals.
Our Health System comprises 21 hospitals including smaller community hospitals (SCH) and larger tertiary care medical centers (TCMC).
For several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage.
This encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of 2 apheresis platelet (AP) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost.
Study Design/Methods: A "Round Robin" (RR) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (RBC) standing orders.
An efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals.
Platelets are transferred at the time of RBC standing order delivery based on a predetermined route schedule.
Each day, 2 AP are delivered to the SCH and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver.
These platelets typically have a 48 hour shelf life remaining.
The same process occurs at the next SCH on the route.
All retrieved platelets from the SCH are delivered to the TCMC which is the last stop on the route.
Thus, the SCH has adequate number of units available for regular transfusion and massive transfusion protocol.
Results/Findings: Review of our RR process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an AP inventory for patients requiring urgent platelet transfusion.
An additional benefit was further decrease in AP waste (Table 1 ) resulting in a cost savings of $50K.
An additional cost savings of approximately $25K was noted due to decreased cost of emergent platelet transportation.
Conclusion: Our novel RR process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of AP waste at our health system from our previous platelet sharing process.
We anticipate additional decreases in AP waste as 237A TRANSFUSION 2017 Vol.
57 Supplement S3 we further streamline our process.
With the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste.
Post Implementation Adjustments of Our Pathogen Reduction Process Jacqueline Carlson* 1 , James R Stubbs 1 , Scott A Hammel 1 and Manish Gandhi 2 .
1 Mayo Clinic, 2 Mayo Clinic-Rochester Background/Case Studies: The implementation of Pathogen Reduction for Apheresis Platelets using CerusV R INTERCEPT System for Apheresis Platelets was a substantial endeavor encompassing many different areas.
As with any process change, adjustments and modifications can occur along the way.
After implementing 100% Pathogen Reduction Technology (PRT) for Apheresis Platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products.
Study Design/Method: Our PRT Validation consisted of 100 Apheresis platelet products.
Each product was tested pre-processing for white blood cell (WBC) content and platelet yield, along with post processing platelet yield.
This data was used to calculate our yield and volume retention during processing.
We anticipated products with preprocessing yields of 3.0, 3.1, and 3.3 x10 11 may end up below a 3.0 in the final storage bag and would need a post-processing sample to ensure the product met criteria at !3.0x10 11 platelets.
Results/Finding: During the validation, we discovered one collection was not leukoreduced and two collections started at a 3.4 yield but ended with a yield below 3.0.
These two discoveries led to adjustments in our PRT platelet process.
With the WBC failure, we reviewed the WBC count on the Sysmex XE-2100D preprocessing report to see if it would alert us to a potential WBC failure.
The review discovered that 99 of 100 results were 0.00 or 0.01x10 3 / mcL with the exception being the WBC failure with a count of 0.29.
Further monitoring of the WBC counts discovered a result of 0.04 which was tested on the ADAM r-WBC for WBC count and determined to not be leukoreduced.
We decided all Sysmex WBC results from the pre-processing Sysmex report would be reviewed prior to processing and a WBC result of 0.03 will be tested on the ADAM to confirm a leukoreduced product.
We also discovered 2 of 4 (50%) of the 3.4 preprocessing yields products ended with a post processing yield <3.0.
We decided to increase the yields requiring post processing samples to include the 3.4.
Conclusion: We are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of !3.0x10 6 platelets and to have enough data to accurately determine our volume and yield loss during processing Background/Case Studies: The University of Kentucky Medical Center (UKMC), a large academic hospital with level I trauma center, is supplied with blood products by the Kentucky Blood Center (KBC) on a consignment agreement-based contract.
UKMC is KBC's largest consumer of blood products.
As platelet usage can vary widely day to day platelet usage projections are provided to KBC by the UKMC blood bank, thereby allowing KBC to act accordingly with a given day's stock (i.e.
import vs export).
Daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs.
This process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy.
Study Design/Method: Daily platelet projection forms from 9/16-12/16 were retrospectively reviewed and compared to actual usage data over that same time.
The prediction system used in the UKMC BB up to that time (estimated clinical need 1 6) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage.
The prediction system was subsequently changed based on this data in 2017; the revised prediction method (estimated clinical need 1 11) was then evaluated retrospectively using the same data sources covering 1/17-4/17 and then compared to the prior method.
Results/Finding: The average number of platelets transfused from 9/16-12/ 16 was 18.2 U/d with a standard deviation of 5.3 U/d; the predicted amount was 13.7 U/d.
The difference between the predicted amount and the number of units used was -4.5 U/d.
79% days (23d/month) were under-predicted (average: 6 U/d).
17% of days (10) were under-predicted by !10 U (average: 12 U; max: 15 U (4x)).
The average number of platelets used from 1/17-4/17 was 17.5 U/d with a standard deviation of 4.4 U/d; the predicted amount was 18.3 U/d.
The difference between the predicted and units used was a 10.8 U/d.
38% days (11d/ month) were under-predicted (average: 3.5 U/d).
One day (1%) over this period was under-predicted !10 U (11 U).
Conclusion: Review of clinical platelet usage over this time identified a relatively stable average daily clinical demand.
Adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages (17% a 1%), reduced the number of days under-predicted (79% a 38%), and decreased the discrepancy on those under-predicted days (5.9 U a 3.5 U).
These improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining KBC's supply flexibility or severely limiting UKMC clinical settings.
Rapid Implementation of Zika Virus (ZKV) NAT Blood Donor Screening Joan Dunn Williams* 1 , Maria Noedel 1 , Nancy Haubert 1 , Kenneth Hudson 1 , Larry Morgan 1 , Robert Shaw 1 , Tracy Fickett 1 , Jamie Jue 1 , Valerie Winkelman 1 , Sally Caglioti 1 , German Leparc 2,3 and Phillip C Williamson 1 .
Background/Case Studies: On 08/26/16, FDA issued a guidance document for "Reducing the Risk of ZKV Transmission by Blood and Blood Components".
In response, a plan was implemented for mandatory ZKV testing for all Clients with locally acquired mosquito-borne cases of ZKV within 4 weeks; nationwide in 12 weeks.
This organization performs testing for Clients (blood centers, hospitals) across the country.
We report on 1 of 2 manufacturers' (Sponsor) provided Investigational New Drug (IND) protocols.
A single Project Management (PMO) system was used to control all required processes.
Study Design/Method: Project focus included: clinical trial requirements, Client onboarding, lab operations (Labs).
Our objectives were to implement ZKV testing for 44 Clients within 4 weeks, and an additional 21 Clients within 12 weeks.
To minimize the impact to Labs a staggered implementation was used with tracked/streamlined communications from stakeholders: Vendors, Institutional Review Board (IRB), IT (client and lab based), Client Services and Labs.
Results/Finding: Clinical trial requirements increased the complexity of implementing an unlicensed test.
Documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting.
Multiple IRB documents were required.
To ensure accuracy in IND commitments a Principle Investigator was assigned to Labs with Client sub-investigators.
Deliverables were multiple including Client requirements, Vendor responsibilities and Labs.
Client onboarding included confidentiality agreements between Client and Sponsor.
An immediate ZKV based webinar provided materials and understanding of Sponsor protocol, lab test system, and Client/donor based responsibilities.
To facilitate and ensure effective communication, twice weekly conference calls were held.
Clients sent questions which were facilitated by Labs and directed to Sponsor.
Specific to Clients were IRB documents, IT updates/validation for ZKV test ordering and result receipt.
Labs were multifaceted: vendor instruments, assay materials, package inserts, staff training.
Assessments included: ZKV sample volume, throughput, instrument capability/capacity.
Work requirements included Vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting.
All Clients were provided with ZKV testing within required timeframes.
Conclusion: The success in meeting a rapid implementation of ZKV testing was largely due to a centralized PMO system which provided a controlled process for Sponsor, Client, Vendor and Labs.
Within lessons learned strength was found in a multi-Client onboarding process.
A weakness was in understanding instrument test volume capacity throughput which was exceeded during the 4-week period but overcome during the 12-week cycle.
Red Blood Cells Baby Units Traceability and Discard in Kuwait Central Blood Bank and Five Hospitals Marwa Moemen Al Deeb* 1 , Hala Samuel Boules 1 , Fatemah Saleh Al Matroud 1 , Rabab Hussien Ali Dashti 1 , Hanan Alawadhi 2 and Reem Al Radwan 3 .
1 Kuwait Central Blood Bank, 2 Kuwait central blood bank, 3 Kuwait central Blood Bank Background/Case Studies: Ill children are more likely to receive Red Blood Cells (RBCs) transfusion than any other patient age group.
RBCs are the component most often transfused during neonatal period.
Small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk.
Traceability is the ability to trace each individual unit from donor to recipient or disposal.
Blood component should be fully traceable from collection to final disposition.
The Kuwait Central Blood Bank (KCBB), is preparing baby units and distributing it to all hospitals all over the country.
KCBB, being accredited by the American Association of Blood Banks (AABB), is following the AABB's regulations in tracing every component.
Study Design/Method: This is a retrospective study to assess final deposition and the percentage of discard of prepared packed RBCs baby units in the KCBB and five hospital blood banks (HBB).
Also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products.
Methods: A total of 3000 RBCs baby units were randomly chosen to be traced to their final deposition from the year 2012 till 2016.
Half of them (1500 units) were traced in KCBB.
Tables showing the numbers of the chosen units were distributed to the five governmental HBB (60 units for each year of the study period).
Results/Finding: Preliminary results show that the tracing of RBCs baby units in the KCBB is 100% efficient.
Results from other hospitals are under process.
Statistical analysis of the traceability will be done as soon as the data is collected.
The study will analyze the usage of the baby units in different departments and the percentage of discarded units.
The traceability of RBCs baby units in the KCBB is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing.
Most of the Kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels.
The percentage of discard of the baby units in the hospitals is very high.
This may be due to the practice of using fresh blood (<5 days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure.
The creation of a national policy for using RBCs baby units is highly recommended to reduce the discard of such units.
We also calculated the number of false positive results.
The study traced all products through mid-March 2017.
Results/Findings: A total of 339 products were tested.
Fifteen units (4%) had a false positive result and could not have their life span extended.
Of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations.
Cross-reactive antibodies were identified in all 15 by the vendor and none were true positives by re-culture.
Of the 324 units that were successfully tested, 200 were tested again on day 6 for use on day 7(62%).
There were 166 platelets transfused (51%) and 158 expired after day 7 (49%).
The cost to test the products including controls was $12,970 and our calculated cost to produce 324 products would be $77,436.
If we had needed to import products to meet needs, the cost would be roughly $91,300 without shipping costs which are estimated at $14,815.50.
We averaged 40 expired platelet products per month (range 6-67) before Verax testing and 26 (range 9-40) after implementation.
Conclusion: Using Verax point-of-care testing saved 166 platelet products from discard.
The cost savings were $93,145.50 from importing and $64,466 from producing a replacement for those 324 products.
The average discard rate per month went from 40 to 26 after Verax implementation.
Extending platelet shelf life to 7 days more than paid for the cost of testing and ensured products were available for patients who needed them.
Secure Text Messaging in Transfusion Medicine: Can Texting Decrease Wastage?
Melanie Estrella* and Elsie Lee.
George Washington University Hospital Background/Case Studies: Secure text messaging in hospital settings allows for quick, easy, and HIPAA compliant communication between members of patient care teams.
It works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications.
Secure texting has potential to be a useful management tool in Transfusion Medicine in reducing blood product wastage.
For example, it provides a relatively low-burden means for busy clinicians to provide feedback to the Transfusion Service about scenarios of potential wastage.
This information can be used to identify areas in which management strategies could be developed.
It also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage.
The goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage.
It is hoped that the results will identify secure texting as a useful management tool in Transfusion Medicine.
Study Design/Method: Wastage records that were investigated without the assistance of secure texting from July to December 2016 were reviewed to identify the most common scenarios of preventable blood product wastage.
Wastage records from January to April 2017 were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician.
Results/Finding: For 2016 data, 129 units were investigated without the use of secure texting.
Of these, 118 units were identified as preventable wastage, and 11 wasted units were considered beyond the control of the clinician.
The categories for preventable wastage were defined as follows: 1) Product not released after procedure/ or when patient stabilized (42) 2) Product returned outside of appropriate temperature range (40) 3) Clinician unaware product was assigned (36).
Thus far in 2017, wastage records have identified 31 units of preventable wastage.
Secure texting was used by a Transfusion Service physician to investigate.
Twelve responses provided useful feedback for future management strategies, 11 responses thanked the Transfusion Service for the information, and in 8 instances, the message was read with no reply.
Conclusion: Secure text messaging has the potential to improve communication in Transfusion Medicine.
It is easy to use, HIPAA compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the Transfusion Service.
Sequence of Reagent Adding for Cryopreservation Freezing Solution Guoling Chen*, Xu Zhao, Andrew Tiss, Sasha Turner, Devin Emerson, Manijeh Shemirani, Sharon Novak, David Garvin, John Eng and Wanxing Cui.
MedStar Georgetown University Hospital Background/Case Studies: Dimethyl sulfoxide (DMSO), plasmalyte-A (Plas-A), Human Serum Albumin (HSA) are widely used to prepare cryopreservation freezing solution.
Some use autologous plasma instead of Plas-A and HSA.
This study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution.
Study Design/Methods: Materials: 99.9% DMSO, Plas-A, 25% HSA, autologous plasma extracted.
Containers: transfer pack (bag) and polystyrene tubes.
The Freezing Solution recipe used in this study is (volume ratio) 99.9%DMSO: Plas-A : 25%HSA51:2:2.
Plas-A and HSA are kept at room temperature (20-258C, RT) and refrigerated at 48C, plasma at RT (to simulate the end-of-centrifuge temperature), DMSO at RT (due to high freezing point 18.58C).
Different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken.
Total 14 tests.
At least 10 minutes cooling after DMSO, before adding the next reagent.
See table: (1) After directly adding 99.9% DMSO alone to bag, the bag turned from transparent to white, so DMSO should not add first.
(2) In tube, autologous plasma first, DMSO next, powder-like precipitates.
(3) In tube, DMSO first, HSA next, precipitated instantly, a layered appearance.
(4) & (5) In tube, Plas-A first, then HSA, DMSO at last, precipitates formed; RT Plas-A and HSA combination formed a thicker precipitate than those kept at 48C.
(6)&(7) In tube, HSA first, DMSO next: precipitation formed heavily, sculpture shape.
Precipitation in the 48C group is slightly milder/slower than RT group.
So HSA should not be added first.
(8)&(9) Trace of HSA(<1ml) was mixed into the Plas-A bag (500ml).
In tube, such "HSA-contaminated" Plas-A was added first, then DMSO, small fragments of precipitates formed, so DMSO should not add last.
Background/Case Studies: Maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals.
However, daily blood product use is difficult to anticipate.
Platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store.
Due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration.
Sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage.
Study Design/Method: We have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-month period.
Using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors.
These include day of week, number of abnormal CBC, location-specific hospital census data, and other less important factors.
We exploited this relationship to develop a mathematical model to guide collection and ordering strategy.
Results/Finding: This model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below 10 in our model.
Compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5% to 3.2%.
With an annual platelet usage of approximately 13,000 units, this reduction equates to approximately 950 units saved from expiration annually.
Depending on platelet pricing in different regions, this accounts for annual savings between $450,000 to $650,000, per institution.
Conclusion: To our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand.
Thawed Plasma Implementation: Signficant Cost Savings and Decreased Plasma Wastage Morvarid Moayeri* 1 , Russell Thorsen 1 , Rosaline Ma 1 , Antonio G Insigne 1 , Amy DeCourten 1 , Florence Panganiban 1 , Patricia McKean 1 , Cyril Jacquot 2 , Sara Bakhtary 1 and Ashok Nambiar 1 .
1 UCSF Health, 2 Children's National Medical Center Background/Case Studies: Plasma (FFP, PF24, PF24RT24) stored at 1-6C outdates 24 hours after thawing.
If collected in a functionally closed system, it may be relabeled as Thawed Plasma (TP), extending expiration to 5 days from the thaw date.
Although coagulation factor levels decrease over this period, they remain above hemostatic levels.
As TP can be safely used for the vast majority of patients requiring coagulation support, we implemented use of TP in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage.
Study Design/Methods: The massive transfusion protocol at our instiution already allowed the use of group AB TP.
Following a review of literature and practice at other large centers, the Transfusion Committee extended the approval of TP to all patients.
Neonates (<4 months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which We also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units.
Conclusion: In many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited (24 hr) shelf-life.
TP has an extended shelf-life, and can be used interchangeably with FFP and PF24 for most patients.
Implementing TP in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource.
The Merging of Immunohematology Reference Lab's (IRL) Inventories-Using Technology to Create Advanced Search Functions Alexander Delk 1 and Richard Gammon* 2 .
1 OneBlood, 2 Oneblood, Inc. Background/Case Studies: Immunohematology reference labs (IRLs) must maintain diverse inventory of antisera to aid in antibody identification, antigen type RBC units, and meet regulatory requirements.
When our current organization was established, two IRL sites had independent inventory management systems.
Although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (IFUs) were not.
Our IRL developed a synergistic method to organize and store antisera coupled with in-house designed custom Excel spreadsheet to organize and search antisera and view IFUs.
Study Design/Method: A list of similarities and differences was constructed.
Best practices of both methods were identified.
We determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening.
Sequential lab assigned numbers were given to antiserum for each category: S (rare Sera) and B (Bulk sera).
A dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth.
Antisera assigned to a box remained in that box, but may be moved within the box.
The box itself may be moved among freezers.
To track boxes, location and movement within the box, a custom Excel spreadsheet was created.
Its location tracking feature allowed for two different storage methods to function in one spreadsheet.
The spreadsheet had a tab for S and B antisera categories.
ABO group, desired and unwanted antibodies filters allowed quick search for appropriate antisera.
The spreadsheet also had hyperlinks to scanned IFUs.
Results/Finding: Sequential lab S and B numbers were assigned to new additions using a dynamic/static storage system.
An Excel spreadsheet with scanned IFUs (hyperlinks) was used.
Pre-merger systems, it took on average 5-8 minutes to choose an antiserum and obtain the appropriate IFU.
Post-merger system was reduced to on average 2-5 minutes.
(Table) Conclusion: The merging of two IRL's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked IFUs.
This process saved valuable technologist time and organized the antisera more efficiently.
ABSTRACT continue to flash until they are removed from shelf and their status updated in our database.
'Units Allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < 4 hours.
The XM/HLA Platelets tab provides patient names and status of units allocated.
A 'TRXN/XMPLAT' tab lists pending transfusion reactions and platelet cross match reports.
Dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from 34% to 14%).
Conclusion: Using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service.
The dashboard is stable, customizable and requires little maintenance.
Initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products.
Over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage.
Use of Deglycerolized Red Blood Cells for Hospital Transfusion Service Inventory Management Ronnie L. Hill*, Jason Corley and Lizabeth Ostiguin.
US Army Background/Case Studies: Regional blood shortages have been documented across the United States during the winter holiday timeframe.
Deglycerolized Red Blood Cells (DRBCs) have been shown to be an effective alternative though more expensive to manufacture.
This study looks into the fiscal and inventory efficacy of using DRBCs to meet the needs of transfusion services during times of blood shortage.
Study Design/Method: On three separate occasions, a medium sized DoD donor center used its frozen blood inventory to produce type O DRBCs to meet the needs of two regional transfusion services.
All frozen red cells were manufactured by an offsite facility with the Haemonetics ACP-215 using the low glycerol (40%) freezing method and frozen at -658C within six days of collection.
Thawing occurred in a 328C water bath in the following order: 7 O Positive and 1 O Negative on 3 January 2017; 7 O Positive and 1 O Negative on 7 February 2017; and 8 O Negative on 22 February 2017.
Deglycerolization occurred on site using the ACP-215 with all units passing internal QC requirements.
DRBCs were shipped the same day to a hospital transfusion service, allowing for 13 days of shelf life prior to expiration.
Results/Finding: During the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type O red blood cells (RBCs).
O Positive RBCs were only available through NBE at $240-280 and had the limitation of arrival on the next business day.
Collection and processing time of liquid RBCs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling.
DRBCs cost the DoD on average $400 per unit to produce and distribute.
DRBCs have a shorter shelf life, 14 days versus the 211 days for other RBCs, but are washed during deglycerolization and thus produce fewer transfusion reactions.
One tech can operate up to four ACP-215's and deglycerolize four units at a time.
In January and February 2017, it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment.
Conclusion: While not as readily available as traditional RBCs, DRBCs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory.
Collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe.
Based on this, DRBCs can be ready faster than freshly collected units of blood.
There is an increased cost associated with manufacturing FRBCs which is compensated for by the longer available shelf life of 10 years.
Having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type O red blood cells for hospital transfusion services.
Validation of a Human Anti-Tetanus Toxoid Immunoglobulin Assay Performed on the Abbott c8000 Izekial Butler* 1 , Karen Leighton 1 , Scott Jones 1 and Rachel Beddard 2 .
1 QualTex Laboratories, 2 BioBridge Global Background/Case Studies: Plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools.
This testing serves as a quality control test and helps estimate the antibody potency of the product.
The Binding Site, Human Anti-Tetanus Toxoid Immunoglobulin Liquid Reagent Kit is for use on a turbidimetric analyzer.
The aim was to optimize and validate the Human Anti-Tetanus Toxoid Immunoglobulin Liquid Reagent Kit for use on a photometric analyzer.
Study Design/Method: Experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-Tetanus Toxoid immunoglobulin kit utilizing the Abbott c8000 instrument.
Precision of the new assay parameters was determined by testing 10 replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run.
The panel samples were created by spiking appropriate amount of a WHO tetanus antibody standard into Sodium Citrate plasma.
Accuracy was determined by testing a series of samples ranging from 1 IU/mL to 60 IU/mL of tetanus antibody.
The samples for the accuracy study were created by diluting an appropriate amount of a WHO tetanus antibody standard with sample diluent from the reagent kit.
Linearity regression was determined by using the accuracy study values within the range of 2.0 to 45.0 IU/mL.
Stability of samples was determined by testing samples stored at 2-8 0 C and -20 0 C in triplicate at various time intervals.
Results/Finding: The %CV for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from 1.2855 to 1.3142.
So, precision was acceptable since the %CV for all samples tested was 5%.
The mean values for the samples tested in the accuracy study were all 610% of the expected value which was much lower than the acceptance criteria which was 615% of the expected value.
The linearity of the assay was acceptable with a R2 !
99.0%.
The linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration.
The sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to 14 days at 2-8 0 C and up to 1 month at -20 0 C. Conclusion: The data presented shows the successful optimization of the Human Anti-Tetanus Immunoglobulin Reagent Kit for use on a photometric analyzer.
Validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for 14 days at 2-8 0 C and stored up to one month at -20 0 C. A Deep Dive Audit of Intravenous Immunoglobulin Use for immune Thrombocytopenia: Is Its Use Inappropriate?
Jiajia Liu*.
University of Toronto Background/Case Studies: Intravenous immunoglobulin (IVIG) is a generally safe and effective therapy for immune thrombocytopenia (ITP) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated.
Due to concerns regarding adverse effects, cost and resource availability, an IVIG request form was implemented in our jurisdiction in 2010 to track utilization and appropriateness.
A recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of IVIG was broadly inappropriate (Shih et al, 2017) .
As such, we aimed to conduct an extensive chart review of patients who received IVIG for ITP at our institution to assess appropriateness of use.
Study Design/Method: We conducted a retrospective chart review of all patients with ITP who received IVIG in our institution from April 1, 2014 to March 31, 2015.
Local research ethics board approval was obtained.
Results/Finding: 40 patients received IVIG for ITP at SMH over the study period for a total of 76 unique IVIG infusions.
The most common indications for IVIG within currently accepted guidelines were: active bleeding (13, 17%), pre-operative or antepartum care (22, 29%), a platelet count of less than 10 and contraindication to corticosteroids (8, 11%).
Additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy.
Indications that fell outside of guidelines included: use of IVIG as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic ITP despite a platelet count between 30-50 x 109/L.
6 patients received IVIG for a likely diagnosis ITP while 245A TRANSFUSION being investigated for alternative explanations for thrombocytopenia.
Three patients were refractory to all other therapy for ITP and were dependent on regular IVIG infusions.
18/76 (24%) of infusions consisted of 2g/kg over 2 days; the remainder of infusions consisted of 1g/kg.
Of those who received 2g/kg,3 of patients (17%) had evidence of partial remission after a first 1g/kg dose.
IVIG was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines.
Conclusion: We found, at our institution, that use of IVIG for ITP was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations.
We believe that IVIG remains an important treatment for ITP particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing.
Detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate IVIG use in multiple settings.
We believe that there is an opportunity to promote administration of a single 1g/kg dose to minimize unnecessary utilization of IVIG amongst hematologists who manage ITP.
A Process for Improving Crossmatch Bench Ergonomics Janet Dornfeld*, Sheng-Chung Cheng, Ann Eggebrecht, Beth Greer, SavannahSue Rondeau, Brian Rognholt and Beth Taylor.
Mayo Clinic Background/Case Studies: A mission of our institution is to reduce the risk of work-related injuries.
Accordingly, each year an ergonomic survey is undertaken as a component of a general Department of Laboratory Medicine and Pathology safety audit.
Our 2016 survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches.
Study Design/Methods: A seven item ergonomics survey of the working environment was sent to 32 staff members in early February of 2017.
Twenty-two technologists responded for a 69% response rate.
The below table below reports the survey items and responses.
Results/Findings: The most problematic area was the available workspace.
Of the respondents, 81% indicated that workspace size was insufficient and 71% that the chairs at the fixed height benches were problematic.
Problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height.
Our laboratory Lean Team Operational Support Group was tasked to aid with the bench redesign and to choose products for improving the workspace.
Our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space.
The configuration of the new workspace was guided by the survey findings.
Adjustable height workstations were recommended to replace our fixed height bench.
We worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work.
The benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface.
The opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing.
Conclusion: The survey was effective in identifying working areas for improvement.
Employee comments have been positive for the new workstations.
An effectiveness assessment will follow, using the original survey, to assess the success of the project.
A Retrospective Study of Emergency Department Initiated Type and Screen Testing: Were Patients Transfused after Testing?
Sandra Lamm* 1 , Neil Bangs 1 and Kimberly Sanford 2 .
1 VCU Health System, 2 Virginia Commonwealth University Background/Case Studies: Type and screen (T&S) testing is often ordered on patients presenting in the Emergency Department (ED).
If the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation.
If the patient does not need a transfusion of red blood cells (RBCs), the testing and second phlebotomy is an inefficient use of resources and time.
Study Design/Method: As part of a Performance Improvement initiative in Transfusion Medicine, we performed a retrospective study of all T&S orders that were initiated in the ED from 1/1/2015 to 6/30/2015 to determine if testing was subsequently followed by transfusion of blood products.
Patients were stratified by ED department, time from T&S draw (TSD) to transfusion (<4 hours, > 4 hours < 24 hours), and if a second sample was required.
Results/Finding: A total of 3144 T&S orders were initiated from the ED in this time period.
2787 (88.7%) patients were not subsequently transfused any type of blood product within 4 hours of TSD and 2584 (82.2%) patients were not subsequently transfused any type of blood product within 24 hours of TSD.
A total of 2119 (67.3%) patients required a second sample.
Of these patients requiring a second sample, 1960 (92.5%) were not subsequently transfused any type of blood product within 4 hours of TSD and 1886 (89%) were not subsequently transfused any type of blood product within 24 hours of TSD.
Conclusion: Routine ordering of T&S testing is not an efficient use of resources and time as many patients are not subsequently transfused.
Ultimately unnecessary T&S and second sample collection and testing for those patients not subsequently transfused within 24 hours of TSD amounted to an estimated $699,706 in unnecessary patient charges and approximately 628.7 nursing hours for phlebotomies in a six month period.
Anti-D from Alloimmunization Versus Rh Immune Globulin: Detective Work in the Blood Bank and Transfusion Medicine Services (BBTMS) Margaret DiGuardo* 1 , Debra Berry 1 , Yunchuan Delores Mo 2 and Gay Wehrli 1 .
1 University of Virginia Health System, 2 Children's National Medical Center Background/Case Studies: The Institute for Healthcare Improvement Triple Aim incorporates enhancing patient satisfaction by providing high quality, safe care.
Towards these goals the BBTMS is charged with communicating to obstetric physicians (OBs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk.
Thus, when anti-D is detected in a female of childbearing age, it is critical to determine whether this represents Rh immune globulin (RhIG) or alloimmunization (alloanti-D).
Review of a patient's electronic health record (EHR) helps quickly identify RhIG administration, but if this documentation is missing, then it is easy to assume presence of alloanti-D. RhD alloimmunization impacts mom, fetus, newborn and future pregnancies.
Therefore, without a national, comprehensive health information exchange (HIE) system, it is imperative to investigate beyond the on-site EHR whether a patient received RhIG at an outside hospital (OH).
We report an IRB approved (exempt) case series where detective work revealed RhIG administration at OHs.
Study Design/Method: Over a two month time period, anti-D was identified in four pregnant women.
Review of their EHRs did not reveal a history of ABSTRACT RhIG administration; nor did subsequent direct communication with their obstetricians (OB) reveal a history of RhIG.
Based on each patient's home address, the BBTMS of any nearby OHs were contacted as was a primary care physician if listed in the EHR.
Results/Finding: Investigations beyond the EHR and OBs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites.
Through phone calls to the BBTMS of OHs, a history of one or more RhIG administrations within the preceding three months was found for each patient.
Our BBTMS records and EHR were amended to reflect the presence of a passive anti-D due to RhIG, rather than alloanti-D.
The changes were also directly communicated with the OB caring for each patient.
Conclusion: When a new anti-D is identified in a pregnant female, investigation is required to determine whether it is passive RhIG versus alloanti-D.
When neither the EHR patient history or OB reveal a RhIG history, it remains in the patient's best interest to investigate further.
Through phone calls to OH we revealed a history of RhIG administration in four patients.
Finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent RhIG administrations when indicated, at our institution.
Future strategies for avoiding similar situations include expanding our national HIE for critical information such as BBTM history and allergy history and expanding use of wallet-size patient identification cards with RhIg and alloantibody histories.
Auditing Massive Transfusion Protocol Colleen A. Aronson* 1 , Elizabeth Halperin 2 , Sharon Breining 2 and Mona Papari 3 .
1 ACL Laboratories/ Advocate Hospitals, 2 Advocate Health Care, 3 ITxM Background/Case Studies: A large Midwest hospital system with 5 Level I trauma sites evaluated how to audit the Massive Transfusion Protocol (MTP).
The possibility of real time audits is impractical due to the unpredictability of these events.
A search of the internet found an example from New Zealand for post process evaluation.
This was shared with a team as a starting point and then adjusted for system specific priorities.
To start the audit, the initiation of the MTP needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders.
Study Design/Method: The Transfusion Service (TS) was determined to be the source of truth for all of the MTP events.
A tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage.
This was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event.
The focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (Surgery (OR), Emergency (ED), Labor and Delivery (L&D), etc.
), type of event, use of tranexamic acid (TXA), calcium chloride (CaCl), temperature monitoring and pre/ post lab results.
A trial was started and 3 months of data were evaluated that contained 29 events.
Results/Finding: There was an equal number of events that were initiated in the ED and the OR (12).
Male patients were involved 69% of the time and 31% of time the patients expired.
Trauma of some type was the majority of the cause but 13.8% of the cases involved GI Bleed and only 6.9% were obstetric cases (see chart).
The lowest hemoglobin (Hgb) was found to average 7.1 with the post Hgb average of 9.7.
Ratios of 1:1 for Red Blood Cells (RBC) to plasma as well as RBC to Platelets (PLT) and Cryoprecipitate (CRYO) were also determined with a target of 4:1.
It was found that the RBC: plasma was 1.9:1, RBC: PLT was 5.9:1 and RBC to CRYO was 7.4:1.
Use of TXA was only 24.1% and CaCl was utilized in 58.6% of cases.
Conclusion: Although this data is for a short period of time it has pointed out several opportunities for improvement.
The use of MTP in GI cases was not previously understood but opens up a new group of people for which education and understanding of the MTP process is needed.
The low use of TXA needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the MTP process.
The product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement.
The process will now be expanded to the Level II trauma sites in the system and routine evaluation will be shared with all sites.
Automated Report Significantly Reduces Turnaround Time for RBC Antibody Alert Jessica L Dillon* 1 , Jody A Barna 1 , Donald E Ulinski 1 and Nancy M. Dunbar 2 .
1 Dartmouth Hitchcock Medical Center, 2 Dartmouth-Hitchcock Medical Center Background/Case Studies: Clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion.
At our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (EMR).
An interpretative comment is also entered by the Transfusion Medicine Service (TMS) Physician after the antibody work-up has been reviewed (this may be up to 2 weeks after the antibody is identified).
This comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units.
Since clinicians may not always review these results, the TMS Physician also simultaneously adds an "Allergy to Red Blood Cells" alert in the patient EMR at the time the interpretive comment is entered.
Study Design/Methods: In July 2016, we implemented an automated report to reduce the turnaround time (TAT) for entry of the allergy alert.
The report contains all detected red cell antibodies in the prior 24 hours and is provided to the TMS Physician during daily morning rounds (Monday through Friday) for manual entry of allergy alerts.
This study describes a three month comparison both before and after the automated report intervention, to evaluate the TAT for allergy alert entry into the EMR.
Age ( ABSTRACT Results/Findings: Between August 2015 and November 2015 (pre-implementation) , newly identified clinically significant antibodies were resulted for 56 patients compared to 51 patients between the months of August 2016 and November 2016 (post-implementation).
The TAT for allergy alert entry for both periods is shown in Table 1 .
We observed that 57% of allergy comments were performed within 24 hours in the post-implementation period versus only 30% pre-intervention (p50.0067).
Using the new process, nearly all of the alerts were entered into the EMR within 72 hours of antibody resulting and none of the entries were missed.
Conclusion: There was a significant improvement in the TAT for allergy comment entry following implementation of an automated report.
This project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification.
Blood Bank Verbal Tool Implementation for Cardiovascular Surgery Rita Louie* 1 , Shailesh Macwan 1 , Nancy Nikolis 1 , Arline Stein 1 , Janelle Richardson 1 , Manju Bagu 1 , Lennart Logdberg 1 , Alexander Indrikovs 2 , Vishesh Chhibber 1 and Sherry Shariatmadar 1 .
1 North Shore University Hospital, 2 Northwell Health Background/Case Studies: Our institution is a tertiary care facility performing over 1500 cardiovascular surgeries (CVS) in 2016, an increase of 117% after the healthcare system CVS integration in 2015.
Transfusion support of these patients includes preoperative preparation of PRBCs according to a maximum surgical blood order schedule.
Additional blood components are issued as orders are placed.
Until December 2016, the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication.
After 2 reported events in Q3 2016 that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements.
In collaboration with CVS, the Blood Bank implemented a new workflow process to enhance communication with the CVS team, reduce turnaround time and improve patient safety.
Study Design/Method: 1.
Open discussions and collaboration between blood bank and CVS nursing teams 2.
Mapping the process using flowcharts for additional blood orders from CVS.
3.
Identify bottlenecks and brainstorm solutions.
4.
A verbal CVS order process and form was implemented to improve communication between CVS and Blood Bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders.
A Read Back was also documented for verification of the order.
5.
The blood bank staff immediately processes this order while waiting for the written order to arrive.
Upon receipt of the written order the blood is issued to the OR.
6.
Follow Plan-Do-Check-Act.
The Transfusion Safety Officer reviews each order for the following parameters: number/type of products, turn around times (TAT), wastage/returned products and overall efficacy since implementation of this process.
Results/Finding: A significant improvement was noted in communication and TAT after implementation of the process described above.
For the period 12/23/16-4/7/17 the blood bank has received 327 verbal orders with varying product combinations.
The table below represents average turn-around times to issue blood products: Conclusion: The introduction of the verbal order tool for CVS has streamlined the blood ordering process leading to increased efficiency and lower TAT.
Effective communication between the OR team and transfusion service is the key to timely provision of blood products for these critical patients.
Challenge of Blood Type Testing for Multiply Transfused Sickle Cell Disease Patients Jayanna Slayten* 1 , Tracie Ingle 1 and Heather Vaught 2 .
1 Indiana University Health, 2 Indiana University Health (IU Health) Background/Case Studies: We report our Midwestern, University Transfusion Service challenge of obtaining the correct blood types in RBC exchanged Sickle Cell Disease (SCD) patients tested by our primary testing method, solid-phase red cell adherence analyzer ECHO (Immucor.
Norcross, GA).
The ECHO Operation Manual in Chapter 12-6 and Appendix D it states: "Warning: The Galileo ECHO cannot reliably detect hemagglutination reactions that are graded as 11 or less in tube methodology.
The Galileo ECHO does not generate as interpretation of mixed-field.
Such a mixed-field reaction will be interpreted as positive, negative, or equivocal."
We report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused SCD patients.
Study Design/Method: Two SCD when initially tested by the ECHO as O, D Negative; however, each patient was historically O, D Positive.
Both patients had received a RBC exchange transfusion with 8-11 O, D Negative red blood cells over 30 days previously.
Repeat testing of the samples was completed by the VISION (Ortho Clinical Diagnostics.
Raritan, NJ), NEO (Immucor.
Norcross, GA), and by standard ABO/Rh manual testing (anti-A, Anti-B, Anti-D Series 4, Anti-D Series 5, A1 cell and B cells.
Immucor.
Norcross, GA).
The repeat testing was compared to verify the patient's ABO/Rh typing and the results were entered into the computer system to allow for assigning the patient's ABO/Rh typing and electronic crossmatch.
Results/Finding: Table 1 summarizes the initial and repeat testing with the two patient samples.
Although the ECHO failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of O, D Negative blood with the detection of mixed-field in the D typing or by failing to interpret the ABO/Rh as not type determined (NTD).
The VISION and manual ABO/Rh typing yielded the easiest mixed-field to interpret macroscopically.
Conclusion: Our results agree with the findings of Summers et al (TRANS-FUSION 2009; 49:1672 -1677 who reported the challenge detection of mixed-field with the use of the ECHO compared to improved detected with automated gel column agglutination.
When the samples were tested by multiple automated and manual ABO/Rh methods, the expected mixed-field was detected.
The failure of the ECHO to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the ABO/Rh when there is not a historical ABO/Rh to compare.
To avoid this risk, it may be appropriate to re-type first time SCD patients by other methods rather than the ECHO to avoid this challenge.
Consistent with Summers do not account for regional distribution.
Many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on ODR and BSR, but we hypothesized that these are crude key performance indicators (KPIs) requiring redevelopment.
Study Design/Method: KPI redevelopment occurred in a large tertiary care hospital blood bank in Canada, responsible for 75% and 20% of transfusions in the region and province respectively.
RBC supply, inventory, and disposition data were retrospectively assessed from February 2014-June 2015 as the baseline period.
A "demand-driven inventory planning policy" (DDIP) was instituted to assess and implement the optimal RBC reorder quantity based on the difference between the historical maximum and minimum RBC inventories during weekdays; that would not lead to blood shortages.
Shelflife inventory (SLI) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (ABT) and received (ABR).
Iterative simulation modeling (R statistical software) was then performed to optimize SLI in a post-implementation period from June 2015-October 2016.
Results/Finding: Modeling predicted observed RBC disposition.
Through simulation, optimization of SLI was found to occur by optimizing a set of KPIs for each ABO blood group (Table 1) .
This led to a reduction in observed overall SLI (7.2 6 1.8 days vs 6.0 6 1.5 days, p<0.01) and ODR (0.9% vs 0.5%).
The BSR was not significantly increased during the postimplementation period.
Conclusion: Optimization using simulation modeling of multiple factors other than BSR and ODR led to further efficiency gains in a large tertiary care hospital blood bank.
Hospital blood banks should use an integrative approach with a set of KPIs to optimize the supply chain.
This approach requires validation in other blood banks and jurisdictions.
(6)) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing.
The hospital must maintain records of this Lookback notification as part of the patient's medical record.
Paper records of Lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost.
To facilitate the Lookback process and reduce paper documentation we sought to use the Electronic Medical Record (EMR) to perform and document notifications.
Study Design/Method: Representatives from Transfusion Medicine (TM) and Information Technology (IT) worked together to define minimum and optimal EMR solutions.
Minimally, a completed paper packet could be scanned into the EMR.
This solution had no advantages in terms of ease of use, process control, or transparency.
Desired optimal functionality includes the ability to send letters in the EMR, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received.
The EMR system at our institution, Epic (Epic Systems Corp., Verona, WI), has a function called "Letters" with the capacity to do all these tasks.
A series of five templates were developed: HIV and HCV letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters.
Templates are opened within the patient's EMR and demographic information is automatically populated by Epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g.
RBCs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender.
The completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic In Basket as well as in the patient's EMR.
Physicians may electronically complete and return the response form within Epic, or print it and return the form by fax.
Results/Finding: Between January 2014 and December 2016 thirty-five (35) notifications were sent to physicians using Epic Letters and of those, fourteen (14) responded to the Epic Notification and five (5) used the provided electronic response form.
For these cases the time to mail or handdeliver paper notifications was avoided.
The remaining 21 cases required follow-up paper notification, but the electronic Letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician.
Conclusion: Lookback notifications within the EMR makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors.
Secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters.
Evaluation of Ordering Practice in the Operating Rooms and Its Impact on Product Wastage Alexandra Budhai* 1 , Denden Benabdessadek 2 , Annu George 2 and Alexandra Jimenez 2 .
1 Westchester Medical Center, 2 New York Blood Center Background/Case Studies: Blood product wastage is an issue that many hospitals aim to address.
The OR was identified to have the highest rate of wastage within our hospital.
In this study, we assessed the appropriateness of the product order and utilization by the OR to understand its impact on wastage.
Study Design/Methods: Data on product orders, issue, and return for two months were analyzed.
The hospital CPOE and product requisition forms were used to collect this data.
The surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (MSBOS).
Trends for inappropriate orders for products by physicians were evaluated.
Results/Findings: A total of 941 orders were reviewed.
Approximately, 30% of these products were issued to the OR.
We found that the physician orders were within the guidelines of the MSBOS for most cases (89%), but of the issued products, all were returned to the blood bank in 40% of cases.
We observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal.
In addition, all of the products ordered for C-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases.
Conclusion: The data analyzed demonstrates that the majority of surgeons are adhering to our institutional MSBOS guidelines.
It was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible.
Our analysis revealed that the hospital's MSBOS does allow for an excess in blood ordering for some surgical procedures.
The MSBOS should be updated to reduce the suggested maximum product order.
In general, the data does not imply that the blood product wastage in the OR is due to the ordering practices of the surgeons.
A larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties.
Background/Case Studies: The VisionV R and VisionV R Max (Ortho Diagnostics, Raritan New Jersey) are ID-MTS TM Gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> 50 type and screens (T&S) per day] blood banks 1 , respectively.
Our laboratory which serves a large 1278-bed multispecialty academic hospital and receives 275-300 T&S specimens per day needed to replace three ProVUE analyzers prior to the availability of the VisionV R Max.
We implemented three VisionV R analyzers to work with our existing NEOV R and ECHOV R (Immucor Inc, Norcross Georgia).
A recent multicenter field application trial of the VisionV R reported a mean turnaround time (TAT) for T&S and ABO, Rh typing (ABO/Rh) of 32.2 6 4.5 and 27.5 6 5.6 minutes 2 , respectively.
The objective of this study was to determine VisionV R TATs under routine daily high-volume practice.
Study Design/Methods: One VisionV R was in operation during a five-week period (phase I), and then two additional analyzers were brought into service (phase II).
TATs are defined as the time when the order is received by the instrument to when the test is completed and available for review.
Three-cell screen and ABO/Rh TATs, and number of VisionV R antibody panels were collected for a nine-week period.
The TAT for the screen was used as the TAT for the T&S because the screen is the rate determining step.
All testing was performed using in-service analyzers on routine patient samples by trained technologists.
Samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time.
Results/Findings: Under the high volume conditions of our laboratory with three VisionV R analyzers, the mean T&S TAT was 30% longer and had a larger standard deviation (S.D.)
than the published trial result of 32.2 6 4.5.
TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT During phase I VisionV R 1 performed 263 panels.
During phase II VisionV R 1 performed 351 of the 361 VisionV R panels.
Conclusion: Our VisionV R analyzers are used under high volume conditions more suitable for the VisionV R Max.
When balanced with the testing menu, including ability to perform select cell panels, our TATs using three analyzers were satisfactory.
The large standard deviation indicates that opportunities remain for improving TATs through workflow improvement.
From West Nile Virus to the Emergence of Zika Virus: A Nationwide Survey of How Regulators Are Keeping the Blood Supply Safe and Available Falisha Atwell* 1 , John Roback 2 , Ronald Arkin 1 , Michael Bartlett 1 , Robert Geiger 1 and Jaxk Reeves 1 .
1 University of Georgia, 2 Emory Hospital Background/Case Studies: With the emergence of ZIKV in the United States, it is important to assess the FDA's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases.
This research compares the responsiveness of the FDA during West Nile Virus (WNV) and Zika Virus (ZIKV) outbreaks to evaluate our current preparedness.
Study Design/Methods: The literature review was conducted to analyze FDA's response time during the WNV crisis and determine if it was effective and efficient.
The research survey was performed to determine if the Donor History Questionnaire (DHQ) adequately screens donors for ZIKV as the sole preventive method (as per the February 2016 Guidance for Industry: Recommendations for Donor Screening, Deferral and Product Management to Reduce the Risk of Transfusion-Transmission of Zika Virus) and to determine if the current regulatory practices (including the August 2016 Guidance for Industry: Revised Recommendations for Reducing the Risk of Zika Virus Transmission by Blood and Blood Components) are perceived to be effective and efficient in the face of the current ZIKV outbreak.
Survey Monkey was used and participation was anonymous.
Over 4,000 emails and web-links were sent to members of AABB, SCABB, SEABB, and personal network with a 10% target response rate.
Participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter.
Results/Findings: The literature review revealed that the FDA's response was slow during the WNV outbreak, while the ZIKV response is efficient thus far.
A total of 317 participants responded to the survey (7.94% response rate).
Statistically, participant agreement with FDA's decisions was performed by "t" test (with n-15317-15316 df) of the null hypothesis that the mean50 vs. the alternative that the true mean is> 0.
Overall participants had favorable opinions of the FDA's decisions.
Statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way ANOVA models were used with Likert-scale question responses as if they were continuous.
The F-statistic and P-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable.
There were no significant differences in the years of experience and profession variables for participants.
Region was determined to be unreliable due to undefined states for each region listed.
Conclusion: The research revealed that industry experts conclude that the current system of DHQ and FDA guidance documents, if issued timely, are adequate.
Background/Case Studies: When evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab.
There are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time.
Study Design/Methods: The study involved a current state to a future state comparison of testing processes with an instrument ORTHO ProVueV R (PV) and manual testing vs. an instrument ORTHO VISIONV R (OV).
Data collection methods included direct observation, time studies, and interviews.
The PV bench performs Type & Screens (TS) on the PV and manual AbID/selected cell panels in the gel test.
All other testing; cord blood(CB), DAT, unit confirm(UC), patient type confirm(PC) and crossmatch(XM), etc.
are done manually in tube.
The future state incorporated the OV.
TS, AbID and UC were evaluated in both states.
Cycle time(CT) was averaged based on 3 run cycles.
CT was comprised of 3 metrics; instrument time(IT), standby time(ST) and labor time(LT).
ST may be comprised of 2 components, time that could be utilized as "walkaway" time or vigilant time (VT) which requires operator presence but not operator action.
For automated instruments, VT for each cycle was measured as instrument access unavailable.
Instrument daily maintenance (DM) CT was evaluated as well.
Similarly, timing of manual tube test processes used these metrics.
For repetitive activities within a process, such as UC or XM, a time per individual process was captured and then multiplied per unit.
Results/Findings: Table 1 provides details about the metrics of current state and future state processes.
Tube based test timing is as follows: PC (2:50), XM (32:23), CB (13:18) and DAT (10:00).
By implementing the future state, an average $1.3 min.
LT and VT is saved on each sample loaded for TS equating to a 73% labor reduction over the current state.
A 19% improvement in TAT on the TS was achieved in the future state.
Moving from manual AbID to automated processing resulted in a 59% LT reduction.
On average, a 38 min.
continuous walk-away time is achieved for each automated AbID.
UC had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics.
Conclusion: Based on the metrics evaluated and compared between current state and future state, the OV has demonstrated improvement in lab operations to both the labor required and result TAT delivery.
Opportunity exists to automate workflows on other tests that are still manually performed.
Background/Case Studies: High throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $100 type and screen samples per day.
The ErytraV R (Grifols) is a fully-automated walkaway analyzer utilizing 8-column gel cards for pretransfusion testing.
The blood bank validated and implemented the use of ErytraV R for ABO/D typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform.
The blood bank also validated automation of donor unit retypes.
The instrument has bidirectional interface to the blood bank lab information system (LIS), HCLL TM (Hemocare Life Line, Mediware).
Instrument validation and implementation were done in conjunction with the software version upgrade of HCLL TM and an interface system change to Maestro TM .
Study Design/Method: Correlation testing of the ErytraV R results with the manual tube testing (PEG IAT; reference method) was performed on 100 patient samples for ABO/D typing and antibody screening; of which at least 10 had a positive antibody screen.
Out of the 10, 5 had known antibody specificities.
Forty-two RBC units were also tested for ABO/D confirmation; of which 17 were D(-) and 25 were D(1).
Calculations of concordance, sensitivity, and specificity were performed.
Precision studies were also done.
Interface testing of ErytraV R , HCLL and the hospital's information system using the Maestro TM interface system was performed and validated.
Results/Finding: Concordant results between both methods were obtained in all of the 100 patient and 42 donor samples tested (100% concordance).
All 10 samples with positive antibody screens were obtained by both methods.
All clinically significant antibodies were detected by both systems.
ErytraV R gave 100% sensitivity and specificity.
The precision studies showed that both methods gave the same type and screen results for 5 samples at 3 different testing events.
After validation of the LIS upgrade and interface system change, a bidirectional interface with HCLL TM was established.
The instrument has been operational in our lab for over 3 months.
Conclusion: ErytraV R was found to be reliable and accurate and can handle the high workload of our lab.
Users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable.
The validation of the the instrument is straightforward.
The major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring LIS upgrade and migration of the data integration system.
A post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential.
Implementation of a System-Wide Platelet Inventory Report Optimizes Platelet Utilization and Reduces Unit Wastage Elly Landolfi* 1 , Craig Fletcher 2 and Peter Millward 1 .
1 Beaumont Hospital, 2 Beaumont Health System Background/Case Studies: A sufficient number of blood components should be available to meet routine and emergent hospital needs.
This must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short 5-day shelf-life.
We report the results of a quality improvement project implementing a custom computerized Platelet Inventory Report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates.
The report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location.
All system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier.
Study Design/Method: The study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data.
The report went live October 2014 and quality data was reviewed from August 2013 to December 2016.
The collected data was then analyzed using descriptive statistical methods.
Results/Finding: Data from 2016 indicates platelet wastage comprised 1% of total received platelets and 79% of these wasted platelet units were due to expiration.
Other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility.
The mean of monthly wasted platelet units 12 months preimplementation of the report was 13 units, compared to 11 units 12 months post-implementation and 5 units 24 months post-implementation.
Wastage rates improved from 6% (wasted yearly platelets/total received yearly platelet units) in 2014, the year of report implementation, to post-implementation rates of 3% in 2015 and 1% in 2016 (see table) .
Importantly, this occurred despite a greater than 30% increase in platelet inventory between 2014 and 2016 and resulted in cost savings of over $60,000 in this period.
Conclusion: Study limitations included restricting data collection to one campus.
The option to transfer expiring platelet units to another blood bank was available to all 4 participating sister hospitals.
It would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices.
Aside from lowering platelet wastage within 2 years of implementation, additional benefits to the report included facilitating ordering from the blood supplier.
Cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units.
We have shown achievement of this end is facilitated by a customized daily Platelet Inventory Report -an efficacious and easily adaptable tool with demonstrable gains.
Valerie Halling* 1 , Lisa Marie Button 2 , Lori Scanlan-Hanson 2 , Karen Koch 2 , Janet Finley 2 , Deepi Goyal 2 and Camille van Buskirk 3 .
1 Mayo Clinic-Rochester, 2 Mayo Clinic, 3 Mayo Clinic Rochester Background/Case Studies: Transfusions in the Emergency Department of a Level I Trauma Center were ordered using a handwritten order form.
The Transfusion Lab's (TL) management team and Medical Director met with Emergency Department (ED) leadership and IT resources in 2014 to define the needs of a successful electronic blood transfusion system.
The handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected.
The potential error sources included clerical errors involving the patient's name or medical record number (MRN), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (STAT or Routine), or not including the prescriber call-back information.
The number of ED reported transfusion related events in 2013 and 2014 were 63/1187 (events/ED transfusions 2013-2014).
Study Design/Method: Electronic ordering for the ED was implemented March 31 st 2015.
Any transfusion orders generated from the ED are now electronic, unless in the case of electronic downtime.
The system electronically fills in the patient's name and MRN, controls for the type of blood product being ordered, requires an order priority and provides service contact information.
It was designed to accommodate transfusion ordering needs for adults, pediatric patients <35kg and pediatric patients >35kg.
252A TRANSFUSION orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate.
The electronic system was designed so that an order cannot be submitted unless all critical fields are completed.
Results/Finding: The electronic ordering system has been in place for 2 years (April 2015 -March 2017), and during that time there was 1 instance of blood being ordered for an unintended patient 0.09% (1/1081).
This was because a previous patient's medical record was accessed rather than the intended patient's medical record.
There have been no instances of clerical errors (name misspelled or MRN transposition etc.
), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order.
Electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance.
Prior to electronic order implementation, in 2013, 17/651 (2.61%) units were transfused in the ED but not charted in the patient's medical record.
In 2014, 18/536 (3.36%) transfusions were not charted.
However, in 2016, the first full year of electronic transfusion order capability, only 4/462 (0.87%) transfusions were not charted in the patient's medical record.
Conclusion: Electronic ordering in the ED has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians.
It allowed the order to be processed more quickly by TL, resulting in a faster turnaround time.
Improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against.
Implementation of Blood Bank Automated Attendant Lok Tse*, Gerald Motta and Maria Aguad.
Brigham and Women's Hospital Background/Case Studies: The Blood Bank receives numerous nonemergent phone calls on a daily basis.
These calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred.
The Hospital is categorized as a Level 1 trauma center, with over 700 inpatient beds and over 50 operating rooms.
A proposal to implement a Blood Bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (MTP) and emergency release of blood products.
Study Design/Method: The first step was to categorize the types of phone calls received by the Blood Bank by creating a phone log.
Data were collected and analyzed for four weeks.
The blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls.
It was very important to maintain patient safety and quality of service at the same time.
The automated attendant consist of: Option 1 (Urgent) for trauma, emergency release, MTP and obstetric hemorrhage emergency release; Option 2 (Verbal) for verbal orders and coolers set up; and Option 3 (Staff) to speak with staff member.
Instructions were also given for specimen inquiry and product availability in the hospital information system.
Results/Finding: The data in Table 1 showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries).
Most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability.
There was an overall decrease in phone calls by 68% with the implementation of an automated attendant.
Conclusion: With the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders.
The decrease in phone calls freed up team members to perform other critical tasks in the department.
Improved Detection of Wrong Blood in Tube Errors: Implementation of a Two-Sample Blood Type Verification Process Ariana King* 1 , Steven Zibrat 1 , Geoffrey Wool 2 and Angela Treml 2 .
1 University of Chicago Medicine, 2 University of Chicago Background/Case Studies: Our organization used a blood bank identification (BBID) band system for pre-transfusion testing and detection of Wrong Blood in Tube Errors (WBIT).
Additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available.
The BBID bands were prone to clerical errors and excessive specimen rejections, and believed to miss some WBIT errors.
In 2015, Blood Bank accounted for 48% of all rejected clinical laboratory samples, yet comprised only 5% of total laboratory volume; 88% of rejected Blood Bank samples were due to BBID band issues.
The WBIT error rate detected by BBID-based system was 0.006%.
Study Design/Method: A multidisciplinary workgroup was formed to review data and best practices.
The decision was made to discontinue BBID bands and implement a two-sample verification process, in keeping with Standards.
A new laboratory test order was created in the EMR system and embedded into the existing T&S order.
Providers are prompted to order the ABO verification test only when no previous ABO/Rh typing results are found.
Education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically.
The new process went live in September 2016.
Results/Finding: In the five months following implementation, four WBIT errors were detected with the second sample.
These may have been missed using the BBID band system.
Improved detection revealed a WBIT error rate of 0.128%, three times the national average of 0.043%.
Under the new system, rejected Blood Bank samples decreased from an average of 50% to 28% of all rejected laboratory samples, a 43% decrease.
Implementation of the new process produced a net savings of $55.8K.
Conclusion: Replacing the BBID band system with two-sample verification successfully improved our ability to detect WBIT errors among patients who lacked historical blood bank results.
Additionally, discontinuation of the BBID system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization.
Next steps are for Blood Bank and Laboratory Quality leaders to partner with nursing leadership to drive down WBIT error incidence.
253A addendum with the final culture results.
We used a student's t test to determine whether there was a statistically significant difference in the mean TAT for result addendum entry in the post-implementation period compared to the pre-implementation period.
Results/Findings: In the pre-implementation period, we cultured 19 residual products for suspected STR.
The TAT for final culture result entry into the patient's EMR was 5-78 days (mean 19 days, SD 20).
In the postimplementation period, we cultured 22 residual products for suspected STR.
The TAT for final culture result entry into the patient's EMR was 5-12 days (mean 7 days, SD 2; p50.0082).
There were no positive cultures during either study period.
Conclusion: Our study demonstrates that TAT for documentation can improve with the use of information technology to notify the Transfusion Medicine physician when results are available for documentation in a patient's EMR.
Improved Turnaround Time of Type and Screen Samples Michaelene Hultman* 1 , Marcus Holme 1 , Johnathan Bakst 1 , Gunta Musa 1 and Angela Treml 2 .
1 University of Chicago Medicine, 2 University of Chicago Background/Case Studies: The primary test performed in the Blood Bank with regard to pre-transfusion testing is the Type and Screen (TYS).
The current target for this institution's blood bank is an 80 minute turnaround time (TAT).
In April of 2016, the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method.
The average number of outliers increased 104%.
TAT analysis of a representative one week sampling per month showed an increase in outliers from 28 per month to 57 per month.
Average monthly TYS samples performed is 2758.
These numbers did not improve even upon returning to the original facilities.
Study Design/Method: Two Ortho Clinical Diagnostics VisionV R Analyzers (Raritan, N.J.) were purchased for the blood bank.
The instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests.
Batch testing was eliminated allowing samples to be run as received.
The results were auto interpreted, and transmitted to the laboratory information system (LIS) based on predetermined rules.
Only results in need of manual review or interpretation were held back.
Final verification of results was performed by the technologist within the LIS.
Reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run.
Key quality indicators including TAT continued to be monitored throughout implementation.
Data was monitored for significant changes and improvements in patient care.
The go-live date was 12/20/ 2016.
Results/Finding: The average number of outliers decreased 61% from 57 per month to 22.
Further benefits include a reduction in the number of technologists needed to perform TYS testing.
Additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $134,000 for fiscal year 2017.
Conclusion: The use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in TAT over manual gel method.
Improvements in the Timely Reporting of Final Product Culture Results in the Patient's EMR.
Barbara A. Hewitt*.
Dartmouth Hitchcock Medical Center Background/Case Studies: In certain transfusion reactions it is required that a culture of the returned blood product be performed.
These cultures are reported in our Cerner operating system but those results do not cross over to the patient's EMR .
The finalized product culture results are entered into the patient's EMR as an addendum to the transfusion reaction clinical note.
A review of the Transfusion Reaction database revealed that there were occasions when the final product culture results were not entered into the patient's EMR in a timely manner.
It is important to the patient's care for the Transfusion Medicine Service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health.
This information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility.
Study Design/Methods: A review of a specific 7 month period revealed that the timeframe in which the finalized product culture results were entered into the patient's EMR ranged from 0-65 days with a mean of 13.64 days.
It was determined that this was not in the interest of improving patient care.
In collaboration with Laboratory Information Services a report was created in which once product culture results were finalized an email would be generated notifying the Medical Director and the Transfusion Safety Officer that results were available.
Results/Findings: Data was collected for 7 months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's EMR improved to a range of 0-7 days with a mean of 2 days.
Conclusion: Improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions.
This process can be made easier when the correct tools are used.
Omer Ilyas* 1 and Randy Levine 2 .
1 Northwell Health, 2 Lenox Hill Hospital Background/Case Studies: Transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening Graft Versus Host Disease (GVHD).
After noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank.
Study Design/Method: The project was separated into three parts.
In the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit.
The variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank.
All patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood.
The second part of this project was an educational intervention.
Residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor.
Residents were also instructed to order irradiated blood for all patients on the oncology unit.
In the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful.
Results/Finding: Pre-intervention, 67 units were transfused on the oncology floor with 38 units (57%) requiring irradiation and only 22 of those 38 units (57%) ordered as irradiated.
Since the blood bank occasionally issues irradiated blood without a specific order, 9 additional irradiated units were issued (31/67; 46%).
Post-intervention, 29 units were transfused on the oncology floor with 15 units (52%) requiring irradiation and all 15 of those units (100%) ordered irradiated specifically to prevent GVHD.
Eight additional irradiated units were ordered with no requirement for irradiation; thus 23 of the 29 (79%) total units were ordered as irradiated.
Again, 4 additional irradiated units were issued (27/29;93%) without a specific order by the blood bank.
The results are summarized in the accompanying table.
Conclusion: This quality improvement project demonstrates that educational intervention can succeed in changing clinical practices.
Continued monitoring of ordering practices will ensure that compliance continues.
We plan to expand the quality improvement project to other settings, including the emergency department and surgical floors.
We expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events.
Samantha Ngamsuntikul* 1 , Charlotte Van Dyke 2 , Dina Garza Van Hoose 2 and Rachel Beddard 1 .
1 BioBridge Global, 2 South Texas Blood and Tissue Center Background/Case Studies: At our blood center, apheresis platelets and red cells are collected on Trima Accels and double red cells on Haemonetics 8150s.
In addition to routine quality control (Qc), Qc is performed for instrument flags on collection instruments.
Quality control for apheresis platelets includes: volume variance and rWBC; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass.
Study Design/Methods: During the period of January 1, 2016 to April 19, 2017, 2,097 total collections were flagged for additional Qc by our Trima Accels and Haemonetics 8150 instruments.
Quality control at our center is tracked by our quality control software management system, HemaTerra's HemaComply which allows the ability to track and retrieve this information.
The majority of products flagged for instrument Qc pass and are released for distribution.
A small percentage, however do fail Qc leading to loss of product.
Quality control data can be retrieved and monitored for trends using a quality control software management system.
Background/Case Studies: In 2015, The Centers for Medicare & Medicaid Services (CMS) rolled out a plan for implementing IQCP (Individualized Quality Control Plan) as a new quality control option based on a risk management plan for CLIA laboratories performing non-waived testing.
This plan was meant for CLIA approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples.
Study Design/Method: CLIA Clinical laboratories can either follow traditional clinical CLIA QC requirements according to the regulations or Implement IQCP.
While we perfrom traditional QC assessments on all the tests we perfrom on our cellular products we did decide to implement the IQCP program within in our Quality Control Laboratory.
We followed the IQCP process for assessing some of our QC tests used to assess the safety, purity and potency of our cell based products.
One test in particular where we applied this tool was in the review of our QC sterility testing method and found it to be a very useful in improving the overall process.
The tool walks you through three process requirements: 1) Risk Assessment, 2) Quality Control Plan and 3) Quality Assessment for the preanalytical, analytical and post analytical phases of testing.
ABSTRACT Conclusion: The integration of the IQCP into the Quality Control Laboratory was determined to be a success.
The IQCP tool was successful in identifying gaps within the sterility testing process.
This tool will be used on additional quality control tests and manufacturing processes.
The implementation of the IQCP program ensure regulatory QC requirements appropriate for testing performed.
We were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error.
Objective Performance of Massive Transfusion Protocols at a Single Institution Gustaaf de Ridder* 1 , Rachel Jug 1 , Kimberly Ingersoll 1 , Nicholas Bandarenko 2 , Nicole Guinn 3 and Jessica Poisson 2 .
1 Duke Health Pathology, 2 Duke University Hospital, 3 Duke Health Anesthesiology Background/Case Studies: Hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients.
Using a balanced 6:6:1 transfusion ratio (TR) for massive resuscitation is recommended based on trauma data.
Objective performance during Massive Transfusion Protocol (MTP) activations is poorly studied and there may be differences based on site or medical service of MTP initiation.
With the impending release of a unified, redesigned Exsanguination Protocol (ExP) at our institution, we established baseline performance characteristics for our existing MTP and Obstetric Massive Transfusion Protocol (OBP).
Study Design/Method: Following Institutional Review Board approval, we performed a retrospective study on blood product utilization and outcomes of MTP and OBP activations from July 2015-December 2016.
Data were manually collected from transfusion service paper records, electronic (Safe-Trace) records, and an automated data report from the electronic medical record (Epic).
Conclusion: We observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation.
Trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells.
LOS and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion.
We have identified an opportunity for improvement in MTP transfusion ratios observed in trauma cases, the specific environment from which the 6:6:1 ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious.
Patient Identification Improvement Strategy to Help Reduce Unacceptable Specimens Arline Stein* 1 , Nancy Nikolis 1 , Linda Benison 1 , Ruthmire Thelusca 1 , Renee Liberty 1 , Sherry Shariatmadar 1 , Alexander Indrikovs 2 and Vishesh Chhibber 1 .
1 North Shore University Hospital, 2 Northwell Health Background/Case Studies: Our blood bank (BB) processes approximately 60,000 specimens per year.
BB specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation.
In such cases, a new specimen is requested to be drawn as per protocol.
Our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the BB supervisor for completion.
Following completion, the report was sent to the Nurse Manager of the patient care unit (PCU) for follow-up and investigation with the staff members involved.
This process was cumbersome, taking a few days before the staff member of the PCU was alerted to the deviation in protocol.
At times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services.
Study Design/Method: In June 2016, a Patient Identification Improvement Strategy was implemented jointly by the department of nursing and the BB to address mislabeled, unlabeled and unsigned specimens as part of a Patient Safety Initiative.
Currently, following this strategy, when an unacceptable specimen is received, the Nurse Manager (NM) of the PCU is immediately notified by BB Staff.
The NM promptly initiates a debrief process with the staff involved in drawing the specimen.
A debrief form (tool) was created to guide the discussion.
This process is followed 24/7.
The NM will also engage other available staff in a huddle to review the incident and reinforce the policy.
The debrief form is then submitted to hospital QA and the BB with preventative actions included.
We believe in using the JUST CULTURE MODEL to help us understand the reasons why the staff did not label the specimen according to policy.
Just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care.
Results/Finding: The table below represents the percentage of unacceptable specimens identified by the BB since the second quarter of 2016.
The implementation of this new process has led to a decrease in the number of unacceptable specimens up to 30% quarterly following its implementation.
The opportunity for direct intervention by the NM with the staff involved has risen from 75% to 96%, due to the immediate debrief process.
ABSTRACT Conclusion: The Patient Identification Improvement Strategy allows for real time engagement of the BB and PCU staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection.
The heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience.
Platelet Transfusion Practices Among Pediatric Oncology Patients: A Single Institutional Experience Nicole M Crews* 1,2 , Morgan Rockwell 2 , Joseph Hagan 1 , Jun Teruya 1,2 and Shiu-Ki Hui 2 .
1 Texas Children's Hospital, 2 Baylor College of Medicine Background/Case Studies: Despite advances in adult platelet transfusion (PTx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses.
Therefore, PTx are commonly guided by local institutional recommendations (IR).
The aim of this study was to determine the degree of adherence of PTx practice to IR at a pediatric tertiary institution.
Study Design/Method: Retrospective review of PTx practices including transfusion thresholds, responses and dosages were collected.
Platelet counts within 24 hours pre and post transfusion were evaluated.
Patients (0-18 years) receiving prophylactic PTx from July to December 2016 admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included.
For prevention of volume overload, the IR for PTx were < 10ml/kg for patients < 35kg and one apheresis unit (AU) for patients >35kg; therefore, patients were separated into 2 groups: < 35 kg and > 35 kg.
A significant proportion of orders for both < 35 kg and > 35 kg did not meet patient platelet threshold criteria (p<0.001).
Conclusion: PTx threshold above IR for both groups were 31 ( 35 kg) and 48% (> 35 kg).
Most common reason for above IR threshold was an invasive procedure or low molecular weight heparin therapy.
Greater than 10% of PTx dosage in both groups were above IR, however the platelet response did not increase significantly (p>0.05) with a higher dose vs. IR dose.
This study demonstrated that there were still considerable deviations from IR in PTx practice among pediatric oncological patients.
In addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events.
Each institution should conduct a quality assurance review to determine PTx practice.
Pre-Surgical Sample Process Improvement to Enhance Patient Safety and Compliance Lisa Marie Button*, Stephanie Saathoff, Jered Luedke, Benjamin Colvin, Umalkair Amare and James R Stubbs.
Mayo Clinic Background/Case Studies: Our institution provides the option for presurgical samples (PSS) to be drawn up to 56 days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding 3 months from the date of PSS collection.
When PSS patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the PSS draw to the day of surgery/possible transfusion.
Study Design/Method: An electronic fix was designed that was applied to the surgical intake process.
A new set of questions was added to the A.M. Admit questionnaire that must be completed prior to the patient's surgical procedure.
The questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank.
The blood bank techs review the alert and inactivate the patient's PSS based on the new transfusion/pregnany information.
One year post-implementation of the electronic fix, Transfusion Lab performed a retrospective review of all PSS alerts generated during a three-month period.
Results/Finding: The results of the review were analyzed and are displayed in the Table.
It was determined that only 2.1% of patients with a PSS Alert had an active sample requiring inactivation.
Conclusion: Implementing an electronic solution that requires documentation about PSS eligibility upon return for surgery has resulted in an estimated 3220 (805x4) PSS Alerts in the blood bank each year.
Of these Alerts, it is estimated that approximately 68 patients (17x4) per year are identified as no longer eligible for PSS status.
Once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and PSS status.
While the benefit of having fewer false positive PSS alerts (97.9%) was recognized as an ideal future state, it was not compatible with the institution's current IT project of implementing a new Electronic Medical Record (EMR) system.
The safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new EMR with the intention of targeting only patients with an active PSS in the blood bank, rather than all surgical patients.
Weill Cornell Medicine, 7 Columbia University School of Medicine Background/Case Studies: Blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays.
Risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation.
Given the complexity and high-risk nature of blood ordering a proactive risk assessment (PRA) for blood product ordering using the FMEA methodology was conducted.
The goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering Study Design/Method: To evaluate the electronic blood ordering redesign process, a PRA was completed using the FMEA methodology.
The team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the Risk Priority Number (RPN).
All RPNs with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode.
Results/Finding: The group scored the identified failure modes by categories used in root cause analyses.
The electronic blood order process has internal logic and alerts that improve communication and reduce the risk score.
Several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the RBC order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system.
A transfusion history will be available to providers when ordering blood products to further reduce communication risks.
Categories for failure modes included Clinical,Communication,Equipment People,Process and System.
The average overall failure mode RPN was reduced by 29% with the communication category average RPN having the greatest reduction of 72%.
Conclusion: An FMEA of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration.
Accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood.
Reducing Turn Around Time for Type and Screens in the Blood Bank Kimberly Ouellette* 1 , Karen King 1 and Joseph Sweeney 2 .
1 Rhode Island Hospital, 2 Lifespan Academic Medical Center Background/Case Studies: Expeditious turn-around times (TAT) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner.
The Blood Bank at Rhode Island Hospital, a level 1 trauma center and teaching hospital associated with Brown University, was originally designed to accommodate tube testing by all technologists.
The original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third.
As technology changed, the blood bank adopted first the manual gel station and then the automated gel system (Ortho ProvueV R ) but did not adapt the space.
The second section of the blood bank contained the manual and subsequently automated gel stations with no other changes.
The process of sample receipt through completion of testing and issuing of units remained segmented and inefficient.
The average TAT for type and screens was 80 6 32 minutes.
Study Design/Method: The Blood Bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology.
The first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory.
A wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt.
The third section remained, but was repurposed for teaching medical technology students and residents.
In addition to the remodel, the blood bank retired the Ortho ProvueV R for the Ortho VisionV R , which is considered a true continuous feed machine.
Although the inter-device TAT is not significantly different (30 minutes for the ProvueV R and 28 for the VisionV R ) the VisionV R is built with a scheduler that effectively handles the system and processes samples efficiently.
The VisionV R is also equipped with two centrifuges to process samples, which further reduces TAT when multiple samples are onboard.
A bi-directional interface was designed to allow for test orders (type and screens) to go to the VisionV R and test results to go directly from the VisionV R to the LIS without the need to manually order the tests or transmit the results.
Data on TAT were collected and analyzed using independent t tests and chi square.
Results/Finding: The mean TAT pre-and post-reconfiguration and implementation of the Ortho VisionV R and a fraction of samples with TAT over 90 minutes are shown in the table.
The results show a reduction in TAT by 14 minutes with a 20% reduction of TAT greater than 90 minutes.
Conclusion: A combination of new technology and space remodeling can lead to a significant reduction of TAT of testing in the blood bank.
Caleb Wei-Shin Cheng* 1,2 , Lorna Orengo 3 , Monique Scott 3 and Christopher A Tormey 1,2,3 .
1 Yale University School of Medicine, 2 Yale-New Haven Hospital, 3 VA Connecticut Healthcare Background/Case Studies: The type and screen (T&S) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients.
Despite this, erroneous blood product administration may occur as much as 1 in 19,000 blood transfusions.
To prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management.
Hemolysis rates for T&S specimens are reported to be as high as 25% prior to interventions, but may potentially be reduced to as little as 1.5%.
However, there is little published data on non-hemolysis-related type and screen rejections.
An initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of <5%, with a particular emphasis on non-hemolysisassociated forms of rejection.
Study Design/Method: A root cause analysis (RCA) was performed over the preceding 6 months to obtain a baseline understanding of the errors involved.
T&S submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously.
When a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection.
Following RCA, an intervention was created to resolve the most common issues documented that resulted in rejection.
Approval for the intervention was granted by the Department Chair, Transfusion Committee, Forms Committee, and the Medical Executive Committee.
After implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention.
Results/Finding: Over the study period, the T&S rejection rate averaged 8.2%.
Reasons for specimen rejection were divided into 5 groups: 1) hemolysis, 2) blood bank witness collection form errors, 3) quantity not sufficient, ABSTRACT 4) duplicate sample, and 5) specimen tube labelling errors.
The highest percentage of rejections was due to improperly-filled witness forms (Table 1) .
After multiple form redesigns and approval by appropriate committees the new form was implemented.
Preliminary data collected thus far demonstrates a 6.8% rejection rate with only 1 rejection relating to witness form errors.
Conclusion: Rejected T&S specimens are an impediment to safe clinical care as it may delay medical/surgical management.
Rejection rates could be reduced through simplification of blood bank specimen collection forms.
Care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error.
Future quality initiatives will aim to simplify complex healthcare processes without compromising patient care.
Reduction of Failed Whole Blood Donor Testing Runs on the Roche Cobas s 201 System Christopher Shahan* 1 , Christina DeJesus 1 , Mosi McCall 1 , Fallon Hampton 1 , Tangi Herring 1 , Judy Davis 1 , Anjali Patel 1 , Sonya Gomillion 1 and Bonnie Maltby 2 .
1 QualTex Laboratories, 2 QualTex laboratories Background/Case Studies: As part of our quality control program, we track the number of technician related failed runs observed on the Roche cobas s 201 System.
This system is used to test whole blood donor samples for Human Immunodeficiency Virus (HIV) RNA, Hepatitis C Virus (HCV) RNA, Hepatitis B Virus (HBV) DNA and West Nile Virus (WNV) RNA.
Technician related failed testing runs can cause the laboratory to report results outside of the contractual 10-12 hour turnaround time.
Failed runs also cause retesting which increases reagent utilization for the system.
Currently 42% of whole blood donor testing turnaround time delays are due to issues and failed runs on the s 201 system and we have 3 technician related failures per week.
A Lean Six Sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s 201 system.
Study Design/Method: The number of technician related failed runs on the s 201 system were tracked from 10/11/2015 thru 12/11/2016.
A Pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures.
The 5 Whys were performed to determine root causes of technician related failed runs.
A Gemba walk was performed on all of the lab testing processes to help identify areas for improvement.
The process Improvement team talked, met, observed, and worked directly with staff that operate the s 201 system.
Roche was also contacted to provide guidance on how to help decrease technician related failures.
Results/Finding: The main root cause determined was that there was no current process flow map for whole blood donor testing using the s 201 system.
Counter measures implemented included creating a two phase process map.
One phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples.
Roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s 201 system.
After counter measures were implemented, the number of technician related failed runs decreased from 3 to 1.2 failures per week, which was a 58% decrease.
Conclusion: A Lean Six Sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s 201 system.
This lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by 58%.
patients who were transfused for pre-transfusion hgb >7g/dl with resulting post-transfusion hgb >9g/dl were reviewed.
Demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by 11 volunteer internal medicine residents.
Group discussion for each case ensued before determination of transfusion appropriateness occurred.
Principal investigator/attending physician then made final determination of appropriateness of RBC transfusion.
Results/Finding: 265 patient charts were reviewed.
91 were excluded for bleeding and cardiovascular instability.
106/174 (60.9%) were determined to be transfused inappropriately.
There was no difference in appropriateness of transfusion with respect to age or sex.
Patients with solid tumors (76.19% vs 55.73%, p50.0181) and anemia of chronic disease (76.47% vs 54.1%, p50.006) were more likely to be inappropriately transfused.
Patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb 7.7 g/dL vs 7.3 g/dL, p<0.0001).
Inappropriately transfused patients also had higher median post-transfusion hemoglobin (9.9 g/dL vs 9.4 g/dL, p<0.0001).
Moreover, lab evalutions revealed association with lower folate levels (median 8.1 nmol/L vs 15.7 nmol/L, p50.029).
29/106 (27.3%) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between.
7/64 providers were responsible for 32.3% of all inappropriate transfusions.
1/68 appropriately-transfused patients experienced an FNHTR.
3 deaths unrelated to transfusion occurred (1 in appropriate, 2 in inappropriate group).
Conclusion: Physicians in training are interested in promulgating optimal RBC transfusion practice.
This study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion.
Beyond CPOE, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions.
Successful Implementation of a Blood Bank Information System in a Small-Scale Caribbean Blood Bank: A Structured Step-Wise Approach.
Luigi Sille* 1 , Willem Martin Smid 2 and Ashley John Duits 1 .
1 Red Cross Blood Bank Foundation, 2 Sanquin Consulting Services Background/Case Studies: An important tool for complying with GMP quality standards is the effective use of a blood bank information system (BIS).
Validation and implementation of a BIS is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment.
.
Small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention.
For the introduction of a BIS at the blood bank of the Dutch Caribbean island of Curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting.
Study Design/Method: The Red Cross Blood Bank Foundation Curaçao is the sole provider of labile blood components for the Dutch Caribbean islands of Curaçao, Bonaire and Sint Maarten.
After selection of the BIS provider for implementation ISBT and BCSH guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures.
These procedures were meant to evaluate and validate the features of a BIS (e-Delphyn, Hemasoft America, Miami, USA) before introduction.
The outcome of the approach was entered in worksheets that were evaluated by the implementation team and management.
From this the implementation plan was designed and implemented.
An external auditor (Sanquin Consulting Services, Amsterdam, Netherlands) was invited to evaluate the implementation and validation plan and its practical implementation.
The evaluation was performed according to risk assessment of critical process steps.
Results/Finding: Based on the ISBT and BCSH guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a BIS was designed.
Comparison of the current processes and procedures were compared to the BIS characteristics making use of 8 worksheets.
With these worksheets the existing gaps with the BIS procedures were carefully described.
These gaps and the appropriate procedural changes for BIS or blood bank were effectuated.
The worksheets also provided the basis for staff training in a separate training environment before BIS introduction.
During the early validation phase all procedures and processes were audited by an external auditor.
With the feedback of the expert several improvements were added for the validation and subsequent implementation processes.
Conclusion: With the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a BIS in a small scale Caribbean blood bank.
The program as designed seems well suited for small scale blood banks contemplating introduction of a BIS.
Time and Cost Savings through Implementation of a Remote Blood Fridge Jessica Peters* 1 , Dee Dee Cassidy 1 , Jed B Gorlin 1,2 and Nancy L Van Buren 1,2 .
1 Hennepin County Medical Center, 2 Innovative Blood Resources Background/Case Studies: Rapid delivery of emergency release group O red blood cells (RBC) are vital to patient care.
Commercial remote blood fridge packages are available but have large upfront and maintenance costs.
We implemented a remote blood fridge directly in our Emergency Department (ED) using an under counter fridge requiring ID access, and a selfdeveloped IOS application that scans, tracks and real-time alerts Transfusion Service (TS) to products used and to whom they were dispensed.
Prior to ED fridge implementation, RBC units were verbally requested and an ED blood runner would pick up and return the cooler.
Given that our ED is located in a separate building from the TS, this meant 10 or more minutes may be required for transit of units often released in less than 5 minutes.
The net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required.
Implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release RBCs more quickly, with the observed benefit of decreasing wasted staff time.
Study Design/Method: The remote blood fridge was implemented in July 2016.
Data for RBC requests in coolers, RBC returns and RBC transfusions from the ED was collected and compared.
Baseline data included January 2015-June 2016, and post change included August 2016-December 2016.
July 2016 data was excluded as it included both the pre and post processes.
Results/Finding: Baseline data shows that the ED requested an average of 99 RBC/month in coolers.
Post change this dropped to 62 RBC/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge.
Baseline data also shows that an average of 54 RBC/month were returned (55%).
Post change, the average RBC/ month returned was 27 (44%), this represents an absolute 50% reduction in number of returned products.
Each RBC dispensed and returned takes approximately 20 minutes to complete paperwork and transport, therefore this change saved an average of 540 minutes per month.
It was also noted that the average RBC/month transfused was 44 for baseline and 57 post change.
This confirms that the decreased requests and returns were not due to decreased patient volume or severity.
The fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of 15-20 minutes (estimated) was reduced to 3-5 minutes.
Conclusion: Implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient.
This change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns.
Implementing a blood fridge can also be done at a reduced cost through homegrown processes.
Transfusions are everyday procedures and over 250 Patent-applications have been filed related to "Transfusion Medicine" and over 400 related to "Transfusion Alarm", during the last 30 years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions.
The aim of this contribution is to present a developed low-cost real-time individual Intravenous Blood-Transfusion monitoring system, based on the Internet of Things.
Study Design/Method: The designed system is based on a commercially available Pan-Tilt-Zoom (PTZ) camera, employing an 1/4 inch color CMOS Sensor, providing effectively 1.0 MP, a 3.6 mm Lens, IR-cut, Day/Night Minimum Illumination 0.1 Lux/F 1 and 808 Viewing Angle.
The camera is focused on the droplets and acts as VIS/IR Detector with a 24 Hz sampling-rate.
Custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g.
clotting, blood 260A TRANSFUSION 2017 Vol.
57 Supplement S3 ABSTRACT or other suspensions depletion etc.)
and enables, if necessary, wide-angle image-capturing.
The Video Image-Audio Settings provide for Compression H.264, Video Frame Rate (FPS) 1-30/s, Refresh Rate 50 Hz and Audio Input, through bidirectional built-in Microphone.
The acquires an IP-address, the connection mode is Wireless, the Network Interface is Wi-Fi/802.11/b/g, the supported Protocols include DHCP, TCP/IP, UPNP, HTTP, SMTP and P2P is provided.
Typical 5V Power-Supply, sized 165x125x101mm and weighing 370 g. Client Software is required.
The IR range is 10-15 m; IR-Cut Filters, Remote Access, Dual Stream, Motion Detection, Day/Night and IR Night Vision Distance of 10 m are offered.
Two-way radio-link is provided, as well as, Trans-Flash (TF) recording and storage on a 32 GB SD-Card.
Pan/Tilt-Horizontal 355 o and Pan/Tilt-Vertical 90 o movements can be performed.
The system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as NIBP, ECG, and SPO 2 , if present.
Results/Finding: The system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation.
Conclusion: The system can measure infusion-speed with a deviation lower than 6 3 %.
The developed IoT-system takes advantage of the existing Hospital Wi-Fi networked environment and offers a low-cost solution, under 20 $ for each monitoring-set.
It allows for even multi-platform (IOS, Android, Windows) smart-phone, short-range connectivity, for up to 4 participants, for example Nurse, Physician etc.
Two Potential Approaches for the Quality Control of BacT/ALERTV R Culture Media Using Various BioBall TM Organism Preparations Patricia Rule*, Michelle Keener and Christine Crawford.
bioMerieux Inc. Background/Case Studies: The BacT/ALERTV R BPA and BPN culture bottles are used with the BacT/ALERT Microbial Detection System for rapid screening and detection of microbial contamination in Leukocyte Reduced Apheresis Platelets (LRAP).
Recent changes in the CLIA quality control guidelines and AABB accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth.
A study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel.
Study Design/Method: The general protocol consisted of three replicates each of each organism inoculated into two lots each of BPA and BPN by two different analyst.
The study was two part in that Aspergillus brasiliensis, Candida albicans, Bacillus subtilis subsp.
spizizenii, Pseudomonas aeruginosa, Escherichia coli, Clostridium sporogenes, Staphylococcus aureus and Streptococcus pyogenes were prepared from BioBall SingleShot (30 CFU), MultiShot 550 CFU or HighDose 10K organism preparations at a low level (< 50 CFU) and evaluated on the same day of preparation as method validation.
The second part of the study utilized Escherichia coli and Staphylococcus aureus prepared and frozen at a higher level and then evaluted over a 14 day study as a stream line approach to routine quality control testing of the BacT/ALERT culture bottles.
Inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point.
Inoculated BPA and BPN bottles were loaded into the BacT/ALERT Microbial Detection system at 36 C for automatic monitoring of growth.
Negative BPA and BPN bottles were included in duplicate at each day of testing.
Results/Finding: Escherichia coli, Staphylococcus aureus, Streptoococcus pyogenes and Bacillus subtilis subsp.
spizizenii were positive in both the BPA and BPN culture bottles.
The aerobic Aspergillus brasiliensis, Candida albicans, and Pseudomonas aeruginosa grew and were reported positive in only the BPA aerobic culture bottle as expected.
While the obligate anaerobe, Clostridium sporogenes was positive only in the anaerobic BPN culture bottles.
Bacterial cultures were positive in the BacT/ALERT BPA and BPN bottles < 1 days and the fungal organisms in < 2 days.
The overall agreement was 99.8 % in 698 bottles tested here.
No significant differences were observed in the time to detection between the different lots or between the different analyst.
Conclusion: The BioBall prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of BacT/ALERT BPA and BPN culture media.
The method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory.
Use of an Electronic Patient Identification System for Blood Banking Specimen Labeling Found to be Superior over Historical Armband Approaches Annie Newton* 1 , Diane Schafer 2 , Debra Brown 1 , Jesse Cox 3 , Scott Koepsell 3 and Sara Shunkwiler 3 .
1 Nebraska Medicine, 2 The Nebraska Medical Center, 3 University of Nebraska Medical Center Background/Case Studies: Anticipating the implementation of the new (30 th addition) AABB standard concerning the confirmation of patient ABO blood typing of Type and Screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands.
Continued use of the armbands would require a second sample for ABO confirmation of patients that did not have a historical blood type on file.
Concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction.
Moreover, 2 nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern.
Study Design/Method: Current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well.
An in-depth evaluation, including a failure modes and effects analysis (FMEA) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart.
An alternate process for specimen labeling and ABO confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system.
Extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process.
Alerts were congruently built into the electronic health record (EHR) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use.
Results/Finding: Within days of implementing the new process (September 11, 2016), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling 4 in 6 months post implementation compared to 144 in the 6 months prior.
In addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential 2 nd specimen collections needed for ABO confirmation.
Conclusion: Use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for 2 nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction.
Background/Case Studies: Based on a few small randomised controlled trials (RCTs) performed in the late '90s and in early 2000, intravenous immunoglobulin (IVIG) use has been suggested as a potential treatment to avoid exchange transfusion (ET) for Rh hemolytic disease of the newborn (HDN).
This treatment modality is now routinely used for Rh-HDN and has been extended to HDN caused by ABO incompatibility or by other red blood cell antibodies.
However, larger RCTs performed since 2010 have shown that prophylactic IVIG did not reduce the need for ET, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions.
The primary objective of this study was to describe the usage of IVIG for HDN at a tertiary academic referral hospital.
Study Design/Methods: A retrospective chart review was performed of all neonates who received IVIG for HDN in the neonatal intensive care unit (NICU) from January 1, 2005 to June 30, 2016.
Data collected included patient demographics features and diagnosis, indications for IVIG, neonatal laboratory results, treatment details, adverse events and patient outcomes.
Results/Findings: Ninety-seven neonates received IVIG during the study period: 57% were female and 7% were less than 34 weeks of gestational age.
None had co-existing G6PD deficiency, pyruvate kinase deficiency or spherocytosis.
All neonates received phototherapy prior to IVIG treatment.
Indications for IVIG were ABO-HDN (41%) and Rhesus-HDN (59%).
Antibodies most often implicated in Rh-HDN were anti-D (22/57), anti-D and anti-C (22/57) and anti-c (5/57).
Sixteen infants with Rh-HDN had received intrauterine transfusions.
The mean cumulative dose of IVIG was 1 g/kg (range from 0,3 g/kg to 3,8 g/kg).
Neonates received one to four IVIG administrations.
Table 1 shows the number of patients receiving IVIG during two time periods.
Three adverse reactions were noted during IVIG administration: cutaneous rash, hypotension and fever.
Of all neonates, 14 required an ET for Rh-HDN and 3 for ABO-HDN.
Forty-five (46%) patients needed top-up transfusions during hospitalisation and until three months of age: 8 with ABO-HDN and 37 with Rh-HDN.
The mean number of transfusions was three (range:1 to 7).
Conclusion: Although initially described for RH-HDN, ABO-HDN is now one of the most frequent indications for IVIG in neonates.
The optimal use of IVIG in ABO-HDN needs to be better characterized.
Our study shows a wide variation of IVIG dosing and a significant proportion of neonates requiring top-up transfusions.
Further research is required to evaluate whether anemia in ABO-HDN might be exacerbated by hemolysis from IVIG isohemagglutinins and if it is dose-dependent.
Background/Case Studies: Background: One of the most serious adverse reactions to transfusion is the development of graft versus host disease.
Symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia.
The clinical course is rapid with an over 90% case fatality rate.
The patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of HLA-matched cellular blood products and the recipients of cellular blood products donated by blood relatives.
The basic etiology of TA-GVHD is the inability of the transfusion recipient to mount an effective immune response against donor T-lymphocytes.
Treatment options for TA-GVHD are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication.
Study Design/Methods: Most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components.
However, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components.
Following a "near-miss" situation, our hospital information department developed a 4-part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components.
Results/Findings: Our blood products ordering system has been redesigned to include 3 specific queries to identify those patients who required irradiated cellular blood products.
First, physicians have been notified to include the need for blood product irradiation in the patient Problems List.
Once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the Problem List is modified by the clinical staff.
Second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list.
Finally our system will automatically default to request irradiation on all cellular products ordered for children less than 4 months of age to comply with local irradiation policies.
Conclusion: We believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm.
We believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate.
Using Lab Information System and a Dynamic Dashboard for Labeling and Tracking Coolers Russell Thorsen, Rosaline Ma, Peter Suslow, Gina Giannarelli, Sara Bakhtary, Ashok Nambiar and Morvarid Moayeri*.
UCSF Health Background/Case Studies: Our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ORs, ICUs, Cath-Lab, etc.
Coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites.
A robot that can hold one cooler delivers products to locations not served by the pneumatic tube.
On average, 30 coolers are issued every day.
Cooler set-up is a multi-step, labor-intensive process.
Transfusion Service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage.
We developed a lab information system (LIS)-based solution to manage cooler labelling and tracking more efficiently.
Study Design/Method: Nine cooler test batteries were built; the batteries for RBC, plasma, platelet and cryoprecipitate (2 each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and RBCs with variable expiration times) is slightly different.
The second battery in each pair was built to avoid duplicate test cancellation by LIS when a second cooler (for same component type) is being set up for the same patient.
Each battery consists of tests capturing the following information: cooler location, cooler ID, number of units issued, and expiration.
Custom barcodes representing each test battery and 45 different locations can be scanned from a 'quick-pick list', avoiding need for manual entry.
When coolers are returned, a final entry is made in the test battery, updating LIS.
A dynamic cooler tracking dashboard with live-feed from LIS displays data captured in the test battery.
Elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in LIS) is captured automatically.
Color codes alert users to coolers that have less than 1 hour before expiration.
A flashing alert pops up for coolers that have expired.
Results/Finding: We replaced our manual process (hand-write patient information and expiration time on 2 separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel LIS-driven labeling and tracking system.
Each time a cooler is set up, a test battery is ordered and resulted in LIS by scanning the related custom barcodes.
A single LIS-generated label is printed and attached to each cooler.
Cooler expiration is defaulted to 10 hours (per our current cooler validation) from the time the test battery is ordered during cooler set up.
Techs pay attention to expiration of each product they place in a cooler.
If an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown.
Color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction.
Conclusion: Using LIS for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient.
These tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking.
Improving Cryoprecipitate Collection Operations Using Operations Research and Analytics-Based Methods American Red Cross, 2 Georgia Institute of Technology AP72 Reduction in Unnecessary Use of Type O-Negative RBCs in a Level I Bellevue Hospital-NYULMC Our hospital is a level I trauma center serving a diverse predominately non-Caucasian population.
Historically we stocked our trauma blood bank monitored refrigerator with 2 O-negative RBCs.
Trauma requested that we stock additional RBCs to be able to initiate a MTP for multiple patients at the same time.
Believing that most of our trauma patients are male, elderly, or Rh-positive, we agreed add 2 type O-positive RBCs to the stock.
Rules for determining which units to use were established.
O-positive RBCs are to be given to 1A) all adult males (AM), 1B) women of non-childbearing age (WNCBA), and 1C) if both O-negative RBCs were used but not yet restocked, and O-negative RBCs are to be given to 2A) women of childbearing age (WCBA) and 2B) children until the patient's ABORh type are determined.
We sought to assess the impact of this change on our usage and purchases of O-negative RBCs.
Study Design/Method: All patients issued emergency release trauma RBCs following the addition of O-positive RBCs were assessed 3%) would have needed to be O-negative.
The addition of O-positive RBCs to our trauma refrigerator will enable us to markedly reduce our purchases of O-negative RBCs.
AP73 Saving Apheresis Platelets through Use of Verax Point of Care Testing Jennifer Rhamy* 1 and Rebecca Wride 2 .
1 St. Mary's Regional Blood Donor Center, 2 St. Mary's Regional Medical Center Background/Case Studies: Our rural hospital-based blood center serves 17 hospitals and a diverse patient population including acute trauma.
Because of the varying need for platelet products (varies between 0 and 8 per day in 2017), we investigated the use of the Verax point-of-care test to better manage our valuable inventory Barrett Lawson 2 and Jun Teruya 1,2 .
1 Texas Children's Hospital, 2 Baylor College of Medicine AP127 Vision Titers --Easier or Problematic?
(Table 1) .
Results/Findings: Post INTERCEPT, T had volumes of 261-320 mL, with 98 6 4% hemoglobin (Hb) recovery.
T had 10-fold less extracellular protein than C. After 35 days of storage T had higher ATP and Na 1 than C while lactate and hemolysis were lower.
Hct, pH, K 1 and glucose were equivalent between T and C on D35.
D35 hemolysis for T was 0.08-0.31%, while for C it was 0.10-0.57%.
T and C ATP was >2mmol/g Hb, the level of ATP associated with effective RBC viability, throughout storage (Table 1) .
Hematocrit (Hct, %) 58.5 6 2.5* 56.3 6 1.7 60.8 6 2.7 61.8 6 2.9 Hemoglobin (g/unit) 59 6 7 6 0 6 4 not measured Hemolysis (%) 0.02 6 0.01* 0.06 6 0.04 0.16 6 0.07* 0.29 6 0.14 pH (378C) 6.9 6 0.1* 7.3 6 0.1 6.7 6 0.1 6.6 6 0.1 Total ATP (mmol/g Hb) 7.7 6 0.5* 5.0 6 0.4 4.6 6 0.6* 3.9 6 0.5 K1 (mM) 1.5 6 0.3* 5.7 6 2.5 53.
TOTAL TESTED TOTAL PLTS ISSUED FEB 87 121 64 48 24 185 181 MAR 226 516 342 276 133 858 757 TOTALS 352 730 471 375 174 1201 Table: 2.
Resident reports to the intranet "drop box" increased from 53.7% to 69.3% to 100%, each over 2 month time spans.
Conclusion: Safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time.
Safe ordering prevented recurrent allergic reactions in our patient population.
The TSO plays a pivotal role in ensuring the full circle of communication occurs.
Processes that integrated the pathology resident improved with PDSA cycles and impacted the quality and timeliness of hand off.
Finally, the data provided from the residents enabled efficient participation in hemovigilance.
Decreasing Results/Finding: The main root cause determined was that there was no standard work process.
SOPs were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow.
Counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the Batch Release Department.
Specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing.
After counter measures were implemented, the number of exceptions decreased from 17.4 per day or 10,370 DPMO to 6.3 per day or 3,539 DPMO.
This is a statistically significant difference since the p-Value calculated was 0.002.
Conclusion: A lean Six Sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory.
This approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process.
Background/Case Studies: Our Blood Bank processes approximately 4,400 specimens per month.
Since 1998, the requirement of having a second blood type on record was met by:1.
Utilizing the historical blood type and the current specimen, OR 2.
Having second type performed on same specimen by different technologist, AND 3.
Each type and screen specimen signed by 2 staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.To comply with the AABB Standards 29 th edition, #5.16.2.2 a decision was made to change our practices.
We considered challenges encountered at other hospitals and collaborated with nursing and IT to create a streamlined and safe process.
In April 2016, the second specimen procedure was implemented addressing the following: II.
Extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively.
Results/Finding:1.
There was a minor adjustment period with more phone calls made to blood bank to explain the process.
2.
There was minimal impact on turn around times for release of components.
3.
ABORh retype workload decreased from 1500 to 950 (35% to 20% of T&S volume) per month.
4.
Unnecessary blood draws minimized, improving patient experience.
5.
No emergency release requests due to absence of a second specimen.
The second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services.
Overall feedback from staff on the process has been positive.
Our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution.
Background/Case Studies: The hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome.
With this in mind, AABB and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as 15 to 48%.
Computerized Provider Order Entry (CPOE), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment.
The objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion.
Additionally, factors associated with inappropriate transfusion were examined.
Study Design/Method: In our 926 bed hospital, a retrospective chart review was performed (07/01/15-12/31/15) on hospitalized internal medicine patients.
CPOE with hospital guidelines for RBC transfusions were in place.
Transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies.
Charts for Background/Case Studies: Our Midwestern University-Based Transfusion Service (TS) evaluated the appropriateness the automated platform VISION (Ortho Clinical Diagnostics.
Raritan, NJ) for prenatal titration studies.
It has been established from previous publications that the micro-column assay, of which the VISION is based, may lead to higher titer results compared to standard tube titrations.
This study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective.
Study Design/Method: Twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the VISION.
The samples were manually tested with a standard two-fold serial dilution.
The titer was reported as the last tube to demonstrate a 11 reaction by macroscopic observation.
The titer studies were then repeated using the VISION.
The results of the manual and automated processes were compared and categorized as "< 2 grade" or "> 2 grade" difference between endpoints.
This analysis is similar to the acceptable ranges used for evaluating College of American Pathologists (CAP) proficiency survey challenges.
A cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer.
Results/Finding: Table 1 demonstrates a summary of the samples tested by manual titer study and VISION titration method.
The VISION titer results (mean, median, and mode) were higher than the manual tube titer results.
Less than half of the samples (48%) were > 2 titer results higher, while the majority was 2 titer results different (52%).
The cost analysis is summarized in Table 2 .
The indirect cost (labor) was significantly lower with the use of the VISION.
The reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the VISION completing the titration as part of the profile of the titer study of the analyzer.
Conclusion: With an estimated 41% decrease in the cost of a VISION titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank.
However, the VISION demonstrated the expected increase in titer results compared to manual tube titer results.
This would impact the critical values currently utilized.
An impact assessment for clinical staff would be necessary to adequately implement the change in method.
Consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values.
In addition, as part of changing to the VISION an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to VISION titer results which would be higher and may be interpreted as a significant change for clinical care of the patient.
What Is the Best Practice for Testing Residual White Blood Cells in Blood Components for Monthly Routine Quality Control?
Janja Pajk*.
General Hospital Celje Background/Case Studies: We wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components.
Our aim was to validate the Adam device for counting thne number of residual white blood cells (WBC) in leucocyte depleted and in non-leucocyte depleted blood components (BC) and to compare with standard counting method by microscopy in Fuchs Rosenthal chamber (FRC) used in GHC and with Flow cytometry (FC).
Study Design/Method: After 23 samples of red blood cells (RBC), platelets (PLT) and fresh frozen plasma (FFP) (leucocyte depleted in top and top (T/ T) bags and non-leucocyte depleted in top and bottom (T/B) bags) were stained with propidium iodide (PI) on r-Slides; Adam -rWBC device was messured fluorescent images of stained WBC nucleus.
Data were analised by image analysis software and later compared with results of testing samples in FRC by microscopy and with FC.15 samples of BC were microscopic tested in FRC at Department of laboratory medicine in GHC; another 8 samples were measured with FC in UCC Maribor.
Results/Finding: 15 samples (6 RBC, 3 PLT, 3 FFP-all leucocyte depleted and 3 non-leucocyte depleted FFP) were tested in triplicates on Adam and with FRC once.Coefficient of variation of (KV%) of samples measured on Adam for leucocyte depleted BC varied for: RBC from 44,10 -173,21; PLT from 86,60 -100,00; FFP from 86,60 -173,21; and for non-leucocyte depleted FFP from 0,69 -8,85 (Table 1) .
8 samples (2 RBC, 2 PLT, 2 FFP -all leucocyte depleted and 2 nonleucocyte depleted FFP) were tested in triplicates on Adam and with FC once.KV% of samples measured on Adam for leucocyte depleted BC varied for: RBC from 91, 56; PLT from 78, 21, FFP from 66, 21 ; and for non-leucocyte depleted FFP from 11,17 -15,91 (Table 2) .High percentage of KV was noticed in samples with low numbers of WBC (in leucocyte depleted BC; low percentage of KV in non-leucocyte depleted FFP, with higher amount of WBC was observed.
Conclusion: All samples tested with Adam met expected criteria for WBC in BC in European Union (less than 1x10 9 /unit for leucocyte depleted or 1x10 6 / unit for non-leucocyte depleted) and were comparable with those tested with FC; the correlation with microscopy in FRC was worse.With use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.From January 2017 we changed our protocol for testing residual WBC in BC with Adam device and we advise it as the best practice for monthly routine quality control.