U.S. FOOD AND DRUG ADMINISTRATION

+ + + + +

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

+ + + + +

BLOOD PRODUCTS ADVISORY COMMITTEE

+ + + + +

89th MEETING

+ + + + +

FRIDAY,
APRIL 27, 2007

+ + + + +

		The meeting convened at 8:00 a.m. at the Hilton Washington D.C. North/Gaithersburg, 620 Perry Parkway, Gaithersburg, Maryland, Frederick P. Siegal, M.D., Chairman, presiding.

COMMITTEE MEMBERS PRESENT:
FREDERICK P. SIEGAL, M.D.
Chairman
JUDITH R. BAKER, M.H.S.A.Consumer Representative
ADRIAN M. DI BISCEGLIE, M.D.Member
WILLARDA V. EDWARDS, M.D., MBA          Member
MAUREEN A. FINNEGAN, M.D.Member
LOUIS M. KATZ, M.D.
Non-Voting Industry Representative
HARVEY G. KLEIN, M.D.Temporary Voting Member
MATTHEW J. KUEHNERT, M.D.Member
COMMITTEE MEMBERS PRESENT (continued):
CATHERINE S. MANNO, M.D.Member
KENRAD E. NELSON, M.D.Temporary Voting Member
GEORGE B. SCHREIBER, Sc.D.Member
SIMONE A. GLYNN, M.D., Msc., M.P.H.Temporary Voting Member
IRMA O.V. SZYMANSKI, M.D.Member
DONNA S. WHITTAKER, Ph.D.Member

FDA PARTICIPANTS:
DONALD W. JEHN, M.S.Executive Secretary
JAY EPSTEIN, M.D.
Director, Office of Blood Research and Review
BASIL GOLDING, M.D.
SHERYL A. KOCHMAN
Chief, Devices Review Branch, DBA/OBRR/CBER
HIRA NAKHASI
MARIA RIOS, Ph.D.
DETTD/OBRR
DOROTHY SCOTT, M.D.
OBRR/CBER
MARK WEINSTEIN, Ph.D.OBRR/CBER
ALAN E. WILLIAMS, Ph.D.Director, Division of Blood Applications, OBRR/CBER

GUEST SPEAKERS:
RICHARD J. BENJAMIN, M.D., Ph.D.Chief Medical Officer, American Red Cross Biomedical Headquarters
CELSO BIANCO, M.D.
America's Blood Centers
EILEEN FARNON, M.D.
Division of Vector-Borne Infectious Disease, Arboviral Disease Branch, CDC
GUEST SPEAKERS: (continued)
JERRY HOLMBERG, Ph.D.Executive Secretary, Advisory Committee on Blood Safety and Availability, DHHS
STEVEN H. KLEINMAN, M.D.University of British Columbia
RAVINDRA SARODE, M.D.Director, Transfusion Medicine and Hemostasis Reference Laboratory, University of Texas Southwestern Medical Center
SUSAN L. STRAMER, Ph.D.Executive Scientific Officer, American Red Cross
DAVID F. STRONCEK, M.D.Chief, Laboratory Services Section, Department of Transfusion Medicine, Clinical Center, NIH

		This transcript has not been edited or corrected, but appears as received from the commercial transcribing service.  Accordingly, the Food and Drug Administration makes no representation as to its accuracy.

	C O N T E N T S

Committee Updates             PAGE

Summary of August
30-31, 206 Meeting of the
DHHS Advisory Committee 
on Blood Safety and
Availability
Dr. Jerry Holmberg............. 10

Summary of December 
13, 2006 Meeting of the
Transmissible Spongiform
Encephalopathies Advisory Committee
Dr. Dorothy Scott.............. 18

Q&A Session.....................32

Dr. Mark Weinstein..............39

Q&A Session.....................49

Summary of September 25-26,
2006 FDA Workshop on Molecular
Methods in Immunohematology
Dr. Sheryl Kochman..............50

Topic II:  Transfusion Related
Acute Lung Injury (TRALI)

A. Introduction, Alan Williams
Ph.D............................67
B. Clinical and Laboratory 
Aspects of TRALI
Dr. David F. Stroncek..........80

Q&A Session...................108

C. Current Use of Transfusable
Plasma
Dr. Ravindra Sarode.............117

Q&A Session....................137

D. Review of REDS-II LAPS Study
on HLA and Granulocyte
Antibody Prevalence
in Blood Donors
Dr. Steven Kleinman............142

Q&A Session....................167

E. American Red Cross
Experience with TRALI
Dr. Richard J. Benjamin........180

Q&A Session....................195

F.  America's Blood Centers
Experience with TRALI
Dr. Celso Bianco...............206

Q&A Session....................222

Open Public Hearing............n/a

Questions for the Committee....234

Topic III:  Issues Related
to Implementation of West Nile Virus 
Testing

A.  Update on WNV Epidemic 2006
Dr. Eileen Farnon, CDC.........255

Q&A Session....................270

B.  Issues for Testing
Dr. Maria Rios, FDA............276

C.  Data in Support of the 
Current ID-NAT Triggers
American Red Cross 
Dr. Susan Stramer..............301

Q&A Session...................330

	P R O C E E D I N G S
		DR. SIEGAL:  Good morning.
		MR. JEHN:  Let's everybody sit down and we're getting ready to start.  I have a brief statement to read on the conflict of interest, an addendum from yesterday.
		This brief announcement is in addition to the conflict of interest statement read at the beginning of the meeting on April 26, and will be a part of the public record for the Blood Products Advisory Committee meeting on April 27, 2007.
		This announcement addresses conflicts of interest for the discussions of topic II, Transfusion   Related Acute Lung Injury, TRALI, and topic III, issues related to the implementation of blood donor screening for infection with West Nile virus. 
		For the discussion of Topic Three on West Nile Virus, Dr. Adrian Bisceglie has received a waiver under 18 U.S. Code Section 208(b)(3).  A copy of the written waiver may be obtained by submitting a written request to the Agency's Freedom of Information Office, Room 12A30 of the Parklawn Building.
		Dr. Louis Katz is serving as the industry representative, acting on behalf of all related industry and is employed by the Mississippi Valley Regional Blood Center.  He receives consulting fees from firms that could be affected by the discussion.
		Dr. Katz is also the medical director for Scott County, Iowa, Health Department, who has a contract with an affected firm.   Industry representatives are not special Government employees and do not vote.
		The Agency has determined that the information provided by the guest speakers is essential.  The following information is being made public to allow the audience to objectively evaluate any presentation and/or comments made by the speakers.
		Dr. Richard Benjamin is employed by the American Red Cross.  Dr. Benjamin received consulting fees from firms that could be affected by the discussion.
		Dr. Celso Bianco is employed by the Americas Blood Centers.
		Dr. Eileen Farnon is employed by CDC in Fort Collins, Colorado.
		Dr. Steven Kleinman is employed by the University of British Columbia.  He receives consulting fees from several firms that could be affected by the discussions.
		Dr. Ravindra Sarode is employed by the University of Texas, Southwestern Medical Center.  He is a scientific adviser for a firm that could be affected by the discussions, for which he receives a fee.
		Dr. Susan Stramer is employed by the American Red Cross.  She is the principal investigator on a study from a firm that could be affected.  She also was a speaker for an affected firm.
		Dr. David Stroncek is employed by the National Heart, Lung and Blood Institute at NIH.  As part of his official Government duties, he is a scientific adviser for a NHLBI-funded grant on TRALI.
		This conflict of interest statement will be available for review at the registration table.  We would like to remind participants that if the discussions involve any other products or firms not already on the agenda, for which an FDA participant has a personal or imputed financial interest, the participants need to exclude themselves from such involvement and their exclusion will be noted for the record.
		FDA encourages all other participants to advise the committee of any financial relationships that you may have with any firms that could be affected by the committee discussions.
		Mr. Chair.
		DR. SIEGAL:  So good morning, it's Friday, and since it's Friday, I'd like to stick to our time, if we can, so that we can get out at a reasonable hour, unlike yesterday.  So I'll try and encourage people to stick to their allotted time.
		We're first going to have some committee updates.  The first is Jerry Holmberg, executive secretary, Advisory Committee on Blood Safety and Availability, summarizing the August meeting of the DHHS Advisory Committee on Blood Safety and Availability.
		Dr. Holmberg.
		DR. HOLMBERG:  Thank you, Mr. Chairman.  Disclosure.  I am employed by Health and Human Services and am the senior adviser for Blood Policy and also the executive secretary for the Advisory Committee on Blood Safety and Availability.
		Our last meeting was in August, the end of August 2006.  We did not have a meeting in January, and at that meeting our primary topic was whether the United States needed to move towards a biovigilance system.  The questions that were asked of the committee were what are the essential components of a basic element of biovigilance?  Should biovigilance be considered part of a comprehensive quality standard as expressed by CGMP, CGTP, or CLA?  What characteristic--and I should just identify what those are--current good manufacturing practices, current good tissue practices, or the clinical laboratory improvement amendment.
		What characteristics of a biovigilance system are already in place within the United States, and also to look at the impact--and I know this is a little difficult to read--Does the U.S. need a biovigilance system?  What would be the strengths, weaknesses, opportunities and threats of a biovigilance system?  How would a biovigilance system contribute to and integrate into, with the transformation of the health care system within the United States?  And should a biovigilance system integrate with international systems?
		In the above questions, how does this integrate, what does this integration mean to the committee as far as standardization of data elements, platforms, data sharing, analysis, and forums to discuss the analysis of the data collected?
		We also asked the committee to take a look at responsibilities.  What is the responsibility of the Federal Government and the private sector in a biovigilance system?  What recommendations, or recommendation or recommendations, does the committee have in regards with the Government's role and function in the development, operation and support of a national biovigilance system?
		The committee came back with a recommendation to the assistant secretary, and to the secretary,that the safety of the U.S. blood supply is a principal activity of the Advisory Committee and the inclusion of efforts to improve organs and other tissue safety and availability also needs to be considered.
		We recommend that the secretary coordinate federal actions and programs to support and facilitate biovigilance and partnership with initiatives in the private sector.
		The committee also went on to make a definition of biovigilance as a comprehensive and integrated national patient safety program to collect, analyze and report on the outcomes of collection and transfusion and/or transplantation of blood components, and derivative cells, tissues, and organs.
		The program should be outcome-driven, with the objective of providing early warning systems of safety issues, exchanging of safety information and promoting education in the application of evidence for practice improvement.
		The committee went on to recommend that there be a formation of an HHS and Public Health Service biovigilance task group for the identification of the vision, goal and process needed to advance these objectives, and the PHS task group should perform several tasks that included a gap analysis, the need for mandatory versus nonmandatory, or regulatory versus nonregulatory reporting, the scope of reporting, database centralization, database governance, format, and standards for data reporting, coordination with non-U.S. safety reporting systems, funding, design, and feasibility of suitable pilot programs.
		Since that meeting and since those recommendations were forwarded to my boss, Dr. Aquinobi, there have been several action items.
		The first action item was that the Advisory Committee for Blood Safety and Availability's charter was revised and renewed.
		I am pleased to say that Secretary Leavitt signed it the first part of October and as with most of the Advisory Committees, these are under the sunset rules, and so that it has to be reapproved every two years.
		The charter was expanded to include the scope of transfusion and transplantation safety.  Along with that expansion of the scope of the charter, there was also an expansion of the nonvoting government membership to the committee and that is that we do have representation now from another office within CBER and that is of Cells, Tissues and Gene Therapies, and also within the Health Resources and Services Administration, HRSA, the Department of Transplantation.
		There has been a working group formed within HHS operating division and the operating divisions that I'm referring to are the National Institutes of Health, the FDA, CDC, and CMS, and I'm probably leaving one out but I can't remember, right off the  top of my head, to develop a gap analysis, and the co-chairs of that working group are Dr. Kuehnert who is on this committee, and also Dr. Goldsmith from the FDA.  I believe that that's all I have for you.  If there are any questions, I can take those now.
		I apologize for not having a handout available to the committee.  This PowerPoint will be available to the executive secretary  and if you'd like, copies will be available and will be posted on the Web site.
		DR. KUEHNERT:  I just wanted to make a quick comment.  Just given the discussion we had yesterday about Chagas, I think it's clear that attention to and the coordination of transfusion and transplantation safety issues are missing in the United States somewhat, and we're behind other developed countries in these efforts, other countries throughout the world already have programs for hemovigilance and, increasingly, biovigilance, to include organ and tissue.
		So for us, an integrated platform for surveillance will allow us to catch up, and perhaps lead in these efforts.  So we're, at CDC, very excited to be involved.
		DR. SIEGAL:  Thank you, Dr. Kuehnert.
		The next speaker will be Mark Weinstein.  Well, it appears, I guess, Dorothy Scott and Mark Weinstein, summarizing the December meetings of the Transmissible Spongiform Encephalopathies Advisory Committee, and FDA's Risk Communication on Plasma Derived factor VIII and Factor XI.
		DR. SCOTT:  Thank you.  On December 15th of 2006, we had a one-day TSA Advisory Committee meeting, and the purpose of that was really threefold.  Dr. Anderson, from the Center For Biologics at FDA, presented the draft quantitative risk assessment for vCJD risk potentially associated with the use of human plasma-derived Factor VIII, manufactured under U.S. license from plasma collected in the U.S., and this was a long time in the making because there were a number of meetings that discussed the inputs to this risk assessment.
		It is a highly complex document, and rather than trying to summarize it here without the benefit of Dr. Anderson's presence, I would refer you to the FDA Web site where this risk assessment now is published in its draft form.
		At any rate, the risk assessment was presented and it was presented as the amount of potential risk associated with the plasma-derived Factor VIII manufactured as U.S. products. 
		The additional issues, besides the presentation itself, were the risk communication, which Dr. Weinstein is going to tell you about, and also a discussion on the experimental clearance of TSE infectivity in plasma-derived Factor VIII products.
		So why do we care about the clearance? W ell, obviously clearance of infectious agents is very important in manufacturing processes.  It has been shown to be so for viruses, and it's presumed to be so, should TSE infectivity be present in the plasma of donors.
		But this risk assessment actually gave us new insight into the impact of clearance.  This is something that Dr. Anderson and Hong Yang did called the importance analysis, which is a way of looking at how much the different inputs into the risk assessment make a difference in the outcome, which is a risk in Factor VIII products.
		And this is not really numerical but everything is relative here.  What you can see, right off the bat, is that the log of manufacturing reduction of the variant CJD agent has a major impact on risk.  That is, the greater the reduction, the lower the risk.
		This is compared with some of the other inputs for the risk assessment that are also important but don't reach this level of impact.
		Those include the amount of Factor VIII used per year by an individual patient, the prevalence of variant CDJ in the United Kingdom.  So the prevalence of variant CJD possibly present in U.S. donors is prorated to that United Kingdom variant CDJ prevalence, and of course it's much lower in the U.S.
		The efficiency of transmission of this agent by the intravenous route, which is a real scientific question, the amount of infectivity in human blood of variant CJD, the truth is, we don't have any idea, what that amount is.
		What we do know is that there have been reported transfusion transmissions of this agent in the United Kingdom.  The quantity of infectivity in blood, we have had to estimate from animal studies of other TSE agents, the yield of Factor VIII from plasma and the efficiency of the donor deferrals that we have to try to limit the number of donors who may have been exposed to bovine spongiform encephalopathy, the agent of human variant CJD.
		I'm showing this really, though, to show you this big impact of clearance.
		So the amount of clearance by manufacturing processes is a major driver of risk.  More clearance; less risk.  The TSE Advisory Committee had discussed TSE clearance already on September 19th of last year and in that discussion, they affirm the importance of using bioassays in TSE clearance studies rather than binding assays for the abnormal Prp protein.
		And they also discussed the limitations and advantages of the model that we used to study the amount of TSE clearance during manufacturing processes.  In particular, they discussed spiking of plasma with a TSE agent, infectious preparation from brain, versus a use of endogenously-infected plasma from animals as starting material for the TSE clearance studies.
		However, we asked them in that meeting, would a minimum TSE agent reduction factor, studied by manufacturers in scale-down experiments, enhance the vCJD safety of products?  In other words, would the definition of a minimum clearance level enhance the safety?
		And we also asked them what TSE agent reduction factor would be appropriate for these products.
		The committee at that time had not seen the draft risk assessment and they preferred to respond to these questions later, when they had also had the chance to review the draft risk assessment and see it presented.
		So this all happened in September and that's what brought us to a meeting in December.
		I just want to mention a few things about TSE clearance studies.  They are done in an analogous fashion to viral safety studies, that is, an infectious agent--and these studies are frequently done and well-defined--the infectious agent is spiked into plasma or the manufacturing intermediate, and then a manufacturing step or series of steps is performed, just as it would be done in the manufacturing facility, and the removal of the infectious agent is assessed		 at the end of that manufacturing step or steps.
		So how much viral clearance does one like to see in a manufacturing process?  Well, you obviously want to clear at least the maximum amount of virus that you expect to be in your starting material, but also it's going to be useful to have a margin of safety, in part, because it's difficult to know exactly how much is the maximum amount of virus in your starting material.
		We know what's been published, but we can't be sure, and of course these processes aren't robust, they're not going to remove exactly the same amount of virus every single time because these are complex matrices that are used or that exist as manufacturing intermediates and you cannot control, precisely, every single parameter that goes into  precipitation, for example.  	You have a range of controls.
		So the margin of safety seems like a very good idea.  Now how much TSE infectivity do we estimate might be present in plasma?  This is the other thing that was important, I think, for the committee to know.
		Unfortunately, all of our estimates are based on animal models, for animals that are infected and the amount of infectivity in their blood or plasma has been measured.
		Based on these studies, two to thirty infectious units per mil has been estimated as what is likely or what is present in the plasma of animals, and what we guess might be present in the plasma of people.
		And if you take a plasma unit, collected by plasmapheresis, what you find is that you can estimate perhaps 3.2 to 4.4 logs of infectious agents might be present in a plasma unit from somebody who is incubating variant CDJ.
		Like many of the inputs to the risk assessment, there are a lot of caveats obviously to this estimate, and you can see this is only one example of many cases where the risk assessment had to take a range of possibilities, based on the best available data, and use those as inputs for the model.  Well, this is the kind of outcome that we got from the model and this is excerpted from a much larger table, which again you can access from Dr. Anderson and Hong Yang's risk assessment that is on the Web.
		So this is an example, I'm really showing to you, so that you can see how the levels of clearance impact the real outputs of the risk assessment.  In these cases, we're looking at definition of subjects who episodically receive plasma-derived Factor VIII, who do not have inhibitors, so they're not getting super high doses, and with the assumption that the prevalence variant CJD in the United Kingdom, based upon epidemiological modeling, is about 1.8 persons per million.
		And very briefly, for 7 to 9 logs range of clearance, the estimate, or the output from the risk assessment is that the risk will be 1 in 3.2 billion to a patient.  If you to 4 to 6 logs of clearance, you have one in 9.4 million.
		So you can see that your orders of magnitude differ already, and if you have 2 to 3 logs of clearance, one in 21,500.  I'm showing you the point estimate but, actually, ranges were also described in the risk assessment, which is appropriate.
		Likewise, these are subjects that again have episodic treatment, no inhibitors, but one estimates the U.K. variant CJD prevalence to be one in 4,225.  This estimate is based on a tonsil and appendix tissue survey that was anonomized and done in the United Kingdom to look for evidence of the abnormal prion protein in those issues.
		And you can see this number, one in 4,225, is quite a bit different from 1.8 per million people incubating this disease.  This is a matter of scientific debate and I think as time goes by and as more surveillance studies are performed, we'll find out what is actually the case.
		And of course these will be asymptomatic people and this number is based on the presence of clinical cases.
		So going down, I'm really showing you the same kind of numbers.  For 7 to 9 logs of clearance, where you think--this is the U.K. variant CJD prevalence, one in a 100 million, for 4 to 6 logs, one in 105,000, and for 2 to 3 logs, one in 159.  So, again, you see, if there's a lot of clearance, there's a very low risk, and if there's quite, a little, or a bit of clearance, there's a substantially higher-looking risk.
		I would also like to mention here, that the available data suggests that all of our U.S. licensed products are likely to have TSE clearance of greater than or equal to 4 to 6 logs, based on the studies that we have, using the best available model.
		In this session, the manufacturers, through the Plasma Protein Therapeutics Association, also reported the status of their clearance studies and the kinds of numbers that they are getting in their TSE clearance studies for plasma-derived Factor VIII.
		In these studies, they spike the TSE agent into the starting manufacturing material.  They use bioassays or binding assays as a readout for TSE infectivity.  The types of steps that were generally studied were precipitations, chromatographic steps, and filtrations, and as I said, the U.S. products seemed to have around 4 logs of clearance or more with a series of steps or a number of different steps.
		There were varied study designs, and the reason for this is that there are lots of ways to prepare your TSE agent preparation.  You can study various numbers of steps, a single step or a sequence of steps, and the readouts were different.
		So this is a question we asked the Advisory Committee, and I'm very nearly finished here, it's a long question, and in fact the committee did us a favor of adding a few things so that they could refine the question.
		Based on available scientific knowledge, would a minimum TSE agent reduction factor measure by bioassay, demonstrated using an exogenous spiking model in scaled-down manufacturing experiments, enhance variant CJD safety of the products?
		And the committee voted 15 yes and two abstentions.
		Then we asked them, if so, what TSE agent reduction factor is most appropriate, and for this they had both the calculation I showed you with how much agent might be present in plasma, in addition to the risk assessment, which suggests the impact of clearance factors and the amount of risk that results, or is estimated.
		But the committee, at this time, did not feel confident in identifying a minimum TSE agent reduction factor, in the main, because they had uncertainties about the current TSE clearance model.
		I think the problem here is mainly that we do not have a good idea of exactly what form the agent takes in blood and plasma and so the spiking experiments are a model, the endogenous experiments are another model, but of course you're using animal and not human plasma there, and they didn't feel that they had enough scientific information to be certain that the model used to come up with a reduction factor would be as ideal as possible.
		So here I'm going to finish.  Thank you for your attention.
		DR. SIEGAL:  Thank you very much.  Are there any questions?
		DR. NELSON:  You discussed a production process of Factor VIII, but I understand there's been a report from Dr. Rohr and his group on filtration.  Was that discussed at the meeting and how much does that reduce the infectivity?  Do you know?  Are you familiar with that?  Or was that discussed--
		DR. SCOTT:  Well, I'm familiar with several filtration kinds of steps but I think what you're referring to is a more novel method of filtration, which was not actually discussed at this meeting, because that's sort of an up front filtration for blood or plasma.  But it is something that could be very important, certainly, in trying to prevent exposure right at the start.  I know that's in development and we look forward to hearing more about it.  It's certainly a scientific advance, potentially.
		DR. NELSON:  What proportion of hemophiliacs get recombinant as opposed to plasma-derived?
		DR. SCOTT:  I'll probably defer this to Dr. Weinstein.  The number that comes to mind is 80 percent.  80 percent are taking recombinant and about 20 percent are using plasma-derived.  The reasons for using plasma-derived, clinical reasons, reasons that people cite, but this does bring up the option, or the concept that there are treatment options for people that they might want to consider in the context of how they feel about the risk assessment.
		DR. KLEIN:  To my knowledge, there's never been a reported case of CJD in a hemophiliac using Factor VIII.  Is that right? 
		DR. SCOTT:  That is correct.
		DR. KLEIN:  So in the United States, if there's 15- to 20,000 hemophiliacs and the risk is one in a million, even, the chances are that we would never see a case, even if there was a risk of that magnitude, if the majority of the people, as you say, are taking recombinant.
		DR. SCOTT:  Well, I think that another point that can be made is so far, there are no known cases in the United Kingdom either, where the exposure was much greater, and up until the late 1990's, when this risk was recognized, they were using United Kingdom plasma for the manufacture of their experiment products.
		DR. MANNO:  There are some compelling reasons why people still recommend the use of plasma-derived products, although the recombinant products, as we all know, are severalfold more expensive than plasma-derived.  Those plasma-derived products that retain von Willebrand factor have specific indications.  So I don't know that we've seen the end of plasma-derived recommendations for use.
		DR. SCOTT:  That's absolutely right, and I should have made that point, that people with von Willebrand disease are essentially obligate users of plasma-derived Factor VIII products.
		DR. MANNO:  But there are other emerging indications for those Factor VIII products that retain von Willebrand factor.
		Your suggestion that they're less likely, when initially used, to be associated with inhibitors--
		DR. SCOTT:  Inhibitors; yes.  And that's certainly another reason--
		DR. MANNO:  And for immune tolerance therapy, some people would prefer to use plasma-derived products to induce tolerance rather than recombinant product.
		DR. SCOTT:  Absolutely.  Those are two of the other commonly cited reasons for wanting to use plasma-derived Factor VIII.  Thank you.
		DR. KLEINMAN:  Yes.  I wanted to ask, in your risk assessment, as incidence figures you use the input of variant CJD risk in the U.K.  But I thought you're talking about product that's derived from U.S. sources.
		So why didn't you adjust an input calculation to what the risk of a donor in the U.S. for carrying vCJD would be.  I assume it would be orders of magnitude lower.
		DR. SCOTT:  That's quite right, and actually when--it's more complexity than I was thinking of going into for the sake of this presentation, because the full presentation by Dr. Anderson gives you a substantially greater explanation of this.
		The way of estimating variant CJD potential prevalence in U.S. donors is to look at the U.K. risk and to estimate how many donors will have been exposed to BSE in the U.K. or in Europe, that might be donating here.
		So it is actually prorated.  We're not taking the U.K. risk, we're using that U.K. risk to calculate the residual risk in donors in the U.S.  So these will be donors that have been in the U.K., or European countries for some time period, below that time period of deferral, but still may have been exposed.  So that's a residual risk and it also includes donors that perhaps ideally should have been deferred but were not deferred because these donor questions are very complex, and you're not going to get a 100 percent perfect deferral.
		So that is how that residual risk is calculated and we do show how much risk we think is in the U.K., because we need a starting number to look at the residual risk.
		DR. KLEINMAN:  So the numbers you put up on the slide were the U.K. numbers--
		DR. SCOTT:  That is correct.  There's pages and pages of calculations about this in the risk assessment, that explain this in substantial detail.
		DR. KLEINMAN:  Okay.  Well, I guess we have interesting bedtime reading, then.
		DR. SCOTT:  It's a very sophisticated risk assessment, I think, perhaps the most sophisticated one that's been published for this kind of risk estimate for a TSE agent in blood, and so it is actually very good reading and you can appreciate the complexity and how we had to address the things that we don't have exact numbers for, and that's why we had the two estimates for the U.K. prevalence.  But there are many other things along the same line that were done, use of ranges in scientific estimate.
		DR. SIEGAL:  Thank you very much, Dr. Scott.
		The next speaker, Sheryl Kochman, DBA, OBRR, FDA, will summarize the FDA Workshop on Molecular Methods in Immunohematology.
		Oh, I'm sorry.  We still have Dr. Weinstein.  My apologies.
		DR. WEINSTEIN:  Thank you.  Dr. Scott has given you a summary of the discussion that took place at the TSE Advisory Committee meeting in December regarding the clearance of TSE infectivity from plasma-derived Factor VIII products.
		I'll give you an update on our risk communication efforts with respect to variant CJD and U.S. plasma-derived Factor VIII that was presented at that meeting and subsequently modified upon advice from the committee and input from other sources.
		I'll also talk about the risk communication regarding an investigational Factor XI product that was made from U.K. donor plasma.  Topics that I'll be discussing include the development of key message points in question-and-answer documents, our communications strategy and progress with our Factor XI risk communication.
		First of all, with regard to the development of our risk communication messages, the public health messages in the form of key points, and questions and answers, were developed with the input from our sister Public Health Service agencies, including the NIH and CDC.  We also enlisted the help of special Government employees who are patient advocate, as well as experts in risk communication.
		The patient advocate SGEs were asked specifically for their comments regarding whether the interpretive documents such as the key points in questions and answers adequately represented the findings of Dr. Anderson's risk assessment.
		They were asked whether they felt the documents would be easily understood by the targeted audience and whether they had suggestions to improve the clarity of these documents.
		They were also asked whether they had suggestions with regard to how the information was to be delivered to patients and patient family members.
		We are extremely appreciative of this input and we feel that the contributions made a significant difference and improvements in the clarity and delivery of the risk communication messages.
		Now we developed three key message points.  These encapsulated the major take-home messages with regard to the risk assessment. 
		The first of these key points summarizes, you know, why we did the study in the first place.  I'll just read it off.
		In recent years, questions have been raised concerning the risk of variant CJD to hemophiliac A and von Willebrand disease patients who receive U.S.-licensed plasma-derived Factor VIII products.
		The second key point summarizes our conclusions with regard to the risk assessment.  Based on a risk assessment, the U.S. Public Health Service, including FDA, CDC and NIH, believes that the risk of variant CJD to hemophilia A and von Willebrand disease patients who receive U.S.-licensed plasma-derived products is most likely to be extremely small, although we do not know the risk with certainty.  vCJD risk from other plasma-derived products, including Factor IX, is likely to be as small or smaller.  
		That latter sentence there is with regard, particularly, to patients with hemophilia B, who we feel would also be interested in this risk assessment.
		The third key point gives information about where patients may receive information, further information.  Contacting a specialist in hemophilia or von Willebrand disease at a hemophilia treatment enter is a good way to learn about new information as it becomes available.
		In other parts of the document, we go into other sources of information, but we felt that the hemophilia treatment center might be the primary source of information.
		Now, in addition to the key message points, we prepared an additional part of this document that gives further information.  Again, this slide summarizes some of those key informational, or additional information topics.
		We talk about why FDA has conducted the risk assessment, actions that we have taken to reduce the potential of variant CJD, the risk, we talk about the uncertainties in the risk assessment, and again we give suggestions to patients and health care providers about further actions to take and where they can obtain more information.
		The last point is that we give current status of the variant CJD risk.
		In addition to the key points and additional information document, we prepared another document that is in the form of questions and answers.  This is another way of conveying information to interested parties.  I'm not going to read all the questions here that we prepared.  They are present in this slide and the following slide.  But it gives you a sense of the different topics that were discussed, and of course answers are provide in these documents.
		The communications strategy that we had, then, was again to develop these assessments or communication with regard to the risk assessment.  We contacted the hemophilia treatment centers, there are about 140 or so in the country.  We had a conference call inviting all the hemophilia treatment center medical directors, and other interested parties, to hear about our risk assessment.
		They provided input and are willing to disseminate information with regard to this risk assessment.
		We also had on that conference call patient advocacy organizations and they are publicizing this risk communication through their newsletters and other media.
		We have done outreach to trade and physicians organizations, and again, the key points in question-and-answer documents list sources for further information and answers to questions.  Our primary means of relaying information now is through this Web page that we posted in March of this year.  This gives you the address of that Web page, and on that Web page you can find the key points, additional information, the actual risk assessment, and again, we were talking about whether it's good bedtime reading.  Well, you can get the full document here, the risk assessment and the appendix, the question-and-answer document.  You can also find links to guidance documents regarding donor deferral related to classic CJD and variant CJD, further links to other sources of information including the CDC Web site, and there is a list of patient organizations that people can contact.
		The second part of today's discussion includes our Factor XI risk communication efforts.
		As you may recall, this has to do with the possible health risk to about 50 individuals who, between 1989, and 2000, received an investigational product, a plasma-derived Factor XI that was made in the U.K. to treat deficiencies of Factor XI.
		This plasma-derived Factor XI was made using plasma from donors in the U.K., where variant CJD, where the disease, variant CJD, has occurred.
		It's very important to note that the product was not made from the plasma of anyone known to have developed the disease and no one who has received this product is known to have become infected.
		However, although the product was not made from plasma of anyone known to have developed the disease, it's still possible that a donor, who felt well at the time of donation, might have been carrying the disease and so the recipient may have been exposed to--there's a potential of exposure to the recipient of the product.
		Our response to this situation is that we also made a computer model of a risk assessment.  We reported the preliminary risk assessment results to the TSE Advisory Committee in February 2005, and following the information, received from the committee at that time, and also in October of 2005, we have revised the risk assessment.
		The members of the committee also advised the FDA to consult with SGEs, including patient advocates, to obtain input on the risk assessment and communication materials.
		So this risk assessment, the preliminary risk assessment was posted on a Web site in 2005.  We have subsequently revised that risk assessment and we have finalized communication materials regarding the risk assessment and have received input from patient advocates and communication experts.
		We are now in the process of communicating that information to the individual IND holders, to share information with them, to answer any questions, and we strongly suggested they contact their patients and give them this information.  Once this contact has been achieved between the IND holders and the patients, we will be updating our Web page with the finalized risk communication materials and the risk assessment.  We will also be contacting hemophilia treatment centers and patient advocacy organizations about this updated Web page.  Thank you.
		Any questions?
		DR. SIEGAL:  		Well, again, my apologies.  Are there any questions?
		DR. NELSON:  I just wondered whether there's been any update on the data on the prion prevalence in tonsils and appendices that have been studied, that was going on in the U.K., and that was published some years ago, to try to estimate what proportion of the U.K. population might be carriers.  They had just one or two out of 12,000 or something.
		I understood that there was an update that hadn't been published.  Was that presented at the meeting?
		DR. WEINSTEIN:  I haven't seen that.  		MR. ASHER:  No.  
		DR. WEINSTEIN:  No.  I guess that's David Asher.
		DR. SIEGAL:  Then let's move on.  Thank you.  All right.  Now we have Dr. Kochman summarizing the Workshop on Molecular Methods in Immunohematology.
		MS. KOCHMAN:  I'm summarizing a workshop that was held in September of 2006.
		It was generously co-sponsored by DHHS, Office of Public Health and Science, and NHLBI, National Heart, Lung and Blood Institute.
		Many would ask why did we need this workshop.  There's a growing body of knowledge on the basis of blood group genotypes that's being published in the literature, and we know of growing use of various molecular methods in transfusion medicine.
		I should mention that molecular methods have already been widely used in HLA typing and now we're just beginning to see the use being applied to red blood cell antigens.
		Tests for IBD use are available in Europe but here, in the United States, it's currently home-grown and research-use-only testing.
		These techniques are showing promise of addressing current problems in transfusion medicine.  So what are those current problems?
		I'm going to go through this pretty quickly.  A lack of reagent grade antibodies, both polyclonal and monoclonal.  You might think that monoclonal antibiotics would not present a problem, but, in reality, science has been unable to create a monoclonal antibody for every antigen of interest in the red blood cell systems. 
		There's been variability of reactivity of monoclonal antibodies as compared to each other, as well as the reactivity as compared to polyclonal antibodies, most notably anti D's.
		They have varying reactivity with variant D's.  We're finding that this is true of other antibodies in the Rh system.  We're also noticing weak reactivity of clinically significant antibodies.  Most recently, big E and Kel have risen to the list of things that are becoming more difficult to detect.
		Jka and Jkb have been consistently difficult to detect.  The concern here is that if there's a failure to detect an antibody to one of these antigens, it may erroneously allow the patient to qualify for the electronic cross-match, thus a serologic cross-match may not be performed to detect incompatibilities, and we are aware of fatalities related to these and other antibodies that we're looking into.
		There is also weak expression of the antigen, both on donor and patient cells, as well as reagent red blood cells used to detect those antibodies.
		There's a lack of a single universal test method for antibody detection and identification and we know that the different methods are optimum for different antibodies. There is no single method that detects all of the antibodies of interest optimally.
		So different transfusion services use different systems, based on what they believe is best for their patient base.
		We also know that there are inherent limitations in the human glutination test.  There's limited detection on the antibody.
		There's clearly a subjective nature of the test performance, reading and interpretation.  Proficiency in this area may be a problem.
		There are usually single anolytes per test, meaning that you can only test for one antibody or antigen at a time, in general, and we're not always able to automate these serologic methods to allow for mass scale testing.
		The goals of the workshop were to provide FDA with sufficient information to frame a dialogue with manufacturers of these kits wishing to proceed to market, to identify potential issues of importance for those manufacturers, and to identify potential issues of importance for users of molecular methods.
		Those would be those in blood establishments, transfusion services and reference laboratories.
		The agenda is basically reprinted here.  It was broken down into the international experience and the Americas experience.  From a point of view of the international experience, some of this includes sites in the U.S.
		The International Society for Blood Transfusion, and the International Committee on Standardization in Hematology, have provided international workshops and proficiency testing on molecular blood group genotyping.
		There was a presentation on a project that is going on in Europe called the BloodGen Project.  
		There's a great deal of genotyping in Germany, so we had an update on blood group genotyping in Germany and we had a presentation from one of the European manufacturers on molecular genetic blood group typing by the use of PCR SSP techniques.
		For the Americas experience, this sort of includes Canada and South America as well as the United States, there was an overview of molecular methods provided, there was a summary of pheno blood group genotyping, a presentation on the Rh complexities, both serologically and in DNA genotyping, the Kidd blood group system, the Duffy system, the Kell and Kx blood group systems.
		A group called the Consortium for Blood Group Genes, CBGG, this is a group of investigators in Canada, the U.S., and South America, who are interested in furthering the use of these methods.  They're looking into issues related to standardization, proficiency testing, and that sort of thing.
		We had an American company that makes a kit called the Human Erythrocyte Antigen or HEA B chip, do a presentation.  We had a presentation on potential applications of genotype analysis for the quality assurance of reagent red blood cells, applications of blood group antigen expression systems for antibody detection and identification.  In other words, is there another way that we can detect antibodies and antigens, then, using red blood cells?
		There was a talk on the potential use of donor genotyping.  Instead of simply patients, some time spent on proficiency testing for molecular assays and overcoming limitations in current pre-transfusion compatibility testing methods using phage display.
		We also presented work on current FDA processes for bringing products such as these to market and a review of the current FDA guidance that applies to molecular testing.
		The key points that came out of the meeting were that using molecular methods in donor screening will allow testing more donors for more antigens, because of automation and multiplex testing.
		This will assist in the management of rare donor units.  Using molecular methods in patient testing will allow testing when cells are sensitized with antibodies.  Right now, in many cases, it's difficult or impossible to do that with serological methods.
		Also, it will allow testing when there are multiple cell populations.  For example, when a patient has already had one or more transfusions, and clearly, they're going to have cells of different phenotypes circulating. 
		There are serological methods, they're difficult, they're cumbersome, they're not totally reliable, and so far, the testing is indicating that this method may be useful here, because you can test a sample other than red blood cells.  You can test saliva.  You can test epithelial cells.
		It's been extremely helpful in resolution of unusual serologic findings, and has been helpful in determining a more rational approach to transfusion practices involving multi-transfused and transfusion-dependent patients.
		The focus switches from antibody detection and then transfusion of compatible units to genotyping, and then providing genotype matched or closely-matched units.
		We also heard that using molecular methods for fetal genotyping aids in prediction and management of HDN.  This has been used in many centers for a number of years now.
		Using molecular methods in the manufacture of reagent red blood cells could provide the possible genotype when antisera are not available to determine the phenotype, and that is becoming more and more of a problem.
		And it may also help assist in selection of homozygous cells to increase the chance of detecting weak but clinically significant antibodies, particularly of Jka and Jkb.  What we also heard, the molecular methods cannot completely replace serological methods.
		Antibody detection and identification currently cannot be done through any molecular methods.  There's a strong feeling that we will probably need to confirm the serological phenotype, at least for a while, to confirm that the molecular genotype is giving us the information that we need, and we will need to keep the cross-match, at least for some time, but we don't know for how long.
		Some of the concerns that come out when you look at some of the data that were presented, the first ISBT/ICSH workshop in 2004 included testing of a number of DNA samples.  There were 40 participants, six DNA samples.
		These samples were tested for about 23 different antigens.  There were 34 errors, which was an error rate of 5 percent.  I don't know the total number of actual tests in this system or in this particular workshop, but it is clear that there were 34 errors.  It calculates out to 5 percent of all samples.  Most of those errors were in the Rh system, probably due to its complexity.  There were some clerical errors.  So molecular methods are not unexpectedly going to help us deal with clerical errors.  There was also a failure to detect silencing SNFs, especially for D and DY.  So that workshop came away with seven recommendations for use of controls and various testing schemes. 
		So at the 2005 quality assurance exercise, there were two DNA samples distributed to 29 laboratories, that antigens covered are listed on the screen.  In this case we know there were 496 tests performed.  Only three were clearly incorrect results, which brings the error rate down to less than one percent.
		So it would appear that some of the recommendations that came out of the first workshop have been extremely helpful in making the testing more reliable.
		But there was another workshop in September of 2006, immediately prior to this workshop.  Forty-one laboratories participated; six samples were distributed.  I don't know the total number of tests.  The data that were presented were preliminary.
		There were approximately 52 errors at that time, and the percentage of errors is not known.  We also heard a lot of details about the BloodGen Project or the gene chip in Europe.
		This was a three year project that ended in 2006.  Interestingly, it as funded by a group called the Framework of Five of the EU. They put in 2.5 million Euros and a manufacturing company called Progenica put in 1 million Euros for the study. 
		They are the company that will be manufacturing the gene chip when it's ready for use.
		The major goal of their study is a demonstration project to look for a mechanism for high-throughput molecular testing.
		They did include ABO and D in their study, which appears to have been problematic.  when they actually went out to do some of the clinical testing, out of 685 samples that were analyzed, 154 of the samples showed a discrepancy between genotype and phenotype.
		That's an average error rate of 22.4 percent and the range between the sites was as low as 10.9 percent, which is still awfully high, to as high as 40 percent.  They don't know entirely what these errors were due to.
		They're looking into whether or not it was  DNA quality issue or whether it's software issue.  So software is going to prove to be a huge problem as we go to these methods as well.
		So there are some questions remaining, the biggest one being how much premarket testing is needed to evaluate these methods.
		The numbers cited for the BloodGen Project are consistent with FDA's draft guidance for field trials, but that guidance was written with serologic methods in mind and has manufacturers using three to 5000 randoms, plus known selected variants for ABO and D, and one thousand randoms plus selected variants for all of the other specificities.
		I suspect that that number is not going to be sufficient for molecular testing because there's not the history behind the methodology.
		I think we don't know enough about the limitations to adequately inform the user through the package insert.  There were problems in the early development and use of monoclonal antibodies and there's a question as to whether or not we can do something to avoid those same kinds of problems.
		We need to figure out how the technology should be used.  Will this be something that the FDA mandate or will this be something that individual laboratories take on voluntarily.
		Will the use in a blood establishment versus a transfusion service versus a reference laboratory be different?  Will it be in every transfusion service?  Will it be in every blood center?
		Should we make it mandatory for manufacturers of reagent red blood cells?
		Clearly, we're going to need to educate users in how to perform and interpret the tests and how to recognize when something needs further investigation, and we need to determine what's going to be the best way to do that.
		But did we meet our goals?  Partially.  We have had discussions with one kit manufacturer.  It is clear that proficiency testing will be needed, and it is also clear that tests for some red blood cell antigens are closer to marketing than others.
		ABO and Rh are far too complex at this point.  I don't see them coming in to the FDA any time soon, whereas the other antigens listed below appear to be much better defined and much closer to market at this time.
		And the presenter slides are posted on one CBER Web site and the transcripts for each day are on different Web sites.  Both are listed up here.  Thank you.
		DR. SIEGAL:  Okay.  Any questions?  		[No response] 
		DR. SIEGAL:  All right.  Then let's move on and begin our discussion of TRALI.  I hope at the end of these presentations we'll be able to vote on the "rose of Tralee."  And we will be able to elect someone.
		But we'll start with Alan Williams, PhD, from FDA, to introduce the topic.
		DR. WILLIAMS:  Good morning and welcome to the discussion on transfusion-related acute lung injury, also known as TRALI.
		I'm just going to provide an overview of the session and most of the areas that I'm going to highlight are going to be developed in considerably more depth by the speakers that will follow.
		The basic issue for discussion is FDA seeks to be advised whether available scientific data support the development of FDA policies and methods to reduce the incidence of TRALI.
		Just very briefly, the TRALI criteria, as focused by a Canadian consensus conference in 2004, clinical criteria defined as acute onset of acute lung injury during or within six hours of transfusion, with clinical evidence of hypoxemia, bilateral infiltrates on a frontal chest radiograph, no evidence of circulatory overload, and importantly, absence of other attributable causes, because acute lung injury itself is not uncommon in the patient population and there are other factors contributing to comorbidity which tends to complicate the diagnosis.
		The risk per transfusion of morbidity related to TRALI is estimated to be on the order of one in 2500 to one in 5000 transfused products.  Estimates do vary widely, in part, because of the variability in the clinical definition.
		TRALI is treatable when recognized with supportive care; however, if not recognized quickly, it can be fatal and is currently, and has been, for the past three years, the leading cause of post-transfusion fatalities reported to FDA.
		Shown here is a table outlining reported fatalities for 2004, 2005, 2006. Focusing on year 2006, there were 35 reported TRALI fatalities, constituting 50.7 percent of all of the reports, followed by hematologic incompatibilities with clinically significant antibodies related to some of the detection problems just defined by Sheryl Kochman.
		ABO incompatibility, bacterial contamination, and instances when a clear cause was not identified, the transfusion could not be ruled out, for a total of 69 reported fatalities.
		Mechanisms of TRALI will be discussed in detail by Dr. Stroncek, but 45 to 60 percent of cases appear to be associated with neutrophil-specific antibodies, also known as NSA, in the donor.  Donor antibodies to HLA class one or class two antigens also have been implicated, although to a lesser extent, and it's known that these allotypic leukocyte antibodies are stimulated both by pregnancy and transfusion and there will be a talk discussing the prevalence of these antibodies and some of the potential origins.
		Of the 2006 reports, the reporting reflects largely a relationship with components that have a high volume of plasma, with 24 of the cases associated with transfusion of fresh frozen plasma.  Six cases were associated with red cell transfusion.  While there is a low level of plasma contained in intact red cells, this is a little bit aberrant in this particular year because red cells are not typically the component most frequently associated with TRALI.
		Two cases were reported with single donor platelets or plateletpheresis.  Two cases with a combination of red cells, NFFP, in one case red cells plus a cryoprecipitate for plasma.
		Keep in mind in looking at these numbers, that the denominators vary considerably.  Transfusion of plasma occurs, about 4 million transfusions per year, red cells about 14 million per year, and single donor platelets, a little more than 8 million per year.
		I believe Dr. Benjamin's going to present some odds ratios for some quite well-characterized data within the Red Cross system, which I think will help elucidate some of the relative risks.
		The Blood Products Advisory Committee discussed TRALI, in depth, in June of 2001, and it was I think a very extensive and interesting discussion, but the general consensus of the group was that due to a focused clinical definition of TRALI, and a real dearth of information regarding the underlying mechanisms that cause it, and the factors in the donors that might be related to development of TRALI, the committee did not recommend regulatory interventions at that time to identify donors or donations with an increased risk for producing TRALI in the recipient by a vote of one yes and thirteen no.
		But it sent a strong message, that there was clearly a defined need for increased surveillance, focusing of the clinical definition and production of better data on which to define future interventions.
		Importantly, though, following this meeting FDA issued a physician letter to help physicians to understand TRALI, to recognize it and provide the support of care which would help reduce mortality.
		And this appears to probably have been quite an important public health measure and I think it clearly increased the amount of reporting in to FDA leading to an assumption that cases were being recognized on a more reliable basis.
		There have been some recent observations regarding TRALI, quite a few of them.  The suspected hazards of transfusion analysis in the U.K. and a subsequent intervention study, showed that transfusion incidence was five to sevenfold higher following administration of high-volume plasma units, and in an intervention, the U.K. minimized use of FFP in buffy coat-derived platelets from female donors on the basis that multiparous women tend to have higher levels of allotypic antibodies.
		This was done in the fall of 2003, and preliminary reports indicate that the incidence of TRALI in the U.K. has declined dramatically.
		There was a consensus conference in 2004 in Canada.  This conference introduced standardized TRALI definitions for TRALI and possible TRALI, and also provided a recommendation that blood collection agencies assess the value and cost of TRALI interventions, and consider implementing interventions to reduce the morbidity and mortality.
		Additional observations.  A recently  published paper by the American Red Cross, which will be presented by Dr. Benjamin today, objectively assessed 550 systemwide probable TRALI cases between 2003 and 2005, found a strong association with plasma administration in 63 percent of the probable TRALI fatalities, and provided odds ratios related to this product.  		Plateletpheresis were associated with five of the 38 probable TRALI fatalities or 13 percent.  Female donors were disproportionately implicated in development of TRALI, and this publication proposed that limiting plasma from female donors might reduce as many as six recipient deaths annually in the Red Cross system.
		In an association bulletin issued in 2006, the AABB recommended but did not, at this point, make into a standard, interventions related to TRALI, and these interventions that were recommended included that blood collection facilities should implement interventions to minimize the preparation of high plasma volume components from donors known to be leukocyte-alloimmunized or at increased risk of leukocyte-alloimmunization.  That blood transfusion facilities should work toward implementing appropriate evidence-based hemotherapy practices in order to minimize unnecessary transfusion.
		In other words, clearly, some of these interventions may have an impact on supplies of not only plasma but single donor platelets, and that as well as focusing the interventions to have the best benefit, one should look at the use of the products and try to optimize that as well, particularly in the plasma arena.
		And third, blood collection and transfusion facilities should monitor the incidence of reported TRALI and TRALI-related mortality.
		There are some voluntary interventions being discussed and many in fact have already been implemented among the blood collection community.  These include, really going back several years now, since the prior BPAC discussion, deferral of donors who have been implicated in previous TRALI cases, preferential use of male plasma for transfusion, selected donor testing for neutrophil-specific and HLA antibodies.  
		For instance, group AB female donors of plateletpheresis.  There's been a focus on review of evidence supporting appropriate use of plasma, and a lot of research has been started regarding mechanisms of TRALI pathogenesis, the prevalence of associated antibodies and other factors, and the role of previous transfusion in white cell alloimmunization.
		So for the agenda for today's discussion, Dr. David Stroncek's going to lead off with an in-depth discussion of clinical and laboratory aspects of TRALI and this will be followed by Dr. Ravindra Sarode.  I'm sorry.  David Stroncek's with the National Institutes of Health.  Dr. Ravindra Sarode is with the University of Texas, Southwestern Medical Center, and he's going to discuss current use of transfusable plasma.
		Dr. Steven Kleinman from the University of British Columbia will discuss some very fresh and very useful data derived from the REDS-II LAPS study on HLA and granulocyte antibody prevalence in blood donors, and some of the cofactors.
		Dr. Richard Benjamin from American Red Cross will discuss the American Red Cross experience with TRALI, and Celso Bianco, Dr. Celso Bianco from America's Blood Centers will discuss America's Blood Centers experience with TRALI.
		So what we're trying to attain here is not only a scientific review of the field but also a perspective on some of the potential blood supply impacts of potential interventions, and this is an important area for the committee to keep in mind.
		Questions for the committee   Question one.
		Do current scientific data support the concept that the following interventions will reduce the incidence of TRALI?
		This includes use of predominantly male plasma for transfusion, the nonuse of plasma for transfusion from donors with a history of prior transfusion, and selective donor screening for anti-neutrophil or anti-HLA antibodies.
		And then question two.
		Based on the available data, please comment on the effect of the U.S. plasma supply of the following same three interventions.  Use of predominantly male plasma for transfusion, nonuse of plasma for transfusion from donors with a history of prior transfusion, and selective donor screening for anti-neutrophil or anti HLA antibodies.
		So that's the end of the introduction.  I think we have an outstanding set of speakers assembled for the presentations and I look forward to a very informed discussion.
		DR. SIEGAL:  Are there any questions for Dr. Williams?
		[No response] 
		DR. SIEGAL:  All right.  Then let's proceed.  Dr. Stroncek.
		DR. STRONCEK:  Thank you.  I'm from the Department of Transfusion Medicine at the clinical center at the NIH, and the views that I express are those of the presenter and they do not necessarily represent the position of the NIH or the Department of Health and Human Services.
		And my second disclaimer is that nobody else may have these same views either.  TRALI's controversial.  So I'm going to go over the definitions of TRALI and the clinical features, and I want to spend some time about what's known about the pathophysiology, including female donors, leukocyte antibodies, leukocyte activating agent, patient factors, and then finally summarize about tests available that people might consider using to screen for donors that would be at risk for causing TRALI.
		Again, Alan Williams mentioned this, but TRALI clinically has been defined as severe shortness of breath within four to six hours of transfusion, no signs of fluid overload, and pulmonary infiltrates on chest x-ray.
		This definition's been around for quite a while and it was interpreted in different ways by various centers, so some consensus definitions were developed.
		As Alan said, there was a Canadian consensus group but there was also an NHLBI group and the NHLBI defined it as TRALI is a new onset of acute lung injury within six hours of the transfusion of a plasma-containing blood product.  Again, it shows bilateral pulmonary infiltrates.
		They also included a measure of-- again, pulmonary artery preclusion pressure's less than 18, or a lack of evidence of left atrial hypertension.  And then they also included a measure of change in the oxygenation of arterial oxygen to inspired O2 ratio of less than three hundred.
		And it's difficult to get blood gases but it's much easier to get hemoglobin oxygen saturation by oximetry, so they also included a fall in oxygenation to less than 90 percent done room air.  And it's also emphasized that this is a clinical diagnosis.  There's no need--it's based on what's happening clinically, it's not dependent on tests for antibodies or other patient tests, donor tests.
		The incidence of TRALI is quite variable.  It's anywhere from one to a thousand to one to ten thousand units transfused.  I think some of the more recent studies that have looked at this more closely have found the incidence is more closer to one to a thousand transfusions than one to ten thousand.
		Again, all blood products have been implicated but plasma-containing products such as FFP and platelets tend to be more commonly involved with TRALI.
		What is interesting is it's been reported that solvent detergent plasma does not cause TRALI.  Several years ago, solvent detergent plasma was developed and this is made from pooled plasma, and was used in the United States for a while, and it's still used in a few places in Europe, and the places where it's being used have been reported, that it's not associated with TRALI.  It's not sure why this is but it's speculated that the pooling of the plasma dilutes out any leukocyte antibodies present in the plasma.
		Again, the clinical features.  The patients usually become dyspneic and hypoxic during the transfusion or shortly afterwards.  They may experience fever.  They often become either hypotensive or hypertensive, and then the x-ray showed bilateral pulmonary infiltrates.  On occasion, an x-ray will show a whiteout type picture.
		Treatment is usually just supplemental treatment with oxygen therapy.  A number of these patients required intubation and mechanical ventilation.  If they're hypotensive, they made need intravenous fluids or agents to increase blood pressure, often because acute lung injury is often treated with corticosteroids.   These patients are given steroids.  It's not clear if that makes any difference.
		Typically, the symptoms resolve with 24 to 48 hours, so usually with supportive care, this resolves fairly quickly.  Sometimes the symptoms will even resolve before the diagnosis is fully established.  But despite that, the mortality rate of this syndrome is really pretty high.
		It's been reported to be anywhere from 10 to 50 percent.  So it remains a serious problem.  So now a couple minutes on the pathophysiology.
		You've heard that a number of centers are excluding females from donating plasma for transfusion, and why the concern for female, about female donors originally.  And this study is about five years old now and it's from a Scandinavian group.
		But this is a prospective, randomized study of plasma from multiparous donors compared to control donors.  So they had a 100 ICU patients and they randomized them into two groups.  One group got control donor plasma first, followed a couple hours later by a unit of plasma from a multiparous female.
		The second group was randomized the other way, to get the multiparous donor plasma first, followed in a couple hours by control donor plasma.
		After each transfusion, blood gases were measured as well as blood pressure, heart rate, temperature, and then they compared.  So they were comparing the effects of the control plasma versus multiparous plasma on these variables, and what they found is on blood gases, you'd expect the control plasma, it really shouldn't do anything to oxygenation, and the arterial oxygen to FIO2 ratio didn't change with control plasma.
		However, with multiparous plasma, the oxygenation fell and this suggests that again the multiparous plasma was causing some pulmonary dysfunction.
		Similarly, you'd expect with a plasma infusion you'd get some volume expansion and blood pressure increase, and with the control plasma that was the case.  The mean arterial pressure increased slightly, but that wasn't the case with multiparous plasma.
		So this data suggests there is something different about multiparous plasma that causes some cardiopulmonary problems.  Unfortunately, this group didn't test all the donors for leukocyte antibodies, so it's unclear what caused this.  What they did do, though, is they noted transfusion reactions, and out of these 100 patients, there's one case of TRALI and that case was associated with a transfusion of a multiparous donor FFP unit.
		They tested that unit for leukocyte antibodies and a granulocyte antibody was found but no HLA antibodies.  So this does support that granulocyte antibodies can cause TRALI.
		They also had four mild reactions.  One was just a febrile reaction and that was from a control unit.  They had three pulmonary reactions.  They didn't meet the criteria for TRALI but the patients did have some shortness of breath.
		All three of those units were associated with multiparous donors and one of the three units had a granulocyte antibody but not HLA antibodies were detected in the units.
		There's been a recent study from the Mayo Clinic that looked at TRALI in intensive care unit patients, and this again suggests a role for plasma-rich products.  Again, this is a single institution retrospective case-controlled study.
		What they did is in some of their intensive care units, they looked at all the new cases of respiratory failure within six hours of a transfusion.  They identified 24 TRALI cases, 25 cases of fluid overload.  Then they took and matched, found matched patients in the intensive care unit and they identified 124 of those patients, and they compared the transfusions in the patients with TRALI and the controls, and they found that TRALI patients were more likely to get plasma-rich products and they did an estimate of the volume of plasma they were infused, and the TRALI patients had larger volumes of plasma.
		These were the primary end points.  So then they looked at secondary end points, which would be looking at how many of the plasma donors were female donors, and they had a higher incidence of female plasma donors in the TRALI group.  But as they pointed out, that study really wasn't designed to test this issue.  So this is a hypothesis-generating finding and further study should be undertaken for that.
		Okay.  So there's some other studies that point out that female donor plasma may be bad but some of the other presenters will go over those, so I won't mention those.  What about specific causes of what people have found that cause TRALI?
		I first want to go over the data that supports the role for leukocyte antibodies in TRALI, and this issue goes back to the 1950's, and the first real case of, really a good case of TRALI in leukocyte antibodies was reported in 1957, and Brittingham was studying the effects of alloimmunization in blood transfusion, and they infused subjects, three subjects with plasma from--plasma that had leukoagglutinins in.  In one patient, they infused 50 mls of blood and they stopped the transfusion.  This blood with leukoagglutinins caused vomiting, diarrhea, chills, fever, hypertension, tachypnea, dyspnea, cyanosis and leukopenia within 45 minutes.
		These are classic symptoms of what we know cause TRALI.  The symptoms resolved the next day but they did a chest x-ray and it showed bilateral pulmonary infiltrates and a small pleural effusion and they resolved by two days.  So really a classic case of TRALI.
		I only mention that.  In this report, they also transfused two other patients with 250 mls of plasma containing a weaker leukoagglutinin and these patients didn't have reactions.
		So, again, not all leukocyte antibodies cause transfusion reactions.
		At this time, they had identified leukoagglutinins but they didn't know what they were.  These have been described as HLA antibodies and neutrophil-specific antibodies.
		Neutrophil-specific antibodies that have been reported to cause TRALI include human neutrophil antigen I, II and III, and both HLA class I and class II antibodies have been associated with TRALI.
		Again, in the '60s and '70s, there are several case reports of TRALI associated with the transfusion of leukoagglutinins, but you have to remember about case reports--and this is well-summarized by Thompson in 1971--that our case reports suggest that acute pulmonary edema was related to leukoagglutinins but such a relationship was not established, and interestingly enough, in 1971, they were suggesting that we should avoid transfusing plasma from multiparous donors.
		In the 1980's, the idea that leukocyte antibodies causes TRALI was firmly established and a definition of TRALI was, at least the clinical definition we've been using for many years, was by Popovsky and Moore, and they reported five cases of transfusion reactions or TRALI in 19 implicated donors, and one donor in each case had an HLA antibody.
		They had another case of more patients, two years later.  They described 36 cases.  They found leukocyte antibodies in 89 percent of, at least in one donor from 89 percent of the cases, and HLA antibodies in 65 percent.
		So this is interesting but they're not controlled studies, and what you have to remember is oftentimes a patient that's getting transfused, they may not just get one product, they may get two red cells, for example, four units of FFP and one unit of platelets.
		And the incidence of HLA antibodies in any one blood donor has been reported to be 4 to 7 percent.  So if you get transfused with 6 or 7 units, just by chance, there may be 20 to 30 percent of the donors may have HLA antibodies.
		So it's really very difficult to interpret what it means to find HLA antibodies in units of blood that cause TRALI, without having a control group, and with neutrophil antibodies it's a little different, because a number of studies have found that less than one percent of blood donors, or even about .1 percent of blood donors have neutrophil antibodies.
		So if a neutrophil antibody is found in one of these units, it's more suggestive that it is causing TRALI.  That said, there is evidence that leukocyte antibodies can cause, does cause TRALI, and there's been a number of reports of the transfusion of both HLA neutrophil antibodies associated with TRALI and leukopenia, and if you're transfusing an antibody and it's caused leukopenia, and then have a transfusion reaction, it's very suggestive that these are all related.
		One case of transfusion of neutrophil antibody to antigen 1B was reported by Yomtovian in 1984.  And then there have been three cases of the transfusion of HLA class I and II antibodies causing leukopenia and TRALI, followed by another group of three cases of the transfusion of HLA antibodies causing leukopenia and TRALI.
		Dr. Fadeyi, in our group, has found that the transfusion of a neutrophil antibody, HNA-2a, causes leukopenia and pulmonary symptoms, shortness of breath, but not TRALI.
		So there's good evidence that these are all suggestive, that it's not a good thing for the lungs to transfuse leukocyte antibodies.
		There's been some look-back studies, much like the infectious disease literature, where, if a unit, a donor's blood is implicated in TRALI and you find they have an HLA antibody, you'll go back and look at the previous transfusions and see how many have been associated with TRALI, and Akopko reported that antibodies to the neutrophil antigen 3a frequently caused TRALI.  
		They had one donor who was involved with 36 previous transfusions.  Fifteen of those caused at least a reaction and eight of those were severe, really pretty severe TRALI.  So that suggests that this leukocyte antibody was pretty potent in causing a pulmonary reaction.
		Another group had an antibody with the same specificity from one donor who donated 25 times, but they didn't see any reaction.  Again, Fadeyi had an antibody to neutrophil antigen 2a, that donor donated 39 times, twelve were associated with reactions, but they were more mild and none with TRALI.
		In contrast, with HLA, antibodies to HAL class I and II antigens, there have been four reports.  The only one that really has found TRALI, of these approximately 140 units, was a report by Pearl Toy, they had one reaction and it was TRALI.  So it looks like the HLA antibodies are slightly less potent in causing transfusion reactions than neutrophil antibodies.
		There's been a recent report in Vox Sanguinis that supports this.  This is a group in Poland that looked at a thousand blood donors.  They looked at 633 were previously pregnant women, 410 were male, they tested them all for HLA neutrophil antibodies.  No neutrophil antibodies were detected in any of these thousand donors.  No HAL antibodies were tested, or found in the males.  HLA antibodies were found in 9.8 percent of the females.
		They then went back and looked at approximately 60 females who had HLA antibodies and they had donated 211 components, and they found of these components, one of them was associated with TRALI and that was a red cell from a woman with multispecific antibodies to HLA class I and class II antigen.
		Again, about one in 200 units with HLA antibodies was associated with TRALI, which is higher than about the one in a thousand to one in ten thousand incidence reported from any unit.
		Finally, there's animal models that are associated with transfusion of neutrophil antibodies and HLA antibodies with pulmonary injury that is similar to TRALI, and all of these models require not only the antibody but neutrophils must be present, and one of them requires that complement must be present.
		So what about other factors people have implicated in TRALI?  One is Silliman, from Denver, has done a lot of work with bioactive lipids.  These are lipids that accumulate in blood products during the storage of cellular blood products.
		What they do is they prime neutrophils.  So what do we mean by priming?  Is that prime neutrophils, they don't really do anything on their own until they're stimulated with an activating agent, and when they're primed, the response to the activating agent is greater than if a nonprime neutrophil is stimulated, and they've that these prime new factors, specifically the bioactive lipids, they're given to animals before an insult, that they will enhance neutrophil-mediated lung injury. 
		And then they went on to do clinical studies, and there's been prospective and retrospective studies that found a greater level of bioactive lipids in TRALI-implicated units or post-transfusion sera from TRALI patients, when they compare these with controls.
		Again, these have been small studies and they've been typically single institution studies.
		Similar, recently, a soluble CD40 ligand has been implicated in TRALI.  It's been found that soluble CD40 ligand is released by platelets during storage.  This is also a neutrophil priming agent.
		A number of animal models have shown that CD40 and CD40 ligand system incudes acute lung injury in animals, and in this particular study published in Blood, there was a case control study where they compared soluble CD40 ligand levels in units that were implicated in TRALI with control units, and there were higher levels in the TRALI-implicated units.  Again, this was a single institution study with a relatively small number of patients involved.
		Patient factors have also been reported to influence transfusion, TRALI, and Brenda Moore anecdotally reports that he sees more of it at the Mayo Clinic in surgery patients, and Silliman gain had a case control study where they looked at TRALI, and they found TRALI was more likely to occur in hematological patients, patients with hematological malignancies or patients with cardiac disease.
		So the fact that there's multiple factors that have been implemented in TRALI has led to a so-called "two hit model" for TRALI, and in this model, patient conditions are thought to lead to the activation of pulmonary infiltrates, which leads to sequestration of neutrophils in the lungs.
		When these neutrophils are stuck on the endothelium in the lungs, they become prime, and then infusion of a leukocyte antibody or a CD40 ligand, or a bioactive lipid, then stimulates these prime neutrophils and they cause pulmonary damage, capillary leak, and pulmonary edema and TRALI.
		Finally, I want to conclude with a few minutes on possible testing and issues associated with that for TRALI, and what are the types of things you might want to test for to eliminate TRALI would be HLA antibodies, neutrophil antibodies, bioactive lipids or CD40 ligand.  Testing for antibodies would be fairly straightforward, just like we test or many viruses now.
		We test donor serum and we test them at the time of donation.  Testing for bioactive lipids in CD40 ligands is not the same. 
		Because they accumulate during storage, we'd have to test the product and they'd be tested at the time of transfusion.  So that makes that a little more complicated.
		The other issue is for HLA antibodies the techniques are well-established. There's solid phase assays to do it and they're commercially available.  Testing for neutrophil antibodies is more difficult.  These assays require intact neutrophils and no commercial kits are available.  This is similar to the same situation with bioactive lipids.
		CD40 ligand can be tested by ELISA and there are kits available but they're for research use only.
		A little more specific, testing for HLA antibodies, the antigens that are available are usually isolated affinity chromatography or they're produced by recombinant technology.
		These antigens can be adhered to solid surfaces, so there's multiple different types of assays out there.  There's ELISA kits.  There's flow cytom--they put the antigen on beads and use flow cytometry to detect the antibody.
		There's also a system available, a modified flow cytometer, to do more rapid testing.
		So testing for HLA antibodies does lend itself through high throughput testing, and most--a problem, though, with this is if you start testing for HLA antibodies, is most of the products we find with HLA antibodies won't cause TRALI.  Neutrophil antibody.  Again neutrophils are needed.  The problem with this is neutrophils have a short life span, so you literally have to test the cells the same day you isolate them.  There's a variety of cellular assays that have been described, agglutination, immunofluorescence or flow cytometry, and monoclonal capture assay and a mixed pass of hemoglutin test.
		In general, these require all fresh granulocytes.  People have wanted, and worked on developing solid phase assays.  The problem is is that one of the antigens that's commonly implicated in TRALI, 3a, hasn't been characterized on a molecular basis, and there's not even monoclonal antibodies to this antigen.
		So antibodies to 3a can only be tested with intact neutrophils at this time.
		Bioactive lipids.  Again, people are assaying these using intact neutrophils.  Typically what they do is they incubate neutrophils with plasma or a lipid an then measure the respiratory burst compared to a control, and if neutrophil priming is present, then the respiratory burst of the neutrophil should be higher.
		One of the problems with this is even if you set up the assay, a threshold for causing TRALI's not known.  So, really, it's not been defined, how much neutrophil priming would you need to cause TRALI.
		CD40 ligand.  Again, ELISA assays are available but it's only available as a research tool, and then the threshold again, what's the threshold of CD40 ligand that would cause a transfusion reaction of TRALI?  That's not yet well-established.  So it'd be difficult to start to test for this agent and exclude blood products.
		So, in summary, for HLA antibodies, testing donor samples is straightforward and readily available, but a positive test result has a low predictive value of TRALI.  Testing for neutrophil antibody require working with fresh neutrophils but a positive result has a higher predictive value for transfusion reaction.
		Testing for bioactive lipids also requires working with fresh neutrophils and testing of the products at the time of the transfusion would be challenging.
		And then with CD40 ligand, again, it's a straightforward test, but then again we'd have to test products at the time of transfusion.
		And in conclusion, donor product and patient factors have all been implicated in TRALI.  Unfortunately, no single factor is highly predictive with TRALI.  Testing for HLA antibodies and CD40 ligand is feasible.  Testing for neutrophil antibodies and bioactive lipids is possible but more difficult.
		Alan did ask me to just take a minute and talk about the practices at the Department of Transfusion Medicine at the clinical center, and we have now gone to transfusing only plasma from male whole blood donors.
		We are a little bit short of AB plasma, so we do have a small number of females that donate AB plasma by apheresis but we screen them for neutrophil antibodies and HLA antibodies.
		We know for sure we defer them if they have a neutrophil antibody.  We haven't found any with HLA antibodies but we see what the strength of the antibody is and if they've been involved with transfusions before we necessarily exclude them for having an HLA antibody.
		And then our policy is if we have a donor that's been implicated in TRALI, if we identify a antibody to a characterized neutrophil antigen, then we defer them for donating.
		And then we do have a protocol that's been recently approved and we're investigating the incidence of transfusion reaction in recipients of plasma components with and without HLA antibodies.  Thank you for your attention.
		DR. SIEGAL:  Thank you, Dr. Stroncek.  Are there any questions?
		MS. KOCHMAN:  Sheryl Kochman, FDA.  We have cleared an HNA test within the last year and you can find the details on our Web site.
		DR. STRONCEK:  Is that a genotyping test or a phenotyping test?  Or testing for an antibody screen?
		MS. KOCHMAN:  I think it's a typing test for the antigen.
		DR. SIEGAL:  Given what little we know about the pathogenic mechanisms, I take it there is no simple pharmacological approach to prevent, like giving Benedril or something?
		DR. STRONCEK:  Well, I don't think so.  I mean, if there was a good commercial solid phase test for testing for neutrophil antibodies, I think all of us would be doing that.  There's a number of other speakers that have data and thoughts on that, but HLA antibodies, in my view, they're more likely than a random unit to cause a transfusion reaction.
		But the problem is if we start eliminating donors with HLA antibodies, that we will lose a lot of donors that aren't causing any problems.
		DR. GOLDING:  Can I ask a question?  Basil Golding, FDA.  One of the things you said that struck me as being important is solvent detergent treated plasma is not associated with TRALI and I'm not aware of any IGIV products being associated with TRALI.  So a simple explanation to me would be a dilution factor, and that if you have a high titer, it gets diluted out.  So my next question is what do we know about quantitation and is that going to be important?
		In other words, it's not just knowing that there are antibodies but what is the titer that is important, and do the current tests assess that?
		DR. STRONCEK:  Well, I mean, not really, not much is known as far as titer and quantitation of antibody in causing TRALI.  A couple reasons is it's a rare incidence, so it's difficult to study, and there have been a lot of assumptions that just antibodies are bad, so people haven't really thought about looking at what are the important factors that--what are the factors about those antibodies that are important in causing TRALI?
		DR. KLEINMAN:  Just a comment.  There has been a report of a IVIG TRALI case but it was from an experimental IVIG reported by Patrick Mollison, which, by the way, can be found in the 11th edition.  It was made by a very small pool, so pooling may indeed have a role.
		DR. SIEGAL:  Dr. Szymanski.
		DR. SZYMANSKI: I have a question.  It seems to me--I write that you need the antibody, regardless, if you have a bioactive substance or not.  So why to test for both, if the antibody's always needed, even if you need a bioactive material?
		DR. STRONCEK:  I think the groups that work with--Silliman's group, that's done the work with the bioactive substance, would say that you don't need an antibody necessarily to cause TRALI in some cases.  That the CD40 ligand or the bioactive lipid would be sufficient on its own.
		DR. SZYMANSKI:  But bioactive liquid is coming from granulocytes or lymphocytes or whatever, white cells?
		DR. STRONCEK:  They are thought to be released by stored platelets and red cells, so then when they're transfused, they react with the granulocytes circulating in the patient.
		DR. FINNEGAN:  I was delighted to see that you actually looked at the patient component, because in reading the materials that we have, there seemed to be very little assessment of the comorbidities of the patients who develop TRALI.  And one of my concerns is if we have a national disaster and we not allowing females to give plasma, we're going to run into some problems.
		Do you have a sense, other than the hematological or immunocompromised patients, are there patients who really did not seem to develop TRALI if they were given the granulocyte?  I'm sure there are because, obviously, the number of TRALI cases versus the number of multiparous women who donated, do not match up, so there have to be patients who can get it and it doesn't bother them.
		Do you have any sense of, is that younger? is that trauma patients?  Do you have any sort of sense of that?
		DR. STRONCEK:  Not really.  It's really--again, there's been mostly single institution studies and we all have our, you know, different patient mix.  But almost all patients have been described to have TRALI, even patients without antibodies who, you know, if you really believe it's all neutrophil-mediated, that they shouldn't have TRALI, but some of those patients do too.
		DR. SZYMANSKI:  I also wanted to bring out the idea that there are many, many female donors who have donated platelets many, many times, and plasma many, many time, and they have never been associated with any TRALI event.  So they're sort of biologically proven to be safe females. 
		What do you think about that?  I mean, why do you have to even test them?
		DR. STRONCEK:  Excuse me but I couldn't hear the question.
		DR. SZYMANSKI:  The question is that let's say in my institution we have many females who donate repeatedly platelets, you know, many times a year, and they have never been associated with any TRALI kind of incidents.  To me, they are biologically proven safe donors.  I haven't seen where they can just be accepted as such, without any kind of testing.
		DR. STRONCEK:  I think that's a good point.  I think the problem is is that it's hard to know if they're completely safe.  Again, I agree that there's many women that have HLA antibodies and when you transfuse their products they don't cause TRALI.  But it's unclear if there's a subset of people with HLA antibodies that will cause TRALI, but it's just a real low incidence, and then other donors are completely, have HLA antibodies but will never cause TRALI.
		So I think that's one of the important questions.  You know, we and other groups are trying to address that by having prospective studies, to actually start comparing the incidence of transfusion reactions and the type on people with HLA antibodies and without, and try and get an idea, is titer important?  Is it only certain specificities? 
		Is there something about the patient that's really the critical factor, and it's not the donor?
		DR. SZYMANSKI:  Man of the females have not even been pregnant, they're young women, they cannot have any antibodies--
		DR. STRONCEK:  Sorry?
		DR. SZYMANSKI:   There are many of these biologically safe women, have not even been pregnant once.
		DR. STRONCEK:  Yes.  I think even in multiparous donors, women, the incidence of HLA antibodies is--well, if they've had three or more pregnancies it's about 20 percent.  But there's a number of women that don't have HLA antibodies.
		DR. SIEGAL:  Dr. Di Bisceglie.
		DR. DI BISCEGLIE:  I guess you've identified the series of risk factors associated with TRALI but what might be useful is some quantitation, some relative quantitation of the risk, and maybe the incidence is so low that you have--it's not possible to calculate, say, an odds ratio, or a relative risk or a rate per thousand or something like that, that would allow a prioritization of the risk factors.  Are such numbers available?
		DR. STRONCEK:  Not yet but, again, you know, the Red Cross has begun looking at this in a systematic way, and the REDS group will be able to look at large cases of numbers of TRALI over several institutions.  So I think that data will be coming but it's not available at this time.
		DR. SIEGAL:  The next speaker, please.  This will be Dr. Sarode.  Current use of transfusable plasma.
		DR. SARODE:  Good morning.  You know, when I got this e-mail about transfusable plasma, I had no idea what I was supposed to talk, but I figured out, since it's TRALI, you basically want to know how much plasma we are misusing.  So that's why the topic of my talk is misuse of plasma in clinical practice.
		Now in United States we transfuse more than 3 million units annually, and this is very old figure from 2001, and I can assure you, it's more than that now, because there are seven changes in the way laboratories, they are doing testing for correlation factors.
		Now most of these plasmas, they are transfused before any procedure is performed, a surgical procedure is performed, or for patients who have an abnormal coagulation test in ICU setting, and which is very common.
		Unfortunately, the pre-surgical hemostatic assessment is mostly performed by using laboratory tests, rather than good old-fashioned clinical testing.
		So if we don't take a good clinical history about bleeding, rapidly we are doing screening tests for coag and not bothering about the history.
		So many times you get abnormal coagulation test and we act on these lab results.
		Now this particular slide shows the ratio of red cells to FFP transfusion in different countries.  If you look at United States and Germany, we are transfusing one unit of plasma for every three red cells, as opposed to Finland, U.K. and France, where they're transfusing only one unit for six units of red cells.
		So we are transfusing almost twice more plasma.  Dr. Dzik from MGH, he published this study about request for plasma in their hospital.  So they wanted to know why physicians order FFP, and guess what?  Most of the time they ordered plasma to correct an abnormal test result, that's INR, before any procedure, and there's about 15 percent of patients, they get plasma to reverse warfarin effect.
		So almost 50 percent of plasma used to correct an abnormal coagulation test.  Now you should look at literature.  Not a single study had shown any good correlation between prolonged coagulation test results and efficacy of plasma in treating those numbers, or having an effect on hemostatic efficacy when the coagulation tests are only mild to moderately abnormal.
		This study from Cleveland Metro Hospital showed that when they had 80 patients of cirrhosis, who had slightly elevated INR, they gave them plasma, 41 percent received for prophylaxis, and 60 percent who were already having some bleed, they got anywhere between two to four units, and actually 25 percent, they got more than four units of plasma.
		That's more than a liter of volume in a patient who is cirrhotic, and, I mean, you can imagine, pouring one liter of plasma in two to four hours.  It can be a disaster.
		And what were the results?  90 percent of patients failed to achieve the goal the had.  So they wanted all these clotting tests to be within three seconds of upper limit of normal and 90 percent of these patients who got plasma, even more than four units, they did not achieve the goal.
		So their conclusion was very simple.  FFP frequently failed to correct INR.  This is another study which was done a long time back.  It looked at bleeding time at the site of liver biopsy, and compared with prothrombin times.  		And you can see that the bleeding times, they're all over, even when the patients, they had near normal PT, that longer bleeding times as opposed to when they had longer prothrombin time, and they had no bleeding.
		So this again shows that even in 1981, we knew that there was no good correlation between prothrombin time and bleeding tendencies.
		Now this table, which is crowded, basically has the same message.  Unfortunately, there's not a single, well-designed, randomized, control study in the literature which has looked at efficacy of plasma in either correcting an abnormal PT and PTT and its efficacy in preventing bleeds.
		So these various studies, they have looked at--of course these are retrospective and observational studies--but none of these studies found any predictive value for either PT or PTT as regards bleeding, post procedure.
		Now what happens when we have hemoglobin of 11 grams.  We don't transfuse red cells.  When the platelet count is mildly reduced, we don't transfuse platelets.
		When creatinine is 2.1, we don't do dialysis.  You tell a nephrologist to do a dialysis at 2.1, you're going to get yelled at.
		So why do we transfuse plasma, when now PT is one or two seconds prolonged, and we want absolutely normal PT?  Is there any evidence for that? 
		Now if you look at evaluation in hemostasis, in our body hemostasis is a very complex phenomenon. If you look at it the way normal hemostasis works, you have procoagulant factors, factor I through XIII, except VI and XII, which are not important.  These clotting factors, they are kept under check by natural anticoagulants, antithrombin, protein C, protein S, and now TFPI or tissue factor pathway inhibitor, and then once you have clot in the system, you have very efficient fibrinolytic system which dissolves the clot, and of course you have platelets and endothelial cells, which are important players in maintaining normal hemostasis.
		Now not a single test assesses this whole hemostasis.  
		Now PT and PTT.  These tests, they were done to diagnose patients who had a bleeding disorder, meaning when patients had hemophilia A and B, people really want to know why they bleed.  So they came up with a test called PTT.
		Similarly when cows in Wisconsin, they were bleeding after eating plants which had warfarin, they wanted to know why they were bleeding, and they found out--PT.
		So these tests, they were developed in patients, or cows who have bleeding tendencies.  So these tests, if you perform in patients who have bleeding tendency, you have very high test--probably you are finding them abnormal.  They have never been shown to assess bleeding risk in nonbleeding patients.
		If you take 100 patients in your hospital and you do PT and PTT, and you find slightly abnormal PT and PTT in 10 percent, there's no guarantee that all these ten patients, they are going to bleed after surgery or during surgery.
		Now most of the plasmas, they are given for abnormal PT.  Now let's look at what PT is.  It's a very simple test.  You take patient plasma and you add a reagent which has tissue thromboplastin and calcium chloride.
		Now in the past, the sources of tissue thromboplastin included brains, which were human brain, rabbit or goat.  We use human placenta because they are all rich in thromboplastin.
		Now we have recombinant tissue thromboplastin that is used.
		However, in clinical practice, you will hear the term INR, and if you ask what is INR, most clinicians, they don't know what INR is.  They know this is a nice number, which is associated with PT.  Now this INR, or international normalized ratio, was developed to monitor patients who were on anti-coagulation or warfarin.
		So INR is the prothrombin time ratio that, by calculation, would have been obtained, though regional WHO-referenced thromboplastin had been used to perform the PT.
		Now the reason we needed INR was that if you have tissue thromboplastin coming out of one brain and used in one lab, it can last only maybe up to six months.  But after that you will prepare another reagent, and therefore the sensitivity of each brain for different, Vitamin K, different factors, was different.  So you had highly variable PTs.  And this was even worse when you had patient going from one hospital to another one.  The reagent was totally different.
		And most of these tests, they were done in '80s by using manual or semiautomatic techniques.  So changing one or two seconds really created havoc.  So we needed a method which then was provided.  That's INR.
		So the way INR is calculated is you need a prothrombin time ratio where patient's PT is divided by control, so, for example, if PT is 24, control 12, you get a prothrombin time ratio of two, and to calculate INR, you raise that prothrombin time ratio by ISI.
		Now this is the key.  ISI, the International Sensitivity Index, which is given to each tissue thromboplastin reagent, when it is compared and standardized and calibrated against WHO standard.
		So in this case, if the ISI, the reagent is two, your INR will be four.  So that's how you get INR.  Now the problem with tissue thromboplastin used in '80s and '90s was that they had very high ISI.  So to maintain INR between two and three, your PT was between 16 and 18.  Now that's where we all got trained, is '80s and early '90s, that if the PT as 16 or 17, patient was coagulopathic, because that was the--INR was very high.
		But the problem was if you had PT of 15, patient was subtherapeutic and if PT was 19 or one second above, was supratherapeutic.
		So it was giving nightmares to the clinician to manage anti-coagulation treatment for the patient.  They had to adjust doses every now and then.  So we did slightly better by improving the sensitivity of tissue thromboplastin to two and to maintain INR between two and three, now we had slightly wider safety range, between 17 and 21.  But again, it wasn't ideal.  So we went to recombinant tissue thromboplastin or some of the other tissue thromboplastin which have ISI close to one, and to maintain INR between two and three, now we have PT between 23 and 35.
		But what happened?  We got stuck on these PT values.  And that's what still happens.  So surgeons who are used to having bleeds when patients had PT of 16 and 17, in '80s and '90s, they still want 16 and 18 here, which is actually equivalent of somewhere here--28.  So the problem here is we have not communicated with the clinicians all these changes that have happened in the clinical labs.
		And this is one of the reagents that we use, it has very sensitive PT reagent, and it causes very short, PT's normal, but it's too sensitive to Vitamin K-dependent factors, especially VII.  So what happens now is even slight decrease in factor VII leads to prolongation of PT/INR, and we react to those by giving plasma.
		Now if you look at the hemostatic levels for various factors, or important factors, you basically need anywhere between 15 to 30 percent or 40 percent, and you can perform any surgery.
		When we did sensitivity of our PT reagent for all these factors, we found that when the factor VII was 49 or less, PT got prolonged.  But you need only 15 percent for major surgery.  So coming to indications for plasma, the absolute indication is still TTP until we find recombinant Lnt is thirteen.  Patients who have coagulopathy with bleeding, patients who have coagulopathy during surgery, and a reversal of warfarin overdose.  However, the ideal product would be to use prothrombin complex concentrate, which is recommended by American chest physicians, British hematologists, Australian and European societies.
		However, we don't have a prothrombin complex concentrate in this country licensed for this particular effect, and of course for real congenital factor deficiency for which we don't have beautifed concentrates.
	However, plasma is often misused to correct mild to moderate abnormal PT/INR, without bleeding, and volume expanded.
		Now the published data provide no evidence-based guidance for use of pre-procedure SFP, or even platelet transfusion among patients with mild to moderate abnormal results.
		So what we did, we talked to our neurosurgeons, and when they wanted some plasma for slightly prolonged PTT, we did a study.  We looked at, we stored their patients' plasma who had slightly prolonged INR, we gave them the plasma at that time and looked at hemostatically important clotting factors.
		And if you look at the results the medium PT was slightly prolonged as compared to upper limit of normal.  Similarly, INR was slightly prolonged.  PTTs were normal.  Factor VII was normal, and Factor VIII actually was very high, which is shown in the next slide.
		These are certain selected cases.  Now whenever we do PT and PTT, everyone looks at PT/INR, and others, they ignore PTT, and I don't know why.  Look at these highlighted PTTs.  They have very high levels of Factor H.  A PTT of 90.  Even though the INR is 1.4, which surgeons thinks has risk for bleeding.  If you look at the Factor VIII, 547.  Factor VIII is a known risk factor for clotting.  It's a known prothrombotic risk factor.
		So by looking at PT and PTT, this patient didn't need any plasma.  So I presented this particular study at Neurosurgery Grand Rounds, and there was a drastic reduction in FFP orders for prophylactic plasma use, 85 percent.  We followed up after a year and it still remains 75 percent less than pre study, and I still keep working with these surgeons to remind them.
		Now in Parkland, we have a very active transfusion medicine practice.  It's a county hospital.  Blood Utilization Review Committee, or so-called Transfusion Committee, has established evidence-based transfusion criteria, and since 2003, transfusion medicine residents, they are involved in each and every request for plasma and platelets that do not meet the guidance.
		And the goals were to reduce unnecessary transfusion and advise appropriate products, because we always try to treat everything with plasma and forget about cryo or other products like DDVP, prothrombin complex concentrates.  So that's what we do because all these factor derivatives, they are kept in blood bank and we like it.
		And of course to reduce the incidence of TRALI.  I won't go into details but this is a triage sheet for blood bank technician to call a resident when to give plasma, and similarly for platelets.
		Now since 2000, when I came there in the end of 2000, I started taking part actively in 2001, you can see that the number of red cell transfusions has remained the same because we haven't intervened in that particular practice yet.
		But if you look at plasma, it has started around 11,000 and now it's dropped to less than 4800.  So there's a 60 percent reduction in plasma use in four years.
		A similar thing happens for platelets, decreased from close to 5000 to less than 2500.
		However, the admissions, they have remained the same, surgeries, they have remained the same, and trauma has remained the same.
		Now we have a master transfusion protocol established for our trauma center, and the goals were to provide rapid blood component therapy, to prevent or treat coagulopathy earlier, than waiting for it to happen, and reduce blood wastage.
		And this is our shipment protocol for different products.  Whenever a protocol is initiated, we send five red cells to plasma, in the first shipment followed by five red cells to plasma, those are platelet and small dose of NOVA 7, and the third shipment goes with car.
		And if you look at the blood products used, pre and post MTP, we have reduced the use of red cells by seven per incident, so instead of using 24 we use now seven.  Seventeen red cells.  But look at the plasma. It has decreased by 50 percent, and same with platelets.
		So what's happening is we are trying to prevent or treat the coagulopathy earlier rather than waiting till it develops and try to pull all these factors.
		So it seems that appropriate products utilization is the goal.  We should recommend when the cryoprecipitate is indicated.  All these patients who are getting plasma, they may have low fibrinogen, they may have dysfibrinogen anemia, which is common in patients with chronic liver disease or cirrhosis.
		Use of antifibrinolytic agents is encouraged.  Patients with mild gum bleeds and all, and they need platelets, we recommend Amicar.  Similarly, a reversal of heparin-induced bleeding, many surgeons, they still want to give FFP, which is actually contraindicated, in my opinion, because it supplies antithrombin and makes the bleeding worse.
		We have started using prothrombin complex concentrate, which is not an ideal one, but still, it has reduced our plasma use by 50 percent.
		So the take-home message is that there's still significant misuse of plasma in clinical practice.  Blood banks should practice transfusion medicine, rather than function as simply blood-dispensing units.  That's what's happening in almost 99 percent of hospitals in the country.
		Whenever there is a request for any blood products, they are dispensed without any question.
		There's a greater need for clinicians and medical students education in transfusion medicine and hemostasis.  And most importantly, they like it.  If you tell them what's the real stuff, they like it and they respond to it.  Thank you very much.
		DR. SIEGAL:  Thank you very much.  Are there any questions?
		DR. SZYMANSKI:  I just want to commend you on this wonderful presentation because it's really a great problem in this country, and the smaller the hospital, it seems to be even greater.  I recently inspected a blood bank at a relatively small hospital and they use plasma for slight increase in PT.  They never check if the PT changes after plasma transfusion, you know, prior to surgery, and they really don't have proper guidelines for any of the blood components, and I think if you can reduce plasma and limit infusions, you know, by having good guidelines, like you have presented, it would also reduce the incidence of TRALI.
		And I really liked your talk very much.  Thank you.
		DR. SARODE:  I mean, that's my goal.  I mean, I have been going to different departments, giving Grand Rounds, and they are responding very positive because these changes in the laboratories, they have not been communicated to clinicians and they still think that, you know, a PT of 16 is as bad, you know, as it used to be.  But that's not true.  We have changed practices in radiology, intervention radiology, hepatology, neurosurgery, 
		[No response]
		DR. SZYMANSKI:  Yes.  The surgeons particularly need a lot of education in this area.
		DR. MANNO:  I have a question about your recommendation for a PCC as opposed to FFP for reversal of coumadin.  Is that based on ease of use, because you don't have to wait for the plasma to thaw, or efficacy? 
		DR. SARODE:  I think both.  The first thing--
		DR. MANNO:  Are there data to demonstrate this?
		DR. SARODE:  Pardon?
		DR. MANNO:  Are there data, have there been studies of this, or is this your preference?
		DR. SARODE:  Yes.  European data is there, and we have our data coming out soon.  The advantage of PCC--now there are two types of PCCs.  People get confused.  One is--
		DR. MANNO:  I don't.
		DR. SARODE:  I know.  But there are many hematologists who are still confused.
		There is one called activated PCC or autoplex, which is not available in U.S., and FEIBA.  And there are PCCs which are nonactivated clotting factors, so it has purified factors II, VII, IX, and X, and these PCCs, they are used to treat hemophilia B.  The advantage of four factor PCC, where you have all Vitamin K, different factors, actually some of the products in Europe, they also have protein C and protein S.  So it's much more complete PCC.
		And you can transfuse PCC within five minutes, the dose, 15 -- for the worst INR with bleed, and you will find correction of INR within five minutes, as opposed to FFP, when you are trying to give FFP, you are thawing the plasma, it takes half an hour to thaw the plasma, and maximum you can transfuse is one unit every 15 to 30 minutes, and that leads to a volume overload, because the majority of the patients who come with warfarin-related bleed, they are elderly, on warfarin for atrial fibrillation or cardiac condition.
		So both safety reason, I think it's very good, and rapidity is excellent.  And the current PCC that's available in U.S., called Profile 9, has II, IX, and X.  It doesn't have factor VII, and that's why we had to use FFP to correct that, because INR doesn't get corrected and surgeon really wants INR.
		DR. MANNO:  Some of course would argue about the expense of a PCC as well as the old reports of thrombosis, particularly in patients who have preexisting liver disease, or in the neonate.
		DR. SARODE:  But most of these thrombotic complications, they were seen in patients with hemophilia B and who were getting PCCs at regular interval for a long time.  So if you give daily PCCs to hemophilia B with liver disease, you are replacing factor IX by PCC, but they still have their own factor II, VII, and X, and by giving PCCs every day, you are raising their other clotting factors.
		So they had prothrombotic state, and that's why they had more complication, not significantly more but they thrombotic complications.  But when you treat PCC for warfarin reversal, you give only one dose, and you give all these factors, so risk of thrombosis is significantly less, and the European studies have shown there's no evidence that it's thrombogenic.
		DR. SIEGAL:  We're going to have to move on.  Thank you, Dr. Sarode.
		Dr. Steven Kleinman from the University of British Columbia will talk about the REDS LAPS study on HLA and granulocyte antibody prevalence in blood donors.  
		DR. KLEINMAN:  Thank you.  So I'm going to address, give a preliminary report on the Leukocyte Antibody Prevalence Study, which the acronym is LAPS, and since we have a second study planned, the acronym is LAPS I.
		This is part of the retrovirus epidemiology donor study, second iteration, so REDS I was a 15 year study.  REDS II is now a different study.  I'm one of the members of the REDS II group and I'm presenting this on behalf of many other investigators.  They actually have an old slide projector control here and an old slide projector.  I haven't seen that for a long time.  It'd be a page down.  Yes.
		So the structure of REDS II.  This is a study that was funded beginning in 2004 by NHLBI as a five year study.  It has multiple project areas.  I'll just be talking about the leukocyte antibody prevalence study today.
		There are six blood centers that are participating in this study.  They are the blood center of Southeast Wisconsin, the American Red Cross New England Region, along with the American Red Cross Southeast Region, and Emory University, Hoxworth Blood Center from Cincinnati, Institute for Transfusion Medicine from Pittsburgh, and the blood centers of the Pacific, San Francisco.
		The coordinating center for this study is Westat, located locally, and the central laboratory is Blood Systems Research Institute in San Francisco.
		The objectives of this leukocyte antibody prevalence study.  There are several objectives.  The main objective is to determine the HLA class I and II antibody prevalence in blood donors, and correlate this with the number of pregnancies, the lifetime history of transfusion, the time from the last immunizing event, and to compare these alloexposures with a baseline group that has no evident alloexposure, so never pregnant or never transfused.
		So in addition to determining the prevalence of these antibodies, another goal is to actually identify the HLA antibody specificities, and the third goal is to determine the presence of antibodies to neutrophils, and because neutrophil antibody testing is complex and expensive, our goals are a little more modest here.  We don't think we can test the whole cohort for neutrophil antibodies, and so we don't know if we'll have the power to relate neutrophil antibody prevalence to these epidemiological factors, pregnancies and transfusion, but we are planning to do some neutrophil antibody testing, first in those with HLA antibodies because we think those will be higher rates of neutrophil antibodies as compared to some of our donors without HLA antibodies.
		So in doing a statistical power calculation as we began this study, we estimated from existing data in the literature, and there are some papers indicating the rate of HLA antibody prevalence in donor populations, that we would need to enroll about 5100 female donors to have greater than 90 percent power to detect differences in HLA antibody prevalence, both by number of pregnancies and the interval from the last pregnancy.
		So that was our sample size calculation for female donors.  With regard to male donors, the preliminary data from the literature suggests that the--and in very small numbers--that the rate in transfused male donors and untransfused male donors of HLA antibody is not every different, and so we could not power the study to actually find a difference in rate, and we decided that we would look at a thousand donors, a thousand male donors with a lifetime history of transfusion, a thousand never-transfused males, and the purpose here was to try to tighten the confidence intervals around the prevalence estimates that are currently in the literature.
		So a little bit about the protocol.  We give everybody enrolled into LAPS a questionnaire that--well, it includes both transfusion history, and pregnancy history for the female donors.  The questions for pregnancy history are taken from the National Health and Nutrition Examination Survey, NHANES.  These have already been validated.  It's a series of six questions.
		They ask the donor whether they've ever been pregnant, and if so, the number of pregnancies, and divide this between number of live births, still births, miscarriages or abortions, and then we ask for the date of the last pregnancy and the transfusion history consists of a question, Have you ever received someone else's blood? and if yes, then the date of the last transfusion.
		One of our first tests was to decide what assay we would use to measure HLA antibody.  The potential assays that are out there are flow cytometry assays, both flow PRA, and Luminex, which I'll talk about in a minute.  There's also, as Dr. Stroncek mentioned, solid phase ELISA assays, and then there are the classical methods of lymphocytotoxicity, usually now done with human, anti-human immunoglobulin enhancement.
		We decided that in this research study we should use the tests that had the highest sensitivity for HLA antibodies and based on the high sensitivity of Luminex and flow PRA, and the high throughput that we could achieve with Luminex, since we did have such a big study, the assay chosen was the--we chose the Luminex platform which is a specialized flow cytometer, and we used the LABScreen reagent from One Lambda, a company in California.
		We decided we would perform both supplementary screening--I'm sorry.  In addition to screening assays, we would perform supplementary testing with their single antigen assay to confirm the screening results, and I'll get to that a little bit more specifically in a few slides..
		So the LABScreen reagent that we used is called the LABScreen mixed reagent.  It's a screening assay that uses multiple beads coated with purified HLA antigens.  Each bead contains purified antigens from five to six cell lines.  The assay consists of five beads that have class I antigens, three beads that have class II antigens.  There are a total of 54 different HLA class I antigens and 32 class II antigens involved in the initial screening assay.
		The company has also developed a couple of beads for a new antigen system that's been discovered, about five to ten years ago, called MICA, or MICA--I'm not sure how it's pronounced--which stands for MHC Class I related antigen.
		This antigen is present on endothelial--this antigen system, which actually has multiple specificities, just as HLA does, is present on endothelial cells but is present on a number of other cells in the body as well, and we thought this would be of interest to look at because endothelial cells have been implicated in TRALI.
		So we use the Luminex flow cytometer and the assay measures, laser-based light emission, through the binding of reagents that are conjugated to antibody, which is, in turn, bound to the class HLA 1 and 2 antigens.
		And here is a schematic provided by the company.  There are, as I said, multiple beads here that fluoresce at a difference intensity and then each bead is covered with different purified HLA proteins, and basically, the principle of the assay is the Luminex bead has a bound antigen to it, or a series of antigens.  If the patient has alloantibody it will bind to the antigen, and then there'll be a photochemically-tagged anti-lgG.  If that binds, then there'll be emission of light and you can see that in the flow cytomer, and you can actually know which bead it's coming from, and therefore you can decide which antigens are implicated, whether they're class 1 or class 2.
		So this is a screening assay.  One could go beyond this to actually try to identify the antibody specificity.  Their common way of identifying the antibody specificity is similar to the old ways, and that is that you would use a sort of panel reactive antibody.  You'd have multiple beads and you'd have to try to dissect what was happening.
		But for purposes of this study, the company also has a single antigen assay which isn't used as frequently because it's more expensive, but we are using it for this study.
		So in this assay, each bead contains one and only one recombinant HLA antigen and that's characterized well out on the molecular level.  So, for example, you can have an A2 but we have multiple A2s on different beads.
		So this mix contains 94 class 1 and 52 class 2 antigens, each on a single bead.  The specificities included are within class 1 are the AB, BW, and C loci, and within class 2 cover DR, DQ and DP.  All of the common antigens are represented.
		Obviously, there are some rare antigens that are not included on the panel.  And so the idea here is if you get reactivity, you can be much more sure of the specificity or the multiple specificities that might be present in a given donor.
		But because HLA antibody interpretation is complex, even with this system, we've adopted, and most of the investigators in REDS II are not HLA experts, we've established a review panel of three HLA experts, and each confirmatory testing result will undergo review by at least one expert and any problematic case will be reviewed by a panel of experts in order to assign both interpretation of the confirmatory test and the specificities.
		So that's a little bit about the lab study.  I wanted to give you a little bit of background about the data that we have overall in REDS II, cause I'm going to show some of that in a moment.
		And so we have accumulated in a central location, all of the donation and donor data that are accumulated by the six blood centers.  So we have one complete year of data for 2006.  This represents 1.2 million successful donations.  So it probably constitutes about 7, 8 percent of donations in the U.S.
		So we have all the basic demographic information on each donation made by each donor.  We know the gender, the age of the donor, that sort of thing.
		We know what type of donation was made, whether it's whole blood of apheresis.  And in addition, we have two additional pieces of data that aren't usually obtained by blood centers.
		We've added questions at these six centers to the usual donor questionnaire.  So we have the pregnancy history.  We ask a simpler question of all donors than we do to the LAPS enrollees.  So we ask, Have you ever been pregnant, and if so, how many times?  And we give them a option of one, two, I think up to six, actually.  And then we ask a question, if they're ever been transfused.
		So I want to show you some data from the donation database here.  This may have some implication for thinking about male versus female plasma issues.  So you can see here, for whole blood donors, whole blood donations, it's basically a 50/50 mix between donations coming from male donors and female donors.
		On the other hand, if you look at platelet pheresis donors or donors from whom we collect platelets and another component at the time of apheresis, you can see that these are skewed to be greater than 60 percent male and less than 40 percent females.  So we have a lot more male platelet pheresis donors.  Plasma pheresis donors, same thing, but about 55 percent male and I just threw this in, it's not particularly relevant, but for red cells collected by automated apheresis, for which you have to have higher hematocrits, in general, almost 90 percent of those procedures are done in male donors.
		Now as I said, we collect the transfusion history data from all of these donors and it's summarized here.  This is on a per donor basis rather than on a per donation basis.
		So you can see that amongst our male donors, about 2.6 percent indicated that they had a previous lifetime history of transfusion, and just to remind the committee, we generally ask all donors if they've been transfused in the last 12 months, and if they had, they're not allowed to donate until those 12 months are over.
		But if they've been transfused greater than 12 months ago, they are eligible as a donor.  So these are people who were not transfused in the last year because they're eligible as donors but had a previous transfusion.
		And in female donors, you can see that the number is about 4 percent.  Interestingly, between 2.5 to 4 percent of the donors, depending on gender, answer that question as "not sure," whether they've ever had a lifetime transfusion.
		If we were to do this per donation rather than per donor, based on our experience in REDS I, some of these donors turn out to be multiple time donors and at least in REDS I, where we had a similar percentage of about 4 percent of donors who had a previous history of transfusion, they accounted for about 5 percent of donations.
		So that's the background on history of transfusion.
		Now returning back to the specific protocol of LAPS, here's our enrollment to date, and our enrollment is almost complete.  Rather than 5100 female donors, we've been so aggressive, that we have almost 6000 female donors who have been enrolled in this study.
		We have 1241 males who have not been transfused, they're easy to enroll, but we have, so far, only 749 males who have been transfused, and as you can see, if only 2 percent of our, 3 percent of our males have been transfused, it's hard to find these people when donors come to mobiles.
		So we've had to do recruitment through special efforts to find male donors with a previous history of transfusion.  That's why the enrollment is ongoing.  Whereas the enrollment of the first two groups, we just approached donors coming in at multiple collection sites determined by the individual blood centers, and these were based on ease of logistics as well as trying to get a representative demographic mix of donors that do visit those particular blood centers.
		Now we've analyzed the questionnaires from these enrollees and I just want to go through the parity history in the female donors that we've enrolled.
		So, again, close to six thousand donors.  And what I've represented here is--the green bar represents the parity history of all the female donors, not the LAPS enrollees, but the greater than 400--I think 300,000 female donors in the database for 2006.
		And this is coming off the question that we ask each female donor at the time that they come in for their donation.
		 The blue bar is also from the REDS donation database.  It represents the apheresis female donors and then the gray bar represents the enrollees in LAPS.
		A couple of things to point out there.  Number one, the number of never pregnant females, this is on a donor basis, so of our female donors, about 44 percent have never been pregnant, which is actually much higher than what has been reported in previous papers in the literature about the U.S. donor base, and of course these papers are ten to fifteen years old, and  think what we might be finding is the demographics of pregnancy have changed, at least in blood donors, if not in the population, in general, and so we have a lot more females who have never been pregnant.
		We haven't completed the demographic analysis, but preliminarily, it looks like this is, as you would expect, related to age, that the younger donors are less frequently pregnant than women who are older.
		You can see, there's a bit of a discrepancy between apheresis donors, in that there are fewer never-pregnant women donors, and then we have the LAPS donors who happen to be mostly whole blood donors but their pregnancy history reflects a little bit more that of the apheresis donors.
		And then we have about 11 percent of donors who've been pregnant once, about 20 to 22 percent that have been pregnant twice, and then anywhere in the general population of donors, about 25 percent that have been pregnant three times or more, and then even more in apheresis donors.
		And so this is important when we actually get HLA antibody prevalence and our enrollees will have to do a weighted average to say what it is would be in the overall database.
		So I want to give you the status of the actual testing now.  We've completed the screening assay, HLA screening assay on about 4700 samples.  The supplementary single antigen assay testing is in progress.  We only have very few results on that and the data analysis is still in progress.
		But I do want to give you some preliminary observations.  It's interesting, the package insert for the One Lambda LABScreen test has a statement, that they suggest using the cutoff, their normalized cutoff ratio of 2.2, but at the same time, they say that each individual laboratory may need to determine their own cutoff.
		It's also interesting that the data from previous studies, which comes from generally in-house validations, and they go to a population, they expect not to have any HLA antibodies, so a non-alloimmunized population and get the cutoff value, btu those populations are much, much smaller than the group we've screened from, from LAPS. 
		And so another thing to recognize is the cutoff, as recommended by the manufacturer, was developed in the context of using this assay system to maximize sensitivity, particularly in the organ transplant setting.
		 So they would screen patient sera, and they were looking for patients who had HLA antibody, and clearly, if a patient who had HLA antibody was then given an organ with the corresponding HLA antigen, you would be worried about an anamnestic response, so that any indication of previous immunization would lead to the potential for organ rejection.
		But I want to point out that this is very different from the way we might be using an assay to determine the safety of a donor in a TRALI situation, because here, we're talking about passively transfused antibody causing the pathology.
		And so it may not be important to detect antibody at very low tiers, cause it's all going to get diluted out in the person's own bloodstream.  So here's all I can say about the data right now.  Using an NBG ratio of 2.2, we're finding HLA antibody in a significant proportion of apparently non-alloexposed persons.  So non-transfused males and never- pregnant females.  And interestingly, if you go back to the one paper that everybody quotes about this phenomenon, which was published in about 2000 out of St. Louis, in apheresis donors, they used lymphocytotoxicity and they showed a 7.8 percent incidence of HLA antibodies in never-pregnant females, and our findings at this lower NBG ratio are in that order of magnitude.
		However, we haven't yet done confirmatory testing, and as I said, if we choose a higher NBG ratio, so set the cutoff higher, than the rate significantly decreases, and we're doing some analyses to see where the cutoff really should be set, and this is highly dependent on the confirmatory assay results.
		And same thing as you would expect with any assay, that again, depending on where we set the cutoff, we'll also see a different rate of HLA antibodies in females, depending on their level of pregnancy, and we are seeing a correlation, as we would expect, of the greater the number of pregnancies, the greater the amount of HLA antibody, regardless of whether the donor is--regardless of the cutoff we use.
		So our thoughts now are that the same concerns about assay sensitivity, which we're demonstrating in the Luminex system, which is how sensitive do we want to be, will also apply to other HLA detection systems.
		And so as of now, it's unclear if antibody with low signal strength is of significance for the safety of transfusion recipients, and it's unclear how such information should be used to make donor deferral decisions, even if we're adopting the precautionary TRALI risk reduction policy that says we want to minimize transfusion from HLA-immunized or leukocyte-immunized donors.
		One other aspect of this study I wanted to mention is we are establishing a repository.  We have anywhere from four to six aliquots of plasma frozen, two aliquots of whole blood, and in many cases two aliquots of serum.  These are stored at minus 70 degrees Centigrade.  We'll be able to access these for neutrophil antibody testing and maybe neutrophil antigen or HLA antigen testing, if we decide to do it.
		We can also access these for HLA antibody testing using other test systems, provided we get the funding to do that, and we can actually recall donors if we want to.
		And then my final slide is to tell you what we're planning to do.  In addition to analyzing the HLA antibody data, we will do neutrophil antibody testing.  We are planning to do some HLA titering studies to see how strong some of these antibodies are, we'll look at the MICA data, and we may do alternate HLA tests.
		And finally, we are far advanced in a planning stage of what we're calling LAPS II, which is a clinical study in which we will identify previous products donated by these persons with HLA antibody, and then go back to recipients of high plasma volume components from these donors, well, not to the recipients but to their charts, over the last few years, and through our triage protocol of looking at their x-rays first, decide whose medical record to review, and see if we can document whether people with leukocyte antibodies resulted in the production of more TRALI than donors who did not have leukocyte antibodies.  So that's in the planning stage currently.  Thank you.
		DR. SIEGAL:  Thank you.  Are there any questions?
		DR. KLEIN:  Steve, thank you for sharing the data with us.  I'm not surprised about the frequency of the female, nonparous women with this sensitive Luminex assay.  In fact, I'm surprised it was as low as it is.  But we know that history is sometimes misleading and spontaneous abortions occur.
		Do you have any data to share with us about male screening at this point, because clearly, that's a big issue, especially for apheresis platelets?
		DR. KLEINMAN:  Yes.  A couple of preliminary observations.  If you look at any specific cutoff right now, we've looked at two, a ratio of 2.2 and a ratio of 10, and if you take a preliminary look at the screening data and look at the frequency of antibodies in never-transfused females, never-pregnant males, never-transfused males, and transfused males--now we only have data on about 250 transfused males--the numbers in those three groups are, at a particular cutoff are fairly comparable to one another.
		So it doesn't look like transfusions--again this is preliminary--but it doesn't look like transfusions in the male donors has caused much additional HLA antibody to the background rate of transfusions of antibody in never-transfused males.
		So it doesn't look like transfusion's a very powerful stimulus, at least in the amount that our donors have been transfused.  But this is preliminary, Harvey.
		DR. KLEIN:  But it looks like about 5 percent of your males are positive by this assay transfusion in this; correct?
		DR. KLEINMAN:  Well, it depends where we set the cutoff.  If we set the cutoff down at 2.2, then it looks like again, without confirmatory data, somewhere about 7 to 8 percent.  But, again, confirmatory data will probably bring that down, and we haven't looked at the strength of antibody.  If you go up to a much higher ratio, in the ten range, then we're down to 1 to 2 percent.
		DR. KATZ:  Steve, do you have the year of transfusion in these histories, or--
		DR. KLEINMAN:  We do have--
		DR. KATZ:  --whether they got LR, or what they were transfused--
		DR. KLEINMAN:  We do have the year of transfusion.  We haven't analyzed on it yet.  And again, we only have data on 250 transfused males.  We have 750 enrolled,.  We're continuing with enrollment and hope to get to a thousand.  So, again, it's a very partial data set and, you know, we hope to have a lot more data in the next few months.
		DR. SIEGAL:  Are you looking at the women in terms of how many sexual partners they've had as--
		DR. KLEINMAN:  No; there's no history of sexual partners.  We're not taking a history of sexual partners.  It brings up an interesting question that we've tried to ask the experts, and if you really haven't been alloimmunized by pregnancy or transfusion, is it possible to have HLA antibody based on immunization, based on sexual contact with partners?  I mean, is that an immunizing event for HLA antibodies that could explain this background rate?  Apparently nobody knows the answer to that.
		Or could this background rate be cross-reactivity against other antibodies, perhaps through exposure to vaccines that might have been grown in cell lines, etcetera, and, you know, we know that's possible, people have reported that as possible, but we really don't have an expla--or is it just, you know, false positive stuff that nobody, as with many assays, that nobody's ever able to identify the cause?
		So we really don't know at this point.  But we don't have that data to be able to evaluate it.
		DR. NELSON:  This is really a fantastic study.  It seems, though, that the sample size is going to be too small, probably, if the rates are one in a thousand and one in ten thousand, and you've got 5000 people to actually identify, link antibodies with TRALI cases, and I wondered if there was thought about doing a case control study in the REDS, where you might be able to save or collect, since the event occurs within a few hours of the transfusion, and actually link the antibodies with the TRALI event?
		DR. KLEINMAN:  Yes.  Two comments on that, Ken.  With regard to the sample size and the look-back study, depending on the assumptions you make, and obviously that's always true for sample size calculations, we think that we might have an adequate sample size to do the study, if you think that TRALI actually occurs at some of the higher rates that people are predicting.
		With regard to doing a case control study, going forward, there is an NHLVI-funded SCCR, you know, Specialized Clinical Center of Research, or something like that, that Pearl Toy's group has at UCSF, in conjunction with Mayo Clinic, and that is one of their protocols.
		They have, I think, gone through a year of enrollment of TRALI patients, and controls, where they're actually collecting ever donor unit and analyzing it, and I think the reason they're not here is because it's just too early to have enough data to be meaningful.
		But I think that's the stud that will look at it in the appropriate scientific control fashion.  The look-back is the best we think we can do within the REDS context.
		DR. GLYNN:  Steve, could you give us the estimates for the women who were pregnant, who have been pregnant before?
		DR. KLEINMAN:  Well, you know, it's a dilemma on how much data to present because we really haven't, you know, it's really an analysis in progress, and if this meeting was two months from now, I think we'd feel much more confident about presenting actual numbers.
		But, again, depending on where you set the cutoff--I guess a few things I can say.  It's more common to find HLA class 1 antibodies than HLA class 2 antibodies by about threefold, and it's actually not that uncommon to have both HLA class 1 and class 2 in the same person.
		With regard to numbers, they certainly are comparable, if not higher to what's been reported in the literature, and that has been about 15 percent in people with one to two pregnancies, and greater than 25 percent in people with three or more pregnancies.
		So, again, if we use the lower cutoff value, we're finding numbers that are as high, probably higher than that.  But we really would like to have our confirmatory data analyzed, because I think if somebody has antibody on the screening system and then, quotes, it's positive on the supplemental test, while it doesn't necessarily confirm it, it tells us that there's reactivity against a specific HLA antigen and not other HLA antigens, and whether that makes it HLA antibody or cross-reactivity, we still don't know, but I think that's really the crux of the data that'll be more solid.
		DR. WILLIAMS:  Alan Williams, FDA.  Two quick donor management questions.
		What has been the experience in administering the NHANES pregnancy question?  Put differently, is it feasible in a blood donor setting, and secondly, are donors being given the results of the antibody testing?
		DR. KLEINMAN:  So the first question about administering the questionnaire, I really can't answer.  I don't think it's been a problem in--well, it has been easy to administer in the research setting but whether that would be practical to ask six questions in the normal donor setting, I don't know, because they're actually given a questionnaire that they fill out, a separate questionnaire.
		Clearly asking the two additional questions at the time of donor interview, which we've done in REDS I and REDS II, that I think has been a very transparent and easy process.
		So if you just add a question, have you ever been pregnant?, and then, if so, how many times?  But that doesn't go into sort of the details between miscarriages, abortions, and the advantage of NHANES is you're telling people that you're really interested in not only live births but, you know, any other pregnancy episodes, and we're not doing that on the short form.  So I don't know how feasible that is.
		With regard to giving results back to donors, that was sort of an issue of great debate at the time of taking the protocol through to IRBs and the decision was left to each center, but I think at the time that the research study was approved, most people felt that HLA antibodies results would not be meaningful to give back to donors because they really had no health significance, and at that time they had no consequence on your donation eligibility.
		But, on the other hand, if we found donors with granulocyte antibodies, especially certain specificities, most people felt that donors should be told that, so the consent that donors sign has said you may be given your test results if deemed to be of medical significance.
		Thus far, since we don't have confirmatory data back, except on a handful of people, we haven't actually given any donors' results yet.
		DR. SIEGAL:  Ms. Baker.
		MS. BAKER:  So the questions on pregnancy history in the REDS database, they are or are they not the six NHANES validated questions?
		DR. KLEINMAN:  No.  The NHANES validated questions are only for the 6000 women who enrolled in the LAPS study.  The other, the three to 400,000 women who've been asked the pregnancy questions, I don't remember the exact questions but I think they're essentially, number one, have you ever been pregnant? and number two, how many pregnancies have you had?  And I think they give either an oral answer or fill in something.  So it's less substantial in the NHANES.
		DR. SIEGAL:  Dr. Kleinman, thank you very much.  I think perhaps the other questions could be private.  We're running an hour behind.
		DR. NELSON:  Just one quick.  Did you ask about the mode of delivery, C-section versus--
		DR. KLEINMAN:  No.
		DR. NELSON:  Cause that might be interesting as to when sensitization might occur.
		DR. KLEINMAN:  Sure.  Well, I mean, it's the same issue, you know, how many questions can you ask people in a quick intake interview and we just tried to hit the highlights.
		DR. SIEGAL:  I'd like at this moment to suggest that we take a break since we're a half an hour behind and continue.  We don't, at the moment, have any open public session speakers, so we may be able to make up some time.  But we are an hour behind, so let's make this break quick.  Are there any questions to that?
		[No response] 
		DR. SIEGAL:  Okay.  Let's do it.
		[A recess was taken from 10:59 a.m. to 11:13 a.m.]
		DR. SIEGAL:  All right. All right.  So to restart, we have Dr. Richard Benjamin from the American Red Cross, talking about their experience with TRALI. 
		Dr. Benjamin.
		DR. BENJAMIN:  Good morning, and thank you to the committee for the opportunity to present the American Red Cross's experience with TRALI.  In the beginning of 2003, the Red Cross instituted a nationwide hemavigilance program that we call the Donor and Recipient Complication Program.
		The single most common adverse event reported to this program is TRALI.  In the period from 2003 to 2005, there were some 550 reports of TRALI, with increasing incidence during this period.  This data is now published in Transfusion this month by Dr. Anne Eder and most of my presentation is taken from this publication.
		Of those 550 reported TRALIs over three years, there were 72 fatalities, and we can see here that in 2003 we had 17 fatal, suspected TRALI fatalities reported.  It rose to 33 in 2005, and we had another 33 in 2006.
		We did an analysis of these fatal cases.  We focused only on the fatal cases because the data tended to be more complete and the investigations were better in the fatal cases.
		We did a retrospective review using three of our transfusion medicine specialists to go through the 72 cases, and basically they classified them as probable TRALI or unrelated cases.
		They decided, without looking at the antibody results, that about 38 of them were probably TRALI.  We then looked at the antibody data and about 75 percent of these probable TRALI cases, there had been a antibody-positive female donor involved in the case.
		I should point out that the antibody testing in the study was not standardized.  It was performed in multiple labs, in multiple hospitals, and, in fact, only in a minority of cases were the recipients actually typed.
		So only about in 20 to 25 percent of cases did we actually have cognate recognition.  So I would not use this data to prove that antibodies cause TRALI.  That's my point here.
		Anyway, 75 percent did have antibodies, and we document that here.
		When we looked at the components that were involved in the TRALI cases, and I think about a third of them, there was a single unit transfused within six hours of the onset of TRALI, so a good third, that clearly we could say that unit caused the TRALI.
		The units most commonly involved were plasma units, 24 cases out of the 38, and 18 of those cases had a antibody positive female donor involved.
		That's where we get our conclusion, that if we intervene with respect to plasma, we could prevent six fatal cases a year from the 18 cases shown in this figure.
		There were five fatalities implicating apheresis platelets and seven red cells, or with red cells.
		Clearly, we transfuse many more red cells than we do plasma and platelets, so we converted these data into rates, and the rate, clearly, for plasma, was much higher than any other component.  But one in 200,000 distributed units of plasma was implicated in a TRALI fatality.
		About one in 300,000 apheresis platelets and for the other components, it was in the range of one in two million components implicated in a fatality.
		We looked at the odds ratios comparing plasma to red cells with an odds ratio, highly significant, of 12.5.  Apheresis platelets versus red cells, odds ration of 7.9.  The difference between plasma and pheresis platelets was not significant but clearly a strong trend.
		We then correlated that with the amount of plasma in these components and plasma and apheresis platelets both contained about 250 or 300 mls of plasma.  The other components are clearly less than 50 mls, on average.
		We also looked at the proportion of female donors in these components and most of our components have about half and half male and female, except for apheresis platelets, that are 65 percent from male donors and 35 percent from female donors.  A conclusion from these data was that fatality appeared to be correlated with the amount of female plasma exposure in the system.
		The Red Cross felt that they needed to intravene and do something about these fatalities, and clearly, we wanted to focus on the plasma and apheresis based on these data.
		We're also very aware that we transfuse way more red cells than we do plasma and platelets, and that we had no way of intervening to do anything about red cells at this point in time.  So if we intervened on plasma, at best, we could address about 60 percent of the problem.  We are not going to completely avoid TRALI by any intervention around plasma or platelets but we could get to the majority.
		So it's going to be a partial solution, whatever we do.
		What were the possible solutions?  I think you've seen this slide before.  Well, clearly, Dr. Sarode talked about the appropriate use of products.  We've spoken about the selective use of male products, donor histories, testing, pool and store.  All blood-derived platelets have been apparently less frequently involved in TRALI.  We're talking to manufacturers about platelet additive solutions to reduce the amount of plasma in apheresis platelets, and we recognize that solvent detergent plasma could be attractive from the TRALI point of view.
		I do want to emphasize what Dr. Sarode said about appropriate use of blood clots.  Of the 24 fatalities that implicated plasma, twelve of them, the plasma was transfused for reversal of coumadin, and in six of those twelve, there was no indication that the patient was bleeding at the time.
		So when we did a clinical correlate, we tried to look at the clinical correlates of these fatalities, the only thing that stuck out was coumadin reversal.  So I do want to emphasize that that is probably the single best intervention to reduce TRALI.  Maybe to reeducate our physicians about the use of plasma for coumadin reversal.
		Having said that, we embarked upon asking the question for plasma, at least, could we selectively use male products?  Within the Red Cross system, we produce just over 5 million units of plasma from whole blood.  We transfuse about a million and a half as transfusable plasma and the rest are sent to fractionation, either as less than 24 hours or greater than 24 hours plasma. 
		We have this vast pool of plasma for fractionation less than 24 hours, and it occurred to us that we could start simply swapping out the male plasma that was sent to frac, at least initially with the FP24, the 24-hour plasma that we were making, and later on, with the FFP.
		So we undertook a pilot, where, in October of last year, in three of our divisions, where we attempted to make predominantly male plasma, FP24, plasma frozen within 24 hours, and we did this really to assess the manufacturing issues, not the clinical efficacy.
		Essentially, we labeled the products at donation with a blue sticker or a pink sticker, or an M or an F, you know, a very crude system, and when they came into manufacturing, we triaged them for fractionation versus useful transfusion.  It was instituted as a business practice.  The SOP was one page long.  The final product was not labeled, nobody was notified, and if we had shortages of plasma, which did occur around the West Nile season, and around AB plasma, we would use female plasma.
		We did succeed in getting to greater than 95 percent male plasma in the divisions where we tried this, quite successfully, and we continue to do that for FP24.  The system is feasible in the Red Cross system.
		So with the discussions that we've had with the AABB and the ABC, and the bulletin that came out last November, we are moving to address the issue of plasma in the Red Cross system.  Right now, 95 percent of our plasma comes from whole blood donations and about 5 percent from apheresis.
		For the whole blood plasma, that includes FFP, FP24, and cryo pool plasma.  It is our intention to move to greater than 95 percent male by November of this year.  We would like to get to the goal of a 100 percent but we recognize that availability is going to be an issue, and, in fact, right now, we only collect enough AB plasma to get to 92 percent male.
		So we are certainly going to have to do some selective recruitment and some active intervention to get to 95 percent.
		It is our intention to move to more FB24 as opposed to FFP.  We could, indeed, make male-only FFP but there are significant costs involved in doing that and one of the drivers from the start here, has been to do this at minimal cost to our hospitals.
		So in our hands, in fact, FFP will become a specialty product.  We do not intend labeling any products with donor gender.  For apheresis plasma or the Auto-C, we will be, we have a few regions that produce quite a lot of Auto-C plasma, and they will be going forward with a donor history and testing strategy for apheresis platelets and the concurrent plasma, which is a very small part of our plasma.
		We will be looking at history and testing, but, again, for November of next year, not this year.  So that's the strategy we're taking at this point.
		I want to emphasize this change of moving from FFP to FP24.  Right now, we're at about 55 percent FFP, 45 percent FP24.  We have shifted, over the last five years, from 27 percent FP24.
		We are going, by November of this year, to have to go to the majority of our plasma to be FP24.  I want to point out that of our 35 regions, seven regions at this point in time, distribute a 100 percent FP24, including our second largest plasma distributor, which is our Greater Chesapeake and Potomac Region, which is Baltimore and Washington, only distribute FP24 at this point in time.
		So some major institutions, such as Johns Hopkins, have found this perfectly acceptable.
		What about apheresis platelets?  Well, this shows all of our apheresis procedures with platelets, platelet products and platelet plasma products.  We are at about 67 percent male on our apheresis platelet donors at this   point, so we like to think we're already at predominantly male platelets, but clearly, we could like to do something about the female donors here and it is our plan to go to a history and testing strategy for platelets.
		So for apheresis products, for platelets, free storage AquaDose, whole blood-derived platelets have been piloted and the pilot is being successful, and is coming to an end as we speak, so we will be developing that as an alternative to apheresis platelets. 
		As we lose donors, we may have to fill up the gap with AquaDose platelets.  For apheresis platelets, we assume we must test and we assume history will play a role.  But having been involved in the lab study, we really do believe we need this data before we make decisions around what to do, concerning transfusion or pregnancy.  I'm particularly concerned about any data that might suggest that untransfused males or females may have a background of antibody.  If so, would that mean, ultimately, we need to test everybody?  I don't know.  I think this needs to be resolved and REDS II, the lab study is very timely and we will wait for that data before making firm decisions, and I do hope the committee will too.
		In terms of testing, we are faced with the issue that there are no licensed tests available for donor screening, that are licensed for donor screening for HLA or HNA antibodies.
		In fact I'm not aware of any suitable technology for routine screening for neutrophil antibodies on a large scale. We are working with manufacturers for HLA antibodies.  We are aware of at least two that are developing automated systems for this, and that may facilitate the HLA screening.
		But there will be uncertainties.  What is the appropriate screening strategy?  How do we define a relevant positive?  Are low titer, low, verity antibodies relevant or not?
		What's the appropriate donor deferral?  Right now, we intend switching donors that are positive to red cells and fractionated plasma.  We don't intend turning anybody away and we're certainly not going to evoke the wrath of female donors by, in any way, turning them away.  In fact, we're encouraging them to donate as much as possible.
		If you find an antibody-positive unit, what's the appropriate recall or withdrawal strategy?  Again, we don't know.  And final slide, in summary, we do believe, at this point, that prudent measures to reduce patient exposure to alloantibodies may reduce risk and are wise.
		We do recognize that availability issues around AB plasma and apheresis platelets argue against gender labeling on the use of only male products and we want to emphasize that there are uncertainties still around etiology of TRALI, the lack of antibody screening technology and the understanding of specific antibodies, that really does argue against universal history or antibody screening at this time.
		The interventions we're putting forward are only going to intervene for 60 percent of the fatal TRALIs that we've seen, since we are not yet addressing the red cell issue.  So we need to be aware that we can't get to zero, we need to do the best we can with what we've got.  Thank you.  I'm happy to take questions.
		DR. SIEGAL:  Thank you, Dr. Benjamin.  Any questions?
		DR. KLEIN:  Richard, I know for many members of the committee, the concept of FP24 is probably a normal one.  So could you tell us whether there are clinical implications of switching to FP24 from FFP.
		DR. BENJAMIN:  FP24 essentially--let's first understand what the difference is.  To make FFP, you have to get the blood back from a drive and frozen within eight hours, which means that if you're running a blood drive, you have to shuttle the blood back to your manufacturing center, and you have to have people waiting on that box of blood to process it immediately and freeze it.  There are costs involved in that.
		Blood centers prefer to wait till the end of the drive, get the whole box of blood back into the second or third shift of the day, and process everything in one go, and generally the blood is frozen within 16 hours of collection.
		We know from studies, that the only factor that is decreased in FP24 is Factor VIII.  From the time of collection to eight hours, you lose about 20 percent of your Factor VIII.  By 24 hours, you lose about another 17 percent.
		So there's about a 17 percent difference in Factor VIII levels between FFP and FP24.  We still have greater than 60 percent Factor VIII.  According to the circle of information, they are used interchangeably, except for the replacement of Factor V and Factor VIII.  Well, we don't use plasma to replace Factor VIII, and I think the Factor V description is antiquated.  It's quite clear that there's no difference in Factor V levels in FFP and FP24. 
		Many centers use 24-hour plasma interchangeably with FFP, and some cities, as I said, Baltimore, are at a 100 percent and see no difference at this point in time.
		DR. KATZ:  I think there's one other thing, particularly for the clinicians to understand, that some of us were waiting till January to switch to FP24, which my center has done, because we were waiting for the data on Adams PS and TTP.  For many of us, the really clear indication for plasma transfusion has been TTP. 
		The confirmation that Adams PS13 levels are preserved in FP24 was critical.
		DR. SIEGAL:  Okay.  Is there a comment in the back?
		DR. SARODE:  At what temperature do you store whole blood when you're transporting for FP?  Is it 4 degrees?
		DR. BENJAMIN:  Excuse me?
		DR. SARODE:  Whole blood when you're transporting from collection site to the blood center.
		DR. BENJAMIN:  We are required to place the blood in a container, so that it moves progressively towards storage temperatures which are, you know, four to ten.  So it's placed on ice.  How long the blood actually takes to get down to 4 degrees, I don't know that.
		DR. SARODE:  So are you going to collect pooled platelets from that particular product, because for that you cannot keep your whole blood on ice.
		DR. BENJAMIN:  Right.  So if you're making random whole blood-derived platelets, you'll have to treat them differently.  You absolutely have to shuttle those back.  But then you're using some of the plasma to make the random go in a platelet.  So that's not the ideal product from to make FFP.
		DR. SARODE:  So basically you'll get two products out of whole blood.
		DR. BENJAMIN:  You are, but you're taking some of your plasma for the random donor platelet.
		DR. SARODE:  And your comment about decreasing Factor VIII, I mean, we had done the study that showed that Factor VIII is the only factor which reduces for, you know, 4 degrees, significantly.  Factor V is stable for up to five days.  So it's not an issue.
		And most of these patients who need, actually, plasma, they have very high levels of Factor VIII because it's an acute phase reactant.  So all these questions with cirrhosis and all, they have Factor VIII, 200 and 300 percent.  So that's how we convince our clinician to use plasma.
		DR. BENJAMIN:  Thank you for the slide you showed this morning of a number of patients that had such high Factor VIII levels.  I will be asking your permission to use that slide because it's a wonderful demonstration that Factor VIII is an acute phase	 reactant, that it goes up in ill patients, it doesn't go down.
		DR. WILLIAMS:  A comment and a question.  The comment is I believe that most of the plasma frozen within 24 hours, that was being referred to as whole blood-derived, which is mentioned in the circular of information, can also be prepared from automated or apheresis procedures, but this currently is not a licensed product, and we encourage submission of data to help validate this, and have it become a licensed product in the future.
		The question is looking at the differences in the incidence of TRALI between single donor platelets and plasma, where the amount of plasma is similar, most likely the gender differences play a role, but I don't have a good sense for the deferral of donors with prior, implication in prior TRALI cases, given that the apheresis platelet donors are a very multi repeat donor population.
		Could that possibly be a factor as well, or is it probably too small?
		DR. BENJAMIN:  I haven't looked at the data but my gut feeling is it's such a small number, that it would have no impact.
		DR. WEINSTEIN:  I was wondering, do you know if the von Willebrand factor is also affected by being in contact with cells for up to 24 hours? 
		As I understand it, you do not separate the plasma from cellular material, potentially up to 24 hours.  It's not only the Factor VIII that can be affected.   It's also von Willebrand factor multimers, and this can potentially have an effect on the manufactured product that you're making from this material.
		So has that been examined?
		DR. BENJAMIN:  I'd have to go back and see which assays they actually used in the published work, whether they were actually measuring Factor VIII or von Willebrand, but I'm assuming that it would go down together with Factor VIII.
		DR. WEINSTEIN:  [off-mike] and affecting multimer size, in particular  You may find that that's a factor.
		DR. EPSTEIN:  Thank you, Dr. Benjamin.  You mentioned the potential use of screening for HLA or HNA antibodies for the subset of donors for whom you need an apheresis platelet, and you flagged that you might make that decision, or at least look at it based on the result of the REDS II LAPS study.
		So this is really a question for Steve Kleinman, which is in that study, if I understood it correctly, you're only going to be looking for the anti-neutrophil antibodies in subjects who already have a positive HLA.
		And those two things may not correlate very well, and based on what we heard from Dr. Stroncek, the "bad actor" is more likely to be the anti-neutrophil antibody.
		So my question to you is whether there's been any consideration within REDS of adding testing for anti-neutrophil antibodies and, again, I would mention that Sheryl Kochman pointed out that FDA has cleared at least one assay for HNA antibodies, including HNA 1, 2 and 3.  So there's at least one approved assay that could be used and I'm sure there are other experimental assays.
		So I'm just concerned that we "might be missing the boat," you know, that we're going to learn a lot about HLA from REDS II LAPS, but it's relevance to TRALI might be constrained for lack of studying the anti-neutrophil antibody, and then given the Red Cross interest, you know, maybe there's role for cooperation.
		DR. KLEINMAN:  Yes.  Jay, just a couple comments on that.  That is really a question of active debate within the REDS group and it really just, it comes down to money.  You know, with the budget--and I don't know if the NHLBI people are still here, but if they are, that'd be great, because the budget doesn't allow testing for as much neutrophil antibody as we would like to do.
		And so the original plan was really based on two issues, to only screen a portion of the subjects for neutrophil antibody, was based on the fact that it's a fairly low throughout assay, so logistics, but it would just take more time, and then it was based on cost, and I think that we have also evolved to the idea that neutrophil antibody testing is probably more important than we might have thought originally.
		The original plan, when we--the way we solved this problem in the original planning, when we realized we couldn't afford it, was we said we would put down this repository, and then as techniques got better, you know, we might not be able to do this in the same timeframe as HLA.  We could access the repository.  So I agree with you.  I think it would be helpful if we could screen the whole repository.
		The reason we decided to start with the HLA antibody positives is based on the supposition that women who form, or men who form HLA antibodies would be better immune reactors and we might see a higher neutrophil antibody rate in those people versus people without HLA antibodies.
		I think there's some data to show that's true but I don't think it's really that solid.  And then if we found a very low rate of neutrophil antibodies in people with HLA, well, based on that rate we could decide what our yield would be from the non-HLA positives and then decide if it was worth it.
		But I totally agree, I actually agree, that I think if we could screen more people for HNA, that would be a big contribution.
		DR. SIEGAL:  Okay.  Thank you very much.  Dr. Bianco will speak next, representing the America's Blood Centers experience with TRALI.
		DR. BIANCO:  Thank you very much for the opportunity to be part of this discussion.
		I want to raise a few points with limited amount of data, but try to add some perspectives to the discussion that we are having today.  First is I think that we should think a little bit about what triggered this meeting, these discussions, and all that, and essentially all of us are trying to comply with an ABB set of bulletins and standards that have asked that we address the issue of TRALI, and obviously based on fatality data from FDA, but--and other data that I'm going to discuss a little bit.
		But even if these are not really standards from the ABB and there is no regulatory mandate, there is a consensus, I think, among all of us in transfusion medicine, that we should be doing something about it.
		The other important thing was the experience in the United Kingdom, and we didn't talk much about it, but since the end of 2003, based on data that they obtained in their Serious Hazards Of Transfusion, or SHOT, there is a hemovigilance system that was created in England and spread to the U.K. and all of Europe, today, and the system is a voluntary system that has about ten years of experience at this point.
		They decided to use mostly male plasma for transfusion in recipients, in an attempt to reduce the incidence of TRALI.  But they have special issues regarding plasma, that are very different from ours.  First of all, they don't use their plasma for fractionation.  They only use plasma for transfusion and not for all the transfused patients.
		For instance, people, patients that are 16 years old, or younger, will receive plasma that has been imported from the United States and actually has been treated by methylene blue, and because of their concern with variant CDJ.
		These slides were loaned to me, nicely, by the National Blood Service, and I just wanted to emphasize a couple of things from the SHOT data.  First of all, we are making an effort to move to mostly male plasma, or predominantly male plasma, as written in the briefing document.  But if we look at what the Brits have done after essentially three years of experience, is that they have kept in the yellow line, more or less their distribution of plasma at 90 percent, because even with all the effort and the experience, they have not been able to maintain the supply of AB plasma, if they don't work at the 90 percent.
		They had a substantial reduction in TRALI that has been attributed to plasma.  They have a slightly different definition than we have in terms of TRALI.  We, based on the Canadian's consensus conference, and then in HLBI, we used the six hour timeframe, and when they started all these SHOT studies, they have defined TRALI as pulmonary acute lung injury happening within 24 hours of transfusion.
		This may have lessened the specificity of the definition but according to what they say in many of the discussions we had, I think that the vast majority of the cases are within the six hours.
		And they had, in the last several years, a substantial reduction, from 20 highly- likely cases in 2003, to 10, and to three cases in 2005.
		If you look at the cases, of reported cases of TRALI and the deaths associated with TRALI, again, that over this ten years experience, or nine years experience, there has been a substantial decline in deaths, and while the number of cases reported continues to be about the same, but here, those cases are all combined.  The highly likely, the probably, and the suspected cases.
		So these drove a lot of our thinking today in terms of one intervention that could be used to reduce the incidence of TRALI.  Their results were very encouraging.  They had fewer reports of TRALI following FFP and platelet transfusions, fewer total reports of suspected TRALI, and fewer deaths.
		However, there are many issues that we should be aware, as we analyze, as we interpret these data.  First of all, it's a passive reporting system.  There are no denominators in the SHOT data.  Second, they have focused their definitions, they have done antibody studies and the "highly likely" cases that they call are cases where they identify an antibody through HLA antigens.
		And mostly to HLA, not to much to neutrophil antigens. 
		The other bullet is that there has been a substantial decline with a lot of education and a big effort placed by the National Blood Service, to rationalize utilization, and there has been a reduction of about 15 percent in the use of blood products in the last five years.  The 15 percent was measured in terms of red cells but there was a reduction of plasma.  I'm not sure about the amount in the reduction in plasma.
		A substantial number of cases continue to be reported, and also, with all the discussion about TRALI, the clinician awareness has increased, the diagnosis is made earlier and the interventions in terms of approaching the patient have been better in recent times.
		Obviously, we had a very healthy discussion, and particularly because of the data, or the studies that Steve just presented to us, but we still have a lot of unresolved questions in terms of antibodies that we would like to raise.  The actual proportion of TRALI reactions. 
		It appears from the data presented by Dr. Benjamin, that about 60 percent of the reactions that he was reporting, were associated with males and antibodies, but we don't know exactly what is this proportion.  We are still discussing assays, cutoff, and more importantly, if we go to some screening program, what antibodies and what amounts of antibodies have clinical significance?
		If we are talking about 25 percent of multiparous women having antibodies to HLA, we didn't see that high number of TRALI reactions that could be attributed to that, and actually, there are some studies, in more recent times, and I did the references there, that showed that a lot of the TRALI, a lot of it, the products with antibodies to HLA, when transfused, will not necessarily induce TRALI even when there is a cognate antigen in the HLA system.
		We talked a little bit about the biologic response modifiers, but, again, yes, there may be a combination of both events, the antibody and the biologic response modifier, but we don't know what is the proportion, really, in which the biologic response modifier plays a role versus the HLA.
		With the approaches, the interventions like the male plasma, we are looking more at the HLA antibody and neutrophil antibody than to the biological response modifier.
		We also talked a little bit about donor-related questions and issues.  The questioning about pregnancies, miscarriages, abortions, and the importance that each one of these events have in triggering the antibody response, how we would question donors, how we would approach these as subsequent donations.  We have issues of donor notification that also were raised very appropriately.
		We all know that if we defer a donor, for instance, for being implicated in cases of TRALI, something like that, that the donor must be notified.
		But do we need to notify donors if we, for instance, start using mostly male plasma?  Do we have to tell people?  Do we need to tell donors that we need the antibody testing for HLA, when there is no significance to their health?
		And do we have to notify an antibody-positive donor if we are not deferring a donor?
		There are many issues that would affect timeline for implementation.  Dr. Benjamin discussed them, in detail, so I'll go very quickly, that is, in a short period of time, interventions like moistly male plasma increase proportion of the plasma, 24 hour frozen plasma, and platelets from whole blood, pooled platelets, probably can be implemented.
		Changes in donor mix, in terms of proportion of male versus female donors, for instance, in apheresis, and other procedures, will be a much more difficult role to achieve.
		Knowing that I was going to be here, I asked ABC members through a survey, a little bit about their plans on how they intended to response to the ABB bulletins.
		And here, I asked what were they considering in terms of moving female plasma to recovered plasma, and moving to 24-hour plasma.  And I saw that probably by the end of this year, 90 percent of the members of ABC plan to have predominantly male plasma implemented at different times, and about half of them were planning to increase, substantially, the proportion of 24-hour plasma versus FFP.
		I think that there is still a concern that the customers, the hospitals, may not receive the--since most of the plasma that has been distributed by ABC members is fresh frozen plasma, there is still some concern that hospitals and physicians may not accept it without a good, extensive educational program.
		What their members in smaller numbers are considering, and because of the many issues that we discussed here, is the use of antibodies to, either in a selective mode, or for certain populations, to detect antibodies to HLA, or to ask specific questions of donors, because of more difficult issues that they will have to do.
		This will happen more slowly and there is a lot of concern about the technologies available, automation, and even issues of cutoff that were well-discussed a few minutes ago.
		And finally, in terms of how to deal with the changes in platelets, many of the centers plan to supplement some of the apheresis platelets with whole blood platelets, but again, there is more of a tendency of looking to some intervention with the apheresis platelet donor, that would allow many of them to continue donating, if they are shown to be safer donors.
		And this is consideration for antibodies and deferrals.  Members are considering mostly to look for an antibody assay that would be feasible, and that would allow the selection of the apheresis donors.
		I'm coming to the end and I wanted to express some of the concerns of the members.  When TRALI became a part of the agenda of BPAC, obviously they all woke up and were concerned about what they thought that would be premature regulatory actions that may remove flexibility in TRALI risk reduction measures.  Strict approaches.  We know that they will not eliminate TRALI and will affect product availability.
		Suppose that we had a decision that we should only use male plasma, for instance, and certainly that's not the tone of the discussion here today.  The other thing that concerns a lot our members, and ourselves, is that we are creating somewhat unrealistic expectations, with all the talk and all the effort that we are applying to TRALI, that we are going to substantially reduce or eliminate it.
		The British saw a reduction of 30 percent between 2004 and 2005, and we forgot, over the years, the experience that we had with the ALT test, for instance.
		When we introduced ALT in the '80s, we didn't expect more than a reduction of 20 to 30 percent in non-A, non-B hepatitis, and we thought that this was a great thing then, an achievement, and we were able to talk about it.
		Today, when we implement regulatory measures, we think about the last case that we want to prevent with either a question that there's no way out, that a person has to say yes or no, or if we do the 120 days for West Nile virus deferral, if the person has--because we are looking--or in the malaria question, because we are looking to an absolutely black and white.  And I think that it would be wise, at least in the case of TRALI, to think again that partial measures are going to get us there.
		We have a wish list, and the wish list, some of it is happening as more research.  We need help from both manufacturers and from FDA in terms of computer software.  We need faster changes, because we need logistics to be able to manage these donors and products and those changes.
		Most of the computer systems available in the news today do not link the release of a unit to the gender of the donor.
		Yes, we can use simplified systems like Dr. Benjamin proposed, or an M or an F, but we need systems that are more precise.
		For the practicing physicians we need more education, we need clearer definitions of TRALI and TACO, which is a picture that is still confusing for many of them.
		We need better assays for antibodies to HLA, and to neutrophils.  We need practical assays for detection of biological response modifiers.  There's one lab in the country that does it.
		We need practical approaches to reduction of biological response modifiers.  We need clearly regulatory definition.  We heard, I was glad to hear from Dr. Williams that this is under consideration by the Agency, and we need also some funding for hemovigilance systems, and so that we can involve more, hospital involvement in hemovigilance so that we can measure the impact of what we do.  I thank you very much.
		DR. SIEGAL:  Thank you, Dr. Bianco.  Are there any questions?
		DR. EDWARDS:  Maybe it's just that I'm hungry but what's TACO?  Willarda Edwards.
		DR. BIANCO:  Transfusion Associated Circulatory Overload.  There's too much fluid leading to the--
		DR. EDWARDS:  Thank you.
		DR. KLEINMAN:  Steve Kleinman.  I just wanted to make a couple comments on Celso's presentation, and for the committee, but not from the perspective of REDS II but in my role as senior medical adviser to ABB, and so I wanted to make a couple of comments about the ABB bulletin.
		And the first comment is there are three recommendations in that bulletin, not just one.  Everybody is focused on the one recommendation which says we need to find ways to change the transfused component to minimize risk.
		But I can tell you, the committee that formulated that bulletin felt just as strongly, if not more strongly, about the second recommendation in the bulletin, and that is we need to get clinicians to use these products appropriately and only to transfuse when indicated by evidence-based medical guidelines as was mentioned by Dr. Sarode earlier.
		And I was very encouraged to see how much he could modify the use of FFP in his hospital.
		Now the reason we didn't make that the primary recommendation is because it's kind of a motherhood recommendation.  The ABB and Red Cross and ABC has been saying this for years.  We need to get clinicians to minimize the use of unnecessary transfusion.  Unfortunately, we haven't been successful at doing that nationwide, and that's why we felt we needed to come up with some other recommendation, we couldn't rely on that.  But I really want this committee to also consider this as an action.  
		I don't know that FDA can have any role in minimizing inappropriate transfusion, but I think it's very important that we, as a community, recognize that that's a prevention mechanism, not just for TRALI, but for all adverse transfusion reactions.
		And so one of my comments on both Celso and Richard's presentation is that as their centers go out and try to educate the community that FB24 is equivalent to FFP, please include in your educational efforts that most of the times you transfuse FFP, you don't need to do it, and we have an alternative product you can use, but you know what?  You don't really need it.
		And I don't know if we can get that into the educational message, but I think it's a very important one.  The second point that I wanted to--and then the third recommendation, which is difficult to put into practice, is we need to be able to monitor what's happening with TRALI as we take these interventions, and that of course in order to monitor cases of TRALI, we need good clinical recognition, good reporting to the blood bank, good reporting to the blood center, and that's a whole separate topic, but, you know, we shouldn't lose sight of the fact that that's important.  Otherwise, we don't know if what we did really had a positive effect.
		The other issue I wanted to address is why the ABB came out with a recommendation to say that we should try to do something in terms of modifying our components, and I think it's all been alluded to but it's worth saying again, that we had to make a decision based on the best data that's out there, and the data that was out there was a combination of what you've heard today.
		The SHOT experience, which suggests, strongly suggests, I think, that making this change affects the incidence of TRALI, the Red Cross experience that you heard Dr. Benjamin present, which says it looks like if they were not transfusing female plasma, they would have prevented cases of mortality, the FDA data that says there's really a lot of mortality out there, and then one additional item that wasn't addressed today, and that is if you look at the clinical series of cases of nonantibody-mediated TRALI, it seems to be a much more mild condition.  Many fewer patients go on to mechanical ventilation and the fatality rate is essentially zero from nonantibody-mediated TRALI, whereas for antibody-mediated TRALI, a high rate of mechanical ventilation and, you know, maybe a 10 percent mortality.
		So we recognize we're not going to--TRALI is multifactorial and these interventions are not going to prevent TRALI that comes from biological response modifiers, but if, in fact, what we think is true, turns out to be true, and that is that the more severe cases are from antibody-mediated TRALI, that's another reason to take a step now and recognize that we can only solve part of the problem, not all of the problem, but that as Celso said, it's a significant part of the problem and if we can reduce TRALI, the severe cases, by 20, 30, 50 percent, that's a realistic goal and we should take it.
		So that's the reasoning that went behind the ABB recommendations.
		DR. SIEGAL:  Thank you, Steve.
		DR. BIANCO:  I added a couple of slides, at the end, that I didn't talk about, that were slides about use and misuse of blood products, and a few references, just to encourage people to think about it just following your recommendation.
		DR. SIEGAL:  Okay.  I think the hour is late.  Dr. Epstein.
		DR. EPSTEIN:  A quick question for Celso and then a quick question for Dr. Stroncek.
		For Celso, you showed on one of your graphs, that centers expecting to consider selection of products and donors would reach 90 percent by fourth quarter of 07.  Are we talking about predominance of male plasma?
		DR. BIANCO:  Yes.
		DR. EPSTEIN:  So we'll have essentially a uniform system in the country by fourth quarter 07, at least--
		DR. BIANCO:  That's the hope.  They are trying to comply with the ABB recommendation that was for November, actually.
		DR. EPSTEIN:  Right.  Thank you.
		And my question for David.  In reviewing the available technologies, I was struck by these are all detection methods, and I was wondering if anybody's looking at a compatibility type method, in other words, where you would, you know, mix donor serum with recipient cells and look for some kind of reaction, presumably something related to agglutination.  Is that feasible?  Is that thinkable?  Is anybody working in that direction?  Because it would have the advantage of being testing done at the time of release in the transfusion service and wouldn't require a lot of prior typing work.
		DR. STRONCEK:  Nobody that I know of is thinking about that.  One of the issues would be you're looking at--part of the problem is like with red cells, the plasma is diluted, and then you'd have to use cellular isolation methods.  Or leukocyte isolation methods.  But there are reagents that are available, that you can isolate leukocytes fairly quickly.  So I guess it's feasible.  People just haven't really thought about doing that.
		That might be a way--agglutination assays tend to pick up higher, tighter antibodies, so that might be a way to only screen our products that are more problematic.
		DR. KLEIN:  If I could just comment on that, what goes around comes around, because some 30 years ago NIH did precisely that with platelet transfusions using a leukocyte agglutination assay, sent off thousands--I should indicate that's when the National Cancer Institute was running transfusion of platelets and had a lot of money.
		We got a lot of data on leukocyte agglutination and one of the problem is that certainly by the technologies that are not available, you get a lot of agglutination related to HLA antibodies, and so what happens is that you find a lot of individuals who appear incompatible and yet there's no problem when you transfuse the component.
		DR. SZYMANSKI:  About the U.K. study, I'd like to ask you, those cases that were not prevented by male plasma, what was the reason for the death or other kind of symptoms?  Were they female plasmas?
		DR. BIANCO:  No.  There were some cases where there were HLA antibodies in males and then there is a proportion of cases in which no antibodies were identified and no obvious reason for the TRALI could be determined.
		DR. MANNO:  I wonder, Dr. Benjamin, or Bianco, if there's been any reinvigorated interest in solvent detergent plasma, with the better understanding of the pathophysiology of TRALI, and I mentioned earlier that it wasn't recognized, following the transfusion of SD plasma.
		DR. BIANCO:  Well, there has been a substantial interest in solvent detergent plasma in Europe, and to my knowledge, the manufacturer in Europe, that is Octapharma, is distributing over a million units a year in Europe at the present time, and I know that because some of our members provide that plasma.
		I think that there are other issues that are not directly related to the product itself, that prevent it's manufacture in the United States.  It's more a question of manufacturers and patents than the product itself.
		DR. SIEGAL:  Dr. Kleinman.
		DR. KLEINMAN:  I just wanted to comment again on the question about the U.K.  In my understanding, their continued TRALI cases do not come from high plasma volume components from females.  They primarily come from red cells, and I don't think they had any cases since they implemented their female, their predominantly male plasma program, and since they, a number of those cases in 2004 were still caused by female plasma that was collected and still in inventory.
		But I think since they've actually made the change, they don't have any cases from female plasma containing FFP or buffy coat platelets.
		DR. BIANCO:  Yes, that's correct, Steve, but the numbers are very small.  They went down from six to three.
		DR. SIEGAL:  We're running very late, so we'll have one last comment.
		DR. SARODE:  My comment is about S/D plasma in United States.  I think the way it was prepared is slightly different than European, and we had a lot of thrombotic complications because this particular plasma had diffuse levels of natural coagulants and there was also decreased amount of alpha-20 plasmin, that led to thrombotic complication in patients who were getting a lot of S/D plasma.  So that could be a concern for physicians in United States who use the same product.
		DR. SIEGAL:  It's now time for the open public hearing.
		Is there anyone who wishes to participate in the open public hearing?
		[No response] 
		DR. SIEGAL:  		If there's no one who wishes to speak, we can proceed to the questions for the committee.
		DR. WILLIAMS:  Okay.  So question one for the committee.  Do current scientific data support the concept that the following interventions will reduce the incidence of TRALI?  The first part.  Use of predominantly male plasma for transfusion.  Second.  Nonuse of plasma for transfusion from donors with a history of prior transfusion, and third, selective donor screening for anti-neutrophil or anti-HLA antibodies.
		I was asked if this could be taken as a yes/no question.  I think I'd give a qualified yes response to that.  I think there may be some variants between the responses to those subsections, so if you could vote those individually, and we would like a sense from the committee of the degree of yes-ness or no-ness with respect to their value as preventions.
		The second question is based on available data.  Please comment on the effect on the U.S. plasma supply of the following interventions, and then again use of predominantly male plasma for transfusion, nonuse of plasma for transfusion from donors with a history of prior transfusion, and selective donor screening for anti-neutrophil or anti-HLA antibodies.
		DR. SIEGAL:  Okay.  Do we want to entertain discussion on the first question?
		DR. KATZ:  I had a question for Alan, very quickly.  The circular of information is being revised by an organizational group convened by ABB.  That brings FD24 and FFP into essentially the same set of indications.
		My understanding was that the language had been submitted to FDA and was interested in what is FDA's current thinking about the equivalence or lack, or not, of those two products?
		DR. WILLIAMS:  I can't comment specifically because if that's under review, I think it really hasn't been discussed extensively internally.  I think basically the concept in the prior circular of information, FFP, has been indicated for all plasma therapies, including the labile components, Factor VIII, Factor V.
		Whether these are then being modified to have the same clinical indications between FFP and 8-hour product and a 24-hour product I think would need to be data-based.
		DR. SIEGAL:  Any other comments?
		[No response] 
		DR. SIEGAL:  Then as they say in the Congress, let's have an up or down vote on the first question, which is do we think that scientific data support the concept that the following intervention will reduce the incidence of TRALI, and that's the use of predominantly male plasma for transfusion.
		DR. DI BISCEGLIE:  I think I heard Dr. Williams indicate that he'd be willing to hear a vote on each of the three parts separately.
		DR. SIEGAL:  That's what I'm advocating that we do.  First question.  So do we believe that there is enough evidence for the first question?  All saying yes?  Can I have a show of hands.
		DR. DI BISCEGLIE:  The first question being what?  The first part of the first question?
		DR. SIEGAL:  First part of the first question.  Male donors.
		I'm sorry.  I didn't hear you.
		DR. SZYMANSKI:  Can we comment on that issue before you take yes/no vote?
		DR. SIEGAL:  Yes.  Please, no filibusters.
		DR. SZYMANSKI:  No; okay; okay.  Listen, I think the evidence shows that the male plasma is associated with a lesser amount of TRALI cases.  However, I think there are very many good female donors that should not be eliminated from platelet transfusion, particularly, because otherwise you have enough pheresis platelets who are good donors, and they should be, you know, permitted to keep donating platelets, especially if they have not had a history of transfusion or pregnancy, or if they are totally antibody-negative.  So that's my qualification for that question.
		DR. SIEGAL:  Thank you, Dr. Szymanski.  Any other points?
		DR. FINNEGAN:  I would support that comment in a Texas form, to say the answer is yes but this is similar to using a nuclear weapon for a fire anthill.
		DR. KATZ:  It's actually not.  With the conversion to--if people will accept FP24, this is simple, this is a doable thing, that at least the British experience suggests has observable impact in a short period of time.
		DR. FINNEGAN:  And I agree with that but if you look at it from scientific point of view, what we're doing is we don't understand the problem, there's pathophysiology for this problem, we have only looked at one side of the pathophysiology, we have no idea or very little idea bout the recipient, and the recipient is probably at least a reasonable component of the pathophysiology and we have no data on that.
		So that, in fact, yes, it will solve the problem for now, but, in fact, it's going to have some secondary consequences, some of which we won't be happy about in a year or two, especially if we have a major disaster, and we need some science on the other half of the problem.
		DR. SIEGAL:  So the question is whether we believe there's enough evidence to support that.
		DR. SZYMANSKI:  Can we way support yes, support with qualifications?
		DR. SIEGAL:  Shall we have a show of hands or go around the room, do it person by person?  Let's go around the room.
		DR. RIOS:  So answer to 1A?  Mine is yes.
		MR. JEHN:  Yes.
		Dr. Klein.
		DR. KLEIN:  Yes.
		MR. JEHN:  Dr. Nelson.
		DR. NELSON:  Yes.
		MR. JEHN:  Dr. Schreiber.
		DR. SCHREIBER:  Yes.
		MR. JEHN:  Dr. Szymanski.
		DR. SZYMANSKI:  Yes, with qualifications.
		MR. JEHN:  Dr. Whittaker.
		DR. WHITTAKER:  Yes, predominantly; not exclusively.
		MR. JEHN:  Ms. Baker.
		MS. BAKER: Yes, with the qualifications previously indicated.
		MR. JEHN:  Okay.  Dr. Di Bisceglie?
		DR. DI BISCEGLIE:  Yes.
		MR. JEHN:  Dr. Edwards.
		DR. EDWARDS:  Yes.
		MR. JEHN:  Dr. Finnegan.
		DR. FINNEGAN:  Yes, with qualifications.
		MR. JEHN:  Dr. Kuehnert.
		DR. KUEHNERT:  Yes.
		MR. JEHN:  Dr. Manno.
		DR. MANNO:  Yes.
		MR. JEHN:  Dr. Siegel.
		DR. SIEGEL: Yes.
		MR. JEHN:  Okay.  And Dr. Katz, do you have an opinion?
		DR. KATZ:  No.  Yes, I do.  I have an opinion.
		DR. SIEGAL:  Okay.  Let's move on to the second part of the first question.
		Is there enough evidence that the nonuse of plasma for transfusion from donors with a history of prior transfusion?  Anybody want to speak on this issue?  
		Dr. Finnegan has an opinion.
		DR. FINNEGAN:  I don't think we've seen any evidence that that is in fact a good way to help this problem.
		DR. KUEHNERT:  I was just wondering, I mean, if there may be other things that are more, that have higher relationship to this.  So I mean, I guess that's what bothers me a little, because I'm not sure this is the next strongest risk factor, and so it may have some ability to reduce the incidence but it wouldn't be the most effective risk factor, possibly.  I don't know.
		DR. KLEIN: I think we've seen virtually no data to say that it would reduce the incidence or the frequency, and the data that we've seen related to antibodies suggests that there's no significant increase in antibodies.  So there's virtually no data to support doing anything regarding people who have been previously transfused, regarding TRALI.
		DR. SZYMANSKI:  Besides, usually the transfusion are leuko-reduced, so you don't get leukocyte antibodies that much, I would think.
		DR. SIEGAL:  Are there any other comments?  Then let's go around the room again.  Don.
		MR. JEHN:  Dr. Glynn.
		DR. GLYNN: No.
		MR. JEHN:  Dr. Klein.
		DR. KLEIN:  No.
		MR. JEHN:  Dr. Schreiber.
		DR. SCHREIBER:  No.
		MR. JEHN:  Dr. Nelson.
		DR. NELSON:  No.
		MR. JEHN:  Dr. Szymanski.
		DR. SZYMANSKI:  No.
		MR. JEHN:  Dr. Whittaker.
		DR. WHITTAKER:  No.
		MR. JEHN:  Ms. Baker.
		MS. BAKER:  No.
		MR. JEHN:  Dr. Di Bisceglie
		DR. DI BISCEGLIE:  No.
		MR. JEHN:  Dr. Edwards.
		DR. EDWARDS:  No.
		MR. JEHN:  Dr. Finnegan.
		DR. FINNEGAN:  No.
		MR. JEHN:  Dr. Kuehnert.
		DR. KUEHNERT:  No.
		MR. JEHN:  Dr. Manno.
		DR. MANNO:  No.
		MR. JEHN:  Dr. Siegal.
		DR. SIEGAL:  No.
		MR. JEHN:  Any comments, Dr. Katz?
		DR. KATZ:  No.
		MR. JEHN:  All right. 
		DR. SIEGAL:  Then finally, the third component of question number two, which you can call read.  Selective donor screening for anti-
neutrophil or anti-HLA antibodies.  Comments?
		Dr. Schreiber.
		DR. SCHREIBER:  I think we're just starting to look at some of those relationships and try to understand them.  From what we heard, some of them are a lot more complex than we think.  So I would say that it's just too early to tell.
		DR. SIEGAL:  Anyone else?
		DR. KLEIN:  I would agree with that.  I think the data that we've seen suggests that neutrophil antibodies may well play a role but we've also heard that the tests are not yet really robust, certainly not for screening purposes, and we've seen no data on screening.
		This is a compound question, of course, and we've seen some data on HLA antibodies but none to suggest that it's a helpful technology at this point in time and I suppose those data need to mature.
		DR. SIEGAL:  Okay.  Do we need to go around the room on this one?
		DR. GLYNN:  So could we divide the question or you really want to have the anti-neutrophils with the NG/HLA?  It doesn't matter.
		DR. SIEGAL:  I think we probably have a consensus on that one without actually taking a vote.  Do you agree?  Can we get by without that?  Okay.
		Dr. Szymanski.
		DR. SZYMANSKI:  I think it's a good idea, but I think we need more data, more information as to what antibodies to screen, and what methodologies to use.  But I think it is basically good idea.
		DR. SIEGAL:  Presumably we'll get those data, eventually, certainly from the REDS study about HLA.  Okay.  Shall we go on to question two?
		DR. WILLIAMS:  So question two has to do on the impact on supply.  Based upon available data, please comment on the effect on the U.S. plasma supply of the following interventions.  And they're the same three interventions.  I think we'd probably also welcome any comments related to apheresis platelets in the same thing.
		DR. SIEGAL:  All right.  We're going to take this one section at a time.  Okay.  Comments on question one, 2A?
		DR. KATZ:  Well speaking as a collection facility person, a representative of the industry, handled appropriately the male plasma is eminently doable.  The cell that is required to hospitals and clinicians, FP24 versus FFP, has turned out, at least in my experience with 55 hospitals, to be much easier than I ever thought.  I was certain the surgeons would tell me, when my patients start bleeding FP24 I'll transfuse it.
		And they haven't.  And the data, the in vitro data, it's not clinical data, it isn't a clinical trial of FFP versus FP24 for a variety of complex coagulation disorders.
		But the in vitro data says the level are maintained and to the degree that we know what FFP does, FP24 should be equivalent for almost all indications, and so I think as long as we have the ability to use FP24, where required logistically, it should not have an important impact.
		DR. SIEGAL:  Anyone else?  Harvey.
		DR. KLEIN:  I would agree with that and I think in some extent it's moot, because I think most of the U.S. plasma will be predominantly male by the time anyone gets around to making any kind of recommendation, in addition to which I think that like whole blood and red cells, the education will not be all that difficult.  We don't use whole blood anymore, for very good reasons, and I think we'll probably use FP24 predominantly for equally good reasons.
		DR. SIEGAL:  Dr. Szymanski.
		DR. SZYMANSKI:  I think it will affect the platelet availability.
		DR. SIEGAL:  Dr. Schreiber.
		DR. SCHREIBER:  I'm not convinced you can separate the first and the second parts.  I agree that it's probably adequate now, but if you do away with transfused males and you drop somewhere between, I think the estimate is 4 to 7 percent of the population, I think we'd have a tough time filling all of the plasma needs with females.
		DR. GLYNN:  But George, it's predominant, it's not all.  I mean, that's a big difference.
		DR. NELSON:  I think the word "predominant" is key here, because if there's a shortage, critical shortage, it means that it can be filled with a female donor.
		DR. DI BISCEGLIE:  If I might comment, another factor that affects availability, we've already touched on, which is use or misuse.  As a hepatologist taking care of patients with liver disease, who have coagulopathies, I'm quite comfortable with the strategy outlined of not misusing plasma.  My problem is my colleagues.
		If I want to get somebody to operate on one of my patients, or a cardiologist to do a heart catheterization, they won't do it unless I fix the prothrombin time.  And so what I'd really like is if there are guidelines widely promulgated, that I can point to, to tell them to back off.
		DR. KATZ:  The Clinical Transfusion Medicine Committee at ABB has started the process of producing a definitive guideline for the use of plasma for transfusion.  It's an extraordinarily complex process as I know you're aware, so I would guess the output's, I don't know, a year away or what; but it's going to be a while.
		DR. SIEGAL:  Any other comments?
		All right.  Let's go around the room again.  I think on one and two, if that's agreeable to people, since we've discussed them both.
		MR. JEHN:  Dr. Glynn.
		DR. GLYNN:  I'm sorry.  We have to vote on this?  Exactly what kind of vote?
		DR. SIEGAL:  All right.  So we don't have to vote.  Okay; fine.  So then we've commented.
		So let's go to question three, or 2C.  Again, it's the selective donor screening for antibodies.
		DR. FINNEGAN:  I think once the technology's there, that's going to be the answer for helping solve the problem, but I still reiterate that we need to figure out what the recipient's problem is as well as what the donor's problem is.
		DR. KLEIN: I think that the data are clearly too immature to make much more comment than to say if one wanted to screen for anti-HLA antibodies, it would be a very big problem in terms of the availability of plasma.  So again, you could divide those, and say for anti-neutrophil, that you probably can't do it, but it wouldn't be a big problem for HLA antibodies. With the current technology you could probably do it.  You wouldn't get much benefit and you'd lose a lot of plasma. 
		DR. SIEGAL:  All right.  Dr. Schreiber.
		DR. SCHREIBER:  Actually, I think that if we had adequate technology, we might be adding back a significant portion of the women.  As Steve Kleinman said, if we're running about 30 percent, or so, elevated HLAs, and that's for the whole population, and we're getting rid of 50 percent of the donors that are women for the plasma, we might have a net gain of 25 percent.
		DR. KLEIN: I think you're assuming that HLA is actually related, and I would point out that in the large study that was quoted by 
Palthey, et al, it was predominantly women who'd have multiple pregnancies; it was multiparous women.  And they didn't look for antibodies.  So I'm not sure that that's what we want to hang our hat on.  I'd like to wait for the data on that one.
		Maybe we're looking at the wrong antibodies by the particular technology that we're using.
		DR. SCHREIBER:  I agree.  I think we really need the data, but I think we might be pleasantly surprised.
		DR. SIEGAL:  Are there any other comments?
		[No response] 
		DR. SIEGAL:  In that case let's declare this session over and we'll reconvene in 45 minutes.  Is that agreeable to everyone?
		[Whereupon, a luncheon recess was taken from 12:33 p.m. to 1:18 p.m.]

	A F T E R N O O N  S E S S I O N
		DR. SIEGAL:  Okay.  Let's start.  Do we have a quorum?  Probably not.  Okay.  Topic three.  Issues related to implementation of West Nile virus testing.  We're first going to have an update on West Nile epidemic 2006, by Dr. Eileen Farnon from the Centers For Disease Control and Prevention.
		DR. FARNON:  Good afternoon and thank you for inviting me to speak today.  I'll be presenting an update on West Nile virus epidemiology in the United States from 1999 to 2006.
		West Nile virus is a mosquito-borne flavivirus that is transmitted in an enzootic cycle between bird and mosquitoes.
		It causes epizootics in which birds and mammals are affected by bridge mosquitoes.  Horses and humans are usually considered "dead end" hosts because they don't develop a high enough viremia to infect mosquitoes, and thereby they don't perpetuate the cycle.
		This maps shows the approximate global distribution of West Nile virus.  Prior to 1999, West Nile virus was found predominantly in parts of Africa, Asia and Europe, with the closely related Kunjin virus occurring in Australia.
		In 1999, West Nile virus was introduced into North America and since then has been found in parts of Central and South America.
		Human disease has been detected in the Cayman Islands, Mexico, El Salvador and Argentina.
		CDC developed ArboNET, the U.S. arboviral national electronic surveillance system, in 2000, in response to the 1999 detection of West Nile virus in the U.S., in New York City.
		ArboNET is a passive surveillance system that collects data on West Nile virus and other domestic arboviral infections in humans, as well as ecologic data regarding infected mosquitoes, birds and other animals.
		The information that I'll cover today is based on reports to ArboNET and all the data are current as of April 12th, 2007.
		The human West Nile virus syndromes reported to ArboNET include West Nile fever, West Nile neuroinvasive disease, including meningitis, encephalitis, and acute flaccid paralysis, and other clinical syndromes, as well as unspecified illness.
		This series of maps shows West Nile neuroinvasive disease incidence by county in the U.S. from 1999 to 2006.
		Neuroinvasive disease incidence is thought to reflect the burden of human West Nile virus disease more accurately than total case counts since West Nile fever reporting varies widely from state to state.
		Counties highlighted in green on these maps had ecologic activity in mosquitoes, birds, or animals, and the dots represent incidence per million of West Nile neuroinvasive disease in humans.
		In 1999, West Nile virus was first recognized in the U.S. in the outbreak in New York City.  In 2000, there were cases found in neighboring states.  In 2001, West Nile neuroinvasive disease incidents increased in the Northeast and spread to the Southeast.
		In 2002, high West Nile neuroinvasive disease incidence was seen in Central, South-Central and Western Plains states.  In 2003, the highest incidence was seen in the Western Plains states.
		The incidence in 2004 was lower overall but continued to be high in the South-Central, Central and Western states, and increased in the Southeast and along the West Coast.
		In 2005, incidence increased in the South-Central, Central and Western Plains states.
		In 2006, incidence increased again and remained highest in the South-Central, Central, and Western states.
		This map shows the cumulative West Nile virus activity and human cases by county, from 1999 to 2006.  Counties that had nonhuman West Nile virus activity are depicted in blue and counties with human cases are depicted in red.
		All of the Lower 48 states have had evidence of ecologic West Nile virus activity and only Maine, Hawaii and Alaska remain free of human cases.
		Since 1999, almost all counties in the Lower 48 state have reported some West Nile virus activity, sparing only a few counties which are mostly in the Northwest.
		The onset of human West Nile virus cases in the U.S. has changed from 1999 to 2006.  The West Nile virus season has steadily lengthened, and in 2005, and 2006, the dates of onset of human disease span almost the entire year, from early January until mid December, with states in the South having the longest West Nile virus season.
		We wanted to see whether some states have had persistently high incidence of West Nile neuroinvasive disease over the past five years, to see whether there might be "hot spots" of West Nile virus activity in humans.
		This map shows the cumulative incidence of West Nile neuroinvasive disease by county in the U.S. from 2002 to 2006, with the red counties having the highest cumulative incidence and the letter color counties have lower cumulative incidence.
		Harding County, South Dakota, has the highest cumulative incidence of 241 per 100,000.  Twenty-five counties in six Western states have a cumulative West Nile neuroinvasive disease incidence of over 100 per 100,000.
		Nicole Lindsey, in our group at CDC, recently examined how many of the counties with high cumulative West Nile neuroinvasive disease incidence also had consistently high annual rates of West Nile neuroinvasive disease incidence, and therefore might be "hot spots" where West Nile virus activity may continue to be high in the future.
		This is a scatterplot that compares the median annual West Nile neuroinvasive disease incidence rank with the cumulative West Nile neuroinvasive disease incidence rank for each country from 2002 to 2006, in order to determine whether counties with high cumulative incidence had persistently high annual incidence in the five years from 2002 to 2006.
		Since some counties have very few West Nile neuroinvasive disease cases and small populations, Nicole limited the analysis of county incidence to 447 counties where the cumulative incidence rate was, where the 95 percent confidence interval for the cumulative incidence rate was more than zero.
		This plot shows that most counties did not have consistently high annual incidence.  There were only a few counties that were "hot spots," shown in red, orange and light blue, with high cumulative incidence and persistently high annual incidence.  Most counties with high cumulative incidence had only one or two years with high annual incidence, which can be seen at the end of the x axis.  Over there.
		This analysis also indicates that counties that have had low incidence of West Nile virus transmission, in dark blue, over here, tend to maintain low transmission rates.
		Looked at geographically, this map shows the "hot spot" counties with persistently high West Nile neuroinvasive disease incidence from 2002 to 2006.  The counties in the top 5 percent of both cumulative and median annual incidence for West Nile neuroinvasive disease are shown in red, the ones in the top 10 percent in orange, and the ones in the top 20 percent in light blue.
		These "hot spot" counties cluster in the Western Plains states and at the eastern edge of the Rocky Mountains, with a few in the South-Central states.
		This chart summarizes the total reported West Nile disease cases in humans in the U.S. from 1999 to 2006.
		The columns show the total number of cases, West Nile neuroinvasive disease cases, West Nile fever or other cases, and deaths, and the rows show the year.
		The number of human cases reported to ArboNET peaked in 2003, declined by almost a fourth in 2004, and have been increasing again, for a total of 4,261 human cases reported in 2006 to ArboNET, and almost 24,000 cases, to date, since 1999.
		The need for West Nile virus blood screening became apparent in 2002, when 23 cases of West Nile virus transfusion-associated transmission, or TAT, were documented.  In 2003, FDA initiated screening of the blood supply with nucleic acid amplification test or NAT.  Mini-pool NAT or MP-NAT is done on pools of samples of six to sixteen units, and units from positive units are then tested individually by individual NAT or ID-NAT.  Blood banks report presumptively viremic donors or PVDs, to local health departments and remove infection blood products from circulation. 
		Public health departments then report these cases to ArboNET, perform clinical follow-up on the cases, and perform traceback investigations along with partners including blood banks, tissue banks, organ procurement organizations, and FDA, as appropriate, in order to identify other potential cases infected by the same donors.
		This chart shows the number of West Nile virus presumptively viremic donors by year.  In 2003, after screening of the blood supply for West Nile virus began, 818 donors were identified, followed by 224 in 2004, 417 in 2005, and 361 in 2006.  A total of 1820 PVDs have been identified by this system over the past four years.
		This map shows the number of presumptively viremic blood donors by their state of residence in 2006.  These numbers correlate fairly well with the states that had the highest incidence of neuroinvasive disease cases in 2006.
		Over 8 million blood donations were screened for West Nile virus in 2006.  Of the 361 PVDs reported to ArboNET in 2006, 23 percent developed West Nile fever, 0.1 percent developed West Nile neuroinvasive disease, and 0.1 percent developed other unspecified illness from West Nile virus infection.
		These percentages are similar to those seen in West Nile virus serosurveys.
		33 percent of the PVDs reported in 2006 were from three states--Nebraska, Idaho and Texas.  Two suspect cases of West Nile virus transfusion-associated transmission that occurred in 2006 were found to originate from a donor whose multiple NAT screen was negative.
		The blood bank testing his donation had not yet reached their trigger for ID-NAT, although other blood banks had also found PVDs around the time from the same region.
		The first case was an 82-year-old male resident of South Dakota who had recently received a kidney transplant.  At four days, post transplant, he received two units of packed red blood cells.  At 21 days post transplant, he developed encephalitis.  West Nile virus IgM was detected in his serum and CSF.  
		Pre-mortem serum from the organ donor tested negative for West Nile virus by serology and PCR.  The recipient of the organ donor's other kidney was asymptomatic. and his serum, post transplant, tested negative for West Nile virus infection.  The case patient had also received blood products from six different donors before he developed encephalitis and all six donors were tested for West Nile virus infection.
		One of the blood donors was positive for West Nile virus IgM.  He was from rural South Dakota and had been asymptomatic before and after donating blood.
		His sample had tested negative for West Nile virus RNA by multipool NAT.  A traceback investigation of this blood donor revealed that a second immunocompromised patient had also likely been infected by a donation from the same donor.  Fresh frozen plasma from his donation had been transfused into a 60-year-old male resident of Iowa who was five years post-kidney transplant.
		He had undergone a spinal fracture repair in South Dakota and received 15 blood products, including six units of FFP from the West Nile virus infected blood donor.
		Eleven days, post-operatively, he developed encephalomyelitis and West Nile virus IgM was detected in his CSF.
		Both of these cases are considered to be suspect cases of West Nile virus transfusion-associated transmission.  Both had been hospitalized for at least to weeks before the onset of West Nile neuroinvasive disease and were unlikely to have acquired the disease via mosquito bite.  These cases were described in an MMWR article that was published in February, which discussed the issue of how ID-NAT triggering mechanisms differ across the U.S.
		The occurrence of these cases indicates that despite enormous successes in screening the blood supply for West Nile virus, it may be possible to improve on current triggering mechanisms.
		This could be approached by examining how different triggering methods would have worked, using either existing data or using theoretical models of West Nile virus outbreaks.
		In summary, West Nile virus transmission now occurs throughout the continental United States.  States and counties in the Western Plains and the eastern edge of the Rocky Mountains seem to have the highest incidence of West Nile neuroinvasive disease.
		The dates of onset for human West Nile virus disease now span almost the entire year.  Two cases of transfusion-associated transmission occurred despite multipool-NAT testing in a region where PVDs were diagnosed simultaneously by separate labs.
		Because of these cases, new approaches to triggering ID-NAT are being considered in order to further lower the risk of transmitting West Nile virus through the blood supply.
		I would like to thank the following people and organizations for their assistance providing the data I presented today.  Thank you.
		DR. SIEGAL:  Thank you very much.
		Are there any questions for Dr. Farnon?
		DR. NELSON:  I was interested that 23 percent of the people have fever.  Presumably this was fever that occurred after the donation, because they would have been excluded from donating if they had a fever at the time of donation.  Is that correct? 
		DR. FARNON: That's absolutely correct.  They should be excluded, if they had a fever at the time.  I'm not entirely certain about last year's numbers.  I know in the past, there have occasionally, in published reports, been cases of people who donated despite having had a recent history of fever, and so hopefully at this point, all of the cases that subsequently, all of the PVDs subsequently developed fever before or on the day of their donation.  I don't have, unfortunately, that information.
		DR. SIEGAL:  Harvey.
		DR. KLEIN:  Is there any speculation as to why West Nile seems to have settled into the Western states where you showed the prevalence for a couple of years now?
		DR. FARNON:  Right.  Especially in the Western Plains states, there's a lot of irrigation of large plots of land for agriculture, and it's thought that that kind of practice actually is sustaining the epidemic in those areas.  So particularly, say, Nebraska and so on.
		DR. NELSON:  Have vectors been studied as well in that area, because I think, as I understand it, Culex tarsalia is much more of a human pest, biter, human only, and I wonder does this explain it or--
		DR. FARNON:  That is also one theory, that Culex tarsalia is a more aggressive human biter than pipiens in the Northeast, say.  Again this is a theory and we like to latch on to it, but from what I understand from entomologists, it seems more likely that just the agricultural practices and topography of the land, and so on, may be playing more of a factor than the mosquito itself.
		Obviously, there are a number of different factors that play into this and it's complicated to figure out what the reasons are.
		DR. SIEGAL:  Yes, Dr. Szymanski.
		DR. SZYMANSKI:  A question.  How long after infection does the NST test remain positive and when do the antibodies appear?
		DR. FARNON: I'm sure there are people in the audience who could respond to this better than I can.  I believe the NAT test itself can--although live viremia, in terms of virus isolation is thought to last on the window of, say, three to five days, maximum, the NAT test can remain positive for much longer than that, up to I think about two or three weeks.
		DR. RIOS:  It has been found up to 104 days, positive.  104 days.  That's the extreme end of the--but within a month or two will still be positive.
		DR. SIEGAL:  Any other questions?  Thank you very much, Dr. Farnon.
		DR. KLEINMAN:  Will you take some from the audience?
		DR. SIEGAL:  Yes; sure.
		DR. KLEINMAN:  I have two questions.  The early onset cases in the early part of the year, prior to April, can you give us, in the last two years, can you give us some idea of how many cases come on in the first quarter and where they're from and how well-documented they are.
		DR. FARNON:  Sure.  They do tend to be well-documented, because generally speaking, the first cases of the year attract some attention, both at the state health departments and at the CDC, and we believe that they are true cases, confirmed cases.  Mostly in the Southern states are where you will see the first cases of the year, and it's varied from year to year.  One year, the first case was found in LA County and one year it was in Texas.
		So this year, we may be having our first cases now in Mississippi but, generally speaking, there are all these in the South of the U.S.
		DR. KLEINMAN:  Okay.  And my other question.  Have people at CDC speculated as to what they might expect for 2007?
		DR. FARNON:  People like to say in arbovirology that you never really know what to expect.  But that being said, I think that's part of the reason why they wanted to do this study that I presented, that Nicole Lindsey did, looking at "hot spots," and I think the feeling is that the highest incidence areas will remain in the Western Plains states and possibly increase in the West Coast as well.
		But that being said, we all think that West Nile virus is now endemic throughout the U.S., throughout the Lower 48 states.
		DR. SIEGAL:  Okay.  Let's move on.  Thank you.  The next speaker is Maria Rios, PhD, of FDA, speaking on issues for testing.
		Dr. Rios.
		DR. RIOS:  Thank you, and after Eileen's presentation, my presentation will be actually much better, much clearer, I hope, and I will be talking to you about issues related to implementation of blood screening for infection with West Nile virus.
		FDA seeks comments from the committee on issues to implementation of donor screening with NAT for West Nile virus with regards to screening itself, additional testing, and donor counseling, and that's what we would like the committee to discuss.
		As Eileen already posed the background, West Nile was identified as a threat to the blood supply in August 2002, and efforts among DHHS agencies and test kit developers and blood establishments led to the rapid development and implementation of a nationwide screening under FDA-approved IND in July 2003.
		So was nine months, exactly, as she stated, after the initial identification of a threat, West Nile is a threat, and today, this number is higher than what report in CDC because CDC reporting is lagging behind.  It's done through the State Department.  But we know that it's over 2000 units that have been intradicted due to the West Nile reactivity in the United States.
		And that may have led to the prevention between of 2000 and 6000 potential transmission by blood transfusion since the implementation of ID--oh, since the implementation of NAT under IND.
		In this slide, what I wanted to point out to you is that since 2002, there have been increasing number of cases of neurological disease, and as Eileen pointed out, it started very low and it peaked in 2002 to 2003, and since 2002 there has been higher than one thousand cases of neurological invasion of West Nile, and at least a 100 deaths, and more.
		Based on the estimation made by CDC, early estimation that one in a 150 infections will lead to neurological invasive disease, and more recent data published in 2006 with new data collected during this testing period, that one in every 350 infections will end up causing neuroinvasive disease.  We can estimate that it has been in the lower end at 1.5 to 3.5 million infections in the U.S. during the last eight consecutive years.
		So West Nile epidemic has reoccurred, causing almost 24,000 human disease and almost one thousand deaths, as shown by Eileen.  These titers of assays since implementation, FDA license in 2002, the first NAT for volunteer donor screening in its Procleix assay, manufactured by Gen-Probe and distributed by Chiron, and in March 2007, FDA licensed the first fully automated system for volunteer blood donors screening, and it's the Procleix assay running the TIGRIS system manufactured by the same manufacturers and distributions.  Alternate assays are currently under IND. 
		You have seen this, Eileen showed you, and this slide she kindly provided me last year, and it's just to show that from 1999 the awareness grow, growth of awareness, the identification of human cases of West Nile increased.  So did the extent of the epidemic.  So now we have year-round human infections which is sustaining 2006.
		So we know that it's from January of December.  So West Nile became endemic in the U.S. with peak during spring to fall, and it's a reportable disease to the CDC, and since 2002, we have observed that there have been higher than 1000 cases of neuroinvasive disease, meaning encephalitis, meninges encephalitis, and the meningitis and the acute flaccid paralysis, and this makes a parallel with the Japanese encephalitis in the Far East, that has been for many decades causing at least a thousand encephalitis in the Far East.
		And we also have seen that there have been at least a 100 fatalities a year since 2002.
		So it is an issue.  FDA is considering whether or not--whether blood establishments should screen West Nile by minipool NAT year round, because we have seen activity from January to December.  With the  begotten test, in two thousand--this was already mentioned to you--but in 2003, six cases of West Nile transmission after minipool tests and these units were tested as negative by minipool NAT and they were transfused and transmitted the infection.  Retrospectively, studies were performed and voluntarily by blood establishments, and they observed that 75 percent of the infectious units would be detected but 25 percent of units would be undetected.
		That led to the voluntary implementation by the blood centers in 2004 of ID-NAT, which would be used in individual donating testing, which would be used in areas during the peak epidemic with high West Nile activity, that led to additional identification of units that wouldn't be detected in minipool NAT and increase the safety of the blood supply.  And the same was used in 2005.
		So ID-NAT was implemented based on the criteria that the most common was one in a thousand reactive donation, or two minipool reactive or positives a week, whichever would come first during the epidemic.
		You're going to see these models from Dr. Stramer, that will be in the next presentation.  So since selecting ID-NAT, there have been three confirmed cases of West Nile transmission by transfusion.  One was in 2004.  There had been no cases documented in 2005, but I want to remind you that we don't test all the blood recipients.  In West Nile, not a 100 percent of cases will cause symptoms.
		So in the--two cases in 2006.  So FDA is considering, we are considering whether blood establishments should implement ID-NAT in  areas with high West Nile activity and uniform criteria to initiate ID-NAT is desirable, and there is a fully automated system licensed right now.  However, there is a paucity of data to define unifying criteria for search implementation of uniform ID-NAT.
		The AABB has voluntarily recommended they publish the position and it's published in the Bulletin 0702, if anybody wants to take a look at it, and we are considering whether blood establishments should define and validate criteria to trigger ID-NAT and to revert back to minipool NAT when the peak season is passed.
		The current algorithm used is that the blood donations are pooled into six or sixteen minipools, depending on the kit manufacturer, and if the minipool tests negative and the units are suitable for transfusion, then they are released for transfusion.
		If the minipool NAT is reactive, then each one of the units that compose the minipool is tested individually to identify the unit or units, or specimens that led to the pool reactivity.
		Once that you identify that the unit's negative by ID-NAT, if suitable, they are released, and the ones that are reactive, they are discarded, donor-deferred for 120 days, and additional tests are performed with the purpose of counseling.
		These additional tests are repeat the NAT, either using the same NAT that was used for screening or an alternate NAT with greater or equal sensitivity of the screening assay, and to perform West Nile antibody or presence of antibody to West Nile virus.
		I would like you to note that flavivirus family has high rate of cross-reactivity among the various members.
		In studies that soon we will release for you, that has been collected by the blood agency, has shown that a combination of a repeat NAT plus presence of antibody has a positive predictive value of 98 percent and sensitivity of 98 percent, and she will give you details of this.
		So we are considering whether blood establishments should retest donation by ID-NAT, using either the same screening assay or an alternate NAT of equal or higher sensitivity, and test ID-NAT initial reactive donations by antibody to West Nile virus.
		I would like to call your attention again that antibody for West Nile virus can cross-react with other flavivirus and vice-versa.
		With regard to additional test, if a NAT assay is specific for flavivirus but not for West Nile, discriminatory of West Nile, we are considering whether blood establishments should perform West Nile specific discriminatory assay in order to determine West Nile virus infection.
		In the case of additional testing, as I mentioned to you before, the minipool NAT is tested by individual donations testing, and the individual, the reactivity of the individual donations are repeated by ID-NAT using the same alternate assay and antibodies performed.
		If these initial reactive repeat positive, then the specimen is considered positive and the donor is considered positive, regardless of antibody result.
		But if the antibody is present, and both are present, then it's clear that it's antibody to West Nile or at least to the flavivirus and you consider the donor positive.
		However, if the NAT is nonreactive but the antibody's positive, then you would consider positive based on the antibody and the same thing with the antibody negative but not reactive.
		These specimens which are ID-NAT reactive and the repeat, and the repeat NAT is nonreactive, and the antibody are absent, it's considered false positive or meaning through negative.  However, Susan will show you these data again, that 2 percent of the true positive on follow-up are actual, the false positive are actually true positive upon follow-up.
		And overall, close to 10 percent of the initial repeat NAT, that it's nonreactive in the repeat NAT, they are true positive based on antibody test.
		So we are considering whether it is appropriate initial reactivity specimen not be regarded as false positive based solely on negative test results on additional testing in the index donation, regardless of the NAT to antibody solely, or whether it's appropriate for a donor to be considered true positive based on repeat reactive NAT, or West Nile antibody positive.
		And again I would like to call attention that the assays, they will not discriminate between West Nile and other, Japanese encephalitis, other group of virus.  It's not really appropriate to consider these individuals as true positive for West Nile virus.
		Regarding donor counseling, due to the potential for false negative result, it is desirable to inform donors with initial reactive ID-NAT about the possible infection with West Nile virus.  Donors with initial reactive ID-NAT may be counseled and invited for follow-up test performed using ID-NAT and antibody assays, at least 30 days after the initial reactive donation.
		And initial reactive ID-NAT donations may not be released for transfusion and donors should be deferred for 120 days.
		We are considering whether it's appropriate that container label and instruction circular to reflect the results of West Nile NAT, consistent with labeling for infection disease markers, and also West Nile reactivity units not to be shipped or used except as provided in FDA-approved program and/or research, autologous use only.
		And such units should be labeled with appropriate warnings.
		With regard to donor deferral and reentry, product retrieval and recipient notification, we are considering maintaining the recommendation as is stated in the June 2005 guidance, which is in the Web site.  Thank you.
		DR. SIEGAL:  Thank you.  Questions?
		Well, I have a question, just to ask you a couple of things.  The total, the epidemiology of this virus is that it's infected over a million people in the United States.
		DR. RIOS:  I'm sorry.  I didn't hear you.
		DR. SIEGAL:  It's quite prevalent, this virus.  You go outdoors and you get bitten by the wrong mosquito, and you're likely to get infected, and looking at the epidemic spread and the wave-like process that took us across the United States and the very low prevalence now, makes me think probably there's a lot more prevalence to this virus and it actually may have saturated the population to some extent.
		Well, even so, if there are, say, 2 million people infected in this country, how many transmissions have we had by blood transfusion?
		DR. RIOS:  We don't know.  We know confirmed, documented cases.  We don't know how many transmissions there has been, because not all West Nile infection cause, lead to infec--to serious outcome or symptoms, and also we don't know if one unit is not detected as positive because of the limited detection of the assay, would cause some mild febrile symptom that can be considered as normal reaction for transfusion.
		So, in fact, we don't really know.  We have--what we don't, we can't prove, is how many really are, and we know which ones were documented but we don't know how many there were in fact.
		DR. SIEGAL:  Well, what I was getting at is just to play the devil's advocate, if it's so easy to get infected in the ambient environment, relatively speaking, and there's an awful lot of transmission going on anyway, and most of the people who are getting this are the "canaries in the mine," the immunocompromised hosts that have clinical manifestations.  Maybe it isn't justified.
		If you look at Mike Bush's analysis of cost-effectiveness, to spend all the money that it costs to interdict the few transmissions by blood transfusion, when the virus is out there anyway.
		DR. RIOS:  I understand and I agree with you, but the public health issue is to prevent transmission by transfusion to people that need to be taken care of.
		DR. KUEHNERT:  I just wanted to say that I think if you want to look at the number of transfusion transmissions that likely occurred before screening, it would be best to look at the models rather than the actual cases that were reported, cause, you know, there's a definition that needs to be met, and, you know, all the hurdles that we talked about for all the other diseases we've talked about at this meeting.
		But anyway, you know, either the model is wrong and there are less cases, or there's massive underreporting, and that's what we think is going on.  If there are 23 cases in that first year, and, you know, I think the modeling suggested hundreds or thousands.  I can't remember the exact numbers now.
		I guess what I'm saying in answer to--I mean, I think you're trying to get at what the utility of the test is and looking at cost and benefit.
		DR. SIEGAL:  What's the incremental benefit that society will have compared with the natural vector transmission?
		DR. KUEHNERT:  And for that I think you really have to try to estimate what the actual number of transmissions are.  Otherwise, you're starting from a--it's a false premise.
		DR. SIEGAL:  Just thought I'd ask.
		DR. DI BISCEGLIE:  I think I'm missing something in what you're asking us.  I've seen the phrase used, to trigger ID-NAT, but I don't know what that means, and as a sort of part of that question I think, can you tell us the absolute sensitivities, approximately, of the assays NAT used in screening, and then you alluded to more sensitive assays.
		DR. RIOS:  What I can tell you is that since the identification, or the finding that only 75 percent of the infected units were detected in the minipool, and the limitation of availability of reagents at that time, and logistics at the blood centers and automation, etcetera, we knew that the viral load in West Nile is not so high that the pooling, unlike HAV and HCV would much be affected, West Nile has a lower viral load. 
		So what I'm calling the trigger is what would tell people to please start testing because human cases are rising in these particular area anyway, and what has been in the news and was a volunteer determination from blood centers that have done a series of studies--and Susan will address that I hope--if not, if you could please--is that when they see one reactive unit in a thousand, they implement ID-NAT in that particular region because West Nile is very focalized.  It's not spread.  Regionally, it's very localized and actually they can pinpoint area of activity.  Pockets.
		Or having too many pools reactive and then they start doing ID-NAT, and that's what we say trigger, is to start the implementation of ID-NAT instead of minipools.
		DR. DI BISCEGLIE:  And that's permanent thereafter or it's for the rest of that season, or what?
		DR. RIOS:  But that's why I said we hope to have the blood centers validating their implementation, reverting back for minipool, and what has been used to revert back, Susan will approach, but it's one week without any reactive and they go back.  But Susan will give these detail.
		DR. NELSON:  In areas where there's epidemic, there'll be a certain proportion will have a very low level of viremia, so they go from the pools of six to sixteen down to a single donation, hoping to pick up those with lower levels, which would still transmit a virus if it was transfused.
		And the trigger now is one in a thousand or two minipool NATS in a week.
		DR. RIOS:  Reactive.
		DR. NELSON:  In what, a county?  In a county or a state or a city, or what geographic--
		DR. RIOS:  I would like Susan to address that because this is their algorithm for triggering.
		Dr. Nakhasi--
		DR. NAKHASI:  I just wanted to make some clarification about the trigger, a little bit.  You know, as Maria pointed out, that even though these assays are very sensitive, some of them are, you know, approximately ten copies per mL detection, there are, you know, even at--the viral load, as such, for West Nile is much, much lower, so with minipool you may miss some of them and that's why you need to trigger that to ID-NAT and what the trigger, which was--you know--because as we were testing them, blood establishments wanted to find out, you know, to catch all those cases, most of the cases, so they arbitrarily, depending upon, on this, so one in thousand or two in minipool, meaning two minipool NATs in a particular area, it was started, and turned out to be that, you know, in 2005, there were none, in 2004 there was one case.  However, as you saw in 2006, in spite of that trigger there were two cases which held true.
		So the question is do we need to really tighten that trigger and I think Sue will tell you some of the studies, what can be done to tighten that trigger.  And also you have to remember that it doesn't remain permanently.
		If the blood establishments don't see for seven days, continuously, no positive in that area, they will revert back to minipool NAT testing.
		DR. RIOS:  Dr. Nakhasi, I would like to just make a correction, that the claim for the assay is a 100 copy per ml., not 10 copy per ml.
		DR. NAKHASI:  Yeah.  I know the claim is different but it can detect as low as ten.
		DR. DI BISCEGLIE:  And what's the level of infectivity?  What's the amount of viremia that we think is infectious, or--
		DR. RIOS:  This is not known and the reason is because we would need NHLBI to fund as a study of effectivity, and to define what is the viral load required for infection.  As you know, when the concentration is low in one area, and you use a small animal, you cannot simulate a human blood transfusion.
		We know that units that have been detected, or tested nonreactive in minipool, were capable of transmitting infection to the recipient.
		DR. NELSON:  Presumably, if it's transmitted by a mosquito bite, if you find any in a unit of blood you're going to probably transmit it, I think, if there's any virus there.
		DR. KUEHNERT: I just wanted to just clarify.  I'm hearing two cases in 2006 and it was two infections from transfusion transmission but it was from one donor.
		DR. RIOS:  I agree, Matt, but if a mosquito bite, two people, it's two transmission.  It's not one mosquito only.
		DR. KUEHNERT:  Right; right.  It's two transmission from one--but it's one minipool NAT.  That's the only point I was making.
		DR. KLEIN:  But Matt, if I understand it correctly, the second recipient who had meningeal encephalitis, would not have been detected without the look-back.  In other words, they would not have known that this was due to West Nile, which again goes to the issue of it's probably underreported, because you would guess that something as impressive as encephalitis would not go underreported, but clearly it does because people don't think of West Nile.
		DR. KUEHNERT:  You're absolutely right, and also that these happen to be two transplant recipients who are at manyfold greater risk of developing complications and these complications were so unusual, that it prompted the clinicians to seek out--the one clinician to seek out why it happened.
		The others, it still did not prompt an investigation until the look-back.  So I think those are points well-taken.
		DR. SIEGAL:  Any other questions for Dr. Rios?  In the back.  Okay.  Then I think we have come to Dr. Stramer, data in support of the current ID-NAT triggers, American Red Cross again.
		DR. STRAMER:  Good afternoon.  I'm back.  So to continue along the presentations that have already been made by the CDC and FDA, this is like the blood center rebuttal, or whatever, I'm covering two major areas.  One is West Nile confirmation, which we really need to understand for appropriate donor counseling, and this will be based on data that we collected nationally from the periods of time, from 2003, when testing began, through 2005.
		And then also data in support of West Nile ID-NAT triggers, as advertised in the agenda.
		The first series of slides comes from an AABB presentation that has been modified for the use in this presentation, and also through the work of the West Nile Task Force, which is a small group from the blood centers  who work through the AABB.  It also includes AABB staff, members from the FDA and members of the CDC.
		So we have gone through all of the concepts that I will be presenting to you.  So this should not be new to many individuals, but anyway.  So this first portion deals with confirmation.  So just to give the committee some background, donor screening for West Nile RNA by NAT began in June 2003, prior to the onset of the national epidemic for that year, and we all heard about the magnitude of the 2002 epidemic and the short period of time it took for test kits to be developed and implemented.
		During 2003 through 2005, all blood programs in the United States performed investigational NAT for West Nile in minipools, or individually, designated as MP or ID, during the epidemic periods and in epidemic locations.
		And we converted from minipool to ID-NAT, which was dependent on site-specific triggers.  We already mentioned that there were a number of triggers used through the United States.  But, for example, one such trigger, as Maria mentioned, was two positive cases and a frequency of one in a thousand positive donations, and I'll go through the derivation of the triggers and standardization of the use of triggers in later slides.
		For this particular portion of my talk, three blood collection testing programs contributed data, which represented greater than 80 percent of blood collected in the United States, or over 4 million donations per West Nile season, and covered all geographic regions.
		And even though West Nile testing occurs throughout the year, in all U.S. areas, to focus on incidents and new cases reported each year, this report covers only the epidemic periods of the mosquito season for a given year.
		And that's defined as the date of collection between the first and last West Nile confirmed positive blood donor.
		The screening tests used were either the Gen-Probe test, as I describe on this slide, or the Roche test, which I'll describe on the next slide.
		Gen-Probe, which is now FDA licensed on both platforms, that is, their semiautomated platform and an automated platform, was used by some participants in the study.  The test is distributed by Chiron, it's transcription mediated amplification, which is an RNA amplification system, in minipools of sixteen, using either, as I said, the semiautomated or the TIGRIS system, both of which are now FDA licensed.
		The sites included the American Red Cross, United Blood Services, and all contract facilities for both of those blood systems, and it represented again all geographic areas within the United States.
		The second test used was the Roche investigational test.  This is in minipools of six, using the Taq screen, that's the commercial name of the test, and it included the 12 sites that are listed here.  I will read them.  They're listed in your handout.
		For confirmation, which is a really important component of this talk, TMA or PCR, depending on the test, if they were initially reactive, they were considered confirmed positive, if they met one of the following criteria.
		If the initial test repeated as reactive in the original or modified test format, and a modified test is called Alternate NAT or ALT NAT, and preferably, this was from an independent sample from the index donation, such as I talked about yesterday from the retrieved plasma unit.
		The index donation tested West Nile antibody that is IgM, or IgG positive, and the way the sites use antibody testing was program dependent.  There was a test available from Abbott laboratories for the first year, Focus has tests, and the Safe Public Health Labs also do antibody testing.  And the other criteria for confirmatory was if the donor follow-up samples were tested and they were reactive by either repeat NAT, that is, TMA and/or PCR or antibody.
		And in most programs, if we did have a positive PCR, that was followed by a quantitative test that was performed at National Genetics Institute.
		This is the results of the first three summers of testing.  So this includes a total of 13 million donations.
		The number of donations that were screened during this period of time, individually, were 3.8 percent--actually, there was some individual donation testing that we performed in 2003, when we realized quite a large local epidemic in Nebraska and Kansas.  So that total percent is 4 percent.  These are the date ranges.  So pretty reproducible form--this was the earliest year, the beginning of May, but we've seen cases all the way through the end of the November into the first day of December was our last case.
		So what the data set will now focus is on the 1329 confirmed positives.  So if you look at the frequency for 10,000 donations overall, we see a frequency of one in ten thousand, which is actually ten times higher than the one in one thousand trigger that we'll talk about.
		And here on the bottom we just show the CDC numbers in comparison.  I mention this is only about 80 percent of collected blood in the United States contributed data to this study, so this is consistent with what was reported to CDC which should be all.
		When did West Nile occur in those three years?  This is the epidemic curve, showing you 2003, the beginning.  The upslope of the epidemic and then the tailing through the first week of December.  Then 2004 came up  a little bit earlier, and 2005 actually came up a little bit later.  2006 was somewhere between 2003 and 2005 and isn't on this map.
		Where West Nile occurred, you already heard that in the first presentation from CDC, but again, in this corridor that's east of the Rocky Mountains, where we see the highest number of cases, we've also now seen in the last couple of years very high numbers in the state of California.
		So the increasing color here indicates numbers that are increasing and the numbers in the states obviously indicate the number of positive donors.
		So in Nebraska, for example, we've had 298 viremic donors that have been identified.
		Looking at the characteristics of these 1329 donations, if you start from the earliest period of time, these are those donations that are ID-NAT reactive only, followed by those donations that could be detected by minipool, obviously would be ID-NAT reactive as well, but the difference here is these are ID-NAT only, minipool NAT negative.  Then we have minipool NAT positive, both cases being antibody negative.  Then with the appearance of antibody, we still have donations that are detected by minipool and are antibody positive.  But then we have this long tail of donors that are detected only by ID-NAT, that have antibody.
		So if you look at the data in aggregate, and look at those donations for which triggering is important, that's the top row and the bottom row, that total is, as Maria said, about 25 percent, but in this study of all of our data for the first three years, it turned out to be 22 percent of NAT reactive donations required ID-NAT for detection.
		That is, if we maintain minipool NAT, those would not be detected.
		Also if you look at the difference in antibody positivity, the vast majority, or I should say three-quarters of what we see are antibody negative donations versus 26 percent that are antibody positive.  The smallest percentage here being minipool, NAT, and antibody test positive for this category.
		If you now look at viral loads, which are indicated on the x axis here again--this is from the National Genetics Institute--and you divide the samples into those that are antibody negative and antibody positive, you can see, in red, these are the antibody positive samples.
		The antibody positive samples have the lowest viral loads, as I showed on the prior graph, that more of the antibody positives are detected by ID versus minipool NAT.
		Only 76 percent of the total had adequate volume and went through the algorithm for quantitative testing.  But in any event, you can see that the large numbers of antibody negative donors here have the highest viral loads.
		But even so, the highest viral load of any sample detected was only 580,000 copies, which is considerably lower than what we see for HIV or HCV, which is why, for West Nile, we have to have a strategy of ID-NAT triggering as opposed to just minipool NAT testing because we would miss a considerable number of donations.
		So one can think of minipool NAT as kind of a surveillance system until the epidemic hits, and then we convert to ID-NAT.
		If you now look at all donations that were detected and divide them into both minipool reactive and individual donation reactives, but look at those that are false positive versus those that were confirmed positives, and then look at the signal-to-cutoff ratios, which everyone, as I said yesterday for Chagas, everyone wants to know what the meaning of a signal-to-cutoff ratio is.
		For those that confirm positive by minipool, which are in red, those are the highest signal-to-cutoff ratios.  Those that are confirmed positive but were only detected by ID-NAT, as I showed you in the prior slide, have lower viral load, so their distributions across the S to CO values are much broader.
		And if you look at an S to CO value in the case of the Gen-Probe test which we use, at least at the Red Cross, and blood systems that is predictive for whether a sample will confirm, we use an S to CO of seventeen, and we have found that 88 percent of those samples that have an S to CO equal to or greater than seventeen will confirm.
		So it's very useful as we identify NAT-reactive donors.  In contrast, the false positives all have low S to CO values, and they're comprised of those to be detected, either ID-NAT or minipool NAT.
		And the algorithm we use specifies, if an ID-NAT result is positive, we take actions on the donor and the products, versus minipool NAT where you have two rounds of testing before you do doom a donor, so to speak.
		So both the minipool and individual donation NAT have to be positive.  So if you just base positivity on one test result, we're obviously going to see more positive hits with ID-NAT than minipool NAT.  So one of the downsides of triggering and converting to ID-NAT is we lose more donors because each ID-NAT reactive is a deferred donor, and their products are destroyed.  Looking at false positive across the three years of study, I think the individual years is not what is necessarily important, but the bottom line message here is that 70 percent of what we see for false positives, as I just mentioned, occurred during the ID-NAT season, and the frequency of false positives, overall, is .55 per 10,000 donations.
		So 500 of 722 false positives seen in the three years were detected during the ID-NAT season, or as a consequence of triggering.  Now focusing on the confirmed positives, I've divided them in different colors here.  So the first column here, before we go through the rows, is seroconversion of follow-up.  How many donors were followed through follow-up and confirmed as positive?
		The other criteria here, these now represent the index donation--how many were antibody-positive at index, either by IgM or IgG?  And then the other index criteria is how many showed repeat reactivity by the same, or an alternate NAT at index donation.  So these reflect what we did at index versus donor follow-up.
		So of the 200 in this category here, that were confirmed as repeat reactive, and indexed by alternate NAT, for most, we didn't have a follow-up sample.  But in the further slides where I'm going to talk about positive predictive value, etcetera, we would consider six of these eligible to go on for further study, because they did show RNA positivity at follow-up, so we could reproduce the fact that they were West Nile infected in a subsequent sample.
		Now looking at either NAT reactivity at index in the presence of antibody, or NAT reactivity at index in the presence of antibody--these are addended index, so these are antibody-positive at index, split into NAT-positive at index or NAT-negative at index.  We have the two numbers here but those that will go into further study are 83 and 156.  So of these 128, we followed 83, and all 83 had zero converted.  So we will carry these numbers on into further calculations.
		Of the 217 that did confirm by two independent methods of index, at follow-up 156 of those followed did zero convert.  So in each of these cases, one could argue you could take all of these, because we had a 100 percent agreement between those in these categories that were confirmed by follow-up.  But for the purposes of analysis, we will deal with these six, this 83, this 156, and then we come to these yellow boxes.
		 Here we have donations who did seroconvert on follow-up.  This first category here is those that were antibody-negative or NAT-negative at index.  So for these ten donors, as Maria already mentioned, the only way that we confirm them as positive was by follow-up.  So these would represent false negatives at index and influence the sensitivity of the confirmatory algorithm.
		These 764 that were repeat NAT-reactive at index also seroconverted.  So again, these will be the numbers that we take through
on the next slide.
		So now looking at the calculations of how good a confirmatory algorithm is, based on index testing and/or follow-up testing, we have no donors who were confirmed positive in index, as I indicated on the previous slide, but were not antibody-negative at--but were antibody-negative at follow-up.  We had ten in the category I just showed you, that were confirmed only by follow-up testing, and were negative at index.
		Then, if you added the six, if you add all of these numbers together that I indicated, you come up with 1009.  So these were the confirmed positive at index, and we confirmed them again at follow-up to corroborate the index test results.  And then I showed you the 540 false positive results, and these were based on negative results at follow-up and these individual donors.
		So these 1009 split into the two groups that I showed you, 239 that were confirmed at index based on antibody and antibody at follow-up, and the 920 of the larger group that were confirmed positive at index based on repeat NAT and antibody.
		So going through the various calculations, firstly, we have 1019 confirmed positives over the 1559 initial reactives.  That's all comers.  So the positive predictive value of the screening assay is 65 percent.  The negative predictive value here, how many of these false positives were over the denominator of total reactives, for which we need a follow-up to resolve, there were 540 over 550, adding in these ten, so we have a negative predictive value of the screening test of 98 percent.
		And then based on the positive predictive value of index confirmation, all of these 1009 that were confirmed at index also were confirmed at follow-up.  So our positive predictive value was 100 percent.  Sensitivity, we did have the ten false negatives, so that drops that to 99 percent.  Sensitivity of repeat NAT, which is this calculation over the 1019, 90 percent, and a sensitivity of antibody at index is 23 percent.
		So overall, the sensitivity of index confirmation is 99 percent but it's made of these two components.  You can just repeat NAT for 90 percent sensitivity or you can do antibody as well, to increase that or get the extra 20 percent, which, because there's overlap, this adds up to 99 percent.
		Now one question that we ask is, well, some people repeat the same NAT method, or some use an alternate NAT method.  Is there a difference in sensitivity? 
		So of the 1196 samples that were tested at index by both a primary and an alternate NAT, here we had 86 percent detection, or 86 percent detected with the alternate NAT assay, and about 87 percent actually no significant difference between these numbers by the primary NAT assay.
		And the discordance solves the vast majority of these were antibody-positive anyway, so even if you use only one NAT, rather than using two NATS, we have the benefit of doing the antibody test, which we'd recommend that you do both repeat NAT and antibody.  So we would recommend only one NAT test, their primary or alternate be adequate.
		So what do we conclude from this?  The positive predictive value of the screening algorithm, as I mentioned, 65 percent, indicates the need for confirmatory testing, and I did mention the 69 percent of false positives drive the lower PPV obtained during the periods of ID-NAT.  The positive predictive value of our index donation confirmatory algorithm was a 100 percent, using follow-up as our gold standard.  All donors who were confirmed positive based on the index donation results have been accurately classified as West Nile infected, that is, no false positives were observed.
		The sensitivity of the confirmatory algorithm based on the index samples testing approximates that based on follow-up testing, indicating very little additional value is obtained by follow-up testing.  I showed you a 99 percent sensitivity that was divided into 90 percent by repeat NAT only, and 23 percent, if you add the antibody--well, 23 percent alone by antibody, giving you a total of 99 percent.
		Now if you balance this against follow-up, the disadvantage of follow-up is a confirmatory algorithm, as we've seen, requiring follow-up testing, will never have 100 percent sensitivity, in practice, because not all donors will participate in follow-up.
		And then lastly, the few true positive donors who would not be classified as confirmed positive based on the index test results, would already have been counseled for possible West Nile infection.  They have been deferred for 120 days and components from their donations would have been quarantined.  Thus, there is no adverse impact on blood safety by eliminating follow-up testing.
		Now let me switch to triggering.  The need to trigger, that is, convert from minipool testing to ID-NAT, during epidemic periods, is based on the low viral loads that we have discussed for West Nile as compared to HIV or HCV.
		I mentioned the 22 percent of West Nile NAT-positive samples detected required ID-NAT for detection.  Or another way of saying this, or looking at the data, where the 26 percent of the detected samples were antibody positive, of which the majority, 81 percent, required ID-NAT.
		Most systems have implemented some type of trigger, it's not standardized, no method exists for site-to-site communication.  So triggering has been successful; however, as we've heard, there have been two West Nile breakthrough cases that occurred in 2006, and these numbers you've seen before, so I'll skip ahead.
		So based on--I say "need for improvements," I should say that in quotes, we can always make improvements.  But based on the two cases that we saw in 2005, the AABB West Nile Task Force that I mentioned already, representing the blood industry, developed an association bulletin that was released on April 3rd.  We did receive input from the CDC and FDA on that bulletin, and the recommendations involved the use of a minimum trigger that has been shown to be feasible and has relatively high effectiveness.
		And a comparison of multiple triggers were published by Brian Custer and his co-workers at Blood Systems and Transfusion in 2004.  We first validated this triggering algorithm by 2002 retrospective studies, based on the frequency of West Nile clinical disease, which was one in a 1000 in the 2000 year, and the observation of one minipool negative unit for four minipool NAT-positive units that we observed in those retrospective studies, and those data were published.
		So what did we say in the association bulletin?	  We recommend a minimum criteria based on initial reactive donations and rapid time to respond, that is, within 24 hours, due to the short duration of the ID-NAT-only- window period.  And for ID-NAT, that's about two days.  The entire minipool NAT window period is about seven days.  And after seven days without seeing a repeatable ID-NAT reactive, or an antibody positive, sites can revert back to minipool testing.
		The other portion of the association bulletin focuses on a communication plan and a communication plan based on the existing testing sites that have entered data already into the AABB Web site, and therefore have had communication plans in place for their institutions and their customers.
		So institutions already have such communication plans, but what we don't have is communication between facilities.  So contact information and states for which collected donations are tested is provided as an attachment to the association bulletin.
		Sites for which collections occur in adjacent or overlapping areas should be communicating.  That's what we're trying to accomplish.  And there are many tools for tracking activity.  You can use your site-specific data, the data that's entered into the West Nile map that's on the West Nile biovigilance map.  That's on the AABB Web site, and those donors are entered by residential zip code.  Maps provided by the CDC.  CDC reports of avian and mosquito activity.
		So all of these factors should be used in determining whether you trigger or not.  But once you met the minimum criteria, which I'll describe, triggering is required.  So these tools can be used, as I said, as part of planning activities within facilities and between facilities.
		I plan on sending out a weekly "blast" e-mail, actually, to all the facilities that share our borders, to facilitate easier communication this year, so it won't be just within the Red Cross.
		So the minimum criteria, I'm going to describe those now, they have to be feasible or they won't be done.  They have to be realtime, because as I said, the ID-NAT window period is only about two days.  It's based on two West Nile NAT reactives and a rate of one in a thousand, and then we've gone into some further criteria to define, if sites have lower numbers of collections, how they should be tracking their rates.
		Also, as far as defining a geographic area, it's really impossible or impractical to say it's within a zip code or within a county.  So the way that we have defined geographic areas is by the number of collections per--what's feasible is the number of collections.
		Some facilities segment their facility into quadrants, so that they don't have to trigger within the entire facility.  But we've said if your number of collections is low, that's really not advised.  And then we recommend growing periods of time for calculating rates, depending on your collection numbers, or another method of doing this is just to monitor between your first and second NAT reactive, and trigger if less than 2000 collections have occurred between that interval.
		So, again, there are just a number of tools available, clinical cases by county, clinical, or avian cases reported by CDC.  Again, clinical cases, West Nile-presumed viremic donors, presumptive viremic donors, and then we have the AABB tool, which is the most useful tool, because it's updated, at least last year, weekly.  We're going to try to update this more frequently this year.
		Donors are entered by zip code.  So for last year, and all the maps for 2006, we see 439 confirmed positives.   We had 64 false positives.  We also track by date, when donations occurred.  This is again on the AABB Web site.  We provide site-specific information.  This is the Red Cross map for 2006.
		So actually to tie this up, or to conclude, the logistics, West Nile ID-NAT is a balance between sensitivity and capacity.  The largest labs may have capacity for a thousand samples per day or 1200 samples per automated instrument per day, but of course that involves that the sites may have to have multiple instruments and it assumes that the instruments are working all the time, which they're not when they're constantly challenged with the high number of samples.
		Reagent performance issues or other sources of false positivity may cause sites to artificially trigger early, or extend the time of ID-NAT which is problematic.  So with that, I just show you that ID-NAT and a blood system, year round, does occur for multiple regions.  This is Nebraska, the site that we trigger every year in, and we see prolonged West Nile activity, and just as an example, for last year we did 135,000 individual donation tests during the West Nile season.
		So with that I'll conclude and answer any questions.  Thank you.
		DR. SIEGAL:  Thank you, Dr. Stramer.  We're open for questions.
		DR. KATZ:  Sue, can you talk about the two ends of the ID-NAT windows and where transmission occurs, the ramp-up and the AMBI-positive.
		DR. STRAMER:  Well, all of the cases we have seen of the twenty-three, and the subsequent nine cases, have been in antibody-negative individuals, and that's the shortest period of time, that's the two-day window, versus that longer tailing period that Maria said can extend up to 104 days. 
		When we've looked at, at least for Red Cross data, viral clearance occurs for 99 percent of individuals at 56 days, but as we've observed from blood system studies, they did have one donor that went out to 104 days.  None of those--we're not saying that those aren't capable of transmission, but none of those have ever been implicated in transmission.
		So the prolonged, or the tail-end of the ID-NAT trigger period, it's important that we're able to get out of ID-NAT triggering promptly, without prolonging it for extensive periods of time due to false positives.
		Lou, is that kind of what you wanted me to address?
		DR. SZYMANSKI:  I'm interested as to what the ID, the individual testing, NAT testing, is more often false negative.  Does it mean that the material there is in a lesser strengthen than that of the true positives, and therefore it's diluted better in the minipools?  And what would that material be?
		DR. STRAMER:  Okay.  I tried to explain it but let me do this again.  When we do minipool testing, if you've a positive result we do resolution testing, which means--
		DR. SZYMANSKI:  [inaudible]
		DR. STRAMER:  Right.  So by statistics, false positives, which are random events, may occur, let's say, at a rate of one in ten thousand.  So you have a one in ten thousand event happening at a minipool stage.  So then, when you do resolution testing, that one in ten thousand event would have to happen again.
		So statistically, if something's a false positive, it's very unlikely that it would repeat two times in one, for one donation.  So if we have an ID-NAT positive, that one in a thousand event once, that defers the donor.
		DR. SZYMANSKI:  But what is that material that gives the false positive?  Do you know what--
		DR. STRAMER:  Pardon?
		DR. SZYMANSKI:  What kind of material is that, that reacts in that, you know, individual, for false positive?
		DR. STRAMER:  Why it's false positive?
		DR. SZYMANSKI:  Yes.  What material is it?  Is it something--
		DR. STRAMER:  Oh, I see what you're saying.  The way we perform the NAT tests, although many of them are automated, what happens with NAT, unlike serological tests in which we have biological false positives, in NAT, we have, unfortunately, contamination, and that's the cause of false positives. 
		Whether it's from a strong positive contaminating an adjacent sample due to aerosolization, or less likely due to external controls, or the assay calibrators, although those are much lower viral load samples.  But if there's one high titer sample, very frequently you can trace neighboring samples that have been contaminated.
		DR. SZYMANSKI:  Okay; thank you.
		DR. STRAMER:  And it doesn't matter how good the technique is, there's always going to be false positives with NAT.
		DR. KLEIN:  West Nile virus testing has really become an industry.  I mean, there are meetings, and there are all kinds of communications, and triggering mechanisms.  It really is pretty complex.  I could understand that in the days when it was semiautomated, but now, with automated technology, I know issues of logistics and cost are the main drivers, but it seems to me that there must be an argument for simply ID testing across the country.
		You've gone pale.
		DR. STRAMER:  No; my pocketbook--well, the Red Cross's pocketbook has gone pale.
		DR. KLEIN:  So perhaps we should know what the tradeoffs would be.  Is it only money, or are there other--
		DR. STRAMER:  Well, it's certainly false positives, loss in donors, and it's a temporary loss in donors.  Donors can come back at 120 days.  We do follow-up testing at donor request, but it's certainly the loss of associated products.  Cost is certainly a giant issue.  Even though we have automation of ID-NAT triggering, we still have a capacity within each blood center, that even if we have multiple instruments, we sometimes have to balance tests between other labs to balance our capacity.
		So even with automation at 1200 samples per day, if we have multiple regions trigger within one area, such as Nebraska, Kansas, Oklahoma, you know, all that area, we've exceeded the capacity of that particular lab, and we would need probably two or three times the number of automated instruments to do this continuously, year round.
		And I would argue what the benefit is, because I believe--or the data demonstration that the ID-NAT triggers, although there are limitations, and that we did have two cases this year--or two cases this past year, I believe the system is pretty sensitive, and if we watch it carefully, and are able to communicate and use multi-site triggers, there is no reason that this can't be almost, well, close to the sensitivity of ID-NAT year round.
		In the association bulletin, we also mention validations, that, actually, FDA recommended that we put in there, that some sites	validate the use of this multisite trigger, and triggering just in general, to see how sensitive, and if there were prior donations that were missed.  So with some of our regions, we're going to trigger early, let's say after one NAT reactive, to see what we would have missed between the threshold of one and two, to try to answer the question, Are we missing anything? and, really, what the benefit of year-round testing would be.
		DR. SIEGAL:  Dr. Rios.
		DR. RIOS:  Yes.  I would like to ask you to comment on your false-positivity slides, that you had ten samples that did not repeat NAT.  Nevertheless, you considered them as positive.  If you would have applied your algorithm that you need either transmission or repeat NAT, and not perform follow-up, how would you have identified, if had ten additional cases that were true positive and would have been considered false negative?
		DR. STRAMER:  Well, these donors are deferred, and in our materials we do tell donors that--we counsel them such, that the possibility of West Nile infection cannot be excluded, and we do invite them, if interested, into follow-up.
		So all of the follow-up that you've seen was voluntary, and our system, we won't change that, but yes, there is that chance, that if those ten donors didn't come in for follow-up, yes, we would have missed them.  But in our counseling materials, we don't tell anyone that you're unequivocally uninfected, for any agent.
		DR. KUEHNERT:  How do you know they weren't otherwise infected in the interim, between the time of donation and follow-up for the ten?  Was that a--
		DR. STRAMER:  They were IgM positive, relatively short--in a short interval after their viremic donation.  Our follow-up is pretty rapid, within one to two weeks.
		DR. KUEHNERT:  Okay.
		DR. SIEGAL:  Dr. Epstein.
		DR. EPSTEIN:  While we're on these ten confirmatory false negatives, have you been able to figure out why it happened?  In other words, is it correlated with low titer in the index sample?  Is it correlated with the relative sensitivity of the secondary NAT?
		In other words, were you using the original NAT?  Were you using the alternate NAT?  Were you using a different manufacturer's NAT?  Etcetera, etcetera, etcetera.  Because one of the puzzlements here is you found those samples from minipool screening.  They were initial reactive on ID-NAT--
		DR. STRAMER:  Well, those ten were actually from ID-NAT.
		DR. EPSTEIN:  Okay.  Well, that's another important point.  Okay.  So the question is do we know the attributable cause?
		DR. STRAMER:  Well, and actually, the way we were doing index testing, we were not only repeating the same NAT but we were also doing alternate NAT.  So in this case, the index not only scored negative when we did--I mean, it was initially reactive when we did primary screening, but then, on retesting, it was nonreactive by the same method, an alternative NAT method, and antibody.
		So the only thing I can attribute it to is the one out of twenty.  It was just the luck that we found it, because these are such low level due to stochastic.
		DR. EPSTEIN:  So another way of looking at it, this is that the--
		DR. STRAMER:  A chance effect.
		DR. EPSTEIN:  --negative predictive value of the confirmatory algorithm is higher on samples that are found first on positive minipool than on samples that are found first on IDT NAT, and the reason is obvious.  It's because you have the ability to pick up lower-titered samples by IDT NAT.
		But, you know, if you were to stratify it, in other words, if you were to do that calculation for the ones found first by minipool versus the ones found only by IDT, you'd come up with a different set of answers for the predictive values of the algorithm.
		DR. STRAMER:  That's true.  I mean, are you saying you want me to--well, we can look at those.  We could look at that calculation.
		DR. EPSTEIN: I mean, I think the results are very impressive and I'm not quarreling with them at all.  I'm just trying to understand what we know and what we don't.
		DR. SIEGAL:  Dr. Finngean.
		DR. FINNEGAN:  Can I ask you what you think it would do to your bottom line, if we followed up on Dr. Klein's suggestion, but instead of doing it universally, that you did it during the endemic period in those counties where you know you have a much higher risk, and dropped it down to the first positive case or one in five hundred instead of one in a thousand.
		DR. STRAMER:  Well, actually, one in a thousand is more sens--yes; right.  Well, I think, you know, what I tried to say in the last slide that I had with text was we've got to balance the logistics with the sensitivity, and I think for this season--well, I know for this season we're going to keep it as we have and then look at several regions that we know trigger each year, and I will trigger those early, like Nebraska, Kansas, and we'll look at what would happen if we instituted a more sensitive trigger, that was, as I said, an FDA request to validate, in a way, the use of this, and the only way to do that is to do more individual donation testing to determine what we're not picking up.  So we will look at it in a validation mode, but until we see data that supports that, I don't think we're ready to convert to a more sensitive trigger criteria.
		I think what we need to do first is convert the entire country to a standardized trigger criteria and to make sure that we're all communicating, so that if I have one NAT reactive and you have one NAT reactive, and we're collecting in the same region, at least we know that together we trigger, and that's the kind of data that would have prevented, if we lived by lessons learned, that would have prevented the two transmissions in 2006.
		DR. SIEGAL:  Are there any other comments?
		DR. DI BISCEGLIE:  You may not be able to answer but maybe Dr. Farnon can.  The donors begin to be positive at the beginning of May and go through the first week of December. But we heard from the CDC, that there are cases being diagnosed in every month of the year.
		So why the disconnect?
		DR. STRAMER:  We don't see positive blood donors--I mean, one limitation may be minipool testing.  Not to contradict the CDC, but the one from California, the first case in January of last year turned out to be--unless we're talking about different cases--an antibody positive that was a carryover from the prior year.
		So the clinical case that was observed in January turned out not to be a new infection but recognition of an older infection.  We haven't seen cases to support testing--you know, one of the questions that we ask as an industry was do we even need to test, as they do in Hema-Quebec, they suspend testing during the non-mosquito season.  I mean, you need mosquitoes to transmit, and in areas where there are no mosquitoes, perhaps we don't need to test.
		But we have agreed with travel, and because not all of the United States does lose mosquitoes, but we, as a country, will continue to do minipool NAT, year round.  But I can't explain the disconnect other than delayed reporting for those clinical cases, because we don't see it in blood donors.
		DR. KUEHNERT:  I just wanted to make a comment about the false positives for ID-NAT.  It looked like, from the chart, I mean, certainly, when you have a low S to CO, I mean, it indicates that, you know, it's probably a false positive, but not for sure, and even when you get up to the higher numbers for ID-NAT, you could have an S to CO over thirty and it could be a false positive.
		So it seems like it's really hard to figure it out, and it particularly has implications if we're talking about moving towards ID-NAT more often, and also for organ and tissue testing, because they can't do as much, you know, resolution, can't follow up with the donors.
		So it makes it more difficult.  I just wondered if there's any more research, along the lines of what was asked before, about what produces a false positive, particularly in the setting of ID-NAT and trying to further discern a false positive?
		DR. STRAMER:  Regardless of how complex West Nile is, or complex NAT is, the answer to false positives is extremely simple.
		It's contamination.  So for sites who don't cram through 5000 tests a day, who have technologists who are highly proficient, and in many of the facilities, who just perform organ and tissue testing, I think, you know, those are probably smaller numbers and plagued with less proficiency than the giant blood centers who do this as their "apple pie," so to speak.
		And also I will comment that doing organ and tissue, they do them individually, so the chances of having a false positive do increase.  And then I'll also comment that not all lots are created equal, and there's some magic, sometimes, into driving the enzyme or probe into solution, and if there is any precipitate left in some of these solutions, those lots become very highly prone to--I don't want to say some type of biological false positive, not related to the sample, but related to the dynamics of the test.  So it's a tricky business, and the organ and tissue folks do have a formidable challenge.
		DR. SIEGAL:  Dr. Rios.
		DR. RIOS:  Sue, I hate to bring the ten samples again, but the ten specimen that did not repeat NAT, what is clearest to me--and I don't mean to talk about tissue because we are discussing blood--but how would you know that the false positive rates are so high, if you don't have follow-up of a 100 percent to see sort of conversion.
		And I understand, and I know you can't do it, but when we claim claim that it's false positive, you have to have the basis of at least having a follow-up.  Lack of follow-up does not indicate false positivity.
		So if you have a donor that was reactive and did not repeat and didn't come for follow-up, they should not be regarded as false positive.
		DR. STRAMER:  Well, as I mentioned, our donor letters don't necessarily tell them, you know, we give them all possibilities, we do say you are likely false positive, because the data do support that they are likely false positives.
		And the 540 carried on into the additional analysis were followed, and shown by two types of NAT, and antibody, that they were false positive.
		I know those ten will occur, but we have to also recognize, even if we require follow-up, not all donors are going to participate in follow-up.
		It happens to be amazing.  We looked at all of our--for purpose of writing a reentry chapter, we looked at all of our follow-up for donors and reentry numbers, and the marker that we have the highest success rate for on donor follow-up happens to be West Nile.
		Each year, it's about 75 percent, the donors, without really twisting their arm or hitting them over the head with a bat, that you have to come in for follow-up, they do participate, and, you know, why isn't that high for HIV or HCV?  And the only thing that we can surmise, which is off the topic, is that this doesn't have a behavioral stigma.
		I mean, there's nothing wrong with getting socially--it's socially acceptable to be bitten by a mosquito, even though some of our HIV-positive donors tell us that's how they were infected.
		But in any event, we're not going to get all donors to participate in follow-up.
		DR. RIOS:  I do appreciate and understand your point and I am not questioning about the safety of public health, that you have to cover everything, but the scientific basis by which you nominate them false positive, that I'm questioning.
		So maybe it's more appropriate to say that it's inconclusive than call them false positive, if you lack follow-up.
		DR. STRAMER:  Well, our letters actually do use "inconclusive," they use kind of innocuous words such as those.
		DR. RIOS:  Thank you.
		DR. SIEGAL:  Dr. Schreiber.
		DR. SCHREIBER:  Sue, I understand, I think I understood that part of the trigger system is dependent on the individual testing labs putting their information into a third party Web site, AABB Web site, and this then can be used to decide whether a trigger takes place, for example, if it's by zip code or geographic area.
		It seems to me that when you have a big system like yours, where you're controlling the labs, that's a pretty good system.  But when you're dependent on others to have to put something in, without any mandatory requirement, that seems to me to be a little bit of a weak link in the system.
		Is there anything being done now to try to work towards that?  I know you said that the communication link was a problem.
		DR. STRAMER:  It's not a problem; we've just never encouraged it before.  I mean, we have encouraged--Barbie Whitaker's microphone, she'll address for AABB--but we've never--none of this is mandatory, but I think we all want to do the right thing.  I mean, we all want to protect the safety of our recipients, and the only way for us to be able to--well, one way for us to be able to identify what's happening in the United States is for us to have a very active Web site for which all of these cases are reported.
		Within the Red Cross, I'm going to do something in addition to the Web site, as you mentioned George, but I have the ability to do that.  I can send out to every institution that's also on the AABB Web site.  I can send them out a weekly update.  I'm going to include them in our con calls when we trigger, so that there are no secrets, to make this completely transparent.
		But, you know, yes, it is voluntary.
		DR. SIEGAL:  In the back.
		DR. SCHRIEBER:  It's voluntary but many centers are making it SOP, and, you know, that's as close to mandatory at my center as you can get, is it's an SOP that says within x number of hours of a presumptive positive, it's going to be on the AABB Web site.  I'm hoping that most places are doing it now.
		DR. STRAMER:  Yes.  We have it in, yes, the SOP as well.
		DR. WHITTAKER:  Just to add to that, Barbie Whittaker, AABB, last year it was not required and we had 430 PVDs confirmed positive, that were entered into the Web site, which is more than were reported to the CDC, and the average number of days--and this wasn't required--it was ten days from date of collection to date of entry into our Web site.
		So I think it speaks pretty well for the compliance, when it wasn't even a required system.
		DR. STRAMER:  And the association bulletin now--well, it requires within 24 hours of reaching the trigger, that you actually initiate testing.  But I think we used pretty strong language in the association bulletin as well, to which the speed you enter into the Web site.
		DR. SIEGAL:  Dr. Kleinman.
		DR. WHITTAKER:  And we can send our reminders too, to the members, to do that.
		DR. KLEINMAN:  I just wanted to add one point on ID testing, to kind of address the question that Dr. Di Bisceglie had asked before about infectivity.  So I think Sue mentioned this but just to reiterate it.  Number one, the people that we pick up by ID-NAT, when we do the quantitative testing, usually have less than a 100 copies per mL, which is the limit of the quantitative assay to detect.
		And then, secondly, if you actually look at transmissions from ID-NAT positive units, you can have two kinds of ID-NAT positive units.  You can have the ID-NAT positive unit at the very beginning of infection, where there's no antibody, and then the person goes through peaks of viremia, starts to lose their antibody, starts to lose their virus, and antibody comes up at that point in time.
		And so far, we don't have any examples of transmission from a unit, that I'm aware of, that was both viremic at low levels, only detectible by ID-NAT, in association with transmission, which kind of mimics Hepatitis A situation, as an example.
		And so most of the units that we identify by ID-NAT are units that have antibody.  So the additional value we get from ID-NAT, in terms of preventing transmissions, may not be 22 percent.  The additional yield we get is 22 percent but not all of those units may be infectious.  It may only be that initial window.
		If it's correct, that the initial window is the important period of time, which we all believe, then obviously it's important to trigger very quickly because once the epidemic is spreading through the community, that's when you're going to have people acquiring infection, presumably in an initial wave, and if you wait too long to trigger, if you wait a couple of weeks to trigger, when the mosquitoes are no longer perhaps transmitting the disease, you'll still pick up a lot of people who are ID-NAT positive but they're probably not the most infectious people.  
		So just a couple of additional comments about how to think about ID-NAT.
		DR. SIEGAL:  Dr. Kuehnert.
		DR. KUEHNERT:  I wasn't going to bring this up but since it's been brought up again, I just want to urge some caution about stating that transmission doesn't happen with--
		DR. KLEINMAN:  I said we haven't detected it, Matt.  I didn't say it didn't happen.
		DR. KUEHNERT:  Okay.  But the denominator is very small.  I mean, we're talking about less than thirty, because, for instance, in the 2006 cases, we didn't even have a sample to test for antibody, to look back.  So that doesn't even add to the data, and we sort of have to be cautious about that.
		And the other point is that in the organ transmission case, that was last reported and given, that's organ transmission, the donor was antibody positive and NAT negative.
		So it does happen in the organ transplant world, although I agree that's very different from blood, but we just have to--but I just wanted to give that caveat.  But I agree with what you said.
		DR. KLEINMAN:  I think that's true, that it's not an absolute statement, but I think we should regard units in the initial period as perhaps more infectious or more likely to be infectious than units, once a person's developed antibody.  It's not an "all or none" phenomenon, probably.
		DR. KUEHNERT:  Sure.
		DR. RIOS:  I appreciate the discussion and I respectfully disagree with you, Steve.  Of course.  Why would it be fun?  But there are two things that happen here.
		One is that the absence of evidence is not evidence of absence, first of all.  And we do not definitely test every single recipient during the period of West Nile season.
		Second, we have published that in primary human T-cell, monocyte culture, antibody does not make any difference in the infectivity.  Second, there is the information about the organ and donors, and then we are publishing a paper now in Clinical Infectious Diseases, where we show, in collaboration with Dr. Stramer, that red cell attach to--West Nile attach to red cell and it can be as high as one log higher than in the plasma.  I would like to remind you that we use plasma to test.  So if the red cell compartment is saturated, you cannot say that because of the absence of the low viral load in the plasma, wouldn't be infectious in the red cell.
		In the presence of antibody, I would like to remind you that some flavivirus has AD, that it's enhancement of infectivity by the presence of antibody.  Second of all, neutralizing antibodies, performed in a completely official system, using kidney cell from green monkey, and, you know, cultivated in lab, so it's not a natural situation.  I would like to caution you to evaluate this as lack of absence, or the evidence be evidence of absence. 		Another point is if you are testing low viral load in plasma, in pool, unlikely that you detect those ones, that the red cell units may have high viral load.  So you may trigger much later, if you have one rate, or one in a thousand, you may have to drop this to one in five thousand, or one in ten thousand.  I don't know.
		DR. DI BISCEGLIE:  I have one more discrepancy I'd like to explore, and that is this issue of informing the other blood centers in the region that a test is positive, I think is very important, and I guess I heard that at some centers, within hours, a positive is reported, and then I heard from somebody on the floor that the mean time was ten days for this to be reported on the Web site.  Can you clarify for me.
		DR. STRAMER:  That was last year, when we just brought up the Web site.  We didn't talk about using the map or the Web site as a tool for communication.  That was just a tool for reporting.  So there was no encouragement to rapidly report results, which this year is very different, and the AABB Web site is actually only one way for labs to communicate.
		DR. DI BISCEGLIE:  I hear you but what I'm getting at here is whether there is a need for the Agency to regulate the timing as opposed to relying on voluntary reporting.
		DR. STRAMER:  Well, as Dr. Katz also mentioned, if it's in our SOP, you're required, by law, to follow our SOPs.  So I would argue that that's the same.  But I think what we need to do is review the association bulletin and if we can tighten up that language, we can certainly encourage members to enter data within a given period of time to speed up the process.
		I think it's in everyone's interest to do it quickly and I do believe that blood centers understand that, at least the ones that I've talked to, and since the association bulletin has come out, I've had lots of calls and lots of e-mails.
		DR. SIEGAL:  Is there any more discussion?  If not, I believe we can adjourn.  Any objection?  Have a nice weekend.  You had a question or a comment?
		DR. NAKHASI:  That all of you had a chance to comment because, you know, we ask specifically, when we talked about on slides, Maria talked about we are considering this, we are considering that, that you didn't have any other discussion on that, because I know you focused mostly on the ID-NAT trigger, but did you have any comments on the positive predictive value of the, you know, false positives and other things?
		So I just wanted to make sure, because I think, you know, sometimes, because we didn't ask the questions, I just wanted to make sure that there are no comments.
		DR. SIEGAL:  Anyone?
		[No response] 
		DR. SIEGAL:  All right.  Thank you all very much. 
		(Whereupon, at 3:04 p.m., the meeting was adjourned)