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.







     79th Meeting of:













March 19, 2004


  Holiday Inn

        2 Montgomery Village Avenue

Gaithersburg, Maryland








Reported By:


    CASET Associates

10201 Lee Highway, Suite 160

Fairfax, Virginia  22030

    (703) 352-0091




Open Committee Discussion

FDA's Current Thinking on Product Standards, Quality

Assurance, and Submission Requirements for Platelets,


- Introduction and Background, Alan Williams, PhD     1

- FDA Update: Collection of Platelets Pheresis by      6

  Automatic Methods - Current Thinking Including

  Quality Control - Sharyn Orton, PhD

- Laboratory Evaluation of Platelet Components Submitted       26

  to CBER - Betsy Poindexter

- Strategies for Quality Assurance Monitoring      61

  - Alan E. Williams

- Blood Center Perspective on Platelet Pheresis Quality 86

  Control - German Leparc, MD


Open Public Hearing 99


Open Committee Discussion

- FDA Current Thinking and Questions for the Committee      107

- Committee Discussion and Recommendations 108


Review of Site Visit of the Laboratory of Hepatitis and

Related Emerging Agents and the Laboratory of Bacterial,

Parasitic and Unconventional Agents

- Introduction and Overview - Kathryn Carbone, MD    130

- Overview of Office of Blood Research and Review 133

  - Jay Epstein, MD

- Overview of Division of Emerging Transfusion,   155

  Transmitted Diseases - Hira Nakhasi

- Summary Presentation - Edward Tabor, MD      172

- Summary Presentation - Gerardo Kaplan 185

- Summary Presentation - David Asher, MD       202



KENRAD NELSON, MD, Chair. Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland

LINDA SMALLWOOD, PhD, Executive Secretary. CBER, FDA

PERLINE K. MUCKELVENE, Committee Management Specialist. Scientific Advisors and Consultants Staff, CBER, FDA


JAMES R. ALLEN, MD, MPH, American Social Health Association, Research Triangle Park, North Carolina

CHARLOTTE CUNNINGHAM-RUNDLES, MD, PhD, Mount Sinai Medical Center, New York, New York

KENNETH DAVIS, JR, MD, University of Cincinnati Medical Center, Cincinnati, Ohio

DONNA M. DI MICHELE, MD, Weill Medical College and Graduate School of Medical Sciences, Cornell University, NY, New York

SAMUEL DOPPELT, MD, The Cambridge Hospital, Cambridge, MA

JONATHAN GOLDSMITH, MD, Immune Deficiency Foundation, Towson, Maryland

HARVEY KLEIN, MD, Magnuson Clinical Center, NIH, Bethesda MD

SUMAN LAAL, PhD, New York University School of Medicine, NYC

JUDY LEW, MD, University of Florida, Gainesville, Florida




MICHAEL STRONG, PhD, BCLD, Puget Sound Blood Ctr, Seattle WA





LIANA HARVATH, PhD, NHLBI, NIH, Bethesda, Maryland

BLAINE F. HOLLINGER, MD, Baylor College of Medicine, Houston, Texas

JAY HOOFNAGLE, MD, NIDDK, NIH, Bethesda, Maryland

KATHARINE KNOWLES, Health Information Network, Seattle, WA

T. JAKE LIANG, MD, NIDDK, NIH, Bethesda, Maryland

JEANNE V. LINDEN, MD, MPH, New York State Department of Health, Albany, New York

DANIEL MC GEE, PhD, Florida State University, Tallahassee FL

KEITH C. QUIROLO, MD, Children's Hospital and Research

Center at Oakland, Oakland, California

GEORGE  B. SCHREIBER, ScD, Westat, Rockville, Maryland

DONNA S. WHITTAKER, PhD, Lt. Colonel, United States Army, Brooke Army Medical Service, Fort Sam Houston, Texas



P R O C E E D I N G S   (8:00 a.m.)

DR. SMALLWOOD:  Good morning.  Yesterday I read the conflict of interest statement that pertains to this meeting.

I will not do so today.  However, if there are any declarations to be made regarding any items on the agenda, that the committee members may want to make, please do so at this time.

Also, any other participants that are speaking are reminded that, when they speak, they are to divulge any affiliation that they may have regarding the item that they are speaking on, and you will be reminded again during the open public hearing.

At this time, I will turn over proceedings of this meeting to the chairman, Dr. Kenrad Nelson.

DR. NELSON:  Thank you, Dr. Smallwood.  .this morning, the first item is discussion of quality assurance and submission requirements for platelet pheresis.  Dr. Alan Williams will open the discussion.

Agenda Item:  Open Committee Discussion.  FDA's Current Thinking on Product Standards, Quality Assurance, and Submission Requirements for Platelets Pheresis. Introduction and Background.

DR. WILLIAMS:  Good morning. As Ken mentioned, the title of this session is platelet pheresis, product standards, quality assurance, and submission requirements.

The purpose of the session is that FDA intends to develop updated policies regarding product standards and quality assurance for the manufacturer of aphoresis platelets, the formal product name being Platelets Pheresis.

In order to define a regulatory policy that best addresses recent development in the field, CBER seeks the advice of the committee on FDA's current thinking regarding donor selection, component collection, process validation, quality assurance testing, and standards for licensure applicable to aphoresis platelets.

What I am going to do is just take a couple minutes, give a little bit of background, and then introduce the agenda.

Just to give a little bit of scope for some of the new members of the committee, allogeneic whole blood collection in the United States is performed by 1,002 collection facilities that are actually FDA licensed for interstate shipment, collection facilities being individual sites for blood collection. There are actually 146 individual licensed holders, and then they hold the responsibility for multiple collection facilities operating under their SOPs.

These licensed facilities collect about 92 percent of the nationwide blood supply.  There are also 786 registered-only facilities.  Typically these are hospitals, blood banks and transfusion services.

They collect about eight percent of the supply.  They tend to be lower volume operations and, because they are not licensed for interstate commerce, distribute interstate only.

Regarding this session, there are 707 establishments that are registered for manufacture of platelets by automated phoresis.

I didn't do a break down between licensed and registered, but I think it is safe to assume that most of the license holders would, in fact, conduct platelet aphoresis.

Aphoresis procedures are used to collect red blood cells, platelets or plasma components.  This can be done selectively for a single component or in a variety of combinations.

Platelets pheresis production, in a facility of moderate size, would be approximately 800 to 1,000 per month, but there is a wide range of production volumes between different types of facilities.

Nationally, there certainly is a trend toward increased use of single donor platelets collected by aphoresis as opposed to random donor platelets. However, FDA, itself, doesn't track national production volumes.  The AABB and certainly some of the large collectors might be able to supply some of that information.

FDA, specifically the division of blood applications, does receive and review licensed supplements.  Regarding manufacture of aphoresis platelets in 2002, we received 77 submission.  Thirty-six of these were prior approval supplements, and 41 in a category known as CBE 30, which is just a little different category of supplement submission.

In 2004, to date, we have received 10 PASs.  That just gives sort of the range of license supplements that are reviewed within the agency.

The Division of Blood Applications administers these applications, and they are largely reviewed by the division of hematology.

As with any regulated manufactured product, there are minimal product standards established for content and quality.

These can both be derived from the labeling of the devices that are used to produce the product -- i.e., the automated aphoresis machines -- and there can also be in our regulatory and industry standards for the manufactured blood component itself.

Conformance to the standards is assessed by quality assurance monitoring, and this can be defined at several different levels.

Clearly, today we are talking about the FDA input into quality assurance monitoring, but industry also maintains standards which may be more rigid, and there are local SOPs that can vary and be more rigid than the regulatory standards.

The goal for this session is to look at the range of quality control options, and I think you will hear some interesting divergence of perspectives on the role and the burden of quality control for this product.

FDA's interest is to find the least burdensome approaches that are scientifically and statistically sound, and that is what we are asking for your help in.

The agenda for the session, the next talk with be by Dr. Sharyn Orton in the Division of Blood Applications, who will provide an outline of FDA's current thinking regarding collection of platelets pheresis by automated methods.

The next talk will be by Betsy Poindexter in the Division of Hematology.  As many of you know, CBER receives platelet components for quality control testing in CBER's own lab, and Betsy is going to review the procedures in their laboratories, and some of the findings on submitted components.

I am going to discuss some sort of general aspects of quality assurance monitoring, the role of sample sizes in different strata of the manufacturing process, and provide some of the current thinking as far as statistical process control and the evolution within the agency with respect to blood components.

Finally, as a scheduled speaker, Dr. Herman Leparc from Florida Blood Services is going to give a blood center perspective on quality control for platelets pheresis.

The question for the committee is a little bit broad and, prior to the question being presented by Sharyn, she is going to just recap some of the quality control elements that we want to put forward for your consideration.

The questions for the committee will be:  Does the committee agree that the proposed recommendations for quality control testing are adequate to assure quality of platelets pheresis.  If not, please comment on alternate approaches to quality control for the product.  Thank you.

DR. NELSON:  Dr. Sharyn Orton from FDA.

Agenda Item:  FDA Update:  Collection of Platelets Pheresis by Automated Methods:  Current Thinking; Quality Control.

DR. ORTON:  Good morning. I am going to be talking about the collection of platelets pheresis by automated methods, the current thinking, including quality control.

I am going to talk a little bit, give you some background on the format of the guidance document that is in draft right now within the office of blood.

As you know, this draft guidance has been being worked on for several years.  I want to mention that Betsy Poindexter has spent a tremendous amount of time working on this document, as have several individuals in the division of blood applications in the blood and plasma branch.

Back in the fall, when I was doing a detail in the blood and plasma branch, I had the opportunity to take a look at this document, and thought that I could help with the document because, for those of you who don't know, I am an ex-med tech with a lot of experience in validation and quality control of these products from the many years that I worked in blood centers.

Right now, the format of the guidance, it covers quite a few things.  We tried to be as inclusive as we could on things that we thought were important.

So, it covers donor selection, collection and management, and breaks out the selection, the donor management component of collection and management, it talks about dedicated donors and required medical coverage.

It covers information provided to donors, including informed consent.  It breaks out process validation to a separate category, and includes our recommendations for what should be included in a validation protocol, equipment device installation qualification, the operator performance qualification, component performance qualification, and re-qualification of the process.

There is another section on quality assurance and monitoring.  This section will cover component QC, equipment and ancillary supply quality assurance, operator quality assurance, SOPs and record keeping, and there is a fairly large section of the SOP specific, either requirements or recommendations.

When I say requirements and recommendations, it is not uncommon for some of the requirements that are in the regulations to actually not be included in some of our SOPs.  So, we are giving some bulleted guidance to make sure people include them in these SOPs.

There is donor monitoring, including donation intervals and total collections per year, adverse events, things like that, and then the use of quality systems audits.

It covers processing and testing, labeling, registration and licensure, and it goes into the different submission types, what to do if you want to submit for an alternative procedure.

It includes what documents need to be included in the submission. It talks about, if you are going to have concurrent component selection of red cells or plasma, what would be needed in an additional submission, and the component submission, and the submission to CBER for quality control, what those requirements are, and shipping information.

Now, this document has a few things that I wanted to point out that are different from the 1988 guidance document.

One of the things that this document does ask for is predonation platelet count for all collections.  One of the big concerns the division of hematology has voiced in the use of the default mode on these automated instruments.

So, in trying to find a way that would avoid that, we determined that the easiest thing to do would be to recommend that a count be done on all collections.

Now, we do allow for a post-donation count to be used. It did come out, what about mobile blood draws where someone shows up for the first time, and we do make provisions for that as well.

There is the addition of deferral for taking plavix, and we do give some guidelines for targeted platelet yields for both double and triple collections.

One of the other concerns voiced by the division of hematology is that, when components have come in, particularly the doubles and the triples, there is concern that the target count hasn't been high enough. So, in fact, some of the bags are split. Some of the bags have counts that are less than 3.0 times 1011 as their absolute platelet count.

The criteria for red blood cell and plasma loss has been updated in this guidance document.  The criteria for the validation protocol, which there is a lot of information in the guidance document, also includes incorporation of tolerance limits as designated by the manufacturer, and it will include specific numbers to be tested for each parameter and the allowable process failures for that.

There is reference to the use of failure investigations and determining whether something is a process or a non-process failure, and the use of quality system audits.  It does actually make a few recommendations for specific audits.

Now, for the component performance qualification, or what we know as validation of the actual component, the recommendations are going to include the absolute platelet count, pH, volume, and residual white blood cell counts.

What we are recommending for the number to be collected is, for single collections, it would be 60 consecutive collections, for doubles it would be 30 consecutive, with both bags being tested and, for triples, it would be 20 consecutive, with all three bags being tested.

We are also making a recommendation for sterility or bacterial contamination being done on 100 consecutive collections.  In fact, the 1988 guidance document does recommend sterility testing during validation.

Within the criteria, the absolute platelet count would not only include a minimum of 3.0 times 1011, but also that the manufacturer's designated maximum also be assessed.

When we do get some of the quality control in, there are a few times when the absolute platelet count per bag is exceeded, and we would like to have this assessed more carefully during validation.

For pH, we are recommending not just assessment at 6.0, but also at a pH of 6.2.  There is a lot of literature that addresses the fact that, below a pH of 6.2, you begin to lose viability of the platelets and, below 6.0, we know that there is a lot of damage and the platelets are not as viable. So, we would like to see both of these assessed.

For volume, we are including the minimum and maximum per the manufacturer but, in the volume split, for a double, that 50 percent, plus or minus five percent, be in each bag and, for triples, essentially a third, plus or minus five percent, in each bag.

For the residual white blood cell counts, it would be per the manufacturer's specifications, because they do vary quite a bit from manufacturer to manufacturer.

For the quality control testing, right now there are three documents, or three pieces of information that we use.

One is a recommendation.  This is the revised guidance for the collection of platelets pheresis 1988.  It recommends that the platelet content of each unit should be determined.

FDA has determined this to be the daily product specification check.  We know that the absolute platelet count is determined on every platelet pheresis collection that is drawn.

For regulations, 21 CFR 640.25(b) states:  Each month four units prepared from different donors shall be tested at the end of the storage period as follows:  platelet count, pH of not less than 6.0 measured at the storage temperature of the unit, and the measurement of the actual plasma volume.

FDA has interpreted this to mean that these four units apply per collection site, per machine type, and per collection type -- single, double and triple.

So, for each collection site and each machine type, we want to see four single, four double, four triple. At the end of the storage period, we have interpreted that to include at time of issue.

In the memoranda, recommendations and licensure requirements for leukocyte-reduced blood products in May of 1996, the recommended QC testing includes that these be performed using a sampling plan that includes one percent of monthly production, or four per month for establishments producing less than 400 units per month, and that all units tested meet less than 5.0 times 106 residual white cells.

FDA has accepted one percent, or a minimum of 20 units per month, on the collection, or essentially the parent bag.

Now, the acceptable limits have been at issue or outdate, the absolute platelet count, a minimum of 75 percent, have an absolute count greater than or equal to 3 times 1011, and then 100 percent have a pH greater than or equal to 6.0.

For the residual white counts that are done after collection, that the residual white count be 100 percent less than 5.0 times 106 residual white cells.

Our current thinking is that the absolute platelet count and pH requirements will remain unchanged. The residual white cell acceptable limit recommendation has been modified to 100 percent having less than 5.0 times 106 residual white cells or, per manufacturer, if the claim is less than 100 percent at 5.0 times 106.

It turns out that, two of the manufacturers that we have, the manufacturers at a 5.0 level do not claim 100 percent, and that is why this modification has been added.

We are also recommending the following:  Like with validation, the maximum absolute platelet count per bag, 100 percent of them meet the manufacturer's specification, and the same with the minimum or maximum per bag, per the manufacturer's directions.

The calculation, again, with the volume separation for doubles and triples, the calculation should be done so that 50 percent, plus or minus five percent of the volumes, be in doubles, and 33 plus or minus five percent for the triples.  Again, assessment of pH at 6.2.

QC failures should be evaluated as process or non-process failures.  What I mean by that is, as an example, we know that with the leukoreduction filters, there are times when the filter does not filter, in sickle cell trait, for example.

You could, in fact, have your process completely in control and actually have a failure in the residual white count that is related to the donor.  That would be considered a non-process failure.

However, if someone does not draw a platelet count properly or something like that, that would be considered a process failure.

So, when you have a QC failure, a very thorough investigation should be done as to why you didn't meet the minimum targets.  It is also very important, and we are recommending, good tracking and trending.

Now, for double or triple collections, where the component you have tested for QC does not meet acceptable limits, what I mean by that is, you have a double pheresis, you have taken one of the bags -- we will call it bag A -- you have done your QC and it doesn't meet your minimum target.

We are recommending that the corresponding component, which would be in the case of a double bag B, would need to be tested and found to be acceptable.

We are also recommending the use of quality system audits.  Specifically, I wanted to mention two that we are going to recommend.

One is the volume separation.  I know that some blood centers are doing QC on both bags for doubles and all three bags for triples, and it is not being done consistently across the blood centers.

One of the concerns, and one of the reasons this has been requested, in some cases, is the fact -- and Betsy will give you some data on the QC of components that come into the division of hematology, is that there can be some widely varying volumes between bag A and bag B on a double, or A,B and C on a triple.

There has been some concern that these are, in fact, being split properly.  If the target value hasn't been set high enough, you could, in fact, end up with a platelet count that is acceptable in one bag and not acceptable in the other.

So, we are recommending, for an audit that, based on the volume of pheresis that you draw, that you do an audit of volume separation.

So, you can do larger sample sizes, to really be sure that the empty bad is tared properly, the actual calculation for the volume is done correctly, that the split is done accurately.

The second audit that we are recommending is ongoing sterility testing or bacterial contamination.  This has to do with, the regulations state that platelet pheresis, or platelets are exempt from sterility testing, but there has been a huge amount of literature, as we all know, about the problem with bacterial contamination.

We feel that it is prudent to recommend, at least in an audit system, ongoing sterility testing or bacterial contamination testing.

Now, I want to stress that we believe the regulations represent a minimum standard for QC.  FDA is still considering other scientifically and statistically found QC plans, while also considering the burden to blood centers.

As Alan mentioned, he is going to be speaking about statistical sampling plans. Specifically, I believe he is going to touch on the residual or white blood cell testing.

You will notice that, when I talked about QC, I didn't mention any number that you have to test. That is because I believe he is going to address this more thoroughly.

Now, two documents that we have used to help us with this guidance document -- and I think it is very important for blood centers to do the same when they are developing their protocol, both for validation, setting up their quality control and monitoring plan -- is the guideline on general principles of process validation from May of 1987, and the guideline for quality assurance in blood establishments from July of 1995.  These can be found on the CDRH web site.

I also want to, while I have the opportunity, mention one other thing. Because both the division of blood applications and the division of hematology are dealing both with the manufacturers and the blood centers, recently there has been a lot of confusion when it comes to telephone calls coming in and out, and kind of who is talking to who and when and where.

I wanted to point out to everybody that CBER has an SOPP 8104, and here is the location of that document.  It is specifically called, telephone contact with regulated industry.

So, we are asking, and what I want to stress today, is that all contact with CBER should be set up through the regulatory project manager or consumer safety officer.

In that way, we can assure that the right people are involved, that all the people that need to be involved in the conversation are there, the minutes of these telephone conferences are documented and you get a copy, and things like that.

So, for the blood centers, if you need to talk to us or to the division of hematology, please call me, and my phone number is here, and I will makes sure it gets set up with the appropriate consumer safety officer, and individuals from the division of hematology if necessary or appropriate.

For the aphoresis device manufacturers, prior to or related to a submission of a device, Dr. Sayah Nedjar is the branch chief in the regulatory project management branch.  He would do the same thing. He would make sure that one of his staff gets the meeting set up and includes all the appropriate individuals.

For devices that have already been cleared and the manufacturers want to talk to us about upgrades, again, that would come through the blood and plasma branch. So, again, call me, and I will be sure that someone from the blood and plasma branch gets the meeting set up with the appropriate individuals, including individuals from the division of hematology, if necessary or appropriate.  I think that is it. Thank you.

DR. NELSON:  Any questions for Sharyn?

DR. LINDEN:  Sharyn, thank you very much for the overview.  Could you please elaborate on the internal audit system of the volumes that is separate from the initial component qualification, and separate from the QC?

Is the guidance going to give recommendations for the number of samples and the frequency of this testing of the volumes?

I am gathering that what you are saying is that there is supposed to be testing of a certain number of volumes and this is going to be analyzed as some sort of audit.

DR. ORTON:  Right, and the sample size for an audit should be relative to your collection size. So, outside of -- we can say you should use a statistically sound plan to do that.

We don't have specific numbers in there, again, because the numbers that are drawn at different sites varies dramatically, but it should be ongoing, again, it should be statistically sound.

The volume split has to be done and calculated appropriately in order to get the absolute platelet count. So, that has to be done during the validation or the qualification of the component, in order to get the absolutely count, and the same for QC.

So, essentially, you are doing, for the single, 60 during the validation and the 12 per month at each site.  This is in addition to that.

Clearly, if you are drawing hundreds of these, looking at four or even 12 really is not -- we are not sure that it is getting an adequate picture of whether this is actually being done properly.  No, we didn't give specific numbers more because of different collection sizes at facilities.

DR. LINDEN:  And the frequency of these audits?

DR. ORTON:  It should be ongoing. Certainly, I can only speak from audits I have done in the past.  We would set up monthly audits.  If we found that a process appeared to be in control over a period of time, then we would go to random auditing.

Most places now, under a quality program, have an audit system, or should have an audit system in place.  What we are saying is that this kind of evaluation should be incorporated into that quality program.

DR. SCHREIBER:  Is the goal to have process evaluation as opposed to product evaluation?  For example, on pH, from the paper that was in the package, there is certainly a lot of deviation or variation, even at much higher pHs, and you have set your cut at 6.2, which seems reasonable.

It looks like, from the graphs that are in there, there is a significant number of units that have much lower recoveries at higher pHs, and also the half times are a lot lower.  So, there is considerable variation that you are not testing for.

DR. ORTON:  The pH and viability issue, over time, really is more an issue for validation of the bags. One of the other things that I believe is in that paper, over times there have been different ways to store the platelets, whether it be on a rocker or rotator, and a lot of the variation had to do with that as well.

Most of the more recent literature will show you that the pHs on these, in general, are quite high and they are maintained over time.

However, again, I think Betsy will show you, with some of the data that they have from the division of hematology, she is finding what she considers an unusual number that are lower than 6.0.  Because the literature does address the viability at 6.2, we thought it was prudent.

Yes, overall, we want to look at the entire process, some of which, if it has to do with the validation of the bag and really isn't within the process validation that is going on at the blood center itself.

DR. LINDEN:  A related question, still on these audits, you are auditing the products. You are weighing the products. You are not like looking at the scales and revalidating the scales.

In an audit process, your scales should be validated.  There is a whole equipment qualification section to this.

When we are talking about the volume split, what we believe is a problem is the empty bag isn't tared properly.  The split is not done accurately.  We don't know why it is. I don't know what every blood center is doing.

The idea is that, on a day to day basis, there should be some method so that this is being done properly. The only way to do it is to either make sure that you document every single one, or that you audit the process and the operator and the fact of, was the tare done properly, was the split done and weighed.

So, you are saying that it is the entire process. You are looking to see if your personnel are following the procedure properly, are the scales working properly, as well as weighing the product.  So, it is the process as well as the products.

DR. ORTON:  Correct.

DR. LINDEN:  Okay, that wasn't clear to me.

DR. QUIROLO:  Can you just elaborate on the sterility testing and what your thinking is on that?

DR. ORTON:  Could you be a little bit more specific?

DR. QUIROLO:  I can't be, because you just said -- there is one line in this about sterility testing.  What is the guidance going to be for that?

DR. ORTON:  The guidance for sterility testing is that, for validation, 100 consecutive units should be tested by a cleared device.

We know that, as I said, the regulations have exempted platelets from being sterility tested over time. I actually went back, got a copy of the 1975 preamble to see why that was decided.

It was decided because the platelets are drawn in a closed system.  So, they determined that sterility testing wasn't necessary.

Now we know that that isn't entirely true, that between arm contamination and perhaps asymptomatic septicemia in the donor, we are seeing bacterial contamination.

We know the AABB has moved forward with bacterial contamination being a standard for them.  So, we thought at this time it was prudent to not necessarily specify a number, but incorporate that as an ongoing monitoring.

In fact, the blood centers are going to be doing that.  So, this was a good time to incorporate it as well, and because we thought it was scientifically important.

Now, again, as far as the size, the numbers that we want, a lot of that has to do with the size of the collection facilities.

So, we are trying to give the collection facilities some control over determining the appropriate sample size for themselves.  We will give some guidance to it, but not specific detail.  Does that answer you?

DR. NELSON:  You don't specify the method of testing for bacterial contamination. It is just a method; is that it?

DR. ORTON:  Like I said, I didn't get into every detail of the document because it is very large at this point.

If a device is cleared for QC for bacterial contamination, we clearly want them to use a cleared device.  So, it is not just any method. There are recommendations in there for the method.

DR. SCHREIBER:  There is one statement in the recommendations that says, the FDA has accepted one percent or a minimum of 20 units per month on the collection, parent.  I don't quite understand what that means.  The above part talks about a minimum of four.

DR. ORTON:  There isn't a regulation for residual white count.  The guidance document that is in place, you want the residual white counts to be done before the product is labeled as leukoreduced.

So, the devices that are cleared generally have 24 hour or 48 hours, these need to be done.  Those numbers, as far as one percent or 20, is what is in the guidance document.  There are no regulations that talk about residual white counts.  That is why the four doesn't necessarily apply.

DR. STRONG:  I just had a question about any of your rules that say 100 percent. It seems like to be perfect is asking too much.

DR. ORTON:  I think you guys are perfect. What do you mean?  That is certainly open to conversation.  We do believe that if a manufacturer says, this is the target you should be meeting, if it really should be 95 percent, we certainly could discuss.

I think the idea is more being that the manufacturers and some of these operators manuals are really quite specific about what the specifications are and what you should be meeting as a target.  I think it is more that we are trying to mirror that than demand perfection.

DR. NELSON:  Next is Betsy Poindexter, who will discuss laboratory evaluation of platelet components submitted to CBER.

Agenda Item:  Laboratory Evaluation of Platelet Components Submitted to CBER.

MS. POINDEXTER:  Good morning. Obviously, Sharyn prefaced some of what I was going to say.  We will be talking about some of the instrumentation that is available for platelet pheresis collection and other product collection, the products that are available from those devices, quality control as the centers might do it, and how we do it at CBER, some overview of the sample submission over the last eight years, some conclusions and some references that have already been cited by Sharyn.

There are a number of manufacturers with a variety of instrumentation out there.  These are what are currently available in the United States.  They collect a variety of products.

I am only going to concentrate on the platelet products that can be produced.  Many of them produce single products.  A few produce singles and doubles, and there are a couple of devices that produce single, double and triple platelet pheresis products, some leukocyte reduced by the end process, leukocyte reduction by the centrifugation and gradient separation of the products, and other products that need to be leukocyte reduced by filtration.

There are a variety of types of leukocyte reduction in place at the blood centers. There are integral in line filters with some of the kits that are used for aphoresis collection.

They are integral in the continuous flow filtration, for example, on the Hemanetics filtration plus devices.

There are post-processing filters that can be attached with a sterile connecting device to filter the whole product at the end of the procedure, and then there is the end process leukocyte reduction.

Product quality is defined in 21 CFR 210.1, the manufacturing, processing, packing and holding of a drug to assure that the drug meets the requirements of the act as to safety, and has the identity and strength and meets the quality and purity characteristics that it purports or is represented to possess.

Blood centers routinely perform volume platelet count, which allows them to then calculate the product yield, pH and white blood cell count, to determine the residual white blood cell count on the leukocyte reduced products.

We also encouraged blood centers to do white blood cell counts on their non-leukocyte reduced products, to determine whether the collection process was adequate or not.

At CBER, we do all of our quality control testing at expiration.  As Sharyn mentioned earlier, the blood centers frequently are doing their quality control at the time that they are issuing the products to hospitals for transfusion.  So, that QC frequently occurs at day two or day three.

On rare occurrences -- maybe more than I am aware of -- they do the quality control on expiration.  I believe that, due to the cost of these products, that is probably a rare event.

They do pH at -- we do pH at expiration.  We do the platelet count at expiration. We re-weigh all of the products and calculate the bag weight specific for the device that is being used, to subtract that and do the calculations.

We do not do residual leukocyte counts, because we don't have the product as fresh as what it needs to be to do that, plus we don't have the upgraded image at hand.  We have the one that was taken off the market.

In addition to those parameters, we measure the temperatures, particularly those that are very cold or very warm to the touch as we box them.

We inspect the products for clumps or fibrinous material, for red blood cell contamination, evidence of swirling, excessive air in the products, proper product code, expiration date, proper product name, placement of labels -- and that is important because there was a time when the labels were being placed on the back side of the bag, in addition to the base label that was already on the front side, which then reduced the oxygen transport through the bag, the obliteration of the license number of the facility, unless they have a CBE and a comparability protocol that would allow them to ship within 30 days after notification.

We compare our data to the blood center's data, and we check how many containers might have been used to store the particular product, since many of the manufacturers specify when you need to actually store a single product in two containers.

This is some data.  I have gone back through our data base, and these are the product failures. This is not meant to only produce the bad news.  A couple of slides later, we will have the good news.

This tells you the number of samples we received, the pHs that we were measuring, the concentrations, the volumes that failed.

That means, in the volume column, that means that we were off 25 to 68 ml per container, and the number of products that we received that were out of the 20 to 24 degrees centigrade shipping or storage conditions.

There are occasions that we received products that are five to 10 degrees that obviously suffered some cold effect.  We have also, in the summertime, received some products that were in the 26 to 28 or 29 degree range.

You can see that, in 1995, we had 18 that failed on pH, 22 that failed on concentration, and 42 on volume.  My division director asked that I put the average and the standard deviation so that you could see what those ranges might be.

In 1996, we had more fail on pH and in 1997, we also had a good number fail on pH.  Frequently this is associated with either them coming in very warm, or they are overwhelming the bag system that the manufacturer has provided by putting too little volume, too much concentration in one particular container.

Again, you can see that we are still having some concerns about the concentrations and the volume discrepancies.  This year was a bumper crop year for receiving hot and cold specimens.  We will move on from there.

This gives you data for 1998 and 1999, as well as 2000.  These numbers appear to increase dramatically in 1998.

I believe it was the introduction of different technologies and different storage containers for some of the blood centers, but they were using it as they had used it in the past.  They were overwhelming the system.

We also had a huge number of concentration differences, and this was because centers that had already been licensed for single product collection were not opting to collect double and triple products and, because of volume products, or perhaps distribution between part one and part two, were having problems meeting the minimum concentration range.

You can also see that we had an increase in the products that failed to come in at a proper storage temperature.

In 2000, we see a reduction.  This is because, in previous times, when we got products where the volumes were off tremendously, and that compromised their calculations for their concentrations, we were asking them for their SOPs and their procedures for weighing and counting the products. Then we were asking them to re-submit.

Once they appeared to learn that lesson, those numbers have dropped considerably. You can see, even though they passed on concentration, we were still having volume issues.

So, we have, in the past number of years, irrespective of the fact that the concentration might have passed and the concentration might have passed, we are failing them because their volume measurements are not plus or minus 10 percent and, on some occasions, like this particular one, where they half of the volume of total collection on one bag.

The transfusion service that might have received that would have pooled those two containers into one product, and written down on the patient's chart that they had received 219 mls of volume where, in reality, they had received closer to 450 mls.  In the year 2000, we had still some volume problems.

In 2001, you can see we are still operating on some products not meeting the concentration. We still have an apparent volume issue. In 2003, that volume issue seems to be resolving itself, again, because we have become more heavy handed about what is acceptable and not.

Consider the fact that these products are collected by the blood centers, and they are running the donors to the FDA.  So, these are data from our data bank, 2002-2003 time frame.

These were the pHs done at the blood center proximal to the time of shipping their products to CBER.  Our guidance document, since 1981, has asked the blood center to report what the pH is, at the time they are getting ready to ship it to us.

Eight to 10 percent didn't report the pH at all.  So, we have no idea what they thought they were starting with.

Sharyn mentioned we are having some concerns about high pHs.  These are pHs that are in what one might consider above the normal range. Note the 8.3 that came in on a number of products. This was not a one-time event.

A good number of them are falling well within what would be considered normal for the collection process, the storage bags, and the concentrations that are stored in those bags.

There is still a sizeable number that are coming in at values that are -- this would be between 6.5 and 7.0, but those would be still acceptable pHs.

These were values that were on the tie tags from the blood centers, that the pH, at the time that they measured it, prior to shipping to us, was between 6.14 and 6.5  That would lead one to believe that perhaps their method of evaluating the pH is not accurate.

The concentrations or yields at the time of shipment to CBER, these are the blood centers' results again. This is that same data base.

There were 70 that were not reported to us.  So, we had no idea what the blood center thought they had in the container.

Four actually sent us products with substantially less and, on a previous edition of these, I had the ranges, but they ranged from about 1.74 to 2.75.

So, they sent us products that didn't meet the minimum standard to start with, expecting that they might pass after we evaluated them?

In reality, of those four samples, three of them did pass.  The yields that the blood center calculated were incorrect.  One of them, the one that was 1.74, truly did not meet the minimum standard.

A good number of them, approximately 10 percent, come in at this cut off range, 3.0 to 3.3 times 1011.  These numbers include singles, doubles and triple products.

So, you can see where, if you had a double or a triple perhaps, that if this is where you thought you were when it left the blood center, that after a couple more days of storage it might not quite meet the mark when CBER is evaluating it, and these are the other concentration ranges.

Those that were extremely high for single products -- these are single products -- the blood center just ran it, my guess is in a default mode -- with someone who had a 450,000 platelet count, and ran it for the maximum amount of time, maybe 60, 75, 90 minutes, and really skewed the curve.

The good news.  Of that same data set, these are the passing products and the mean pHs and the standard deviations of those products, and the mean concentration or yield, and the standard deviation on those products.

Although the last couple of slides were referring to the product failures, and perhaps process failures within the blood centers, in reality, a good portion of the samples that are submitted here to CBER do pass.

Can the yield or concentration, at the time of distribution from the blood sample, its distribution or submission to CBER, predict its outcome?

Again, those four that came in with less than three times 1011 platelets, three of them actually passed on concentration.  One of them failed.

Of those that came in at what I will call a borderline concentration, 64 of 300 failed on concentration.  Eighteen of those 64 were volume related, 19 of those being single products, and 45 of them being portions of double or triple product collections.

Those that are over the edge and probably quite substantial products, a little less than 10 percent of them failed, probably closer to eight percent of them failed on concentration.

Nineteen of those were singles. Again, we have this bag weighing issue. Twenty-six of them were portions of doubles and nine of them were triples.

These nine triples were three sets of triples where all three of the products did not meet the minimum criteria.

Sharyn alluded to this.  This is a problem with the volume distributions between parts A and parts B of a double collection.

These were the volumes that we calculated by weighing the total bag and taring -- subtracting the bag weight from each of these, and doing the calculations, and these are based on our counts rather than the blood center's count.

You can see there was a 47 ml variation here, and the effect that it had on the final yield.  Although both of these products passed, one patient should have gotten a better effect than the other.

This is a situation where there was a 50 ml, approximately a 50 ml, volume difference. You can see the effect on the concentration.  One is a perfectly acceptable product, and the other one is a less than standard dose.

This is not to say this can't be used for transfusion, but the blood center, if they know that concentration is 2.5 times 1011, they should put a different label saying this is different than standard content, and put a tag on the product that tells the physician that this product contains 2.5 times 1011, a 15 percent difference from a standard product. So, if they don't get quite the bump from their patient, they will know why.

This is my slide just to say that, here is our target, and what a difference one bag weight or two bag weights might make at a blood center, if they thought they had 260 ml, but they had forgotten to tare for both of the bags.  They would have thought they had a 3.9 where, in reality, they had a 3.0.

The same if they had forgotten to tare for the one bag, the difference that it will make. This is assuming that they thought they had a 200 ml collection where, in reality, they didn't tare for one bag or two bags, and the drastic differences in the concentration.

Each of the storage containers by the manufacturers have a particular weight.  Some weigh more than others, and the scales or balances should be tared for the particular bag that is in use.

So, this is sort of commentary to what the blood center should be looking for.  They should investigate their processes and investigate their product failures.

They should listen to comments from their processing staff, or listen to comments from other blood centers, who might be having similar problems, particularly in state organizations.

If they are having problems meeting the target goals, ask the device manufacturers for assistance. They are more than willing to help you through this process.

They should review their training practices to assure competency of the staff, performing each step of the process.

A particular situation comes to mind where a major collection facility transferred the weighing of the products -- weighing and labeling of the products -- from a nurse to a processing room person.

The nurse knew, by rote, that she needed to tare the weights for the bags that were being used, the collection.

When the blood center submitted their products to CBER, all their weights were off by 30 to 60 mls, grams. I know that they are not the same but let's, for this conversation, say they are.

The nurse had not written that step into the 1, 2, 3 processing for the processing room person.  So, all of their weights were off by one bag weight or two bag weight.

It wouldn't have been bad if this was just one site, but this was a collection center where they had a number of collection facilities, all of their products coming back to a main site for processing.

It turned out that all of their products, irrespective of whether they were from city A, B or C, were coming back to D for weighing, and all of their weights were off.  They had to go back and retrain their people.

This is sort of an off take on Sharyn's quality control, should the testing interval be redefined.  We have talked about this for leukocyte reduction.  Should monthly become weekly, so that we perhaps capture these processing errors sooner rather than later, to perhaps reduce the potentially unsatisfactory products, if a product is found to be out of control.

Certainly, if it is out of control, determine the root cause.  Is it a device malfunction, or is it a human problem?

So, in conclusions, centers should be carefully following the operator's manual. They should equally distribute the collection volume between the storage containers.

They should tare the balance for the correct number of storage containers, and that is a frequent problem.

They should adhere to the manufacturer's limits as to the product yield or platelet concentration, and the volume limitations of the particular storage containers recommended by the manufacturer, and they should enter real donor data rather than running in default mode.

Sharyn mentioned some guidance documents, and these are reiterated here, and the draft guidance for leukocyte reduction also.  These are points of contact and e mail addresses for people in our testing lab.  Thank you very much.

DR. NELSON:  Thank you.  Any comments?

DR. HARVATH:  Betsy, I wondered if you would comment, if there is a correlation in the pH problems observed in the testing lab, between the introduction of leukocyte reduced products, whether you have seen any correlation between residual leukocyte count and a pH problem in the platelet products.

DR. POINDEXTER:  From our data, there would appear to be a relationship between the earlier slides, where there were higher numbers of pHs that did not meet the minimum criteria, but that was prior to leukocyte reduction being in place in most of the instrumentation.

We have also seen a correlation between the increase in the pHs over time, up to -- the highest value we have gotten is 7.99 in products that were collected after leukocyte reduction had pretty much gone across the board in the three major device manufacturers.

DR. CHAMBERLAND: I may have missed it, but can you review for us what the procedure is for submission of samples to FDA for testing, what the requirements are?  Does each licensed facility have to send you X number of units per year?

DR. POINDEXTER:  No, a licensed facility that wants to change from one type of collection process to another, either a device change, Baxter to Hemanetics, or a product change, from a single to a double and/or a triple, is required to submit a license amendment to us.

One of the requirements of that amendment is that they ship two products, two singles, two doubles, both portions of the doubles, or two triples.

If they are coming in from singles, doubles and triples, we ask them for two singles and two triple sets. It is one time, until the next time that they decide that they want to buy the new little red car, I was telling my division director last night.

DR. CHAMBERLAND:  So, this is not any sort of national sampling or anything like that.


DR. EPSTEIN:  I wanted to make a similar point, that we are looking at new facilities, new procedures, et cetera, but the related question, then, Betsy is, do we have any insight how the failure rates that you are finding with changes in procedure, change in facility, correlate or correspond to the failure rate that is being found in routine quality control on site by facilities that have been approved?

DR. POINDEXTER:  The only quality control that we receive from the blood centers, with their submission, is two months worth of quality control.

We do call them into question occasionally where they have pHs that all read 6.5, 6.5, 6.5, 6.5.  One would think, perhaps, that they weren't running them, or they were running them on a method that was less sensitive.

We don't see their validation data.  That is one thing that we may ask for with the new guidance document.  The quality control that they submit to us, generally, is spot on but, again, we know we have weight problems and, when we question them about that, they do try to go back to their centers and retrain their processing room staff.

DR. ALLEN:  Does this data get fed back to the FDA field staff who go in to do on-site inspections in the blood centers?

DR. POINDEXTER:  Not that I am aware of.  Actually, we have had some conversations with people recently where, when we do request, through review letters, that the center send us their method for weighing and send us their methods for determining pH, that we might send those letters to the office of compliance, so that the field investigators would know to look for that.

One of the concerns that we have is that they make a paper correction and send us what we want to hear, but perhaps haven't actually put those corrections in place across the board.

DR. CHAMBERLAND:  If you find deviations from what is acceptable as part of these new application processes, do you require, then, a subsequent submission from the blood collection center, to see that they have remedied the problem?  I presume they have to do some sort of an investigation and try and determine what the root cause was.

DR. POINDEXTER:  If they fail on concentration, frequently it is because they have targeted a range that is too low, perhaps, to adequately meet that target.

The targets are, at best, plus or minus 15 percent, according to most of the manufacturers.  You have to have the best possible use of the device.

So, we do allow them to re-submit samples that might have failed.  So, if they fail on a double, they re-submit another double.

We have just had discussions as to whether, after a certain point -- for example, the second submission that might fail -- whether we should just to back to them and say, okay, if you have your license number on these products, you should not be sending these products out of state, and you have to do a thorough failure investigation.

Choosing two products out of 1,000 products that you might produce, and sending them to CBER, you may have actually randomly selected products and they just happened not to meet the bar.

When you are particularly putting a donor on the machine with the intention of sending it to CBER, one would hope that you were trying to meet the mark.

DR. STRONG: This is one of the most frustrating areas for blood centers, in dealing with QC for platelets, particularly now with the advent of new clinical hematology analyzers.

The analyzers that we used for platelet counts were not designed for this kind of quality control, and many of them don't work in the ranges of concentrations that we use.

So, if you look at proficiency data across different counters, there is an enormous difference from one counter to the other.

I know for many centers it has been very difficult to try to standardize that.  So, despite the fact that we go through competency assessments on a regular basis, proficiency testing with various CAP and other kinds of control measures, we get inspected by CLIA, AABB, FDA, everybody in the world, and somehow we pass those, there seems to be still an enormous number of samples that fail quality control when they get to the FDA.

This raises the issue of how do we standardize to do better. Some of this data, I suspect, has to do with the variability, just in the kinds of measurements that we are using.

So, I am wondering about, for example, what kind of platelet counter you use. I know the one we recently bought, we are having a hard time getting the same numbers as other people get, because it tends to count on the low side.

We have been using Baker's over the years, and that is no longer available, and that gives a totally different count than the newer analyzers.

Most of them are using flow cytometry principles and they have software that varies, and it is a secret how they are calculating the measurements.

I think this is one of the areas that is really frustrating for blood centers. I am just wondering about how you standardize your laboratory.

Do you go through CLIA inspections and proficiency examinations and all that sort of thing?  How do we get to a standard in which we can actually compare?

DR. POINDEXTER:  Two things.  One, I, too, was using a Baker, we were using a Baker from 1988 up until this January, when it literally had a stroke and we pushed it out the window.

In anticipation of that, and in anticipation of the center becoming more ISO oriented and standard oriented, we did buy an ABX Pentra 60C-plus, I believe, is what the number is on it.

We have particularly selected machinery that has been cleared through CDRH to count platelet rich plasma, not whole blood clinical samples from patients in hospitals, but particularly cleared to count platelet rich plasma and platelet rich concentrates in the anticoagulant solutions that are available from blood bank samples.

We did side by side runs of our Baker 9000-plus dif that we had, and had excellent use of it for 15 years, and the ABX, until the Baker died.

We ran a correlation -- we were running them side by side.  The same sample was being run in both devices in tandem, and we found that our counts were five to 10 percent, within five to 10 percent on each device, not one counting higher than the other or lower than the other.

There were some samples -- and I believe that probably has to do more with the mean cell volume of the platelet, whether they were really tiny platelets maybe cut off by the lower threshold of some of the monitoring devices, and larger platelets that occur infrequently in some donors, but frequently occur in lower pH products, maybe count eliminated by the higher thresholds that are available on some of the devices.

The agency, or the center, over the last year or two, two years, has been trying to set up ISO standard type labs.

We do run controls with all of our samples that are provided by the device manufacturer.  On the Baker 9000, we participated in some best committee studies, where they were doing just what you were speaking about, comparisons of the various devices that were out there.

I know for a fact that centers that were using Baker devices, our counts are nearly superimposable. So, if you got 1495, I might get 1475.  They were very compatible.

I think some of it also has to do with devices, as to whether they can use your product as a straight shot, meaning absolutely no dilutions need to be done.

So, the linearity probably has to go up into the two to three million range so that, for both your random donor platelet concentrates and your platelet pheresis products, you have a counting range that is compatible with your products.

When you have to do a one to three or a one to five or even a one to ten dilution, then you are depending on the accuracy of that dilution and that operator.

I know, from my work in clinical hematology labs, sometimes when you are out of range, somebody is taking a 10 microliter or a 100 microliter sample, and putting it into a 10 ml volume.

All you need is a drip on the end or wiping off the edge of the pipette and slurping out just a little bit to throw those counts way off.

DR. STRONG:  That is part of the problem. The platelet count is notoriously variable.  The CVs are not good.

When we are depending on licensing a platelet product and you have got a 10 percent variability on your side and a 10 percent variability on the blood center's side, and they are working with a hematology analyzer that they have to dilute because it is not in the linear range, these make enormous differences in the outcome.  So, do you think that that is part of the reason that we have so many failures?

DR. POINDEXTER:  I don't have that data with me. What I did do was to go back and back-calculate all of our yields and the blood center's yields and found that, for the most part, our concentration, our platelet counts, were really fairly similar to each other.

Where the problems came in were with the volumes being five, 10, 20, 40, 80 mls off.  Our counts can be the same, but if our concentrations, once we have done that calculation, that is where a lot of the variability comes in.

Also, on those that have low pHs at the end of the counting period, at the end of the storage period, frequently you have minute clumping that is going on, or even massive clumping that is going on, that will tremendously offset your platelet count.

DR. STRONG:  Which is, of course, part of the problem with storage as well.  Over time, sometimes you do get some micro aggregation that can't even be seen.

If you are using flow cytometry principles, they can count those. There may be two or three platelets stuck together that can be counted as one.

It seems to me that we have a problem here with quality control measures in and of themselves, in addition to adding to, how do we qualify platelet products that can be licensed.

The other question I had has to do with qualifying platelets from every single site. Most places have been essentially forced to use a single manufacturer because we don't want to QC every single instrument and, if we have more than one instrument, that just doubles the burden of quality control.

We are ending up now putting more and more people into quality control laboratories. They all have to be certified and tested and competency assessments made and all of that sort of thing.  It is becoming a huge burden.

Why do you take the position that we have to qualify every site in which we collect platelets?  That is a very large burden.

DR. POINDEXTER:  If every site was running exactly the same procedure, exactly the same procedure, with full donor information and always targeting 4.0, and you knew they could weigh the products correctly -- I am not being glib about this, I honestly mean that -- you could probably get away from the facility to facility.

When you have main sites that have all the collection and all the testing parameters there on site, where you can actually do a platelet count, you can actually do a hematocrit or a CBC on your donor, and off site mobiles and school gyms and Lion's Club, where you don't have that technology available to you, so they put a donor on and they target 4.0, but they have a low platelet count, and maybe they are just never going to get there, or they are targeting a double at 6.0.

If you target at 6.0, your chances of getting there are plus and minus 15 percent, and that is with full donor information.  If you don't have full donor information, it is a shot in the dark.

If collection facilities were all running exactly the same protocol -- and the machine manufacturers have protocols available where you can target a particular thing, but you actually need donor information in there to get there, then probably we wouldn't need facility by facility.

If your processing rooms -- assuming you have multiple collection facilities that are all returning products back to the main site for final testing and labeling and all that sort of thing -- if those people were then all sufficiently trained that they could accurately weigh your products at the end of those collections and prior to fully labeling them and writing those little numbers on the label, those are areas that we have great concerns about.  They are all solvable.  It is just a matter of getting there.

DR. STRONG:  Are you saying that you are allowing variances for places where there may be multiple sites, but all the QC is done in one place, that you wouldn't require platelet counts for all those sites?

DR. POINDEXTER:  The collection sites need to have certain information available to them.  They need to have the donor's pre-count.  They need to set the machines.

The products that are coming back to the main site for counting, if the one site is doing all the counting and weighing, and if they know that each of their collection facilities are collecting identical products, given donor variations, then we could probably get away from that.

Perhaps if you have a historical record that you have been submitting for the past year, five years, 10 years, and you have always had acceptable products, then perhaps we could get away from that. If you are a repeat offender, maybe not.

DR. STRONG:  A lot of centers -- you made a comment on moderate size. I think our center, for example, is in the moderate size case.

We have 10 collection sites. They all follow the same SOPs, they all use exactly the same instruments, they are all trained in the same way, the counts are all done at a single QC lab.  Yet, we have had to qualify platelets for every one of those sites.

It is a huge burden and it is a lot of platelets that get wasted.  Are now, of course, living in an era where, because of bacterial contamination and testing, that it really puts the pressure on the platelet inventory.  So, losing platelets to QC is also a problem.

DR. POINDEXTER:  Are you testing at the out date, or are you testing as you would release them to the hospital?  My understanding is that very few centers are actually expiring products.

DR. STRONG:  It varies on the platelet supply, which is a constant pressure now, with essentially only three-and-a-half days of storage time.

In our particular case, I don't think we are standard because we are a large transfusion service that services 20 hospitals from our centers, as opposed to being a stand alone transfusion service inside a hospital blood bank, which is quite a different circumstance.

DR. HOLMBERG:  Mr. Chairman, I have three questions.  Do we have time for all three?

DR. NELSON:  If they are quick. We are actually getting behind, so if you can be brief?

DR. HOLMBERG:  Betsy, I am hearing what you have to say about your evaluation process. It appears to me that there are really two aspects to the evaluation for license of this product, which would be able to then go across interstate boundaries.

It appears to be the logistics part of that and also the quality control part of it, where you would actually test in the laboratory.

I bring that up because you mentioned that there were different times when the temperature of the product varied.

To me, that would indicate a logistic problem in the transport. You and I have had this discussion before, when I shipped platelets from Okinawa, Japan to you 15 years ago.

What do you do when somebody comes back, or when you receive a product that is out of temperature?  Is it then reported back to the facility to then go through their process control on correcting that logistics?

DR. POINDEXTER:  The products that we get are shipped overnight, express, by Fedex generally, but by other carriers on occasion.

Most frequently, the reason for the temperature failures is that they put a couple of platelet pheresis packs in a blood box that has a half inch of polyurethane foam in it, no other insulating materials.

It is rare that they come in, properly packed, and fail temperature. Most of them spend at most 16 to 18 hours in transit.  It is not like your products from Okinawa, that took a couple of days to get there.

DR. HOLMBERG:  So, if they are shipping platelets long distances, but they may have a problem in their process.

DR. POINDEXTER:  That is right, and that is one of the areas that we will consider in the revised draft guidance, is to validate shipping.  I don't believe, in some centers, that is being done, both for the cold and for the hot.

DR. HOLMBERG:  Another question. In your preliminary slides, you mentioned about evaluating the product for excessive air.


DR. HOLMBERG:  I have never seen any numbers associated with excessive air. That seems very qualitative. Is this something that you measure or is this something that you would require in your validation process of the facility, to actually pull out a syringe and measure the amount of air that is in a unit?

DR. POINDEXTER:  Some air is always going to be there because of the lines that are in the aphoresis sets.  We have seen bags that were literally little balloons that had 100 mls of air in them.

We have discussed this with the manufacturers and most of them have, in their operators' manuals, that you are supposed to push back the air in the samples or in the products back into the collection containers or one of the other containers that is connected to the bag.

My understanding is that air activates platelets, and that might actually then go on to cause -- particularly, then, when they are in agitation with this large interface of air.

DR. HOLMBERG:  But there is no quantitative guideline.

DR. POINDEXTER:  No, we were doing it initially because we went from seeing virtually no air, or just a small bubble at the top of the bag, maybe five of six mls of air, to seeing huge amounts of air.

I actually had one manufacturer tell me that it wasn't there when it left the blood center, it must have happened during the flight.

At that same time, I got three or four other boxes of products and none of them had air in them, and they had all gone through St. Louis or wherever Fedex meets midway and then transfers out.

In reality, it was just a processing situation on their part.  We did go in with syringes and actually measured this air for a period of time. We are not doing that currently.

DR. HOLMBERG:  I just bring that up as a point. Maybe it might be wise for the guideline to actually have as part of the process or validation, if you think it is a significant issue.

My third question, again, goes back to the issue of pH.  Do you recommend any instrumentation for determining pH?

We all know now, with the advent of trying to reduce the risk of bacterial contamination in products, a lot of facilities are using pH from dip stick.  Is this an acceptable method for you?  Do you advocate any instrumentation in your guideline?

DR. POINDEXTER:  I would encourage people to use an honest to goodness pH meter, whether it is bench or whether it is hand held, rather than dip sticks or nitrozine paper, mostly because the pH nitrozine paper, and perhaps the dipsticks -- I haven't seen a dipstick in a long time -- may not have the range that is necessary for evaluating platelet products.

The storage of the nitrozine paper, in particular, if it is in a damp place, it is going to take on different properties than it might have had, had it been stored properly.

Nitrozine paper is frequently at whole pH units, 5.0, 6.0, 7.0, 8.0 or half unit, so that if you get one that only goes from 6.5 to 8.0, you may not capture those products.  That might fail.

DR. HOLMBERG:  I also believe that the protein would affect that reading.

DR. POINDEXTER:  It is an off label use and we are not in agreement with that use.

DR. DAVIES:  Betsy, one question. Could you comment a little bit more on the validation procedures you use in your laboratory for validating your instruments and your procedures?

DR. POINDEXTER:  The validation procedures that we have used have been to take products that have been collected and submitted, either from a site that we get research products from, and to do repeated counts on them, to check its reproducibility and to also do dilutional counts, serial dilutions, to get down below and to actually spin down and concentrate the sample to get above the linearity range, and to do repeat counts on those.

In the past we have used some calibration devices from various scientific companies that were linearity checks that were available for ranges of 500, 700, one million, million and a half, two million.

We run the controls that the manufacturer has available for the systems that we have used, and used both the high, the low and the mid-range check.

DR. FITZPATRICK:  Betsy, getting back to Mike Strong's question, I thought I had heard Sharyn say that the guidance has some provisions for sites doing mobile collections and that the donor parameters of pre-count and that might be covered in the guidance.  I am not sure from your answer that I got that follow through there.

DR. POINDEXTER:  I think it is more difficult for mobiles. If you have repeat donors and you have their post count from their most previous procedure, then that post-count can be used to qualify them and to run that procedure.

The product yield may be dependent somewhat -- people's platelet counts will drop somewhere between 50 and 125,000 depending on the type of collection procedure that they endured.

So, if they had a 300,000 platelet count on Monday and now it is Wednesday or Thursday, and they are coming into a mobile for another procedure, and you are using their platelet count that says they are now 225 or 250, the machine might stop short, and you may not get quite the product, or it may actually overwhelm.  You may actually get more than what you were bargaining for.

If you set it at 4.0, you might get 5.0 or 5.5 because perhaps, depending on donor variability, their platelet count may have almost come back up to their baseline again.

DR. FITZPATRICK:  When they come back to the center and do the count and determine whether or not to do a split at that time, they will make those decisions.


DR. FITZPATRICK:  So, they still have to be able to run the instruments on the default parameters on the mobile.

DR. POINDEXTER:  That is not default. Default is running it purely at the manufacturers, that it will run at a 250,000 platelet count, whether the donor's platelet count is there or not.

It will run for, say, 60 minutes. It will run at a 1.4 million platelet concentration.  That is what we mean by default values.

By putting the donor's precount from the most previous procedure in there, that is essentially as real a donor as you can actually get without doing today's platelet count.

DR. FITZPATRICK:  If they are a repeat donor.

DR. POINDEXTER:  If they are a repeat donor. If they are not, then perhaps you shouldn't -- perhaps what one should target is a single collection and put it in that 4.0 range or something like that.  You are probably assured, then, of meeting 3.0 and you may actually end up with a chubby single.

DR. ORTON:  Mike, I just wanted to clarify, for a first time donor coming to a mobile, we were making provisions for no platelet count being available when you put them on the machine, if you don't have a way to count.

DR. NELSON:  Okay, next, Dr. Williams?

Agenda Item:  Strategies for Quality Assurance Monitoring.

DR. WILLIAMS:  I am going to speak to challenges in applying statistical process control to blood component manufacturing, with an emphasis, of course, on aphoresis platelets and measuring residual white cells following leukoreduction.

I added Tony Lachenbruch and Jay Epstein as co-authors of the talk because, in fact, this has been a small working group that has been looking at some of these statistical aspects and apply them to the blood component quality control process.

So, what I am going to do is pick apart the topic a little bit.  Why statistical process control?

It provides an objective, reproducible framework for assessing product conformance to a defined standard, and one big advantage is, this framework can be adjusted, based on considerations of safety implications of a non-conforming product, the baseline rates of non-conformance, and the manufacturing burden.

I think it is fair to say that statistical process control really is an evolving concept for biological products in general, and particularly blood and blood components, and I am going to address some of the reasons why this is both complex and challenging.

So, what are the challenges?  As mentioned, production volumes vary widely across manufacturers.  Non-conformance is, in fact, rare, and the power to detect it is low based on small sample sizes, which are often necessary to preserve product, or make the quality control process reasonable, from a burden standpoint.

Some non-conformance, due to biological factors, may not be fully controllable, and I put wither validation.  For something like an add on leukoreduction filter, if the device being used doesn't provide validation data for things like changes in temperatures or time that the product is held until it is filtered, the blood center needs to do that to align with its own operating procedures.

It is something that both is dependent upon the characteristics of the device as well as the process that ultimately produces a blood component.

A few additional challenges.  Non-conformance has  a range of safety impacts. This can, in fact, vary across different indications for a single product.

For instance, in platelet content, one has considerations of the therapeutic effects, and whether there is sufficient content to that product.

The patient may, in fact, after a course of transfusions, need an additional product that they might not otherwise had, had the product been up to standard in all the prior transfusions.

Referring to multiple indications, again referring to leukoreduction, there are patients, particularly immunocompromised patients, who really need a leukoreduced product.

So, the quality control and the standards for that product need to reflect the fact that there can, in fact, be morbidity if a product is poorly leukoreduced and given to an immunocompromised patient.

On the other hand, as most of you are aware, the concept of universal leukoreduction is still somewhat hotly debated, as far as the advantages to the general patient recipient population, and perhaps that indicates a different impact of a non-conforming product.

The time to the detection of products out of conformance, and if one detects it the same day, one can obviously stop the production.

If you have a sampling cycle that requires a month or a quarter to meet a certain cut off for detecting a process out of conformance, there has been product produced along the way that probably shouldn't have been distributed.

Optimal uses of available resources, that has already come up today and I think it is particularly applicable to the platelet aphoresis consideration.

There are many process control points. These can either be considered individually, depending on their importance to the process itself and the impact of a failure at that point, and there needs to be consideration that process control may, in fact, require or result in sacrifice of the product, either because the product needs to, itself, be tested and entered to do that, or it could be a timing issue.

If you are testing products at the end of their useful shelf life, you may not, in fact, have an opportunity to distribute and make use of the product.

So, there are high production volume considerations.  A large blood establishment may produce several hundred components a day by a variety of procedures.  A systematic error may result in the release of a large number of non-conforming products in a short time.

Low production volume considerations.  A small blood establishment that manufactures 100 components per week by four different procedures, if they choose to do QC according to each procedure, they may have only 25 components per week available for quality control testing and sampling within that group.

Low volume production facilities have the lowest numbers of process of control, but may need it the most due to less frequent use of the procedures.

If you have a tech that does something one day, doesn't do it for two or three days and does it again, there might be an indication for increased surveillance of that procedure.  A systematic error may result in release of non-conforming products over an extended period.

Non-conformance rates are generally expected to be low.  As a rough guideline you would consider them to be generally less than 10-2 for a manual procedure, and less than 10-3 or even less than that for an automated procedure.

Frequently, if one has a bad lot of a certain reagent or some other material, or a certain technologist that has been improperly trained, some of these failures may, in fact, be clustered.

Irrespective of whatever framework, or no framework, that is used for the sampling, the power to detect non-conformance is going to be low for small samples.

I referenced that some biologic variables can't be controlled, and this was mentioned.  Examples would be a cold bacteremia, and sickle cell hemoglobin in a donor, which results in, in most cases, a blocked filter but, in many cases, that do go through, about half the time the unit sufficiently filters, but white cells are insufficiently removed and are left in the product.

Manufacturing burden is a consideration.  Each blood component is considered an individual lot.  Therefore, the labor and the cost of process control are major factors, particularly if the product is sacrificed.

The burden may, in fact, lead to increased uniformity of the manufacturing process.  It may be desirable in some respects, but it may have negative impacts in other respects.

For instance, use of multiple devices for something like leukoreduction helps to ensure supply.  If a certain manufacturer develops problems, one has an alternate source on line to divert production.

In the case of aphoresis, automated machines, it was mentioned that optimization for an individual donor might be best obtained by having multiple machines available for that process.

If there is a push toward a single machine at a single site to reduce the QC burden, that has some negative impact in producing the product.

There are issues of complexity and diversion of resources that can be counterproductive.  I think one also needs to consider that one doesn't necessarily tailor all quality control procedures to available technology, because creation of a new market can, in fact, stimulate a new technology which would meet that need.

Process control points in the procedure, the control itself may be best suited to an individual SOP that varies by different machines, different protocols or different locations.

This contributes to a lower sample number available for quality control, based on that individual control point.

There is power gained by lumping those control points and considering the entire process, either for a given site or a given manufacturer.  So, that gives increased power to detect non-conformance.

Where does the FDA fit into this?  FDA provides a minimal standard for local quality control procedures, which are ultimately defined by the blood establishment SOPs.

These SOPs are reviewed by FDA prior to license, or license supplement approval.  Then, compliance to the center's own SOPs are reviewed at pre and post-licensure inspection.

I think, from the regulatory side, the burden on us is to define practical strategies for very large and very small facilities, and every one in between.  Again, industry or local standards can certainly be more stringent than the regulatory standards.

I am going to end on consideration of the evolution into considerations of statistical process control.

There are several things to consider, and this goes back to somewhat Dr. Strong's question. What is 100 percent quality control?

In fact, there are some reasonable indications where you would want 100 percent quality control, and perhaps measuring every product to ensure that it meets that standard.

One would consider aphoresis platelet counts, bacterial contamination, which is the current American Association of Blood Banks' standard, to measure every unit, and leukoreduced products for immunocompromised, CMV-susceptible patients.

This was an element of the draft guidance proposed in January 2001 for leukoreduction, that all products destined to go to immunocompromised patients should be counted for residual white cells.  So, practical aspects aside for the moment, there are some situations were that might be appropriate.

However, in most instances, one would benefit from a sample based quality control approach and, really, for some time, the mantra for this in the regs and in current practice has been the count of four products per month or one percent, whichever is greater.

Now, if one has a 100 percent standard and you count four per month, have you met the standard?  Well, yes and no.  What is your level of confidence, counting such a small number of products.  So, that is where development of a framework to allow characterization of the process fits in.

This is an example of this from the memorandum that was cited recently, one percent of monthly production or four per month. All units tested should meet a count of less than five million residual white cells. If a failure is observed, the label must be revised and the process investigated.

Now, this was revised a little further in the evolution in the January 2001 draft guidance. We are in parallel with the best working group recommendation.

FDA recommended a quality control process based on a binomial distribution, and a standard such that 95 percent of products would meet a defined standard 95 percent of the time.

The standard in this case was also changed to one million residual white cells, but that still remains an issue, because technology may not be able to meet that standard currently.

I think what I want to emphasize is that this is a little bit more well defined statistical plan.  This can be met by pre-defining a count of 60 products and finding zero failures, or similarly, pre-defining a count of 93 with one failure.

Within the guidance it mentioned that, if you find a failure within your count, you should assess the root cause of that failure, and ultimately revalidate with 60 consecutive counts.

This is the subject of a blood products advisory committee a couple of years ago.  The discussion really was centered on the fact that this would potentially lead to an endless cycle of quality control and revalidation, and was unlikely to be workable in its present form.

Some of the advantages and disadvantages of the binomial approach are listed here.  It does define parameters of performance. It is conceptually feasible, at least within the agency.

The disadvantages are, it does require a pre-defined static sample. Additional sample cycles may be needed to determine if an increase observed in a single cycle is true.

There may be clusters of failure at either end point of the samples that could mask it through non-conformance, and the operational feasibility was questioned by industry.

What I am going to end on is some of the current thinking within the agency, based on a technique known as scan statistics.

Scan statistics are quite well known in areas like particle physics, and I think in the past several years they have been used increasingly for identification of increases in infectious diseases around the world, identification of potential BT, CT events, based on observed patterns of activities.

What it is, is a system that identifies clusters of events in time or space.  What I think, in considering using this for biological process failures, it is based on the fact that many product failures are non-random.  It could be due to bad reagent or soft goods, faulty machine or software, or some sort of staff error.

This is something that actually came off the net, and the reference for it I will include in the last slide. It is a pictorial description of what scan statistics actually does, without showing the math.

You see here a variation in a procedure with a peak roughly in the center, and each of these pictures is the same, from that perspective.

That is your total count of samples that you are looking at.  The red bar represents a window of definable size which, at each time you take the sample, moves along this track.

In doing so, you begin to pick up a signal when you get into the area of higher observations or non-conformance, such as the case might be.

There is a little stop light here that shows that the first three here are actually still in the green zone and, as one begins to get into this higher phase, you get a yellow and then, right in the middle, you actually get a signal that indicates you have a non-performance.

This is known as scan statistics. The SW is the number of observations, maximum number of observations, within that window.

As you might imagine, the math behind this is really quite challenging.  I am not even going to walk you through this.  Tony Lachenbruch is here, for those who understand it enough to ask a question.

It basically results in an odds ratio comparison between the probability of events that you might expect normally and the observation of events that meet a certain threshold, which can also be defined, such as three times baseline, five times baseline, et cetera.

The variables that result are reflected here, where N is the total number of tests to be done, i.e., the whole range of that chart. M is the window size, K is the observed failures, p is the probability of one observed failure -- i.e., background -- P probability of two or more failures -- i.e., non-conformance.  Delta is an adjustable variable for the threshold above baseline, and the power is the ability to detect this delta threshold.

So, just in one theoretical application to counting residual white cells, we took some numbers which aren't necessarily a recommendation, but would appear to be feasible for measuring this sort of process.

So, if one had a window of 233 observations in an overall sampling frame of 1,200, one observed two failures, had a p of .005, a P of .049 and power levels, the translation would be, when counting a moving window of N equals 233 within a group of 1,200 tests, observing another failure in that window would be detecting an overall failure rate at three times baseline, with an 84 percent power, with no more than a five percent chance of falsely determining an in-spec process to be out of spec.

So, what this reduces to potentially is a table, or certainly a computer program, which would allow you to change variables to meet a certain situation and provide a scan statistic which, hopefully, would be compatible with ongoing operations.

So, in summary, process control for blood products is complex, and often consists of trying to detect rare, non-conformance events with small samples.

Current thinking within the agency is that, in the future, FDA will recommend statistical parameters for process control as appropriate.

In doing so, FDA will provide at least one acceptable procedure that can be used in a user friendly format.

Certainly, alternate quality control approaches will be considered, as submitted to the agency, that meet defined parameters that are recommended.

This references the graphic that I used, and one general overview of scan statistics as published in 2001, which is very mathematical, but has a good introductory chapter.

DR. NELSON:  Thank you. Comments?

DR. MC GEE:  Tony, I happened to be around when you did the binomial thing which, if people thought that was complex -- this seems a little -- maybe Mary -- I don't know, CDC has been doing scan statistics for years, and there is a problem with this window definition.

Do you want to comment on it?  You know what I am talking about.  Like the example you just showed --

DR. WILLIAMS:  You mean the 233?

DR. MC GEE:  The picture he showed, until you centered that blip, you missed the thing.  So, the actual definition -- I think they went through a lot of stuff on the exact -- 233 is just a number, but where do you define that 233?

DR. LACHENBRUCH:  The 233 window size was chosen to control the error rate at five percent. That is how that came in there.

We also looked at various plans where you could say, well, two failures is not sufficiently convincing.  We might go to three, and that would change the window size. As the window size gets bigger, your probability of getting a hit gets bigger.

DR. MC GEE:  That wasn't my question. That much I understood.  It is the definition of the window.  Is one end of the window at the second or the third failure?


DR. MC GEE:  So, it is a closed end window where the second failure occurs within the last 233.

DR. LACHENBRUCH:  I am not sure I understand you.

DR. MC GEE:  These come temporally.

DR. LACHENBRUCH:  Yes, these are coming temporally and we are just moving the window along.

DR. MC GEE:  The end point of that is the 233rd sample.

DR. LACHENBRUCH:  We get to 233 and then we move it over one, and then we move it over one.  So, we are getting multiple blips.

DR. MC GEE:  But you are always using 233.


DR. MC GEE:  My point is, the right side of that window is the second failure. Is that what you are saying?

DR. LACHENBRUCH:  Not necessarily, no, it could be anywhere in there.

DR. MC GEE:  Where do I trigger the process examination?

DR. LACHENBRUCH:  As soon as you find the second failure within a window of 233.  If you found two failures in your first two, you would stop it right there.

DR. MC GEE:  What if I find one on the first one and a second on the 233rd?  I would stop it again.

DR. LACHENBRUCH:  Right, if you find one on the first one and nothing until the 235th one, you are free, you keep going.  If you then find another one on the 245th, that is when you stop.  You are going back roughly to the 10th through 243rd, that would be a failure.

DR. MC GEE:  Just as a comment, people are going to have to have some reasonably careful data management.  You could actually give them software.

DR. LACHENBRUCH:  Basically, what you are doing is recording the results of your tests in temporal order.  When you get the second -- the first time you hit the second failure, find out if that occurred in a window of 233.  That, I don't think, is going to be terribly tricky, but I think we can work on it.

DR. MC GEE:  Can I ask you guys one more question that relates to the earlier question?  There are two terms being used here. One is process control.  This is actually product control.  You are just looking at product failures.

DR. LACHENBRUCH:  I am sorry, I was having a side bar here.

DR. MC GEE:  There are two things being talked about here. One is process control. This is a method for just product control.  So, it would have nothing to do with process.

DR. STRONG:  One of the problems we have are these random errors which include primarily our donors. What we are having to do, in order to conform, is to eliminate donors who don't conform, and that is becoming more and more problematic.

So, for example, with a platelet count, when you have a donor that comes in with a higher platelet count, they may generate a product that exceeds the conformance of the product container.

Therefore, you have to eliminate the donor.  It doesn't say that the donor platelets are non-functional, but we can't meet the QC limits by that.

DR. ORTON: Our concern about exceeding the maximum isn't that you can't use that donor any more but that, in fact -- I know that the operators manuals don't give you much guidance on if you have a donor that does exceed it in that situation.

We actually give some recommendations in the guidance document for sterile docking and other bags, so that you can split the components.

So, you don't have to, in fact, not use the donor again but, as soon as you determine that the count exceeds the limit by the manufacturer, that you do something with that product that, in fact, it is stored properly.

DR. STRONG:  Sometimes it comes down to the fact that, for example, a donor with a very high platelet count exceeds the capability of the instrument to control white cells, for example.  You immediately overcome the white cell control.

Now you have got a failed QC and, if we have a single event failed QC, then we have to do root cause analysis, and 60 continuous -- it gets really excessive.

DR. ORTON:  That is why, I think, in the guidance we very specifically talk about whether it is really a process failure or something that is really out of your control and is not a process failure, which I think you are talking about.

DR. WILLIAMS:  I guess I would add, if you identify it as something out of your control and it doesn't reflect the process control, that doesn't affect your QT scheme. It is a process failure if you identify it.  Now, there is the trick.

DR. POINDEXTER:  Could I make a comment, and I don't work for any of the manufacturers, I work for the FDA, but you are correct.

Some donors with high platelet counts will track their white cells over, but the longer you run them, the greater change you have that that tracking will occur.

So, if you have identified a donor with a high platelet count, run them for a shorter procedure.  Run them for a 3.0 or a 3.5 or a 4.0 -- and I am not advocating a 3.0 -- but don't try to get a double, don't try to get a triple from that person, if you know their white cells track.  Then you can store their single in the two containers that are provided by the sets.

DR. STRONG:  That is all well and good, except sometimes it takes two or three or four collections before you realize that that has happened. Meanwhile, you have failed QC two or three times.

DR. EPSTEIN:  You might hit an alert level in the QC monitoring scheme but, when you investigate it, you are going to discover that you have a non-conforming donor, if you will.

You can address how to manage the non-conforming donor, and then it won't be part of the calculation of whether process control was out of spec.

DR. STRONG:  My only comment was that that is one of the variables that we struggle with, because we do have donors that don't conform.

DR. EPSTEIN:  This is true but, again, another point here is that there is an expected frequency of non-conformance of an under control process.

We went very quickly through that, but that was the significance of p in the model, that your historic experience in a state of the art center enables you to preset an expected non-conformance rate of an under control process.

Then the model is built on looking for something above that, the thing we call delta, which is three times above that, five times above that.  So, the beauty of this model is that it allows for a certain background rate of non-conformance.

DR. STRONG:  That gets back to my other point about 100 percent QC.  It is not 100 percent of the products being QCd, but rather, always having 100 percent compliance because of those non-conformities.

DR. EPSTEIN:  Again, I think we mixed two different ideas.  Whether the quality control test should meet a specification 100 percent of the time is a different question than whether 100 percent of products should have a quality control test.

I think when Alan was talking about, for example, leukocyte reduction to be used in lieu of CMV antibody testing for a CMV at risk recipient, the suggestion, which was made in our 2001 draft guidance, was that, for those units you actually want to quality control each unit.

In that instance, you do want 100 percent of units to meet a minimum standard, but again, the two different issues are, you could have a 95 percent conformance standard and want 100 percent of units tested, or you could have a 100 percent conformance standard but only want a sample tested.

So, there are these two different things going on, but there are settings in which you would want 100 percent testing and 100 percent conformance.  That is if you think the deviations are life threatening.

DR. LINDEN:  I have a question for tony Lachenbruch. I thought I semi-understood this and it sounded like a reasonable approach, compared with the one we recently heard about a couple of years ago, but now I am confused.

If you are looking at 233 and have one non-conformance, I thought you would stop at that point.  Why are you going up to 245 and finding a second non-conformance.  That is what I am confused about.

DR. LACHENBRUCH:  Let's take something that I can handle with easier numbers.  Let's say we set a window size of 10 and the first one was non-conforming.

You went on up to the 10th one and there were no non-conforming lots.  You continued, and the 15th test showed a non-conformance. You would not stop there, because it is two non-conforming lots within that window width.

So, you look at 15 and you go back 10 unites, that would have started at the 6th one, and there was only one non-conforming lot between the 6th and the 15th donation. You keep going.

At the 20th donation, you look back to a window beginning at donation 11 going up to 20, and you find there were two non-conforming lots there.  Now you have found two lots within a window.  That is when you pull the trigger.

DR. LINDEN:  So, you are saying the counter starts over again as soon as you start and find one more non-conformance.

DR. LACHENBRUCH:  Yes, all you are doing is sliding the window along.

DR. MC GEE:  You don't start over.  The window of 10 keeps moving.

DR. LACHENBRUCH:  And it says, for every window, do I ever see two bad lots in that window at any point in time.

DR. ALLEN:  Alan, I applaud the intent of the FDA to move to this sort of statistical analysis. I think, by the discussion we heard here today, as well as the earlier presentations that indicated there are difficulties both with methodology in terms of platelet counts, as well as even getting technicians to account for the weight of two or three extra bags on the scales, I think we have some real challenges, as you pointed out.

I hope, as the FDA moves forward with these plans, that certainly there will be close collaboration with AABB and perhaps selecting a few centers where one might do pilot testing of this and look at how, in fact, it can be applied and rolled out most effectively, to assure that, when it is implemented, it will be done so effectively.

Again, knowing that there are multiple computer programs and systems that different blood centers use for this extraordinarily complex task of tracking the collection, the donor information, all of the laboratory tests before one can release a product for transfusion and all, are there statistical programs for quality control that can be integrated into some of these?

Is this a whole new area of software development that perhaps will have to be developed as one moves forward in this?

DR. LACHENBRUCH:  I think I can't comment on integration.  There are plenty of programs available for quality control.

I know all the major programs -- SASS, Stata, SPSS, mini-tab -- have quality control routines built in.  In fact, what this would involve is essentially importing some data into Excel and just looking and seeing how wide it was.  If you ever got two bad ones in a window of 233 or whatever.  You can tailor these appropriately.  Do you want to have five percent false positive or one percent false positive?  You can do it that way.

I should also point out that there are some combinations of p and P and N and stuff like that, that you can't find any way to quality control them adequately.

If you are down at the 0001 adverse outcome level, it is either an immense window or a very low power or whatever.  So, we have to understand that, too.

DR. WILLIAMS:  To address the other part of the question, these approaches were just presented conceptually today.

I think as we move further down the line, obviously, we will get a little more specific and have a chance to either discuss it again, or most certainly issue recommendations in draft format, at which point there would be an opportunity for pilot testing.

I think if industry were to come to us to discuss a potential pilot program, that would probably be a good way to approach it.

So, yes, I think that would be a valuable way to roll something like this out but, in fact, you don't have to know how the car works to drive it.

I think this can probably be reduced to a quite comprehensible format to be managed with both available software and even manually as needed.

DR. WHITTAKER:  Is there any way, with this program, to actually look at trending?  If a non-conformance occurs, then you are already out.  Is there any way to look at upward counts?  With a control chart, you can see tracking and trending.

DR. LACHENBRUCH:  In fact, a lot of control charts are based on scan statistics.  For example, in this case, we used the example of two non-conforming lots.

If you had said, I want to wait until I see three non-conforming lots, you could say, oops, I have seen two non-conforming lots.  This is my warning limit and I am going to do a little stronger monitoring before I go through any sort of a revalidation process.

I think the other issue that was brought up -- I am sorry, I have forgotten your name -- Dr. Strong -- is that whenever we see something, we investigate the cause of failure.

Even if you had two events happening, 500 apart, if they were for the same reason, you might say, what do we have to do.

I wouldn't presume to tell people what to do in that case, and I go into a lab and I burn holes into my clothes, but yes.

DR. NELSON:  Okay, I think we will move on.  Dr. Leparc from the Florida Blood Center.

Agenda Item:  Blood Center Perspective on Platelet Pheresis Quality Control.

MR. LEPARC:  Thank you.  I think my role here is to kind of give you a snapshot of what all these rules mean in the real world of blood banking.

There are a lot of rules and regulations that are intended to work one way, but sometimes they just don't achieve what we intend, and may backfire  With that, I welcome the opportunity, and we have heard some good, stimulating discussion.

I just would like to put things into context. We collect approximately 1,200 aphoresis -- we perform 1,200 aphoresis procedures.  That yields about 2,000 transfusion doses per month.

The last I looked, the AABB reported in 2001 about 400,000 aphoresis collected nationally.  So, that kind of puts things into perspective as to where we are.

Our service area, our demand for platelet transfusions is approximately 3,000 transfusion doses a month.  We have to make up the difference, which is about a third of the platelets that we transfuse with whole blood dry platelets.

So, there is a constant pressure to collect more aphoresis because it is the preferred component. However, we do not have enough available to satisfy the demands in our region.

Our region uses approximately 1.2 percent of the nation's blood supply.  It has a cancer center and a children's hospital that have very active bone marrow transplant programs. So, that drives the demand for lots of platelets.

We have nine collection sites.  They are all fixed sites.  We don't go on blood mobiles or gymnasiums or any places like that.  We collect them in designated sites.  We have a total of 32 instruments.

We use only one instrument, and you will see later on why we made that decision, which I think is an unfortunate one.

I think it is one that we have been pushed into, but the fact that you use only one instrument puts the manufacturer of the instrument in the driver's seat.

There is a very high cost of switching instruments, regulatorily and practically.  There is a very high cost of maintaining QC on more than one instrument.

I think it puts a big burden on the regulators.  Let's say that tomorrow you decide to take regulatory action on Baxter.  A lot of people are using only that instrument, and that is going to affect drastically the supply of platelets that are collected that way.

So, the lack of diversity in an institution of providers puts pressure on everybody, plus the manufacturer of that product.

We do quality control on a daily and monthly basis.  On a daily basis, we perform on every platelet we collect by aphoresis, a complete -- a CVC, including platelet and white blood cell count.

We do bacterial culture for aerobic bacteria and fungi, and we measure the volume in mls, and give the total number of platelets that are in that container, by calculating the concentration for unit volume and total volume of the bag.  We calculate the number of platelets, and that goes on the tag that goes with each unit if blood.

That is done on every single unit.  Even when we have doubles and triples, we will count each one of the siblings, so to speak, and we give that tag on each one of them.

On a monthly basis, we do the regulatorily mandated four units per month for each component site.  That is, for every single, double and triple dose collection, for each collection site and for each collection instrument.

Now, we won't do it on every instrument every month, but we rotate so that, in some sites where we have only one instrument, we do it every month, but if we have two or three, we will rotate so that we cover every instrument, which is something that I think some people may or may not do.  It is not required.

Then you may select an instrument that will give you better numbers with certain donors, and then you are cheating the system.  You are not achieving what you are supposed to do.

This results in our quality control lab doing approximately 60 to 75 CVCs on products. This is on top of all the CVCs we do on donors themselves.  This is just exclusively product testing, 60 to 75, and bacterial cultures, 40 to 50, depending on the collections.  That is our daily QC work load.

How about monthly?  Well, monthly, we decided to abide by the letter, or at least the spirit, of the regulation.

Since we issue platelets, two, three, four days, rarely five, because nobody will accept any platelets that are on its last legs, we do test on the fourth day.

That is kind of worst case scenario time of issue, and we perform a platelet count in 100 percent of the cases. It has to be at least three times 1011.  We use a cell dying instrument. I think it is a 4700. It had a validated by us linearity range of up to five million platelets.

The white blood cell count is performed and that is done by flow cytometry using a Beckton Dickensin instrument, and they have a special kit to measure residual white blood cells by flow cytometry.

We measure pH using a bench pH meter.  I agree with the comments about dip sticks or paper strips. Those are not reliable measurement devices.  They may be good for urine, let's put it that way, but it is not good for platelets.

We do this for each type of component.  Again, we have four singles, four doubles and four triples, and we did that after long discussions with staff at CBER.

So, basically, we hold these units until day four of storage, test them and, when QC is passed, we make them available right away, ut that has an effect on our outdating rate, because you can't get rid of them fast enough.

The end result is, when we looked at the statistics, an average of 72 doses of aphoresis platelets are just thrown away because they expire on the shelf.  That is about 34 percent of the phoresis that are set aside for QC, a big burden.

What this amounts to is, if you do the math of the four doubles or four singles, four triples, nine sites, at 24 components for QC per site, it is 216 components for QC per month are held in storage.

So, again, when you look at the CFR you have probably what I call one hand statistics. Somebody says, what shall we do.  It is four.  There is no statistical significance to doing four. I don't know how that magic number came out, but this is what it means to us.

It used to be a lot worse. I say that because Florida Blood Services was born about 12 years ago out of a merger of three blood centers in our region, and we had a hodge podge of instruments.

When we first started and looked at the nightmare QC, even though we were collecting only on four sites, we had all these different instruments.

Just doing in six sites -- a third less than what we have now -- we were doing 288 QCs per month because as long as you have an instrument that rule applies to every single instrument.

Well, what we are doing now, I told you what the total, the cost in phoresis, is.  What about dollars?  What does it cost in real dollars to meet these requirements?

Well, monthly costs for platelet counts is around $750, and I am not putting there the cost of machines. You can count beans in many different ways. I am not going to put all those depreciation tables and whatever, just what it takes to get a body of reagents to do what is already there.

$750 a month for counts.  Flow cytometry is about $6,500, pH $20.  The component update is -- outdate is about $3,500.  I am not counting bacterial infection or hematological QC.

That adds up to $42,000 a month and you put it times 12, $500,000, half a million dollars in quality control, but that is done.

So, there is a price tag for every measure that we do.  Now, I know that this group is not supposed to be concerned with cost, but there are things that we have to live in the real world with that, unfortunately, also deal with costs.

So, questions to consider on my side, from the regulated world, do current QC requirements offer a significant additional assurance.

I am all for quality control.  We started doing bacterial contamination detection a year ago, in March 2003.  Why?  There is a cost, there is complexity involved, but it adds a measure of quality to the product that makes it to me worth it, no matter what the cost.  I shouldn't say no matter, but the cost that it takes to do that is a reasonable prudent measure to do.

The question is, is this other stuff that we are doing worth the half a million dollars in work that we do.  I leave it up to the committee to decide.

Another thing that bothers me, and I am very pleased that we are now talking about using real statistics, is that you know, the four sites, four machines, that does not make any statistical sense.

Now, I know it is in the books and we need to do it, and I will do it as long as -- and I will be pleased to do it as long as -- I have some reassurance that there is a purpose to it and there is a goal.  We are not doing it for the process, we are doing it for the outcome that we seek.

Lastly, beyond QC, I just question the interpretation of why do we have to do each location where platelet phoresis are collected regarding separate license.

I am going to give you a prime example we are living in now. We are in a very rapidly expanding region of the country.

We are opening a new donor center in the suburbs.  One donor center that we had for many, many years is not a good producer of aphoresis donations.

So, what we plan to do, we think we have the demographics, we looked at zip codes of donors and stuff.  We are going to move the machine, the couple of machines we have there, we are going to have the same people go there. All those platelets are going to go to the same QC lab.  The SOP is going to be the same. Yet, I still have to reapply like I never collected a phoresis in my lifetime.

I know it is in the book, but you know, please understand that you are making us jump through hoops -- I would jump through a thousand loops again, as long as I know I am doing it for a good reason, for a rational reason.

Licensing each site requires submission of components to the FDA for each type of component.  We saw the problems of different instrumentation, different techniques, transportation.

Sometimes I think I would like to paraphrase an old saying.  We all know God is everywhere, but God does QC only at FDA.

I know -- we all are trying hard and we try to do our best, but there may be third party independent labs that can do it regionally or locally, or even the blood bank can be audited.

When the FDA field staff comes to our place, they spend a month and they turn over every little piece of paper that is there.  So, you are certainly free to look at our procedures.

Last, as I understand, collection systems go through a very difficult licensing process, and they are licensed to be used with certain procedures and the bags, everything is licensed as one package to serve a purpose.

So, if we license a bag to keep platelet for five days, why do we have to measure everything at the end of five days.  As long as you follow manufacturing instructions, it should perform.

Now, we still do QC and all that, but we will expect, without having to send to the FDA all these things, to perform in the way it was intended and, as long as the blood center does the proper QC to demonstrate that the device performs as intended, that should be sufficient.

So, I just would like to leave you, then, with this snapshot of what we have to experience very day as a provider of this very valuable product, that every donor who spends an hour on a machine expects it to go or improve the life of a patient.

Sometimes, unfortunately, it ends up in the QC bin instead of ending up in the veins of a patient that really needs it.  Thank you very much.

DR. NELSON:  Thank you, Dr. Leparc.  Questions?

DR. SCHREIBER:  Your biggest cost appears to be waste because of outdate.  Would there be any reason that it couldn't be that you could do your QC for your average storage time before you ship your specimens out of units out?

DR. LEPARC:  It could be. Again, those are the areas, the gray areas where you get guidance on how to tackle a problem, and that is the answer we get. We do that.

There are certain things that definitely don't need to be kept to the end of four days. The white cell count, the white cells don't go anywhere.  Actually, you are better off doing the count at the very beginning. So, yes, we could do that.

The rationale that was given to us was, well, what is the time span when you issue stuff.  Getting it back from the hospital for QC purposes is not practical.  You will never get it back.

DR. ORTON:  I just want to clarify that, George.  The regulation says at outdate, so we have interpreted that to mean at issue.

Even if it was at issue from the blood center to the transfusion service, that isn't always practical. I can tell you, I set up one of the first programs for Dr. Leparc, and it was easier to have a set date that you would do it rather than try to do it at issue when, in fact, the products are going out in the middle of the night.

In order to incorporate getting every site, every machine type, every machine over a period of time, that was not practical.

Now, whether we need to reconsider at issue to be more flexible in validation, we have become more flexible with the validation being done, a certain part of the components, during the first part of the expiration, the middle, the end, again, to spread it out, and perhaps that is what we need to consider as well.

DR. EPSTEIN:  I just want to comment on the issue of validating additional facilities under the same establishment license.

We are considering a concept of reviewing comparability protocols, which would then function more or less in the following manner:

If a licensed blood establishment that has been approved for platelets wants to extend platelet manufacture to additional facilities under the license, if they have a valid protocol which has been approved and accepted by the FDA, that they can, under that protocol, validate additional facilities without sending products to the agency.

Now, the full concept has not evolved yet, because we need to get a very concrete idea of what we need to see in the comparability protocol, and what quality control data would be submitted to the FDA, because we would still want to look at data.

So, that concept exists, and we are working on it. We just don't have all the Is dotted and the Ts crossed, but we understand the problem that you and Mike Strong have been talking about and we would like to move toward a rational scheme.

I think that the other side of the coin is that we do have to respect the fact that the laboratory testing being done at the FDA does show non-conformances.

They are not all just due to shipping.  They may be at a higher frequency when you have new facilities, new equipment, new operators, and that is to be expected.

The question is, how much of that should be done by the FDA as part of preapproval. How much of that can be done by looking at data where it has been done operationally by an already licensed establishment. That is what we are trying to sort out.

DR. LEPARC:  I thin that would be a most welcome move.

DR. STRONG:  Jay, perhaps what you ought to consider is licensing on the basis of the analyzer.  The big differences in the platelet counts seem to be between analyzers.

So, if you have more than one analyzer, if you have an analyzer at every center, then maybe you do have to qualify it, because the analyzer is different.

DR. EPSTEIN:  We are open to suggestions and, when we republish guidance, we will get comment again.

DR. NELSON:  Okay, I think we will take a break now until 11:00 o'clock.

[Brief recess.]

DR. SMALLWOOD:  We will reconvene, and we will be moving to the open public hearing.

Agenda Item:  Open Public Hearing.

DR. NELSON:  We will continue on this platelet topic. The first person who wanted to speak is Dr. Michael Fitzpatrick from the Americas Blood Center.  Is he here?  Okay, we will reverse the order. Kay Gregory from AABB.


MS. GREGORY:  Thank you.  My name is Kay Gregory and I am the director of regulatory affairs for the American Association of Blood Banks.

The American Association of Blood Banks is the professional society for over 8,000 individuals involved in blood banking and transfusion medicine, and represents approximately institutional members, including blood collection centers, hospital-based blood banks, and transfusion services, as they collect, process, distribute and transfuse blood and blood components and hematopoietic stem cells.

Our members are responsible for virtually all of the blood collected in the country and more than 80 percent of the blood that is transfused.

For over 50 years, the AABB's highest priority has been to maintain and enhance the safety and availability of the nation's blood supply.

The AABB would like to commend FDA for recognizing the need for new platelets pheresis guidance.  This is a matter of extreme importance to our members.

As a matter of fact, it was the issue mentioned most frequently when we queried our members for issues to discuss with the FDA at our upcoming FDA liaison meeting in April.

The current guidance, Guideline for the Collection of Platelets, Pheresis Prepared by Automated Methods, was issued in 1988 and is woefully out of date.

There have been tremendous improvements in the automated equipment used to prepare platelets pheresis.  All recommendations for platelets pheresis, not just those related directly to quality control, need to be addressed in updated guidance, including a complete list of items required to be submitted for product licensure.

Unfortunately, the AABB is unable to provide substantive comments concerning the FDA proposal described today.

FDA does not make this information available until it is published as meeting materials on the BPAC meeting agenda web site.

This material is typically not posted on the web site until a day or two prior to the meeting, and does not always include sufficient details or reflect the proposed discussion.  This does not allow adequate time for us to review the material and receive feedback from our membership, and it creates a real problem.

With regard to platelets pheresis, however, we note the following concerns we would like to see addressed in the new guidance, and I want to stress, this isn't everything in there.  We just picked out some of the more critical items.

First, FDA should not require separate license applications for each location preparing platelets pheresis. This current requirement is unnecessary.

Once the establishment has obtained approval for one location for a particular collection device, with specific SOPs, additional license supplements should not be necessary.

The exact same device is used for collection with the exact same SOPs and, in many instances, with the exact same personnel.

More important, not all aphoresis platelets are collected in fixed sites.  Many establishments have successfully implemented such collections as part of their mobile blood drives.

As long as an establishment has received one license approval, separate applications for each location become merely additional paperwork and expense without added safety benefits.

The requirement to submit actual product to FDA as part of each license supplement application is inappropriate.

Submission of quality control records, coupled with observation during on-site inspections, should serve the same purpose.

Submission of actual product for license supplements, especially when required for each location, results in significant loss of product that would otherwise be available for patient therapy.

It is clear from the discussion this morning that the FDA laboratory is not subject to the same accountability as the blood center laboratories.

We don't know, but we assume they are not CLIA approved.  They are not inspected by the AABB.  They are not inspected by ALCP or a number of other myriad organizations that come and look at us in the blood center.

It is not clear to us exactly what kind of quality control they are doing.  It is not clear that they belong to any kind of proficiency program.  It is also not clear exactly what kind of validation they are doing. Yet, we are expected to rely on those laboratory results.

Required quality control for split products should be clearly identified in the guidance, and should not necessarily require full quality control of each product.

Today's technology allows for the collection of product that can be split into two or three full dose products.

Emphasis should be placed on the criteria used to determine the number of full dose products that can be made from the collection, including the methodology for preparing these splits.

We encourage FDA to consider adopting recommendations that will be consistent with the existing AABB standards.

The Standards for Blood Banks and Transfusion Services delineates two specific standards relating to platelets pheresis.

Standard states:  Platelets pheresis components shall be prepared by a method known to yield greater than or equal to three times 1011 platelets.

Validation and quality control shall demonstrate that at least 90 percent of units sampled meet these criteria.

The second standard, states:  The platelets shall be suspended in sufficient plasma so that the pH at the end of the allowable storage at the appropriate temperature is greater than or equal to 6.2.  Validation and quality control shall demonstrate that at least 90 percent of units meet this criterion.

Finally, while not the major focus of today's discussion, AABB standard states:  The blood bank or transfusion services shall have methods to limit and detect bacterial contamination in all platelet components.

The AABB anticipates that new guidance will be issued as draft guidance so that the AABB members and others of the public will be able to comment on it before it becomes final.

This is especially important, given the limited opportunity to prepare for this presentation to BPAC.  Any recommendations should be based on sound science and clearly articulated.  AABB requests specifically that the recommendations be practical and not unduly burdensome.

DR. NELSON:  Thank you.  Any comments?

DR. KATZ:  I am Dr. Louis Katz, president of America's Blood Centers. I think most of the committee knows that we are the association of independent, not for profit, community blood centers, collect, process and distribute about half the blood supply.

We always appreciate FDA inviting us to the meetings and allowing us to comment on new guidance and issues of regulatory interest.

This new guideline is badly needed, and the off-the-cuff analysis that Key Gregory has presented we endorse, recognizing that the information we were provided before the meeting did not exactly reflect the content of what we heard this morning, and that written guidance, draft guidance, will eventually be provided, upon which we can make formal comments.

We are committed to providing meaningful comments, suggestions and complaints, where appropriate, to the FDA.  To do so, with the benefit of input from, and reaction from the BPAC, we need the information in a more timely fashion.

We think that the input of this committee to FDA remains a very important aspect of their deliberations and, in the usual process of written comments, it is not clear -- certainly in my tenure on the BPAC, we didn't always get the written comments.

So, when we have a draft guidance available, we will be providing very detailed written comments, and would ask the chair and the agency to provide those to the blood products advisory committee for their review.

We think that this would enhance the ability of the blood products advisory committee to understand the real world situation in which blood collection facilities operate, and understand the perspective of the regulated community.

Finally, Betsy Poindexter presented some provocative data regarding QC failures on products sent to the FDA lab.

We desperately need more detail, more meat on the bones of that presentation, primarily to begin to form an estimate of the generalizability of the data, and whether this represents repeated failures by individual organizations or an endemic problem across the blood community, so that we can provide feedback to our membership and enhance the ability of FDA to assure safety, security and potency.  Thank you for your attention, and I can answer any questions if there are any.

DR. NELSON:  Thank you, Dr. Katz. Are there any other people who want to comment during the open public hearing?  If not, Sharyn, are you going to re-state the issues for discussion for the committee?

Agenda Item:  Open Committee Discussion.  FDA Current Thinking and Questions for the Committee.

DR. ORTON:  Okay, just as a recap, the current thinking is that the monthly QC performed on four units from different donors at issue or outdate per site per machine type per product site of single, double, triple.  The current absolute platelet count and pH requirements are unchanged, as I noted earlier.

We are going to add to the residual WBC acceptance limit per manufacturer's specification, if the claim is, in fact, less than 5.0 times 106.

We are going to look at the maximum absolute platelet count per bag per the manufacturer's specifications, also the minimum maximum volume per bag, again per the manufacturer's specifications, and that there be guidelines for volume separation of double and triple collections.

There will be assessment of pH at 6.2, and the use of quality system audits to actually look at volume separation issues, and sterility testing or bacterial contamination, these being on an ongoing basis.

We discussed components sent for QC testing to the division of hematology at CBER.

So, the first question is, does the committee agree that the proposed recommendations for quality control testing are adequate to assure quality of platelets pheresis and, if not, please comment on alternative approaches.

DR. NELSON:  So, I guess there were two issues.  One is the criteria for measurement of these variables at the pheresis center, and then the second is the samples that are sent to FDA for testing.  Should we comment on these separately?  I think they are somewhat separate issues.

Agenda Item: Committee Discussion and Recommendations.

DR. NELSON:  Why don't we start with the criteria, the guidelines that FDA was proposing in the guidance document for the criteria for the centers, or for the pheresis centers, the testing that is done at the centers, rather than the FDA.  Any comments on that?

DR. LINDEN:  I certainly commend FDA for looking at this guidance that is over 15 years old. It certainly needs to be looked at, and for looking at these issues of quality control, testing aphoresis platelets, which really definitely needs to be reassessed.

I am really concerned about this approach that was outlined for us, because it strikes me that it is not thoroughly thought out yet.

What was given to us was not thoroughly described to my satisfaction, anyway, and seems to be internally inconsistent, in that not all of our questions were answered.

So, I really think it needs some more thought. I am not in a position of saying, I think this is fine. I can't say that.

Particularly, the internal audit part really needs a lot of thought.  If there is a problem with the volumes, I think there may be some other ways of going at that, short of introducing a whole new procedure here that is going to be one more regulatory burden for the blood centers to be having to comply with.

If that, indeed, is an issue, I think there might be some other way of getting at that. I would encourage the agency to think about that, short of introducing some whole new procedure there.

I am also concerned about what you are framing as a different question, of the samples being tested by the FDA, because I am not convinced that we have really seen data that there is a big problem here.

I mean, Mike really didn't get a good answer to his question about the lab at FDA that was doing these tests, and how they are certified, and what kind of lab this is.

I didn't hear that these products, these components that went there, are representative of the components at all, because a lot of them were shipped under sub-optimal thermal conditions and they were tested at outdate, and really, they aren't remotely representative.

I am reluctant to draw conclusions, really, of a problem with the components on the shelf. I am not sure what problem we are addressing here. I would like to see data that show me that there is a problem with the components on the shelves in the blood centers, before we completely revise the entire algorithm.

There may be, and I certainly agree that we need to look at this whole issue, and the whole guidance needs to be looked at.

I don't think that one set of data are all that we need to look at.  I mean, we need to look at other things.  I think that today this committee has not seen all that we need to look at, and I think this entire thing needs to be really given some more thought by the agencies.  That is my opinion.

DR. NELSON:  I am not a blood banker.  So, I am free of a lot of the perhaps nuisance problems.  When the FDA audits or reviews records at a blood center, how many of the criteria, or what sort of comparison is there with the record at the blood bank, in terms of platelet count, volume, bag, that kind of thing.

In other words, are there records at the blood bank that could be used, probably more complete records, in some respects, and maybe some variables missing in others.

DR. ORTON:  Ken, are you asking about the components that are going for QC testing at CBER?

DR. NELSON:  No, I am asking, when records at the blood bank are used on platelet pheresis, what kind of records are there for the FDA to look at?

DR. ORTON:  If they are field inspected?

DR. NELSON:  You proposed like nine different criteria, volume, number of white cells, platelet count, all this kind of thing. Are these records all or most available on each unit at the blood center?

DR. ORTON:  All.

DR. NELSON:  So, there are data for comparison, to get to your question about whether the samples that are sent to CBER are representative and might they be, because of outdate and shipping and other things, not representative?  There are data that one could look at that; right?

DR. KLEIN:  There aren't really data.  Those data that you just mentioned, Ken, are certainly available.  That tells you that what the center is doing is well documented, and within specs.

It doesn't tell you that they are doing everything properly but, of course, an inspector can look at that, and does look at the actual procedure being carried out when they are inspected.

I have to agree absolutely with Dr. Linden that, if specimens -- if there is a problem -- and I think we need to establish that -- if specimens are to be sent somewhere, they absolutely need to be sent to a laboratory that is using state of the art equipment, that is CLIA approved, and is really standardized in the way that every laboratory is standardized that produces diagnostic procedures or components for transfusion.

I must add to that, that at the National Institutes of Health where, for many years, research laboratories produced results on their equipment that were used for patient decisions, all of those laboratories now need to be CLIA approved before any result can be used for a patient-related diagnosis.  I think the same must be said about components that are going to be transfused.

DR. NELSON:  You addressed certainly the second part, the issue of the testing at CBER.  Now, some of the issues are certainly new since 1988.  The white cell reduction and the pH, I guess, was always there, but there are other issues that have changed, that would be included in this new guidance document.

I was a little concerned about the 100 percent criteria on the platelets of -- of white cells of less than five times 106, given the fact that there are patients and other problems in reality, if you were to test all of the samples rather than a small sample.  You would find it is not 100 percent.

I thought initially that there was some -- that it was somewhere, 95 percent or something met those criteria and, when they don't, the unit isn't used or something.

DR. ORTON:  Ken, let me just clarify.  On the devices we have cleared through CBER, the majority of them, particularly the newer ones, have two levels where they give the specifications for the residual white count.

They have a percentage at 5.0 and a percentage at 1.0.  One of the limitations is the counting devices. So, the 5.0 has been the standard.

Now, what we have accepted in the past was 100 percent.  As I indicated, some of the devices, their clearance isn't even for 100 percent, and that is one reason why we have re-looked at it.

The devices are sufficiently cleared and the new ones go to the lower level of 1.0.  So, they do give parameters. That is why we have really included what the manufacturer's specifications are, to be considered in QC and validation.

DR. NELSON:  Essentially you are measuring how good the device is at measuring this level.

DR. ORTON:  If the device is cleared to do a certain thing, we want to be sure that that is, in fact, what you are getting.

DR. STRONG:  I think, actually, to agree with Dr. Linden, that we have an opportunity here, because there are other issues that need to be addressed in regard to platelets.

We are really kind of in a new era.  We have got new equipment, we have got new machines, we have got new issues to deal with, and the bacterial testing one, I think, is one that is really critical.

The AABB now has a standard that requires everybody to test. I think FDA hasn't exactly caught up to that yet, and that needs to be addressed.

Along with that would be the extension of the dating period from five days to seven days, which used to be licensed and isn't now because of bacterial testing.

There is a disconnect in terms of the availability of testing equipment for whole blood platelets.  There is a meeting to be held in May to address what the standards should be for acceptability for platelet survival, recovery, et cetera.

Perhaps this is a time when all of those things could come together and a more complete package could be provided.

DR. EPSTEIN:  I just want to make a comment. In a more ideal world, we would have had a cleared draft document that would have been published that would have been available to the public, shared with the community, and we could be looking at all the specifics.

Since we are not there yet, we felt it would be useful, nonetheless, to talk about some of the broad concepts.

So, we appreciate that, in this forum today, we are not in the position to talk about the details, because we have not presented them.

What we are looking for is a general sense of where are the big problems, and is the thrust of the direction that we are going the right direction or the wrong direction?

So, there are limitations today, and we do expect to issue a draft guidance and get formal comment and, if it seems necessary to reexamine the document with the BPAC, we will do that, too.

DR. ALLEN:  Jay, I think those were helpful comments, and they are in the introductory materials that were provided also.

Certainly we are dealing with a unique situation here.  If we were talking about a standard manufacturing process, humans would come nowhere close to being acceptable as a source material, in terms of the absence of uniformity, and the fact that platelet counts vary, and all the rest of it.

That is what we deal with because, obviously, dealing with the real product is the best thing. How does one then establish, at diverse blood collection centers and transfusion centers across the country acceptable quality manufacturing standards for a product that is now being outdated in five days, with the bacterial monitoring, should probably go up to seven days, although that does create additional problems in terms of pH and all the rest of it.

Yes, these parameters do need to be measured.  I was baffled and a bit concerned by statements that -- I hope nobody is still using a dip stick to assess pH in collected platelets.  Based on what I heard today, it sounds as though that is still being done.

There are a lot of issues that need to be addressed. I certainly think that the introduction of the bacterial monitoring -- that was discussed at an earlier BPAC meeting -- has been implemented.

There are several different processes that are in place.  Those clearly need to be looked at as part of this whole process.

I think the industry, in terms of the comments that they offered, have provided some guidance.  AABB, I think, in particular should be worked with very closely in terms of developing this.

This is a good start.  We have got, I think, a long way to go and certainly it would be easier for the committee to comment on something specific if we did have that draft document in place, and Jay, I understand the problems of getting that done.

I understand the issue of resources at the FDA in order to carry out these processes.  We have a strange situation where, obviously, congress expects that we will be doing things in absolutely safe fashion without regulation, or burdensome regulation of industry, and yet they don't give the agencies the resources, either personnel or financial, to carry out the work that needs to be done.

So, we are in a difficult situation, and keep moving forward.  There is a lot of important work to be done here, and we look forward to discussing it, I am sure, in the future.

DR. CHAMBERLAND:  I am also not a blood banker. So, I know that I don't have as good command on the level of details that a lot of other people do in the room.

I think what I am struggling with qualitatively is, the question is asking us to assess, kind of in an up or down fashion, yea or nay, if the proposed recommendations are adequate.

I think it is difficult to assess adequacy without the level of detail and the full guidance.  I guess, in some ways, I think this lends itself more to one of these questions for the committee, more qualitative discussion-y comments, you know, that kind of guidance, rather than an up or down vote.

Potentially, I think what you have heard is that a lot of people are all for taking a look at the guidance and really making some much needed revisions and updating, but it is just going to be a struggle to vote yea or nay.

DR. KLEIN:  Jay, I do think you are going in the right direction. I think you have identified the obvious things we need to measure, I think the actual levels, although there may be some modifications in the details.

I certainly applaud the idea of having a statistically based product evaluation. I think it is absolutely critical and is an order of magnitude more important than what we have done in the past in terms of blood quality and product safety.  I just encourage you to continue and give us something more specific that we can deal with.

DR. LINDEN:  If it is going to be considered, I would also encourage the agency to reconsider the four per month issue.

I think the proposal is to basically keep that the same. I think we heard from the blood collection agency that that is problematic and things have changed, that there is a lot more standardization. I think that could be reconsidered, and maybe some other sort of approach, considering the amount of standardization. I would urge a reconsideration of that issue as well.

DR. NELSON:  How about the issue of monitoring by the CBER lab in terms of the second part of the question?  In other words, now there are certain specific guidelines, how many samples based on machines and collection sites, and when the sample is sent, and what the FDA does in measurement.

There was some discussion by Kay Gregory and others about this issue. Does the committee want to make any comments about this, other than comments that have already been made?

DR. STRONG:  Once again, I think this really is an opportunity for dialogue, and it is another good instance of how we can work together to perhaps solve these problems.

It is not that blood centers are opposed to QC.  We do a lot of QC and we are certainly always concerned about the quality of -- I hate to call it product, but what we put into people.

I think there ought to be a way that we can sit down and come to a meeting of the minds and resolve some of those issues.

DR. NELSON:  I wonder to what extent the AABB, which also produces guidelines for the members, and the FDA regulations or FDA criteria, are in harmony at the moment.

It seems like the criteria are similar, as far as I can tell, but I am not sure to what extent there are any differences at the moment.

MS. GREGORY:  Some of the AABB requirements come directly from FDA, like three times 1011 and some of those things.

However, I think where there is some difference is that we have imposed a standard that 90 percent of the time or 90 percent of the samples tested, and that is not certainly the end of where we want to go. That was just a beginning that we felt we ought to get started on.

Certainly, AABB is very happy to work with FDA and, as a matter of fact, FDA does have liaisons to our standards committee.

So, we do try to talk to each other and make sure that we are going in the same direction as much as we possibly can.

DR. NELSON:  I think AABB has recently produced some guidelines for the options for bacterial testing, for instance, and the options for -- I don't know about the options for pH measurement, but some of these have been more specifically addressed recently; is that right?

MS. GREGORY:  We have.  Because the standard for bacterial detection is fairly new, we have issued some fairly detailed information about that particular aspect.

I don't know that we have anything recent about pH or any of the other testing.  Certainly, we are available to do that sort of thing, if people would find that helpful.

DR. ORTON:  Ken, I just want to also qualify, with the bacterial testing issue, as I mentioned earlier, platelets in the CFR are exempt.

Now, whether we believe that that is appropriate or not, it is in the regulation, and we are trying to get around that to get scientifically where we want to go, and even issues about pH, how we can incorporate pH without, in fact, violate what is already written in the regulations.            Again, we are getting scientifically to where we want to go in that area.

MS. GREGORY:  I guess I had forgotten that. AABB does have a pH requirement of 6.2.

DR. ALLEN:  You asked specifically for comments on submitting product to the FDA.  I can certainly see arguments for why that needs to be done.

I understand the industry's issue with regard to the cost and an appropriate sampling scheme, which I am not sure is in place.

On the other hand, I absolutely agree with Dr. Klein and Dr. Linden in terms of the need for assurance of the FDA laboratory standards and CLIA certification on this.

I would urge that this whole area be looked at very carefully in terms of what is hoped to be accomplished are the processes in place in order to ensure that there is a reasonable degree of confidence that that will be accomplished.

If product isn't sent, what are the implications of that?  For example, simply sending records, if, as Dr. Klein pointed out, the records don't, in fact, because of the devices being used to measure quality control and assurance on the laboratories where the data is being collected, if those are inadequate, then it doesn't matter how large the volume of records submitted. It doesn't really tell you what is happening.  So, this is an area that does need to be considered very carefully.

DR. ORTON:  I do want to clarify that a little bit, too. In the validation protocols that we get, we ask for SOPs that are pretty much associated with every part of the process.

So, we are not just getting two months worth of QC data and absolutely no other information on how the QC is done and quality assurance or anything like that.

In fact, the new guidance documents give much more specifics in terms of quality review oversight and things like that.  So, it is not done really quite as simply as you just stated.

DR. EPSTEIN:  I just wanted to comment that there is an initiative in CBER to bring our laboratory functions up to the ISO standards or ISO 17025.

Funding for that has been extremely limited, and so we haven't been able to make the progress we would like to make, although we are well aware of the issue.

DR. KLEIN:  Even getting all of the SOPs doesn't tell you that someone is actually taring the bags.  That is the point I wanted to make.

DR. ORTON:  Right, we can only hope.

DR. SCHREIBER: Just a point of clarification for me. There were a couple of things in the document that I found confusing.

One is, I am not quite sure of the extent of the QC program that you are talking about.  Right now, if I understand, it is just when there is an application or an application change, samples are sent to the FDA.

I guess I was not under the impression that the FDA was going to initiate a standard QC program across the board, that at periodic points in time all centers would send so many samples for testing.  So, it is really just the same -- it is not really an overall QC program that we are talking about for the FDA.

DR. ORTON:  We give guidelines for the overall QC program at the center, and then the products that come in are associated with specific types of licensing applications.

DR. SCHREIBER:  I think that is a big difference. I think when you look at licensing applications and the FDA testing something, that is quite difference than having FDA measure you on quality control.

I would tend to go one way with that but, if it were the other situation, on overall FDA quality control, I think I would be a little bit more skittish.  In terms of the application process, that doesn't bother me as much as the quality control process.

DR. ORTON:  In fact, when the licensing application comes in, not only do the products come in, as Betsy alluded to.  Two months of quality control come in.

So, we are seeing a combination of the product coming in and what they have been getting for two months at the center.

DR. SCHREIBER:  For a limited time period.

DR. ORTON:  Correct.  We have, in fact -- Betsy can confirm this or not -- sometimes when we had some questions, either the data that comes in is incomplete or we are seeing some miscalculations, we may actually ask for an additional couple of months worth of data just to be sure that we are seeing good representation.

DR. LINDEN:  Just to be clear, in terms of the way the questions were phrased to us in writing, A and B on the four per month and the internal audit system, I am recommending reconsideration of those.

On C, the consideration of bacteriologic testing as part of the QA monitoring, I am in favor of the agency considering that as part of considering this whole guidance.

DR. ORTON:  I don't think we would drop anything that is there. It is more putting it into a plan that is scientifically and statistically sound.

DR. HARVATH:  Having not seen the guidance, I am going to ask FDA to consider, perhaps in the preamble to the guidance, some background information regarding how it is that platelets pheresis have this unique historical perspective of being tested at FDA, whereas other types of cellular components do not.

If there were, I think, background information provided, I think it would help the reader, if you still intend to continue that program.

I also think that it is going to be very critical that, if FDA does continue having this testing program, that they have to be resourced to support the level that is needed to have a laboratory that has the staffing, the space, the instrumentation, all the costs that are associated with running that type of laboratory have to be provided to the agency in order for them to be able to comply with the requirements that everyone else has raised here.

I just think that it would help the reader to have that historical perspective of how did this platelet testing program originate?

What was its original intended purpose, why is it still necessary, if so.  I think that will help put this guidance in a better context.

DR. NELSON:  I think that is a good point.

DR. KNOWLES:  I think what you just said was excellent. I think to just take it a little bit further, it becomes a credibility issue, overall.

DR. ORTON:  Is the answer, keep working on it?

DR. NELSON:  I don't think this is easily susceptible to a yes, no, or maybe vote.

DR. ORTON:  I also want to thank everybody. When we drafted this, we drafted, particularly the quality control section, using the regulations that are in place and what has always been done.

As we started talking more about the statistical sampling that had to do with the scan statistics, it started making us think, well, what does this really mean overall, and that we are dealing with very high tech instruments compared to the manual methods that were done.

We certainly are thinking that perhaps we need to think very much out of the box and, in fact, intend to meet next week to discuss your concerns and perhaps how we can get there in a better way.

Also, keeping in mind that the regulations do restrict us in some ways and we are trying to work around that as well.

DR. ALLEN:  Let me second that. I think that is a very good approach and take it one step further, that implementation and training for implementation, when you get to that point, needs to be very important.

You know, I am not a statistician.  I did do an MPH with an emphasis on epidemiology and took as many statistics courses as I thought I could pass.

You know, it astounds me how many people get confused even on calculating a percentage and a percentage reduction, and how they count numbers and don't look at rates and that sort of thing.

I think the training for implementation is going to be very, very important as you move toward trying to implement a statistical analysis for quality control manufacturing.

DR. ORTON:  It even goes down to a very basic level of understanding, when you say calculation, understanding how to calculate a volume from a weight and a specific gravity.  It gets as simple as that as well.

DR. NELSON:  Well, I think we are not too far behind today.  Maybe we can come back at 12:45 and then in the afternoon we have presentations on the laboratory of hepatitis and related emerging agents, and the laboratory of bacterial, parasitic and unconventional agents.  So, 12:45.

[Whereupon, at 11:40 a.m., the meeting was recessed, to reconvene at 12:45 that same day.]

A F T E R N O O N  S E S S I O N (12:45 p.m.)

DR. NELSON:  Thank you, Dr. Smallwood. This afternoon we are visiting the site visit of two labs, the laboratory of hepatitis and related emerging agents, and the laboratory of bacterial, parasitic and unconventional agents at CBER.  For an introduction and overview, Dr. Kathy Carbone.

Agenda Item:  Open Committee Discussion.  Review of Site Visit of the Laboratory of Hepatitis and Related Emerging Agents and the Laboratory of Bacterial, Parasitic and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, OBRR, CBER.  Introduction and Overview.

DR. CARBONE:  Good afternoon. I am Kathy Carbone.  I am associate director for research, acting, at CBER.  I always find that funny, because I wonder if I am up for an academy award at the end of the year.  At any rate, I am going to be brief because the real issue here is the review of some of our scientists.

Those of you who may be familiar with intramural programs, of course, we don't officially participate in any kind of NIH RO1 grant writing and grant review which, in many academic centers, serves almost as a de facto review body on a particular investigator's work.

So, what we hold are internal site visits, where we bring in members of the advisory committee and experts from outside the advisory committee, who are experts in the field of the scientist being reviewed, and ask them to comment on the research programs.

We also ask them to comment on specific personnel issues, of promotion and conversion to permanent civil servant status and those will be discussed, of course, in a closed session because those are personnel issues.

We have a large effort going on in research within CBER, because most of our reviewers within the organization who deal with the regulatory issues, also conduct research at various levels -- laboratory, computers, statistical, et cetera.

In addition, we have the excellent support who perform full time review activities at CBER, and these groups work integrally.

In many cases, the belief is that the importance of maintaining an active research group that has expertise and hands on experience, is defined by the nature of the complicated products with which we work, as you may know, from having participated in these sessions.

Today we will have presentations by the individual laboratories and scientists on their research, a brief presentation.

Much more extensive presentations were given to the subcommittee, the site visit subcommittee, and then that will be followed by questions, and then we will follow that with a closed session, where the committee will discuss the report that has been submitted by the site visit committee.

I think Dr. Epstein has some specifics about the organization of the office, but does anybody have any questions for me?

DR. KLEIN:  Could I just ask you, how does a research project get initiated, and what project does it go through to the point where it starts to get done?

DR. CARBONE:  The offices are in charge of directly administering their research programs. I have oversight, essentially, on the entire center's programs.

Generally, the office has a series of experts in a particular field that is of interest or need to the center.  That expert is a reviewer and becomes very aware of the critical problems in science, or lack of science knowledge, that provide blocks to their review process.

The projects are often designed around critical gaps. I don't know how many of you are aware of the FDA Commissioner's release of the critical path, research white paper, at Research America! talking about the specific types of research that we do.

Those research projects have been discussed with their supervisors, and their supervisors provide them with resources in order to do what they can to make the strides that they can on those areas. So, that is a within office prioritization process.

Agenda Item:  Overview of Office of Blood Research and Review.

DR. EPSTEIN:  My purpose in the next few minutes is to give you some orientation regarding the office of blood research and review and then, following my presentation, Dr. Nakhasi, who is the director of the division of emerging and transfusion transmitted diseases, will give you an overview of the scientific operation within that division, two components of which are the subject of these two site visit reports.

Let me apologize in advance.  A number of you were at the orientation that we had last October, and the things that I am going to say are a little bit duplicative, but those of you who have been on the committee for along time and already know these things, I guess it is a refresher.

You can't read this, although it is in your handout.  This is the organization chart for the Center for Biologics Evaluation and Research.

What you see here is that we have about eight different offices, but three of them are directly involved with product review.

Those are my office, the office of blood research and review, the office of vaccines research and review, which is under the acting directorship of William Egan, and then the office of cellular tissue and gene therapies, which has been under Dr. Naguchi. However, he is going to be moving within the organization and there will be an acting office director in place.

So, the structure of the office of blood research and review is as follows.  We have the office of the director, which has about 18 staff.  We then have three divisions which I will be showing you in a subsequent set of slides:

A division of blood applications, which deals with blood component review, but also deals with our administrative process for tracking of all of our regulatory documents, including applications and supplements, guidance documents, letters and the like.

A division of hematology which, not surprisingly, is focused on the hematological products per se, and I will go into some detail;

The division of emerging and transfusion transmitted diseases, which has the primary responsibility to deal with control of transfusion transmissible diseases, and deals with the donor screening tests that are used to screen for etiological agents, as well as the scientific basis for our donor selection policies.

In terms of size and funding, we have a staff ceiling of 167.  We have been operating, in the last year or so, at a height of 162 full time equivalents.

We also have contract hires, which are, for the most part, fellowships, Fogartys, ORIs and ERDA fellows, who serve within the laboratories and assist our investigators.

We have, in a typical year, about a base budget of $1 million operating dollars, some of which is garnered by, if you will, underutilizing our FTE ceiling.

In other words, we get designated dollars based on a staffing limit, and we sometimes hire fewer staff than we are allowed to, so that we can convert those salary dollars to operating dollars.  That is sort of a dilemma that we face annually, is what can we afford by way of personnel versus operation.

This is now significantly supported by outside grants.  We are allowed to compete for various forms of external funding, which include interagency transfers and some competition for agency funded grants, such as from the NIAID.

Now, this is the chart that shows the division of emerging and transfusion transmitted diseases. Dr. Nakhasi is the director, Paul Mied is his deputy.  Here, if you will just raise your hands so people can identify you, and you will be the next speaker up.

Then we currently have three laboratories within this division. The division is based on the NIH campus, although there is a significant component at the Nicholson Lane research facility, specifically including Dr. Asher's laboratory of bacterial, parasitic and unconventional agents, which is the subject of the site visit.

Then we also have some administrative staff and product reviewers who are at yet another facility, Woodmont Office Complex, which is a couple of miles north of the campus.

The laboratory of molecular virology, under Dr. Hewlett, has been reviewed by this group on a previous occasion.

The third laboratory which is, again, the subject of the current discussion and site visit, is the laboratory of hepatitis and related emergency agents under Dr. Gerardo Kaplan.

Just for completeness and for the sake of future reference, the division of hematology, under Dr. Golding, has a laboratory of cellular hematology, laboratory of hemostasis, which currently has the vacancy of a lab chief, although we have a superb senior investigator in that group, laboratory of plasma derivatives, laboratory of biochemistry and vascular biology, and a clinical review branch, which does not do wet laboratory work, but is involved with clinical trial design issues.

Now, the division of blood applications, which is then our third division, is directed by Alan Williams, and that branch has a plasma branch, blood and plasma branch, a regulatory product management branch, which is the administrative nucleus for managing our entire review process, managed review, milestone tracking for our user fee funded programs for pharmaceutical drugs, and also for devices.

Then we have a devices review branch, although some of the devices, such as aphoresis machines, are reviewed in the hematology division.

So, the mission statement for the office of blood research and review is as follows:  We provide regulatory oversight of the safety, efficacy and availability of blood products and retroviral diagnostic tests.

In the past, we had a direct product responsibility role on human tissues, but now we have only a consultative role, since the establishment of the office of cellular and gene therapy.

So, the functions that we provide under this mandate, we establish the policies and standards for the regulated products.

We review applications for investigational and marketing or commercial product use.  We conduct a lot release program, which is both for the test kits and also for certain of the injectable products.

We perform, in cooperation with the office of compliance, establishment inspections.  We engage in product investigations, when there are problems with the products, and we assist in compliance actions, whether they be warning letters or revocations or consent decrees, to ensure that current science is being properly integrated into our safety assessments.

Also, along that line, we make health hazard assessments when there are product deviations or when there are medical adverse event reports.  It is the scientists in the office who make the assessments of the health significance and, of course, we lean especially on our medical staff.

Then, additionally on this list, we carry out mission related research, and it is the mission related research that you are here to review.

I am going to take you on a brief tour of the spectrum of products that are evaluated in this office.  We review blood and plasma components for transfusion, also blood components such as source plasma, that are used further in manufacturing.

We review and approve plasma derivative products, donor screening tests, blood grouping reagents, the devices that are used in blood collection, storage and processing, some of which may be also approved in the Center for Devices and Radiological Health, for other uses.

So, for example, an aphoresis machine for therapeutic aphoresis would be reviewed and approved in our sister center whereas, if it is used to collect components from a donor, then it would be additionally reviewed, or independently reviewed, in our center.

We review blood bank and related computer software and, because of an inter-center agreement that dates back to 1984, we are the center that reviews all HIV and other retroviral diagnostic tests, not solely related to use in blood banking, but also for their general medical use.

That is why, at some times, this committee has been asked to look, for example, at HIV rapid tests, because they are retroviral tests, even if they are not necessarily labeled as donor screens.

I am not going to read all this.  You have it in your handout.  These are some detailed lists of the kinds of products that are regulated in the divisions.

Again, the focus here, within the division of emerging and transfusion transmitted diseases, is on infectious disease control, and so we have scientific programs for many of the etiologic agents that are of public health concerns, such as retroviruses, such as hepatitis, such as parasitic agents, and we are concerned with the test technology infrastructures.

So, we are concerned with methodologies, such as nucleic acid amplification, or looking a little bit to the future, perhaps gene chip technology.

In the division of hematology, as I said, we are focused on the plasma derivatives.  Plasma derivatives are not all of human origin.  We deal just as well with animal derived antisera, for example, antivenoms, often made in horses.

We have some progress toward developing plasma products made from transgenic animals out of blood or milk, and examples would be, for example, bovine thrombin, as a kind of product that we might regulate.

Then, as you might imagine, program responsibility of the cellular blood products, but also plasma volume expanders, hemoglobin and non-hemoglobin based oxygen carriers, and the related devices such as aphoresis equipment.

Then, in the division of blood applications, it is not solely an administrative unit.  There is the scientific expertise and the review responsibility for the blood components for transfusion.

These would be, then, the more standard components, blood components for further manufacturing, including source leukocytes, collection and storage, solutions and containers, the anticoagulants, the additives, the rejuvenation solutions, freezing solution, blood establishment computer software, blood grouping reagents, and there is actually a small web lab that deals with some product testing in that area, the blood processing devices, blood warmers, centrifuges, scales, and donor deferral criteria.

Also, there is a strong group here under Alan Williams, who is, as you know, a highly trained epidemiologist, to look at the epidemiological issues that affect the blood donation and the blood system.  So, for example, Alan was responsible for developing monitoring systems for shortages.

Now, you are all aware that the scientific work goes concurrent with regulatory work. Each individual that is involved as an investigator has responsibility for some share of the regulatory load.

The message that I want to convey to you here is that the review work load is large.  This is a summary of our review in 2003.

As you can see, we had nearly 100 original INDs or investigational device submissions, as well as more than 1,000 amendments.

The premarket reviews themselves come in a variety of flavors, original applications for biologic licenses, again, a modest number, but these represent an enormous work load extending over about a one year period, and then about 1,000 supplements which often have extensive clinical data.

Premarket applications are somewhat smaller in number, new drug applications, accelerated new drug applications and traditional 510(k)s.

I guess I am not showing you a figure, what this represents in terms of an FTE burn, but let me just assure you that it is extensive.

So, why do we do research at CBER?  We often need to explain this, both to the public and to sources of government funding.

There is the general concept that FDA regulates, NIH does research, and CDC does epidemiology and surveillance.

The reality is that there are some special needs which lead one, especially in the area of biological products, to support a research endeavor and, in actual fat, the laboratory program for biologics goes all the way back to 1902, to the first biologics act, and is more in the tradition of biologics regulation than otherwise.

Just to try to explain it, we do research to address existing product safety and efficacy concerns.  The products are not perfect. They have their technical limitations, their toxicity profiles and, in the case of screening tests, problems with perhaps sensitivity, specificity, reproducibility.  So, we are concerned with current products and the gaps in perfecting them.

We also, as I mentioned, are involved in product investigations.  When there are unexpected events affecting the products -- product failures particularly -- our laboratories can be mobilized to shed scientific light on causes of problems.

For example, this committee heard, in the last year, about white particulate matter that was found in blood containers, and a lot of the investigation was done in our own labs, and that is an ongoing saga.

At any point in time, we could tell you at least four or five product specific investigations that are ongoing because of problems that have been reported.

Of course, we respond to new safety threats. In the division of emerging and transfusion transmitted diseases, the chief concern here is emerging infectious disease.  There can be other forms of threat as well.

We conduct research to facilitate product approvals and improvements, largely through the development, which comes here, of product standards and controls, but also to lay the scientific foundation that will underpin the requirements that we establish, either at the investigational level or in the course of the phase II, phase III trials and applications.

You know, we are the portals through which the companies must come to get marketing approval.  So, what we require, by the way of preclinical or clinical evidence, including trial design and statistical methodology, is then the determinant of what it takes to get to market.

Without research laboratories, we would be hindered in investigating the scientific framework that enables us to establish the review standards above and beyond specific biologics standards and control per se.

We also use the research to anticipate public health need, as well as to support science based policy and decision making.

Because of this, we have a very broad mind set of what is appropriate and mission related. So, for instance, in work on etiologic agents for which there either are, or we think there might need to be, control programs for blood safety, you might find us doing pathogenesis research, and we would consider that appropriate in trying to understand the public health dimension related to the etiologic agent.

Finally, although not a trivial matter, having laboratory programs enhances our ability to attract in, and retain, high quality scientists who also bring into the agency cutting edge knowledge and skill, which then keeps us at the forefront and enables us to serve our mission optimally.

So, in broad brush, these are some of the areas of current mission related research -- safety, efficacy and standardization of clotting factors and immune globulin products.

For example, yesterday you heard about the whole issue of standards for approving immune globulins specific to hepatitis B.

Novel viral detection -- and really, this shouldn't just be viral, it includes parasitic and other agent detection, prion, for example, NAT.

Toxicity of oxygen carrying compounds, which have been a barrier to progress in the development of alternatives to red cells.

TSE detection, pathogenesis and removal and contamination, which I think you will hear about shortly. Structure, function and storage issues related to platelets.  You heard a lot about platelet variability.  These turn out to be very delicate, cellular elements and a lot of the management of the platelet product in blood banking would be improved if we could solve some of the underlying scientific problems related to platelet storage and activation.

The epidemiology related to viral variance.  For example, new subgroups of HIV, so-called silent hepatitis B.

Diagnostic tests for bacterial and parasitic diseases and, as I said earlier, emerging infectious diseases, and we have a very wide list.

I mean, in the last two years alone, this includes bioterrorism agents, west nile, SARS, we have had a program of smallpox vaccination, we had an outbreak of monkey pox.

So, we are actually kept very busy, and there is a long list of things that perhaps have not hit your radar screen that we are also looking at, things like chlamydia, HHBA, nanobacteria, and some of the things we think are resolved, TTZ, senzi, HGV.

So, at any given point in time, again, there is a relatively long list and, more often than not, there is some reflection of laboratory activity.

I think, because this group is very highly familiar, I am going to quickly skip over a slide set of background on the blood industry.

I think you are well aware of the dimension and significance of the blood system. So, if we could just pace through the slides?

I think it is worth dwelling on this slide for a moment, current risks of transfusions.  This is now risk per million.

You can see that we have had some major triumphs in the areas of HIV and hepatitis C, with rates around one and two million.

Hepatitis B is now one of the more frequently transmitted agents, only because we have managed to reduce the other risks to such a remarkable degree.

There is the issue over clinical significance of parvovirus B19. However, a major effort to control infectivity of parvovirus from plasma derivative products, through mini pool NAT testing of donors, and then the evolving effort to monitor and reduce bacterial contamination of blood products, but most specifically including platelets on account of their room temperature storage and the risk that they, therefore, present.

Among the non-infectious risks of transfusion, we are focused in three ways primarily, hemolysis, which is either acute or delayed, which is largely related to medical errors, wrong unit released, and the whole effort to improve medical monitoring and control systems in hospitals and transfusion services.

Then TRALI, which is still a difficult scientific problem being addressed.  Although we have made efforts to raise awareness, control measures are really not yet in hand, although at least in the United Kingdom they are thinking about at least some donations by multiparous women to at risk patients.

We have a several level approach to ensuring blood safety, which includes donor selection, again, science based using epidemiology, use of deferral registries, infectious disease testing.

Quarantine holds, while we have a highly validated system to check the results and control the units before release, monitoring, corrective action, and then removal and inactivation, mostly for derivatives with progress anticipated but not yet at hand for the cellular components of blood.

Let me just quickly touch on a couple of initiatives that occupy a great deal of our time and effort.  Since 1997, we have had a blood action plan, which has focused in a number of ways.

Updating regulation is one, improving responsiveness to emergencies is another.  Probably the area here that has garnered the most attention is on monitoring and improving the blood supply, because of all the stresses that have come in recent years on maintaining an adequate donor base.

We have two user fee programs, which have compelled us to implement milestone tracking, more or less, an assembly line mind set on moving products through the system.

There is a major effort to accomplish quality of review, while putting it all on time lines. That is, of course, the necessary predicate for us succeeding in that program, while meeting the bench marks that are required for us to get the additive funds.

Then, just to mention a few issues of current concern.  Counterterrorism, emerging infections, completing the implementation of NAT screening for HIV and HCV, but then looking forward to the possibility to roll that out to hepatitis B.

Updating donor suitability criteria. You heard recently about revised questionnaires, the uniform donor history questionnaire, and then the question about whether we can move toward modifying test requirements as we move toward technology improvements.

Standards for new blood products, can we develop safe and effective, and I guess also cost effective pathogen inactivation.

Looking at frozen cells, for example, can we make better use of long term stored products. The open question on universal leukocyte reduction.

As I mentioned, hemolysis related to medical errors in matching donor and recipient is only part of a much larger effort focused on medical errors in the wake of the IOM November 1999 report, To Err is Human.

One recent initiative there was the final rule on bar coding, which includes requirements for machine readable code for blood components.

Then, disease control for cellular and tissue products, analogous to the kinds of programs that have been built for blood, and where the blood program serves as a consultant.

I think this is the last slide, just the approach to medical error which, again, is multi-faceted, technology solutions, automation, software, standardized labeling, patient identifier -- for example, radio frequency ID on the wrist band.

An upgraded guidance on requirements for reporting fatalities.  You heard at a recent blood products advisory committee of this apparent increase in donor fatalities, which we can not yet verify, and it has led to a call for more standardized reporting of fatalities.

Rule making on labeling standards, we had a recent proposed rule, a patient safety rule, that contained updated requirements on reporting a serious adverse reaction to transfusion, which are currently exempted from the agency reporting, and an initiative on trying to introduce the medical event reporting system for transfusion medicine, that was developed by Al Kaplan and Jim Battles under an NHLBI contract.

I think that is the last slide. Oh, counterterrorism also looms large.  For example, anthrax, products to address medical needs like vaccinia immune globulin, and then the systems issues of outreach and coordination.  I hope that is the last slide.  In any case, we will stop the slides.

I know I have spoken at some length, but I feel that, since the advisory committee is called upon, on multiple occasions, to review all the different components of our program, that it would be worthwhile for you to have at least a bird's eye view of the entire program, both with respect to the scope of the scientific activity and the regulatory activity.

So, I appreciate your patience, and I will invite, with Kenrad's permission --

DR. NELSON:  Any questions or comments?

DR. STRONG:  I have a couple of questions. Jay, the issue of cell and tissue, I thought at one point, was to come to this committee, but I don't know if there is another committee to deal with that.  If there is overlap there, I wonder if you could clarify.

DR. EPSTEIN:  I don't believe that that has been resolved yet.  When the cellular and tissue program was divided between the office of therapeutic products and the office of blood, we dealt with most of the donor eligibility standards and processing issues, for products that might not be subject to independent licensing, as therapeutic products.

Now that we have this new office of cellular products and gene therapy, we have not yet clarified which advisory committee would be used, unless Kathy can tell you something that I don't know.

We still do have experts on bone and tissue, on the blood products advisory committee. Committee members can be used on other committees. So, we don't lose anything having members here who could be, on occasion, coopted into other committees or joint meetings.

The responsibility still lies wit the biologic response monitoring committee, the modifier committee, which was the advisory committee to the office of therapeutics, which, as I think you know, was consolidated into the center for drugs, and the blood products advisory committee.

So, the expertise is still resident on those same two committees, and we haven't quite sorted out which issues we will bring where.

DR. STRONG:  Then another question about parvo.  We have heard rumors that there is going to be a requirement for parvo testing for plasma products.

DR. EPSTEIN:  Well, we haven't moved to a requirement in the sense of regulation. We had a blood products advisory committee which endorsed the concept that we should treat parvo virus screening as in process testing, in the manufacturing of plasma derivatives.

It was pointed out that there might be a narrow range of circumstances in which you might want to temporarily defer a donor, for example, a frequent aphoresis platelet donor.

The driver for screening for parvo virus remains protecting the safety of plasma derivatives.  There has been transmission, of course, of parvo virus by factor 8 and 9, and there is, of course, concern that there not be transmission to pregnant women, especially, who are getting RH immune globulin.

So, most of the thrust has been in that direction because, as you know, parvo virus, being non-enveloped and heat stable, is not easily removed.

So, the combination of screening and assuring an adequate neutralizing antibody titer in the pool has been the strategy.

For whole blood, the pressure to screen the donor is coming from the industry standard on acceptability of plasma for fractionation, and FDA has not yet developed a guidance.

If we were to follow the advice of the BPAC, it would be that we, even in that setting, not treat it as a donor screen requiring notification, but that certainly, if you could interdict high titer units, you should do that.

Then there would be an open question, whether the industry would want to move toward screening a subset of collections, so that positive units, or high titer units, could be averted from use in high risk recipients, for example, people who are immune compromised or who have underlying hemolytic conditions or other complications to the erythron. So, we are not quite there yet, but we are nowhere near requirements.

DR. LAAL:  Who looks at the non-retroviral diagnostic tests related to blood transfusions?

DR. EPSTEIN:  The Centers for Devices and Radiologic Health has the primary responsibility at FDA for medical diagnostic tests.

The only exceptions are blood donor screening tests and retroviral diagnostic tests, which are in the center for biologics.

DR. NELSON:  Dr. Nakhasi is going to give us an overview of the division of emerging transfusion transmitted disease.

Agenda Item:  Overview of Division of Emerging Transfusion Transmitted Diseases.

DR. NAKHASI:  Thank you, Dr. Nelson. I will just give you a brief -- I won't go in detail about the whole process, because Dr. Epstein gave you a very nice overview of the whole office, and some of the elements that I am going to be talking about with regard to our division, that is the Division of Emerging and Transfusion Transmitted Diseases.

As Dr. Epstein mentioned briefly, I will just give you the organizational chart.  The division has the office of the director, where we have several people, including myself, Dr. Mied and other policy related and regulatory related people, who are responsible for that.

Then the division is sort of arranged into four major labs, the lab of molecular virology, Dr. Indira Hewlett is the chief of that laboratory, and I will talk about the areas of research, basically, the HTLV, HIV, west nile, molecular virology, immunopathogenesis, and regulation they are doing, and also I will talk about their regulatory responsibilities.

Then the other laboratories, the laboratory of hepatitis and related emerging agents, Dr. Gerardo Kaplan is the chief of that, and the area of the pathogens that is the responsibility of that laboratory is hepatitis B, hepatitis C, hepatitis A and other emerging CT agents like hemorrhagic viruses and things like that.

That lab will be reviewed today and Dr. Tabor, who is part of that laboratory, even though he is directly associated in the immediate office of the director, the office director, his laboratory is in this laboratory, and he will be presenting his site visit report, as well as Dr. Gerardo Kaplan, whose lab was also site visited recently, will be presenting his program.

Then we have the laboratory of bacterial, parasitic and unconventional agents.  Dr. David Asher, who is the chief of that laboratory, will be presenting his part of the work, which is the TSE pathogenesis and detection.

In that also we have the section on parasitic bacterial pathogenesis detection.  In particular, my lab is situation in it and I am not going to be presenting, because you heard last year in our site visit report on that.

Then we have the product testing staff, which is basically a major area of our activity, which is involved in the testing both of the serological and nucleic acid based test kits for these agents, HIV, HTLV, hepatitis B and C, and now maybe soon west nile or other agents which we will be looking at.

The mission of the division is, as you heard from the overall mission from the office of blood.  Our mission is to really plan and conduct basic and applied research in the development, manufacture, pathogenesis and testing of blood borne agents, such as viral agents, which include HIV, HTLV, hepatitis, and west nile.

Then, things such as leishmaniasis, malaria, chagas, bacterial and then BT agents such as plague, anthrax, hemorrhagic viruses, smallpox also, and obviously the transmissible spongiform encephalopathy agents by prions.  So, it is a broad range of agents which we are looking at, many of them emerging and many of them already emerged.

Then we have, in addition to conducting the basic and applied research on that, we have the expertise in our group, we also have a major work load which ensures the safety of the nation's blood supply by reviewing, validating and recommending actions on several biological application, premarket approvals, INDs, IDEs, 510(k)s for blood screening and diagnostic tests for retroviral diagnostics, not for all, I shouldn't say that, it is only the diagnostic testing for the retroviral agents.

As Dr. Epstein clarified, hepatitis and other diagnostic agents are reviewed by the CDRH, our sister center.

We also develop and devise FDA guidances for users of blood screening and diagnostic products.

In addition to that, we perform lot release testing, as I said earlier, and also investigational tests.  I think many times there are certain areas where we are asked to look into the particular test performed, or where a particular donor transmitted with a particular agent.  So, we are given the samples and we look at those samples. So, there is that investigational type of research.

Then we also develop reference materials for lot release testing.  As you much have heard, last year we went through the whole litany with the west nile virus epidemic, developing these reference materials for this, so that we can validate the sensitivity and specificity of those tests.

We provide scientific and technical advice to other agencies and government components. We interact through the public health service agencies.  We also interact with other agencies, CDC, NIH and DOD.

We present issues, like this one here, at advisory committee meetings, on tissue, spongiform encephalopathy, at advisory committee meetings.

The personnel and the budget for the fiscal year 2003, we now have a total staff of 59 in the division, and there are 11 senior investigators, which are supposed by biologists, staff fellows, staff scientists, regulatory scientists, which are full time reviewers, they don't do any research.

Then we have administrative staff and some of the post doctoral fellows, both funded internally and externally.

This number here may be a big number but, as Dr. Epstein said, the majority of this money is funded from the grants that we get through interagency and congressional initiatives, so that was last year's approximately $800,000 total money.  The division published 41 original articles and some book chapters.

With regard to regulatory activities, as you can see, we have a significant regulatory load, a total of 374 applications, which included all applications in different colors and varieties and flavors.

We also did quite a bit of lot release testing, and performed a lot of inspections and did the laboratory investigations.

So, that gives you the dimension of the regulatory work load that we have, in keeping with our scientific expertise at the same time.

The areas of research, as Dr. Epstein mentioned, that I will discuss in a little bit of detail, what areas we are working in this division are basically what we have set up the division into two major research areas.

One is to look at the pathogenesis of these blood borne viral bacterial agents, and then the second one, I will talk in a minute about the development of tests which could detect these agents.

So, basically, as Dr. Epstein mentioned earlier, this allows us to look at the agents as in how they cause the disease, how they can be tackled, so that we have in-house knowledge and understanding of these pathogens when we are reviewing these applications.

As an area of research, the pathogenesis of HIV, drug resistant HIV, genetic variants of HIV, HTLV, west nile virus, which you heard last year was a major effort in the division, HHVs and other herpes infections in AIDS, pathogenesis in malaria, leishmaniasis and chagas.  As you heard, this last year we had a significant outbreak of leishmaniasis in the soldiers who are at the moment stationed in Iraq and Afghanistan.

So, at the last blood advisory committee, we presented regulatory as well as research areas which are important in that area, needed to look at the donor deferral policies and things like that.

We are also having a research program in the pathogenesis of the bioterrorism agents, both bacterial and viral agents.

We also have a major emphasis on the hepatitis related pathogens, and you will hear more when Dr. Tabor and Dr. Kaplan give their presentations, and also the pathogenesis of the TSE agents and their detection, as well as bacterial agents, and Dr. Asher will give you more detail about the progress about those agents.

As I said, the other research aspect of our division is the blood donor screening test development. That is another part of our bread and butter here.

We all have constant efforts going on in the development of new technologies for the detection of retroviruses, such as use now of the micro array and technologies, and pushing the frontiers of these new technologies because, more or less, they will be soon at our door, and we have got to be prepared in advance to know how these things work and what are the nuances in those kinds of techniques.  So, therefore, we do ourselves the research in those areas.

Then the identification of the HIV-1 and 2 variants, in the diagnostic area, drug, therapy and vaccine development.  Obviously, you will not hear anything about this one, as Dr. Hewlett presented last year their report.

We also develop standards for new technologies, which include HIV, west nile virus, hepatitis C.  The major effort is also in the pathogen chip, the development of the pathogen chip, using micro array DNA technology.

The purpose of this really is an important thing because, as more and more agents are emerging and they are threatening the safety of the blood supply, we cannot afford the detection of one agent at a time.

As you heard from other previous BPACs, we need to have an effort where we could be possibly looking at several of these agents together, such as multiplexing.

So, one of the ways is to do this micro array technology. Therefore, we have a significant effort going on in the division which is looking at multiplexing several of these viral and bacterial agents, and how many can we multiplex, what is the effect on the sensitivity and the specificity when you are multiplexing those things.

The same thing, we have a strong effort on the development of detection and validation methods for TSE agents, and you will hear more from Dr. Asher's presentation, and also in the area of detection of bacterial agents in blood.

In addition to that, the other agents that we are looking at, emerging and re-emerging agents, we also have, in the last few years, initiatives on the counterterrorism initiatives, such as I mentioned, the detection of those agents, the bioterrorism agents, that can threaten the blood supply.

Therefore, what we are trying to do is to develop laboratory expertise in these new technologies, use all the expertise from the evaluation of related submissions from industry.

We could look at them and, if we have developed a technology internally, can we transfer that to the development in industry.  If these products come to us or the detection comes to us, we should be ready for lot release testing of that and, more important, we should understand how these pathogens work and how they can work in the blood system.  I think this is a perspective, looking a head of time, how we should be ready for tackling such situations.

The last slide, actually, Dr. Carbone mentioned to you that there is a big initiative going on, on the critical path research in the FDA.

To really understand what that means, is the kind of research that is important where you bridge the research with the regulatory mission.

The areas which other outside people may not be interested in, how we can do that research and really perform in those situations.

An example, I have given you several examples, what we have been doing in the pats and what we will be doing in the future, is really some of the examples here.

For example, we last year were very heavily involved in the development of standards for west nile standards testing, and panel development and areas like that, on analyzing the sequences of the several parvo viruses isolated from both bird and human, to figure out whether the sequence differences between those west nile viruses may have any effect on the detection.

Our group has been involved in looking at this smallpox and blood safety, where people are vaccinated, how long there will be any viremia, if there is any viremia in those people, at what stage can those donors be reinstated, and how the current testing methodologies may interfere in detection of those things.

I think that kind of research and how we could be using that information when we can provide to the industry, they will be up front and provide for those kinds of questions.

The same way with the HIV surveillance, the new variants coming up and there is a collaboration going between Dr. Hewlett's group and in The Cameroons, that they are looking at these different variants of HIV and how it will affect on the detection in these currently available tests.

You must have heard a couple of years back, we did an internal study where we compared the relative sensitivity of the HBSA test that are available, that are antigen tests, versus the HBV NAT and how they are comparing.

Because of that, we now have the HBV NAT system, or the development of NAT is coming through and also the guidance documents which are very important to tell which tests are performing better than the others.

So, these kinds of comparisons and these kinds of studies really is the type of research which is important for the regulatory aspect of it.

For example, the other one, this one we call opportunities here.  These are the stories and these are the opportunities, and the opportunity areas, we are looking ahead, like as I mentioned, the development of the micro array, which allows us to detect in multiple systems for multiplexing various bacterial, viral and parasitic pathogens based on the micro array.

So, if we have that system going here in house and we know the nuances, it is a kind of proof of concept, so that the industry can pick up on that, and they are aware that the FDA is looking in those kinds of areas.

Similarly, the other areas, for example, in the vaccine strategies for parasitic and viral agents and looking at the pathogenesis of these agents in the blood, we are developing certain concepts which would be important, even though we may not be able to develop those vaccines.

We are looking at those agents where we can develop a proof of concept and industry can take it from there.  Thank you. I think that at this point I will stop and take questions.

DR. LINDEN:  In light of Dr. Epstein's explanation of the office's role in tests for blood donor screening and retroviral assays, can you elaborate on your division's role in tests for bioterrorism agents?  Would that be just for blood donor screening assays?  I wasn't clear what you were referring to.

DR. NAKHASI:  That was mostly related to blood donor screening, yes.

DR. KLEIN:  If someone comes to you from your division with a great idea about developing a screening test for TSE, how do you decide, first of all, that that is what you are going to do, and how do you decide how much you are going to resource it?

DR. NAKHASI:  I think as you heard that problems -- I am just giving you a broad range. I am not focusing on TSE. I think David will.

The areas such as, how did we decide on the west nile?  The problem came up and we were ready, because of the things we were doing in house.

We dealt with HIV and HCV before.  The concept of universal screening using investigational NAT, well, where did that concept from?  The concept already was tested in the HIV area before.

That is how I think then we see on the horizon things coming up and we really start working in those areas.  For example, SARS.

In the beginning of the SARS epidemic, we were not sure whether there is a viremic phase. We developed a guidance. However, once we came to know that, up to now, there may not be an asymptomatic phase kind of a thing where there is a viremia, because we could emphasize, if there was, we would immediately embark on, we should have that area of research going so that we can develop the technology for testing that.

DR. KLEIN:  Is the research primarily externally driven?

DR. NAKHASI:  I think both primarily externally driven, but we cannot afford to have, every time, a new investigator on each area.

So, for example, my research group is working primarily in leishmaniasis, because that is my expertise I came from.  However, that expertise, tomorrow if it is chagas, we are ready to tackle that area.

For example, we have a malaria group, which unfortunately you will not be able to hear the presentation because the person had to rush to India because his father is seriously ill.

That is the area where we are developing the areas. So, there are certain areas where we can have expertise and have the resources to do it, and certain areas where we just leverage it.

DR. KLEIN:  I don't want to beat this to death, but I am just sort of interested in how you get an idea, how you prioritize the idea, then decide that it is a quality idea, and then decide how much you are going to resource it.

DR. NAKHASI:  I think that is a good question.  What we do basically is look at what is threatening the blood supply.

Then, depending on how much impact it will have and how much we should really emphasize it.  We do internal discussion about it and, therefore, based on that, prioritize it.  I think that is the only way to do it.

DR. CARBONE:  Let me elaborate just a little bit. I think one of the things that has come about in CBER is an extremely thin level of coverage of multiple areas.

We have an extremely broad group of activities to cover and very few people to cover it with and very little resource money to do so.

The decision on where to act is often one that has to be done quickly in response to an emergency.  We do take a lot of input from the outside world, but one of the advantages in our regulatory activity is we have a chance to see, across products, across areas, across agents, issues coming down the pike that seem to be common themes.

So, we tend to orient first to the urgent issue. If there is something urgent and unknown, then there really isn't time for the formal process, you might have a review, a discussion of formal applications.

If something is identified by consensus, immediately all the resources may be directed for a short period of time on that project, and current projects, the more continuing underlayer of projects, just simply stops, and everybody gets redirected.

That would be the west nile example, where there really isn't time for a formal review, because this is an urgent issue.

As far as the ongoing programs, those decisions are also made based on external products we know that are coming down the pike.  Many of these products are in development for years.

So, we see an area coming down, a large issue coming down and resources are redirected. We are talking really about a very small amount of resources that get redirected into projects.

Every year, every PI reports to the research program reporting, and that is reviewed by their superiors, their current activities and their accomplishments in every area.

One can have a proposed area of research, but if no accomplishments are forthcoming, then it must not continue, or the problem, the reason for no accomplishments, must be identified and fixed.

We also use site visits to direct research programs.  We take very seriously the site visit report. It will go to the division director and he will review the site visit report.

Site visits occur every four years, which is a reasonable amount of time for somebody to develop success in their program.

Since I have only been in the job for about a year, in the new site visit guidelines, we have now formally instructed the investigators to talk about their accomplishments, but then put in a section of what their future plans are, so that the committee can review those as well as their superiors and discuss with them.

I am speaking with Dr. Nelson this afternoon about initiating office wide site visits for more research program management.

So, we take this very seriously, because we do have very limited resources, and they must go into very carefully directed places.

Now, that said, we also have, as I mentioned, have investigators on the front line.  They initiate the idea, but the ability to work on it really requires review and careful monitoring of it, because of resources.

Pardon me for jumping ahead on this, but part of the idea with the office site visits is to get input from the advisory committee members as well as external experts, in devising specific priority paradigms, with the understanding that we often simply have to react urgently and drop everything to work on a specific problem.

DR. NELSON:  I think that is a fair amount of what you do. I think as a background, too, the other thing -- I have been on a couple of site visits -- is the expertise and the interest of the investigators.

I mean, many things are related to the safety of the blood supply, but also determine what expertise the staff has or the researchers have.

I think that is also a consideration, where they can be productive, in addition to responding to the acute issues.

DR. NAKHASI:  I just wanted to add, just one more thing, what Kathy said, obviously it is driven from what is outside, what is coming down the pike, the emerging pathogen, west nile.

Then we present to the other body, like yours here, these ideas and where do we go from here. So, we get input from both this advisory committee, the site visit committee and, in that process, there is a culling process.

If they feel that this particular research is not going where it should be, or it is not mission related, then that is how it is prioritized and channeled in a different way.

DR. NELSON:  I want to move on. So, Dr. Tabor, are you ready?  Dr. Ed Tabor will talk on the viral pathogenesis section, laboratory of hepatitis and related emerging agents.

Agenda Item:  Summary Presentation.

DR. TABOR:  Good afternoon. I am Ed Tabor. I am the associate director for medical affairs in the office of blood research and review, and I also have a small laboratory group that I brought with me from NCI when I came back to CBER.  This is my second period of time working in CBER.

I brought them in 1995, and have continued to work, as a small part of my responsibilities, with that laboratory group.

I want to thank the site visitors for spending the time to go over the research program in my laboratory,  Dr. Linden, who is here on the committee, and Dr. Blaine Hollinger, who is a former committee chairman, former BPCA chairman, who couldn't be here today because he is recovering form an illness, but he is present by telephone.

DR. NELSON:  Dr. Hollinger, can you hear us?

DR. HOLLINGER:  I am with you, Ed.

DR. NELSON:  I am glad to hear you are doing well.

DR. HOLLINGER:  I am looking forward to seeing your slides.

DR. TABOR:  I also want to thank Dr. Smallwood and her staff for facilitating the site visit and facilitating my presentation to you today.

My laboratory group is in the laboratory of hepatitis and related emerging agents, which is in the division of emerging and transfusion transmitted diseases.

This small laboratories is perhaps one of the most productive laboratories in the division of emerging and transfusion transmitted diseases.

In many years, this tiny laboratory has contributed more publications to the CBER annual report than any other DETTD laboratory group.

In the five years between the two site visits, from January 1998 to May 2003, I published 48 publications, including 20 in reviewed journals, most of which were original laboratory-based research.

In addition, from the month of May 2003, when the site visit took place, I have published seven more papers, which include several papers reporting the results of studies that were reported to the site visit.  Almost all the projects that were reported at the site visit have either been published or are in press.

This is a very high output for such a small laboratory. I had only three doctoral level people, now reduced to two, as of about seven months ago, because of budget cuts that went cross CBER, and we just happened to be part of a broad spectrum of laboratories that were hit by these budget cuts.

So, I have got two post docs working 50 percent of their time on research, the other 50 percent on regulatory work.

In addition, 80 percent of my time is devoted to my policy and regulatory duties, as associate director for medical affairs.

I want to just briefly tell you what some of those other responsibilities are.  I represent the office of blood on many CBER committees, such as the medical policy coordinating committee, and the international policy coordinating committee.

I have played an active role in the development of the nucleic acid testing regulatory policy.  I am the chair of the PHS committee that monitors and discusses emerging infectious diseases that could affect the blood supply.

For this particular duty, the existence of my laboratory has been very important. When there have been problems with emerging infectious diseases, that you will hear about in a few minutes, that no other laboratory was able or interested in pursuing, we did pursue them, and I focused the resources of my own laboratory in order to solve those problems.

I am now the coordinator of the office of blood counterterrorism activities, and I conceived and initiated a program to develop draft guidance documents for all potential biological and nuclear terrorism incidents, with regard to the deferral of blood donors.

The research blue print for this laboratory is to investigate issues of hepatitis B virus and hepatitis C virus that relate to transfusion, and to be ready to tackle any new emerging transfusion transmitted agent that threatens the blood supply when it first appears.

I am now going to briefly outline some of the projects that were presented to the site visit committee.  I will be going over them much more quickly than I did at the site visit presentation.

The first was a study of the drift in the hypervariable region of the hepatitis C virus in two individuals over a 27-year period.

We had the unique collection of serial serum samples from a patient and a nurse, who was also infected by a needle stick accident involving that patient, samples collected over a 27-year period that we could study.

With this unique collection of samples, we not only had perhaps a longer collection of serial samples to evaluate quasi-species evolution, but we had samples that came from two people who, in 1973, at the time of the transmission, should have had identical virus populations.  It was a really unique opportunity.

Now, it turned out that our investigations -- I won't go into it now -- that this nurse was almost certainly infected by this patient. It had not occurred as we had originally thought, based on the history.

This study was important for the public health, in that quasi-species differences in hepatitis C virus may be responsible for differences in outcome.  We don't know.

How else would this study have been done, if this laboratory had not done it?  No one else has serial serum samples from chronic hepatitis C virus over a 27-year period, or beginning with two quasi-species populations in two separate individuals.

The plans for this study?  This study has been completed, it has been published, and the project is now discontinued.

We also conducted another study of quasi-species evolution, this time in individuals followed over a 10-year period in a village in Japan.

In doing so, we also looked at what the minimum number of clones was that needed to be sequenced, in order to obtain the maximum information about hepatitis C virus quasi-species.

This village in Japan had a 27 percent prevalence of hepatitis C virus.  We had a really great collection of specimens.

We noticed that all the publications, or almost all the publications of hepatitis C virus quasi-species in the literature, reported the sequencing of only three to five clones per specimen.

Well, if you sequence three clones, and you detect a different quasi-species in each clone, you might draw the conclusion that this patient had three quasi-species circulating in his blood.  If you sequenced 10 or 20 clones, you might find that he really had 15.  So, we investigated that.

Again, quasi-species are important to the study of HCV, and sequencing too few clones may give you the wrong information about the number of quasi-species in a patient's blood.  How else would this study have been done if we had not done it?

No other lab had evaluated the best number to sequence, and almost all, if not all, publications of HCV quasi-species had reported sequencing only three clones per sample.  This study has been completed, it has been published, and the project is now discontinued.

We conducted a study to compare the relative sensitivity of HBV NAT as conducted by the two leading manufacturers, as well as a number of old and new, licensed HBsAg tests, and newer HBsAg tests that are in the pipeline and are more sensitive.

We compared the HBV NAT with the HBsAg test. We compared single donation NAT with mini pool NAT, and we compared the older HBsAg tests with the newer HBsAg tests.

This was a very important study to do for blood safety.  It allowed us, at least theoretically, to delay implementing or starting to work toward implementation of HBV NAT testing at the time a study was done, until a more sensitive mini pool method or single donor method format was feasible.

It indicated that perhaps the time was right to increase the sensitivity requirement on the HBsAg lot release panel, and a movement to do that is in progress and has been discussed previously as BPAC.

Would anyone else have done this study if we had not done it?  The resources to do this study -- that is, access to all the manufacturers and their tests, were really unique to CBER.

More important, the initiative was unique to CBER.  The fact is that no one else would want to do this study as much as we would in FDA, because we can see the whole problem with regard to the sensitivity of assays for screening blood.  This study is completed, it has been published, and the project has been discontinued.

We conducted a study to compare HBV DNA levels and infectivity levels in titered inocula derived from human chronic carriers of HBV.

In this study, we had three well defined inocula from human plasma that had been carefully titered by myself and others in chimpanzees many years ago.

Using Taqman methodology, we evaluated the actual viral load in these samples, and we wanted to correlate HPV viral load, infectivity titer, and HBsAg titer for each of the HBV subtypes.

This study had significant importance for the public health. It can provide data that would allow us to set an end point for the sensitivity that we would seek in the future of assay development for screening blood.

It might conceivably provide us information that would allow us to use NAT testing to reinstate anti-cor positive blood donors.

If you use NAT testing and the NAT testing is negative, can you reinstate the donor?  Well, the question is, has infectivity been ruled out.  This study will help.

How else would the study have been done?  These inocula and inocula like them are only available to NIH and FDA and no one else had done this study, and we did.  This study was completed, a manuscript has been drafted, and the project has been discontinued.

We did a study comparing HCV, RNA, NAT testing and hepatitis C core antigen.  This was completed, the study was published -- it came out a few weeks ago -- and the project was discontinued.

We conducted studies of HBV cases that cannot be detected using licensed screening tests, so-called silent hepatitis B virus infections.  These studies were completed, these studies were published, and the project has been discontinued.

Now, we also looked at emerging infectious diseases. This is the laboratory of hepatitis and emerging related diseases, and we are one of the main components looking at emerging infectious diseases.

One of those was a study of SV40 in blood donors. This was a good example of our response to a public health situation involving an emerging infectious disease of blood.

An article appeared in Cancer, reporting the finding of SV40 DNA in the buffy coats of 29 percent of normal blood donors in Italy.

We wanted to see if those findings could be replicated and, more important, could they be replicated in U.S. blood donors.

We also wanted to do something that the authors of that study in Italy did not do. We wanted to conduct this study in a way that would rule out the possibility that there had been cross reactivity with a very ubiquitous polyoma viruses, BK and JC viruses, which have about a 60 percent homology with SV40.

This is a potentially very important study for U.S. public health.  As you probably know, SV40 is a cancer virus.  It can cause cancer in animals.  It has also been isolated from several human tumors.

Between 1955 and 1963, 98 million U.S. citizens, mostly children, were exposed to SV40 virus when they took the polio vaccine.

If these individuals today still had SV40 in their blood, it could pose a potentially serious problem for blood recipients.

How else would this study have been done, if our lab had not done it?  As far as I know, no other lab has tried to replicate the published study, or to conduct it in a way that would rule out reactivity with PK and JC virus.

We expect to complete this study this spring. We plan to publish it later in 2004, and we are going to do subsequent studies, which I will discuss in a few moments.

In addition, we looked at another emerging infectious disease, SEN-V virus.  Just briefly, this is a candidate non-A-E post-transfusion hepatitis virus, but it is not known if it causes liver diseases.

If, in fact, it could be shown to cause liver disease, it might be possible to screen for this virus.  It was important for us to get involved in evaluating SEN-V infection.

There are only about two or three other laboratories in the world that are studying SEN-V.  Dr. Hollinger's is one of them, but I think probably in the United States the only laboratories are Dr. Hollinger's, ours, and Dr. Harvey Alter's.

We completed this study.  A manuscript has been submitted for publication, and we have discontinued the project.

We have discontinued the two quasi-species studies in HCV.  We have discontinued our studies of the sensitivity of HBV NAT and HBsAg.

We have discontinued our studies of HBV infectivity and copy number.  We have discontinued our studies of HCV RNA and HCV core antigen.  We have discontinued our studies of silent HBV, and we have discontinued our studies of SEN-V virus.

What are we going to be doing in the future?  We are going to continue working on SV40. When we finish the studies that are currently in progress on SV40, which we expect to finish in a couple of months, we are then going to look at antibody levels to SV40 in blood donors, and we are going to look at the infectivity of any positive samples that we find by inoculating cell cultures with these samples.

We have thought about possibly organizing a workshop to bring together other laboratories that are also interested in SV40 to decide what approach to take in the future.

Another study that I did not present to the site visit committee is a study of chlamydia and pneumonii in blood donors.

There have been reports that this can be found in between five and 25 percent of blood donors. It has also been reported in the mononuclear cells in patients with a number of different cardiac diseases, and we wanted to find out whether this could be found in U.S. blood donors.  That study is in progress and we are going to continue it, particularly if we get positive results.

In addition, we would like to continue other suitable projects related to the hepatitis viruses. Of course, this assumes that there is no further change in personnel levels and no further changes in other resources.

We also want to be ready for any emerging infectious disease that comes along that may threaten the blood supply.

Some of our planned projects are to evaluate the infectivity of anti-core positive blood.  That is blood that has anti-cor detectable, but no detectable hepatitis B surface antigen.

We would like to look at the interaction between hepatitis C virus and oxygen free radicals.  This may be a mechanism by which the hepatitis C virus causes damage to the liver.

We would like to study various aspects of the hepatitis C virus E2 antigen, its role in inducing protective antibodies, and its possible role in circumventing the antiviral effects of interferon.

Again, I would like to thank the site visitors for taking the time to hear our presentations last May, and I want to assure you that, whatever the outcome of the site visit report is, we will do what we can to implement it, and to continue to be a useful laboratory for CBER.  Thank you.

DR. NELSON:  Thank you, Dr. Tabor. I wonder, is Blaine still on the phone?

DR. HOLLINGER: I am still here.

DR. NELSON:  Do you have any comments?

DR. HOLLINGER:  No, not at this time.

DR. NELSON:  One interesting issues is, as you know, one population, namely drug users, injection drug users, commonly have cor antibody but don't have surface antibody.

Theoretically, their blood might be more likely to have infectious virus. So, it would be interesting to include that population in this evaluation, I think.

DR. TABOR:  Thanks for the suggestion.

DR. NELSON:  Okay, next is Dr. Kaplan, Gerardo Kaplan, laboratory of hepatitis and emerging related agents.

Agenda Item:  Summary Presentation.

DR. KAPLAN:  Good afternoon. I would also like to thank the committee for doing a good job.  What I would present today to you is a summary of the site visit review that we had in October of 2003, of my group, which is within the laboratory of hepatitis and related emerging agents.

I would like to give you a little overview of how this lab is organized.  Basically, there are two research groups.

You just heard the talk by Dr. Tabor, and his research group is more clinically oriented, and right now is composed of one staff fellow and one biologist.

My group is more basic research oriented, and my group is now formed by six people.  Erica Silberstein, Krishnamurthy Konduru, Dino Feigelstock are visiting associates.  Cecilia Tami and Roberto Arena are post-doctoral fellows, and Chu Chiah Hsia is a staff scientist.

In addition to the two research groups, we have the regulatory branch, which is coordinated by Dr. Robin Biswas, who also serves as the associate director of our division.

This regulatory branch has two individuals who are regulatory scientists, Susan Zullo and Abdur Tazzaque.  The three parts work pretty closely.  We have common seminars, rotary discussions.

Basically, most of the people do 50 percent or less of regulatory work, except for my post-doctoral fellows, that do not do regulatory work at all.

That is an introduction to my research program.  So, I would like to present you with this slide, basically, on viral hepatitis, which there are five agents, A, B, C, D and E.

A and E have common features.  For instance, the source of the virus is feces, the transmission is fecal-oral and you will note that there are chronic infections, B, C and D, the sources are blood are blood derived fluids.  The transmission is percutaneous and permucosal, and indeed, they induce chronic infection.

Prevention for these agents, hepatitis A, there is a vaccine and also Ig can be used for preventing it.  For E there is no vaccine or very good candidates. The only way of preventing this is by ensuring safe drinking water. It is not a problem in the United States at this point.

Hepatitis B, there is a vaccine and also prevention by Ig.  C, unfortunately, there are no vaccines and there are a lot of people working on them.  So, the only way of preventing it is through blood donor screening and risk behavior modification.  D, the infection is dependent on hepatitis B infection.  So, hepatitis B and Ig will also prevent it.

Basically B and C are well documented blood transmitted agents, and I don't have to emphasize that. However, in hepatitis A, there are a few levels of blood transmissions.

It is probably an issue at the level of blood products, and most blood products are tested for HAV now. There are other issues, for instance, the deferrals and testing.

For instance, we have just had a major outbreak in Beaver County, Pennsylvania.  Three people died from hepatitis A contracted at a Chi Chi's Restaurant, and there was a handful of people that had the disease, and the blood banks there had to implement safeguards and deferrals.

This virus, the biology in general is not very well understood, and some of the mechanisms are less understood.

From all the group, the only very well understood is the hepatitis A, and this is done through work in my lab, where we have identified several receptors that we call hepatitis A virus receptor 1, which was a known molecule that was identified.

It is a member of a family which the importance is becoming more obvious day by day now.  Hepatitis E, there are no receptors known, B no receptors known, it doesn't grow in culture. This is a very interesting area of research.

For hepatitis C there are two candidates, LDLR and CD81.  They are good candidates. There is a lot of work being done at this point.  For D, it is incidental and dependent on B.

So, the major focus of the research in my lab is in hepatitis A and, for historical reasons, I have worked at FDA for more than 10 years now and I started my work on the vaccine side.

So, I was involved in the regulation of hepatitis A vaccines, and I developed there the hepatitis A program and the replication of this novel gene at the receptor.

I moved to the OVRR a couple of years ago. I brought with me my program.  However, we are now developing a hepatitis C program and also responding to the need at the FDA, we are developing a counterterrorism program.

So, basically the department has three components, a minor component of replication of hepatitis C, and Dino Feigelstock is the only person in the lab working at this point.

We have another minor effort in counterterrorism research, and we have one FTE and one post-doctoral fellow working on this.  The majority of the program is in cell entry and pathogenesis of hepatitis A.

I will describe a little bit of what we are doing in hepatitis C.  I will not show you data, but I will show you what we are doing and what we are trying to accomplish.

So, basically, hepatitis A doesn't grow well in cell culture.  People have developed replicon stimulants, a mini-genome that can replicate itself.

These cells, the Huh-7, those are permissive for replication of these replicons. So, what we would like to do is understand why these cells are permissive and others are not permissive and using this to isolate factors that are important for hepatitis C replication.

So, the idea is to develop an expression cloning strategy to make a library of these cells, transfect through a number of permissive cells, and this is undergoing a little bit of change right now.

It seems that, under certain circumstances, for instance, HeLa cells can be permissive to hepatitis C infection, or they permit replication of the replicons.

So, after transfecting it to a non-permissive cell, then we will transfect the replicons calling for a neomycin resistance gene.

This was developed in Germany by the lab of Kurtis Slager(?) and Shelly Rice(?) here in the United States.  So, this cell can take and convert this replicon, will also express the neomycin resistant gene.

So, if we want to select for them, once they are selected, we will be able to isolate plasmids, because this is an episomal library, and then to prove that these plasmids allow replication of the genome, transfect the plasmid back to the non-permissive cell line.  So, using this strategy, we would like to isolate factors important for replication.

Let me tell you a little bit about the counterterrorism program  The main part of the program, as you heard, is developing a pathogen chip, and we are doing this in a shared effort with the laboratory of Dr. Nakhasi.

Basically, his lab is mainly working on bacterial, the transfection of bacterial pathogens.  My lab is working more on virus detection.

So, we have developed this chip based on oligonucleotides. In each spot there is a different probe.  For instance, we have the probe for ebola virus and the marburg virus, which could be important when transmitted to blood, also spots for pox viruses, including vaccinia, for west nile, for VE(?) virus as well.

The idea is to get a blood sample, amplify whatever positives are there with specific oligos and then, in a multiplexing scenario, just throw into the chip to see if we can detect it.

Let me show you some of the data we have now. So, as a model, we have been using hepatitis C and hepatitis B pathogens, the viruses.

Here is the chip, which was hybridized to a blood sample which had a very low copy of hepatitis C.  You can see that the three spots are corresponding for hepatitis C, like that.

The same scenario would use a receiver plan that would contain very low levels of hepatitis B. You can see that these spots light up as well.

Here is a specific spot, and this we have resolved the problem by changing the temperatures, the hybridization temperatures.

Now, we are multiplexing this, and with other pathogens.  So, the idea will be to have one reaction that will detect all possible blood patterns that are not BT related and then other reactions that are BT related.

We have pretty interesting data.  For example, at this point we don't need to see bands in a gel to be sure that they are hybridizing.  So, the level of detection is very good.

I will now describe to you the cell entry and pathogenesis for hepatitis A.  Hepatitis A is a picornavirus, and this is a model based on many other picornaviruses.

There is very little known specifically about hepatitis A, and it has been modeled like polio, or other picornaviruses are easy to work. Hepatitis A grows very slowly and to low levels and everything takes time and effort.

So, basically, after a virus binds to a cell membrane receptor, which I told you about for hepatitis A we discovered, something happens to the virus, and the information will change, frees the RNA genome, and then is translated into functional proteins that replicate the RNA and functional proteins that close the capsids.  The messages and capsids get together, similar to particles, and they are released.

For hepatitis A, most of these steps are black boxes, and it is the black boxes that we are concentrating mainly on today, and I will tell you some of our research in this area.

So, three years ago we identified a seroreceptor that we call hepatitis A virus receptor 1. I am sure it was a receptor for hepatitis A, and then identified its homolog, which we call human hepatitis A receptor 1.

This was based on an expression cloning strategy using a monoclonal antibody that built up cell entry to a specific cell line.

Here I show you a monofluorescence assay. If you treat the cells with this monoclonal antibody, so these are not infected, where if you mock treat them and then infect them, you see the classical fluorescence of hepatitis A.

So, using this monoclonal antibody, we pulled this gene, and I am showing you the sequences, because it has some very interesting characteristics.

It is a class I internal membrane glycoprotein, and it is probably chimeric of an Ig super family and a muscine family molecules, like other molecules of immunological importance.

It is a class I internal membrane glycoprotein. It has a signal to sequence. The first domain is the G domain, with a very distinctive characteristic of having six cysteines.

We don't exactly how this falls, but it is like a blueprint of a family of molecules, and this was the first one to be discovered.

Then it is followed by a mucin-like domain and you see it is highly repetitive here.  There are 27 repeats. This is the monkey one.  It has a PTTTTTL, with small variations, followed by a missing(?) area, transmembrane region, and a cytoplasmic tail.

We think it looks like basically that the immunolo(?) domain is very far from the membrane, which is exposed by the missing domain which is quite extended, transmembrane with a cytoplasmic tail, very different from many other receptors that the picornaviruses have.

We think that it works very differently. Basically, a poliovirus receptor, and it is collapsed very close to the membrane.

Another example would be ICAM that is a receptor for rhinovirus, has five monoclonal domains and is also collapsed.  So, the way this receptor and these receptors work, I think, is very different.

What is the natural function of hepatitis A receptor?  We don't know yet the exact function, but something very interesting has happened in the last few years.

Basically, the group of Delumitsu(?) at Stanford and Gordon Freeman and V.J. Gutra(?) at Harvard, they tried to identify asthma determinant genes.

They found that this gene, the hepatitis A virus receptor, is an asthma determinant gene. We don't know exactly how it works at this point.

They were able to conclude that the gene is important for T cell differentiation and basically the T cells and T helper 1 and T helper 2 are responsive, and T helper 2 is responsible for the asthmatic response.

Something very interesting on this pathway is that, for a long time, it has been known that there is an inverse association between hepatitis A infection and development of asthma, as well that the incidence of hepatitis A in the developed world is coming down, where the incidence of asthma is coming up.

Further work has been able to show that there is a link between the hepatitis A infection and development of asthma in humans.

So, the hypothesis here is that, for hepatitis A infection can induce the development of TH1 response, given a specific antigen, or that hepatitis A can block this transition from Naive T cells to Th2, or it can drive the Th2 cells already present into apoptosis, inactivating the allergic response.

We are showing this mechanism as a corollary to an effort with Delamitsu and Gordon Freeman, who wrote a grant, a PO1 grant to NIH, which is granted.

This grant, I am a collaborator on this grant, and two post-doctorals and funding for the research in this area, from background was awarded to my lab, and this grant will last for about four and a half years, and we hope to understand how this mechanism is working.

One of the big questions here is what are the natural ligands for a hepatitis A receptor.  It is not known.  The only known ligand is for the hepatitis A virus.  So, we want to identify this natural ligand, and we are using an expression cloning strategy.

Basically, transfection of human lymph node episomal cDNA library into dog cells, which are receptor negative.

Enrichment of cell transfectants that bind to soluble receptors for a panning procedures where the plates are coated with the soluble forms of the receptor, and then cells expressing the ligand, they bind so we can select.

This selection of these clones, we have developed a rosetting assay to identify them.  Then, rescue the episomal plasmids and pull out the genes, in a similar way that we did with the receptor a few years ago.

This is work done by Cecilia Tami in the lab.  She was able to identify a cell, a dog cell transfectant with the lymph node library, that is expressing the ligand.

This is the first plan.  Basically we have developed a rosetting assay using beads that are coupled with a solo form of the receptor conjugated to an Fc fragment.

Basically, this clone is combined, these beads are conjugated with the serial form, the serial receptor.  However, if you put a similar construct, but now calling for a poliovirus receptor, these beads don't bind.  This is pretty specifically inhibitive with solo receptors. It will not be inhibitive by the PVR.

Here is a dog cell that was transfected with an Fc receptor.  So, it is possibly controlling for the assay.  Both are binding, the beads with the Fc.

Here is a dog cell, like most of the population of dog cells that we were using, they don't bind the cell receptor.

We have not been able to pull it directly from here.  We are doing a secondary transfection and we are having binding finding positive. I am really optimistic that in the near future we will have a natural ligand.

We are virologists, every interested in how this receptor interacts with the virus itself.  The first question that we asked is, where does it bind.

So, we used dog cells that were transfected, for instance, with a vector, and here we see that, in a binding assay on 96 walls, dog cells do not bind to virus.

Detection of the viruses come with IUNA antibodies, hantivirus. If you put the whole receptor, now it binds.  I will talk only with this one, that if you delete the D1, it has deletion of the immunolo(?) domain.  If you delete it, there is no binding whatsoever.  So, from these experiments we knew that the virus binds to the first domain.  That is required for binding.

We follow up our studies doing these soluble receptors. This is work done by Erica Silberstein in the lab. Basically, these are three fusion proteins that we constructed.  Now we have constructed several others.

Basically, the D1-Fc has only the first domain, D1muc has the same immunolo domain, plus the muscine.  PVR-Fc has the poliovirus receptor instead of the hepatitis A cell receptors. So, these were basically our negative controls. They all share the same Fc.

What we learned here is that, if you treat the virus with PVR-Fc, there is no effect on the virus. If you treat it with D1-Fc, only the modeling domain.  However, if you treat it with the D1muc, both domains, there is a high level of neutralization, about two logs.

If we run these reactions on a sucrose gradient -- this is the bottom, this is the top -- particles treated with PVR, they migrated like this.  This is 16S, this is where the full particles migrate, at 80S, is where the empty particles migrate.

Here is a superimposition with particles treated with D1Fc, the domain alone, and you can see there is just a little shift.

Our interpretation is that these particles may have receptor bonds.  However, very striking were these results where particles were treated with D1-muc, containing the Ig and the muscine-like domain, and you can see that the particles are up here.

We think this is an encoding process.  We haven't been able to encode the particle. To further prove this, some EM analysis, and you can see that the particles treated with PVR have less of an effect.

We found virions that were highly refringent, empty capsids, which have the classical shape.  However, in particles that are treated with the solo receptor, we found these particles, that they internalize the strain. So, they are open, they are permissive to the internalization of stain, and the shapes are very diffuse.

The particles will not allow -- full particles will not allow the internal staining, which is what you see here.

So, we were going to model this cell, and we know that the receptors are mostly on the inside of the particle, it has internalized particles to this place where there is a lot of receptors.

Something happens to the particle, and that is what we are studying, the RNA is into the cytoplasm and the infection starts.

We are also developing a model for pathogenesis of HAV.  The main problem is that primates are the only current models of hepatitis A.

It is very difficult to work with.  So, we were interesting in adapting the virus to mouse cells.  So, Dino Feigelstock did this work in the lab, and basically if you infect mouse liver cells with the parental virus, basically the virus doesn't grow, doesn't enter.

However, we have been able to adapt -- this is a project that has about 10 years in the lab, so I am very keen about it.  Now we have this virus that grows in mouse cells, mouse liver cells.

I think we are closer to developing a mouse model at this point. I think it will be very important for vaccine development, for blood testing, for understanding asthma and learning how to prevent asthma.  So, it could have very important implications.

In summary, we are beginning to understand the mechanisms of cell entry of hepatitis A. We are just beginning.  There are many things that are not known.  I think it would be a great model for all picornas, because there is very little known about this mechanism.

We are in the process of identifying natural ligands of the human, which as you heard before, is very important.

We are studying this inverse association of hepatitis A infection and development of atopy, which is a big problem here in the United States and in all the developed countries.

Finally, we have adapted hepatitis A to grow in mouse liver cells.

I would like to recognize the work of Erica Silberstein, who did the cell entry for hepatitis A virus, Cecilia Tami, identification of natural ligands, Dino Feigelstock, the mouse-adapted HAV and HCV, and Roberto Arena for the development of the pathogen chip.  Thank you.

DR. NELSON:  Thank you, Dr. Kaplan.  Are there any comments or questions?  Okay, Dr. Asher?

Agenda Item:  Summary Presentation.

DR. ASHER:  Thank you, Dr. Nelson. I am going to present a summary of research programs in the laboratory of bacterial, parasitic, and unconventional agents, a laboratory that was established four years ago by a merging of four investigators who were interested in concentrating on spongiform encephalopathies with a small existing bacterial and parasitic diseases staff.

We were joined two years ago by Dr. Sanjai Kumar, who, as you have heard, is unable to be here today to present his own program, because of the sudden and tragic illness of his father.

We were joined one year ago by Pedro Picardo, who is an alumnus of the TSE advisory committee, to establish a new section.

he laboratory is comprised of three official sections, first, the malaria transfusion transmitted parasitic and bacterial infection section under Dr. Kumar, the TSE research program, which is comprised of three functional sections in my part of the laboratory, and then Dr. Nakhasi's section on leishmaniasis, chagas disease, a section which is completely separate, except for administrative purposes, and which was reviewed separately.

Dr. Kumar's section is comprised of two biologists and a post-doctoral fellow.  My section has one very experienced microbiologist, a laboratory technician, and a post-doctoral fellow.

Dr. Picardo and Dr. Taffs, at the moment, are working in splendid isolation. We expect Dr. Picardo to be joined very soon by a professional biologist, well, he is actually a physician, but he is being classified as a biologist.

You may wonder why I bother to list groups of people this small as comprising separate sections, and there are really two reasons for that.

First, each of the nominal section heads is absolutely independent.  That is, neither Dr. Taffs nor Dr. Picardo needs any supervision from me, and have a set of skills and experiences that I do not have.

The other reason, if I can be philosophical for just a second is, in an earlier age, when the agency was more generously supported, both of these sections would have a staff of some kind.

One of the things that is of greatest distress to me is that people like this, who are in early middle age and have a tremendous experience and expertise, have no one to whom to pass that along today, and it is no one's fault.  The agency simply can't support larger groups of people.

I do believe that, in this situation, we are eating the seed corn that would ordinarily have gone to future generations, if you will forgive me for that digression.

To put the goal of our laboratory into context, this laboratory is dedicated to the goal of the Food and Drug Administration, to protect the safety of regulated products, and that means materials involved in producing FDA products, including biologics, should be free of extraneous organisms.

There are several ways of doing that.  We conduct research in all four.  One, to assure a history of low risk.  An example of research in that would be risk assessments.  Second, in testing raw materials and sometimes finished products.

Third, by eliminating contaminating agents that might enter the manufacturing process during manufacturing, and to prevent cross contamination of equipment and establishments that prepare regulated products, meaning cleaning and effective disinfection.

In addition, we have other research that is mission related but not directly regulatory.  Both Dr. Kumar and Dr. Picardo conduct research on the pathogenesis and molecular biology, Dr. Kumar with malaria, Dr. Picardo with spongiform encephalopathies.  Dr. Kumar is also involved in development of novel malarial vaccines.

We believe that such non-directly regulatory work, as Dr. Nakhasi explained, is very important.  First, it provides materials used for regulatory research, it increases the insight of the investigators into issues of regulatory importance. It helps to maintain their expertise.  It certainly helps to maintain their morale, and most of the projects are self supporting.

Perhaps now would be a good time to respond to Dr. Klein's question about how a new project would be supported.

We are fortunate, in the Food and Drug Administration, that at least for most of us, we don't have to worry about our salaries each year.

There is a basic operating budget for maintaining the usual laboratories, offices and equipment.  Everything above that, we have to support with some sort of outside funding.

For example, for spongiform encephalopathies, the research began with a small special grant from the office of the CBER director.

We got one slot. That is how we were able to recruit Dr. Picardo, from the office of the director, as part of the bovine spongiform encephalopathy response plan.

We competed for funding through a collaborative research program through the office of the commissioner.  We got funding from the NIAID as part of the national vaccine program, and got some money, the details of which are perhaps better discussed out in the hallway, providing administrative services for the biotechnology engagement program, and we have recently acquired a cooperative research and development agreement with the American Red Cross on a subtopic of mutual interest.

In addition, we support a lot of the research through the collaborations.  For example, Dr. Picardo is now at the Creutzfeldt-Jakob Disease pathogenesis unit in Edinborough.

They have infected transgenic mice.  He is nominal principal investigator on this international project. They have inoculated transgenic mice with a BSE agent.  They are holding the mice. They observed them. They processed the tissues.  They mount the slides, they stain the slides, all of which they pay for, and then Dr. Picardo reads them, does the summaries, and plans the next part of the experiment.

So, we cobble these experiments together as best we can from outside sources of support.  I don't want to short change Dr. Kumar.  So, let me begin with the projects he has been most involved with.

Everybody is expected to pitch in on the subjects of greatest interest to the division and, of course, bacterial infections of the blood, and particularly of platelets, are very important pathogens, very important transfusion transmitted pathogens.  Somewhat less important in this country, but more important worldwide are transfusion transmitted parasites.

Dr. Kumar has been working on nucleic acid testing for rapid recognition of blood borne bacterial and parasitic infections.  He has developed a simple, PCR-based method for detecting, I think it is more than 12 different bacteria, based on consensus sequences in the 16 S-ribosomal RNA, and a similar study for plasmodium, in this case, falciparum.

Unfortunately, his slides didn't come over into my power point very well. He has also developed a rapid chip speciation system, so that when a nucleic acid has been amplified, it can be rapidly identified for which kind of plasmodium is involved in an infection in a chip format.

Of course, for transfusion safety, you don't really care what the species of bacterium or plasmodium is, because they are not supposed to be there at all.

He has also developed a Taqman assay that has a sensitivity at the moment of about one organism per microliter.  I would think the next step would be figuring out how to concentrate the bacteria or parasites to increase the sensitivity of the assay, although it already compares very well with culture or with fixed smear.

Dr. Kumar's main interest is in malaria, and I wouldn't presume to try and explain the details of the projects that he is involved with.

The main thrust of the projects is this.  It has always been a paradox that people who get malaria get at least transient immunity to reinfection, and that can be mimicked with vaccines that are made with whole, crushed, mosquito-borne parasites, which, of course, is not practical for mass use.

If you try to break the antigens down into which ones are responsible, none of the sub-unit vaccines have ever been successful in mimicking that immunity.

Dr. Kumar's work is involved in, a, trying to find combinations of those antigens that would produce immunity, using genomic approaches for looking at new antigens that haven't been tested that might be protective, and in looking at novel, effective and non-toxic adjuvants that would increase the immune response while still being acceptable.

All of his work involves some collaborations with investigators at the National Institutes of Health, with Walter Reed, and in other places.

At that point, if there are no questions, I couldn't do Dr. Kumar's extensive research work justice in this little five-minute summary.

At this point, I will try quickly to review the activities of the TSE research program which is, of course, my own major interest.

First, the regulatory and measurement sciences section, comprised of Rolf Taffs, who is perhaps CBER's premier expert in statistical aspects of regulatory test design.

He also has acquired expertise in probabilistic risk assessments, working with Steve Anderson of the office of biostatistics and epidemiology.

He also works in product control and release tests.  Kathy Carbone, I know, has collaborated with Rolf.  He works actively outside the office of blood with other offices, and has worked with the center for devices.

His main interest is in the pathology and pathogenesis of spongiform encephalopathies, particularly in variations in the abnormal prion proteins that are associated with various familial spongiform encephalopathies.

I think this is very important, because there has been a tendency lately to over-simplify what appears to be a very complex interaction between the genetics of the host, the genetics or genetic equivalent of the agent and other aspects of the disease.

For instance, there is some evidence that the prion protein changes in disease of long duration, and all of this has to be clarified if one is going to rely on the prion protein for rapid diagnosis, particularly if it is relied on, in the future, for blood tests and, of course, there has been no proof of principle that that will be possible.

He is also interested in transgenic mouse models for familial spongiform encephalopathies, very important because of the long generation times in humans that make it very difficult, and the social sensitivities of investigating familial diseases that are autosomal dominant, and not wanting to disturb the carriers. In many of the kindreds, there is a very high penetrance.

He has joined with me in looking recently at a general approach to testing, to improve the diagnosis of spongiform encephalopathies.

My own part of the program, my own research, main research interests are as I have just mentioned, early diagnosis of TSEs and improved detection of the agent, and validation of spongiform encephalopathy agents decontamination methods.

We also have a program looking at the susceptibility of novel cell substrates to infection with spongiform encephalopathy agents.

This has been supported in the past by NIAID through the office of vaccines, and I have been approached recently to ask if we were interested -- by NIAID directly -- interested in more support.  So, I have great hope that that will develop.

Then, of course, as I mentioned, for the regulatory science section, we have improved risk assessments.

Just one brief reference to improved diagnostic techniques.  Our one and only research fellow, Olga Maximova, is very interested in trying to set up an object basis for histological and immunohistochemical diagnosis, of the spongiform encephalopathies.

There are several properties of the pathology of spongiform encephalopathy that might be amenable to setting up quantifiable morphometric decision criteria.

I won't go into the reasons for that, except to say that it can be very distressing for a regulator to ask whether a given donor has spongiform encephalopathy, and to be told by the same pathologist on a Friday that, yes, I think the donor makes those criteria and then one prepares to drop the axe on the tissue program, only to be told on a Tuesday that, well, probably not, it is not going to be signed out as a spongiform encephalopathy.

I realize that that is life, but it would be better to have quantifiable specific criteria.  We might not always be right, but at least we could attempt to be consistent in a way that could be defended if we were challenged.

The three criteria that are amenable to quantitation are spongiform change, astrocytic proliferation and the accumulation, in cells and parenchyma of the abnormal form of the prion protein.

Of these, the last seems to be the most amenable to quantitation. Dr. Maximova has, with great perseverance, finally adapted a quantitative microscopic morphometric system to do this.

There are two very promising methods, one to quantify the total stained area of a given second and the second, the integrated optical density.

The problem is that this is the area in which the microscopist, the pathologist, has trouble reading it.  Of the two methods, it looks like this one will be most likely to be developed into something statistically significant.

Let me close by reviewing our work with decontamination. As most of you know, FDA regulated products have been contaminated with TSE agents.  They are very difficult to inactivate.  I will show you an example of that.

They are substantially inactivated in the presence of several chemicals. World Health Consultants, in 1999, recommended decontamination procedures for health care environments, using a combination of sodium hydroxide or sodium hypochlorite solutions and moist heat.

Other authorities have doubted the need for that, and both the CDC and the FDA have had to respond to challenges when we asked that the WHO recommendations be followed for decontaminating potentially contaminated facilities.

Just to review, cornea dura mater pituitary growth hormones have infected a substantial number of recipients, and I think to that we may now have to add packed red blood cells.

Fortunately, most of these accidental transmissions have occurred outside the United States, although we have had, at least count, 26 recipients of pituitary growth hormone who have already become ill with Creutzfeldt -- out of 8,000 total recipients, who have already become ill with Creutzfeldt-Jakob's disease. The demonstrated incubation period has exceeded 38 years.

Neurosurgical instruments have also been demonstrated to transmit infection. Those are classes of product regulated by the Food and Drug Administration. There have been five and possibly six cases over the past 40 years that can be attributed to neurosurgical instruments.

The problem with decontamination is the context dependency of the inactivation of the agent.  For example, when scrapie agent is suspended in an aqueous solution, and then heated very carefully with stirring to 121 Celsius, the infectivity falls to undetectable levels in only five minutes.  Unfortunately, at boiling temperatures, it plateaus at 10 minutes, and then doesn't drop further.

At the same temperatures, when agent has been dried onto surfaces, you can heat them at 121 Celsius, 132 Celsius, even 144 Celsius for 60 minutes or even 90 minutes, and some of the infectivity remains.

So, the inactivation properties of the agent are different when suspended in aqueous solution and on dried surface.

For that reason, in 1999, a consultation of the World Health Organization recommended that contaminated surgical instruments be incinerated and discarded when that was possible.

When it wasn't possible, they recommended, in order of more to less effectiveness, six methods that they felt were acceptable.

The first five all involve exposure either to sodium hydroxide or to sodium hypochlorite, and the first four of them, exposure to heat.

The last method they recommended was autoclaving at 134 Celsius for 18 minutes, and it is that method that those authorities, who have challenged the CDC and the FDA about decontamination, have suggested would be acceptable, although we know from animal experiments that treating materials under those conditions certainly don't save hamsters from scrapie.

Concerned about that, the center for devices and our center proposed to the office of science, and were successfully supported, a validation study seeing whether two variations of the WHO recommended procedures would, in fact, successfully decontaminate surfaces that we have intentionally contaminated with scrapie agent.

We developed two basic methods. One of them I had developed a number of years ago, before this issue came up. Both of them used a well known hamster adapted strain of scrapie agent because it propagates in hamsters to very high levels, and produces disease that is recognizable, sometimes in as few as 60 days.

In one method, the infectivity was dried onto glass slips.  In the second method, dried into steel needles, and both of these methods are similar methods that had been described in the literature.

For the glass slip method, a suspension of infected brain tissue is dropped -- we don't really prepare it in this way, but I like the pictures -- are dropped onto glass slips.  The slips are dried.

After they are dried, the slips with the varying infectivity can be exposed to any number of decontamination regimens -- in this case it was potassium permaganate, which does not disinfect the surfaces.

The slips can then be rinsed, because we have demonstrated independently that the infectivity does not come off on exposure to plain, distilled water.  They are then pulverized in diluent, and aliquots of the diluent are inoculated into an assay animal.

The second procedure, steel needles prepared by CDRH, can be exposed to a paste of contaminated brain, and then dried, and the paste can be done in titrations, so you can estimate how much infectivity is on a surface.

Then, either the suspended glass or the needles are then assayed in hamsters and they are observed for -- we observed them for 450 or 500 days.  If they get sick, their brains are examined for evidence of the presence of protease resistant prion protein, which is a sign that they have come down with scrapie.

The basic procedure is to dry the brain tissue on the objects and then to decontaminate with chemicals, either sodium hydroxide or sodium hypochlorite, followed by autoclaving in a gravity autoclave.

Following that, because we were attempting to replicate hospital conditions -- and we had a limited amount of resources, we couldn't do every possible variation -- each object was then cleaned by ultrasound in hot detergent, rinsed in distilled water, and then put through a 20-minute hospital type sterilization, secondary sterilization in the autoclave.

Every experiment had a positive control that is similarly untreated scrapie infected material, a test of 40 replicates, and I didn't put it on the slide, but a negative control with sham normal brain and a similar set up for steel needles.

We have very nice titrations.  This shows the titration on glass that has a 50 percent end point at a dilution of 10-8 and on steel needles, it has a 50 percent end point at 10-7.  So, we have got a lot of infectivity dried on.

These are titrations that are done after the material has been dried on.  This is the infectivity that survives drying, and then the assay procedure.

Because there was a wash in hot detergent with sonication, we were interested in how much the infectivity would drop under those conditions alone. So, we put them in the sonicator and titrated before and after.

We found that there was a substantial reduction in infectivity on both the glass slip procedure and the steel needle of over five logs, but all infectivity was not removed. There was always residual infectivity after the hot wash and sonication.

Here are two of the procedures from the WHO recommended series of procedures, sodium hydroxide with the autoclave, sodium hypochlorite autoclaving, sodium hydroxide at an elevated temperature, and sodium hypochlorite at elevated temperature.

You notice that almost all the infectivity is not detectable, but one animal came down with scrapie and, in the last couple of slides, you will see this pattern again.

Here is a similar experiment with the steel needles.  This was one of the first experiments that we did.  That is why, instead of 40, you see these lower numbers, because we had a higher mortality.  Again, a small number, three animals came down with scrapie.

This summarizes a number of experiments done using either sodium hydroxide alone or sodium hydroxide with detergent, sodium hypochlorite alone, or formic acid.

You will notice that they are very effective at protecting all of the systems for protecting hamsters, but a few hamsters did come down with scrapie.

The explanation for this, and we hope to investigate the reason for the false -- it is an important distinction whether we are getting real false positive, or whether there is survival of scrapie infectivity in a small number of cases.

If it is a false positive due to western blot area, that should show up simply by repeating the western blot and doing infectivity assays of the positives, and then, of course, increasing the numbers.

These are not heavily controlled, because most of them had only four or eight hamsters, sham inoculated hamster brains, tested for controls.

If it is a true positive due to cross contamination in the laboratory or the animal colony, that should show up.

If we increase the number of normal materials that are tested -- that is, objects that don't have scrapie on them -- and if it is a true positivity from resistant infectivity, the sham inoculated control should show that as well.

So, to conclude, the measures that were recommended by the WHO to evaluate, methods have been devised that evaluate the effects of decontamination agents.

Two models, both suggested that the methods recommended by WHO are effective in removing huge amounts of infectivity.  Other chemical treatments may also be effective. However, uncertainty remains.

It is still not clear whether the decontamination procedures are absolutely reliable, and the predictive value of the results from the model for health care and manufacturing situations are also not clear.

Let me just close by reviewing those collaborative research projects that I think we have enough support to continue during the coming year.

We have been awarded, by the office of science, another grant for completing the validation of the decontamination procedures.

In addition to the WHO procedure, there have been very promising results announced in the past couple of years for a phenolate disinfectant and for proteolytic enzymes.  We hope that, by combining these, maybe those last few positives can be eliminated.

We have some funding for testing the infectivity of neuronal cells over-expressing mutations predisposing to familiar Creutzfeldt-Jakob Disease, and the promise of additional money for that.

The studies of the pathogenesis of TSEs are supported both by CRADA and by a very generous collaborative study with the CJD pathogenesis unit.

Probabilistic risk assessment models, of course, are an entirely internal project, no problem with funding there.

We are hoping, with support, to conduct a study developing rapid genotyping of donors who have been deferred because of a familial history of Creutzfeldt-Jakob disease. Of course, these specimens won't be from actual donors, but they will be from families with the mutations that result in deferral.

We have had, in the past, a study supporting Yaroff Vostel(?), who is in the division of hematology, and his very important study on rodent blood components.  We would like to continue supporting him.

With the American Red Cross, who has a BL3 containment facility, we would like to investigate the susceptibility of various transgenic mouse lines to spongiform encephalopathy agents, including the BSE agent, and we would very much like to establish a United States TSE biological reference material and proficiency panel, to use to evaluate candidate tests for diagnosis of the spongiform encephalopathies.  With that, I will close and answer questions.

DR. NELSON:  Thank you.  Any questions?  Thanks, Dr. Asher. Now, Dr. Smallwood?

DR. SMALLWOOD:  At this time, we will be moving into the closed session. I would like to invite everyone who is not a member of the FDA to please leave as quickly and as quietly as you can.  That also includes the audiovisual staff, please, and may I ask that you turn off your cameras and also disconnect any audio device that you may have.

DR. NELSON:  I also would like to thank Dr. Tabor, Dr. Kaplan and Dr. Asher for some very good, informative helpful presentations.

[Whereupon, at 3:05 p.m., the meeting was adjourned.]