1

DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR BIOLOGICS EVALUATION AND RESEARCH

BLOOD PRODUCTS ADVISORY COMMITTEE

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.

Friday, July 23, 2004

8:00 a.m.

Gaithersburg Holiday Inn

2 Montgomery Village Avenue

Gaithersburg, Maryland 20877

PARTICIPANTS

Kenrad E. Nelson, M.D., Chair

Linda A. Smallwood, Ph.D., Executive Secretary

Pearline K. Muckelvene, Scientific Advisors

& Consultants Staff

MEMBERS:

James R. Allen, M.D., M.P.H.

Kenneth Davis, Jr., M.D.

Donna M. DiMichele, M.D.

Samuel H. Doppelt, M.D.

Jonathan C. Goldsmith, M.D.

Harvey G. Klein, M.D.

Suman Laal, Ph.D.

Katherine E. Knowles,

Acting Consumer Representative

D. Michael Strong,

Non-Voting Industry Representative

TEMPORARY VOTING MEMBERS:

Liana Harvath, Ph.D.

F. Blaine Hollinger, M.D.

Katharine E. Knowles

Matthew J. Kuehnert, M.D.

Susan F. Leitman, M.D.

Keith C. Quirolo, M.D.

George B. Schreiber, Sc.D.

Donna S. Whittaker, Ph.D.

C O N T E N T S

PAGE

Update on West Nile Virus, Hira Nakhasi, Ph.D. 6

IV. Hepatitis B Virus Nucleic Acid Testing (NAT)

for Donors of Whole Blood:

A. Introduction and Background,

Gerardo Kaplan, Ph.D., Laboratory

of Hepatitis and Related Emerging

Viruses, DETTD, OBRR, FDA 28

B. Serological Course of Hepatitis B,

F. Blaine Hollinger, M.D.,

Baylor College of Medicine 32

C. Preclinical and Clinical Data for

HBV MP NAT, Steven Herman, Ph.D.,

Roche Molecular Systems 51

Allan Frank M.D., M.S.,

Roche Molecular Systems 65

Open Public Hearing:

Michael Busch, Blood Centers

of the Pacific 103

William Andrew Heaton, Chiron 121

Sherrol McDonough, Gen-Probe 129

Richard Smith, NGI 136

Harvey Alter, AABB 144

IV. Hepatitis B Virus Nucleic Acid Testing (NAT)

for Donors of Whole Blood:

E. Committee Discussion and

Recommendations 154

V. Current Trends in Plasma Product Manufacturing

A. Introduction and Background,

Mark Weinstein, Ph.D., Associate

Deputy Director, OBRR, FDA 223

B. Presentation, Jan M. Bult, CEO,

Plasma Protein Therapeutics

Association 225

Open Public Hearing:

Patrick Schmidt, CEO, FFF Enterprises 258

1 P R O C E E D I N G S

2 DR. SMALLWOOD: May I ask all advisory

3 committee members to, please, take your seats?

4 Welcome to the second day of the Blood Products

5 Advisory Committee meeting. Yesterday I read the

6 conflict of interest statement that applies to this

7 meeting, however, we have a new process now and we

8 will read a conflict of interest statement for each

9 day.

10 So, if you will indulge me, I will read

11 that at this point. This brief announcement is in

12 addition to the conflict of interest statement read

13 at the beginning of the meeting yesterday, and is

14 part of the public record for the Blood Products

15 Advisory Committee meeting on July 23, 2004. This

16 announcement addresses conflicts of interest for

17 topic V.

18 Drs. Liana Harvath, Blaine Hollinger,

19 Matthew Kuehnert, Susan Leitman, Keith Quirolo,

20 George Schreiber, Donna Whittaker and Ms. Katherine

21 Knowles have been appointed as temporary voting

22 members for this meeting

1 Dr. Michael Strong is participating in this meeting

2 as the non-voting industry representative, acting

3 on behalf of regulated industry. The Food and Drug

4 Administration has prepared general matters waivers

5 for the special government employees participating

6 in this meeting who required a waiver under Title

7 XVIII, United States Code 208.

8 In addition, there are regulated industry

9 and other outside organization speakers making

10 presentations. These speakers have financial

11 interests associated with their employers and with

12 other regulated firms. They were not screened for

13 these conflicts of interest. I would just like to

14 remind everyone participating to, please, make

15 known, if you have not already done so, any

16 affiliation you may have and your status with that

17 affiliation prior to speaking.

18 Our committee chairman, Dr. Kenrad Nelson

19 has joined us this morning, and we also have Dr.

20 Blaine Hollinger who will also be part of the

21 committee this morning.

22 I just wanted to announce to those who

1 were not here yesterday that the next date, which

2 is tentative however pretty much firm, for the next

3 Blood Products Advisory Committee meeting will be

4 October 21st and 22nd, 2004.

5 At this time I will turn over the

6 proceedings of the meeting to the chairman, Dr.

7 Kenrad Nelson.

8 Update on West Nile Virus

9 DR. NELSON: Thank you, Dr. Smallwood. I

10 will try to keep awake after the 24-hour airplane

11 ride. I came in last night but I feel really

12 pretty good and I am very interested in the topic

13 today so I think that will help.

14 The first topic is an update on West Nile

15 virus by Hira Nakhasi.

16 DR. NAKHASI: Good morning. I just want

17 to give you an update, as Dr. Kenrad Nelson

18 mentioned, on the West Nile epidemic and donor

19 testing which is happening now, in 2004. First I

20 will try to wrap up last year's things and then

21 come up to 2004.

22 Next slide, please. The topics which I

1 will update you on are, as I said, last year's

2 epidemiology and the investigational West Nile

3 testing outcome of that, and some of the

4 transfusion-transmitted cases, and then the trigger

5 for the ID-NAT testing. Then I will update you on

6 the West Nile donor and product management

7 recommendations with the recent revelations we have

8 got. Then I will update you on the 2004 epidemic

9 and investigational West Nile testing, and also our

10 efforts in-house on the panel development and other

11 scientific issues--you know, the variation among

12 the strains of viruses infectivity of these

13 studies.

14 Next slide, please. If you summarize in

15 one slide the last year's epidemic, it really

16 basically sums up that we had approximately 1000

17 [sic] cases or, to be precise, 9862 cases, human

18 cases, and 264 deaths. And, the proportion of the

19 West Nile meningitis/encephalitis was 29 percent,

20 whereas, the fever was 69 percent in the human

21 cases.

22 Forty-six states, including Washington,

1 D.C., were endemic, and donor testing started, as

2 all of you know, in July of 2003, using two

3 investigational NAT testing. In some cases, a

4 small proportion started in the middle of June.

5 Despite this testing, I think these two

6 investigational NAT testing--these are minipool and

7 the two tests were the Gen-Probe test and the Roche

8 test, and Roche tested, as you know, in pools of 6

9 and the Gen-Probe test involves a pool of 16.

10 Despite testing, there were some

11 transfusion-transmitted cases and CDC had

12 investigated a total of 23 cases. They were

13 confirmed by NAT and IgM reactivity and also by

14 follow-up of both the donor and the recipient. Out

15 of the 23, 6 were confirmed cases. Only 4/6, you

16 may recall, had very low viremia, around 0.1

17 pfu/ml. Eleven cases did not confirm; 3 were

18 inconclusive because of the follow-up situation;

19 and 3 were open investigations.

20 Next slide, please. As I said, since it

21 started on July 1 of last year, screening using

22 minipool NAT and IND, all geographic regions of the

1 U.S. were screening at that time. With that, what

2 happened 1000 units of West Nile infected blood

3 donors were interdicted after screening

4 approximately 8 million donations. So, I think it

5 was a very, very vast improvement over the year

6 before when there was no testing. The last

7 positive donation was reported in the middle of

8 December in 2003.

9 Despite this testing, as you see, the

10 majority of cases were interdicted, more than 75

11 percent, but there was a small percentage which

12 went through because, as you know, this was done in

13 minipool NAT.

14 Next slide, please. This slide is Mike

15 Busch's slide where he showed why we were missing

16 some of these cases, and we knew that minipool NAT

17 sensitivity was such. The areas, you know, where

18 the wrap-up takes place when--you know, he calls it

19 stage I, II, III, IV and V, and in stage I and II

20 they are ID-NAT positive but minipool NAT negative,

21 IgM negative. So, it could be plus/minus. So,

22 during that stage they become IgM positive but they

1 become minipool negative and they are still ID-NAT

2 positive. So, this region and this region were the

3 ones where they went through. But, you know, these

4 were IgM negative and these were IgM positive so

5 the question is what is the infection of these

6 types of samples.

7 Next slide, please. So there was a

8 potential for transmission of West Nile through

9 minipool NAT negative blood of low viremia in some

10 patients. Therefore, what happened at that time is

11 that limited prospective ID-NAT testing started in

12 high incidence areas. If you remember last year,

13 Colorado, Kansas and certain other areas, and

14 Nebraska were hot spots and ID-NAT was triggered at

15 that time, and the trigger was based on if the

16 preceding the rate of 1/200 minipool NAT positive

17 rate of 1/250, then they would start testing with

18 ID-NAT testing. Also, what happened at that time

19 is that there was voluntary withdrawal of the

20 frozen transfusables in the high incidence areas

21 before the ID-NAT was initiated by some blood

22 establishments.

1 Next slide, please. There was also

2 another initiative started at that time. The

3 initiative was to go back to do the retrospective

4 study on the minipool NAT negative samples and test

5 them by ID-NAT to find out how many we missed. It

6 would also let us know what was the low level of

7 viremic high incidence samples in high incidence

8 areas where minipool NAT did not pick them up.

9 The other purpose of the study was also to

10 identify samples which are like minipool NAT low

11 titer, minipool NAT negative but ID-NAT positive

12 for infectivity studies. I told you that we do not

13 know whether those samples are still infectious at

14 low levels, and what is the level of infectivity.

15 So, these samples would be tested in various animal

16 models including non-human primates. Also, the

17 purpose of these samples is to really find out the

18 relative clinical sensitivity of various West Nile

19 investigational testing. I will report in a minute

20 what is happening with the infectivity state.

21 Next slide, please. Based on the

22 observation that we had minipool testing and we

1 missed some of the samples because the viremia was

2 low, and also in the ID-NAT testing in the high

3 incidence areas--based on those studies and based

4 on the logistics issues, the question was what

5 should be the trigger for ID-NAT, and also logistic

6 issues such the availability of adequate resources,

7 recruitment, reagents and trained technologists.

8 So, the discussion about the trigger for

9 ID-NAT was held in collaboration with the AABB task

10 force. By the way, we are very indebted to the

11 AABB task force for the biweekly meetings almost

12 throughout the year, and weekly meetings with the

13 task force during the epidemic to update us and

14 jointly discuss the strategies for how to go

15 forward with the testing performance, as well as

16 the epidemic.

17 So, based on that discussion, which was

18 held in February, the recommendations were the

19 following for the ID-NAT trigger: It was discussed

20 that we should monitor reactive rates by zones

21 daily, enrolled 7 days when the epidemic was

22 starting, which was usually, you know, around the

1 beginning of July and early June even and this year

2 even May some cases were found. The trigger was

3 that if you have 2-4 cases in any geographic

4 area--that is the blood collection, and the

5 frequency of 1/1000. This was based on the fact

6 that every 1/4 would be missed by minipool NAT and

7 require ID-NAT. This was the study done by ARC and

8 BSL and they found out that that would be the

9 trigger. And, you go back to minipool NAT only

10 when you see ID-NAT reactivity and you don't find

11 zero cases in a consecutive 3-4 day period or the

12 rate is less than 1/1000. So, that was the trigger

13 because, you know, we wanted to be prepared this

14 year because last year it was on an ad hoc basis to

15 start ID-NAT testing in those hot areas. So, we

16 wanted to be prepared this year if these areas

17 become hot so that we get the logistics present

18 there so we can start without interruption of the

19 ID-NAT testing.

20 Next slide, please. Now we come to 2004,

21 where are we now? As of July 20, which is a couple

22 of days back--as you see, every week the numbers

1 keep changing. Last week there were 108. This

2 week it is 182 human cases out of which there were

3 4 deaths. There are 2 from Arizona, 1 from Texas

4 and 1 from Iowa. Out of total infections, 74

5 percent of cases are neuroinvasive West Nile

6 illness and 26 percent cases are West Nile fever.

7 At the moment there are 35 states endemic for West

8 Nile. This slide has been kindly provided by Jen

9 Brown, from CDC, and other slides which I will

10 mention later.

11 The total number of presumptive West Nile

12 viremic donors reported to the CDC ArboNet--that is

13 why I highlighted this, is 23. There are more

14 cases than that but, as you know, there is a delay

15 in reporting to the ArboNet from the health

16 departments. So, using minipool NAT as well as

17 ID-NAT in select areas, starting on May 4. Out of

18 these 23 presumptive West Nile viremic donors, 21

19 are from Arizona. The majority are from the

20 Maricopa county near Phoenix, in Arizona; 1 from

21 New Mexico and 1 from Iowa. But this is the tip of

22 the iceberg.

1 Next slide, please. This slide, again, is

2 provided by Jen. You can see the distribution of

3 the West Nile, both the animal, avian and mosquito

4 infection, which is in this color, and the blue

5 color shows you the human cases. You can see it is

6 very high in Arizona and California. I am telling

7 Mike Strong that it is creeping up in Washington

8 soon. So, he has been telling me we don't see

9 anything and I said, well, wait and watch! As you

10 remember, in 1999, how this started and how it is

11 spreading and, you know, it just keeps on going. I

12 hope it will end up in the ocean sometime.

13 Next slide, please. This is just to give

14 you how early the human cases can be detected. As

15 you see from the slide, the earliest one was in

16 April. So, you know, there is an expansion of this

17 epidemic, it looks like. We were told in the

18 textbooks it is mostly in August and September or

19 late July but you can see it as early as April now,

20 and last year we saw it as late as December, in the

21 middle of December. So, you know, it is almost a

22 year-round activity now.

1 Next slide, please. Thanks to all the

2 blood establishments and testing establishments, I

3 got these data from several folks and I will

4 acknowledge them as I speak. The total number,

5 according to my calculations but this may not be

6 right, is 61 presumptive viremic donors reported,

7 starting in May, 2004. As I said, some of them are

8 reported to ArboNet and some of them are not. So,

9 it is not in addition to that; it is inclusive of

10 the ArboNet reports. ARC has told me--Sue Stramer

11 gave the data from June 16 to July 20, 7 hard

12 cases. Again, this is also in the Arizona area.

13 But she says no region has their ID-NAT trigger.

14 Mike Strong gave me this data from Roche.

15 There are 2 positive confirmed by ID-NAT--around

16 300,000 donations screened.

17 BSL, Sally Caglioti and Mike Busch told me

18 that there are 23 confirmed, out of which 16 came

19 from minipool NAT and 7 came from ID-NAT, confirmed

20 positives. There are 14 pending and he was saying

21 that some of them are ID-NAT and would have been

22 missed by minipool NAT. Also, some of them are low

1 viremic and also there are some which are IgM

2 positive. The denominator is around 400,000.

3 Gen-Probe, Leanne Kiviharju, gave the

4 data. These are non-ARC data but I am not sure--I

5 sent an email to Leanne--whether this is also

6 non-BSL but I am not sure; maybe we can find out

7 from here, but 21 confirmed positive and 7 are

8 pending. I am glad that you guys sent me several

9 slides. I was basically trying to summarize what

10 the presumptive donors are and, you know, I really

11 appreciate your sending extra slides.

12 The Department of Defense, Ron Hagey sent

13 me the data which has 8 confirmed out of 62,774

14 since January of 2004.

15 So, you know, this is the majority of the

16 screening going on at this time and there may be a

17 few cases which have not been reported yet, but

18 this is where we stand as of today.

19 Next slide, please. I just wanted to sort

20 of briefly remind you that FDA is still continuing

21 to work closely with the test kit manufacturers and

22 we would like to facilitate implementation of these

1 tests and expediter test licensure. I just want to

2 remind you that we issued two guidances in October,

3 2002 and May, 2003. There are 3 INDs for West Nile

4 minipool-NAT. One is from Roche, one from

5 Gen-Probe and one from ARC. This is public

6 information. FDA is continuing to work with the

7 AABB task force. I think that has been a

8 wonderful, wonderful collaboration with the AABB

9 task force and the people on the task force are

10 really helpful in doing this project together, and

11 with the CDC, NIH help, and to monitor the epidemic

12 and monitor the testing.

13 Next slide, please. Both ARC and BSL did

14 a study, which is unpublished observation. We had

15 a small discussion at the task force on what they

16 found out in some of the viremic donors when they

17 followed up. They wanted to find out what is the

18 rate of the disappearance of RNA when they convert

19 IgM and IgG. As you remember, in the last years

20 before the testing started the literature was that

21 it can go as long as 28 days of viremia. But from

22 their studies, and I don't want to go into detail

1 here because these are unpublished and, you know, I

2 don't want to divulge information--the gist of that

3 was that what they found out in both cases is that

4 the viremia may last up to 49 days in one case and

5 39 days in the ARC study, and in the BSL 49 days,

6 and West Nile RNA may go coexist with IgM.

7 Therefore, this sort of started us thinking. In

8 the guidance document we put 28-day donor deferral

9 and so we may have to rethink the deferral for

10 that.

11 Next slide, please. We have not discussed

12 it with the AABB task force but we will be

13 discussing with the task force that, you know, the

14 integration of West Nile testing information. We

15 are thinking about maybe 56-day deferral for West

16 Nile diagnosis of symptoms, including headache and

17 fever, or 14 days after symptom resolution if it is

18 more than 56 days. Potential reinstatement of

19 donor deferral for West Nile symptoms only

20 following 30 days without symptoms, and negative by

21 West Nile IgM or ID-NAT. Again, this is current

22 thinking. We have nothing in the works yet but we

1 have internal discussions, and we will discuss it

2 at our next regular AABB task force before we come

3 up with a recommendation. Dr. Alan Williams is

4 spearheading this initiative.

5 Next slide, please. With regard to our

6 activities in-house, as I mentioned last year also,

7 we are still working on the panel development. The

8 purpose is to monitor sensitivity of assays to

9 detect viral nucleic acid antibodies, and also

10 trying to isolate and characterize West Nile

11 strains from human samples during 2003 and 2002

12 epidemics. The purpose of this study, which is done

13 by Dr. Maria Rios in our group--and all these

14 studies actually really are done by Dr. Maria Rios'

15 group--is the genetic variation of viral strains;

16 detection by currently available West Nile assays.

17 The purpose is to really see if there is any

18 genetic variation and also infectivity studies

19 using animal models. Currently, the samples have

20 been identified which could be used for infectivity

21 studies. However, there are logistic issues about

22 the animals, baboons, which are being worked out

1 with the Southeast Medical Center. I guess the

2 task force is working on that. Hopefully, we will

3 get some information by fall and we will be set to

4 do those studies.

5 Next slide, please. Briefly, they have

6 two isolates, NY99 in 2002, which have been

7 characterized by genetic sequencing which I can

8 show you in a minute. The viral infectivity is

9 determined by in vitro studies using cell lines and

10 primary human blood cell cultures. Final panel

11 specifications are being established through the

12 collaborative studies, and the range of

13 concentration ranges between 1000-5 copies/ml.

14 Next slide, please. Just a piece of

15 information here that Maria was kind enough to

16 provide to me. You know, she did the comparison of

17 the human 2002 strain and the NY99 flamingo isolate

18 and then passed through the Vero cells. She found

19 there were 20 nucleotide mutations and one

20 insertion. The mutations are distributed all

21 across the region which result in 5 amino acid

22 substitutions. She is characterizing more isolates

1 and she already has 6 from 2002, 11 from 2003 and 6

2 from 2004. So, the purpose is to really compare

3 and to see what the differences are and how those

4 differences impact on our tests.

5 Next slide please. The outcome of the

6 panel testing--six laboratories participated in

7 that. She tells me there were no false-positive

8 results reported. More variability in detection

9 was found towards the lower end of the viral

10 concentration, i.e., 80 percent of the time

11 detected 100 copies/ml member but all laboratories

12 detected 100 percent of the time the panel members

13 of 500-1000 copies/ml. Further testing is going to

14 define the consensus copy number.

15 Next slide, please. This is the important

16 slide. I would like to thank all the people who

17 really helped to make this talk possible. Jennifer

18 Brown, whom I have always been bugging to provide

19 the slides. Thank you, Jennifer. Dr. Sue Stramer,

20 Dr. Mike Busch and Sally Caglioti, Dr. Mike Strong,

21 Leanne Kiviharju, Roland, Maria and all these

22 people--whoever I send an email they are kind

1 enough to respond quickly. Also my colleagues at

2 the FDA, Maria Rios, Alan Williams, Dr. Epstein,

3 Martin Ruta, Indira Hewlett--always helping in this

4 whole project and, last but not the least, the AABB

5 task force. I am really, really grateful to them

6 for providing all the information and helpful

7 discussion. Thank you very much.

8 DR. NELSON: Thank you. Any questions or

9 comments? Yes?

10 DR. GOLDSMITH: Do you have additional

11 data on the level of viremia in these samples that

12 you have been studying? What is the maximum level

13 of viremia?

14 DR. NAKHASI: Which samples are you

15 talking about?

16 DR. GOLDSMITH: The ones that you

17 recovered from the viremic donors.

18 DR. NAKHASI: From the viremic donors, I

19 don't know. Maria, do you know what the levels

20 are?

21 DR. RIOS: Between 10

5 and 106 is the high

22 level of viremia that we have found. Are you

1 asking for the range of viremia or the high level

2 of viremia?

3 DR. GOLDSMITH: I was just curious about

4 the high but it is fine to give the range.

5 DR. RIOS: It varies. It varies. The

6 assays, in general, that use lower volumes do not

7 detect them. Assays that have higher volume and

8 high throughput detect, but do not give accurate

9 quantitation, to 10

6 copies/ml.

10 DR. NELSON: One of your slides had 23

11 positives with 16 by minipool and 7 by ID. Were

12 those 7 not detectable by minipool or was it just

13 that ID screening was triggered and they weren't

14 tested by minipool?

15 DR. NAKHASI: I think they came for the

16 ID-NAT testing. Is that true? Yes. You know, in

17 BSL they had already started ID-NAT testing in

18 Maricopa County. The trigger had started earlier.

19 DR. NELSON: So, they were negative by

20 minipool?

21 DR. STRONG: No, the trigger was activated

22 and they started doing ID screening so they haven't

1 gone back yet, I think, to see if those would have

2 been picked up by minipool.

3 DR. BUSCH: Actually, 7/12 that were

4 picked up in the region that had been converted to

5 ID-NAT, 7 of them had been fully worked up and 5 of

6 those 7 are negative at 1:16 dilutions so they

7 would have been missed by minipool. Of those 5, 1

8 of them is antibody negative and 4 have IgM and

9 IgG.

10 DR. NELSON: Has anybody looked at the

11 characteristics of the donors that have low levels?

12 Are there host factors that might influence whether

13 somebody has high level or low level? I know one

14 feature may be antibody but in those that are

15 antibody negative, I wonder if there are any donor

16 characteristics that influence the level of

17 viremia.

18 DR. BUSCH: Sue has looked at that I think

19 more formally and there wasn't any correlation.

20 These are representative donors of the donor pool

21 in terms of the viremics, non-viremics and low

22 viremics. I think it is just by chance. This

1 phase of early viremia is completely asymptomatic.

2 DR. RIOS: It may have some inherited

3 characteristics that limit the viral replication.

4 The reason why we think that is because we have

5 performed some in vitro studies with human primary

6 macrophages and there is a great variability not

7 only in the day of the viral peak, but some

8 individuals can have a very steady and low titer

9 that doesn't progress to peak. So, that indicates

10 that some inheritance variability may interfere

11 with replication.

12 DR. NELSON: That is interesting. Other

13 questions?

14 DR. LAAL: Unless I misunderstood, I

15 noticed that in 2003 we had a majority of your

16 isolates from people who had fever, and about

17 one-quarter were from neuroinvasive cases. In 2004

18 it is reversed.

19 DR. NAKHASI: Yes, that is an important

20 point. I discussed it with the CDC folks and they

21 said, you know, don't pay attention to that because

22 the fever cases were--you know, this year they are

1 paying more attention so some of the fever cases

2 were not real fever cases. You are right, you saw

3 the switch.

4 DR. LAAL: But then in the isolates that

5 you are picking up now for the genetic studies, are

6 you carefully making sure that you look at both

7 types?

8 DR. NAKHASI: Maybe Maria can say; I don't

9 know.

10 DR. RIOS: The isolates that have been

11 studied so far don't come from patients. Actually,

12 that is the effort we are going to move towards

13 now. They are identified through the blood

14 screening. So, in order to evaluate if there is

15 any isolate that may not be picked up by the blood

16 screening we need to acquire samples from cases

17 that are non-blood donors to investigate this

18 possibility.

19 DR. NELSON: Yes, Mike?

20 DR. STRONG: Just a quick comment on the

21 donors. In the studies that were done last year,

22 many of the donors that were interviewed, in fact,

1 were symptomatic either shortly before or shortly

2 after their donations but the screening questions

3 just didn't pick them up.

4 IV. Hepatitis B Virus Nucleic Acid Testing (NAT)

5 for Donors of Whole Blood

6 DR. NELSON: Thanks. The next topic is

7 hepatitis B virus nucleic acid testing for donors

8 of whole blood. Dr. Gerardo Kaplan will introduce

9 this and give us background.

10 A. Introduction and Background

11 DR. KAPLAN: Good morning.

12 [Slide]

13 I am Gerardo Kaplan, Chief of the Lab of

14 Hepatitis and Related Virus Emerging Agents. I am

15 with the Office of Blood, and I will introduce for

16 you the hepatitis B virus n nucleic acid testing

17 for donors of whole blood.

18 [Slide]

19 The general agenda for this meeting is

20 that after the introduction and background, Dr.

21 Blaine Hollinger will give us an update on the

22 serology of hepatitis G. This will be followed by

1 two presentations from the Roche Molecular Systems

2 and their preclinical and clinical data in support

3 of their application. Finally, I will come back to

4 give you the FDA perspective on hepatitis B MP-NAT

5 and present the questions for the committee. I

6 understand that there will be a break and then a

7 public hearing.

8 [Slide]

9 So, the issue is that the FDA is seeking

10 the opinion of the committee on the performance of

11 the Roche COBAS AmpliScreen HBV test in minipools

12 of 24 samples to screen blood for transfusion by

13 nucleic acid testing, and its proposed intended use

14 as an alternative to hepatitis B surface antigen

15 testing in conjunction with testing for antibodies

16 to hepatitis B core antigen.

17 [Slide]

18 In support of their claims, Roche

19 Molecular Systems performed a clinical trial. The

20 study objectives of the clinical trial were to

21 determine whether the COBAS AmpliScreen test, which

22 is a minipool of 24 samples of plasma from

1 volunteer blood donors, can detect hepatitis by DNA

2 in hepatitis B surface antigen-anti-core negative

3 window period cases. This is the primary objective

4 of the study. In hepatitis B surface

5 antigen-positive donors, who are acutely infected

6 or chronic carries, and in persons previously

7 exposed to hepatitis B as the secondary objective.

8 [Slide]

9 During the clinical trial, Roche Molecular

10 Systems identified two window period cases in about

11 600,000 volunteer whole because donations screened

12 by hepatitis B NAT using their minipools of 24

13 samples.

14 RMS, Roche Molecular Systems, claims that

15 the use of the COBAS AmpliScreen HBV test in

16 conjunction with the anti-core test would reduced

17 the residual risk of transfusion-transmitted

18 hepatitis B, and also that this test, the COBAS

19 AmpliScreen, could be used as an alternative to the

20 commonly used hepatitis B surface antigen donor

21 screening test. I would like to point out that

22 blood in the U.S. is currently being tested by

1 surface antigen and core antibodies.

2 [Slide]

3 So, we would like to request comment of

4 the committee on three questions. Basically, the

5 firs would be do the sensitivity and specificity of

6 the Roche COBAS AmpliScreen hepatitis B test in

7 minipools of 24 samples support licensing of the

8 assay as a donor screen?

9 [Slide]

10 If so, assuming continued use of screening

11 tests for anti-hepatitis B-core, do the data

12 support use of the Roche COBAS AmpliScreen

13 hepatitis B test in minipools of 24 samples to

14 screen blood for transfusion as an equivalent

15 alternative to the surface test? If not, which

16 studies would be required to validated such a new

17 test?

18 The third question is do the data support

19 use of the Roche COBAS AmpliScreen hepatitis B test

20 on minipools of 24 samples to screen blood for

21 transfusion as an added test in conjunction with

22 licensed donor screening tests for hepatitis B

1 surface and anti-core?

2 Now I would like to introduce Dr. Blaine

3 Hollinger. He will give us an update on hepatitis

4 B serology.

5 B. Serological Course of Hepatitis B

6 DR. HOLLINGER: Thank you, Gerardo. It is

7 always a pleasure to come to these meetings and I

8 always tend to learn more than I think anyone here

9 because there is always so much information.

10 [Slide]

11 The talk today is about serology, and

12 serology can be confusing. It is confusing to our

13 GI residents and fellows in trying to determine

14 what happens after hepatitis B infection. The

15 clinical and serologic changes that occur following

16 infection represent a complex interaction between

17 the host, the virus and specific antigens. If you

18 have an understanding of these items, I think it

19 becomes very easy to understand what happens.

20 There are certain changes that occur after

21 infection with all viruses that seem to be very

22 similar. Viruses are either enveloped or

1 non-enveloped. Hepatitis B virus happens outcome

2 have a lipoprotein envelope that contains hepatitis

3 B surface antigen as the envelope protein. There

4 are also some other particles in this serum which

5 are seen in excess of the infectious virion, and

6 these are the small particles here, and these

7 tubular forms which actually come off, in many

8 cases, from the hepatitis B virion.

9 The inside, or the nucleocapsid of the

10 virion is comprised of the hepatitis B core

11 antigen, and this encloses a relaxed, circular,

12 partially double-stranded DNA molecule. Another

13 antigen which is seen also is the hepatitis B

14 antigen, but this is not part of the virion in any

15 way. It is a secretory protein, about 16-17 kd in

16 size, that is secreted during active infection, and

17 is representative usually of high concentrations of

18 virus in the bloodstream. We don't really know

19 what is the reason for this particular antigen.

20 [Slide]

21 This is the HBe genome. It has four open

22 reading frames. One of them is responsible for the

1 hepatitis B-surface antigen and is comprised of a

2 small, a medium and a large protein. A second one

3 is the core gene which expresses the core-antigen,

4 the hepatitis B core-antigen and also the e-antigen

5 as well. There is a DNA polymerase that is

6 important for the replication of this virus, and

7 there is an x-gene here which is very important for

8 initiation and probably maintenance of the

9 infection. It is probably also weakly oncogenic as

10 well. Now, once one understands these things, you

11 can then start to see what happens after an

12 infection.

13 Next slide, please. This slide shows what

14 happens in the typical course--now, I am going to

15 talk about typical things here. Certainly all of

16 us know about the exceptions and we have all seen

17 them at one time or another, but I think it is

18 important to deal right now with the typical

19 changes that can occur. Again what happens, you

20 have a hepatocyte that is infected. There is a

21 very short eclipse phase in which we would not see

22 virus in the hepatocyte. Then viruses start to be

1 produced and are transported out of the cell.

2 Then, one sees initially HBV DNA in the

3 bloodstream. It precedes the expression of

4 hepatitis B-surface antigen which then starts to

5 occur here very early in the course of the disease

6 and peaks usually during the acute phase of a

7 clinical illness.

8 What happens then in this non-cytolytic

9 infection is that there are usually some holes that

10 are punched in the plasma membrane of a hepatocyte.

11 ALT, which is in the cytosol, aminotransferase

12 enzyme which is in the cytosol of hepatocytes, are

13 released into the bloodstream. So, the ALT is

14 elevated in these patients and then follows the

15 clinical symptoms of anorexia, fatigue, jaundice,

16 etc.

17 Usually about the time the ALT begins to

18 appear, HBeAg and IgM anti-HBc are found. IgM anti

19 HBc appears in high titer or high concentration in

20 individuals as a response to the synthesis of

21 nucleocapsids and, therefore, represents active

22 viral replication. The IgM rises during the early

1 courses of infection and then starts to disappear.

2 Usually by 4-6 months or so, or 6-9 months, the IgM

3 anti-HBc disappears, although it may be present for

4 up to 2 years in individuals, if you use very

5 sophisticated research tools.

6 Usually the HBV DNA disappears in the

7 acute infection at the time that a hepatitis

8 B-surface antigen disappears. So, you have a

9 pattern here. HBV DNA first; HB surface antigen,

10 and the HBsAg disappears and usually about that

11 time HBV DNA is gone, or at least is

12 non-detectable.

13 Again, another pattern appears. When

14 HBsAg disappears it is replaced by its antibody,

15 anti-HBs. Now, we know that probably anti-HBs is

16 produced very early in the course of the infection,

17 even probably back in this area here, which results

18 in serum sickness, vasculitis or other things, but

19 you don't detect it because there is so much

20 hepatitis B surface antigen available so it is

21 non-detectable. But eventually the surface antigen

22 goes; anti-HBs then is detected. When HBeAg

1 disappears in the active infection, then anti-HBe

2 replaces it. Anti-HBe lasts in most patients for a

3 long period of time but in about a third of the

4 patients disappears after about 6 months of

5 follow-up.

6 Of course, the IgM anti-HBc--there is a

7 switch-over between the IgM anti-HBc and IgG

8 anti-HBc. What is important to recognize is that

9 the assay, the total anti-HBc assay, detects not

10 only IgM but IgG antibodies. It is not an IgG

11 test; it detects both IgG and IgM. So, whenever

12 IgM is present you ought to see the total anti-HBc

13 test positive.

14 Next slide, please. This is an older

15 slide I have but I think it helps us a little bit

16 understand the relationship of all these assays

17 that we are looking at. The EIA assay and those

18 that are being developed can detect down to about a

19 tenth of a nanogram of HBsAg. HBsAg circulates up

20 to 200 mcg or more per ml of blood. These tests

21 really have an upper level of sensitivity of about

22 1 mcg/ml. So, usually they are at their upper

1 limits of detection for most infections.

2 The second test that came along them were

3 the hybridization assays, the liquid phase and the

4 filter hybridization assays. These could detect

5 down to a tenth to 1 pg of genomic equivalence in

6 the blood. At least, at 24 hours if you have the

7 autoradiographs stay for maybe up to 5 days, you

8 can detect anywhere from 2- to 10-fold more virus

9 in the blood. So, they are detecting about this

10 level here, somewhere between here and here, and 1

11 pg is equivalent to about 300,000 copies/ml. You

12 will see 280,000, 330,000 but let's just talk about

13 300,000 copies/ml. So, 1 pg is here, 300,000,

14 30,000, 3,000 and 300 down at the femptogram

15 levels.

16 The PCR assays, which are somewhere

17 between 700-7,000 times more sensitive than

18 hybridization assays, can detect down to 50 copies

19 or less per ml. So, they are down in this range,

20 here. As I said, this is 300,000, 3,000 and 300

21 copies/ml. This would be 30 copies/ml. These are

22 usually with nested PCRs or other assays, nucleic

1 acid--the tests that are available today.

2 So, you see the differences between these

3 assays. One might ask why is it then that the EIA

4 tests are detecting HBsAg very closely to the HBV

5 DNA. Part of the reason for that is that there are

6 anywhere from 1,000 to 10,000 times more--at least

7 10,000 times more hepatitis B surface antigen

8 particles per infectious virion, and there may be

9 even more naked HBV particles. So, you have a lot

10 more HBsAg produced initially than you do

11 infectious virions. That is the reason I think

12 that there is not a great deal of difference, at

13 least initially, in the detection of these

14 particles.

15 Next slide, please. So, during the course

16 of infection HBV DNA is detected from 2-5 weeks

17 after infection and up to 40 days before the HBsAg

18 is detected. However, there is a wide coefficient

19 here of variation but the mean of that is around

20 6-15 days, and there are some exceptions to this.

21 It rises slowly during the course of infection at a

22 relatively low level, say 10

2 to 104 copies/ml

1 during the seronegative period, that is, before the

2 HBsAg appears.

3 Next slide, please. HBsAg, on the other

4 hand, appears 1-3 weeks before the ALT becomes

5 abnormal or 3-5 weeks before the onset of symptoms

6 or jaundice. It reaches a peak during the acute

7 stage of the disease and then it declines to

8 undetectable levels within 4-6 months.

9 Next slide. IgM anti-HBc is indicative,

10 as I said, of ongoing viral replication and appears

11 at the onset of ALT abnormality at high

12 concentration. It is present but undetectable in

13 some chronic infections and may reappear,

14 therefore, during reactivation of HBV during

15 chronic infection.

16 Now, one of the questions that often

17 arises is when you have acute hepatitis how do you

18 determine that this is not a reactivation of

19 chronic disease compared to an acute disease? That

20 has always been an issue. For all practical

21 purposes, the IgM anti-HBc test can be utilized to

22 determine that. It is at high concentrations.

1 Whereas, patients who have reactivation of their

2 hepatitis B virus infection from chronic disease

3 often have a relatively low concentration of IgM

4 anti-HBc in their test. We use a sample to cut-off

5 ratio and it is usually somewhere between 1-3 in

6 those cases, whereas in acute disease it is at its

7 maximum.

8 In addition, the other way, if you have

9 the means to do this, is that the IgM has a 19S

10 sedimentation coefficient during acute infection.

11 It has a 7S sedimentation coefficient during

12 chronic infection. That is another way that one

13 could use to determine acute from chronic.

14 Next slide, please. The anti-HBs is a

15 neutralizing antibody occurring during recovery and

16 after infection, and in most cases indicates

17 protection against reinfection. However, it may

18 become undetectable in up to 20 percent of patients

19 after several years.

20 Next slide. This slide sort of shows what

21 happens in a patient who gets acute disease and

22 gets over it. The anti-core is a very strong

1 antibody, high concentration of antibody compared

2 to anti-HBs. Over time, as I said, in about 20

3 percent of patients this antibody will disappear.

4 Then you are left with a person who has only an

5 anti-HBc response.

6 Next slide. One of the ways that one can

7 tell that that is present is you can do an HBsAg

8 vaccine challenge. You can give them a vaccine.

9 About 2-4 weeks later you have them come back in

10 for a quantitative anti-HBs test and if the

11 response is greater than 10 milli-international

12 units/ml, that is indicative that this person

13 probably had a prior infection and was immune. It

14 is unusual for a susceptible person or a person

15 with a low-level infection to respond to the

16 hepatitis B surface antigen by 2-4 weeks. Probably

17 less than 3 percent in 2 weeks will respond to the

18 vaccine at this particular level.

19 Next slide. I have talked a little bit

20 about chronic infection. The difference is that

21 arbitrarily, say, if the HBsAg is still positive

22 after 6 months or the person has ALT abnormalities

1 for a period of time, then they probably have

2 chronic infection. In addition, the HBsAg is at

3 very high concentrations through that period of

4 time. It doesn't start to decline. So, if you

5 could dilute these samples as a patient is being

6 followed along, you will find that the HBsAg

7 concentration is declining. Just remember that the

8 upper level of their reference is about 1 mcg/ml so

9 you do have to do some dilutions in order to see

10 this happening. The IgM does disappear but remains

11 usually positive at very low levels. With research

12 tools you can detect it in most patients. It just

13 isn't positive in the way the test is done, which

14 is to dilute the sample about 1:1000 or 1:2000.

15 That is why you don't see it in these individuals.

16 That is how the test is configured. Part of that

17 is because of prozone effects and other things

18 which could make a positive test negative if you

19 did not dilute.

20 E antigen is usually present initially,

21 but over time about 5-10 percent of patients will

22 seroconvert from HBeAg positivity to anti-HBe

1 positive. During that period of time they usually

2 show reactivation of their disease. Often it is

3 just an elevation of the liver enzymes but they may

4 become jaundiced and they may develop symptoms that

5 look just like acute disease. Until we understood

6 this, this was one of the reasons we used to think

7 that 10 percent of patients who had acute disease

8 would become chronically infected. Now that we

9 know that many of these patients were chronically

10 infected to start with, we now feel that probably

11 less than 3 percent of the immunocompetent patients

12 who develop acute disease will actually become

13 chronically infected. Of course, the anti-core and

14 HBsAg remain positive in most patients.

15 Next slide, please. Let me just go

16 through, finally, some of the findings that we just

17 talked about. This is HBVd and a surface antigen,

18 anti-HBc and anti-HBs. You can see in the

19 pre-seroconversion window period--we have a couple

20 of windows here because we used to talk about a

21 window period between the end of the HBsAg phase

22 and the development of anti-HBs in which the IgM

1 anti-core is present during acute disease. But

2 here I am talking about the pre-seroconversion of

3 antibodies or the seronegative, if you will, window

4 period.

5 Then, in the early acute infection both

6 the HBVd and a and HBsAg are positive. The

7 anti-core and the anti-HBs are negative. The

8 presence of HBsAg is indicative of a hepatitis B

9 infection, either acute or chronic. Just the HBsAg

10 alone--you can't tell. But the presence of all

11 three of these together in the absence of anti-HBs

12 indicates an HBV infection, either acute or

13 chronic. Eventually you will lose these markers

14 and you are left with anti-HBc and anti-HBs, which

15 means a previous infection with immunity.

16 Next slide. The other findings are in

17 individuals who have a solitary anti-HBc test

18 present. They have no anti-HBs and no HBsAg. Some

19 of them may contain HBV DNA and these represent

20 low-level carriers which do not have enough surface

21 antigen present in the blood to be detectable by

22 the current assays.

1 The other thing is that if this is

2 negative, this could also indicate an early

3 convalescent period in which this anti-HBc will be

4 IgM. It could represent an HBV infection in the

5 remote past, as we talked about, in which the

6 anti-HBs has disappeared and you could do a vaccine

7 challenge, or it could be in many cases a

8 false-positive reaction. Most of these reactions

9 show anti-core at a very low level, again, a sample

10 to cut-off ratio somewhere between 1-3. The

11 anti-HBe is usually absent in these individuals as

12 well, and they don't respond to the vaccine

13 challenge.

14 Then you have a group that have no

15 markers. This usually excludes an HBV infection in

16 this case. Then you have individuals with anti-HBs

17 only. This represents a vaccine type response. It

18 is really unusual to see something else happening,

19 or you shouldn't see anything other than anti-HBs

20 in a patient who gets the vaccine because it only

21 contains HBsAg.

22 Next slide. This is my final slide. It

1 looks at discordant or unusual hepatitis B

2 serologic profiles requiring further evaluation. I

3 have probably seen all of these at some time or

4 another in my career. For example, I have a

5 patient right now who has HBsAg positive only, who

6 is anti-core negative, with 900 million copies or

7 IU of virus per ml of blood. He has been like that

8 most of his life. It happens in some young

9 children who get hepatitis B and become chronically

10 infected. It is seen in other adults, particularly

11 in Asians. But it is a really unusual phenomenon.

12 So, that is a possibility but really rare.

13 Finding of all these three markers in one

14 patient does occur in about 5-10 percent of

15 patients. It is mostly in drug users or people who

16 have been exposed to different genotypes of the

17 virus. It could be found in people who have been

18 vaccinated and have made antibodies to a different

19 genotype, and it usually is in patients who have

20 more serious liver disease.

21 Anti-HBs or anti-HBc positive only--we

22 have talked about those phenomena. The are not so

1 uncommon. HBsAg negative individuals who are HBeAg

2 positive--you shouldn't really see that but I have

3 seen at least two cases that have been HBeAg

4 positive, HBV DNA negative but do not have the

5 presence of surface antigen present.

6 You could have patients who are positive

7 for both e antigen and anti-e at the same time.

8 That has to do with the nuances of the test and

9 probably nothing else. And, you can find patients

10 who are anti-core negative--I mean, you should not

11 find a patient who is anti-HBc negative but IgM

12 anti-HBc positive.

13 Well, I hope now you are a little bit more

14 familiar with the serology of this disease.

15 Antigens are followed by antibodies. HBV DNA

16 precedes the development of antigens very early in

17 the course and usually remains throughout the

18 disease entity itself. Thank you very much.

19 DR. NELSON: Thanks, Blaine. Are there

20 any questions? Harvey?

21 DR. KLEIN: Blaine, there have been a

22 number of reports of people who have low levels of

1 HBV DNA and have anti-HBs. Do you know if any of

2 those are infectious? Have you ever seen one?

3 DR. HOLLINGER: Well, it is not whether we

4 have ever seen one. The issue I guess is that we

5 have this question all the time about occult

6 infection in HBV DNA even though there are no

7 markers, usually in people who are very sick and

8 would not be donors, for example, liver cancer

9 patients and immunocompromised individuals. So,

10 you wouldn't find these as donors anyway. It is my

11 belief that most of these issues of finding nucleic

12 acid in the presence of antibodies probably means

13 it is neutralized and it is not infectious. You

14 see that with RNA found very late in the course of

15 the disease. In the presence of antibody it

16 doesn't appear to be infectious biologically,

17 clinically or in trials.

18 So, I think that finding anti-HBs at a

19 reasonable concentration and HBV DNA would probably

20 not indicate somebody is infectious. But you just

21 have to do these studies, and no one has really,

22 say, taken a chimpanzee and looked at these. I

1 think Prince did so a few years ago and the data

2 was that they were not infectious.

3 DR. NELSON: You didn't mention the

4 genetic variations in the surface antigen. Could

5 those lead to a false-negative surface antigen test

6 in people who were actually infectious?

7 DR. HOLLINGER: Yes, Kenrad, it is a good

8 question. There are about six or seven genotypes

9 of this disease, from a to h, but because they all

10 have a group a antigen present, they all seem to be

11 detectable by the usual tests which are available

12 today. Now, could it happen? Sure, I think it is

13 possible but I think it would be unusual.

14 DR. NELSON: One other comment too, we

15 have studied hepatitis B in injection drug users

16 and it is extremely common for them to have

17 hepatitis B core antibody only, without surface, at

18 very high levels. They are actually truly infected

19 and it is up to 20 percent of injection drug users.

20 It is possible that co-infection with hepatitis C

21 may play a part in that because they are all

22 infected with hepatitis C as well. It is a very

1 complex interaction. But seeing hepatitis B core

2 antibody only in somebody who actually is a drug

3 user and comparing drug users with others without a

4 history, it is like 20 percent of the drug users,

5 maybe 1 or 2 percent in gay men who have had

6 hepatitis B infection.

7 DR. HOLLINGER: Yes, you see it not only

8 in drug users but you see it in volunteer donors

9 who are found to be anti-HCV positive and then you

10 go do some other tests and they are found to be

11 anti-HBc positive only. They don't respond well to

12 the vaccine. And, we know that HCV interferes with

13 the replication of hepatitis B virus so that is a

14 possibility.

15 DR. NELSON: Other comments?

16 [No response]

17 Thanks. Dr. Herman, from Roche?

18 C. Preclinical and Clinical Data for HBV MP NAT

19 DR. HERMAN: Thank you very much. It is a

20 pleasure to be here to describe to you the results

21 of our non-clinical and clinical performance

22 studies on our COBAS AmpliScreen HBV test.

1 [Slide]

2 I am going to begin the talk with a

3 description of our non-clinical performance

4 studies, and Dr. Frank will conclude with a

5 description of our clinical study results.

6 [Slide]

7 Here is an outline of the talk. I am

8 going to give an overview of the COBAS AmpliScreen

9 system and then I am going to describe the

10 different portions of the non-clinical performance

11 studies and give you their results.

12 [Slide]

13 The COBAS AmpliScreen system is designed

14 for screening of minipool samples or individual

15 donor samples. The samples are put onto the

16 Hamilton microlab pipetting robot which prepares

17 the pools. Then the pooled or individual samples

18 are processed to extract nucleic acid with the

19 generic sample processing method. There are two

20 variations of the method, the multiprep method for

21 the pooled samples and the standard method for

22 individual donor samples, and I will describe them

1 in the next slide.

2 Aliquots of the extracted material can be

3 processed in parallel with our three different

4 COBAS AmpliScreen tests, the test for HBV and the

5 licensed tests for HCV and HIV. The analysis is

6 done on the COBAS Amplicor analyzer which automates

7 the nucleic acid amplification and detection

8 processes. Then, the software for the analyzer and

9 the software for the pipetting machine interact

10 with the data output management system.

11 [Slide]

12 This slide describes the two sample

13 preparation methods, the multiprep method which is

14 used for the minipools, and the standard method

15 that is used for individual donor samples. For the

16 minipool method, 1 ml of the pool is centrifuged at

17 23,500 g to enrich for the viral targets, HCV, HIV

18 and HBV. After the centrifugation, 900 mcL of the

19 supernatant is discarded, leaving behind the pellet

20 and 100 mcL of the supernatant. The nucleic acids

21 are extracted by adding a k-atropic lysis reagent

22 which contains the internal control. After a brief

1 incubation, isopropanol is added to precipitate the

2 nucleic acids. The nucleic acids are collected by

3 centrifugation. The pellet is washed with ethanol

4 and resuspended in a specimen diluent, and then an

5 aliquot of the specimen diluent can be used in each

6 of the AmpliScreen tests.

7 The standard specimen processing method

8 for individual samples is almost identical. All

9 the green steps are identical and the differences

10 are highlighted in red. So, the starting specimen

11 volume is 200 mcL instead of 1 ml, and there is no

12 concentration step. The k-atropic lysis reagent

13 with the internal control is added directly to the

14 200 mcL specimen and then the remaining steps are

15 the same.

16 [Slide]

17 Now I am going to describe the analytical

18 sensitivity studies that were performed. These are

19 the results using the multiprep procedure. We used

20 the World Health Organization HBV DNA international

21 standard and we prepared dilutions of it between

22 100 and 3 IU/ml and analyzed 120 replicates of each

1 dilution, at Roche, using 2 different kit lots.

2 This shows the results. So, we had 100 percent hit

3 rate at 100, 30 and 10 IU/ml and a 95.8 percent hit

4 rate at 5 IU/ml. The predicted 95 percent limit of

5 detection, using the PROBIT statistical method, is

6 4.4 IU/ml.

7 [Slide]

8 This slide shows the results using the

9 standard specimen processing procedure. In this

10 study we analyzed dilutions of the World Health

11 Organization standard from 300 to 10 IU/ml. There

12 was 100 percent hit rate on 100 replicates at 300

13 and 100 IU/ml, and 99 percent on 30 IU/ml, and 97

14 percent at 20, and 95.8 percent at 15 IU/ml. The

15 predicted 95 percent limit of detection, using the

16 PROBIT method, is 16 IU/ml for the standard

17 specimen processing method.

18 [Slide]

19 This slide describes the results on a

20 panel prepared by CBER. The original panel

21 contained 3 members at 100, 10 and 0 copies/ml, and

22 at Roche we prepared 4 additional dilutions from

1 the 100 copy/ml member at 50, 25 5 and 2.5

2 copies/ml.

3 This slide shows the results using the

4 multiprep specimen processing method and on 6

5 replicates, 100 percent hit rate at 100 copies and

6 at 10 copies/ml, then 67 percent and 42 percent

7 hits at 5 and 2.5 copies/ml.

8 Using the standard specimen preparation

9 method, 100 percent hits at 100 copies/ml with 6

10 replicates and 92 percent and 75 percent at 50 and

11 25 copies/ml.

12 [Slide]

13 This slide shows the results of our

14 analysis of the performance of the test on HBV

15 genotypes A through H. These samples were obtained

16 from commercial sources, and we analyzed up to 25

17 individual isolates for each genotype. The samples

18 were quantified using the COBAS Amplicor HBV

19 monitor test or, in the case of genotypes G and H,

20 monitor tests that we have in development. Then

21 the samples were diluted to approximately 2-3 times

22 the limit of detection for the multiprep method and

1 the standard prep method, and then they were

2 analyzed using the COBAS AmpliScreen test. So,

3 with the multiprep method and the standard method

4 all the isolates yielded positive results. So, the

5 test detected HBV genotypes A through H.

6 [Slide]

7 This slide summarizes the results on 40

8 commercially obtained seroconversion panels using

9 the multiprep method and the standard prep method.

10 The slide shows the number of days that DNA was

11 detected prior to detection of surface antigen

12 using a licensed surface antigen test, the Ortho

13 surface antigen test system 3. The orange bars

14 show the results with the multiprep test, and for

15 that analysis the samples were diluted 24-fold to

16 simulate minipool testing. The blue bars show the

17 results with the standard prep on undiluted

18 samples. The panels are sorted by the number of

19 days prior to antigen that DNA is detected with the

20 multiprep method. So, using the multiprep method,

21 in 38/40 panels HBV DNA was detected prior to

22 surface antigen, and in 2/40 panels HBV DNA was

1 detected in the same bleed as surface antigen.

2 Using the standard prep method on neat

3 samples in 39/40 panels HBV DNA was detected prior

4 to surface antigen, and in 1 panel HBV DNA was

5 detected on the same day as surface antigen. I am

6 going to show you the results on one of the panels,

7 this panel, 39.

8 Before that, with the multiprep method, on

9 the 38 panels on which DNA was detected prior to

10 surface antigen, DNA was detected an average of 17