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
Gaithersburg Holiday Inn
2 Montgomery Village Avenue
Kenrad E. Nelson, M.D., Chair
Linda A. Smallwood, Ph.D., Executive Secretary
Pearline K. Muckelvene, Scientific Advisors
& Consultants Staff
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
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
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
Open Public Hearing:
Patrick Schmidt, CEO, FFF
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
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
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
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
22 Forty-six states, including
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
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,
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
21 DR. RIOS: Between 10
5 and 106 is the high
level of viremia that we have found.
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
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
21 DR. STRONG: No, the trigger was activated
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
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
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
viremics. I think it is just by
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
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
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
8 DR. NAKHASI: Maybe Maria can say; I don't
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
19 DR. NELSON: Yes, Mike?
20 DR. STRONG: Just a quick comment on the
21 donors. In the studies that were done last year,
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.
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.
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
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.
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.
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
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.
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
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
blood in the U.S. is currently being tested by
1 surface antigen and core antibodies.
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?
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
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
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.
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
similar. Viruses are either
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.
21 This is the HBe genome. It has four open
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
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
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,
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
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
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
goes; anti-HBs then is detected.
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
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
mcg/ml. So, usually they are at their
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
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
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
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
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
determine that. It is at high
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
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
gets over it. The anti-core is a
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
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
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
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
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
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
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
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
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,
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
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
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
studies on our COBAS AmpliScreen HBV test.
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.
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.
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
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.
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
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.
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
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.
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.
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
at Roche we prepared 4 additional dilutions from
1 the 100 copy/ml member at 50, 25 5 and 2.5
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.
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
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.
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
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
11 days earlier, and with the standard method DNA was
12 detected an average of 22 days earlier.
14 Here are the results on panel 39. This
15 panel had 14 bleeds. This column shows the results
16 with the Ortho Surface Antigen Test System 3, and
17 you can see that this turned positive on bleed 12
18 at day 143. This column shows the results with the
19 minipool test, and you can see that it was positive
20 on the first and third bleed and then positive
21 again at the 11th bleed on day 113.
22 On the previous slide, on this panel it
1 was described as having DNA detected prior to
2 antigen 30 days in advance this one bleed. Even
3 though DNA was detected intermittently earlier than
4 that, we made a conservative interpretation and
5 only counted the number of bleeds that DNA was
6 continuously detected prior to surface antigen.
7 In this column, the results with the
8 standard preparation method on undiluted samples is
9 shown. There were 11 bleeds prior to detection by
10 surface antigen and the HBV DNA was positive in 7
11 of them but did not yield a positive result on
12 bleed 11. So, in the previous graph the standard
13 prep method was described for this panel as being
14 detected on the same day as surface antigen.
16 This just illustrates that even though DNA
17 was detected intermittently earlier, on multiprep
18 method we only counted 1 bleed, which was 30 days
19 in this case.
21 This slide summarizes the results on the
40 seroconversion panels. Using
1 method on samples diluted 24-fold to stimulate
2 pooling, DNA was detected prior to surface antigen
3 in 38/40 panels and on the same bleed in 2/40
4 panels an average of 17 days prior to surface
5 antigen. Using the standard procedure on undiluted
6 samples, DNA was detected prior to antigen in 39/40
7 panels an average of 22 days earlier, and on the
8 same bleed on the 40th panel.
10 This slide summarizes the results of the
11 analytical specificity testing and the testing of
12 potentially interfering substances. For the
13 analytical specificity we looked at 38
14 microorganisms and cell lines, and all these
15 samples yielded negative results with the
16 AmpliScreen and valid internal control results.
17 There was no cross-reactivity observed.
18 For the potentially interfering
19 substances, we looked first at 95 clinical
20 specimens with different diseases in patients
21 infected with these viruses or with autoimmune
disease or with yeast infections, and multiple
1 specimens of each. These specimens were tested
2 both with the addition of HBV target and without.
3 So, the testing with the addition of HBV target was
4 looking for interference. No interference was
5 observed. Testing without HBV target was looking
6 for cross-reactivity and no cross-reactivity was
8 Then we looked at these various
9 potentially interfering substances. Again, they
10 were tested with and without the addition of HBV
11 target. On the tests with HBV target there was no
12 inhibition and on the tests without HBV target
13 there was no cross-reactivity.
15 This slide and the next look at our
16 reproducibility study. This slide shows the
17 results of the multiprep procedure. The study was
18 conducted at 3 clinical sites using 3 lots of
19 reagent. The study was conducted over 5 days with
20 2 operators at each site. Each day the operators
21 analyzed the 6-member panel, blinded 6-member panel
consisting of 2 negative samples and 4 samples at
1 low and moderate target concentrations.
2 Essentially the same results were obtained with the
3 3 kit lots and at the 3 sites. So, there was 1
4 false-positive result with lot 3 and that happened
5 at site 3. At the low copy samples the percent
6 hits were nearly identical among the 3 lots and
7 among the 3 sites.
9 This slide shows the reproducibility study
10 with the standard procedure and the same outcome.
11 Essentially the same results between the 3 lots and
12 between the 3 lights on the negative and the
13 positive samples.
15 Finally, this slide describes the
16 performance of the AmpliScreen test alone or in
17 combination with the licensed anti-core test on 918
18 antigen-positive clinical specimens. I am showing
19 the results with the multiprep test on samples
20 diluted 1:14 to simulate pool testing and with the
21 standard method on undiluted samples. So, looking
at the results of the AmpliScreen test alone with
1 the minipool method, 871 of the 918 samples were
2 DNA positive for sensitivity of 94.9 percent.
3 Using the standard method on undiluted samples, 898
4 samples were positive for sensitivity of 97.8
5 percent. These 20 samples that were not detected
6 by the standard test were among the 47 that were
7 not detected by the minipool test. All the NAT
8 negative samples were sent out for anti-core
9 testing and all of them were positive by the
10 anti-core test. So, using the multiprep method, 1
11 sample had insufficient volume for the testing, a
12 sample that was positive with the standard sample
13 prep and negative with multiprep so we couldn't get
14 a result on that so it was excluded from the
15 sensitivity analysis. So, for the 917 samples with
16 NAT positive results and anti-core results on the
17 NAT negative samples the sensitivity was 100
18 percent for NAT and core combined relative to
19 antigen. On the 1900 samples with evaluable
20 results using the standard method, again, the
21 sensitivity was 100 percent.
1 To summarize the non-clinical performance
2 studies, the analytical sensitivity was determined
3 on the World Health Organization standard. With
4 the multiprep method the limit of detection at 95
5 percent probability is 5 IU/ml and with the
6 standard method the limit of detection at 95
7 percent probability is 15 IU/ml. The performance
8 on the CBER panel with the multiprep method, 100
9 percent hits at 10 copies/ml and with the standard
10 method 92 percent hits at 50 copies/ml. Genotypes
11 A through H were detected by the test.
13 On the 40 seroconversion panels using the
14 multiprep method on samples diluted 24-fold, HBV
15 DNA was detected prior to antigen in 38/40 panels
16 an average of 17 days or earlier, and with the
17 standard method on undiluted samples DNA was
18 detected in 39/40 panels prior to antigen an
19 average of 22 days earlier, and there were no
20 panels in which antigen was detected prior to DNA.
21 There was no cross-reactivity or interference
observed in the analytical specificity and
1 potential interfering substances studies. And, the
2 sensitivity on antigen-positive specimens, in
3 combination with the anti-core assay, was 100
6 I would like to acknowledge all the people
7 who did this work, Yuanfeng Yang and his group at
8 Roche Molecular Systems did an excellent job
9 developing the assay and performing these studies.
10 Larry Pietrelli coordinated the clinical studies
11 and, in this part of the talk, he coordinated the
12 reproducibility studies. They were conducted at
13 the Community Blood Center of Greater Kansas City,
14 Royal Blood Centers, Minnesota and the Gulf Coast
15 Regional Blood Center.
16 Now I think I will turn the microphone
17 over to Dr. Frank who will describe the results of
18 the clinical performance study.
19 DR. FRANK: Thank you, Dr. Herman.
21 It is my pleasure to be here today to
describe to you a prospective study to evaluate the
1 screening of plasma pools from volunteer blood
2 donations for the presence of HBV DNA.
4 A clinical trial summary will focus today
5 on two areas of primary focus of improved safety of
6 blood donations by HBV minipool-NAT. The key focus
7 in that particular area will be looking at the
8 potential window period or pre-seroconversion areas
9 of HBV. A secondary focus will be replacement of
10 surface antigen by HBV minipool-NAT. To focus on
11 that, we will look primarily at the areas of
12 samples that were surface antigen positive that
13 were picked up by minipool-NAT and surface antigen
14 positive picked up by anti-core.
15 This clinical study was initiated in
16 August of 2002 at 5 U.S. sites, and was completed
17 in April of 2003. A total of 704,902 specimens
18 were tested with HBV minipool-NAT, of which 581,790
19 were included in the sensitivity and specificity
20 analysis. The differential between the two numbers
21 occurred because specimens were excluded if no
electronic results for surface antigen and/or
1 anti-core were available from the site.
3 If you look at this chart, which is a
4 schematic of the results listing the analytes
5 anti-core, surface antigen and DNA--if you can't
6 see in the back of the room, the red means they
7 were HBV negative for that particular analyte; the
8 green means they were positive for that particular
9 analyte. The totals are on the far right-hand
10 column, the numbers that had that criteria. As you
11 can see, the majority of the donors were, as
12 expected, negative for all three.
14 We were primarily interested in looking at
15 this group down here, and those were enrolled in
16 our follow-up study, the protocol for which I will
17 be describing briefly.
19 Of these 4 eligible for the follow-up
20 study, the 2 areas we wanted to look at are
21 primarily the last row, which is the potential
window cases, that is, those cases which were HBV
1 minipool DNA positive but negative for core and
2 surface antigen. This represents the type of
3 pattern one would expect in pre-seroconversion
4 window cases, and then the potential surface
5 antigen false-positive results, that is, surface
6 antigen positive but core negative and HBV minipool
7 negative as well.
9 I mentioned the follow-up protocol that
10 was utilized and this is the follow-up protocol for
11 those columns that we followed up, the test index
12 donation was tested by alternate NAT, provided by
13 the National Genetics Institute. If alternative
14 NAT was positive, the result was quantitated and
15 then the subject was offered enrollment in a
16 6-month follow-up study for weekly draws times 4
17 then monthly draws to complete 6 months of
18 follow-up. The analytes tested during follow-up
19 are as listed on your screen.
21 The primary objective was to improve the
safety by use of HBV minipool-NAT for detection of
1 window period cases.
3 As stated previously there were 23 donors
4 that were HBV DNA positive, surface antigen
5 negative and core negative. Of these 23, 14 were
6 enrolled in the follow-up study. It should be
7 noted that 9 donors declined follow-up and for the
8 purposes of our calculations for sensitivity and
9 specificity these were presumed to be false
10 positive and were negative on additional index
11 testing as well.
12 Of the 14 enrolled donors, 2 were
13 confirmed window period cases, the details of which
14 I will be showing next, and 12 were shown to be
15 valse positive on HBV due to persistently negative
16 anti-core, persistently negative surface antigen
17 and persistently negative HBV DNA, and negative by
18 alternate NAT index specimens.
20 This window period case is a 26 year-old
21 male repeat blood donor with no known risk factors.
His HBV DNA was positive for a quantitative level
1 of 2000 on index donation. On day 17 post index
2 donation the surface antigen was positive, and on
3 day 48 anti-core was positive.
5 The next window case is a subject who is a
6 49 year-old female repeat blood donor, healthcare
7 worker, with a history of HBV vaccination. This is
8 an interesting case because she had a negative
9 anti-surface antibody result 2 months prior to her
10 index donation when she donated blood previously.
11 What is interesting is between that time, where
12 this result 2 months prior was negative, and here
13 index donation was 2,340--now, this normally
14 wouldn't be done during her blood screening but she
15 was HBV DNA positive and she quantitated out at 200
16 copies/ml, which would be an infectious dose. So,
17 this is a window period case of an individual who,
18 on follow-up, never did convert for surface antigen
19 and did, on day 22, convert for anti-core. What
20 this likely represents is that somewhere between
21 the 2 weeks prior to her index donation when this
was negative and the result of her index donation,
1 she became infected with HBV and had a nice immune
2 system response to her previous HBV vaccination.
4 There are additional window cases I would
5 like to show to you today, and these were detected
6 by sites continuing to use HBV NAT following
7 conclusion of our clinical study. The dates for
8 those 3 sites were April, 2003 to the present, and
9 there are 3 additional window period cases that
10 were detected in the one million donations
13 The first case is a 27 year-old male
14 repeat blood donor who reported high risk male
15 sexual partners. He had HBV DNA positive on
16 donation with a quantitative level of 61,000. On
17 day 7 his surface antigen turned positive and on
18 day 36 his core was repeat reactive.
20 This case is a window period case of a 29
21 year-old male repeat donor who has no appreciable
risk factors for hepatitis B, other than a possible
1 acupuncture treatment for 8 weeks prior to his
2 donations, weekly treatments. His HBV index was
3 positive at a quantitative level of 2,300. His
4 surface antigen turned positive 7 days later and
5 his core turned positive 28 days later.
7 This last case is still on follow-up but
8 this is a 50 year-old male repeat blood donor,
9 positive on index donation with 37,000 copies/ml.
10 On follow-up he remains positive on HBV DNA and the
11 traditional blood banking serologies are still
14 In summary, I thought it was interesting
15 to look at the 5 cases that HBV minipool-NAT picked
16 up. If you look at the left-hand column, and as I
17 have noted in the footnotes of your slides, all of
18 these are repeat blood donors who could normally be
19 expected to be donating blood regularly to the
20 blood pool, and all donated blood units that would
21 have been missed by the current blood banking
algorithms. All of them had
1 concentrations on their index donations. In fact,
2 even the lowest dose at 200 copies/ml, if the unit
3 were divided appropriately, would be sufficient to
4 infect everyone in this room.
6 Well, what is the yield for HBV
7 minipool-NAT? If you look at the clinical study
8 yield alone we had 2 cases that I showed you out of
9 approximately 0.7 million donations for a yield of
10 1/350,000. If we look at the continuing site data,
11 which I also showed you for the 3 cases in
12 approximately 1 million donations, the yield is
13 similar, 1/330,000.
14 Well, what does this mean in perspective
15 of what we are already doing in blood banking to
16 protect the nation's safety of the blood supply?
17 The yield for HBV minipool-NAT is approximately
18 equal to the yield for hepatitis C minipool-NAT and
19 the yield for HBV minipool-NAT is far greater than
20 the yield for HIV minipool-NAT.
If we look at this data
that was presented
1 by Mike Busch in Paris of this year, this
2 emphasizes what I have just stated, that for HBV
3 the yield is approximately 1/340,000, based upon
4 his presentation. For hepatitis C, 1/230,000, but
5 for HIV, 1/3.1 million. Although these aren't
6 quite comparable, West Nile Virus was included for
7 because there aren't a plethora of antibody and
8 antigen tests available yet implemented for West
9 Nile Virus but that yield is 1/5,000.
11 This brings us to a sensitivity and
12 specificity calculation. You can see how we
13 assigned HBV status, on the screen here, for
14 positives, negatives, the HBV status unknown for
15 those that were surface antigen negative but core
18 So, again, in summary, here are the totals
19 and the results of the initial testing. These are
20 the potential surface antigen false-positive
21 results. These are the potential window cases.
This last row I have gone into detail on, the row
1 above, the 4 and the 3 will be going into the
2 secondary objective.
4 The final status determination is here, on
5 the fir right-hand column. The ones that could not
6 be determined are yellow, for those of you in the
7 back of the room. The green is HBV positive. The
8 red is HBV negative. The window cases that were
9 proven in the clinical study are shown here as 2.
10 The remaining 21, which will be accounted for in
11 sensitivity and specificity calculations, were
12 counted as HBV positive but final status HBV
13 disease negative.
15 To that point, our specificity
16 calculations, if you look at the total number of
17 subjects for which we have data--the difference
18 between the numerator and the denominator is the 21
19 cases that I just spoke about a second ago to yield
20 a specificity calculation of 99.9964 percent, with
21 the ranges noted on your screen. For sensitivity
we looked at the positive AmpliScreen minipool HBV
1 results in specimens with positive HBV status and
2 divided by the total number of specimens with
3 positive HBV status. Again, these calculations are
4 for HBV minipool-NAT alone and the sensitivity here
5 is 89 out of 105 or 84.7 percent, with a range of
6 74.4 to 91 percent.
8 So, the primary objective conclusion is
9 that COBAS AmpliScreen HBV test has identified
10 individuals in pre-seroconversion window period
11 that would otherwise have been undetected by the
12 blood banking system. The COBAS AmpliScreen HBV
13 test is suitable for blood screening with minipool
14 strategies presented today, and these data indicate
15 that HBV minipool-NAT will increase the blood
18 Let's turn now to the secondary objective,
19 and that is consideration for the HBV minipool-NAT
20 and anti-core for replacing surface antigen and
1 The index donations of interest for this
2 discussion then would be those that were anti-core
3 negative/ surface antigen positive and we will
4 discuss those first. Those donors fitting this
5 category were 7 out of the total number for which
6 we have data, or 0.0012 percent. Those that had
7 core-negative/surface antigen positive HBV DNA
8 negative were 3. Of those 3, 1/3 enrolled and
9 converted to anti-core. The other 2 remaining
10 donors declined the follow-up study. These were
11 determined to be HBV final status positives. For
12 DNA negatives, that is DNA negative, core negative,
13 surface antigen positive donors, 2 out of these 4
14 enrolled in follow-up. Two of the remaining
15 declined to enroll in follow-up but we do have
16 additional data that has been available and I will
17 make available and show to you today. All 4 of
18 these were assessed to be HBV negative. Let's look
19 at some of those details.
21 This first subject was HBV DNA negative,
surface antigen positive, core non-reactive. On
1 follow-up this subject remained core non-reactive
2 throughout the follow-up study. On surface antigen
3 the subject has non-reactivity on follow-up, and
4 HBV DNA remained negative, as did the other
5 follow-up indicators.
7 This would indicate a case of an initial
8 surface antigen test which did not repeat on
9 follow-up, and all other negative serology results
10 indicate this patient had an initial surface
11 antigen positive that is consistent with
12 contamination or carryover.
14 The next case is a little bit more
15 interesting. This case is surface antigen positive
16 again on index donation, HBV negative, core
17 non-reactive. It should be noted that the surface
18 antigen was positive and repeat reactive throughout
19 follow-up but that the surface antigen was negative
20 on neutralization for all of these testing time
21 points. The donor also states a history of HBV
vaccination. Of note, the core
never did react
1 throughout the course of follow-up and HBV DNA was
2 negative throughout the course of follow-up. The
3 anti-surface antibody was positive and that we
4 attribute to his vaccination status, and the repeat
5 reactive, again, was negative on neutralization
7 In summary for this case, the anti-surface
8 antibody is explained by the donor's vaccination
9 history. And, the persistent surface antigen EIA
10 repeat reactivity, which is negative on
11 neutralization, combined with all the other
12 negative results such as anti-core, is consistent
13 with a false-positive surface antigen result due to
16 We have additional data for the 2 donors
17 who declined the follow-up protocol. Of the
18 remaining 2 donors, 1 donor retested 1 month
19 following his index donation and was negative for
20 surface antigen, anti-core and anti-surface
21 antibody. The other donor was retested a year and
half post index and was negative for surface
1 antigen and for core antibody.
3 This slide may look somewhat familiar.
4 This is a look at the total HBV infected, HBV
5 surface antigen positive specimens. In our study
6 there were 103 specimens fitting that
7 categorization. If we look at sensitivity from
8 that perspective alone, HBV detected 87 of the 103
9 specimens and was negative on 16, for a sensitivity
10 as a stand-alone of 84.5 percent in this clinical
11 study. However, the proposal of HBV minipool-NAT
12 plus anti-core antibody yielded positive in all 103
13 of 103 HBV positive cases and missed none of the
14 cases, for a sensitivity of 100 percent. This
15 parallels the non-clinical data shown to you
16 earlier today by Dr. Herman.
18 What are the secondary objective
19 conclusions? All 105 HBV positive donors were
20 identified by anti-core and HBV minipool-NAT
21 combined. These 105 include 2 window case donors
that would have been missed by anti-core and
1 surface antigen combined, and 103 surface antigen
2 positive cases that were identified by the HBV
3 minipool-NAT and anti-core as an alternative.
4 These data then suggest that HBV minipool-NAT
5 combined with anti-core provide an alternative to
6 combined surface antigen and anti-core screening.
8 In summary conclusion today, I offer that
9 the COBAS AmpliScreen HBV test improves the safety
10 of blood transfusion. I offer that COBAS
11 AmpliScreen HBV test combined with anti-core may be
12 considered as replacement for surface antigen
13 combined with anti-core. This is supported by the
14 clinical trial data, and also supported by the
15 non-clinical trial data, specifically
16 seroconversion panel data shown here today and the
17 other HBV positive clinical specimens shown here
18 today by Dr. Herman.
20 With that, I would like to acknowledge the
21 blood centers that participated in this large
study, as well as the Roche Molecular personnel.
1 Thank you.
2 DR. NELSON: Thank you very much.
3 Questions or comments? Yes. Harvey?
4 DR. KLEIN: On the two window period cases
5 that you detected with HBV alone, what was the
6 antigen assay that was used for those?
7 DR. FRANK: In the two in the clinical
8 study, the first window period case was Ortho Test
9 System 2, and all remaining four window cases that
10 were presented were Abbott Auzyme.
11 DR. KLEIN: And, do you by any change have
12 any data with head-to-head comparisons with PRISM?
13 DR. FRANK: I don't believe we ran this
14 test against any test that was not approved by the
16 DR. KLEIN: Even in Europe?
17 DR. HERMAN: We analyzed the
18 seroconversion panels against the PRISM and the
19 Ortho and I can show that data if you would like to
20 see it. That was the European version of the PRISM
21 test that has the Paul Ehrlich Institute approval.
Do you want to see that data?
1 DR. NELSON: Yes, I think so.
3 DR. HERMAN: These are the same 40
4 seroconversion panels that I showed you previously
5 and the same NAT results but compared to the Paul
6 Ehrlich Institute licensed Abbott PRISM test, and I
7 guess this is the lot number. Again, these are
8 sorted by the results with the multiprep test on
9 samples that were diluted 1:24-fold. Those are the
10 orange bars. The blue bars represent the results
11 with the standard test.
13 With the multiprep method DNA was detected
14 an average of 14 days prior to antigen, and with
15 the standard method DNA was detected 19 days prior
16 to antigen.
18 This slide has the summary. Compared to
19 the European PRISM test, with the multiprep method
20 DNA was detected prior to antigen in 34 of the 40
21 panels, and with the Ortho DNA was detected prior
to antigen in 38 of the 40 panels.
On the other 6
1 panels DNA and antigen were detected in the same
2 bleed. Using the standard method compared to the
3 PRISM test, DNA was detected prior to antigen in 38
4 of the 40 panels, and on the same bleed in the
5 other 2 panels.
6 DR. NELSON: Thank you.
7 DR. HOLLINGER: I just have a question.
8 We talk about the mean and the median between the
9 appearance of one antigen for nucleic acid before
10 the other one. But these are not tests that are
11 done every day so the panels do not include--and to
12 say 14 days or 19 days is a little misleading I
13 think in some cases because theoretically if you
14 draw blood today and you drew one 200 days from
15 now, and that is all you had, you would say, well,
16 it would detect things 200 days before one was
17 positive and one was negative. So, you know,
18 unless samples are collected every single day for a
19 period of time, this is somewhat misleading. So,
20 give me some feeling about the days between each
21 one of these sample collections.
22 DR. HERMAN: It
varies between the panels.
1 So, the only data that I have available on the
2 slides here is the one panel I showed you where the
3 interval between bleeds I think was as few as a few
4 days and as large as 30 or 40 days.
5 DR. HOLLINGER: It is not your fault in
6 doing this; it is just that, unfortunately, that is
7 how panels are set up. But I think all of us have
8 to understand that it may be much less than that.
9 It won't be more than that but it certainly could
10 me much less than that.
11 DR. HERMAN: That is absolutely correct,
12 and that is a limitation of these studies so one
13 shouldn't interpret this to conclude that if
14 testing was done every day these would be the
15 numbers. This is an approximation, subject to the
16 limitations of the interval between the bleed dates
17 and the different panels.
18 DR. NELSON: Actually, often
19 epidemiologists would assume a median--a mid-point
20 seroconversion between the two days. So, if you
21 had a sample that was taken on day zero and day 14,
you would assume that the one that the one that was
1 positive on day 14 seroconverted on day 7. That
2 would be one way, a different analysis of the data
3 than probably what you have done.
4 DR. EPSTEIN: Could I get you to clarify
5 whether the apparent window period samples
6 themselves were tested with HBsAg assays with known
7 sensitivities of 0.1 ng/ml?
8 DR. HERMAN: Let me defer to my
9 colleagues. The window period cases from the
10 clinical study?
11 DR. EPSTEIN: Correct, and you say you had
12 five of them. Right? Two from the trials that
13 analyzed sensitivity and specificity and the
14 additional three--you have five available samples
15 that could have been retested with the most
16 sensitive available HBsAg assays.
17 DR. STRONG: I have to declare my conflict
18 here because I am one of the clinical test sites,
19 but we have been able to test four of the five.
20 The most recent one hasn't been tested, but the
21 four have been tested on the Ortho system 3, the
new licensed test. It picked up
one of the four
1 but missed three.
2 DR. KUEHNERT: Could you clarify what
3 those are? You said five.
4 DR. STRONG: The five window cases that
5 are described here. The first four were tested and
6 it picked up the one that you would expect it to
7 pick up, which is the high copy number sample. The
8 fifth sample, which hasn't been tested, also has a
9 relatively high copy number and you might expect it
10 to be picked up as well, with 37,000 copies.
11 DR. NELSON: Are there blood donor
12 policies or recommendations for the interval
13 between receiving hepatitis B vaccine and donation?
14 Because that is a source of a false-positive
15 infection for the surface antigen. Are there any
16 policies on that?
17 DR. EPSTEIN: FDA has no policy for donor
18 deferral after HBV vaccination. I am not certain
19 whether AABB has a voluntary policy, but I think
20 for non-live vaccines they would also not recommend
21 deferring. Someone else would need to clarify
1 DR. KLEINMAN: I think that is right, Jay,
2 except that there have been some instances where
3 people have had false-positive surface antigen so
4 it is sort of unspoken that you might want to defer
5 people for a week or so, but there is no absolute
6 AABB requirement to do that.
7 DR. LEITMAN: Can I ask a different
8 question. I am not sure I am following what is
9 going on in the 16 donors who were HBsAg positive,
10 anti-core positive but multipool-NAT, COBAS
11 negative. Were those individuals individual donor
12 NAT-negative? In other words, are they infectious
13 or are they not infectious? If they are not
14 infectious, do they just happen to have a lot of
15 HBsAg particles circulating but not infectious
17 DR. FRANK: I have to apologize, I can
18 only hear parts of the question because of where I
19 am standing.
20 DR. LEITMAN: I don't think I can do that
21 again. The 16 donors who were HBsAg positive,
anti-core positive but NAT-multipool negative, were
1 they retested in individual donor NAT to see if
2 they have low level viremia or DNA nucleic acid, or
3 are they, in fact, true HBsAg positive but not
4 infectious because they don't have intact virion
5 but just have particles circulating?
7 DR. FRANK: You are talking about these
9 DR. LEITMAN: Those 16, yes.
10 DR. FRANK: The final status here is that
11 they were HBV positive. The question you are
12 posing is are these 16 patients infectious. They
13 have surface antigen positivity and core
14 positivity. I have to defer to my colleague as to
15 whether or not we tried to quantitate with NGI the
16 HBV minipool NATs.
17 DR. PIETRELLI: I am Larry Pietrelli, from
18 Roche Molecular Systems. Yes, on those 16 donors a
19 sample was supposed to be tested by alternate NAT
20 and also by individual donation. One donor was not
21 tested by either assay. Of the 15 remaining, 10
were positive by ID-NAT, of which 7 sere also
1 positive by alternate NAT. Two were negative. One
2 was not done. Of the 5 that were negative by
3 ID-NAT, 2 were positive by alternate NAT; 2 were
4 negative; and 1 was not tested. The viral
5 concentration was low, as expected.
6 DR. LEITMAN: Thank you.
7 DR. KUEHNERT: I have a follow-up question
8 about those 16. They were core antibody positive.
9 Is there any way to quantitate that level of core
10 antibody positivity? Were any of these at an
11 equivocal level? I am just sort of getting at, you
12 know, whether you had greater numbers whether some
13 of these might have been close to a threshold for
14 missing them by core antibody positivity.
15 DR. FRANK: I don't have that data. I
16 don't know whether my colleagues have access to
17 that data today.
18 DR. PIETRELLI: No, we did not collect
19 that information but we could go back to the sites
20 to see what the S to CO was.
21 DR. HOLLINGER: I have a technical
question. Is there are a reason
you used 24,000
1 times g for pelleting? I know others have used
2 40,000 times g for pelleting for an hour at the
3 enhancement stage, so to speak. Also, why did you
4 use 24 samples in your minipool or multipool?
5 DR. HERMAN: I can answer the first
6 question about the centrifugation. It is a
7 limitation of the instrument that is widely
8 available, which is a table-top centrifuge. It is
9 not quite an ultracentrifuge so it is more
10 convenient and easier to use. That was a choice of
11 what equipment would be the most suitable for the
12 multiprep procedure for all three virus targets.
13 DR. HOLLINGER: And the other question?
14 DR. HERMAN: The choice of pool size, I
15 will defer to Mike.
16 DR. STRONG: The pool size is one that was
17 selected for the HIV, HCV trials which were
18 concluded a couple of years ago and were licensed.
19 So, it was just to continue with the logistical way
20 in which laboratories operate to be consistent with
21 the other testing, and it is what the software does
1 DR. NELSON: It is interesting that there
2 are many places, in the Orient particularly, where
3 core antibody testing is not done because of very
4 high rates of positivity. So, that would cast some
5 limitation. Even though the NAT assay would be
6 useful under those circumstances, it wouldn't
7 perhaps be as useful as continuing using the core
8 antibodies as is done in the U.S. and Europe. You
9 had a question, Donna?
10 DR. DIMICHELE: Thank you. I was just
11 wondering if anybody can estimate, based on these
12 data or based on your data, what the residual
13 risk--in using your assay, if your assay was to be
14 implemented with core antibody testing, what do you
15 estimate the residual risk of transmission of HBV
16 would be through transfusion?
17 DR. FRANK: That is a good question, and I
18 can say that we have shown the yield today but in
19 terms of discussion of residual risk, Dr. Kleinman,
20 would you like to comment?
21 DR. KLEINMAN: Yes, Steve Kleinman. I am
consultant to Roche Molecular Systems on this
1 project. The residual risk estimate is dependent
2 upon what you think the risk is now prior to
3 initiating HBV NAT. We have estimated that risk
4 through the REDS study on two occasions, one that
5 was published in 1996 and then we kind of confirmed
6 the same numbers about six years later as about
7 1/60,000. The risk estimate is based on the
8 incidence window period model and the data for that
9 model with HBV are not as strong as they are for
10 HIV and HCV. So, other people have estimated the
11 residual risk now prior to NAT as being lower than
12 1/60,000, being about 1/200,000.
13 At any rate, if you accept the 1/60,000
14 risk estimate, and then you take a look--the
15 question is how much does this new assay decrease
16 the window period. If you draw on data that was
17 published by Robin Biswas and colleagues on their
18 panel testing of decrease of the window period, you
19 get about a 10-day decrease of the window period in
20 that study, and in this study it looks like the
21 window period decreases by 17 to 20 days based on
the panel. But, as Dr. Hollinger
1 is a maximum case because you don't have closely
2 spaced samples.
3 At any rate, if you take a 10-day
4 lessening of the window period and you apply it to
5 what we think the current window period is, about
6 59 days, and you work this through with the window
7 period and the incidence of HBV, you would probably
8 get that the residual risk is still 1/200,000 to
9 300,000 even with this test being implemented.
10 Actually, it may even be 1/100,000. So, you are
11 removing some of the risk but because you are only
12 cutting the window period back 10 days out of a
13 potential 59 days, the likelihood is that most of
14 the risk still remains even with minipool-NAT and
15 that in order to decrease that risk further you
16 have to go to either smaller minipools or
17 individual donation NAT, which would buy you more
18 in terms of lowering the window period. So, you
19 are making an incremental step in decreasing the
20 risk; you are not decreasing the majority of what
21 we think is the residual risk.
22 DR. NELSON:
These data are heavily
1 dependent on what the window period is, which may
2 vary in different populations.
3 DR. STRONG: Yes, they are dependent on
4 the incidence and what you think the current window
5 period is, and that is something that there hasn't
6 really been good data on. We have estimated the
7 current window period as 59 days based on some
8 older work from the TTVS study and then, through
9 REDS, we made a revised estimate of the window
10 period through mathematical modeling to be about 45
11 days now, the infectious window period. So, these
12 are estimates. We don't really know what the
13 current window period is so it is a little bit
14 difficult. We don't know exactly how much we
15 shortened it so that is why I think you have to say
16 that we can't make the kinds of precise estimates
17 that we have made about HCV and HIV.
18 DR. DIMICHELE: Can I just ask you a
19 follow-up question then? What is the
20 estimate--let's say given that residual risk, what
21 is the estimate of the impact of vaccination,
widespread vaccinations now going on in the younger
1 population with respect to when they become blood
2 donors? Does anybody know how the risk of
3 transmission will be decreased just by vaccination,
4 widespread vaccination alone?
5 DR. KLEINMAN: I don't know the answer to
6 that. I will answer kind of a related question,
7 which is what is the risk to recipients? As more
8 recipients get vaccinated, then they should be
9 immune to a challenge, you would think
10 theoretically even if they were exposed. However,
11 most of our recipients are in the older age group,
12 60 and above, and some have compromised immune
13 systems. So, I don't think that vaccination is
14 going to protect much of the recipient population.
15 Your question is different, whether it makes donors
16 less likely to acquire new HBV infection and I
17 don't know that anybody has worked that through
19 DR. ALTER: Harvey Alter, NIH. The
20 mathematical models have been extremely useful but
21 there has always been a little discomfort with HBV
in that you just don't see the cases as frequently
1 as you would have predicted from the model.
2 It seems to me now that if you make the
3 assumption that the real risk would be coming from
4 HBV DNA positive donors, you could actually
5 determine what the actual risk is now by taking the
6 best or maybe all the different HBV DNA assays and
7 testing these samples as single donations. If you
8 are picking up 1/350,000 now in a minipool format
9 you will pick up even more in an individual donor
10 format. That theoretically would be the risk. If
11 we can exclude those donors, that risk would be
12 removed. It seems too simplistic to be true but I
13 would like to hear a counter argument.
14 DR. BUSCH: Mike Busch. I wanted to
15 comment on the vaccine issue. In this study, I
16 think of the yield cases one of the yields that is
17 in the original clinical trial, clearly by history
18 vaccinated person, had an anamnestic anti-surface
19 response, low viremia in the setting of the index
20 sample. I think one of the additional yield cases
21 also had anti-surface on index. It is interesting
that the data from Taiwan is showing that when they
1 have applied NAT to donor screening--this is a
2 massively vaccinated population--they are seeing as
3 these young people, you know, enter adolescence and
4 in their 20s, they are seeing a fairly high rate of
5 the same kind of phenomenon. These are essentially
6 vaccine breakthrough infections. So the vaccine,
7 rather than being sterilizing--still people are
8 getting exposed and infected and, in a sense,
9 boosted from a wild exposure. In their setting the
10 vast majority of their NAT yield are people who
11 have been vaccinated and have anti-surface and
12 never develop surface antigen, never develop
13 chronic infection. So, the critical question of
14 whether these could transmit I think is still on
15 the table.
16 DR. STRONG: Another point to follow-up on
17 what you were commenting on, on the value of
18 anti-core, I think this clinical trial also
19 demonstrated, as has been published, a small yield
20 of DNA positives from the anti-core positive group,
21 roughly in the same area of 5.5 percent or
thereabouts. Also, I think it
1 difference in specificity with the anti-core assays
2 between the two licensed tests. So, there is
3 clearly a large number of false-positive anti-core
4 donors that we have excluded on the basis of
5 false-positive reaction. I think those of us who
6 have been involved in this trial, we have been
7 anxious to try to push the sensitivity of the assay
8 to allow us to reenter a lot of those donors
9 because it is a very large number.
10 DR. HOLLINGER: I just want to follow-up
11 briefly on what Mike said. I mean, this shouldn't
12 really surprise us very much, if you look at most
13 of the vaccine trials and follow patients for ten
14 years or so you will see anti-core appearing in
15 these patients. I just mentioned that anti-core is
16 a marker of viral replication. So, somewhere along
17 the line these patients will probably have DNA in
18 the bloodstream. The issue is whether it is
19 infectious or not and that is, of course, I think a
20 very critical issue with it.
21 Then I just wanted to ask a question
either of Harvey or Steve about how many cases are
1 recorded now of transfusion-transmitted hepatitis
2 B? I don't see very many. I don't want to get
3 back to this argument we had about 20 or 30 years
4 ago when we said there was no hepatitis C or
5 non-A/non-B hepatitis when really there was a lot.
6 DR. KLEINMAN: No, I think that is exactly
7 right, Blaine. There are very few post-transfusion
8 hepatitis B cases reported. As you know, it is a
9 question about whether those people become
10 symptomatic. So, there may be asymptomatic cases.
11 Some may go on to be chronic carriers. That is
12 certainly possible. Secondarily, even if a case
13 occurs, is it recognized by the clinician?
14 It is certainly a correct statement that
15 we don't see the number of HBV clinical cases
16 reported as post-transfusion cases as the
17 mathematical models would predict. So, then you
18 have two potential explanations. One, the
19 mathematical models are wrong and these cases don't
20 occur or, two, the cases occur and they are not
21 recognized and reported. I don't think we have a
way to sort out which one of those hypotheses is
1 correct. Harvey's point is that we would have to
2 take a different approach to estimate risk, that
3 is, we would have to use the most sensitive DNA
4 assay available, hoping we could pick up one
5 copy/ml or even less. By that, we could basically
6 make the equivalence that if we have a DNA-emic
7 specimen that equates to an infectious specimen,
8 and we could do that kind of study that was done 15
9 years ago or I guess 17 or 18 years ago for HIV,
10 but that would be an extremely laborious and
11 expensive study to do.
12 DR. NELSON: I think we will take a break
13 at the moment and then come back for the open
14 public hearing. Maybe 20 minutes.
15 [Brief recess]
16 DR. NELSON: The meeting is now in session
17 again and we will start with an open public hearing
18 on the topic presented of HBV DNA testing. Before
19 we start I will read this statement.
20 Both the FDA and the public believe in a
21 transparent process for information gathering and
decision making. To ensure such
1 the open public hearing session of the advisory
2 committee meeting, FDA believes that it is
3 important to understand the context of an
4 individual's presentation.
5 For this reason, FDA encourages you, the
6 open public hearing speaker, at the beginning of
7 your written or oral statement to advise the
8 committee of any financial relationship that you
9 may have with the sponsor, its product and, if
10 known, its direct competitors. For example, this
11 financial information may include the sponsor's
12 payment of your travel, lodging or other expenses
13 in connection with your attendance at the meeting.
14 Likewise, the FDA encourages you at the beginning
15 of your statement to advise the committee if you do
16 not have any such financial relationships. If you
17 choose not to address this issue of financial
18 relationships at the beginning of your statement,
19 it will not preclude you from speaking.
20 With that, Mike Busch?
21 Open Public Hearing
22 DR. BUSCH:
1 I wanted to just briefly touch on three
2 items. One is just one slide sort of summarizing
3 the different context of some of the issues that
4 were raised in the questions as to the value of HBV
5 minipool-NAT both relative to the other NAT systems
6 and ID-NAT. Then in most of my comments I want to
7 focus on the issue of can we drop either anti-core
8 surface antigen and, if not or if the data doesn't
9 support it at this point, what further studies
10 would be useful to address that. I have Roche's
11 computer here--
12 DR. NELSON: That sounds like a conflict
13 of interest!
15 DR. BUSCH: The committee does have this.
16 This is a published analysis of cost-effectiveness
17 and I am not going to talk at all about sort of
18 cost-effectiveness but I just wanted to show one
19 slide that sort of emphasizes the relevant clinical
20 impact of these three viruses. We tend to talk
21 numbers as if each of these viruses is equivalent
and the NAT yield is equivalent, but what this is
1 showing is the number of quality life years lost
2 per transmission.
3 What you can see is that for HIV it is
4 about 7 quality life years to the average 60 years
5 old transfusion recipient. Transmission of HIV
6 would cost that person 7 quality life years; HCV,
7 0.6 and HBV, only 1.6. This is because most
8 recipients are older people who naturally resolve
9 the acute infection of HBV, and even if chronic it
10 rarely evolves to significant morbidity/mortality
11 over the persistent life span of the average
12 recipient. So, you can see that essentially HBV is
13 one-quarter as clinically important as HCV and 44
14 times less important than HIV.
15 The other point I wanted to make has to do
16 with this issue of going from current serologic
17 testing to minipool versus serologic testing to
18 ID-NAT, and how much of the yield that would be
19 accomplished by going to ID-NAT is detected by
20 minipool-NAT. For HIV we had very sensitive
21 antibody tests in place so NAT by minipool closes
the window by about 5 days and ID closes it by
1 another 5. So, we picked up about 50 percent of
2 what could be picked up but the risk is very low.
3 With HCV, because of the long plateau
4 phase, 95 percent of the window period is detected
5 by minipool-NAT. With HBV, because of the slow
6 ramp-up, at least the model estimates and some of
7 the empiric data like in the Biswas paper would
8 suggest that minipool is only picking up about 23
9 percent or a quarter of what could be detected by
11 Next slide. The issue of anti core and
12 could we get rid of anti core--there are three
13 studies that have been either fully published or in
14 abstract form, actually the Roche data, that have
15 looked at high numbers of donations that were
16 anti-core only reactive--so surface antigen
17 negative, anti-core positive--and subjected then
18 HBV NAT. Each of these studies, as you can see,
19 has given almost identical rates both on a per
20 anti-core and, if you extrapolate, on a
21 per-transfused unit basis of detecting DNA in
donations that were surface antigen negative. So,
1 about a quarter to a half percent in all these
3 What is interesting is that in each of
4 these studies there was quantitation done in one
5 context or another, and all of these viremic
6 anti-core reactive units, surface antigen negative
7 have very low viral loads. In Roche's data, for
8 example, 92 percent based on previously presented
9 work of their viremic anti-core only were negative
10 by minipool but only detected by ID-NAT. So, these
11 are very low viral loads that really would require
12 ID-NAT to detect.
14 This is just a summary and I am not going
15 to walk through this but with respect to low
16 viremic anti-core reactive donations or anti-core
17 only's there are a number of studies that have
18 documented transmissions, rare but there are
19 transmissions from these low viremic units. Again,
20 I don't have time to go into these but we also know
21 that in liver transplantation from anti-core
reactive, surface antigen negative donors very
1 frequently transmits the virus. So, these people
2 who have low viremia do harbor infectious virus
3 and, obviously, the anti-surface status is a
4 factor. But I personally don't think there is any
5 prospect for dropping anti core.
7 Next slide. This was alluded to earlier.
8 There was one study where purposeful transfusion of
9 these anti-core reactive, DNA positive, surface
10 antigen negative units into chimps was done. This
11 was by Fred Prince and was published. These did
12 not transmit. Three patients who had remote
13 hepatitis B, persistent anti-core, negative
14 antigen, low-level DNA. However, the caveat is
15 that all three of these donors had anti-surface and
16 the volume inoculated into chimps was quite small,
17 1 ml. So, I think larger studies with larger
18 volumes may be indicated to potentially explore the
19 infectivity of these units.
21 Next slide. In terms of dropping antigen,
which I think is the more likely and I believe
1 optimistically we will get there, I think clearly
2 for these viruses like HIV, HCV, HBV where there is
3 not only the acute phase but there are chronic
4 carriers and some of these chronic carriers have
5 low-level infectious viremia the optimal paradigm
6 really is the combination of a front-end sensitive
7 direct assay and a serologic test for antibody.
8 For HBV we have both antigen and NAT which could be
9 viewed as redundant, analogous to P24 antigen in
10 HIV NAT. Clearly, on the front end we know, and
11 saw from Roche and other studies, that HBV NAT is
12 clearly more sensitive than antigen during the
13 acute phase.
14 Next slide. But the problem is, as Blaine
15 summarized, HBV is really an unusual virus in that
16 it produces in chronic infections amazingly high
17 levels of circulating antigen in the absence of
18 infectious particles. Therefore, this excess
19 antigen can occasionally be detected in the absence
20 of detectable DNA by minipool or even ID-NAT. I
21 want to present a recent study on that. Therefore,
to really be sure that we are not taking a step
1 back and dropping antigen when we have NAT, we need
2 to do large studies and I think Roche's data is
3 very impressive but whether it is enough is for the
4 committee to decide. But these studies need to
5 look at these issues in a different context,
6 obviously, the front-end window period anti-core
7 negative as well as chronic carriers. But also I
8 think they need to reflect populations with
9 different endemnicity and different routes of
10 transmission; populations that have been vaccinated
11 because we are, as I mentioned earlier, seeing
12 vaccine breakthrough infections, and how would they
13 play out in terms of antigen versus DNA; and also
14 genotypes and mutants.
15 Next slide. Just to contrast, one of the
16 big differences in acute phase versus chronic is
17 the relationship between the DNA load and the
18 antigen reactivity. This is one of a number of
19 studies. This is a Japanese study. During this
20 acute phase these were all anti-core negative
21 front-end infections. They were actually all
detected, 181 units detected as negative by the
1 Japanese particle agglutination antigen test.
2 Actually, 105 of these were reactive by the PRISM
3 assay. So, only a subset, about 40 percent of
4 these remained negative. But what you are seeing
5 here is a very nice linear regression relationship
6 between the reactivity in the antigen test and DNA
7 load. That is typical of the front-end acute
8 viremic phase.
9 Next slide. I want to summarize now a
10 paper that is in press in collaboration with Mary
11 Kuhns and Steve Kleinman from the REDS group, where
12 we took 200 antigen positive, anti-core positive
13 donations in REDS. We first subjected them to
14 quantitative HBV DNA using the Amplicor Roche
15 assay. This assay has a sensitivity of 400 copies,
16 and 64 percent of the antigenic units had DNA above
17 400 copies. Those samples that were negative, the
18 72, were taken through a more sensitive assay that
19 had a sensitivity of about 65 copies, which is
20 probably comparable to minipool type NAT, and 12
21 remained negative even at that level. Those 12
samples were taken to a very high sensitivity,
1 essentially 1 copy/ml assay, high volume, and still
2 there were 6 that were negative. So, we ended up
3 with still 3 percent negative even after taking it
4 through sequential increasing sensitivity assays.
5 So, these are the chronic carriers in whom DNA is
6 undetectable with at least one time point and one
7 high sensitivity assay.
8 Next slide. This is showing the
9 relationship between antigen reactivity and viral
10 load. Unlike that nice linear relationship, here
11 there it is essentially a scatter plot. These are
12 the 3 that were negative by all NAT. These are the
13 ones that were detected only by the high
14 sensitivity. These are the Amplicor negative but
15 single input, not as sensitive, positive. And,
16 these are the quantifiable. You can see that
17 relationship is not observed in chronic carriers.
18 Next slide. That is people who have
19 anti-core. I think Roche did a nice job of sort of
20 saying that if we keep anti-core the issue is not
21 that problem with anti-core positives, antigen
positives having absence of DNA.
It is really are
1 there donations that are antigen positive that lack
2 anti-core that are truly infectious and not
3 detectable by NAT?
4 In the REDS program and in other studies
5 varying 1-5 percent of antigen positive donations
6 lack anti-core. Now, when these are worked up most
7 of these are window phase seroconverters so they
8 have high-level DNA very consistent with acute
9 infection. But in a proportion of DNA negative,
10 and we saw those cases in Roche, the issue is
11 studying enough of those cases to sort out are they
12 true HBV infections, that either there is a
13 mutation not detected by NAT or individuals who
14 have failed to form anti-core, chronic carriers who
15 have low-level DNA and have failed to form
16 anti-core. Do they lack contamination, which I
17 think most of them are? Are they persistent,
18 non-specific antigen reactivity, and Roche showed
19 us an example of that? Or, are they possibly
20 recent vaccinees?
21 Next slide. This is three studies, two
published papers and some unpublished data from
1 REDS that have looked for these people who are
2 antigen positive, anti-core negative and either
3 lack or have very low-level DNA. I am not going to
4 go through these in detail but in Japan one study
5 identified three such donors who had no history of
6 vaccination and who remained low-level PCR-reactive
7 with only a very high sensitivity enhanced assay;
8 remained anti-core non-reactive, so never
9 converted; and, again, remained antigen positive
10 and very low-level PCR-reactivity for over a year.
11 The French group, Couroce's group reported
12 two donors from endemic countries as well, with 0.1
13 percent of their antigen-positive donors who had
14 this pattern of persistent antigen without
15 anti-core and very low-level HBV DNA, and no
16 history of vaccination or immunosuppression and no
17 evidence of mutations to explain the failure of
18 this person to form anti-core.
19 In REDS--this is a cross-sectional study
20 so we weren't able to do follow-up, but we had 20
21 antigen-positive donors who lacked anti-core.
Sixteen of these were positive for DNA and were
1 probably typical acute phase, but 4 were negative.
2 Three of these were probably contaminations. They
3 had no other serologic markers, probably represent
4 surface antigen carryover. But one had anti-e and
5 could represent an atypical carrier.
6 So, just a caution that there are some
7 studies, particularly from endemic settings where
8 there may be people who have antigen in the absence
9 of anti-core and low-level or absent DNA, and I
10 think we just need to study these further.
11 Next slide. Just in conclusion, I do
12 think that we would see a small incremental yield
13 by minipool-NAT but the clinical impact of that I
14 think needs to be considered as well as the yield,
15 and we also need to view the context of what would
16 we get were we to get all the way to ID-NAT.
17 I am optimistic that we will eventually be
18 able, with ID-NAT or very small pools or high
19 sensitivity HBV DNA, to get rid of antigen,
20 however, I think this is going to require further
21 studies. Particularly, we are capturing units
daily that could be worked up to accrue large
1 numbers with high volume of the plasma components
2 from these various sort of atypical patterns.
3 Particularly relevant is that I do think the
4 combination of NAT anti-core is the ultimate goal.
5 Particularly relevant are these samples that are
6 antigen positive, anti-core negative, and really
7 both studying these samples and particularly
8 enrolling and following the donors, as Roche did,
9 to determine whether these are false positives or
10 contamination. Thank you.
11 DR. NELSON: Thank you. Comments? Yes,
13 DR. KLEIN: Mike, we heard from Dr. Alter
14 earlier that, with the problems in trying to
15 calculate what the residual risk is, it might be
16 reasonable as a start to do a study simply by
17 taking 400,000, 600,000 specimens and using a
18 single unit detection system along with the
19 serology. I would just like to know what your
20 opinion on doing that kind of a study might be.
21 DR. BUSCH: Yes, I think that is a good
idea. I mean, we have the Biswas
1 comparing assays on window period panels neat
2 versus minipool. That is kind of these model
3 estimates that minipool picks up only a quarter of
4 what might be detected neat. But, yes, I think
5 such a large-scale study either with neat--you
6 know, one of the issues with Roche's system is
7 their multiprep high extraction volume assay--we
8 saw the data that was comparing the multiprep pool
9 of 24 versus the standard prep neat, but had they
10 applied their high sensitivity multiprep neat,
11 which is what was done in the Biswas paper, they
12 would have seen a greater incremental closure than