1. A preliminary report on the Leukocyte Antibody Prevalence Study (LAPS-I) Retrovirus Epidemiology Donor Study-II (REDS-II) presented by Dr. Steven Kleinman FDA BPAC: April 27, 2007

2. Structure of REDS II Funded in 2004 by NHLBI as a five year study with multiple project areas Participating U.S. Blood Centers: – –Blood Center of Wisconsin – –ARC New England Region – –Emory University/ARC Southeast Region – –University Cincinnati/Hoxworth BC – –Institute For Transfusion Medicine – –UCSF/Blood Centers of the Pacific/BSRI Coordinating Center – Westat Central Laboratory - BSRI

3. LAPS Objectives Determine HLA Class I and II antibody prevalence and correlate with: – –Number of pregnancies – –Lifetime history of transfusion – –Time from last immunizing event – –Baseline group has no evident alloexposure (never pregnant or transfused) Determine antibody specificities Determine prevalence of neutrophil antibodies in those with HLA antibodies and in controls – –More limited in scope due to high cost and low throughput of assays -

4. Study size and statistical power Enrollment of 5100 female donors gives >90% power to detect differences in HLA Ab prevalence by number of pregnancies and interval from last pregnancy Study is not powered to detect differences in HLA Ab prevalence between transfused and untransfused males – –Enrollment of 1000 in each group will give tight confidence intervals around prevalence -

5. LAPS enrollment questionnaire Pregnancy history: National Health And Nutrition Examination Survey (NHANES) validated pregnancy questions – –Six questions including ever pregnant, number of pregnancies (live births, still births, miscarriages, or abortions) and date of last pregnancy Transfusion history – –Have you ever received someone else’s blood – –Date of last transfusion -

6. HLA antibody detection methods Potential assays – –Luminex, Flow PRA – –ELISA – –Lymphocytotoxicity (AHG enhancement) Based on reported higher sensitivity and high throughput, screening assay chosen for LAPS was LABScreen™ on the Luminex platform Supplementary/confirmatory testing with single antigen assay

7. Description of LABScreen ™ Mixed Screening assay uses multiple beads coated with purified HLA antigens: – –Each bead contains purified antigens from 5 or 6 cell lines – –5 Class I and 3 Class II beads with 54 Class I and 32 Class II antigens – –Also 2 separate beads for MICA (MHC class I related antigen) – present on endothelial cells Uses Luminex flow cytometer – –Measures laser based light emission by reagents conjugated to antibody bound to HLA Class I and Class II antigens

8. Principle of LABScreen™ Technology Purified HLA protein Dual-colored bead

9. Basics of LABScreen™ PE anti-IgG Alloantibody Luminex Bead Antigen

10. Supplementary testing with LABScreen ™ single antigen assay Each bead contains one recombinant HLA antigen of a given specificity at the molecular level – –94 Class I and 57 Class II antigens on single beads – –Includes A, B, Bw4/6, C, DR, DQ, DP loci – –All common antigens are represented Each result will undergo review by an external HLA expert and problematic cases will be reviewed by a panel of experts

11. 1.2 million successful donations Includes basic demographic information on donors Includes donation type Additional information obtained from questions on pregnancy history and transfusion history that are not usually part of the donor questionnaire Overview of 2006 REDS-II Donation Database

12. 2006 REDS-II Donation Database: Donation Type by Gender Percent (%)

13. 2006 REDS-II Donation Database: History of Transfusion by Gender Percent (%)

14. LAPS enrollment to date 5978 female donors 1241 untransfused males 749 transfused males with enrollment ongoing Recruitment of first two groups at multiple collection sites, based on logistics and demographic mix Recruitment of transfused males through special mechanisms -

15. Parity History: REDS-II Donors and LAPS Enrollees Percent (%)

16. Status of HLA antibody testing Screening assay completed on ~4700 samples Supplementary single antigen assay testing in progress – –Expert review in progress Data analysis in progress but still needs several months to be completed

17. Preliminary observations While the package insert suggests a particular cutoff (NBG ratio of 2.2), it also states that individual laboratories may need to determine their own cutoffs Data from previous studies and in-house validations on non-alloimmunized populations used to establish cutoffs are much smaller than LAPS

18. Preliminary observations Cutoffs previously used for this assay system were designed to maximize sensitivity for use in an organ transplant setting – –detection of any level of antibody in the transplant candidate is a marker for anamnestic response, leading to potential organ rejection – –in TRALI, it is passively transfused donor antibody that is involved in pathogenesis; low- level antibody may not be important

19. Preliminary observations Using an NBG ratio of 2.2, we are finding HLA antibody in apparently non alloexposed persons (untransfused males and never pregnant females) – –consistent with previous report of Densmore which showed 7.8% by LCT-AHG in small donor population of never pregnant females – –awaiting single antigen assay results – –at higher NBG ratios, this rate decreases HLA prevalence rates in females by parity are also dependent on NBG cutoff

20. Thoughts for consideration It is likely that similar concerns may apply to setting the cutoff in other HLA detection systems It is unclear if antibody with low signal strength is of significance for the safety of transfusion recipients It is unclear how such information should be used to make donor deferral decisions even under a precautionary TRALI risk reduction policy

21. REDS-II LAPS Repository Consists of 4-6 aliquots (0.5mL) of plasma and 2 aliquots each of whole blood and serum (when available) Storage at -70°C Will be accessed for neutrophil antibody testing Can be used for DNA typing for HLA & neutrophil alleles (discriminate auto vs allo antibodies) Can be used for HLA antibody testing using other test systems Linked samples allow for selective donor recall -

22. Ongoing and additional studies LAPS-I – –Analysis of HLA data – –Neutrophil antibody testing – –HLA titering studies – –Analysis of MICA data – –Alternate HLA tests Clinical study: LAPS-II – in planning phase – –Lookback study of the incidence of TRALI in recipients of high plasma volume components from donors with leukocyte antibodies