A preliminary report on the Leukocyte Antibody Prevalence Study (LAPS-I)

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 Abprevalence by number of pregnancies and interval from last pregnancy • Study is not powered to detect differences in HLA Abprevalence 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™ Alloantibody PE anti-IgG Antigen Luminex Bead

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. Overview of 2006 REDS-II Donation Database • 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

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 enrollmentto 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 of4-6 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