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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. Structure of REDS II. Funded in 2004 by NHLBI as a five year study with multiple project areas

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A preliminary report on the leukocyte antibody prevalence study laps i l.jpg

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


Structure of reds ii l.jpg
Structure of REDS II Study (LAPS-I)

  • 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


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LAPS Objectives Study (LAPS-I)

  • 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

      -


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Study size and statistical power Study (LAPS-I)

  • 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

      -


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LAPS enrollment questionnaire Study (LAPS-I)

  • 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

      -


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HLA antibody detection methods Study (LAPS-I)

  • 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


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Description of LABScreen Study (LAPS-I)™ 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


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Principle of LABScreen™ Technology Study (LAPS-I)

Purified HLA protein

Dual-colored bead


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Basics of LABScreen Study (LAPS-I)™

Alloantibody

PE anti-IgG

Antigen

Luminex Bead


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Supplementary testing with Study (LAPS-I)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


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Overview of 2006 REDS-II Donation Database Study (LAPS-I)

  • 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


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2006 REDS-II Donation Database: Study (LAPS-I)Donation Type by Gender

Percent (%)


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2006 REDS-II Donation Database: Study (LAPS-I)History of Transfusion by Gender

Percent (%)


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LAPS enrollment Study (LAPS-I)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

    -


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Parity History: Study (LAPS-I)REDS-II Donors and LAPS Enrollees

Percent (%)


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Status of HLA antibody testing Study (LAPS-I)

  • 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


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Preliminary observations Study (LAPS-I)

  • 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


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Preliminary observations Study (LAPS-I)

  • 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


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Preliminary observations Study (LAPS-I)

  • 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


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Thoughts for consideration Study (LAPS-I)

  • 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


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REDS-II LAPS Repository Study (LAPS-I)

  • 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

    -


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Ongoing and additional studies Study (LAPS-I)

  • 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


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