Transmissibility of B19 by transfusion: evaluation of donor viremia prior to performing a linked donor-recipient study M Busch, representing REDS-II Parvo B19 protocol team

RADAR Repository Established from in seven US cities NHLBI and CDC funded Full enrollment for recipients included a pre (or peri) tx specimen and a follow-up specimen at months RADAR units (mostly RBC) were targeted to surgical patients on specific clinical services at specific hospitals Enrolled recipients got both RADAR and non- RADAR units

Contents of RADAR repository Donation specimens –Linked to fully enrolled recipients (n=13,201) –Unlinked (n=99.906) Recipients –Fully enrolled (n=3575) –Initially enrolled but without f/u (n=1402) There are two plasma aliquots (1.8 ml in first, up to 1.8 ml in second) and one 1.5 ml frozen whole blood aliquot for each specimen

Parvovirus B19 study hypotheses Phase 1:To determine the rate of low, moderate, and high titre viremia and its coexistence with IgM and IgG antibody in blood donors. Assay development Donor viremia study Recipient seroprevalance (for susceptibility) Statistical evaluation Phase 2: To determine the rate of B19 transmission by DNA-positive units and correlate it with donor viral load and antibody status

Testing methods Qualitative PCR: magnetic bead capture kinetic Taqman assay- originally developed by Chiron and adapted by BSRI Quantitative PCR: exact same assay - run in duplicate on an independent aliquot with quantitative standards on same run Commercial EIAs (Biotrin) for IgM and IgG antibody: run in singlicate and repeated for equivocal results

Parvo, IU/mL Mean CT Std. Dev Std. Error Pos/Total Detected Percent (Pos/Total) Minimum CT Maximum CTCV Range CT Median CT / / / / Cut-off: 40 Cycle Threshold Results of 3 Sensitivity Panels tested before amplification of donor samples Sensitivity Panel Testing

Sensitivity Panel Results

CT ≤45CT > 45, Undetected Internal Control NEGATIVEINVALID TESTING ALGORITHM CT: Cycle Threshold REACTIVE CT≤4040 <CT ≤45 PARVO B-19 PCR CT > 45, Undetected INDETERMINATENEGATIVE CONFIRMATORY QUANTITATIVE TESTING, in duplicate

Mean Mean+2SD Mean-2SD Control Chart, 100 IU/mL Mean+/-2SD = /-2.96; data for 56 plates (107 controls)

PCR results: initial and repeat testing Total tested: 5020 Initially reactive: 113 (2.25%) Initially indeterminate: 26 (0.52%) Initially invalid: 56 (1.12%) Total Completed:4982 Confirmed positive: 44 (0.88%) Indeterminate: 5 (0.10%) Invalids pending conf: 38 Low viremia Prevalence= 0.88%; 95% CI, 0.64% - 1.2%

PCR and antibody results on confirmed positives Viral load #CP No ab IgM and IgG IgG only < (5%) 2 (10%) 18 (86%) 20-< (9%) 2 (18%) 8 (73%) < (56%) 4 (44%) < (100%) 0 Total 44 2(5%) 12 (27%) 30 (68%) Of 23 donations with >20 IU/ML, median was 105 IU/mL, (42-481); highest was 1,869 IU/mL

Exposed Recipients tested for: Enrollment specimens: B19 IgG 6M+ specimens: B19 DNA, IgM and IgG Newly acquired infection probably associated with transfusion of an infected RADAR donor unit = [RADAR + baseline] transmission rate Recipient infected pre- transfusion, not infected, or non- evaluable Non- exposed Recipients Random Sample 230 non-exposed recipients tested for: enrollment and 6M+ specimens: B19 DNA, IgM and IgG Recipient infected pre- transfusion, not infected, or non- evaluable Newly acquired infection not associated with transfusion of an infected RADAR donor unit = Baseline transmission rate RADAR transmission rate = {[RADAR + baseline] transmission rate} – {baseline transmission rate} Tests Interpretation Test all (13,201) RADAR donation specimens linked to recipients for B19 DNA Exposed Recipients Tests Interpretation PHASE II