M Busch, representing REDS-II Parvo B19 protocol team

1. 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

2. RADAR Repository • Established from 2000-2003 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 6-12 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

3. 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

4. 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

5. 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

6. Sensitivity Panel Testing Results of 3 Sensitivity Panels tested before amplification of donor samples Cut-off: 40 Cycle Threshold

7. Sensitivity Panel Results

8. TESTING ALGORITHM PARVO B-19 PCR CT > 45, Undetected 40 <CT ≤45 CT≤40 NEGATIVE INDETERMINATE REACTIVE Internal Control CT ≤45 CT > 45, Undetected NEGATIVE INVALID CONFIRMATORY QUANTITATIVE TESTING , in duplicate CT: Cycle Threshold

9. Control Chart, 100 IU/mL Mean+2SD Mean Mean-2SD Mean+/-2SD = 33.36+/-2.96; data for 56 plates (107 controls)

10. 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%

11. PCR and antibody results on confirmed positives Viral load#CPNo abIgM and IgGIgG only <20 21 1(5%) 2 (10%) 18 (86%) 20-<102 11 1(9%) 2 (18%) 8 (73%) 102-<103 9 0 5 (56%) 4 (44%) 103-<104 3 0 3 (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

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