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 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

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 CT CV

RangeCT

Median CT

30 35.408 0.908 0.169 29/29 100.0 33.235 38.016 0.026 4.780 35.236

15 36.306 0.910 0.178 26/27 96.3 34.487 37.886 0.025 3.399 36.245

7.5 37.326 0.917 0.205 20/30 66.7 35.908 39.405 0.025 3.497 37.299

3.75 37.968 0.951 0.224 18/30 60.0 36.387 39.691 0.025 3.304 38.087

Cut-off: 40 Cycle Threshold

Results of 3 Sensitivity Panels tested before amplification of donor samples

Sensitivity Panel Testing

33

34

35

36

37

38

39

40

Uni

ts

30 IU/mL, Parvo Ct 15 IU/mL, Parvo Ct 7.5 IU/mL, Parvo Ct 3.75 IU/mL, Parvo Ct

Sensitivity Panel Results

CT ≤45 CT > 45, Undetected

Internal Control

NEGATIVE INVALID

TESTING ALGORITHM

CT: Cycle Threshold

REACTIVE

CT≤4040 <CT ≤45

PARVO B-19 PCR

CT > 45, Undetected

INDETERMINATENEGATIVE

CONFIRMATORY QUANTITATIVE TESTING , in duplicate

Parvo-B19 Control Chart

25.00

27.00

29.00

31.00

33.00

35.00

37.00

39.00

41.00

43.00

0 5 10 15 20 25 30 35 40 45 50 55 60

B-19

Ct Mean

Mean+2SD

Mean-2SD

Control Chart, 100 IU/mL

Mean+/-2SD = 33.36+/-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

<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%) 0Total 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