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Parvovirus B19 and HAV Screening of Whole Blood Donations

Parvovirus B19 and HAV Screening of Whole Blood Donations. SL Stramer, KL Kane, ML Beyers, RY Dodd, American Red Cross and RIF Smith, National Genetics Institute (NGI) AABB, 2001 Presented at FDA NAT Workshop, December 2001 and modified for

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Parvovirus B19 and HAV Screening of Whole Blood Donations

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  1. Parvovirus B19 and HAV Screening of Whole Blood Donations SL Stramer, KL Kane, ML Beyers, RY Dodd,American Red Cross andRIF Smith, National Genetics Institute (NGI) AABB, 2001 Presented at FDA NAT Workshop, December 2001 and modified for Blood Products Advisory Committee, December 12, 2002

  2. Background – 1 • Manufacturers of plasma derivatives have implemented NAT for nonenveloped viruses and such testing will likely be implemented for recovered plasma • Most parvovirus B19 (B19V) NAT programs target the elimination of units ³1 x 106 copies/mL • Studies of HAV and B19V frequencies in recovered plasma are limited. Dodd et al., 1997 (AABB) from screening pools of 512 (NGI) reported: • 0:20,000 for HAV RNA • 7:10,000 for B19V DNA

  3. Background – 2 • Three-year experience at Vitex for NAT screening final product (2,500 donations, NGI) for HAV or minipools of 100 for B19V (in-house) report: • 1:476,000 for HAV RNA • »1:800 for B19V DNA • ARC has obtained units from positive subpools of 20 from positive minipools of 100 for identification/characterization of B19V-positive units • Of >1000 units tested from 72 positive subpools, only 23 tested B19V positive at NGI (from 16 subpools) • 77% (56/72) were false positive minipool results

  4. Background – 3 Quant. and Ab Levels in B19V(+) Units Copies/mL IgM* IgG* <100 – + 100 – + 200 – + 14,000 + + 54,000 + + 430,000 + + 920,000,000 – – * Biotrin

  5. Background – 4 Quant. Levels in 23 B19V(+) Units Copies/mL <100-2 14,000 2,000,000 100-4 54,000 3,200,000 200-4 96,000 290,000,000 300 160,000 300,000,000 450 430,000 920,000,000 1,150 5 of 16 pools contained multiple low level positives suggestive of contamination

  6. Background – 5 Therefore, we know that: • HAV is infrequent • B19V NAT false positivity may be common • Low level B19V DNA positive, IgG positive samples occur • Individuals with early acute B19V infection have high viral titers

  7. Unlinked study to determine HAV/B19V frequencies in recovered plasma HIV/HCV NAT-neg, seroneg surplus plasma in PPTs ® NGI 2 primers for HAV and B19V, each tested in duplicate at NGI 512,000 donations pool (Tecan Genesis) 100 x 512 pools testundilute test1:1000 dilution* testundilute – + HAV B19V + + • Resolve to individual donation • Quantify • B19V IgG, IgM (Biotrin) Methods * Estimated 95% sensitivity = 1.2 x 107 copies/mL

  8. Results – 1 • 0:100 pools HAV + = 0:51,200 frequency • 3:100 pools B19V + = 1:12,800 frequency@ 1:1000 dilution4+ donations Copies/mL IgM IgG IgG IU/mL 2.6 x 105 *+ – <310 1.0 x 105 *– – <310 4.4 x 109+ – <310 1.7 x 1011– – <310 * Reactive in same diluted pool

  9. Results – 2 • 34:97 pools B19V + = 1:528 frequencywithout dilution95+ donations No. of Samples Copies/mL IgM IgG IgG IU/mL 3 2.9 x 106 1 + +/– (510) 9.2 x 104 1 + 1 + (1,600) 1.2 x 104 1 + 1 + (4,800) 60 102-103 34 + 34 + 26 – 26 + 14 <102 9 – 9 + 2 – 2 – 3 + 3 + 18 QNS N/A N/A

  10. Parvovirus B19 PrevalenceUsing Gen-Probe Assay • 2,547 pools of 16 (April-May 2002 collections, N=40,752) tested at Gen-Probe using a combination B19V/HAV NAT assay • 23 (0.9%) pools of 16 repeat reactive and disc. B19V NAT reactive; no reactives for HAV • Assume one B19V-pos donation/reactive pool of 16 = 23/40,752 = 1:1,772 versus 1:12,800 for NGI study (7-fold higher) • Product loss, due to discard of all members of a reactive pool of 16 = 1:111 (unacceptable) • Based on distribution of quant. results of 23 positive pools, the addition of a 1:1,000 predilution would result in a prevalence comparable to NGI (3/40,752 = 1:13,584)

  11. Parvovirus B19 Titers for23 Gen-Probe Positive Samples (Copies/mL at NGI for Reactive Pools of 16) 100 200-2 300-2 350 3,700 4,100 5,200-2 5,600 6,300 7,100 8,100 9,500-2 9,900 10,000 13,000 17,000 1,300,000 51,000,000 55,000,000

  12. HAV PCR Test Results onManufacturing Pools of Plasma • 3,250-L mfg. pool (11,500 donations) • Time period covered: 05/08/01 - 11/12/02 • 512 pools tested • 6 (+3 pending) positive pools identified • If initial test is reactive, retest two independent samples • If either of two retests is reactive, consider positive and discard pool • Frequency • 6 (9) pos. HAV RNA mfg. pools per 512 mfg. pools tested =1 pos. HAV RNA sample per 981,333 (654,222) donations • Estimated titers of positive samples £1 x 105 to ³1.1 x 107 copies/mL

  13. Conclusions • Blood collectors considering implementation of B19V screening will have to evaluate NAT methods that are relatively insensitive to prevent issues from contamination and detection of low level NAT positives • 1:12,800 frequency using insensitive method • 1:25,600 high-titer, acute viremic donors, IgG-negative • 1:528 frequency using sensitive method • 1:17,000 moderate-titer, IgG+/–, IgM+ • 1:539 low-titer, IgG+, IgM+/–

  14. Conclusions • High-titer screening methods may not capture all infectious B19V-positive units; however, infectivity of Ab-reactive, low-titer positives is unknown • This study defines expected yields of B19V if sensitive and insensitive NAT methods are used • This study also demonstrates the infrequent occurrence of HAV in recovered plasma • 1:476,000 to 1:981,333 million donations

  15. Strategies for HAV/B19V NAT for Plasma for Further Manufacturing Phase I – Outsource testing; process time exceedsthe dating of labile components Identify individualNAT tubes correspondingto recovered plasma Pools of 16(recoveredplasma only) NGI pools to 512and tests by: HAV UltraQual™2000 RT PCR and Parvovirus B19UltraQual™1000 PCR(Following a1:1000 dilution) Following completionof HIV-1/HCV NAT Plasma for Mfg. Neg Resolve to RxPool of 16 Pos Discard all in-date frozen products(no FFP will exist)

  16. Strategies for HAV/B19V NAT for Plasma for Further Manufacturing Phase II –HAV/B19V Testing In-House (commercial kit) • “Real-time” pool testing (pool size TBD) • Reactives resolved to individual donation • No product release unless HAV/B19V NAT neg • B19V sensitivity level initially set for the removal of high-titer units (³106 copies/mL) for plasma only • No “claims” for labile products • Determine needs for recipients of labile products • Donor notification, management of products from NAT-Rx donors’ previous donations and recipient tracing TBD • Timeline dependent on regulatory policy/availability of test kit

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