1. E0202728A 1 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 Blood Products Advisory Committee, December 12, 2002

2. E0202728A 2 Background – 1 uManufacturers 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  1 x 10 6 copies/mL uStudies 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. E0202728A 3 Background – 2 uThree-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 uARC 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 results77% (56/72) were false positive minipool results

4. E0202728A 4 Background – 3 Copies/mLIgM*IgG* <100 –+ 100 –+ 200 –+ 14,000 ++ 54,000 ++ 430,000 ++ 920,000,000 –– Quant. and Ab Levels in B19V(+) Units * Biotrin

5. E0202728A 5 Background – 4 Copies/mL <100-214,0002,000,000 100-454,0003,200,000 200-496,000290,000,000 300160,000300,000,000 450430,000920,000,000 1,150 Quant. Levels in 23 B19V(+) Units 5 of 16 pools contained multiple low level positives suggestive of contamination

6. E0202728A 6 Background – 5 Therefore, we know that: uHAV is infrequent uB19V NAT false positivity may be common uLow level B19V DNA positive, IgG positive samples occur uIndividuals with early acute B19V infection have high viral titers

7. E0202728A 7 Methods uUnlinked study to determine HAV/B19V frequencies in recovered plasma  HIV/HCV NAT-neg, seroneg surplus plasma in PPTs  NGI u2 primers for HAV and B19V, each tested in duplicate at NGI 512,000 donations pool (Tecan Genesis) 100 x 512 pools test 1:1000 dilution* B19V test undilute HAV ++ –Resolve to individual donation –Quantify –B19V IgG, IgM (Biotrin) –+ test undilute * Estimated 95% sensitivity = 1.2 x 10 7 copies/mL

8. E0202728A 8 Results – 1 u0:100 pools HAV+=0:51,200 frequency u3:100 pools B19V+=1:12,800 frequency @ 1:1000 dilution 4+ donations Copies/mLIgMIgGIgG IU/mL 2.6 x 10 5 * +– <310 1.0 x 10 5 * –– <310 4.4 x 10 9 +– <310 1.7 x 10 11 –– <310 * Reactive in same diluted pool

9. E0202728A 9 Results – 2 u34:97 pools B19V+=1:528 frequency without dilution 95+ donations No. of SamplesCopies/mLIgMIgG IgG IU/mL 32.9 x 10 6 1 + +/–(510) 9.2 x 10 4 1 +1 +(1,600) 1.2 x 10 4 1 +1 +(4,800) 6010 2 -10 3 34 +34 + 26 –26 + 14<10 2 9 – 9 + 2 – 2 – 3 + 3 + 18QNSN/AN/A

10. E0202728A 10 Parvovirus B19 Prevalence Using Gen-Probe Assay u2,547 pools of 16 (April-May 2002 collections, N=40,752) tested at Gen-Probe using a combination B19V/HAV NAT assay u23 (0.9%) pools of 16 repeat reactive and disc. B19V NAT reactive; no reactives for HAV uAssume one B19V-pos donation/reactive pool of 16 = 23/40,752 = 1:1,772 versus 1:12,800 for NGI study (7-fold higher) uProduct loss, due to discard of all members of a reactive pool of 16 = 1:111 (unacceptable) uBased 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. E0202728A 11 Parvovirus B19 Titers for 23 Gen-Probe Positive Samples (Copies/mL at NGI for Reactive Pools of 16) 100200-2300-23503,7004,1005,200-25,6006,3007,1008,1009,500-29,90010,00013,00017,0001,300,00051,000,00055,000,000

12. E0202728A 12 HAV PCR Test Results on Manufacturing Pools of Plasma u3,250-L mfg. pool (11,500 donations) uTime period covered: 05/08/01 - 11/12/02 u512 pools tested u6 (+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 uFrequency –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 10 5 to  1.1 x 10 7 copies/mL

13. E0202728A 13

14. E0202728A 14 Conclusions uBlood 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-negative1:25,600 high-titer, acute viremic donors, IgG-negative –1:528 frequency using sensitive method 1:17,000 moderate-titer, IgG+/–, IgM+1:17,000 moderate-titer, IgG+/–, IgM+ 1:539 low-titer, IgG+, IgM+/–1:539 low-titer, IgG+, IgM+/–

15. E0202728A 15 Conclusions uHigh-titer screening methods may not capture all infectious B19V-positive units; however, infectivity of Ab-reactive, low-titer positives is unknown uThis study defines expected yields of B19V if sensitive and insensitive NAT methods are used uThis study also demonstrates the infrequent occurrence of HAV in recovered plasma –1:476,000 to 1:981,333 million donations

16. E0202728A 16 NGI pools to 512 and tests by: HAV UltraQual ™ 2000 RT PCR and Parvovirus B19 UltraQual ™ 1000 PCR (Following a 1:1000 dilution) Strategies for HAV/B19V NAT for Plasma for Further Manufacturing Phase I – Outsource testing; process time exceeds the dating of labile components Following completion of HIV-1/HCV NAT Identify individual NAT tubes corresponding to recovered plasma Pools of 16 (recovered plasma only) Neg Plasma for Mfg. Pos Resolve to Rx Pool of 16 Discard all in-date frozen products (no FFP will exist)

17. E0202728A 17 Strategies for HAV/B19V NAT for Plasma for Further Manufacturing u“Real-time” pool testing (pool size TBD) uReactives resolved to individual donation uNo product release unless HAV/B19V NAT neg  B19V sensitivity level initially set for the removal of high-titer units (  10 6 copies/mL) for plasma only –No “claims” for labile products –Determine needs for recipients of labile products uDonor notification, management of products from NAT-Rx donors’ previous donations and recipient tracing TBD uTimeline dependent on regulatory policy/availability of test kit Phase II –HAV/B19V Testing In-House (commercial kit)