Experience in Testing of Genotypes of B19

Testing of plasma donation samples B19 Virus Testing NAT Testing Operations Testing of plasma donation samples QC testing of plasma fractionation pools Example Talecris B19 virus testing system: Donation mini pool test - Qualitative NAT assay* Detects genotypes 2 and 3 at high-titer QC test for fractionation pool - Qualitative NAT assay* Investigational test - Quantitative NAT assay** * colorimetric readout ** fluorogenic readout

Assessment of B19 virus test performance B19V Testing at Talecris Biotherapeutics (formerly Bayer Biological Products) NAT development and technical operations activities Assessment of B19 virus test performance Quantitation of all elevated samples (2000-2002) against WHO standard (genotype 1) Tracking and trending of B19 virus unit interdictions Investigational assessment of viral loads in fractionation pools Development of reagents for investigation and technology improvements Archive of positive samples Archive of screened mini-pools

Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Talecris Human B19 virus genotypes - Prevalence Study Purpose To determine the potential prevalence of non-genotypes 1 variants (e.g. genotypes 2 and 3) Design Differential detection of variants using NAT Specific primer and fluorogenic probe combinations to include or exclude detection of genotypes Secondary testing of archived materials previously screened for elevated B19 virus genotypes (including 340 individual reactive samples) Archived samples with reduced B19 virus loads (elevated samples removed) NAT testing using primers and detection probe specific for B19V variants Demonstrated to detect synthetic A6, V9, and D91.1 targets Does not detect B19V genotype 1 below 106 IU/ml

Potential for the Detection of B19V Genotypes during high-throughput NAT Testing Talecris Detection Strategy for B19 genotype 1 vs. variants B19 Type 1 Probe Parvovirus B19 5.6 kb Promoter Structural proteins Non-structural proteins 1348 DST-F DST-R B19 Type 1-F B19Type 1-R B19 Universal-R B19 Universal Variant Probe 1611 Donor Sample Test Quantitative Test

Potential for the Detection of B19V Genotypes during high-throughput NAT Testing Detection using the B19 Universal Variant Probe UVP can detect genotype 2 (A6) and genotype 3 (V9) DNA targets UVP does not detect genotype 1 targets below 106 IU/ml Variant Target WT B19 Stds

Manufacturing-scale plasma sample pools Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Study Materials Manufacturing-scale plasma sample pools Pools containing 3840 donations (40 X 96 donation sample minipools) Combine 40 of the 96-donation sample minipools Samples screened between 2000-2002 high-titer samples removed and analyzed using genotype 1 specific test 960 sample pools Combine 10 of the 96-donation sample minipools Samples screened between 2002-2003

Number of Donations Represented Detection of B19 Virus Genotypes 2 and 3 During High-throughput NAT Testing Study Results Pool Size (samples) Number of Pools Tested Detection Frequency Number of Donations Represented 3840 242 0/242 929,280 960 609 0/609 584,640 Total samples/donations tested without detection of high-titer variants 1,513,920

Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Study Conclusions High-titer units containing B19 virus genotypes 2 and 3 are rare among U.S. source plasma donations None of the study materials contained detectable levels of B19 virus genotypes 2 and 3 Analysis of 340 reactive samples did not identify B19 virus genotypes 2 and 3 The prevalence of B19V genotypes 2 and 3 in the U.S. source plasma supply chain appears to be low If B19 virus genotypes 2 and 3 were present in fractionation pools, levels would have been below the detection limit of the utilized assay

Archived study material was representative of 1,5 million donations Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Comments Standardized material unavailable thus use of synthetic DNAs was required Archived study material was representative of 1,5 million donations To accelerate availability of results archived material collected for other purposes was used

Additional Slides

B19 Virus Genotypes Prevalence Profile of viremia Knowledge from the literature and collaborative communication Prevalence Genotypes 2 and 3 not frequently detected in blood or plasma donations; Hokynar et al., 2004, J Clin Microbiol.; 42(5):2013-9. Genotype 1 (47%) and Genotype 2 (2.5%) detected in batches of clotting factor concentrates; Schneider et al. 2004, Thromb Haemost: 92(4):838-45 Genotypes 2 and 3 identified in diagnostic clinical specimens; Sanabani et al., 2006, J Clin Microbiol. 44(2):604-6; Cohen, Gandhi, and Clewley, 2006 J Clin Virol. ;36(2):152-5. Genotype 3 endemic in Ghana; Parsyan et al., 2006, J Clin Microbiol. 44(4):1367-75. Profile of viremia Persistence of variant genotypes (?) Viral loads associated with variant genotypes (?) Pathogenesis associated with variant genotypes (?)

B19 Virus Parsimony Genotypes 2 and 3 vary ~ 5-15% from genotype 1 Servant et al., 2002 J Virol. 76(18):9124-34.