1. SOGAT 25-05-2005 Validation of HCV-RNA detection in small test pools on cadaveric samples Marco Koppelman #, Theo Cuypers #, Mirjam de Waal #, Maarten Koot#, Arlinke Bokhorst*, Noor Holsboer *. # Sanquin Diagnostic Services, Amsterdam; * BIS- Foundation, Leiden, The Netherlands

2. SOGAT 25-05-2005 Tests on blood borne infections for tissue release Bone, ossicle Anti-HIV-1/2 HIV p24 antigen HIV-1 DNA Anti-HTLV-I/II HBsAg Anti-HBc total Anti-HCV HCV-RNA IgG anti-CMV TPPA Tissue: cornea, skin, cardiac valve Anti-HIV-1/2 HIV p24 antigen Anti-HTLV-I/II HBsAg Anti-HBc total Anti-HCV (pos. HCV-RNA) TPPA

3. SOGAT 25-05-2005 HCV-RNA: Bone-tissue and confirmation samples positive in serology NucliSens extractor (BioMerieux), Amplicor HCV test vs 2.0 1 ml plasma input: 95 % hit-rate 60 geq/ml (8.7 IU/ml) 15 % invalid results with neg. IC signal. 0.1 ml plasma input with 0.9 ml negative human plasma: 95 % hit-rate 600 geq/ml (87 IU/ml) #total number of tissues 5188

4. SOGAT 25-05-2005 Tests on blood borne infections for tissue release Bone, ossicle Anti-HIV-1/2 HIV p24 antigen HIV-1 DNA Anti-HTLV-I/II HBsAg Anti-HBc total Anti-HCV HCV-RNA IgG anti-CMV TPPA Tissue: cornea, skin, cardiac valve Anti-HIV-1/2 HIV p24 antigen Anti-HTLV-I/II HBsAg Anti-HBc total (pos. anti-HBs) Anti-HCV (pos. HCV-RNA) HCV-RNA test pools TPPA

5. SOGAT 25-05-2005 Testing tissue donors in test pools of 4 for HCV-RNA NAT NucliSens extractor (BioMerieux), AmpliScreen HCV test vs 2.0. NAT Blood screening for HCV-RNA and HIV-RNA. Tissues: testing in test pools of 4 samples. Input per donor sample 50 ul EDTA plasma. Filling up to 1 ml with 0.8 ml negative human plasma. Sensitivity per donation * : 99 % hit rate 1600 geq/ml (880-4320 95 % CI) 313 IU/ml 95 % hit rate 840 geq/ml (520-1760 95% CI) 165 IU/ml 50 % hit rate 160 geq/ml (120-240 95% CI) 31 IU/ml *Calculated on dilution factor based on analytical validation Jongerius et al. 42, 792- 797 (2002).

6. SOGAT 25-05-2005 Validation of testing tissue donors in test pools of 4 for HCV-RNA NAT. Robustness. 320 non-selected samples from tissue donors of period 6 weeks Composition of 80 test pools -% invalid results, traceability sample(s) responsible for invalid result -% pools with inhibited IC-signal, distribution of IC signals Sensitivity. -Spiking of 26 test pools with 300 geq/ml HCV-RNA and analyses test pools with and without the HCV- RNA spike. 300 geq per extraction = load run control in standard HCV-RNA blood screening

7. SOGAT 25-05-2005 Acceptance criteria testing tissue donors in test pools of 4 for HCV-RNA NAT. Robustness: Percentage invalid results in test pools < 5 %. Invalid test pools breakdown into one invalid donation and three donations with valid result. Total invalid results on tissue level < 2 %. > 90 % test pools OD/CO > 12.5, saturated signal. Sensitivity: Non-spiked test pools negative for HCV-RNA, spiked test pools (300 geq per pool) positive.

8. SOGAT 25-05-2005 Results test pools of 4 tissue donors in HCV-RNA NAT 78 test pools negative for HCV-RNA. 2 test pools invalid results, with negative IC-signal 2 test pools with inhibited IC-signal, one OD/CO ~ 5, one ~ 12.

9. SOGAT 25-05-2005 Retest and breakdown invalid test pools. Two invalid test pools composed once more. Re-test: one test pool invalid for HCV-RNA; the other one inhibited. Breakdown of the two invalid test pools in individual donations, one donor responsible for invalid result.

10. SOGAT 25-05-2005 Wild type and IC signal (OD/CO) test pools spiked with 300 geq/ml HCV-RNA

11. SOGAT 25-05-2005 Conclusions Testing in pools of 4 with 50 ul of plasma per tissue donor yields an acceptable level of invalid results of 2/320 (0.6 %) for the HCV-RNA NAT test in an unselected sample cohort. Test pools of 4 composed in this way yield a robust test, 95 % of the pools have a saturated signal for the IC. Based on the analytical validation, this test has a 95 % success rate for a positive result of 840 geq/ml (165 IU/ml) for samples tested in this dilution. Spiking experiment show that the level of 6000 geq/ml (1175 IU/ml) for the individual sample is guaranteed. Nearly all spiked samples have saturated wild-type signals.