1. SoGAT 14-06-2006 June 14, 2006 HCV-RNA and HIV-RNA detection in small test pools of cadaveric samples for viral safety of tissue transplants Maarten Koot#, Theo Cuijpers#, Marco Koppelman#, Mirjam de Waal#, Arlinke Bokhorst*, Noor Holsboer*. # Sanquin Diagnostic Services, Amsterdam; * BIS- Foundation, Leiden, The Netherlands

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

3. Testing tissue donors in test pools of 4 for HCV/HIV-RNA NAT NucliSens extractor (BioMerieux), AmpliScreen HCV test vs 2.0., Ampliscreen HIV test vs 1.5. System for blood screening of Sanquin. 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 ml donor plasma * : HCV-RNA 95 % hit rate 840 geq/ml (520-1760 95% CI) 165 IU/ml HIV-RNA 95 % hit rate 1280 geq/ml (760-3040 95% CI) 806 IU/ml *Calculated on dilution factor based on analytical validation Jongerius et al. Transfusion 42, 792-797 (2002) and Koppelman et al. 89, 193-200 (2005).

4. Validation of testing tissue donors in test pools of 4 for HCV-RNA NAT. Robustness on frozen samples, retrospectively analyzed. 320 non-selected samples from tissue donors. -Acceptable level of invalid results: 0.6 % of the test pools. Responsible individual sample can be traced. -Robust test, 95 % of the pools have a saturated signal for the IC (OD>2.5). Sensitivity. Spiking of representative test pools with 300 geq (58.8 IU) HCV-RNA -Level of 6000 geq/ml (1175 IU/ml) for the individual sample is guaranteed. Nearly all spiked samples saturated wild-type signals.

5. Validation of testing tissue donors in test pools of 4 for HIV-RNA NAT (1). Robustness on frozen samples, retrospectively analyzed: 166 non-selected samples from tissue donors; 36 test pools. Samples frozen as test pool of four. 13/36 (36 %) test pools significantly reduced IC signal (OD<1.5).

6. Validation of testing tissue donors in test pools of 4 for HIV-RNA NAT (2). Sensitivity. Spiking of 27 test pools with 280 geq (176 IU) HIV-RNA. Samples frozen as test pools of four. 5 pools with significantly reduced WT signal (OD<1.5): one invalid, one false negative and 3 IC signal 0.2 and 1.5.

7. On-line analysis of test pools HIV-/HCV-RNA (1) Sampling and centrifugation (asap) in Plasma Preparation Tubes (B&D). Shipment and storage at 4-8 o C. HCV-RNA period (03-2005 / 05-2006) 202 test pools, 808 donors. HIV-RNA period (12-2005 / 05-2006) 166 test pools, 664 donors. No reactive donors for HIV-RNA and HCV-RNA. No confirmed serological pos. donors for HIV and HCV. 4/808 (0.5%) donors invalid result HCV-RNA 3/664 (0.5%) donors invalid result HIV-RNA 3/664 (0.5%) donors invalid as result of HCV/HCV NAT testing.

8. On-line analysis of test pools HIV-/HCV-RNA (2) HCV-RNAHIV-RNA Result test pool Result breakdown Result test pool Result donor Positive WT>0.2 100n.a. Invalid IC<1.5 84*4* 7&7& 3#3# Negative WT<0.2 IC>1.5 193n.a.159n.a. &2 test pools no break down. * 28 donors negative # 17 donors negative

9. Analysis of test pools HIV-/HCV-RNA; Robustness HCV IC<1.5 4 % IC≥1.5 and <2.5 3 % IC≥2.5 93 % Total202 HIV IC<1.5 4 % IC≥1.5 and <2.5 8 % IC≥2.5 88 % Total166 HCV HIV

10. Conclusions Spiking experiments on test pools of 4 with inhibiting substances: Increase of Cut-off value IC signal invalid/negative OD=1.5 Testing in pools of 4 with 50 ul of plasma per tissue donor for HCV-/HIV-RNA yields an acceptable level of invalid results (0.5 %) for the individual donor. The number of false positive results for test pools is low (0.5 % for HCV-RNA, 0 % for HIV-RNA). The NucliSens extractor AmpliScreen HCV/HIV assay performed on test pools of 4 tissue donor is a robust test, 93 % of the pools have a saturated signal for the HCV-IC; 88 % for HIV-RNA. The HCV-/HIV-RNA assay in test pools is applied for routine tissue release since February 2006.