A multi-centre NAT evaluation study of run and trend control samples Harry van Drimmelen 1, Joe O’Donnellan 2, Rene Bax 1, Henrik Ullum 3 and the Danish NAT study group and Wim Quint 1 1. Biologicals Quality Control Unit, Delft Diagnostic Laboratory (DDL), the Netherlands, 2. Irish Blood Transfusion Service (IBTS), Dublin, Ireland 3. Rigshospitalet, Copenhagen, Denmark.

Background I Recalibration of standards in copies/ml HBV, HCV and HIV-1 analytical and infectivity standards 1,2.3 have been recently recalibrated in copies/ml using multiple bDNA 3.0 assays 4 The recalibrated standards have been used for analytical sensitivity studies 5,6 and TTI risk analysis 7-12 virusCps/IUCps/CID 50 50%-95% LOD range ULTRIOTaqScreen HBV HCV HIV-10.5~ Lelie N. et al. Transfusion 2002:42; Komiya K et al. Transfusion 2008;48: Katayama K et al, Intervirology, 2004, 47, Van Drimmelen A.A.J. Vox Sang 96 Suppl1 ISBT abstract P Assal et al. Transfusion 2009; 49: Assal et al. Transfusion 2009; 49: Weusten J et al, Transfusion, accepted. 8. M. El Ekiaby et al. Vox Sang 96, Suppl1, ISBT abstract; 9. El Ekiaby et al. Vox Sang 96, Suppl1, ISBT abstract; Vermeulen M et al, Transfusion 2009: in press. 11. Kleinman S et al Vox Sang 96, Suppl1, ISBT abstract; 12. Goubran et al. Novartis, ISBT Satellite meeting, Cairo,

Background II Recalibration of standards in copies/ml DDL standard studies WHO standard studies theoretical limit is 1 HIV-RNA copy per TMA assay 63% LOD (cps/assay)

Compare different HBV, HCV and HIV Quality Control standards in a multi-centre validation study of ULTRIO on TIGRIS in Denmark and Ireland Analyze TMA response values on QC standard dilutions to establish suitable run or trend control levels Aim of the study

Methods I Inactivation viral standards (DDL) 1. Lelie PN., Reesink HW, Niessen J., Brotman B and Prince AM. Inactivation of chimpanzee infectious doses of hepatitis B virus during preparation of an heat-inactivated hepatitis B vaccine. J. Med. Virol. 1987:23: Dichtelmüller SW, Prince AM, Brotman B, Huima T. Inactivation of the Hutchinson strain of hepatitis non-A, non-B virus in intravenous immunoglobulin by beta-propiolactone. J Med Virol. 1988: 26: Lelie PN. Reesink HW and Lucas CJ. Inactivation of 12 viruses by heating steps applied during manufacture of a hepatitis B vaccine J. Med. Virol. 1987:23: virusInactivation methodviral reduction RNA, DNA recovery HBV10 hours 65°C, 1:100 diluted in PBS 1 >6 log84% HCV24 hours incubation 37°C with β-propriolacton 2 >3 log100% HIV2 hours 65°C, 1:10 diluted in PBS 3 >5 log62%

Methods III QC samples evaluated NIBSC working reagents Seracare-BBI Accurun range Acrometrix PeliSpy pro range ISS standards DDL bioQControl check and trend controls DDL inactivated standard dilutions

Methods IV Characteristics QC samples SupplierAcroMetrix.SeraCareNIBSCDDL MatrixDefibr. delip. plasma Defib plasma Cryosuper- natant EDTA plasma Inactivation (insert) HBV-DNAHeatNone10h, 65°C HCV-RNAHeatNone β- propiolacton HIV-RNAHeatDefect strain None2h, 65°C QuantificationNo IU/mlcps/ml * Defibrination; removal of cry precipitating proteins

Methods V multi-centre performance evaluation study Five laboratories from Denmark and Ireland tested multiple replicates of QC samples on TIGRIS in validation phase Inactivated viral standard dilutions were tested in multiple replicates (n=52-58) and 95% and 50% detection limits were determined by probit analysis QC samples were tested in 1:3 and 1:10 dilutions (n=30-33) Distribution of ULTRIO S/CO response values on inactivated standard dilutions were analyzed Longitudinal Procleix Duplex S/CO values of IBTS (Ireland) on HCV trend control sample (27 cps/ml) were analyzed

Results I detection limits on inactivated standards virusRepli cates ULTRIO detection limits in cps/ml 95% LOD (CI)50% LOD (CI) HBV58699 ( )55 ( ) HCV5231 ( )3.9 ( ) HIV5220 ( )2.9 ( ) virusStudies, replicates detection limits in cps/ml on native standards (low - high) 95% LOD (L-H)50% LOD (L-H) HBV5, (96-241)28 ( ) HCV4, 8627 (22-35)3.3 ( ) HIV4, 8626 (19-57)3.1 ( )

Proportion of reactive ULTRIO tests (n=33) on diluted run control samples Run controldilutionHBVHCVHIV AcroMetrix PeliSpy Pro388 %100 %91 % AcroMetrix PeliSpy Pro1052 %73%61 % CheckControl 125 cps/ml*1100 % TrendControl 25 cps/ml*593 %94 %91 % NIBSC Working reagent3100 % NIBSC Working reagent1079 %100 %85 % Seracare Accurun series % Seracare Accurun series %85 % ISS standard****100 % ISS standard****97 %94 % * for HBV ULTRIO 1000 and 200 cps/ml ** for HCV 5700 IU/ml diluted to 40 and 12.5 IU/ml and for HIV 4000 IU/ml diluted to 100 and 33 IU/ml

Distribution S/CO results (n=52) for inactivated HCV-RNA standard dilutions cps/ml 116 cps/ml 35 cps/ml 12 cps/ml cps/mlMean S/CO SD saturated

Distribution S/CO results (n=778 runs) on HCV-RNA trend control (27 cps/ml)

Averaged (50) proportion TMA reactive (n=778 HCV Procleix duplex runs) % reactive (mean + 25 and – 25 runs) % saturated (mean + 25 and – 25 runs)

Conclusions and discussion Viral concentration in most QC samples appears to be sufficiently critical for external run controls Run controls should be quantified; consistent concentrations between different batches ULTRIO detection limits on untreated and inactivated viral standards were comparable* A trend control sample of ~25 cps/ml (around the 90-95% detection limit) is more instrumental for longitudinal monitoring of analytical sensitivity of TMA test runs** * For HBV further studies required to prove the correct calibration in copies/ml ** Follow percentage TMA reactive and the distribution of S/CO values over time