1. EQASs for Blood Borne Virus Genomes and BSE Prions from Cattle Brain and INSTAND Reference Materials H.-P. Grunert 1,2, K.-O. Habermehl 1,2, V. Lindig 1 and H. Zeichhardt 1,3 1 Charité University Medicine Berlin, Campus Benjamin Franklin, Institute for Infectious Diseases Medicine, Dept. Virology, Berlin 2 Institute for Biotechnological Diagnostics (GBD), Berlin 3 Institute for Standardization and Documentation in Medical Laboratory (INSTAND), Düsseldorf (WHO Collaborating Center) Collaborating Centers of International Consortium for Blood Safety (ICBS), New York

2. Grunert, SoGAT, Bethesda, 2005 2 SerologyPCR/NAT HIV-1/2CytomegalovirusHIV-1 (RNA)Enterov. (PCR/NAT) HIV-1 p24Epstein-Barr VirusHepatitis B VirusAdenov. (PCR/NAT) Hepatitis A VirusVaricella Zoster V.Hepatitis C Virus Hepatitis B V. Prg. IHerpes simplex V.Cytomegalovirus Hepatitis B V. Prg. IIMeaslesvirusVaricella Zoster V. Hepatitis C VirusMumpsvirusHerpes simpl. V. 1 Hepatitis D VirusResp. Sync. V. AGHerpes simpl. V. 2 Hepatitis E VirusInfluenzav. A/B AGInfluenzavirus A/B RubellavirusTBE VirusParvovirus B19 BSE (PrPsc)HPV (PCR/NAT) Samples total: > 549.000 National and WHO International EQASs in Immunology/Virology

3. Grunert, SoGAT, Bethesda, 2005 3 EQASs in Virology Virus serology since 1988: > 446,000 samples total (20 programs) Virus genome detection since 1997: > 103,000 samples total (12 programs) OrganizationINSTAND (Düsseldorf) Authorized byBundesärztekammer (BÄK) World Health Organization (WHO) International Consortium for Blood Safety (ICBS) Scientific societiesDeutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) Gesellschaft für Virologie (GfV) Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM)

4. Grunert, SoGAT, Bethesda, 2005 4 Experiences from EQAS for HIV, HCV and HBV: Evaluation of quantitative PCR/NAT by median +/- 0.5 log 10 IU/ml versus copies/ml

5. Grunert, SoGAT, Bethesda, 2005 5 EQAS Nov 2004 HIV sample 10054 median: 1,110 copies/ml; 1,033 IU/ml NAT all [copies/ml] NAT all [IU/ml]

6. Grunert, SoGAT, Bethesda, 2005 6 Quantitative PCR/NAT Confidence Interval: Median +/- 0.5 log * without results for bDNA for samples <200,000 copies/ml (Nov. 1999 - Nov. 2000) & without results for bDNA for samples <3,000 copies/ml (Apr. 2004) without results for bDNA for samples <1,220 IU/ml (Apr. 2004) VirusDateNo. of samples Median (copies/ml) (IU/ml) Number of analyses Results in interval: median +/-0.5 log HIV-1Nov 99-Nov 0435700-21,700349893% (88%-99%) Nov 02-Nov 0416380-14,00010587% (57%-100%) HCV*,& Nov 99-Nov 04323,000-1,351,000111780%*,& (67%-91%) Apr 00-Nov 04291,220-400,000268393% & (85%-100%)

7. Grunert, SoGAT, Bethesda, 2005 7 EQAS Nov 2004 HCV sample 12054 median: 28,838 copies/ml; 10,200 IU/ml N total = 137 N copies/ml = 22 N IU/ml = 115 PCR all [IU/ml] bDNA [IU/ml] PCR all [copies/ml] bDNA all [copies/ml]

8. Grunert, SoGAT, Bethesda, 2005 8 EQAS Nov 2004 HCV sample 12053 median: 3,472 copies/ml; 1,217 IU/ml N total = 136 N copies/ml = 22 N IU/ml = 114 PCR all [IU/ml] bDNA [IU/ml] PCR all [copies/ml] bDNA all [copies/ml]

9. Grunert, SoGAT, Bethesda, 2005 9 Quantitative PCR/NAT Confidence Interval: Median +/- 0.5 log * without results for bDNA for samples <200,000 copies/ml (Nov. 1999 - Nov. 2000) & without results for bDNA for samples <3,000 copies/ml (Apr. 2004) without results for bDNA for samples <1,220 IU/ml (Apr. 2004) # without results for bDNA and hybridization for samples <1,000,000 copies/ml (Nov. 1999 - Apr. 2003) $ without results for hybridization for samples <1,000,000 copies/ml (Nov. 2003 - Apr. 2004) § without results for bDNA for samples <500,000 IU/ml (Apr. 2002 - Apr. 2003) VirusDateNo. of samples Median (copies/ml) (IU/ml) Number of analyses Results in interval: median +/-0.5 log HIV-1Nov 99-Nov 0435700-21,700349893% (88%-99%) Nov 02-Nov 0416380-14,00010587% (57%-100%) HCV*,& Nov 99-Nov 04323,000-1,351,000111780%*,& (67%-91%) Apr 00-Nov 04291,220-400,000268393% & (85%-100%) HBV #,$,§ Nov 99-Nov 04338,300-1,668,000239979% #,$ (67%-91%) Nov 01-Nov 04211,350-933,00041974% § (55%-92%)

10. Grunert, SoGAT, Bethesda, 2005 10 EQAS Nov 2004 HBV sample 11055 median: 1,500,000 copies/ml; 439,000 IU/ml PCR (Roche Copies); N=29 PCR (Abbott Copies); N=1 PCR (in house Copies); N=3 nested PCR (in house Copies); N=1 Taqman (Roche Copies); N=3 Taqman (in house Copies); N=13 Lightcycler (Artus Copies); N=5 Lightcycler (in house Copies); N=10 bDNA (Bayer Copies); N=5 PCR (Roche IU); N=3 PCR (Artus IU); N=1 PCR (in house IU); N=1 Taqman (Artus IU); N=1 Taqman (Roche IU); N=17 Taqman (in house IU); N=4 Lightcycler (Artus IU); N=12 Lightcycler (in house IU); N=2 other PCR (Artus IU); N=2 bDNA (Bayer IU); N=5

11. Grunert, SoGAT, Bethesda, 2005 11 National External Quality Assessment Schemes (EQASs) for BSE Testing Cooperations: K.-O. HabermehlInst. f. Biotechnologische Diagnostik, Berlin H. ZeichhardtCharité Universitätsmedizin Berlin H.-P. GrunertCampus Benjamin Franklin Institut für Infektionsmedizin, Abt. Virologie M. H. GroschupBundesforschungsanstalt f. Viruskrankheiten U. Zieglerder Tiere, Insel Riems Financial support by:BMBF, BMGes, DVV Performed:since May 2002 Participants:total 40 - 56 Samples total: > 1000

12. Grunert, SoGAT, Bethesda, 2005 12 National BSE EQASs: Summary of the Results Date SampleCorrect Results No.Properties%No. of analyses May 2002 1neg.10057/57 2neg.98,256/57 3pos. (1:12)89,551/57 4pos. (1:6)96,454/56 5pos. (1:4)96,555/57 Jun. 2003 1neg.92,738/41 2neg.92,738/41 3pos. (1:12)10041/41 4pos. (1:8)10041/41 5pos. (1:6)10041/41 Mar. 2004 1neg.10044/44 2neg.10044/44 3pos. (1:16)95,542/44 4pos. (1:12)10044/44 5pos. (1:12)10044/44 6pos. (1:6)10044/44 Feb. 2005 1neg.97,539/40 2neg.97,539/40 3neg.10040/40 4neg.10040/40 5pos. (1:16)97,539/40 6pos. (1:12)10040/40 7pos. (1:12)97,539/40 8pos. (1:6)97,539/40

13. Grunert, SoGAT, Bethesda, 2005 13 2 nd National BSE EQAS June 2003 Variance Analysis of the BioRad Test Results

14. Grunert, SoGAT, Bethesda, 2005 14 Global Network for External and Internal Quality Control External Quality Control (EQASs) EQASs (Germany, Europe, WHO) with >990 laboratories with all routine tests 3x per year (2006: 4x per year) Internal Quality Control Surplus samples from EQASs "Run" controls daily National reference materials "Run" controls in coop. with PEI daily Identical controls for:  Diagnostic labs  Blood banks  Industries  Licensing organizations (e.g. PEI)  Scientific labs

15. Grunert, SoGAT, Bethesda, 2005 15 National Reference Materials VirusPropertiesCopies/ml HIV-1 Subtype B pos. (++)40.000 adjusted to international reference materials and standards (WHO) pos. (+)4.000 HCV Genotype 1b pos. (++)30.000 pos. (+)3.000 HBV pos. (++)160.000 developed in cooperation with PEI and reference laboratories pos. (+)8.000 Negative control neg. 0 Influenza A pos. Bejing/262/95 (H1N1) available from INSTAND and Institut für Biotechnologische Diagnostik (GBD) pos. Sydney/5/97 (H3N2) Influenza B pos. Yamanashi/166/98 Negative control neg.

16. Grunert, SoGAT, Bethesda, 2005 16 INSTAND Reference Materials (www.instand-ev.de) INSTAND INSTITUTE FOR STANDARDIZATION AND DOCUMENTATION IN MEDICAL LABORATORIES e.V. P.O. Box 250211, D-40093 Düsseldorf, Germany Tel.: +49-211-159213 0, Fax: +49-211-159213 30, E-mail: instand@instand-ev.de Samples for Test Assessment of Virus Diagnostics National Reference Samples (National Working References) Surplus Samples of External Quality Assessment Schemes in Virus Immunology and Virus PCR/NAT As of February 2005