Standardization: Calibration of International Standards, reference preparations and working standards Micha Nübling, PEI
Paul-Ehrlich-Institut Governmental Authority for biological medicinal products Blood and blood products Assessment of IVDs for CE marking (NB) IVD batch release testing WHO CC: IVDs + Blood Products
Standardization of NAT assays WHO Working Group SOGAT Standardisation of Genome Amplification Techniques for the Safety Testing of Blood, Tissues and Organs for Blood Borne Pathagens
Standardization of NAT assays WHO Working Group SOGAT Representatives from......official medicinal control labs (OMCLs)...plasma manufacturers...in vitro diagnostic industry...regulators...universities, diagnostic labs
Standardization of NAT assays WHO International NAT Standards * lyophilised preparations of biologicals * high concentration of target * gold standard for secondary standards and working reagents
Standardization of NAT assays NAT standards: Desired Features * suitable for all NAT assays: PCR, TMA, NASBA, branched DNA,... * reflect measuring range of quantitative NATs * reflect target material * comparability with conventional IVDs, e.g. HIV: RNA / p24 * one unitage (IU / ml)
Standardization of NAT assays WHO NAT International Standards (WHO IS) * definition of candidate materials * collaborative study defining mean titre estimate * assignation of (arbitrary) unitage (IU / ml) * stability studies (real-time, elevated temperatures) * report to WHO Expert Committee on Biol. Stand.
Standardization of NAT assays WHO International Standards Target Concentration 1 IU = code NIBSC HCV RNA 10 5 IU/ml 3-8 cps 96/798 HBV DNA 10 6 IU/ml 2-7 cps 97/746 HIV-1 RNA 10 6,1 IU/ml 0.5 cps 97/650 B19 DNA 10 6 IU/ml 0.8 cps 99/800 HAV RNA 10 5 IU/ml >10 cps 00/560
Standardization of NAT assays WHO / (NIBSC / PEI) Genotype / Subtype Reference Panels Target Geno-/Subtypes HCV RNA 1, 2, 3, 4, 5, 6 HBV DNA HIV-1 RNA B19 DNA A, B, C, D, E, F (in preparation, PEI WHO CC) A, B, C, D, AE, F, G, AG-GH, N, O 1, 2, 3a, 3b (in preparation, FDA, NIBSC WHO CCs) HAV RNA
Secondary standards Working Reagents * to control individual runs e.g. Assay Run Control: 3 x 95% pos c/o * to control sensitivity requirements testing) e.g. 100 IU HCV-RNA/ml (plasma pool * calibrated against WHO standards, if available
Secondary standards Biological Reference Preparation for HCV-RNA (PhEurBRP) Background CPMH requirement for plasma pool HCV-NAT OMCLs confirm manufacturer s testing (batch release) Need for a common reference material
Biological Reference Preparation HCV-RNA (PhEurBRP) WHO IS (96/790) BRP Candidate Material HCV-RNA pos plasma HCV genotype I antihcv pos antihcv neg diluted in neg pool 100.000 IU/ml 4.000-10.000 geq/ml freeze dried (0.5 ml)
Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study * Calibration against WHO standard * Assignation of unitage (IU/ml) * Suitability for OMCLs plasma pool testing
Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study 26 Participants (Europe, USA) OMCLs (9) plasma derivatives manufacturers (11) diagnostic labs (6)
Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study Protocol * 4 vials of candidate material and WHO IS * routine NAT * dilution series (0.5 log10) around end point * statistical evaluation, potency calculation * independant from methodical sensitivities
Biological Reference Preparation HCV-RNA (PhEurBRP) 10-1 10-1.5 10-2 10-2.5 10-3 10-3.5 WHO IS + + + + + + + + + + + + + + + + + + - + + - - - PhEur + + + + + + + + - + - + - - + - - - - - - - - -
#positive/#trials separate regression lines: test (x) standard (+) 1 0.95 PCR Assay (logit method) for data in: euro.hcv[lab == 1, -1] 0.75 0.5 0.25 WHO IS 10 5 IU/ml PhEurBRP Estimated potency: 10 3.55 IU / ml 0-6 -5-4 -3-2 -1 0 log10(dose) PEI, Tue Mar 9 15:04:02 1999
#positive/#trials separate regression lines: test (x) standard (+) 1 0.95 PCR Assay (logit method) for data in: euro.hcv[lab == 17, -1] 0.75 0.5 WHO IS 10 5 IU/ml PhEurBRP Estimated potency: 10 2.85 IU / ml 0.25 0-6 -5-4 -3-2 -1 log10(dose) PEI, Tue Mar 9 15:10:31 1999
Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study Results * 26 valid data sets * 23 par allel curves, 3 non-parallel curves * mean estimated potency 10 2.97 IU / ml for PhEurBRP (independant from inclusion of non-parallel curves)
0 2 4 frequency 6 8 10 histogra 19* 17* 18 14 Mean Potency: 10 2.97 IU / ml = 1.000 IU / ml 11 2510 15 21247 26 8 9 223 13* 235 204 122 166 1 2.2.42.62.83.03.23.43. potency [
HCV-RNA (geq/ml) Ph. Eur. BRP HCV-RNA Stability at Different Storage Temperatures 10.000-20 C 1.000 4 C 100 RT 10 1 37 C 0 10 20 30 40 50 60 70 80 90 Weeks
Secondary standards more recent calibration studies: preferential use of quantitative NATs relative quantitation of WHO IS and candidate material similar relation on different platforms / by different assays?
Secondary standards Ph. Eur. BRP HCV-RNA 10 3 IU/ml Ph. Eur. BRP B19-DNA 10 5,8 IU/ml PEI Ref Prep HCV-RNA 10 4,9 IU/ml PEI-Ref Prep HIV1-RNA 10 4,9 IU/ml PEI Ref Prep HBV-DNA 10 4,9 IU/ml PEI Ref Prep WNV-RNA 10 9,8 cps/ml
Role of WHO IS and Secondary Standards regulations for blood and blood products therapy guidelines standardization of assays proficiency testing schemes calibration of working reagents
Limiting Dilution Estimation of NAT-titers, copies, genome equivalents infectious noninfectious Probability for a negative test result: p (-) = e -cv c = virus concentration v = test volume
#positive/#trials separate regression lines: test (x) standard (+) Detection limits of different HCV-NATs PCR Assay for data in: (limiting dilution of WHO standard hcv.pcrhcv-rna, Probit analysis) 1 + + x 0.95 x x 0.75 63 % + x in-house HCV-NAT x 0.5 x 0.25 + 0 + x Pr95: 2 4.41517 =21.3 IU/ml Pr95: 2 7.36196 = 164.5 IU/ml 0 2 4 6 8 log2(dose) PEI, Tue Sep 15 15:58:32 1998