Standardization: Calibration of. International Standards, reference preparations and working standards. Micha Nübling, PEI

Transcription

1 Standardization: Calibration of International Standards, reference preparations and working standards Micha Nübling, PEI

2 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

3 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

4 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

5 Standardization of NAT assays WHO International NAT Standards * lyophilised preparations of biologicals * high concentration of target * gold standard for secondary standards and working reagents

6 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)

7 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.

8 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

9 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

10 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

11 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

12 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 IU/ml geq/ml freeze dried (0.5 ml)

13 Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study * Calibration against WHO standard * Assignation of unitage (IU/ml) * Suitability for OMCLs plasma pool testing

14 Biological Reference Preparation HCV-RNA (PhEurBRP) Collaborative calibration study 26 Participants (Europe, USA) OMCLs (9) plasma derivatives manufacturers (11) diagnostic labs (6)

15 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

16 Biological Reference Preparation HCV-RNA (PhEurBRP) WHO IS PhEur

17 #positive/#trials separate regression lines: test (x) standard (+) PCR Assay (logit method) for data in: euro.hcv[lab == 1, -1] WHO IS 10 5 IU/ml PhEurBRP Estimated potency: IU / ml log10(dose) PEI, Tue Mar 9 15:04:

18 #positive/#trials separate regression lines: test (x) standard (+) PCR Assay (logit method) for data in: euro.hcv[lab == 17, -1] WHO IS 10 5 IU/ml PhEurBRP Estimated potency: IU / ml log10(dose) PEI, Tue Mar 9 15:10:

19 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 IU / ml for PhEurBRP (independant from inclusion of non-parallel curves)

20 0 2 4 frequency histogra 19* 17* Mean Potency: IU / ml = IU / ml * potency [

21 HCV-RNA (geq/ml) Ph. Eur. BRP HCV-RNA Stability at Different Storage Temperatures C C 100 RT C Weeks

22 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?

23 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

24 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

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26 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

27 #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) x 0.95 x x % + x in-house HCV-NAT x 0.5 x x Pr95: =21.3 IU/ml Pr95: = IU/ml log2(dose) PEI, Tue Sep 15 15:58: