Quantification of HBV, HCV genotype and HIV subtype panels Harry van Drimmelen 1,2, Wim Quint 2, Nico Lelie 3 and the international NAT study group 1. Biologicals Quality Control, 2. DDL Diagnostic Laboratory, the Netherlands, 3 Lelie Research, Paris, France.
Traceability ladder ISO 17511:2003 The metrological traceability chain Level 1: Traceable to SI Levels 2 5: Not traceable to SI Level Traceable to SI Unit Reference measurement method Calibrator Example 1 Yes Yes Yes Electrolytes 2 No Yes Yes HbA1c 3 No Yes No Enzym kinetics 4 No No Yes WHO standard 5 No No No Herpes
Standardisation in nucleic acid copies (SI units) Limiting dilution analysis Real time PCR, Ct comparison True value Evolution over time Competative quantitative tests
Possible drift in IU of WHO replacement standards 1 st standard 2 nd standard same source 3 rd standard 4 th standard True value Evolution over time
Calibration of VQC standard in copies/iu in WHO collaborative study 1 Copies/IU on WHO HIV-1 standard Assay n 1 st 2 nd Drift Abbott real time 15 0.42 0.38 91% Ampl. Mon V1.5 134 0.33 0.43 133% bdna 2.0 69 0.39 0.58 149% NucliSens 1.0 36 0.35 0.19 54% Ampl. Mon Ultra 16 0.43 0.73 170% Overall 270 0.36 0.45 127% 1 Analysis of raw data used for establishing 1st and 2nd WHO HIV-1 subtype B standard by Holmes et al. ( J Virol Methods. 2001;92:141-50)
Why calibration of viral standards in RNA or DNA copies and not only in IU? Traceability to mol/l feasible in bdna 3.0 assay Drift in IU inevitable in WHO replacement standards One HBV-DNA, one HCV-RNA and two HIV-RNA molecules are equal to one potential infectious virus The virus transmission risk in the early ramp up phase starts with one virus transmitted 1 1 Weusten J et al, Transfusion 2011;51:203-15
Available methods for calibration of viral standards in copies/ml Siemens Versant bdna assays, calibrators derived from purified nucleic acid standards quantified by (bio)chemical methods 1 Limiting dilution testing with NAT blood screening assays 2 Comparison of Ct values in TaqScreen s 201 real time PCR assay Three methods available which should ideally harmonise to same concentration 1 Collins ML et al, Analytical Biochemistry, 1995;226:120-29 2 Heermann K-H et al, J Clin Microbiol 1999;37:68-73
Study design Preparation of HBV, HCV and HIV genotype panels calibrated in copies/ml by multiple bdna 3.0 assays and estimation of NAT detection efficiency in limiting dilution analysis multiple HBV, HCV, HIV-1 plasmas of different genotypes 100-fold Gravimetrically controlled dilution Stock solutions (~ 30.000 cps/ml) Cross calibration in repl (6-12x) bdna 3.0 assays Gravimetrical controlled targeted dilution Stock solutions (3000 cps/ml) Gravimetrical controlled dilution Standard dilution panels (300, 100, 30, 10, 3, 1, 0.3 cps/ml) Replicate testing (12-24x) in blood screening NAT assays NAT detection efficiency in probit analysis Determination of 63 % detection limit (95% CI)* and NAT detection efficiency (95% CI)* in Ultrio, Ultrio Plus and TaqScreen * Detection efficiency is 100% at 63% LOD of 1 copy/ml in TaqScreen (1 ml plasma input) and 2 copies/ml in Ultrio (0.5 ml plasma input)
Example: Cross calibration of HBV-DNA genotype standards in multiple bdna assays Genotype Standard n cps/ml 95 % C.I. Genotype standard n Cps/ml 95 %C.I. A2 DDL 30 2.15E9 2.11-2.20 B1 WHO-PEI 12 8.04E6 7.94-9.34 A2 WHO97-746 24 5.33E6 5.29-5.39 B2 WHO-PEI 12 5.22E6 4.90-5.67 A2 Eurohep 18 2.99E6 2.92-3.09 B4 WHO-PEI 12 6.16E4 5.98-6.86 A2 Chimp 6 1.26E6 1.18-1.42 C DDL 6 2.13E9 1.64-2.76 A2 DDL inact 6 7.23E6 7.09-7.42 C Chimp 6 1.85E6 1.75-2.01 A1 WHO-PEI 12.97E06 6.43-7.61 C2 WHO-PEI 12 6.81E6 6.67-7.66 A1 WHO-PEI 12 4.44E6 4.07-4.85 C2 WHO-PEI 12 4.74E6 4.38-4.92 A2 WHO-PEI 12 3.30E6 3.08-3.30 C2 WHO-PEI 12 5.50E6 5.23-5.86 D DDL 6 3.77E9 3.09-4.60 E DDL 6 1.70E8 1.42-2.03 D Eurohep 6 2.60E9 2.51-3.65 E WHO-PEI 12 3.25E6 3.07-3.34 D1 WHO-PEI 12 4.12E6 4.02-4.76 F DDL 6 1.55E9 1.07-2.24 D3 WHO-PEI 12 3.96E6 3.46-4.04 F3 WHO-PEI 12 3.55E5 3.24-3.86 D1 WHO-PEI 12 4.39E6 4.10-4.44 G DDL 6 8.87E6 7.48-10.5 B DDL 6 2.15E9 1.96-2.35 G WHO-PEI 12 5.79E4 5.48-6.14 1 IU = 5.33 copies 1 CID 50 = 3.7 copies
Data sets used for limiting dilution analysis Ultrio EFS France Assal et al, Transfusion. 2009;49:289-300. Margaritis et al, Transfusion. 2007;47:1783-93 Gen-probe Inc. Linnen et al, USA Shabrawishi Hospital Egypt, ISBT Cairo 2009 Blood bank Warsaw Poland, Crabarzcyk et al, ISBT Berlin 2010 Ultrio plus IHTM Poland, Crabarzcyk et al, ISBT Berlin 2010 Gen-probe Inc. Linnen et al, USA NBTC Egypt, ISBT Cairo 2009 Blood bank Munich Ganschow et al, Germany (validation) TaqScreen Assal et al, Transfusion. 2009;49:289-300
100% 95% Poisson distribution Determination of HBV-DNA concentration In Eurohep standard by limiting dilution analysis 1 63% 50% Poisson 1 copy per assay 1 reactive not reactive 1 Heermann K-H et al, J Clin Microbiol 1999;37:68-73
ULTRIO Plus detection efficiency of HIV, HCV and HBV genotypes 200% TMA detection efficiency (95% CI) 180% 160% 140% 120% 100% 80% 60% 40% 20% 0% B B C D AE O 1 1 1 2 3 4 5 6 A A A A B C C D D D E F G HIV-1 subtypes HCV genotypes HBV genotypes The efficiency has been determined on the basis of 63% LOD (CI). At a 63% LOD of 2 cps/ml TMA detects 1 copy per 0.5 ml or 1 copy per assay Data set from P Grabarczyk et al, ISBT, Berlin 2010
TaqScreen detection efficiency of HIV, HCV and HBV genotypes PCR detection efficiency (95% CI) HIV-1 subtypes HCV genotypes HBV genotypes The efficiency has been determined on the basis of 63% LOD (CI). At a 63% LOD of 1 copy/ml TaqScreen detects 1 copy per assay Data obtained from EFS study of Assal et al. (Transfusion 2009; 49:301-310)
NAT detection efficiency on HBV, HCV and HIV geno-,subtypes Marker Assay n studies HBV Average (range) of NAT efficiency N (%) in which 95 % C.I. overlaps 100% efficiency Ultrio 37 14% (4-45 %) 0 (0%) Ultrio plus 49 40% (12-89 %) 5 (10 %) HCV TaqScreen 7 21 % (9-46 %) 0 (0%) Ultrio 37 56 % (8-209 %) 13 (35%) Ultrio plus 23 86 %( 26-188 %) 17 (74%) TaqScreen 6 19% (6-48%) 0 (0 %) HIV Ultrio 24 84 %(35-234 %) 17 (71%) Ultrio plus 19 109% (26-310%) 17 (89%) TaqScreen 4 22 % (18-24%) 0 (0 %)
Summary results of detection efficiency in limiting dilution analysis Ultrio Plus approaches 100% detection efficiency for HIV and HCV and 50-100% for HBV TaqScreen approaches 25% detection efficiency for HIV and HCV and 50% for HBV. There is still variation present between genotypes and laboratories despite using automated testing
Approach for real time PCR Amplification efficiency can be evaluated by plotting 2 log(conc) against Ct value. The slope is then -1. This criterium must be met before Ct value distribution can be evaluated Ct values should be equal for same viral load irrespective of geno, subtype
Ct value HIV-RNA TaqScreen Ct values on HIV group M subtype B standard dilution series 45 Large variation 40 35 30 Slope equals -1.0 indicating PCR efficiency is 100 % low deviation and used for comparison 25 0.1 1 10 100 1000 10000 copies/ml Data set from Assal et al.transfusion. 2009;49:301-310
Comparison potency probit analysis and Ct value on HBV-DNA Genotype Ct value 1000 cps/ml Potency Ct value (95% C.I.) to genotype A potency Limiting Dilution (95% C.I) to genotype A A 29.81 B 30.61 57% (27-121%) 48% (22-101%) C 29.66 111% (43-286%) 89%(42-189%) D 28.45 257% (57-1155%) 189% (86-434%) E 28.24 297% (162-545%) 139% (64-310%) F 31.13 40%(20-80%) 53% (23-112%) G 30.09 82% (40-168%) 39% (17-84%)
Comparison potency probit analysis and Ct value on HCV-RNA Genotype Ct value 1000 cps/ml Potency Ct value (95% C.I.) to genotype 1 potency Limiting Dilution (95% C.I) to genotype 1 1 31.06 2 30.70 128 % (90-182%) 119 % (60-241%) 3 31.02 103 % (66-161%) 175% (89-363%) 4 33.03 26 % (14-48%) 40% (18-81%) 5 32.18 46 % (24-87%) 50% (23-99%) 6 30.39 159 % (89-285%) 102% (51-207%)
Comparison potency probit analysis and Ct value on HIV-RNA Genotype Ct value 1000 cps/ml Potency Ct value (95% C.I.) to genotype A potency Limiting Dilution (95% C.I) to genotype A A 29.06 B 28.60 138% (63-302%) 76% (39-141%) C 28.94 109% (61-193 %) 103% (55-194%) CRF01_AE 27.94 217% (122-387%) 101% (54-190%)
Probability Variation in Ct value; predicted by poisson distribution 40% 30% 20% 10% 0% 33 35 37 39 Ct value Copy input 1 3 7 10 30 70 100
Ct value Observed Ct values in s 201 on HIV-RNA group M subtype B dilution series 45 43 41 39 37 35 Predicted variation all cases with >1% probability or 98 % C.I. 1 10 100 copies input
Conclusion TaqScreen Ct value analysis The amplification reaction is 100 % efficient for most cases The Ct values of different markers on the same copy levels are close to each other Ct value analysis can replace limiting dilution analysis to a certain extent. The variation in Ct value is larger than can be explained by Poisson distribution. More copies input than available in the sample is not possible. So only less copies input after extraction for amplification can explain the larger variation. This would result in more negative results or higher Ct values
Conclusions quantification of viral geno-subtype standards in copies/ml Good agreement between bdna 3.0 and Ultrio/Ultrio plus limiting dilution analysis for HIV and HCV and some HBV genotypes (close to 100% efficiency). TaqScreen limiting dilution analysis for HBV, HCV and HIV confirms relative quantification. Ct value at 1000 copies/ml for most geno-, subtypes is close to 29-30 for HBV and HIV and slightly higher for HCV Absolute quantification of HBV standards needs still to be confirmed, but is likely to be close to true value on the basis of interpretation of TaqScreen Ct values and limiting dilution results between markers, sub-, genotypes. Distribution of TaqScreen Ct values does not follow Poisson distribution, suggesting limiting dilution analysis does not represent the real amount of copies/ml in the sample but a lower amount after the silica extraction step.
Geno-, subtype panels made available The panel comprises all common geno,-subtypes quantified in copies/ml and diluted to the levels of 1000 and 100 copies/ml. The panels are intended to validate NAT assay versions. Level of 1000 copies/ml can be used with real time PCR to evaluate the geno-, subtype detection efficiency. Genotypes with an aberrant Ct value should be further investigated Level of 100 copies/ml is just high enough for consistent positive results in replicate qualitative tests. (Intermittent) negative results should be further investigated Please comment www.bioqcontrol.com