CALIBRATION OF ANALYTICAL STANDARDS FOR HBV-DNA, HCV-RNA AND HIV-1 RNA IN GENOME COPIES BY A REFERENCE METHOD AAJ van Drimmelen, E.R. Bax and W.G.V. Quint, BioQControl (BQC), Delft Diagnostic Laboratories (DDL), Voorburg, the Netherlands Background Accurate quantification of analytical standards for viral nucleic acid tests (NAT) and estimation of the 50% minimum infectious dose in virion numbers and nucleic acid copies is essential for determining the risk of virus transmission by NAT screened blood donations (Weusten et al, Transfusion 2002, 42;537-548). The WHO international standards are useful in providing inter-laboratory comparability and common quantification in assigned International Units (IU's), which is the 4 th metrological level. The continuity of international units is based on collaborative studies involving different laboratories and a variety of assays. The relation between IU and genome copies, the SI unit, differs per assay and enhances the uncertainty with recalibrating replacements standards in IU. Calibration of standards in SI units provides a system traceable to the first metrological level (ISO 17511; 2003) but the required reference method for quantifying viral standards in nucleic acid copies has not been established. The calibrators in the historical Chiron Quantiplex bdna assay have been quantified in genome copies by physico-chemical techniques (Collins et al, Analytical Biochemistry, 226,120-129). Therefore the current Versant bdna 3.0 assays (Siemens) may serve as a reference method for quantification of viral plasma standards in copies/ml. Aims To quantify HBV genotype A, HCV genotype 1 and HIV 1 group M subtype B viral plasma standards in copies/ml using available bdna 3.0 test results from different studies and to determine the conversion factors from historically assigned genome equivalents (geq) to bdna 3.0 copies and IUs. Methods Our database includes quantitative and qualitative test results of proficiency studies, WHO standardization studies and validation studies of NAT blood screening assays. After initial assessment of the quantitative results of a variety of viral load assays the bdna 3.0 assay results were used to requantify the WHO and BQC standards in copies/ml. Finally the 63% detection endpoint of NAT screening assays was expressed in the newly assigned copy numbers in order to compare the viral concentration at a theoretical input level of one viral copy per amplification assay according to Poisson distribution. Results The quantification of the BQC and WHO standards in copies/ml according to bdna 3.0 assay is presented in table 1. The previous quantification in geq/ml is included for comparison. The conversion factors from historical geq to copies were 2.38, 1.40 and 1.39 for HIV-RNA, HBV-DNA and HCV-RNA respectively, whereas the conversion factors from IU to copies were 0.48, 5.33 and 2.73 respectively. Limiting dilution analysis on the Ampliscreen and Taqscreen results (table 2) revealed similar 63% detection endpoints in copies/assay for the different viral standards. The values for Ultrio almost reached the theoretical limitation of NAT sensitivity for HCV and HIV, but not for HBV. Conclusions The bdna 3.0 hybridisation assays may serve as a reference method for calibrating viral standards in copies/ml because the analytical sensitivities of the individual NAT blood screening assays on the different viral standards were similar (except for Ultrio HBV). For HIV and HCV the Chiron Ultrio assay reached the maximum achievable sensitivity, which may confirm the accurate absolute quantification in copies/ml by the bdna 3.0 assay.
Quantification of standards in SI units or calibration against WHO standards.error! Objects cannot be created Error! Objects cannot be created Error! Objects cannot be created The figure depicts the requirements for quantifying in SI units. The presence of an analytical method is necessary. In this study the Bayer Versant bdna 3.0 assays were evaluated on the ability to fulfill this requirement. The metrological levels show when which type of calibrator material is appropriate. Ideally level 1 should be reached. Lower levels cause less harmonisation of test results. Level 4 is always possible as first attempt to standardise testing. This requires a long-term available stable material. Secondary working standards may be needed. Then an analytical method is also required. The bdna assays include secondary calibrators which are traceable to the NIST Phosphor standard. The Phosphor standard in quantified in mol (SI unit). The quantification in copies/ml is within the accuracy of calibration on the Phosphor standard relating to SI units. (Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. M.L. Collins, J.J. Zayetti, B. Detmer et al. Anal. Biochem. 1995, 226:120-129. Quantification of HBV-DNA genotype A standard. Available HBV-DNA standards and their characterisation Eurohep standards for HBV-DNA were quantified in proficiency studies. Value assignment was done in Eurohep units/ml (aiming to be equal to copies/ml) WHO standard for HBV-DNA. The first standard was characterised in collaborative studies and a value in IU/ml was assigned. The WHO standard is a lyophilised 1:500 dilution of the EUROHEP genotype A standard. BQC HBV-DNA standards are calibrated against the EUROHEP and WHO standard and bdna 3.0 testing.
Quantification of HBV-DNA genotype A standard dilutions, expressed in undiluted standard. Assay average Ln (conc) stdev n low up Bayer bdna 3.0 2.15E+09 2.149E+01 0.005 28 2.13E+09 2.17E+09 Chiron bdna 1.0 3.22E+09 2.189E+01 0.003 17 3.20E+09 3.24E+09 Roche Ampl. Mon. 2.11E+09 2.147E+01 0.015 198 2.05E+09 2.17E+09 Sangtec Affig.VL 1.17E+09 2.088E+01 0.732 8 1.96E+08 7.04E+09 Roche Taqman 7.29E+08 2.041E+01 0.203 28 4.80E+08 1.11E+09 Artus RealArt HBV PCR 2.17E+08 1.920E+01 0.752 9 3.67E+07 1.29E+09 Digene HCS 1.63E+09 2.121E+01 0.019 42 1.57E+09 1.69E+09 Digene HCS Ultra 1.30E+09 2.099E+01 0.183 4 5.92E+08 2.85E+09 There is large consensus between the Roche HBV-DNA Amplicor Monitor 2.0 and Bayer Versant bdna 3.0 tests. The results of Sangtec Affigene VL, Digene HCS Ultra are not significantly different while Digene HCS was significantly lower. Roche Taqman confirmed the quantification in 2003 but in 2004 a lower value (5 times) was measured. Error! Objects cannot be created The quantification of the EUROHEP HBV-DNA standards is based on establishing a consensus value between quantitative testing (corrected on a clone traceable to the NIST Phosphor standard) and limiting dilution PCR testing. Both methods aim to quantify in copies/ml. The results varied from 2.2 to 3.0.10E9 copies/ml. By definition limiting dilution results will be equal to real value or lower (when the PCR efficiency is less than 100 %). The quantitative tests may over- or underestimate. In the 1997 proficiency study dilution series of BQC HBV-DNA genotype A standard and EUROHEP HBV- DNA genotype A standard were tested simultaneously in calibration experiments (1996, 1998) and proficiency testing (1997, 1999) using the Chiron bdna 1.0 assay. In 2008 both standards were tested 6-fold in the Bayer Versant bdna 3.0 assay. The WHO standard is a 1:500 dilution of the EUROHEP HBV-DNA genotype A standard. In 2001 WHO and BQC standard dilution series covering the dynamic range were tested multiple times in the Bayer Versant bdna 3.0 assay. Year copies/ml ln (copies/ml) Stdev N low up EUROHEP genotype A standard / WHO standard 1996/98-EH 3.89E+09 2.21E+01 0.019 9 3.72E+09 4.06E+09 2008-EH 3.74E+09 2.20E+01 0.009 6 3.65E+09 3.84E+09 2001-WHO 2.67E+09 2.17E+01 0.063 12 2.32E+09 3.07E+09 BQC genotype A standard 1996/98 3.22E+09 2.19E+01 0.014 17 3.13E+09 3.32E+09 2008 2.71E+09 2.17E+01 0.099 6 2.06E+09 3.57E+09 2001 2.15E+09 2.15E+01 0.092 16 1.77E+09 2.62E+09 The WHO standard quantification is multiplied by 500 to allow comparison to the EUROHEP calibrations. No correction was made for lyophilisation. The expert committee assigned a value of 1.10 6 IU per vial or ml to the WHO standard (An international Collaborative study to establish a WHO International Standard for Hepatitis B virus DNA Nucleic Amplification Techniques. J. Saldanha, W.H. Gerhlich, P.N. Lelie, K.H. Heerman and A. Heath. Vox Sang. 2001, 80: 63-71). The overall value for the BQC standard is 2.15.10 9 copies/ml (2001-2008). We conclude one IU is equal to 5.33 copies. As the 2 nd WHO HBV-DNA standard is manufactured equally and from same source materials the conversion factor is also assumed for the 2 nd WHO HBV-DNA standard. The potencies between the EUROHEP and BQC HBV-DNA standards were 83% (bdna 1.0) and 73 % (bdna 3.0) while we found for the WHO standard 81 %.
Quantification of HIV-RNA genotype B standard. Available HIV-RNA standards and their characterisation 1 st and 2 nd WHO standard for HIV-RNA. In the collaborative study for establishing the WHO HIV-RNA standard both the 1 st and 2 nd WHO HIV-RNA and the BQC HIV-RNA standard were evaluated together. Using the bdna 2.0 measurements we calculated 0.49 copies is one IU for the 1 st WHO HIV-RNA standard and 0.64 copy is one IU for the 2 nd WHO HIV-RNA standard (An international collaborative study to establish the 1 st International Standard for HIV-1-RNA for use in Nucleic Acid-Based Techniques. H. Holmes, C. Davis, A. Heath, I. Hewlett and P.N. Lelie. J. Virol. Methods 2001, 92: 141-150). The quantification in copies/ml is presented in the table below. Quantification of HIV-RNA genotype B standard dilutions, expressed in undiluted standard. Assay average Ln (conc) stdev n low up bdna 3.0 1.05E+08 1.847E+01 0.019 58 1.01E+08 1.09E+08 bdna 2.0 1.09E+08 1.851E+01 0.020 57 1.05E+08 1.14E+08 bdna 1.0 4.71E+07 1.767E+01 0.395 13 1.97E+07 1.12E+08 Abbott 1.95E+08 1.909E+01 0.125 17 1.49E+08 2.54E+08 NucliSens 2.40E+08 1.930E+01 0.028 116 2.27E+08 2.54E+08 Roche Mon.V1.0 2.25E+08 1.923E+01 0.010 437 2.20E+08 2.29E+08 Roche Mon V1.0 mixed primers 1.53E+08 1.884E+01 0.020 63 1.46E+08 1.59E+08 Roche Mon. V1.5 1.35E+08 1.872E+01 0.022 309 1.29E+08 1.41E+08 Roche Mon. V1.5 UltraSens 1.24E+08 1.863E+01 0.024 142 1.18E+08 1.29E+08 The biomerieux and Abbott assays are calibrated against the VQA standard causing a sgnificant differences in quantitation. Quantification of HCV-RNA genotype 1 standard. Available HCV-RNA standards and their characterisation Eurohep HCV-RNA genotype 1 and 3 standards for HCV-RNA were quantified in proficiency studies. Value assignment was done in geq/ml by testing bdna 1.0 (geq and copy are equivalent) 1 st WHO standard for HCV. The first standard was characterised in a collaborative study and values were assigned. The first and second WHO HCV-RNA was derived from the same HCV-RNA, a-hcv positive plasma pool. The third WHO HCV-RNA standard is derived from a HCV-RNA positive anti-hcv negative plasma pool BQC HCV-RNA genotype 1 standard is calibrated against Eurohep standard and the 1st WHO HCV-RNA standard. Quantification of HCV-RNA genotype 1 standard dilutions, expressed in undiluted standard. Assay average Ln (conc) stdev n low up bdna 3.0 6.30E+06 1.57E+01 0.014 27 6.13E+06 6.48E+06 bdna 2.0 9.83E+06 1.61E+01 0.021 43 9.41E+06 1.03E+07 bdna 1.0 8.76E+06 1.60E+01 4 Amplicor COBAS 2.0 3.68E+06 1.51E+01 0.032 100 3.45E+06 3.93E+06 Amplicor MWP 2.0 3.97E+06 1.52E+01 0.060 35 3.51E+06 4.48E+06 Calibration against WHO standard A HCV genotype 1 standard dilution was included in the WHO calibration study for secondary HCV-RNA reagents (Calibration of HCV working reagents for NAT assays against the HCV international standard. The Collaborative Study Group. J. Saldanha, A. Heath, N. Lelie, G. Pisani, M. Nubling, M. Yu. Vox Sang. 2000;78(4):217-24). The preparation containing 2732 copies/ml was assigned a value of 1000 IU/ml. The conversion factor becomes 2.73 copies is one IU.
Comparison quantification BQC HCV-RNA genotype 1 standard to limiting dilution analysis Both BQC and WHO HCV-RNA standard dilution series were used for estimation of analytical sensitivity on blood screening assays. We calculated the 63 % hit rate and corrected this for the amplication input volume from 29 studies. To convert IU to copies 2.73 was used. Only the first and second WHO HCV- RNA standards were used. Both WHO standards are prepared from the same source and processed similar. We compared the WHO and BQC HCV-RNA standards in the unpaired student-t test. The populations did not present a significant difference. 95% Confidence Interval of the Difference Sig. (2- Mean Std. Error t df tailed) Difference Difference Lower Upper 1.939 28 0.063 0.55 0.28-0.03 1.12 The distribution of 63 % hit rates reveals an asymptote at 1.0 copy input. This confirms the bdna quantification in copies/ml as well as the commutability to the WHO standard using 2.73 copies is one IU as conversion factor. The graph depicts the sorted copies input (n=15) 4 3.5 3 copies input 2.5 2 1.5 calc line WHO BQC 1 0.5 0 0 0.2 0.4 0.6 0.8 1 case
Calibration HCV-RNA genotype standards. The other HCV-RNA genotypes were tested simultaneously against the BQC HCV-RNA genotype 1 standard in Bayer Versant 3.0 assay. This was done is two separate HCV-RNA genotype 1 standard. The standards include HCV-RNA genotype 2, 3, 4, 5, 6 and inactivated genotype 3a. To control the quantification by bdna 3.0 standard dilution panels were tested in Novartis Procleix Ultrio and Roche MPX. Using probit analysis the potencies between genotypes were calculated. Comparison Ultrio (n=24) and Roche MPX (n=12) by estimation potencies using parallel line probit analysis for each assay. Ultrio EFS/ARCBS MPX EFS genotype Estimate Lower Upper Estimate Lower Upper 1 2 1.2 0.7 1.9 1.2 0.6 2.4 3 2.5 1.5 4.3 1.7 0.9 3.6 4 0.9 0.6 1.5 0.4 0.2 0.8 5 1.5 0.9 2.4 0.5 0.2 1.0 6 2.2 1.3 3.7 1.0 0.5 2.1 3 2.1 1.3 3.5 1.5 0.8 3.0 2 4 0.9 0.5 1.2 0.3 0.2 0.7 5 1.2 0.8 2.0 0.4 0.2 0.8 6 1.8 1.1 3.1 0.9 0.4 1.7 4 0.4 0.2 0.6 0.2 0.1 0.5 3 5 0.6 0.4 0.9 0.3 0.1 0.6 6 0.9 0.6 1.4 0.6 0.3 1.2 4 5 1.6 1.0 2.6 1.2 0.6 2.5 6 2.4 1.5 4.0 2.6 1.3 5.8 5 6 1.5 0.9 2.4 2.1 1.0 4.5 Conclusion calibration HCV standards The absolute quantification of the HCV-RNA genotype 1 standard is confirmed by limiting dilution analysis. The potency analysis on different genotypes is significantly different for genotype 3 vs 5 in both Roche MPX and Ultrio testing. Further research is needed to establish the correct quantification for these two genotypes. Origin of test results For quantification in bdna 3.0 longitudinal test results obtained in calibration experiments and proficiency testing were used. The limiting dilution analyses were done using literature data. We used the results of head-to-head comparisons between Novartis Procleix Ultrio and Roche MPX (Comparison of the analytical and operational performance of two viral nucleic acid test blood screening systems: Procleix Tigris and cobas s 201.Assal A, Barlet V, Deschaseaux M, Dupont I, Gallian P, Guitton C, Morel P, David B, De Micco P. Transfusion. 2008 Nov 4 and Comparison of two automated nucleic acid testing systems for simultaneous detection of human immunodeficiency virus and hepatitis C virus RNA and hepatitis B virus DNA. Margaritis AR, Brown SM, Seed CR, Kiely P, D'Agostino B, Keller AJ. Transfusion. 2007 Oct;47(10):1783-93)