BioQualityControl

Eurohep and WHO initiatives for standardisation in nucleic acid testing.

The first standardisation initiatives on the detection of Hepatitis B virus DNA and Hepatitis C virus RNA were done by the Eurohep Group around 1992. The Eurohep group (European Concerted Action on Viral Hepatitis) aimed to quantify the standards in copies/ml thereby relating to the first metrological level. For HBV they used limiting dilution (3 laboratories, one averaged value of 8 dilution series) and 2 hybridisation assays testing the undiluted and 2 dilutions of the Eurohep standards estimated that the Eurohep genotype A standard contained 2.7.109(2.1-3.4.109) copies/ml (geometric mean). The hybridisation assays were calibrated on an accurately quantified highly purified HBV clone supplied by Chiron. For hybridisation assays the extraction efficiency should reach 100% for equal detection of the clone and virion. The Eurohep HCV genotype 1 standard was quantified accurately by multiple testing in the Chiron bDNA 1.0 assay and compared to the results found in proficiency testing. The bDNA assay utilises calibrators that are defined by comparison to transcripts. The transscripts themselves were quantified in copy/ml using different analytical methods including phosphor analysis. This relates to the first metrological level.

Later the WHO initiated collaborative studies for defining the International Standards. The bioQcontrol standards were calibrated against the first WHO standards which are sold out by NIBSC. WHO standards are lyophilised dilutions of viral standards. The commutability between different standards is still investigated. Issues like genotype, matrix, effect of lyophlisation etc are not fully understood. Moreover the batch size of the WHO standards for nucleic acid testing was to limited to guarantee a long-term supply. Replacement of WHO standards for nucleic acid testing occured every 3-4 years since their introduction. This implies systematic deviations are present due to the uncertainty of calibration on the previous standard. As an example we present table 5 from the report in which the calibration of the second on the first WHO HIV-RNA standard is described. The table present the log (potency) in IU/ml for the second standard.

* One qualitative result set was rejected for inclusion in the analysis. Table copied from: EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 24 to 28 October 2005 REPORT ON AN INTERNATIONAL COLLABORATIVE STUDY TO ESTABLISH A REPLACEMENT WHO INTERNATIONAL STANDARD FOR HIV-1 RNA NAT ASSAYS H C Holmes, C L Davis, A B Heath and the Collaborative Study Group* National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK.

The table shows significant differences are present between different commercial methods. When IVD manufacturers have to recalibrate their assay on every version of the WHO standard this implies shifts in quantification upto 0.5 log or a factor 3 may occur. We believe the rapid replacements of WHO standards for nucleic acid testing will deminish their importance for standardisation. The WHO HBV-DNA standard is a 1:500 dilution of the Eurohep genotype A standard. Since the Eurohep standard is still available in sufficient quantities a long-term supply of subsequent WHO HBV-DNA standards is possible.

At bioQcontrol we try to fill the gaps by analysing results on our standard dilution series (run controls, validation panels and proficiency panels) and compare results against theoretical limitations. We also aim to collect sufficient quantities to enable long-term supply. The WHO standards are used by regulatory bodies to define sensitivity requirements and for that reason we mention a conversion factor.

NIBSC, the WHO international laboratory for biological standardisation

NIBSC organises WHO collaborative studies for establishing international biological standards. Candidate reference materials are donated to the WHO and are compared in a collaborative study for their suitability to become the WHO International Standard. A report of the collaborative study is prepared and submitted to the WHO Expert Committee for Biological Standardisation (ECBS).The ECBS can accept one of the reference materials to become the International Standard. The standard can then be distributed by either one of the WHO International Laboratories.

Calibration of reference reagents on the International Standards

NIBSC is organising on behalf of the WHO collaborative studies for calibration of secondary standards on the International Standard. Different reference reagents have been calibrated on the International Standards for HCV-RNA and HIV-RNA. The bioQControl standards for HBV-DNA, HCV-RNA, HIV-RNA, HAV-RNA and parvo B19 DNA have been calibrated against the First International Standards.

Overview current WHO standards

• WHO International Standard WHO First International Standard For Hepatitis A Virus RNA Nucleic Acid Amplification (NAT) Assays;
• 2nd WHO INTERNATIONAL STANDARD FOR HEPATITIS B VIRUS DNA NUCLEIC ACID AMPLIFICATION TECHNIQUES, same source as the 1st international standard;
• WHO International Standard 3rd Hepatitis C Virus (HCV) RNA International Standard;
• HIV-1 RNA, 2nd International Standard, calibrated on the first international standard using a different source material;
• WHO International Standard WHO International Standard for Parvovirus B19 DNA for Nucleic Acid Amplification (NAT);
• Second International Standard for HBsAg, subtype adw2, genotype A, calibrated on the first international standard.

Calibration in International Units or SI units (molecules, grams)

There are fundamental limitations in calibration of viral standards. Where possible the bioQControl standards are calibrated in copies and International Units. One has to bear in mind that differences may exist in configuration of the present International Standards and quality control reagents. This may cause variation in outcomes of calibration results in different diagnostic assays.

Calibration of bioQControl NAT standards on WHO International Standards

The bioQControl standards for HBV-DNA, HCV-RNA and HIV-RNA have been historically calibrated in copies by repeated quantification in Chiron Quantiplex bDNA assays. To avoid any degradation of viral particles or nucleic acid the bioQControl standards for viral NAT were not, lyophilised or inactivated. The liquid standard dilutions are made in plasma and are kept frozen during storage and transport. Later the WHO SoGAT group has established International Standards for HCV-RNA, HBV-DNA, HIV-RNA, HAV-RNA and parvo B19 DNA. These plasma-derived viral standards were lyophilised for reasons of stability at higher temperature and ease of transport. The reconstituted WHO standard typically contains 100.000 International Units of viral nucleic acid. The bioQControl standards were calibrated against the WHO international standards for HCV-RNA, HBV-DNA, HIV-RNA, HAV-RNA and parvo B19 DNA. In the table below the average conversion factors from 'WHO International Units' to 'copies' are shown for the different analytes. It must be emphasised that significant differences in conversion factors were found between quantitative assays from different manufacturers. Further studies are necessary to understand the commutability between different standards.

Conversion factors between IU and copies

The bioQControl Parvo B19-DNA and HAV-RNA have not been quantified in copies/ml, but are calibrated in IU/ml.