CREATING CONFIDENCE

In the mid 1990s the Eurohep and VQC-Sanquin HBV genotype A standards were the first reference materials used for evaluation of NAT methods. Thereafter the Eurohep standard was used for preparation of the WHO standards. The Eurohep and VQC-Sanquin HBV genotype A standards were independently quantified in equivalent nucleic acid copies.

A series of standards of different genotypes have been cross calibrated in copies/mL against the VQC-Sanquin standard by multiple replicate bDNA 3.0 assays as the reference method for quantification. The VQC-Sanquin standard has also been extensively calibrated against the 1st and 2nd lyophilised WHO standards and the conversion factors (95% CI) were established at 5.33 (5-11-5.55) and 5.20 (4.61-5.80) copies per IU respectively. The VQC-Sanquin HBV genotype A standard was also calibrated against a chimpanzee plasma of known infectivity and according to this experiment the 50% chimpanzee minimum infectious dose (range) was determined at 4.0 (1.3-12.6) HBV-DNA copies or virions.

The VQC-Sanquin standard has been used for preparation of a pasteurised standard from which the ViraQ Check and Trend Controls are prepared. A lyophilised WHO HBV genotype reference panel has been made available by PEI and again the panel members were cross calibrated in copies/mL against the VQC-Sanquin standard in multiple replicate bDNA 3.0 assays. The results were comparable to the bDNA 3.0 calibration data in the WHO evaluation report.

Over the last two decades we manufactured 10 member dilution panels from these standards. Reference panels of (approximately) 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3 and 0.1 copies/mL of the HBV genotype standards were tested in multiple replicate tests in different NAT blood screening assays in order to determine the 95% and 50% LOD by probit analysis. More recently we manufacture 8 member dilution panels of the same HBV genotype standards starting at 300 copies/mL. Similar dilution panels were prepared from the 2nd WHO 97/750 standard (one of which mimicking the dilutions tested for the analytical sensitivity claims in the package inserts of the Ultrio versions).

There is one package insert for all these HBV reference panels and the proportions of reactive results of multiple replicate tests in different NAT blood screening assays are available for comparison. One can just as well use the VQC-Sanquin standard dilution panels as the 3rd WHO standard for testing the 95% and 50% LODs in IU/mL values because our standard is directly traceable to the 1st and 2nd WHO standards.

HBV-DNA genotype reference panels