Multicenter evaluation of a commercial multiplex polymerase chain reaction test for screening plasma donations for parvovirus B19 DNA and hepatitis A virus RNA.

BACKGROUND: Three European laboratories evaluated the TaqScreen DPX test (DPX test), a multiplex nucleic acid test assay for the simultaneous detection and quantitation of parvovirus B19 (B19V) DNA and the detection of hepatitis A virus (HAV) RNA. STUDY DESIGN AND METHODS: The 95% limit of detection of the test for B19V and HAV was determined using the respective WHO International Standards. The reproducibility of the test was evaluated by testing replicate samples of B19V at log 4.0 and 40 IU/mL and HAV at 5 IU/mL. The accuracy of the DPX test for B19V was evaluated by replicate testing of B19V samples containing log 3.0, log 4.0, and log 5.0 IU/mL. Panels of B19V Genotypes 1, 2, and 3 and HAV genotypes were evaluated. Cross-contamination was evaluated. For comparison of the DPX test and the established tests, the sites tested plasma samples in pools of either 96 or 480 donations. RESULTS: The mean 95% lower limits of detection of the three laboratories for B19V and HAV were 20.30 and 1.85 IU/mL. The test showed good reproducibility with the major part of the variance of the test being attributed to intermediate assay variation. The test showed great accuracy for B19V, especially at log 4.0 IU/mL. Spiking of test pools of 480 donations and manufacturing pools with log 4.0 IU/mL B19 DNA and 4 IU/mL HAV RNA showed that the DPX assay was robust. The test was able to detect the three genotypes of B19V and HAV genotypes. No cross-contamination was seen. Test results of routine samples correlated well with those of the established tests. CONCLUSION: The DPX test is a robust and sensitive test for the detection of B19V and HAV in plasma samples. The quantitative B19V results obtained with the test are accurate, and the test is able to detect all the known genotypes of B19V and HAV and fulfills all the European Pharmacopoeia and Food and Drug Administration requirements for a B19V and HAV test for screening of plasma donations and samples from plasma pools for manufacture.

Screening of blood donors for hepatitis C virus RNA with the MagNA Pure-COBAS AmpliScreen method.

BACKGROUND: This report describes the validation results and the performance characteristics of a new, semiautomated minipool nucleic acid amplification method for testing of blood products to reduce the transmission of hepatitis C virus (HCV) by transfusions. STUDY DESIGN AND METHODS: Minipools of 96 donations were prepared with the Tecan Genesis pipettors. Nucleic acids were isolated from plasma samples with the MagNA Pure LC instrument (Roche Diagnostics GmbH) and amplified and detected with the COBAS AmpliScreen second-generation HCV assay (Roche Molecular Systems, Inc.). Sensitivity of the method was determined with the WHO International Standard for HCV RNA (NIBSC 96/790) as a reference. The risk of cross-contamination during the nucleic acid extraction and postelution steps was studied with a highly viremic sample. RESULTS: Detection limit of the assay (95% hit rate) was calculated to be 11.7 IU per mL. Altogether 2,423 minipools (232,600 donations) were screened with the following performance characteristics: initially false-reactive results (0%), failure of run control detection (0.45%), and failure of internal control detection (0.90%). Two cross-contamination cases caused by a highly viremic sample were found during the validation phase but not in the routine screening period, although nine HCV RNA-positive minipool samples were observed. CONCLUSION: This combination of HCV RNA screening methods showed the detection limit that is well below the sensitivity requirements of the regulatory bodies. Robustness of the validated HCV RNA screening method has proved to be acceptable for routine screening of blood donors on a large scale.