Quantitative real-time detection of parvovirus B19 DNA in plasma

• Published in: Transfusion (Philadelphia, Pa.) Vol. 44; no. 1; pp. 97 - 103
• Main Authors: Koppelman, Marco H.G.M., Cuypers, H. Theo M., Emrich, Thomas, Zaaijer, Hans L.
• Format: Journal Article
• Language: English
• Published: Oxford, UK and Malden, USA Blackwell Science Inc 01-01-2004; Blackwell Publishing
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Biological and medical sciences; Blood Donors; Blood. Blood and plasma substitutes. Blood products. Blood cells. Blood typing. Plasmapheresis. Apheresis; Computer Systems; DNA, Viral - blood; Humans; Mass Screening - methods; Medical sciences; Parvovirus B19; Parvovirus B19, Human - genetics; Sensitivity and Specificity; Transfusions. Complications. Transfusion reactions. Cell and gene therapy; Viremia - diagnosis; Virus; Transfusion; Parvoviridae; DNA; Erythrovirus; Medical screening; Detection; Real time; Virus B19; Parvovirinae; Blood plasma
• ISSN: 0041-1132; 1537-2995
• DOI: 10.1046/j.0041-1132.2004.00610.x

BACKGROUND:  As of 2004, the European Pharmacopoeia demands that plasma pools for production of anti‐D immunoglobulin should not contain more than 104 IU per mL of parvovirus B19 (B19V) DNA. Hence, before pooling, highly viremic donations have to be identified, and after pooling the level of B19V DNA must be determined. The performance of a new real‐time B19V DNA PCR test (Roche, Mannheim, Germany) was studied, using a DNA extractor (NucliSens, bioMerieux, Boxtel, the Netherlands) for isolation of nucleic acid, and using a DNA quantification test (LightCycler apparatus, Roche, Mannheim, Germany) for amplification and detection.
STUDY DESIGN AND METHODS:  Dilutions of the international B19V DNA standard and reference preparations were tested to determine the precision, linear range, and accuracy of the assay and to calculate the factor for conversion of B19V DNA copies to IUs. The internal control signals, invalid test results, and the effect of cryo‐poor plasma were studied as a measure for robustness. Routine performance was assessed by testing 164 manufacturing pools (not screened for B19V) and 1048 test pools of 480 donations each.
RESULTS:  The copies‐to‐IU conversion factor was calculated to be 3.34 (95% CI, 3.07‐3.63). The assay appears linear between 103 and 107 IU per mL. Between 103 and 105 IU per mL, the test can discriminate samples differing a factor two in B19V DNA content. Overall, 0.78 percent of the test results were invalid. Of 127 B19V DNA negative control plasma samples, 7 were contaminated with low levels of B19V DNA. Of 164 nonscreened manufacturing plasma pools, 92 contained B19V DNA (56%); 13 contained more than 104 IU per mL. Of 503,040 donations, 29 contained more than 5 × 106 IU per mL B19V DNA (1:17,346).
CONCLUSION:  The B19V DNA quantification test (LightCycler, Roche ) is suitable for quantitative, routine, in‐process measurement of B19V DNA levels in plasma pools, using the DNA extractor (NucliSens, bioMerieux) for nucleic acid isolation.