Development of a mitochondrial DNA real-time polymerase chain reaction assay for quality control of pathogen reduction with riboflavin and ultraviolet light

Background and Objectives Transfusion is associated with a risk of infection andalloimmunization. Pathogen reduction using riboflavin and UV light (Mirasoltreatment) inactivates pathogens and leucocytes. With increasing adoption of thetechnology in clinical use, regulatory agencies have recommended the introduc-tion of quality control measures to monitor pathogen reduction efficacy. Wesought to develop a real-time PCR-based assay to document the impact of patho-gen reduction on the mitochondrial genome in blood components.Materials and Methods DNA was extracted from platelet and plasma componentsbefore and after treatment with riboflavin and UV light. Inhibition of PCR ampli-fication of mitochondrial DNA (mtDNA) in short- and long-amplicon targetregions, ranging from under 200 base pairs (bp) to over 1800 bp, was measuredin treated relative to untreated components.Results Pathogen reduction of platelets using riboflavin and UV light resulted ininhibition of PCR amplification of long-amplicon mtDNA targets, demonstratingapproximately 1 log reduction of amplification relative to untreated products.Amplification of short-amplicon mtDNA targets was not affected by treatment.Evaluation of 110 blinded platelet samples from the PREPAReS clinical trialresulted in prediction of treatment status with 100% accuracy. Pathogen reduc-tion of plasma components resulted in similar levels of PCR inhibition, whiletesting of 30 blinded plasma samples resulted in prediction of treatment statuswith 93% accuracy.Conclusion A differential sized amplicon real-time PCR assay of mitochondrialDNA effectively documents nucleic acid damage induced by Mirasol treatment ofplatelets. The use of the assay for plasma product pathogen reduction requiresfurther investigation.Keywords: mitochondrial DNA, pathogen reduction, plasma, platelets, quantita-tive PCR.