Vox Sanguinis (2014) 107, 351–359 ORIGINAL PAPER © 2014 International Society of Blood Transfusion DOI: 10.1111/vox.12173 Development of a mitochondrial DNA real-time polymerase chain reaction assay for quality control of pathogen reduction with riboflavin and ultraviolet light S. Bakkour, 1 D. M. Chafets, 1 L. Wen, 1 P. F. van der Meer, 2 J. M. Mundt, 3 S. Marschner, 3 R. P. Goodrich, 3 M. P. Busch 1 & T.-H. Lee 1 1 Blood Systems Research Institute, San Francisco, CA, USA 2 Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, the Netherlands 3 Terumo BCT Biotechnologies, Lakewood, CO, USA Received: 11 April 2014, revised 29 May 2014, accepted 30 May 2014, published online 27 June 2014 Background and Objectives Transfusion is associated with a risk of infection and alloimmunization. Pathogen reduction using riboflavin and UV light (Mirasol treatment) inactivates pathogens and leucocytes. With increasing adoption of the technology in clinical use, regulatory agencies have recommended the introduc- tion of quality control measures to monitor pathogen reduction efficacy. We sought 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 components before and after treatment with riboflavin and UV light. Inhibition of PCR ampli- fication of mitochondrial DNA (mtDNA) in short- and long-amplicon target regions, ranging from under 200 base pairs (bp) to over 1800 bp, was measured in treated relative to untreated components. Results Pathogen reduction of platelets using riboflavin and UV light resulted in inhibition of PCR amplification of long-amplicon mtDNA targets, demonstrating approximately 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 trial resulted in prediction of treatment status with 100% accuracy. Pathogen reduc- tion of plasma components resulted in similar levels of PCR inhibition, while testing of 30 blinded plasma samples resulted in prediction of treatment status with 93% accuracy. Conclusion A differential sized amplicon real-time PCR assay of mitochondrial DNA effectively documents nucleic acid damage induced by Mirasol treatment of platelets. The use of the assay for plasma product pathogen reduction requires further investigation. Key words: mitochondrial DNA, pathogen reduction, plasma, platelets, quantita- tive PCR. Introduction Emerging infections continue to pose a risk of transmis- sion via transfusion, particularly for pathogens with an asymptomatic blood-borne phase and those for which there is no current intervention strategy [1]. Risk management depends primarily on donor questions and screening tests for specific pathogens. However, these strategies are reactive rather than proactive and are ulti- mately unsustainable as the number of questions and tests increases with the discovery of new pathogens that threaten blood safety. Pathogen reduction technologies Correspondence: Sonia Bakkour, 270 Masonic Avenue, San Francisco, CA 94118, USA E-mail: sbakkour@bloodsystems.org 351