Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine
Kanagavel Murugesan, Catherine A. Hogan, Zaida Palmer, Byron Reeve, Grant Theron, Alfred Andama, Akos Somoskovi, Amy Steadman, Damian Madan, Jason Andrews, Julio Croda, Malaya K. Sahoo, Adithya Cattamanchi, Benjamin A. Pinsky, Niaz Banaei
Department of Pathology, Stanford University School of Medicine, Stanford, California, USA, Clinical Microbiology Laboratory, Stanford Health Care, Stanford, California, USA, NRF/DST Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa, College of Health Sciences, Makerere University, Kampala, Uganda, Global Health Technologies, Global Good Fund, Intellectual Ventures Laboratory, Bellevue, Washington, USA, Intellectual Ventures Laboratory, Bellevue, Washington, USA, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA, Faculty of Health Sciences, Federal University of Grande Dourados, Dourados, Brazil, Oswaldo Cruz Foundation, Campo Grande, Brazil, Department of Medicine, University of California, San Francisco, San Francisco, California, USA|2019|Journal of Clinical Microbiology|57: e00782-19 https://doi.org/10.1128/JCM.00782-19.
The impact of sample processing of pathogen cell-free DNA (pcfDNA) in blood and urine is currently not well understood. In this study, the impact of (i) blood collection tube and urine preservative, (ii) processing delay, (iii) processing method, (iv) freezing and thawing and (v) sample volume of pcfDNA on sensitivity of the pathogen pcfDNA detection was investigated. Blood and urine samples from healthy donors were spiked with cfDNA with different pathogens representative of bacteria, fungi and DNA viruses. A real-time PCR was performed using TaqMan probes on the Mic qPCR Cycler and the effects of each preanalytical process was measured using the PCR cycle threshold (Ct) and were compared to observe which methods provide lowest Ct values. The results suggest that large volume single spin K2EDTA-plasma and EDTA-whole urine with up to 24-hour processing delay may optimize pcfDNA detection. Further validation of the suggested sample processing method is required using clinical samples.