Blog
About

5
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Investigation of Preanalytical Variables Impacting Pathogen Cell-Free DNA in Blood and Urine

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA.

          ABSTRACT

          Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus (EBV) and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. The PCR cycle threshold ( C T ) was used to measure amplifiable cfDNA. In spiked samples, the median C T values for M. tuberculosis, S. enterica, and EBV cfDNA were significantly lower in blood collected in K 2EDTA tubes than those in Streck and PAXgene blood collection tubes, and they were was significantly lower in urine preserved with EDTA (EDTA-urine) than in urine preserved with Streck reagent (Streck-urine). Blood and urine samples from TB patients preserved with K 2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis C T values than with the Streck blood collection tube and Streck urine preservative. Processing delay increased the median pathogen C T values for Streck and PAXgene but not K 2EDTA blood samples and for urine preserved with Streck reagent but not EDTA. Double-spin compared with single-spin plasma separation increased the median pathogen C T regardless of blood collection tube. No differences were observed between whole urine and supernatant and between fresh and thawed plasma and urine after 24 weeks at –80°C. Larger plasma and urine volumes in contrived and patient samples showed a significantly lower median M. tuberculosis C T . These findings suggest that large-volume single-spin K 2EDTA-plasma and EDTA-whole urine with up to a 24-h processing delay may optimize pcfDNA detection.

          Related collections

          Most cited references 27

          • Record: found
          • Abstract: found
          • Article: not found

          Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood.

          We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            DNA sequencing versus standard prenatal aneuploidy screening.

            In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Quantitative and temporal correlation between circulating cell-free Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma.

              Recently, cell-free EBV DNA has been detected in the plasma and serum of patients with nasopharyngeal carcinoma (NPC). We studied the relationship between plasma/serum EBV DNA and tumor recurrence. Using real-time quantitative PCR, the median plasma EBV DNA concentration in 10 patients with tumor recurrence was determined to be 32,350 copies/ml, whereas that in 15 patients in continuous remission for a mean period of 2 years was 0 copy/ml. Longitudinal follow-up of 17 NPC patients revealed 6 individuals with tumor recurrence and 11 patients who remained in remission. Significant elevations in serum EBV DNA, sometimes up to 6 months before detectable clinical deterioration, were observed in the patients who subsequently developed tumor recurrence. Continuously low or undetectable levels of serum EBV DNA were observed in the patients who remained in remission. These results suggest that plasma/serum cell-free EBV DNA may be a valuable tool for the monitoring of NPC patients for the early detection of tumor recurrence.
                Bookmark

                Author and article information

                Contributors
                Role: Editor
                Journal
                J Clin Microbiol
                J. Clin. Microbiol
                jcm
                jcm
                JCM
                Journal of Clinical Microbiology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0095-1137
                1098-660X
                11 September 2019
                23 October 2019
                November 2019
                23 October 2019
                : 57
                : 11
                Affiliations
                [a ]Department of Pathology, Stanford University School of Medicine, Stanford, California, USA
                [b ]Clinical Microbiology Laboratory, Stanford Health Care, Stanford, California, USA
                [c ]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
                [d ]College of Health Sciences, Makerere University, Kampala, Uganda
                [e ]Global Health Technologies, Global Good Fund, Intellectual Ventures Laboratory, Bellevue, Washington, USA
                [f ]Intellectual Ventures Laboratory, Bellevue, Washington, USA
                [g ]Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California, USA
                [h ]Faculty of Health Sciences, Federal University of Grande Dourados, Dourados, Brazil
                [i ]Oswaldo Cruz Foundation, Campo Grande, Brazil
                [j ]Department of Medicine, University of California, San Francisco, San Francisco, California, USA
                Johns Hopkins University School of Medicine
                Author notes
                Address correspondence to Niaz Banaei, nbanaei@ 123456stanford.edu .

                Citation Murugesan K, Hogan CA, Palmer Z, Reeve B, Theron G, Andama A, Somoskovi A, Steadman A, Madan D, Andrews J, Croda J, Sahoo MK, Cattamanchi A, Pinsky BA, Banaei N. 2019. Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. J Clin Microbiol 57:e00782-19. https://doi.org/10.1128/JCM.00782-19.

                Article
                00782-19
                10.1128/JCM.00782-19
                6813001
                31511335
                Copyright © 2019 Murugesan et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                Page count
                supplementary-material: 1, Figures: 8, Tables: 0, Equations: 0, References: 35, Pages: 13, Words: 7372
                Product
                Funding
                Funded by: Stanford Global Health Seed Grant;
                Award Recipient :
                Funded by: Stanford Chem-H;
                Award Recipient :
                Funded by: Canadian Institutes for Health Research Fellowship;
                Award Recipient :
                Funded by: European and Developing Countries Clinical Trials Partnership (EDCTP), https://doi.org/10.13039/501100001713;
                Award Recipient :
                Funded by: South African Medical Research Council (SAMRC), https://doi.org/10.13039/501100001322;
                Award Recipient :
                Funded by: Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation), https://doi.org/10.13039/100000865;
                Award Recipient :
                Categories
                Bacteriology
                Custom metadata
                November 2019

                Microbiology & Virology

                liquid biopsy, pcr, preanalytical, cell-free dna

                Comments

                Comment on this article