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Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine.

Author(s): 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

Publication: J Clin Microbiol, 2019, Vol. , Page

PubMed ID: 31511335 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of collection tube type, delayed processing, number of centrifugation steps, frozen storage, and plasma volume on the recovery of spiked-in fragments of M. tuberculosis, S. enterica, A. fumigatus and EBV DNA in plasma. The effects of collection tube and plasma volume on recovery of tuberculosis cfDNA in plasma from TB patients was also examined. Blood was collected from 10 healthy patients collected into K₂EDTA tubes (control), Streck cell-free DNA BCTs, and PAXgene blood ccfDNA tubes. Blood was spiked with 200 bp fragments of Mycobacterium 126 tuberculosis, Salmonella enterica, Aspergillus fumigatus, and Epstein-Barr virus and stored for 0, 6, or 24 h at room temperature before processing. Blood was processed to plasma by centrifugation at 500 x g for 10 min with or without a subsequent centrifugation at 16000 x g for 10 min. To investigate the effects of freezing, plasma obtained by a single centrifugation was frozen at -80°C for 1 or 24 weeks and thawed at room temperature. cfDNA was extracted using the Maxwell(R) RSC ccfDNA Plasma Kit from 0.5 mL plasma. To investigate the effects of plasma volume, extraction was also performed using 0.5 and 3 mL of K₂EDTA plasma. Blood from seven TB patients was drawn into K₂EDTA tubes and Streck BCTs and immediately processed to plasma. cfDNA was extracted from these specimens using the QIAamp Circulating Nucleic Acid Kit. cfDNA levels were assessed by real-time PCR amplification of the spiked-in DNA in healthy specimens and M. tuberculosis in plasma from tuberculosis patients. 

   Summary of Findings:

   Significantly lower levels (i.e., higher CT values) of M. tuberculosis and S. eterica cfDNA were recovered from blood collected in PAXgene and Streck tubes than K₂EDTA tubes (P<0.05). Significantly higher mean recovery of A. fumigatus was found in PAXgene plasma processed after 0 or 6 h by one-step (P<0.01 and P<0.0, respectively) and two-step (P<0.05, both) centrifugation and after 24 h by single centrifugation (P<0.01) than in the respective K₂EDTA plasma, but significantly lower mean recovery of A. fumigatus was found in Streck BCT plasma processed after 6 h (P<0.01, both) or 24 h (P<0.01 for one step and P<0.05 for two-step) than in the respective K₂EDTA plasma. EBV recovery was lower in plasma from Streck tubes processed after 6 h (P<0.01) or 12 h (P<0.01) by single centrifugation and PAXgene tubes processed by single or double centrifugation after 6 h (P<0.05, both) or 24 h (P<0.01, both) than in the respective K₂EDTA specimens. Double-spun plasma had lower mean recovery of M. tuberculosis, S. eterica, and EBV, regardless of processing delay and tube type, but the difference in EBV was not significant in K₂EDTA tubes. A. fumigatus levels were significantly lower in double-spun than single-spun PAXgene plasma subjected to a 0 or 24 h processing delay. Recovery of the spiked-in cfDNA was not affected by frozen storage of plasma for 1 or 24 weeks at -80°C with the exception of S. enterica in PAXgene tubes. Significantly more viral DNA was recovered when 3 mL of plasma was used instead of 0.5 mL (P=0.005). The levels of TB were significantly higher when blood from TB patients was collected in K₂EDTA tubes rather than Streck tubes (P=0.021) and when 3 mL was used instead of 0.5 mL (P=0.03).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • None (Fresh)
   • Streck/BCT
   • PAXgene

   Diagnoses:

   • Other diagnoses
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	Streck BCT PAXgene blood K2EDTA
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared
   Biospecimen Preservation	Type of fixation/preservation	EDTA Frozen Blood collection tube additive PAXgene
   Storage	Storage duration	0 h 6 h 24 h 0 day 1 week 24 weeks
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Biospecimen Aliquots and Components	Aliquot size/volume	0.5 mL 3 mL

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2. Study Purpose

   This study investigated the effects of preservative type, delayed processing, centrifugation, frozen storage, and urine volume on the recovery of spiked-in fragments of M. tuberculosis, S. enterica, A.  fumigatus, and EBV DNA in urine. The effects of collection tube and urine volume on recovery of tuberculosis cfDNA in urine from tuberculosis patients was also examined. Urine was collected from 10 healthy patients into a collection cup and immediately transferred into conical tubes containing 25 mM EDTA (control), 10 mM Tris-EDTA, or Streck cell-free DNA urine preservative. Urine was centrifuged at 16000 x g for 10 min. To investigate the effects of freezing, urine was frozen at -80°C for 1 or 24 weeks and thawed at room temperature. cfDNA was extracted from 1 mL urine using the Maxwell RSC System. To investigate the effects of urine volume, extraction was also performed using 4 mL of EDTA urine.  Urine from twenty TB patients was collected in 10 mM Tris EDTA (pH8.5) and Streck cell-free urine DNA Preservative, frozen at -80°C, and shipped internationally (Uganda to United States). cfDNA levels were assessed by real-time PCR amplification of the spiked-in DNA in healthy specimens and M. tuberculosis in urine from tuberculosis patients.

   Summary of Findings:

   Recovery of M. tuberculosis, S. enterica, A. fumigatus, and EBV were comparable in EDTA and Tris-EDTA urine with the exceptions of lower EBV levels in unspun Tris-EDTA urine stored for 6 h than the matched EDTA urine specimen (P<0.05) and A. fumigatus in centrifuged and uncentrifuged urine stored for 6 h compared to the matched EDTA urine specimen (P<0.05, both). Compared with EDTA urine, Streck urine had significantly lower recovery of M. tuberculosis when centrifuged immediately (P<0.05), stored for 6 h and centrifuged or not centrifuged (P<0.01, both), or stored for 24 h and centrifuged (P<0.05) or not centrifuged (P<0.01), and lower recovery of S. enterica regardless of centrifugation when stored for 0 h (P<0.05, both), 6 h (P<0.01), or 24 h (P<0.01). Recovery of A. fumigatus was lower in Streck urine than EDTA urine when stored for 6 h, regardless of centrifugation (P<0.05, both), or stored 24 h and centrifuged (P<0.05). Similarly, recovery of EBV was lower in Streck urine than EDTA urine stored for 6 or 24 h, regardless of centrifugation (P<0.01, all). Recovery of M. tuberculosis, S. enterica, and EBV cfDNA was significantly lower from unpreserved urine than K₂EDTA urine (P<0.01, all). There were no consistent effects of centrifugation on the recovery of the pathogenic cfDNA.  Recovery of the spiked-in cfDNA was not affected by frozen storage of urine for 24 weeks at -80°C, regardless of tube type. Significantly more viral DNA was recovered when 4 mL of urine was used instead of 1 mL (P=0.007). The levels of TB were significantly higher when urine from TB patients was collected in Tris-EDTA tubes rather than Streck tubes (P=0.035) and when 4 mL was used instead of 1 mL (P=0.01).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen
   • Streck/BCT
   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Other diagnoses
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifuged Not centrifuged
   Storage	Freeze/thaw cycling	1 cycle 0 cycles
   Storage	Storage duration	0 h 6 h 24 h 0 weeks 24 weeks
   Biospecimen Aliquots and Components	Aliquot size/volume	1 mL 4 mL
   Biospecimen Preservation	Type of fixation/preservation	Blood collection tube additive EDTA Frozen None (fresh)

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