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Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome.

Author(s): Van Paemel R, De Koker A, Caggiano C, Morlion A, Mestdagh P, De Wilde B, Vandesompele J, De Preter K

Publication: Epigenetics, 2020, Vol. , Page 1-11

PubMed ID: 33074045 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of delayed centrifugation of blood collected in five different tube types by comparing cfDNA yield, fragment size, NGS metrics, methylation, and cell source in specimens subjected to a room temperature processing delay of 24 or 48 h to specimens processed immediately after collections. Blood was obtained from three healthy patients in triplicate using a 21-gauge butterfly needle into spray-coated K2 EDTA tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes, and Biomatrica LBgard blood tubes. For each patient, draw order was randomized and seven of the fifteen specimens were collected from the antecubital vein while the remaining eight were collected from the contralateral antecubital vein. For each tube type, one specimen from each patient was processed immediately while the remaining were stored at room temperature for 24 h or 48 h before processing. Plasma was obtained by centrifugation at 1600 x g for 10 min followed by 16000 x g for 10 min (Streck and EDTA tubes), 1900 x g for 15 min (PAXgene), or 4500 x g for 15 min (Roche) or by centrifugation at 1900 x g for 15 min followed by 4500 x g for 15 min (Biomatrica). Plasma aliquots were frozen and stored at -80°C until cfDNA isolation using the Maxwell RSC LV ccfDNA Kit. cfDNA was quantified using the Qubit High-Sensitivity Kit and fragment size distribution and quantity analyzed by capillary electrophoresis on a FEMTO Pulse Automated Pulsed-Field CE Instrument. Sequencing libraries were prepared following the cf-RRBS protocol and cleaned by magnetic bead selection and sequenced on a NovaSeq 6000. To ensure accurate comparisons, all libraries were down-sampled to 18 million reads. Promoters were defined as 1000 bp upstream to 2000 bp downstream of transcription start site and regions with agreement (completely methylated or unmethylated) and those with less than 15 reads were excluded. Cell type contributions were determined using non-negative least squares (NNLS) matrix decomposition and the Illumina HM450K reference matrix as well as CelFiE and its reference dataset.

   Summary of Findings:

   While 20 of the 45 specimens had cfDNA concentrations below the limit of detection by Qubit, cfDNA was quantifiable in all 45 specimens using the Femto PULSE. The second and third nucleosome peaks were lower in specimens stored in Roche, Biomatrica, and Streck tubes after 72 h than in promptly processed specimens but the second peak increased in height and additional peaks appeared in EDTA specimens stored for 72 h. When quantified using the Femto PULSE, cfDNA concentrations were found to increase with storage of blood in EDTA tubes and decline with storage in Roche tubes. Sequencing confirmed a 406% increase in DNA concentration when blood was stored in EDTA tubes for 72 h and DNA concentrations were 22% of baseline when stored for 72 h in Roche tubes. An average of 63.66% of reads mapped to the reference genome but the authors report this increased with centrifugation delay in specimens stored in EDTA tubes. Bisulphite conversion was 99.3% efficient with 89.71% of fragments mapping to MspI fragments. Importantly, there were no differences in duplicate percentage, bisulphite conversion efficiency, and MspI mapping between tube types or timepoints. Genome wide CpG methylation increased slightly when blood in EDTA tubes was subjected to a processing delay (from 43.29% with no delay to 49.21% with a 72 h delay), but smaller increases were found with delayed processing when stored in Biomatrica or Streck tubes. However, the absolute number of unfiltered CpGs detected increased with processing delay in EDTA tubes but decreased when stored in Biomatrica, Streck, or Roche tubes. Despite the observed changes, principal component analysis clustered all specimens based on patient, not tube type or processing delay, and the reproducibility as assessed by the ALC was comparable between all specimens. Nevertheless, comparisons between the 0 and 72 h timepoints identified 2 regions differentially methylated in Roche tubes and 51 regions differentially methylated in EDTA tubes (44 increased methylation, 7 decreased), but no DMRs were found between timepoints for specimens in Biomatrica, Streck, or PAXgene tubes and no differences were noted between the 0 and 24 h timepoints in any tube type. Further analysis of the DMRs in EDTA tubes found upregulation of the apoptosis pathway, apoptotic processes, and NK cell activation. Non-negative least squares (NNLS) and CelFie analysis of the sequences found NK cell contribution increased and neutrophil fraction decreased with delayed processing of blood in EDTA tubes but no change in abundance of erythrocyte progenitor sequences. A small increase in NK sequence abundance appeared to occur with storage in Streck DNA tubes but the effect was only observed in specimens from one of the two donors.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

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

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Bisulfite conversion assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K2 EDTA spray tubes Streck Cell-Free DNA BCT tubes PAXgene Blood ccfDNA tubes Roche Cell-Free DNA Collection tubes Biomatrica LBgard blood tubes
   Next generation sequencing Specific	Data handling	Non-negative least squares deconvolution CelFie deconvolution
   Storage	Storage duration	0 h 24 h 72 h
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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