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Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.

Author(s): Roch B, Pisareva E, Mirandola A, Sanchez C, Pastor B, Tanos R, Frayssinoux F, Diab-Assaf M, Anker P, Al Amir Dache Z, Thierry AR

Publication: Clin Chim Acta, 2024, Vol. 553, Page 117711

PubMed ID: 38101467 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated how the yield of cell-free mitochondrial DNA (cfmtDNA) is affected by isolating plasma using a standard protocol (SP) versus a protocol designed to reduce platelet activation (PA; protocol w/o PA) and to assess the effects of removing vesicle-associated cfmtDNA by additional steps involving filtration (0.22 µm filter) or centrifugation (16,000 x g for 10 min at 4°C) immediately or after frozen storage at -20°C. Blood was collected from seven healthy volunteers (five male and two female) into EDTA vacutainer tubes and citrate-theophylline-adenosine-dipyridamole (CTAD) tubes. Plasma was obtained from EDTA blood by the standard protocol (SP), which consisted of centrifugation at 1200 x g for 10 min at 4°C. Plasma was obtained from CTAD tubes using the protocol without platelet activation (protocol w/o PA), which consisted of centrifugation twice at 200 x g for 10 min at room temperature, centrifugation at 300 x g for 10 min at room temperature, addition of prewarmed citrate dextrose buffer and prostaglandin, centrifugation at 1100 x g for 10 min at room temperature, and centrifugation at 2500 x g for 10 min at room temperature. To test the effects of removing vesicle-associated cfmtDNA by filtration and freezing, plasma aliquots obtained by the SP and the protocol w/o PA were subjected to: 1) no additional treatment, 2) filtration through a 0.22 µm filter, 3) centrifugation at 16,000 x g for 10 min at 4°C, or 4) storage at -20°C before centrifugation at 16,000 x g for 10 min at 4°C. cfDNA was extracted from plasma using the QIAamp DNA Blood Mini Kit and stored at -20°C. cfDNA was quantified and integrity evaluated by real-time PCR amplification of short and long amplicons of nuclear KRAS (67 bp and 320 bp) and mitochondrial MT-CO3 (67 bp and 310 bp) and integrity evaluated.

   Summary of Findings:

   In plasma prepared using the SP, cfmtDNA had a ratio of long (310 bp) to short (67 bp) fragments that was close to 1 (0.96-1.07), but nuclear DNA had lower ratios of long to short fragments (0.09-0.48). When plasma obtained by the SP included a filtration (0.22 µm) or high-speed centrifugation step (16,000 x g) the cfmtDNA yield decreased by 98.7% and 99.4%, respectively (P=0.0004, both). The authors state that the decrease is due to removal of vesicle-associated cfmtDNA. Interestingly, plasma that was frozen before high-speed centrifugation had 1.2-fold higher levels of cfmtDNA than plasma that underwent high-speed centrifugation directly after plasma isolation (P=0.01), indicating that freezing releases some of the vesicle-associated cfmtDNA that was otherwise eliminated by high-speed centrifugation. In plasma obtained using the protocol w/o PA, subsequent filtration or high-speed centrifugation reduced the cfmtDNA yields by 80.04% (P=0.012) and 75.67% (P=0.010), respectively; there was no significant effect of adding a freezing step before high-speed centrifugation versus high-speed centrifugation alone. The decrease in mtDNA following filtration or high-speed centrifugation was much larger when plasma was obtained by the SP compared to the protocol w/o PA (64-fold versus 5-fold and 128-fold versus 4-fold, respectively). The proportion of cfmtDNA to total cfDNA was also lower in specimens that were processed without a high-speed centrifugation or filtration step when processed using the protocol w/o PA than when the SP was used (0.5% versus 19.5%). According to the authors, this supports that platelet activation is a confounding factor that must be considered when studying cfmtDNA. 

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Citrate-theophylline-adenosine-dipyridamole EDTA
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Different number of centrifugation steps compared Multiple speeds compared
   Biospecimen Aliquots and Components	Filtration	Plasma filtered through 0.22 µm filter Not filtered
   Biospecimen Aliquots and Components	Blood processing method	Without platelet activation (CTAD blood centrifuged twice at 200g for 10 min, 300 g for 10 min, mixed with ACD and prostaglandin, centrifuged at 1100g for 10 min, centrifuged at 2500 x g for 10 min) Standard protocol (EDTA blood centrifuged at 1200g for 10 min)

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