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Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients.

Author(s): Neuberger EWI, Brahmer A, Ehlert T, Kluge K, Philippi KFA, Boedecker SC, Weinmann-Menke J, Simon P

Publication: Sci Rep, 2021, Vol. 11, Page 13581

PubMed ID: 34193884 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper investigated how cfDNA yield and/or fragmentation were potentially affected in capillary and venous blood by the time elapsed between exercise and blood collection, the duration of pre-centrifugation delays, centrifugation speed and temperature, the duration of frozen storage and the number of freeze-thaw cycles of plasma, and cfDNA extraction method. Venous and capillary (fingerprick) blood were collected from 28 patients with systemic lupus erythematosus before, immediately following and 90 min after a stepwise exercise test. During the exercise test the speed and elevation of the treadmill increased every 3 min until the participants stopped voluntarily. Unless otherwise specified, plasma was isolated from venous blood by immediate dual-centrifugation at 2500 x g for 15 min and from capillary blood by centrifugation at 600 x g for 10 min after an unspecified delay at 4°C. Unless otherwise specified, cfDNA copy number was evaluated by direct real-time PCR amplification of 90 and 222 bp fragments of L1PA2 from thawed plasma diluted 1:10 in DNase/RNase-Free H2O. Potential effects of delayed centrifugation were assessed using case-matched capillary and venous blood specimens from 3 patients that were stored at room temperature for 0, 60 min, 90, and 180 min before plasma separation. To evaluate the stability of cfDNA in diluted plasma, 20 plasma specimens were diluted 1:10 in DNase/RNAse free waterand were analyzed immediately and again after storage for >2 months at -20°C.  To evaluate the effects of centrifugation speed, case-matched blood from three subjects was centrifuged for 10 min at either 600 x g or 2500 x g at 4°C and room temperature. To evaluate potential effects associated with extraction method, cfDNA levels and fragmentation was assessed in four aliquots of the same thawed plasma sample by direct amplification and after cfDNA isolation with the QIAamp Circulating Nucleic Acid kit, NucleoSnap plasma cfDNA kit, and the QIAamp DNA Blood Mini kit.

   Summary of Findings:

   Inter-individual variability in cfDNA levels were higher in case-matched capillary blood than venous blood specimens. When blood was stored at room temperature prior to centrifugation, cfDNA levels increased nonsignificantly with time, but the authors report the difference was not clinically relevant. cfDNA levels were slightly higher in plasma from all three subjects when centrifugation was at 2500 x g compared to 600 x g, but the authors state no effect of centrifugation speed. There was no obvious effect of centrifugation temperature on cfDNA levels. Isolation of cfDNA resulted in some cfDNA loss compared to direct analysis of plasma specimens, with a 12% and 23% reduction in cfDNA levels following extraction with the QIAamp Circulating Nucleic Acid kit and NucleoSnap plasma cfDNA kit, respectively, and a 84% reduction following isolation with the QIAamp DNA Blood Mini Kit. Further, the fragmentation index of cfDNA isolated with QIAamp DNA Blood Mini Kit was larger, indicating a loss of small cfDNA.  The authors report that storing diluted plasma at -20°C for > 2 months did not affect cfDNA concentration, nor did a second freeze-thaw event. Inter-individual variability in cfDNA levels was higher in capillary blood specimens in comparison to venous blood specimens. Capillary and venous cfDNA levels were highest immediately after the patient exercised and declined back toward baseline post-exercise. While capillary and venous cfDNA levels were strongly correlated pre-exercise (R² =0.933, P<0.0001) and 90 min post-exercise (R² =0.974, P<0.0001), they were only moderately correlated immediately following exercise (R² =0.726, P=0.0002). The authors report no difference in cfDNA yield or fragmentation between plasma from patients receiving medication and those who were unmedicated.  Importantly, when specimens were analyzed again, the cfDNA levels were very strongly correlated between measurements (r=0.925, P<0.0001).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Lupus

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of fluid acquisition	Finger/heel prick sampling Venipuncture
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Circulating Nucleic Acid kit NucleoSnap plasma cfDNA kit QIAamp DNA Blood Mini kit
   Storage	Time at room temperature	0 min 60 min 90 min 180 min
   Storage	Storage duration	0 months >2 months
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Multiple speeds compared Multiple temperatures compared
   Storage	Freeze/thaw cycling	1 cycle 2 cycles

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