Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Pre-analytical pitfalls: How blood collection tubes influence exercise-induced cell-free DNA concentrations.

Author(s): Enders K, Hillen B, Haller N, Brahmer A, Weber V, Simon P, Neuberger EWI

Publication: Exp Physiol, 2025, Vol. , Page

PubMed ID: 40033650 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared cfDNA levels in serum, lithium heparin plasma and EDTA plasma that were collected before and 5- and 30-min post-exercise. The eleven participants completed one treadmill exercise protocol the first day of the study and then completed two other exercise protocols on the same day a week later. Blood was collected from eleven volunteers before, and 5 and 30-min after exercising into K₃EDTA, lithium–heparin and z-gel clot activator (serum) Monovette tubes during three exercise tests. During the first visit, exercise consisted of an incremental running workout on a treadmill that started at 6 km/h and increased by 2 kg/h every 3 min and ended at voluntary exhaustion. During the morning of their second visit (a week later), the volunteers warmed up for 10 min at 50% of their anaerobic threshold (calculated based on lactate levels), then ran for 18 min at 90% of their anaerobic threshold, followed by six intervals of 3 min alternating between 110% and 70% of their anaerobic threshold. At noon of the second visit, volunteers warmed up for 10 min at 50% of their anaerobic threshold, then ran six intervals of 3 min alternating between 110% and 70% of their anaerobic threshold, followed by 18 min at their anaerobic threshold. During exercise, air volume and gas were measured continuously by spiroergometry, heart rate was monitored by a 12-channel ECG, and lactate levels were measured in capillary blood obtained from the ear lobe. The rating of perceived exertion was assessed every 3 min on a scale of 6-20. After collection, blood specimens were inverted, and serum tubes were allowed to clot for 30 min. Within 5 min of collection, plasma was separated by centrifugation twice at 2500 x g for 15 min. Serum was separated by a single centrifugation at 2500 x g for 15 min. Plasma and serum were stored at -80°C until cfDNA quantification. Serum and plasma were thawed at room temperature, diluted in DNase/RNase-free water, and used directly for real-time PCR amplification of a 90 bp region of the L1PA2 region.

   Summary of Findings:

   Mean pre-exercise cfDNA concentration differed among the tube types evaluated, with the highest levels observed in serum (41.9 to 77.1 ng/ml), followed by lithium heparin plasma (18.2 to 26.2 ng/ml), and the lowest cfDNA levels observed in EDTA plasma (13 to 14.8 ng/ml), but the significance of the difference depended on the exercise test and the tubes compared. cfDNA levels were highest 5 min post-exercise in all tube types and exercise regime evaluated, but the magnitude of the observed differences were tube type dependent. At the 5 min timepoint following each of the three exercise regimes, the largest fold increase in cfDNA levels were observed in EDTA plasma (9.8-, 9.1- and 14.6-fold, respectively; P< 0.0001 all), followed by lithium heparin plasma (7.1-, 7.8-, and 8.5-fold, respectively; P<0.001, P<0.0001 and P<0.0001, respectively), and small but significant increases observed in serum (4.5-, 4.6- and 3.7-fold, respectively; P<0.001, P<0.001 and P<0.0001, respectively). At the 30 min timepoint following each of the three exercise regimes, the increases in cfDNA levels relative to the pre-exercise baseline were comparably smaller but still significant in EDTA plasma (2.4-, 3.5- and 4.8-fold, respectively; P<0.0001, P<0.001 and P<0.0001, respectively); however, the significance of the increase in lithium heparin plasma was limited to the second and third exercise test (3.2- and 2.9-fold, respectively; P<0.001, and P<0.05, respectively), was limited to the first and second exercise test and in serum (2.1-, and 2.0-fold, respectively; P<0.05, both). ANOVA analysis identified a significant interaction between exercise regime, time point and specimen type on cfDNA concentration [F(2.86, 28.58) = 3.364, P = 0.034]. A significant effect of exercise regime, time point and specimen type on the fold change in cfDNA level was also identified [F(4, 40) = 3.703, P = 0.012]. The absolute change in cfDNA levels at 5 min post-exercise relative to pre-exercise levels were strongly correlated among the tube types (ρ=0.94 for EDTA and lithium heparin plasma, P<0.0001; ρ=0.78 for EDTA plasma and serum, P<0.0001; ρ=0.82 for lithium heparin plasma and serum, P<0.0001). The absolute change and fold change in cfDNA levels at 5 min post-exercise were also strongly correlated with VO_2peak in EDTA plasma (ρ=0.72, P=0.013 and ρ=0.90, P=0.00016, respectively), and lithium heparin plasma (ρ=0.79, P=0.0037 and ρ=0.86, P=0.00061, respectively), and more modestly correlated in serum (ρ=0.75, P=0.0073 and ρ=0.62, P=0.043, respectively). Mean VO₂ was correlated with the fold change in cfDNA levels at 5 min post-exercise in EDTA (ρ=0.81, P=0.0026) and lithium heparin plasma (ρ=0.69, P=0.019) but not serum. The absolute change and fold change in cfDNA levels at 5 min post-exercise were also correlated with energy expenditure in EDTA plasma (ρ=0.70, P=0.016 and ρ=0.91, P=0.00011, respectively), and lithium heparin plasma (ρ=0.75, P=0.0084 and ρ=0.85, P=0.001, respectively). Fold change in cfDNA levels at 5 min post-exercise was also correlated with energy expenditure in serum (ρ=0.65, P=0.028). In the two endurance tests (exercise regimes 2 and 3), energy expenditure was correlated with both the absolute and fold change in cfDNA levels at 5 min in EDTA (ρ=0.49, P=0.0019 and ρ=0.49, P=0.022, respectively) and lithium heparin plasma (ρ=0.44, P=0.04 and ρ=0.44, P=0.041, respectively). Lactate levels were correlated with the fold change in cfDNA levels at 5 min post-exercise in lithium heparin plasma (ρ =0.52, P=0.02) and serum (ρ =0.49, P=0.03).

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   Small molecule	Clinical chemistry/auto analyzer
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube Lithium heparin tube Serum tube
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Lithium heparin None
   Biospecimen Acquisition	Time of biospecimen collection	5 min prior to exercise (baseline) 5 min post-exercise 30 min post exercise First exercise regime-in the morning Second exercise regime- 1 week following first in the morning Third exercise regime- At noon same day as second exercise regime

   View more