Factors affecting stability of plasma brain-derived neurotrophic factor.
Author(s): Wessels JM, Agarwal RK, Somani A, Verschoor CP, Agarwal SK, Foster WG
Publication: Sci Rep, 2020, Vol. 10, Page 20232
PubMed ID: 33214644 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare brain‑derived neurotrophic factor (BDNF) concentrations in K2EDTA, lithium heparin (with and without gel), and sodium heparin (plastic and glass tube) plasma specimens that were collected from healthy men and women during a 12 month storage period at either -20°C or -80°C. Potential effects of freeze-thaw cycling K2EDTA plasma specimens over the course of a year of storage at -80°C on BDNF levels were also investigated.
Conclusion of Paper
BDNF concentrations were significantly lower in K2EDTA plasma than in the lithium heparin (with and without gel) and plastic tube sodium heparin plasma; however, when patient sexes were considered separately, the difference was only observed in plasma from women. The authors report that BDNF levels were not affected by patient age, sex, or menstrual cycle stage in a multivariable linear mixed effects model. BDNF levels increased when plasma was stored at -20°C or -80°C, but the increases were not progressive with time and significance depended on the type of anticoagulant used and the storage duration. For most time-points the concentration of BDNF was comparable in tubes stored at -20°C and -80°C, but higher BDNF concentrations were observed in all specimen types stored for 6 months at -20°C compared to those stored at -80°C. BDNF concentrations in K2EDTA plasma increased progressively and significantly with each freeze-thaw cycle (1-3 cycles).
Studies
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Study Purpose
The purpose of this study was to compare BDNF concentrations in K2EDTA, lithium heparin (with and without gel), and sodium heparin (plastic and glass tube) plasma specimens collected from healthy men and women during a 12 month storage period at either -20°C or -80°C. Potential effects of freeze-thaw cycling K2EDTA plasma specimens over the course of a year of storage at -80°C on BDNF levels were also investigated. To investigate the stability of BDNF in different plasma types, blood was collected from 24 healthy volunteers (14 females, 10 males) into K2EDTA, lithium heparin, lithium heparin gel separator, plastic sodium heparin plastic, and glass sodium heparin tubes and placed on wet ice. Plasma was obtained by centrifugation at 3000 g for 30 min and aliquoted for storage at -20°C and -80°C. After 0, 1, 3, 6 and 12 months an aliquot was thawed at 4°C overnight and BDNF was quantified by ELISA. To investigate potential effects of freeze-thaw cycling, K2EDTA blood collected from 10 healthy women (mean age 34.3±6.1 years) was centrifuged at 3000 g for 30 min within 30 min of blood collection. Plasma was stored at -80°C for < 6 months prior to first thawing and then was freeze thaw cycled two more times in the next 6 months. Plasma was thawed at room temperature before quantification of BDNF by ELISA.
Summary of Findings:
Although the type of anticoagulant used did not affect BDNF concentrations in plasma collected from healthy male volunteers, a significant effect of anticoagulant on BDNF concentrations in plasma of females was observed (P=0.0001). K2EDTA plasma from females had significantly lower concentrations of BDNF than lithium heparin (without gel P<0.001 and with gel P<0.05), and plastic sodium heparin (P<0.001) plasma. When data from males and females were combined, K2EDTA plasma had significantly lower concentrations of BDNF than lithium heparin (without gel P<0.001 and with gel P<0.01), and plastic sodium heparin (P<0.001) plasma. The authors report that BDNF levels were not affected by patient age, sex, or menstrual cycle stage in a multivariable linear mixed effects model. BDNF levels increased when plasma was stored at -20°C, with significant increases noted for K2EDTA plasma after 12 months (P<0.0001), and all other plasma types after 1 month (P<0.05 for lithium heparin and both sodium heparin tubes; P<0.01 for lithium heparin gel tube); but for all anticoagulants, concentrations fell outside the expected range at one or more time point(s) and the increase was not progressive with storage duration. BDNF levels increased when plasma was stored at -80°C, storage at -80C for 12 months led to significant increases and higher than expected mean BDNF concentrations regardless of the type of anticoagulant used (P<0.001 for all). For most time-points BDNF concentrations were comparable among tubes stored at -20°C and -80°C, but higher BDNF concentrations were observed in all specimens types stored for 6 months at -20°C compared to those stored at -80°C; in sodium heparin (both) and lithium heparin (both) plasma stored for 1 month at -20°C compared to those stored at -80°C; and in plastic sodium heparin plasma stored for 3 months at -80°C compared to those stored at -20°C. BDNF concentrations in K2EDTA plasma increased progressively with each freeze-thaw cycle with significant differences observed between 1 cycle and 2 cycles (P<0.05) or 3 cycles (P<0.0001) and between 2 and 3 cycles (P<0.01).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution K2EDTA tube
Lithium heparin without gel separator
Lithium heparin with gel separator
Plastic sodium heparin
Glass sodium heparin
Biospecimen Acquisition Anticoagulant Sodium EDTA
Lithium heparin
Sodium heparin
Storage Storage temperature -20°C
-80°C
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
Storage Storage duration 0 months
1 month
3 month
6 month
12 month