Evaluating the effects of preanalytical variables on the stability of the human plasma proteome.
Author(s): Hassis ME, Niles RK, Braten MN, Albertolle ME, Ewa Witkowska H, Hubel CA, Fisher SJ, Williams KE
Publication: Anal Biochem, 2015, Vol. 478, Page 14-22
PubMed ID: 25769420 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of delayed centrifugation, centrifugation protocol, plasma storage, and freeze-thaw cycling on the plasma proteome and long-term frozen storage on the serum proteome.
.
Conclusion of Paper
Delaying centrifugation of blood by 6 h only led to increased plasma levels of paraoxonase arylesterase (PON1), but when centrifugation was delayed by 96 h at room temperature or 37°C, there were significant increases in the levels of 39 and 56 proteins, respectively, and decreases in the levels of 2 and 27 proteins, respectively. The single centrifugation protocol resulted in 6.7-fold higher plasma levels of thrombospondin-1 (TSP1) than the standard double centrifugation protocol. PON1 levels increased when plasma was stored for 24 h at room temperature rather than processed immediately after thawing. Compared to plasma and serum specimens stored at -80°C for <3 months, specimens stored 14-17 years at -80°C had 1ower alpha-2-macroglobulin levels in plasma and serum and higher complement C1q subcomponent subunit B and plasma protease C1 inhibitor levels in plasma. However, the authors note many of the identified proteins can be non-specifically retained by immunodepletion columns.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of delayed centrifugation, centrifugation protocol, storage of plasma at room temperature, and freeze-thaw cycling of plasma on total protein levels and the abundance of specific proteins by liquid chromatography mass spectrometry (LC-MS) with iTRAQ labeling. 40 specimens were collected into K2EDTA tubes and either centrifuged within 30 min or stored at room temperature for 6 h prior to centrifugation. Centrifugation included spinning specimens at 1500 g for 15 min, transferring them to a new tube, and centrifuging again at 2200 g for 15 min. Specimens were then aliquoted, transported on dry ice and stored at -80°C. The specimens were also used to determine the effects of 1-3 additional freeze-thaw cycles of plasma and storage of thawed plasma at room temperature for 24 h. An additional cohort of 18 specimens were collected into K2EDTA tubes and subjected to the same double centrifugation protocol outlined above or they were centrifuged once at 1200 g for 20 min within 30 min or after 96 h at room temperature or 37°C. Specimens were then aliquoted, transported on dry ice and stored at -80°C. Plasma was immunodepleted using MARS-Hu14 columns.
Summary of Findings:
Storing blood for 6 h rather than 30 min prior to centrifugation, up to an additional 3 freeze-thaw cycles, and storage of plasma at room temperature for 24 h before analysis had no effect on the total plasma protein levels. Plasma levels of PON1 increased 2-fold when blood centrifugation was delayed by 6 h instead of 30 min (q=7.2 x 10^4), but when blood was stored for 96 h at room temperature or 37°C before centrifugation, there were significant increases in the levels of 39 and 56 proteins, respectively, and significant decreases in the levels of 2 and 27 proteins, respectively. The single centrifugation protocol resulted in 6.7-fold higher plasma levels of TSP1 than the standard double centrifugation protocol (q=0.02). PON-1 levels increased 4.3-4.4-fold when plasma was stored for 24 h at room temperature rather than processed immediately after thawing (q<2.5 x 10^-9), but up to 3 additional freeze-thaw cycles did not affect the proteome. However, the authors note that 21 of the affected proteins can be non-specifically retained by immunodepletion columns.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Different number of centrifugation steps compared
Storage Storage temperature Room temperature
37°C
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
Storage Time at room temperature <30 min
6 h
96 h
Storage Post-thaw duration 0 h
24 h
-
Study Purpose
The purpose of this study was to determine the effects of 14-17 years of frozen storage on the proteome of immunodepleted plasma and serum. Paired serum and K2EDTA plasma specimens were obtained from 20 pregnant women (mean gestational age =40 weeks) and frozen at -80°C for 14-17 years with 2-4 freeze-thaw cycles. These specimens were compared to 10 paired serum and K2EDTA plasma specimens from pregnant women (mean gestational age 35.9 weeks) that had been stored at -80°C for <3 months. Plasma and serum were immunodepleted using MARS-Hu14 columns.
Summary of Findings:
A total of 267 plasma proteins and 279 serum proteins were identified, but only 183 plasma proteins and 212 serum proteins were identified in at least 5 specimens, allowing for statistical analysis. Compared to specimens stored at -80°C for <3 months, specimens stored 14-17 years at -80°C had 12.9-fold (q=1x10-7) and 17.3-fold (q=2x10^-11) lower alpha-2-macroglobulin levels in plasma and serum, respectively. Additionally, plasma stored at -80°C for 14-17 years had higher complement C1q subcomponent subunit B (2.9-fold; q=0.02) and plasma protease C1 inhibitor (9-fold; q=0.03) than that stored for <3 months. However, the authors note alpha-2-macroglobulin and plasma protease inhibitor can be non-specifically retained by immunodepletion columns.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform Protein LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration <3 months
14-17 years
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum