Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays.
Author(s): Shen Q, Björkesten J, Galli J, Ekman D, Broberg J, Nordberg N, Tillander A, Kamali-Moghaddam M, Tybring G, Landegren U
Publication: Clin Chem Lab Med, 2018, Vol. 56, Page 582-594
PubMed ID: 29040064 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of storing blood at 4˚C or 22˚C before centrifugation and freeze-thaw cycling of plasma on the protein profile of plasma. Differences in the protein profiles of dried blood spots (DBS) and dried plasma spots (DPS) were compared for changes that occurred with delayed centrifugation.
Conclusion of Paper
In general, protein levels increased with increasing storage duration before centrifugation and changes were larger when stored at 22˚C rather than 4˚C, but levels of MMP7 declined progressively with storage at 22 ˚C; however, no effect of freeze-thaw cycling plasma on the protein profile was found. Of the 41 proteins found to be increased with precentrifugation delay at 22˚C, 38 were found at higher levels in DBS than DPS and the remaining three were not in the assay. Interestingly, MMP7 and some proteins unaffected by storage were also found at higher levels in DBS than DPS.
Studies
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Study Purpose
This study investigated the effects of storing blood at 4˚C or 22˚C before centrifugation and freeze-thaw cycling of plasma on the protein profile of plasma. Differences in the protein profiles of DBS and DPS were compared for changes that occurred with delayed centrifugation. Blood was collected from 16 healthy volunteers into ten 4 mL K2EDTA tubes. Tubes were mixed and then stored at room temperature for 1, 3, 8, 24, or 36 h at 4˚C or 22˚C before centrifugation at 2000 x g for 10 min. Plasma was aliquoted and stored at -80˚C for approximately 1 month. Specimens from 12 of the patients were stored for 1 h at 4 ˚C or 22˚C, centrifuged at 2000 x g for 10 min, aliquoted, stored at -80˚C, and subjected to 2, 4, or 8 freeze-thaw cycles (22˚C for 30 min). Proteins were quantified by the multiplex antibody and real-time PCR based proximity extension assay on the CVD I and inflammation panels. Whole blood from fingerpricks and EDTA plasma from venous blood from six healthy donors was dried on Whatman 903 sample collection cards for 3 h at room temperature. Proteins were obtained from 1.2 mm punches and quantified using the Olink CVD II and inflammation panels for the Proximity Extension Assay.
Summary of Findings:
Generally, protein levels increased with increasing storage duration and changes were larger when stored at 22˚C rather than 4˚C. Using a normalized protein expression (NPX) threshold for change of 0.5 compared to levels in the specimens obtained after an hour at 4˚C, storage for 3, 8, 24, and 36 h at 4˚C resulted in increases in 4%, 6%, 9%, and 15% of data points (139 proteins in 16 specimens/time point), respectively. Similarly, storage at 22˚C for 3, 8, 24, and 36 h resulted in greater than 0.5 NPX increases from the 1 h at 4˚C specimen in 5%, 10%, 16%, 28%, and 31% of data points, respectively. However, after 36 h at either temperature, only 2% of the data points were decreased and only MMP7 decreased progressively with storage at 22˚C. After 36 h at 4˚C, levels of 16 proteins increased and levels of 40 proteins increased after 36 h at 22˚C, including all 16 affected by storage at 4˚C. Although most proteins increased more after storage at 22˚C than 4˚C, the increases in interleukin 16 (IL-16) and NF-kappa-B essential modulator (NEMO) were greater in specimens stored at 4˚C than 22˚C. Further, levels of SRC and AXIN1 declined between 1 h and 3 h during storage at 22˚C and CXCL5 declined between 1 and 3 h of storage at 4˚C, but levels increased significantly with storage after the initial declines. Levels of the 82 proteins examined were unaffected by freeze-thaw cycling and were very strongly correlated between specimens freeze-thaw cycled twice and four or eight times (P=0.998, both).
Of the 150 proteins found in either DBS or DPS, 92 were found in plasma from the delayed centrifugation experiment. Of the 41 proteins found to be increased with precentrifugation delay at 22˚C, 38 were found at higher levels in DBS than DPS and the remaining three were not in the assay. MMP7, which declined during precentrifugation storage, was also higher in DBS than DPS. The proteins that were found at similar levels in DBS and DPS were generally unaffected by storage at room temperature. Although not among the most differently expressed protein between DBS and DPS, there were a few proteins that were higher in DBS than DPS and were not affected by storage, indicating that some proteins in blood could be more slowly released or be instable.
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immuno-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Method of fluid acquisition Finger/heel prick sampling
Venipuncture
Biospecimen Aliquots and Components Blood and blood products Plasma
Whole blood
Storage Freeze/thaw cycling 2 cycles
4 cycles
8 cycles
Storage Storage temperature 4˚C
22˚C
Storage Storage duration 1 h
3 h
8 h
24 h
36 h