Effect of blood sampling, processing, and storage on the measurement of complement activation biomarkers.
Author(s): Yang S, McGookey M, Wang Y, Cataland SR, Wu HM
Publication: Am J Clin Pathol, 2015, Vol. 143, Page 558-65
PubMed ID: 25780008 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effect of anticoagulant type, delayed centrifugation, thawing method and duration, freeze-thaw cycling, and storage of plasma on complement biomarker levels in serum and plasma.
Conclusion of Paper
Levels of all complement biomarkers were comparable in EDTA and citrated plasma, but serum had non-significantly higher levels of C4d and C3a than EDTA or citrated plasma. Complement factors in plasma were stable when EDTA or citrated whole blood was stored for up to 8 h and 4 h, respectively at room temperature or 24 h or 3h, respectively at 4°C, but longer pre-centrifugation storage resulted in increased C4d and C3a levels. Up to 60 min of thawing at room temperature had no effect on complement levels in EDTA or citrated plasma, but levels of Bb in serum increased when frozen specimens were thawed for more than 30 min at room temperature. While complement levels in EDTA plasma were similarly unaffected by thaw duration at 37°C, Bb, C3a, C5a, and C5b-9 all increased in serum and citrated plasma with increasing thaw durations at 37°C. The authors report no effect of up to 3 h of thawing EDTA plasma, citrated plasma or serum on ice. Compared to specimens thawed once on ice, C4d was elevated in serum or citrated plasma that was subjected to 3 or more thaw cycles on ice or once at room temperature and when EDTA plasma was subjected to 2 or more cycles of thawing at room temperature.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of anticoagulant type, delayed centrifugation, thawing method and duration, and freeze-thaw cycling on complement biomarker levels in serum and plasma. Blood from 3 donors was collected into EDTA, sodium citrate, and plain tubes. Plasma was obtained by centrifugation at 4°C at 1000 g for 10 min followed by centrifugation of the supernatant at 4°C at 12000 g for 2 min, and serum was obtained 30 min after venipuncture by centrifugation at 4°C at 1300 g for 10 min. Serum and plasma were aliquoted and frozen at -80°C. The investigation of thawing involved thawing in an ice bucket for 30 min or in water baths (room temperature or 37°C) for different durations and then refreezing at -80°C until analysis. To determine the effects of freeze-thaw cycling, specimens were thawed on ice or in a room temperature water bath and upon thaw, transferred on ice to a -80°C freezer.
Summary of Findings:
Levels of all complement biomarkers were comparable in EDTA and citrated plasma, and levels of Bb, C5a and C5b-9 were comparable to those in serum. However, serum had non-significantly higher levels of C4d and C3a than EDTA or citrated plasma. All complement factors in plasma were unaffected by pre-centrifugation storage of EDTA blood at room temperature for 8 h or at 4°C for 24 h or by pre-centrifugation storage of citrated blood at room temperature for 4 h or at 4°C for 3 h; however, C4d increased when citrated or EDTA blood was stored prior to centrifugation at 4°C for 24 h (1.80 fold) and 48 h (1.58-fold), respectively, and C3a levels increased when citrated blood was stored prior to centrifugation at room temperature for 8 h (1.94-fold) or when citrated or EDTA blood was stored refrigerated for 48 h (1.53-fold and 2.49-fold, respectively). Up to 60 min of thawing at room temperature had no effect on complement biomarker levels in EDTA or citrated plasma, but levels of Bb in serum increased when specimens were thawed for more than 30 min at room temperature. While complement levels in EDTA plasma were similarly unaffected by thaw duration at 37°C, Bb, C3a, C5a, and C5b-9 all increased in serum and citrated plasma with increasing thaw durations at 37°C. The authors report no effect of up to 3 h of thaw time for EDTA plasma, citrated plasma or serum on ice. Compared to levels in specimens thawed once on ice, C4d was elevated when serum or citrated plasma was subjected to 3 or more thaw cycles on ice or once at room temperature and when EDTA plasma was subjected to 2 or more thaw cycles at room temperature.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant EDTA
Sodium citrate
None
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Time at room temperature 0 h
1 h
2 h
4 h
8 h
Storage Storage duration 0 h
3 h
24 h
48 h
Storage Thaw duration 5 min
10 min
30 min
60 min
3 h
Storage Thaw temperature/condition On ice
Room temperature water bath
37°C water bath
Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
-
Study Purpose
The purpose of this study was to determine the effect of long-term storage at -80°C on complement levels in citrated plasma from patients with thrombotic thrombocytopenic purpura (TTP). 88 citrated plasma specimens from 51 patients with TTP were collected over 6 years and stored at -80°C until analysis.
Summary of Findings:
Up to 6 years of storage of plasma at -80°C had no effect on complement C3a, C4d, Bb, C5a and C5b-9 levels.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 years
1 year
2 years
3 years
4 years
5 years
6 years