Circulating VEGF as a biological marker in patients with rheumatoid arthritis? Preanalytical and biological variability in healthy persons and in patients.
Author(s): Hetland ML, Christensen IJ, Lottenburger T, Johansen JS, Svendsen MN, Hørslev-Petersen K, Nielsen L, Nielsen HJ
Publication: Dis Markers, 2008, Vol. 24, Page 1-10
PubMed ID: 18057530 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of preanalytical variables on VEGF levels in serum and plasma from healthy subjects and patients with rheumatoid arthritis (RA).
Conclusion of Paper
Serum had much higher levels of VEGF than plasma, however, for healthy individuals a significant correlation between serum and EDTA-plasma VEGF levels was found. Collection and storage at room temperature tended to increase VEGF levels in plasma while serum VEGF increased after storage at 4 degrees C or room temperature. In healthy subjects, faster centrifugation led to higher VEGF levels in plasma, but had no effect on VEGF in serum or plasma specimens from RA patients. Plasma VEGF levels varied based on supernatant strata level while serum VEGF levels did not. VEGF levels in EDTA-plasma were also affected by exercise and freeze-thaw cycling. Day to day variation in VEGF levels was high and some differences were attributed to collection time.
Studies
-
Study Purpose
The purpose of this study was to determine the effects of delayed centrifugation, centrifugation speed (1200 x g, 1700 x g, 2000 x g, 2200 x g, 2500 x g, or 3000 x g) and supernatant strata on VEGF levels in serum and plasma specimens from healthy subjects and patients with RA. All specimens were frozen at -80 degrees C prior to analysis.
Summary of Findings:
Serum VEGF was much higher than plasma VEGF regardless of the anticoagulant used. Heparin and EDTA-plasma collected at room temperature had 2 fold more VEGF than citrate-plasma or EDTA-plasma collected on ice. Storing plasma for up to 24 h, at 4 degrees C, prior to centrifugation had no effect on VEGF levels, but when plasma was stored at room temperature, VEGF increased with storage in control and RA patients. Serum VEGF increased after storage for 1 h at 4 degrees C (3%) or room temperature (10%). In healthy subjects, faster centrifugation led to higher VEGF levels in plasma but had no effect on VEGF in serum or plasma specimens from RA patients. Plasma VEGF in healthy and RA subjects was higher in the supernatant closest to the buffy coat than in supernatant taken from the upper strata. No effect of strata location was seen on serum VEGF levels.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Rheumatoid Arthritis
Platform:
Analyte Technology Platform Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Citrate
EDTA
Heparin
None
Storage Storage temperature 4 degrees C
Room temperature
Storage Storage duration 30 min
45 min
60 min
2 h
3 h
4 h
6 h
24 h
Biospecimen Aliquots and Components Biospecimen components Top stratum
Medium stratum
Lower stratum
Lowest stratum next to buffy coat
Preaquisition Diagnosis/ patient condition Healthy
Rheumatoid arthritis
Biospecimen Aliquots and Components Centrifugation Multiple speeds compared
Centrifugation delays investigated
Biospecimen Aliquots and Components Blood and blood products Serum
Plasma
-
Study Purpose
The purpose of this study was to investigate the effects of repeated thawing of plasma and serum and long term storage of plasma on VEGF levels. All specimens were frozen at -80 degrees C prior to analysis.
Summary of Findings:
VEGF was higher in EDTA-plasma specimens thawed once compared to those thawed multiple times, but no differences were found in EDTA-plasma thawed between 2 and 9 times or in serum thawed up to 9 times. Thawing 9 times compared to 10 times decreased EDTA-plasma VEGF by 8% and serum VEGF by 18%. A non-significant decrease in VEGF was observed in EDTA-plasma specimens stored for 2 years at -80 degrees C.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
5 cycles
6 cycles
7 cycles
8 cycles
9 cycles
10 cycles
Storage Storage duration 0 years
2 years
Storage Storage temperature -80 degrees C
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
-
Study Purpose
The purpose of this study was to determine the effects of patient age, gender, and condition, collection time and exercise on VEGF levels in EDTA-plasma and serum specimens. All specimens were frozen at -80 degrees C prior to analysis.
Summary of Findings:
A significant correlation between serum and EDTA-plasma VEGF levels was found (r=0.67; p<0.0001), but in healthy volunteers, there was no correlation of VEGF levels to patient gender, diagnosis or age. EDTA-plasma VEGF was significantly lower at 7AM than at 1 PM (27%; p=0.007) or 4 PM (18%; p=0.006), but no consistent differences were observed between specimens collected at 8 AM and 2 PM. Substantial day to day variability was observed in EDTA-plasma VEGF levels (39-66%). EDTA-plasma VEGF levels increased in healthy and established RA subjects immediately after exercise when compared to pre-exercise levels but returned to pre-exercise levels within 1 h. Exercise had no effect on VEGF levels in patients with early RA.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Rheumatoid Arthritis
Platform:
Analyte Technology Platform Protein ELISA Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Preaquisition Patient age 11-20 y
21-30 y
31-40 y
41-50 y
51-60 y
61-70 y
Preaquisition Patient gender Female
Male
Preaquisition Diagnosis/ patient condition Healthy
Early rheumatoid arthritis
Established erosive rheumatoid arthritis
Biospecimen Acquisition Time of biospecimen collection 7 AM
8 AM
1 PM
2 PM
4 PM
Day 1
Day 2
Day 8
Day 15
Day 22
Month 6
Month 12