Circulating VEGF As a Biological Marker in Patients with Rheumatoid Arthritis? Preanalytical and Biological Variability in Healthy Persons and in Patients

Abstract

Background: Soluble vascular endothelial growth factor (VEGF) is a promising biomarker in monitoring rheumatoid arthritis (RA), but studies of pre-analytical and biologic variability are few. Methods: VEGF was measured by ELISA methods in serum and plasma from healthy persons and RA patients. Pre-analytical factors were investigated. A reference interval for VEGF was established in serum and plasma from 306 healthy persons. Diurnal, day-to-day, week-to-week, long-term variability, and impact of exercise were evaluated. Results: Delayed processing time, room temperature, low centrifugal force and contamination of plasma with cellular elements lead to significant increases in VEGF levels, whereas storage for up to 2 years at −80°C or up to 10 freeze/thaw cycles did not affect VEGF levels. Serum VEGF levels were 7–10 fold higher than plasma VEGF levels. Reference intervals for VEGF (plasma: 45 pg/ml (range: non-detectable to 352); serum: 328 pg/ml (53–1791)) were independent of gender and age. Short- and long-term biologic variability included diurnal variation (sampling should take place after 7 AM) and impact of exercise (increased VEGF immediately after bicycling normalised within 1 hour). Conclusions: Pre-analytical factors and biologic variability including diurnal variation and impact of exercise should be accounted for in future studies that include circulating VEGF as a biological marker.

Measuring myokines with cardiovascular functions: pre-analytical variables affecting the analytical output

“…In biobanking and longitudinal studies, samples are collected over time, stored freezers (−20 or −80 ℃), and then simultaneously analyzed in order to minimize inter-assay variability (209,210). However, as described in this article, the biomarkers are subject to variability arising from sampling and storage procedures (169). Erroneous sampling or handling and improper storage may lead to degradation of these markers, limiting their clinical reliability (211).…”

“…On the contrary, VEGF concentrations increased by 15% in both matrices (108). Similarly, Hetland et al found that up to ten freeze/thaw cycles of serum and EDTA-plasma samples from patients with RA, after 2 years of storage at −80 ℃, had no effects on VEGF immunoreactivity (169). These findings were in contrast with previous observations by Kisand and colleagues who showed that VEGF is very sensible to freeze/thaw: 67% decrease already after the first cycle.…”

“…Therefore, serum stored for long periods is not suitable for assessing VEGF (168). Hetland et al observed an increase in VEGF in serum stored at RT but not in EDTA-plasma (169).…”

“…Hetland and colleagues showed that VEGF has a peak at 7 a.m. and two nadirs at 1 and 4 p.m. with no differences among healthy subjects and RA patients. No circannual rhythm was, instead, found (169). In summary, it has been suggested that VEGF levels should be measured in plasma, possibly CTAD (154), since platelets activation during clotting could potentially contribute to VEGF release (139).…”

“…VEGF is affected by exercise, however, but depending on the pathological status: plasma VEGF was increased immediately after bicycling in the healthy persons (16%) and in patients with erosive RA (64%) but not in early RA (169). VEGF is not affected by age and gender (140).…”