Abstract
Background:The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing.Methods:Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated.Results:Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P = 0.006; P < 0.001).Conclusion:The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.
Highlights
Angiogenesis is the physiological process of capillary sprouting and remodeling of an established network of blood vessels, and plays a central role in embryogenesis, the female menstruation cycle, as well as wound healing [1]
Twenty-nine healthy volunteers were included in the study and divided into three groups: 3 subjects were recruited for the comparison of serum and plasma preparation variants; 3 volunteers contributed to the evaluation of blood storage time; 10 were enrolled to evaluate the impact of blood withdrawal procedures and 13 volunteers with a median age of 59 years served as age-matched healthy control for the collective of pancreatic carcinoma patients
vascular endothelial growth factor (VEGF), thrombospondin 1 (TSP-1), platelet factor 4 (PF-4) and platelet derived endothelial cell growth factor (PD-ECGF) fluctuations in different plasma and serum preparations were analyzed by ELISA in blood samples of three healthy volunteers (1 female, 2 male) and three pancreatic cancer patients
Summary
Angiogenesis is the physiological process of capillary sprouting and remodeling of an established network of blood vessels, and plays a central role in embryogenesis, the female menstruation cycle, as well as wound healing [1]. Circulating VEGF values have shown prognostic or predictive potential for various types of cancer [6,8]. The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Results: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4◦C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Conclusion: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4◦C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential
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