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I read with interest the report by Rehman et al. (1) evaluating the circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in obese subjects. Although the investigators should be congratulated by their results on the study of HGF, I would like to underline some methodological concerns regarding the measurement of VEGF levels.
Serum VEGF is not a suitable indicator of circulating extracellular VEGF levels at the time of sampling; VEGF is stored in the α-granules of platelets and is released during blood clotting. As a consequence, VEGF level in the serum is several-fold higher than that in matched plasma samples (2,3). In plasma, platelet degranulation is minimized by adding anticoagulants to the blood samples; in particular, CTAD (citrate, theophylline, adenosine, dipyridamole) plasma is recommended for the measurement of circulating extracellular VEGF (4). The investigators (1) stated that “serum was separated after coagulation,” but they did not report the interval between venipuncture and separation of serum from blood cell; this interval should be standardized and declared. In serum, VEGF concentrations increase in a time-dependent manner (5). In a clinical situation, where blood samples are taken and left for variable times before processing, the contribution from the clotting process would make the measurement unreliable (5). Allowing the whole blood sample to clot for between 2 and 6 h before serum is collected reduces time-dependent, nonuniform release of VEGF (6). In addition, the researchers did not report the conditions of processing (centrifugal force and the length of centrifugation), which are relevant and should be standardized.
Finally, a direct correlation between platelet counts and serum VEGF has been described (6). When serum is used for the measurement of VEGF, it is advisable to correct the results for variations in platelet count and platelet size (3).
In conclusion, meticulous standardization of sampling is a mandatory step in studies on blood VEGF levels. The investigators’ conclusion that “serum VEGF levels were not significantly elevated in obese versus lean subjects” cannot be justified on the basis of the data presented.
- American College of Cardiology Foundation