Department of Obstetrics and Gynaecology, San Martino Hospital, University of Genoa, Italy
*E-mail: simone.ferrero@fastwebnet.it
I read with interest Aigner and colleagues article [1] published recently in Annals of Oncology. Although the authors should be congratulated for their results on the study of pleiotrophin and fibroblast growth factor, I would like to report some methodological issues concerning the measurement of vascular endothelial growth factor (VEGF) levels.
Despite the fact that the authors reported the use of serum for the measurement of various growth and angiogenesis factors in patients with testicular cancer, the blood samples were collected in EDTA-coated tubes. Adding anticoagulants to the blood samples prevents coagulation and, therefore, plasma, and not serum, was obtained after centrifugation of the whole blood.
The difference between plasma and serum is relevant, particularly when VEGF is measured. In serum, the VEGF level is several-fold higher than in matched plasma samples, owing to the in vitro release of VEGF from platelets during blood clotting [2].
In general, plasma should be preferred to serum for the evaluation of circulating extracellular VEGF at the time of blood sampling. However, recent studies suggested that platelet-derived VEGF also reflects the biology of cancer cells and serum should be used for the measurement of VEGF levels in cancer patients. Platelets are a rich source of stimulators and inhibitors of angiogenesis. They may adhere to tumour vessels and release granules that contain potent angiogenesis stimulators. Alternatively, because platelets may scavenge several hormones and proteins, they may also scavenge tumour-cell-released angiogenic stimulators and inhibitors from the tumour vasculature [3].
When VEGF is measured, the conditions of processing should be standardised and declared. Unfortunately, the authors did not report the length of time and force of centrifugation. Spinning the samples at different speeds or for different times may affect the plasma VEGF levels [4].
The authors observed a two-fold increase in the mean VEGF levels of testicular cancer patients when compared with healthy control subjects. However, individual values were scattered widely with serum levels from several cancer patients being in the normal range. EDTA plasma was used for the measurement of VEGF. EDTA influences platelet shape by changing Ca2+ concentration. This may increase the interpersonal variation in platelet activation and affect VEGF determination. Citrate, theophylline, adenosine and dipyridamole (CTAD) plasma should be used to measure circulating extracellular VEGF [5].
In light of these considerations, I believe that it would be interesting to evaluate the VEGF levels of testicular cancer patients on serum samples. Allowing whole blood samples to clot for 2 h at room temperature and standardizing the conditions of processing will increase the clinical value of serum VEGF determination in cancer patients. When plasma is used for the determination of circulating extracellular VEGF, CTAD anticoagulant should be preferred to EDTA.
REFERENCES
1. Aigner A, Brachmann P, Beyer J et al. Marked increase of the growth factors pleiotrophin and fibroblast growth factor-2 in serum of testicular cancer patients. Ann Oncol 2003; 14: 15251529.
2. Webb NJ, Bottomley MJ, Watson CJ, Brenchley PE. Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin Sci (Lond) 1998; 94: 395404.[ISI][Medline]
3. Pinedo HM, Verheul HM, DAmato RJ, Folkman J. Involvement of platelets in tumour angiogenesis? Lancet 1998; 352: 17751777.[CrossRef][ISI][Medline]
4. Hormbrey E, Gillespie P, Turner K et al. A critical review of vascular endothelial growth factor (VEGF) analysis in peripheral blood: is the current literature meaningful? Clin Exp Metastasis 2002; 19: 651663.[CrossRef][ISI][Medline]
5. Wynendaele W, Derua R, Hoylaerts MF et al. Vascular endothelial growth factor measured in platelet poor plasma allows optimal separation between cancer patients and volunteers: a key to study an angiogenic marker in vivo? Ann Oncol 1999; 10: 965971.[Abstract]