J Thromb Thrombolysis (2006) 22:125–131 DOI 10.1007/s11239-006-8422-z Temporal and venepuncture-related decline in circulating endothelial cell capture from mixed venous blood Christopher J. Boos · Deirdre A. Lane · Delene Kang · Patrick K. Y. Goon · Andrew D. Blann · Gregory Y. H. Lip C  Springer Science + Business Media, LLC 2006 Abstract Background: The quantification of circulating en- dothelial cells (CECs) in whole blood has evolved as a novel method for the assessment of endothelial function, although major methodological issues remain. We hypothesized that there is a temporal decline in CEC counts in static venesected blood and that venepuncture itself may lead to increased CEC detachment. Methods: CEC isolation was performed using the im- munobead method. For the temporal decline experiment, we included 52 patients presenting with acute coronary syn- drome (ACS). We performed CEC counts immediately and at 4 and 24 h later. For the venepuncture decline experiment, we studied 40 patients with stable cardiovascular disease (CVD). CEC counts were determined from the first 4 mL of aspirated venous blood and compared with counts obtained from a sub- sequent 4 mL sample of blood after at least 7.5 mL of blood had been collected. Results: Among the ACS patients there was a significant temporal decline in CEC counts in static venous blood over a 24 h period (p = 0.013). Among the patients with stable CVD, the median CEC counts obtained from the initial 4 mL of aspirated venous blood were significantly higher (by 32%) than that obtained from the later 4 mL of aspirated venous blood (p = 0.041). Conclusions: We demonstrated a significant temporal fall in CEC numbers in static venous blood over 24 h following C. J. Boos . D. A. Lane . D. Kang . P. K. Y. Goon . A. D. Blann . G. Y. H. Lip () Haemostasis, Thrombosis and Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham UK, B18 7QH e-mail: g.y.h.lip@bham.ac.uk C. J. Boos Army Medical Directorate, Slim Rd, Camberley, UK, GU15 4NP venesection. Furthermore, we have shown that CEC counts are higher in the initial aspirated blood compared with that as- pirated from the same needle subsequently. These data would have implications for how CEC determination is undertaken by researchers in studies related to ACS or CVD. Keywords Circulating endothelial cells . Endothelial . Decline Introduction Endothelial damage/dysfunction has been shown to represent a vital step in the pathogenesis of cardiovascular disease. Cur- rent methods to assess endothelial damage/dysfunction in- clude the determination of changes in endothelial dependent flow mediated vasodilatation, the use of skin laser flowmetry and the quantification of known endothelial specific vascu- lar markers, such as levels of plasma von Willebrand factor [vWF] or soluble E selectin [1, 2]. The enumeration of blood borne endothelial cells repre- sents a novel advance in the assessment of endothelial pertu- bation. These endothelial cells can be broadly differentiated (mainly by the use of cell surface markers) into two distinct phenotypes: endothelial progenitor cells (EPCs, which are integral to the regeneration and formation of the endothelial monolayer), and circulating endothelial cells (CECs, which are thought to represent more mature endothelial cells that have become detached from the endothelium) [3]. CEC num- bers have been shown to be a specific and accurate marker of endothelial damage/dysfunction, with CEC numbers in- creasing in proportion to the degree of endothelial injury [4–9]. The two main methods used to capture CECs from whole blood are via the use of immunobeads or flow cytometry [3]. Springer