Detection and quantification of mature circulating endothelial cells using flow cytometry and immunomagnetic beads: A methodological comparison

Abstract

Mature circulating endothelial cells (CECs) are novel cellular markers of endothelial damage/dysfunction. The two main techniques of CEC enumeration are flow cytometry (FC) and immunomagnetic bead (IB) isolation. Both quantify CECs accurately, but a direct comparison of both methods has not been reported. We sought to assess the agreement between the two methods in two patient populations, and a group of healthy subjects, with emphasis given to methodological issues. We included 34 patients with acute coronary syndrome (ACS), 60 patients with primary breast cancer (PBC) and 30 healthy controls (HC). We quantified CECs using the IB method [CD146 and FITCUlex europaeus lectin-1] and FC [CD45, CD34 and CD146]. Bland-Altman plots suggested reasonable agreement (<5% of events >2 standard deviations from the mean) between FC and the IB methods for CEC quantification in whole blood in the two disease groups (ACS and PBC), but not among the HCs. There were no statistically significant differences in CEC levels by the two methods amongst all three patient groups. There is reasonable agreement between the FC and the IB methods for mature CEC quantification in whole blood, especially amongst disease groups. The agreement between the two methods appears to weaken in healthy controls, and at lower and higher absolute CEC counts.

Circulating endothelial cells in oncology: pitfalls and promises

“…All these assays have to deal with the low number of CEC in peripheral blood, (typically 0 -20 CEC per ml in healthy donors) rendering such assays highly susceptible to errors in sampling, preparation, and analysis. For instance, several groups have demonstrated the negative impact of venipuncture, as traumatically detached CEC contribute significantly to CEC counts (Goon et al, 2006;Rowand et al, 2007). Because of this low number, enrichment steps are applied in several approaches, which inevitably leads to cell loss and underestimation of the actual CEC number.…”

“…For this purpose, several unique features of endothelial cells can be used. These include uptake of Ulex Europaeus Lectin-1, or UEA-1 (Goon et al, 2006) or the ability of CEC to scavenge acetylated low-density lipoproteins, which can be visualised when labelled with 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethyl-indocarbocyanine perchlorate (Dil Ac-LDL) (Voyta et al, 1984). In addition, immunohistochemistry or immunofluorescence can be used to demonstrate the presence of von Willebrand factor (VWF), or other endothelial surface markers (Table 1).…”

“…Recently, absolute CEC counts obtained by both magnetic isolation and flow cytometry assays were compared (Goon et al, 2006). The reasonable agreement in CEC counts between both methods, in combination with an established CEC phenotype (i.e.…”

“…CD34 þ , CD146 þ , CD45 À ), is suggestive that actual CEC counts are being approached by this flow cytometry assay. However, isolation and validation as done on the magnetically isolated cells (Rowand et al, 2007), was not performed on the CEC as defined by Goon et al (2006).…”

Circulating endothelial cell count: a reliable marker of endothelial damage in patients undergoing hematopoietic stem cell transplantation

“…8,12,[16][17][18][19][20] However, due to their very low numbers and complex phenotype, several published techniques have shown different degrees of variability, reporting a wide range of CEC values (0-7900 CEC/mL) in healthy subjects. 12,[21][22][23][24] We prospectively correlated CEC count changes to GvHD onset and response to treatment in 90 allo-HSCT patients. Moreover, in this study, we used the commercially available rare cell isolation platform CellSearch system to identify and count CEC and potentially translate results into clinical practice.…”