Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol

Abstract

Summary. Background: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. Aim: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. Methods: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. Results: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4°C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. Conclusion: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

Circulating endothelial cells in oncology: pitfalls and promises

“…Although this is a generally accepted CEC immunophenotype, reports are currently not available in which the endothelial origin of cells enumerated by those assays has been demonstrated unambiguously (Duda et al, 2006;Mancuso et al, 2006). As a result, studies on CEC that are in our opinion reliable, are scarce: only three studies are currently available, all based on CD146 driven magnetic isolation of CEC (Beerepoot et al, 2004;Woywodt et al, 2006;Rowand et al, 2007). In the first study, CEC were enumerated by magnetic isolation, followed by visual analysis of CD31, CD309 and VWF expression, determined by immunofluorescence.…”

“…Next to proper sampling, thorough analysis of enrichment efficacy, reported in terms of purity and recovery is therefore mandatory before using such assays based on enrichment in the clinic. The EUROCEC network has provided useful suggestions on how such a validation might be performed in practise (www.eurocec.com) (Woywodt et al, 2006).…”

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

“…Recent approaches to CEC enumeration use immunomagnetic enrichment using paramagnetic particles coated with CD146 mAb in combination with markers to exclude hematopoietic cells (i.e., CD45) (32), or in combination with markers that confirm endothelial origin (Ulex Europaeus Lectin-1 and CD105) (32,33), as enrichment for CD146 also yields CD146 þ T cells. With these approaches, CEC counts in healthy individuals generally do not exceed 20 cells per mL (32,33).…”

“…With these approaches, CEC counts in healthy individuals generally do not exceed 20 cells per mL (32,33).…”

Role of endothelial progenitor cells in breast cancer angiogenesis: from fundamental research to clinical ramifications

“…Since then, many studies have been performed to determine the phenotypical and functional characteristics of EPC [5][6][7][8]. However, as EPC, endothelial cells and haematopoetic stem cells share many cell surface markers including CD45, CD34, CD133 (CD117 for mouse), CD146, CD31, CD105, CD144, vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand factor (vWF) [6,[9][10][11][12][13][14][15] (Table 1), the term EPC may therefore encompass ranging from relative primitive haemangioblasts to more differentiated endothelial cells. As CD133 is expressed in stem cells and/or progenitors of different tissues [16], it is currently accepted that early EPC (localized in the bone marrow or immediately after migration into the circulation) are CD133?/CD34?/VEGFR2?/CD45-cells, whereas circulating EPC are CD133low/CD34?/VEGFR2?/CD45-and begin to express cell surface markers typical to mature endothelial cells, including CD31 and vWF.…”