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Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol
Author(s) -
WOYWODT A.,
BLANN A. D.,
KIRSCH T.,
ERDBRUEGGER U.,
BANZET N.,
HAUBITZ M.,
DIGNATGEORGE F.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2006.01794.x
Subject(s) - isolation (microbiology) , enumeration , protocol (science) , immunomagnetic separation , computational biology , biology , computer science , medicine , pathology , microbiology and biotechnology , bioinformatics , mathematics , combinatorics , alternative medicine
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.