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


OBJECTIVE
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 degrees 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.


CONCLUSIONS
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.
Woywodt
A
A
Department of Medicine, Division of Nephrology, Hannover Medical School, Hannover, Germany. woywodt.alexander@mh-hannover.de
Blann
A D
AD
Kirsch
T
T
Erdbruegger
U
U
Banzet
N
N
Haubitz
M
M
Dignat-George
F
F
D051929:CD146 Antigen / Q000032:analysis; D002452:Cell Count / Q000379:methods; D048429:Cell Size; D042783:Endothelial Cells / Q000737:chemistry / Q000276:immunology / Q000473:pathology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D016130:Immunophenotyping; D037121:Plant Lectins / Q000032:analysis; D015203:Reproducibility of Results; D014652:Vascular Diseases / Q000473:pathology
D051929:CD146 Antigen; D037121:Plant Lectins; C036850:Ulex europaeus lectins