BACKGROUND: Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL). OBJECTIVES: Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. METHODS: CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell μL(-1). RESULTS: The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). CONCLUSIONS: This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.
BACKGROUND: Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL). OBJECTIVES: Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. METHODS: CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell μL(-1). RESULTS: The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). CONCLUSIONS: This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.
Authors: Nick Beije; Jurjen Versluis; Jaco Kraan; Jan W Gratama; Stefan Sleijfer; Jan J Cornelissen Journal: Haematologica Date: 2015-02-20 Impact factor: 9.941
Authors: Margaret M Tropea; Bonnie J A Harper; Grace M Graninger; Terry M Phillips; Gabriela Ferreyra; Howard S Mostowski; Robert L Danner; Anthony F Suffredini; Michael A Solomon Journal: Thromb Haemost Date: 2014-07-24 Impact factor: 5.249
Authors: Christopher E Radecke; Alexandra E Warrick; Gagan D Singh; Jason H Rogers; Scott I Simon; Ehrin J Armstrong Journal: Thromb Haemost Date: 2014-11-20 Impact factor: 5.249
Authors: N Beije; J Kraan; W Taal; B van der Holt; H M Oosterkamp; A M Walenkamp; L Beerepoot; M Hanse; M E van Linde; A Otten; R M Vernhout; F Y F de Vos; J W Gratama; S Sleijfer; M J van den Bent Journal: Br J Cancer Date: 2015-06-04 Impact factor: 7.640
Authors: P Hamberg; M J Boers-Sonderen; W T A van der Graaf; P de Bruijn; A B Suttle; F A L M Eskens; J Verweij; C M L van Herpen; S Sleijfer Journal: Br J Cancer Date: 2013-12-24 Impact factor: 7.640