Flow cytometric quantification of tumour endothelial cells; an objective alternative for microvessel density assessment.
Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly (r=0.84, P<0.001) with the immunohistochemical assessment of microvessel density. Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.
Angiogenesis Laboratory, Research Institute for Growth and Development (GROW), Department of Internal Medicine, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.
C I M
CI
Baeten
J
J
Wagstaff
I C L
IC
Verhoeven
H F P
HF
Hillen
A W
AW
Griffioen
D002452:Cell Count; D004730:Endothelium, Vascular / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D008833:Microcirculation; D009369:Neoplasms / Q000098:blood supply*; D009389:Neovascularization, Pathologic / Q000175:diagnosis