I am required to evaluate circulating endothelial cells by flow cytometry. I plan to look at CD45 low, CD34 positive cells but what would be the best to add in a third color, CD31? CD136? other? Very different methods are used in the litterature leading to very different amount of CEC. What is, in your opinion, the "true" immunophenotype of CEC? Also what are the scatter properties of the CEC: are they really in the lymphocyte region as published or are they larger scattering cells?
Dear Francis,
The combination of CD45neg, CD34++ and CD146/144+ will work, but adding a DNA specific stain is definitely also needed to exclude (large) platelets and microparticles. At least 1 mL of peripheral blood should be analyzed for a prrecise ennumaration of CEC at a frequency of < 1 cell/µL (normal range 5-80 CEC/mL).
See also attached article for further information.
Regards, Jaco
Article A new approach for rapid and reliable enumeration of circula...
Dear Francis,
I am Pathologist and then I have been using the CD34 as specific marker for EC in the Histology of the tissue. So far in my application we can evaluate CD34 the best marker because of there is the morphology tissue could help us often. In your work, I personally think could be better to try a different triplets of CDs to verify the best solution for you.
CD31 and CD 136 could be well. However must be necessary to compare these results in front of some molecular analyses (i.e. mRNA, qPCR and others).
I can confirm you that CD34 is well used in the tissue to verify it's micro-vasular density (MVD).
All the best
Niccola
I would be interested in what anyone with experience in this has to say, also. When I had a 4-colour machine I refused to even consider a CEC project. With a 10-colour machine I might be tempted, but I'd need convincing.
Francis, it sounds like you've seen the range of definitions in the literature. What kind of sample will you be analyzing (peripheral blood?)? Will you be enriching first? If so, how? Do you have access to a sorter to do further downstream analysis of the cells you define?
Good luck!
Thank you for your answers.
Niccola I understand that, as a pathologist, you are helped by the morphology and the structure of the tissue to identify endothelial cells, but you are right, we need more markers to be sure in flow cytometry. Moreover, the expected number of CEC in blood is probably far lower than in your tissue samples.
Elizabeth, yes, I am analyzing peripheral blood. I am afraid that any enrichment method would bring one more variability cause in a method which bring such a large range in the published results. Due to the expected low amount of CEC, I will label and analyze 1 ml of whole blood for each sample either before or after red cell lysis. I am evaluating both methods.
I made a mistake in the antibodies I plan to use: pease read CD146 in place of CD136.
Dear Francis,
The combination of CD45neg, CD34++ and CD146/144+ will work, but adding a DNA specific stain is definitely also needed to exclude (large) platelets and microparticles. At least 1 mL of peripheral blood should be analyzed for a prrecise ennumaration of CEC at a frequency of < 1 cell/µL (normal range 5-80 CEC/mL).
See also attached article for further information.
Regards, Jaco
Article A new approach for rapid and reliable enumeration of circula...
Here's an total dataset for endothelial specific markers in general.
http://www.antibodybeyond.com/reviews/cell-markers/ec-marker.htm
I don't have specific experience on CECs but i guess markers like CD31 or vWF can be lost on the surface of the CECs cause they are molecules related to cell adhesion. This is just my personal opinion.
Jaco, that is a nicely thorough article. Do you think the technique could be extended to circulating epithelial progenitor cells as well?
Thank you Yosep for this useful website.
Jaco, your paper is very interesting and convincing. The gating strategy is very clever. The DNA stain also sound as a good idea to eliminate microparticles. But do you think that platelets could contaminate CD34+-146+ population? I also question about the scatter properties of the CEC population (figure 1) as compared to the scatter of endothelial cells from a dissected vein. (Figure 2). How do you explain that the cell you sorted as CEC have a morpholgy of endothelial cells but scattered like lyphocytes? Thank you to all of you for this discussion.
DNA staining is absolutely crucial and also very informative as it might offer insight into the viability status of the CECs you enumerate. Most CECs in healthy subjects are apoptotic as a result of vessel wall turnover. CD34 expression is weak in apoptotic CECs, so we believe CD45-CD31+CD146+DNA+ is a good definition for CECs. These cells keep the very large majority of VE-Cadherin RNA in blood, and this is one of the most endothelial-specific gene you can measure. Unfortunately, VE-Cadherin is lost when endothelial cells are released in circulation, so this is not a good antigenic marker for flow cytometry.
http://www.ncbi.nlm.nih.gov/pubmed/19118054
Scater properties of CEC are variabel and mostly higher than Lympho's. Gating on scatter schould not be used to define CEC. only to exclude very smal paritcles/debris or at the final stage of the analysis doublets.
Yes, Elisabeth the assay can be extended for other rare event detection assay.
But how to define epithelial progenitors and will the be above a ferequency of > 1/mL.
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