Date: Fri Oct 19 2007 - 03:49:26 EDT
Dear Jasbani, The measurement of the DNA content by flow cytometry on FFPE samples has a relative long history and boomed after the major publication of David Hedley in 1983. However, there are many factors that contribute to a high CV, but it should be possible to get very good results (see attachment of a FFPE cervical carcinoma. DNA was stained with PI). Especially the use of antigen-retrieval techniques significantly improves CVs. Simultaneously intermediate filaments, like keratin and vimentin, can be easily detected. It allows you to discriminate between tumour stromal cells and carcinoma cells in clinical samples. There are some reports in the literature that also cell surface markers can be used. However, we were not able to repeat these findings. CD79a was the only exception. You can contact me when you need more detailed information. For those interested in our work I can send some PDFs. Some recommendations: - cut 50 -60 µm sections - Use antigen retrieval - dissociate, filter and count cells - test antibodies using the same detection method (fluorescence) on 4 µm tissue sections from the same block using the same antigen retrieval method and enzymes used for dissociation - no signal after enzymatic treatment, than it will likely not work in suspension - titrate PI using 1x10E6 cells. In our hands 10 µM PI in 0.5 ml buffer containing RNase) is optimal Best regards, Willem Corver Willem E. Corver, PhD Dept. of Pathology Leiden University Medical Centre P.O. Box 9600, Building 1, L1-Q 2300 RC Leiden The Netherlands Tel.: +31 71 526 6580 FAX: +31 71 524 8158 -----Original Message----- From: Jasbani Singh [mailto:J.Singh@dundee.ac.uk] Sent: woensdag 17 oktober 2007 10:40 To: cyto-inbox Subject: Paraffin embedded tissue sections Hi All, I am working on formalin fixed paraffin embedded tissue sections and trying to get data for DNA content from flow cytometry analysis. I am having trouble with a couple of things. I am getting a very high Coefficient of variation, any ideas of how to bring this down? What controls can I use for the data I am getting? Is there anything better than Propidium Iodide for FFPE tissue? and lastly has anyone used any cell surface markers to distinguish between the different phases of cell cycle in paraffin embedded sections? I have only just started and there is a lot of literature available but after trying different things I am still looking to get better and more robust results. Thank you in advance, will look forward to replies from anyone who is able to help. Jasbani Department of Cytogenetics/Department of Surgery and Molecular Oncology Ninewells Hospital, Dundee Scotland, UK email: j.singh@dundee.ac.uk
