Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.

TLDR

The method enables retrospective analysis of archival tumors to assess the prognostic value of abnormal DNA content via flow cytometry. Paraffin‑embedded tumor sections (30 µm) are dewaxed, rehydrated, digested with pepsin, stained with DAPI, and analyzed by flow cytometry to quantify DNA content. The technique accurately measures DNA content in paraffin‑embedded tumors, with histograms correlating well with those from unfixed tissue.

Abstract

A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry.