Effects of several commonly used fixatives on DNA and total nuclear protein analysis by flow cytometry

Five commonly used fixatives (AZF, B-5, Bouin's, formalin, and Zenker's) were evaluated for their effect on the flow cytometric analysis of DNA and total nuclear protein (TNP) in solid tumors. Data were obtained with the use of colonic adenocarcinoma, squamous carcinoma of the lung, mammary adenocarcinoma, and spleen with a plasma cell leukemic infiltrate. The parameters examined were G0-G1 DNA staining intensity, %G0-G1, percent coefficient of variation (%CV), percent debris, and TNP staining intensity. The results showed that variations in the fixation of solid tumor significantly affected flow cytometric-derived parameters. In this study, paraffin-embedded tissue (PET) fixed in 10% (v/v) neutral buffered formalin (NBF) produced the best results, with a %CV below 4.7, whereas fixatives such as Zenker's and B-5 produced poor %CVs (above 6.0) or uninterpretable TNP and light scatter data. These data suggest that a portion of all tissue samples be fixed in NBF to allow for subsequent analysis by fixative-sensitive assays such as DNA in situ hybridization and flow cytometry.