Effect of fixation with formalin on flow cytometric measurement of DNA in nucleated blood cells

Abstract

A study was performed to determine the role formalin fixation plays in flow cytometric (FCM) analysis of DNA in nucleated blood cells. Blood was frozen and stored at −80°C in a DMSO-citrate-sucrose buffer. When FCM analysis was needed the sample was thawed and added to a large volume of propidium iodide (PI) stain and analyzed after not less than 10 min of staining. No centrifugation steps were necessary. This protocol has been used by us for the discrimination of triploid from diploid Chinese grass carp ( Ctenophary ngodon idella). Fixation of PI-staned nucleated RBCs with a final concentration of 1–2% phosphate-buffered formalin significantly lowered the amount of fluorescence recorded from the sample even when the fixed and stained RBCs were washed free of the fixative. Fixation with formalin prior to staining with PI yielded similar results. Our results show that formalin fixation of samples prior to or after staining with PI may lead to problems in data interpretation not previously appreciated. A FCM study was done to select the most appropriate biological DNA reference standard for grass carp RBCs. In this study DNA histograms of red blood cells from chicken, rainbow trout and diamondback watersnake as well as human nucleated white blood cells were compared to grass carp DNA histograms. Trout RBCs were selected as the most appropriate DNA reference standard for grass carp.