CHARACTERIZATION OF FACTORS IN ROUTINE LABORATORY PROTOCOLS THAT SIGNIFICANTLY INFLUENCE THE FEULGEN REACTION

We investigated the parameters that could affect the cytophotometric a nalysis of cell nuclei stained by the Feulgen reaction. These paramete rs included: the hydrolysis temperature (in the normal ''room temperat ure'' range); the composition of the Schiff's reagent; the speed of ce ntrifugation of the cell suspensions; the mode of preservation [air-dr ying or ethanol-formalin-acetic acid (EFA) fixation]; the fixation tim e; the pronase digestion time; and the concentration of pronase used t o obtain cell suspensions from archival (formalin-fixed, paraffin-embe dded) materials. Relatively homogeneous material was studied: the MXT mouse mammary adenocarcinoma growing in vivo as tumors with both small and hyperchromatic cell nuclei and in vitro as monolayers with larger and less hyperchromatic cell nuclei. The results of these investigati ons demonstrate the necessity for the precise definition of a protocol for such procedures as sampling, fixation, and staining of cell nucle i if computerized cell image analyses are to be objective and reproduc ible. For present purposes this protocol differs depending on whether fresh or archival material is studied. For fresh tissue the protocol i s immersion of the sample in EFA within 10 sec, fixation for 30 min, a nd staining by the Feulgen reaction in which hydrolysis is performed w ith 6 N HCl at 24-degrees-C for 60 min. For archival tissue, the proto col becomes fixation in formol (or EFA), embedding, sectioning at 80 m um, digestion with 0.05% pronase for 2 hr, centrifugation at 1200 x g on glass slides, and staining by the Feulgen reaction in which hydroly sis is performed with 6 N HCl for 60 min at 24-degrees-C.