Stability of propidium iodide stained samples by the coulterR DNA prep workstation for analysis by flow cytometry

Abstract

At the Caulfield General Medical Centre, paraffin-embedded specimens analysed for DNA ploidy are prepared by a modification of the Hedley Technique'. Nuclear preparations are stained by the COULTER^R DNA Prep Reagent System. This is a commercial workstation designed to prepare cell or nuclear suspensions for quantitative determination of nuclear DNA content by Flow Cytometry (FCM). The reagents comprise non-ionic detergents for cell lysing and permeabilisation, Propidium Iodide (PI) for DNA staining, and avian erythrocyte nuclei as an internal reference standard. The manufacturers instructions specify that to obtain optimal coefficients of variation (CV's), samples should be incubated in PI at room temperature (RT) for 15 - 17 minutes prior to analysing by FCM and must bel analysed within 2 hours of staining. This investigation set out to determine the stability of the stained specimens beyond this 2 hour limit. After routine analysis of a group of stained samples, the batch was re-analysed at different intervals ranging from 24 hours to 14 days. Samples were stored at RT and protected from light. The values from the histograms collected and compared over this period included: the DNA Index (Dl) : percent half peak CV's (%HPCV); mean channel fluorescence of the major peak(s) in the histogram; and percent of events found in each peak(s). Examination of the histograms after 24 hours demonstrated no alteration in the Dl of the specimen, an average reduction of 10% in the %HPCV, 10% increase in the mean channel fluorescence and a 5% decrease in the percent of events found in the peaks. Re-analysis over a two week period showed these values to remain stable. Less than 10% of the samples deteriorated and were uninterpretable at the end of 14 days. The data shows that stained nuclear preparations are stable beyond the time recommended by the manufacturers and allows for storage of stained specimens with minimal deterioration.