Comparison of a Visual to a Computer Assisted Technique for Detecting Apoptosis

In many studies, fluorescent dyes (ethidium bromide [EB] and acridine orange [AO]) are used to stain DNA to determine if nuclei are apoptotic. However, there are numerous visual methods for counting these stained DNA that may lead to inaccuracies Measuring apoptosis by the visual counting method may be imprecise because of the variability of individuals’ perception of color. Therefore, the authors compared a visual method of counting chromatin for apoptosis with a method relying on a computer program. They began counting chromatin using the visual method, in which individuals identify the stained DNA using their own visual perception. For comparison, they used a software-based counting method (analySIS software) to determine the color (hue) of the stained DNA. Using the numeric hue values from the software eliminates the variations in human color perception. Intra and interrater reliability of the visual and computerassisted counting methods were evaluated with Spearman’s. The authors found statistical significance in the intrarater reliability (r = 1.0,P = 0.0001 for all chromatin categories) and interrater reliability (r = 0.975,P = 0.005 for both readings) when using the software program. No statistical significance was found for the visual counting method, indicating inaccuracy between and within raters. Thus, the computerassisted counting method of identifying the damaged DNA is more accurate and precise than the individual’s visual perception of color. Based on these data, apoptosis measurements using color staining with EB and AO should be determined using hue values generated by a computer program and not by a researcher’s visual assessment.