Quantitative precision of an automated image cytometric system for the measurement of DNA content and distribution in cells labeled with fluorescent nucleic acid stains

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Abstract
An automated image cytometric system is described for the measurement of DNA content and distribution in cells stained with fluorescent DNA binding or intercalating compounds. The quantitative precision of integrated optical intensity (IOI) measurements using this system was estimated to be 2.0%, based on the coefficient of variation (cv) of the IOI of DNA check beads. Using peripheral blood lymphocytes stained with 4′‐6‐diamidino‐2‐phenylindole (DAPI), the cv of IOI was found to be 3.5%. Slide to slide variability of the IOI for peripheral blood lymphocytes (cv over 10 slides of the mean IOI) was found to be 2%. The most important sources of error in these measurements were glare, illumination instability, image calibration instability, and staining non‐uniformity. These errors were investigated and minimized.