AUTOMATED FISH SPOT COUNTING IN INTERPHASE NUCLEI - STATISTICAL VALIDATION AND DATA CORRECTION

The evaluation of an automated system for Fluorescence In Situ Hybridi zation (FISH) spot counting in interphase nuclei is presented in this paper. Different types of experiments have been performed with samples from known populations. In all of them the goal is to detect mosaicis m of chromosome X in leukocytes from mixtures in known proportions of healthy male and female blood. First the initial results from the auto matic FISH analysis system were obtained and evaluated. Then the analy sis was modified to reduce systematic errors, so that the results are closer to what an experienced human operator would have obtained (syst em calibration step). Finally, an additional control probe of chromoso me Y was used to detect and discard cells where incorrect hybridizatio n or other abnormal situations had occurred. In each step the system s ensitivity was determined by the use of two statistical validation tes ts, so that the improvement brought about by the correction methods co uld. be assessed. The results obtained in the study showed that, using both corrections, the system is able to detect 10% monosomies with a significance level alpha = 0.1%. (C) 1998 Wiley-Liss, Inc.