VARIABILITY OF DNA ANALYSIS BY IMAGE CYTOMETRY

Two laboratories equipped with CAS 200 (Becton Dickinson Image Cytomet ry Systems, San Jose, CA) instruments participated in this study of va riability of DNA analysis of bladder tumor specimens. Formalin fixed p araffin embedded specimens were disaggregated and centrifuged onto mic roscope slides from ten bladder tumor specimens and two specimens of n ormal urothelium. Sources of variability considered were Specimen, Sli de, Run, Laboratory, and Error. Slides were systematically scanned and 200 cells measured followed by the operator selecting 100 nuclei with abnormal morphology. DNA. index (DI) and hyperdiploid fraction (HDF) were calculated from the DNA frequency distributions. For systematic s ampling, 92% of the variability was due to Specimen indicating that di fferences in HDF values between specimens reflect biological differenc es. With selective sampling, only 67% of the variability in HDF is due to Specimen differences. Other factors, Laboratory, Error, and Labora tory x Specimen interaction each accounted for approximately 10% of th e variability. Similarly variability of DI with selective sampling was also higher, and less specimen dependent than systematic sampling. It is important that sampling schemes and selection criteria be carefull y documented in order to control variability. Enriched Cor selective) sampling for abnormal cells has the potential to increase sensitivity but specimen classification based on these measurements must depend on determination of the frequency of such cells in the total population. (C) 1997 Wiley-Liss, Inc.