Combined flow cytometry and image cytometry of the same cytological sample.

Abstract

Flow cytometry and image cytometry, two measuring techniques in the field of analytical cytology, can be used sequentially on the same cytological sample. Cells stained with a fluorochrome for the determination of for example, DNA or RNA content are first analysed in suspension by flow cytometry. The results of the fluorescence analysis of the individual cells are presented after data processing as frequency histograms of the DNA or RNA content of all the cells of the sample. In these histograms certain cell populations such as those with an increased DNA content are defined and these are then selected for further investigation. This is achieved by sorting cells of interest into centrifugation buckets by means of electrostatic deflection of the droplets containing such cells. Sorted cell populations are then centrifuged on to glass slides and stained according to the acriflavine Feulgen-SITS staining procedure, a quantitative method for DNA and protein. Image cytometry of these stained cells is performed with a computer controlled television based image analysis system (LEYTAS). With this system abnormal cells with elevated DNA content or increased chromatin contrast are automatically detected, thereby eliminating almost all artefacts and normal cells. Subsequently detected objects are stored in grey-value memories after the automated analysis for visual examination by the cytologist. The possibilities of combined flow cytometry and image cytometry are illustrated in typical examples in the field of cervical, bladder and mammary cytology.