SAMPLE PREPARATION AND IN-SITU HYBRIDIZATION TECHNIQUES FOR AUTOMATEDMOLECULAR CYTOGENETIC ANALYSIS OF WHITE BLOOD-CELLS

With the advent of in situ hybridization techniques for the analysis o f chromosome copy number or structure in interphase cells, the diagnos tic and prognostic potential of cytogenetics has been augmented consid erably. In theory, the strategies for detection of cytogenetically abe rrant cells by in sim hybridization ate simple and straightforward. In practice, however, they are fallible, because false classification of hybridization spot number or patterns occurs. When a decision has to be made on molecular cytogenetic normalcy or abnormalcy of a cell samp le, the problem of false classification becomes particularly prominent if the fraction of aberrant cells is relatively small. In such mosaic situations, often > 200 cells have to be evaluated to reach a statist ical sound figure. The manual enumeration of in situ hybridization spo ts in many cells in many patient samples is tedious. Assistance in the evaluation process by automation of microscope functions and image an alysis techniques is, therefore, strongly indicated, Next to research and development of microscope hardware, camera technology, and image a nalysis, the optimization of the specimen for the (semi)automated micr oscopic analysis is essential, since factors such as cell density, thi ckness, and overlap have dramatic influences on the speed and complexi ty of the analysis process. Here we describe experiments that have led to a protocol for blood cell specimen that results in microscope prep arations that are well suited for automated molecular cytogenetic anal ysis. (C) 1996 Wiley-Liss, Inc.