High-resolution R-banding at the 1250-band level. 1. Technical considerations on cell synchronization and R-banding (RHG and RBG).

A comparative analysis of variables for cell synchronization was made and led to the description of optimal conditions capable of yielding, in over 95% of cases, a large number of excellent quality cells in prometaphase and late prophase. Thymidine presents advantages over amethopterin as the synchronizing agent. The block was released with either thymidine or 5-bromo-2'-deoxyuridine (BrdU) for which various concentrations were tested. The presence of colcemid was also evaluated. Without colcemid, the optimal length of the release period was precisely determined so that the wave of synchronized cells could be harvested while going through the early stages of mitoses. Subsequently, GTG, RHG and RBG banding were produced on these elongated chromosomes. A comprehensive approach to RHG banding ensured an easier and well reproducible banding technique. Nine interrelated factors which influence banding quality were studied. The analysis of their effects on this banding pattern revealed new data for the understanding of its mechanism. A high-resolution R-banding technique after BrdU incorporation and Giemsa staining is presented; it is simple, reliable and reproducible. Different conditions for the FPG (Fluorochrome-Photolysis-Giemsa) technique were studied in order to obtain sharper borders and higher contrast between positive and negative bands. Optimal conditions for the incorporation of BrdU and the FPG method produced excellent band separation and band contrast even in very elongated prophase chromosomes. They did not decrease the mitotic index, did not increase chromosomal damage significantly, and did not greatly vary from subject to subject nor with the age of the slide (between 1 day and 36 months). Homologue discordance correlated to band range was compared for GTG, RHG and RBG banding.