A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis

The need for improved methodology to facilitate cytogenetic analysis of hemopoietic stem cell populations, particularly in studies of the hemopoietic malignancies, has been recognized for a number of years. Since primitive hemopoietic cells can be stimulated to form colonies of identifiable progeny under appropriate conditions in vitro, it should in theory, be possible to obtain such information. However, hemopoietic colonies of human origin rarely contain more than 1000 cells, and in handling such small samples, cell loss has historically been a major problem. We now describe a method that has allowed from two up to more than 50 metaphases per colony to be obtained from most erythroid and granulopoietic colonies harvested individually from standard methylcellulose assay cultures. Of key importance is the selection of large but still immature colonies and the use of polylysine coated slides, which ensures recovery of 80 - 90% of the sample. The method yields reproducibly high quality metaphases suitable for analysis after G, Q, and fluorescent-reverse banding. Cytogenetic analysis of 58 colonies removed individually from cultures initiated with a mixture of male and female cells showed both male and female colonies to be present as expected. In all instances only one type of metaphase was ever found in a single colony. A procedure for measuring the incidence of sister chromatid exchange (SCE) in the progeny of primitive hemopoietic progenitors has also been established. SCE values obtained for 1-2-week-old colonies derived from normal progenitors were similar to previously published normal SCE values. Preliminary analysis of erythroid and granulocytic colonies from one patient with polycythemia vera (PV) and two patients with chronic myelogenous leukemia (CML) indicate that preparations of equal quality are obtained from such individuals and thus, for the first time, the Ph1 chromosome could be readily demonstrated in erythroid colonies. Further application of this method to the investigation of patients with myeloproliferative disease is now underway.