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To purify mouse Y chromosomes by flow cytometry, a male cell line containing the Robertsonian translocation Rb(9.19)163H has been established by SV40 transformation. Flow karyotypes obtained from these cells exhibit a well-isolated peak of fluorescence corresponding to the single Y chromosome, clearly distinct from that of chromosome 19. From this peak, 650,000 chromosomes were sorted, and two restriction fragment libraries were constructed from the DNA of the sorted chromosomes. The characterization of several Y-specific fragments has shown that the Y DNA was enriched at least 36-fold. Furthermore, given that there are likely homologies between the X and Y chromosomes, we can assume that this calculated value of the purification factor is an underestimation and that the Y DNA was more highly purified by flow sorting.

A method is described for directly hybridizing a small number of sorted chromosomes with specific DNA probes. The chromosomes are analyzed by flow cytometry and sorted by deflecting the droplets containing the desired chromosomes onto a nitrocellulose filter. By using probes specific for the human Y chromosome, it has been possible to unambiguously identify the peak corresponding to the Y chromosome in the flow karyotypes of a variety of male cell lines. The position of this peak was found to vary significantly from individual to individual, correlating with the heterochromatin chromosomal polymorphism of the human Y chromosome. The sensitivity of the hybridization was such that, with a probe for a male-specific repetitive sequence, only 2,500 sorted chromosomes were enough to obtain a clear, positive signal; 10,000 were needed with .a probe specific for a weakly repeated (maximum, 3-fold) sequence of Y chromosome. With this new method, chromosome sorting may be a rapid and efficient way to assign DNA sequences to chromosomes.

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free intervillous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.

The heterogeneous population of newborn rat keratinocytes was separated into different subgroups according to their cell size. The relation between cell size, position in the cell cycle, RNA content, and proliferative potential in culture was examined. A reserve stem cell population of Go/G1 cells, low in RNA, giving rise to colonies of undifferentiated phenotype in cell culture, has been separated from more differentiated transit basal cells. In the fractions of the larger cells, several subgroups, probably corresponding to different stages of differentiation, were identified: G2M cells with low RNA content, large S-phase cells rich in RNA, and small Go/G1 cells low in RNA. The clonogenic cells from these fractions have limited growth potential and give rise to moderately or terminally differentiated colonies. The selective sorting of stem cell populations may be useful for elucidating the mechanism of carcinogenesis in epidermis and other proliferative tissues. Analysis of the relative proportions of cell subpopulations represents a novel approach leading to the refinement of the concepts of epidermal structure in physiological and pathological states. It also could, by extension, shed new light on the behavior of other proliferative tissues.

The short arm of chromosome 11 carries genes involved in malformation syndromes, including the aniridia/genitourinary abnormalities/mental retardation (WAGR) syndrome and the Beckwith-Wiedemann syndrome, both of which are associated with an increased risk of childhood malignancy. Evidence comes from constitutional chromosomal aberrations and from losses of heterozygosity, limited to tumor cells, involving regions 11p13 and 11p15. In order to map the genes involved more precisely, we have fused a mouse cell line with cell lines from patients with constitutional deletions or translocations. Characterization of somatic cell hybrids with 11p-specific DNA markers has allowed us to subdivide the short arm into 11 subregions, 7 of which belong to band 11p13. We have thus defined the smallest region of overlap for the Wilms' tumor locus bracketed by the closest proximal and distal breakpoints in two of these hybrids. The region associated with the Beckwith-Wiedemann syndrome spans the region flanked by two 11p15.5 markers, HRAS1 and HBB. These hybrids also represent useful tools for mapping new markers to this region of the human genome.

Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?