Chromosome Sorting and Analysis by FACS

Many types of cancer and genetic diseases are characterized by chromosomal aberrations. Conventional cytogenetics, the analysis of banded metaphase chromosomes, can be very time consuming and, in many cases, marker chromosomes cannot be identified by their banding pattern alone. For some years there has been interest in a more rapid and objective method of analyzing chromosomes. Flow cytometry offers an alternative machine-based approach in which a suspension of metaphase chromosomes is prepared, stained with one or two DNA-binding fluorochromes, and passed through a flow cytometer. The first human flow karyotypes were demonstrated in the mid-1970s using chromosomes isolated from fibroblast cells (1 ,2) . High resolution flow karyotypes were subsequently obtained from PHA-stimulated peripheral blood lymphocytes (3 ) and lymphoblastoid cell lines (4 ). In practice, chromosomes can be prepared from almost any culture of growing cells. In the case of the single-laser system in which one nonbase-specific dye, such as ethidium bromide, is used, the intensity of fluorescence from each chromosome is proportional to its DNA content. The signal intensity from each chromosome is measured and recorded as it passes through the focused laser beam. Accumulating data from 10,000–50,000 chromosomes and presenting it as a histogram of fluorescence intensity against frequency produces a distinctive species specific pattern of peaks. Such a histogram is known as a univariate flow karyotype. Not all human chromosomes appear as separate peaks.