Flow karyotyping is a method of analyzing and characterizing the number, sizes and shapes of the entire set of metaphase chromosomes of a cell, using a flow cytometer called fluorescence activated chromosome sorter (FACS). Karyotype analysis is an important laboratory diagnostic procedure in cancer genetics, prenatal diagnosis, hematological disorders, and many other diseases.
Chromosomes are not visible in non-dividing cells, even with the aid of histological stain for DNA and electron microscopy. These chromosomes will condense and become visible at the metaphase stages of cell division during meiosis and mitosis. Cytogenetic and karyotyping analyses have, therefore, been done with condensed metaphase chromosomes obtained from dividing cells. The cells that reach mitosis have already progressed through the S phase of the cell cycle, during which DNA replication takes place. The condensed metaphase chromosomes thus obtained are duplicated structures; each chromosome consists of two sister chromatids that are attached at the centromere. Metaphase chromosomes intended for flow sorting are usually pretreated with polyamine or magnesium sulfate in order to allow adequate discrimination between the chromosome types after sorting, and preserve the quality of the DNA.
The first human flow karyotypes were obtained in mid-1970 using metaphase chromosomes isolated from fibroblasts. A typical flow karyotype is obtained by staining metaphase chromosomes with two dyes. The most routinely used dyes are Hoechst 33258 and chromomycin A3, that bind to AT-rich regions and GC-rich regions of DNA, respectively. A large number of chromosomes (10,000-50,000) are then sorted in a dual laser flow sorter that will sort each chromosome depending on the intensity of the fluorescence signal that it produces. The intensity of the fluorescence signal obtained from these double stained chromosomes is influenced both by the DNA content of the chromosomes and their base pair composition. Signals are recorded for each chromosome and the data is presented as histogram of fluorescence intensity against chromosome frequency. Histograms obtained for each dye are often combined to form an isometric plot. The data can also be presented as a dot-plot or contour map where each chromosome appears in a specific peak. This plot shows a distinctive species-specific pattern of peaks and is called bivariate flow karyotype. This is to be distinguished from a univariate flow karyotype, which is obtained in a similar way except that a single DNA-staining dye, usually ethedium bromide or Hoechst 33258, is used. The Univariate flow karyotype is seldom used because it does not allow the distinction of all chromosomes.
Because the chromosomes used in a flow karyotype analysis are pooled from many cells, this analysis provides no information about an individual cell. It can only provide an accurate measurement of the frequency of the different chromosome types. An increase in the frequency of chromosome 21 by around 50% would for example indicate the presence of trisomy 21. The appearance of two separate peaks in a position where there are normally no peaks is indicative of translocations resulting in two derivative chromosomes.
Although rapid and highly reproducible, flow karyotyping has limitations when it comes to detecting some chromosomal aberrations. The derivative chromosomes from a translocation can appear in the same position as other normal chromosomes and become difficult to detect. A reciprocal translocation resulting in two derivative chromosomes that have the same DNA content and base pair ratio as the parent chromosomes will also be difficult to detect. Polymorphisms in the population is an area of major obstacles in interpreting data from flow karyotyping as opposed to conventional cytogenetics analysis. The two homologues of the same chromosome in a normal individual usually appear as separate peaks, making it difficult to ascertain whether two peaks seen in a flow karyotype are the two normal homologues of a chromosome, or whether one of them is abnormal. These disadvantages of flow karyotyping techniques restrict them for use only as a complement to conventional cytogenetic karyotyping.
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