Karyotyping

Short-term blood cultures, bone marrow cells, and fibroblast cultures are most often used to study chromosomes. Blood with an anticoagulant delivered to the laboratory is centrifuged to sediment the erythrocytes, and the leukocytes are incubated in a culture medium for 2-3 days. Phytohemagglutinin is added to the blood sample, as it accelerates the agglutination of erythrocytes and stimulates lymphocyte division. The most suitable phase for studying chromosomes is the metaphase of mitosis, so colchicine is used to stop the division of lymphocytes at this stage. Adding this drug to the culture leads to an increase in the proportion of cells in metaphase, that is, at the stage of the cell cycle when chromosomes are most visible. Each chromosome replicates (produces its own copy) and, after appropriate staining, is visible as two chromatids attached to the centromere, or central constriction. The cells are then treated with a hypotonic sodium chloride solution, fixed, and stained.

For coloring chromosomes, Romanovsky-Giemsa dye, 2% acetcarmine or 2% acetarsein are most often used. They color chromosomes entirely, evenly (routine method) and can be used to detect numerical anomalies of human chromosomes.

To obtain a detailed picture of the chromosome structure, identify (define) individual chromosomes or their segments, various methods of differential staining are used. The most commonly used methods are Giemsa, as well as G- and Q-banding. When examining the preparation microscopy along the length of the chromosome, a number of stained (heterochromatin) and unstained (euchromatin) bands are revealed. The nature of the transverse striation obtained in this way allows identifying each chromosome in the set, since the alternation of bands and their sizes are strictly individual and constant for each pair.

The metaphase plates of individual cells are photographed. Individual chromosomes are cut out from the photographs and pasted in order onto a sheet of paper; this picture of chromosomes is called a karyotype.

The use of additional staining, as well as new methods for obtaining chromosomal preparations that allow chromosomes to be stretched in length, significantly increase the accuracy of cytogenetic diagnostics.

A special nomenclature has been developed to describe the human karyotype. The normal karyotype of a man and a woman is designated as 46, XY and 46, XX, respectively. In Down syndrome, characterized by the presence of an additional chromosome 21 (trisomy 21), the karyotype of a woman is described as 47, XX 21+, and that of a man is 47, XY, 21+. In the presence of a structural anomaly of the chromosome, it is necessary to indicate the altered long or short arm: the letter p denotes the short arm, q denotes the long arm, and t denotes the translocation. Thus, in the case of deletion of the short arm of chromosome 5 (cri du chat syndrome), the female karyotype is described as 46, XX, 5p-. The mother of a child with translocation Down syndrome, a carrier of the balanced translocation 14/21, has a karyotype of 45, XX, t(14q; 21q). The translocation chromosome is formed by the fusion of the long arms of chromosomes 14 and 21, and the short arms are lost.

Each arm is divided into regions, which in turn are divided into segments, both of which are designated by Arabic numerals. The centromere of the chromosome is the starting point for counting regions and segments.

Thus, four labels are used for chromosome topography: chromosome number, arm symbol, region number, and segment number within the region. For example, the entry 6p21.3 means that we are talking about chromosome 6 of the 6th pair, its short arm, region 21, segment 3. There are also additional symbols, in particular pter - the end of the short arm, qter - the end of the long arm.

The cytogenetic method of research allows detection of deletions and other changes in chromosomes only of approximately 1 million bases (nucleotides) in size.

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