Karyotyping

For the study of chromosomes, short-term blood cultures are often used, as well as bone marrow cells and fibroblast cultures. The blood delivered to the laboratory with anticoagulant is subjected to centrifugation for precipitation of red blood cells, and leukocytes are incubated in the culture medium for 2-3 days. Phytohemagglutinin is added to the blood sample, as it accelerates the agglutination of erythrocytes and stimulates the division of lymphocytes. The most suitable phase for the study of 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 that are in metaphase, that is, at the stage of the cell cycle, when the chromosomes are best seen. Each chromosome replicates (produces its own copy) and after appropriate coloring is visible in the form of two chromatids attached to the centromere or central constriction. The cells are then treated with a hypotonic sodium chloride solution, fixed and stained.

For dyeing chromosomes, the Romanovsky-Giemsa dye, 2% acetaminomine or 2% acetazarine are more often used. They stain chromosomes entirely, uniformly (the routine method) and can be used to identify numerical anomalies of human chromosomes.

To obtain a detailed picture of the structure of chromosomes, identification (determination) of 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-bending. A microscopic examination of the drug along the length of the chromosome reveals a series of stained (heterochromatin) and unpainted (euchromatin) bands. The character of the transverse striation obtained in this case allows to identify each chromosome in the set, since the stripes alternation and their sizes are strictly individual and constant for each pair.

Metaphase plates of individual cells are photographed. Individual chromosomes are cut from the photographs and pasted on the sheet of paper in order; such a picture of chromosomes is called a karyotype.

The use of additional staining, as well as new methods for obtaining chromosome preparations, which allow stretching the chromosomes 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's syndrome, characterized by the presence of an additional chromosome 21 (trisomy 21), women's karyotype is described as 47, XX 21+, and men - 47, XY, 21+. In the presence of a structural anomaly of the chromosome, it is necessary to indicate an altered long or short arm: the letter p denotes a short arm, q a long arm, and t a translocation. Thus, when the short arm of chromosome 5 (the "cat-scream" syndrome) is deleted, the female karyotype is described as 46, XX, 5p-. The mother of a child with translocation Down syndrome - a carrier of a balanced translocation of 14/21 has a karyotype of 45, XX, t (14q; 21q). The translocation chromosome is formed when the long arms of chromosome 14 and 21 merge, short shoulders are lost.

Each shoulder is divided into districts, and they in turn - into segments, and those and others are designated by Arabic numerals. The centromere of the chromosome is the starting point for the counting of regions and segments.

Thus, four markers are used for the topography of chromosomes: chromosome number, shoulder symbol, district number and segment number within the given region. For example, the record 6p21.3 means that it is a chromosome of the 6th pair, its short arm, region 21, segment 3. There are additional symbols, in particular pter - the end of the short arm, qter - the end of the long arm.

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

[1], [2], [3], [4]