Karyotypic analysis.
Date: 2015-10-07; view: 427.
To exactly describe of sizes, shape, and quantity of chromosomes the method of karyotypic analysis is applied.
Specified parameters detect by microcopying of cells at a moment of mitosis metaphase, when chromosomes are visible clean. Method allows getting the final diagnosis of chromosome disease.
There are direct and indirect karyotypic analyses.
The material to indirect karyotypic analyses usually the peripheral blood is. More seldom the material may be other tissue. The cells of this tissue don't pass mitosis at the moment.
Practically all manipulates of karyotypic analyses are conducted in sterile conditions. Blood withdrew in dissolve of heparin (preventive fibrillation). Then put into culture broth (cultural medium) and add a mitogen, as a rule it is phytohemagglutinin (PHA). Cultural medium is required for cultivation of blood cells for 72 hours. The temperate of cultivation equals to +72 ÑO Celsius. In this period of time the PHA puts lymphocytes of blood into the mitosis. Then mitosis stops by destroyer of mitotic spindle (for example, colchaminum). Centrifugation conducts after 3 hours of exposition. Then to destroy the cellular membranes the hyrotensive dissolve of potassium chloride is used. Centrifugation conducts again, and cells undergo fixation and dyeing (painting).
There are routine method of dyeing (figure 29. 1) and differentiating one (figure 29. 2).
Figure 29. 1 Routine method of dyeing. Evenly painted human chromosomes (human karyotype).
Figure 29. 2 Differentiating method of dyeing. Unevenly painted human chromosomes (human karyotype).
Then the photomicrograph of chromosomes may be done. After routine method of dyeing chromosomes can be identified by length, position of the centromere and be classified on groups. After differentiating method of dyeing, chromosomes can be identified by length, position of the centromere, banding patterns and be classified on pairs.
Classification of human chromosomes (Denver, 1960)
| Group | Number | Size, mkm | Characteristics |
| A | 1, 2, 3 | 11 – 8,3 | 1 and 3 – metacentric, large 2 – submetacentric, large |
| B | 4, 5 | 7,7 | submetacentric, large |
| C | 6 –12 | 7,2 – 5,7 | submetacentric, middle |
| D | 13 – 15 | 4,2 | acrocentric, middle |
| E | 16 – 18 | 3,6 – 3,2 | submetacentric, small 18 acrocentric, small |
| F | 19 – 20 | 2,3 – 2,8 | metacentric, the smallest |
| G | 21 – 22 | 2, 3 | acrocentric, the smallest |
X chromosome is submetacentric, middle. Y chromosome is acrocentric, the smallest.
The accommodation of chromosomes according their length is named ideogram (figure 29.3).
Figure 29.3 Ideograms of human chromosomes
The method of direct karyotypic analysis includes of
- stop of mitosis by destroyer of mitotic spindle (for example, colchaminum).
- destroying of the cellular membranes the byhyrotensive dissolve.
- centrifugation conducts again, and cells undergo.
- fixation and dyeing (painting).
- identification and classification of chromosomes.
The material is taken from marrow. And the cells that are dividing at the moment by mitosis are used.
| <== previous lecture | | | next lecture ==> |
| Methods of postnatal diagnostic. | | | Dermatoglyphic maps method. Analysis of dermatoglyphics. |