Preparation of a karyogram of a karyotype

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Karyograms are photographic representations of the karyotype of an individual, usually prepared from metaphase cells

Chromosome source

Any population of dividing cells can be used to prepare metaphase spreads. Blood is the most frequently sampled tissue. Blood leukocytes do not divide, but lymphocytes can readily be induced to proliferate, providing a very accessible source of metaphase cells. But, karyotypes can also be prepared from cultured skin fibroblasts or bone marrow cells.

Steps to prepare metaphase cells from blood lymphocytes

Many different protocols are employed to prepare metaphase spreads from peripheral blood lymphocytes. 

Standard series of steps involve: 

1. A sample of blood is drawn and coagulation prevented by addition of heparin.
2. Mononuclear cells (lymphocytes and monocytes) are purified from the blood by centrifugation through a dense medium that allows red cells and granulocytes to pellet. 
3. Mononuclear cells are cultured for 3-4 days in a medium containing a mitogen such as phytohemagglutinin. Phytohemagglutinin stimulates lymphocytes to proliferate.
4. After the culture period when a large population of dividing cells is present, colcemid is added. Colcemid disrupts the mitotic spindles and prevents completion of mitosis, thereby assembling cells arrested in metaphase.
5. Lymphocytes are harvested and treated briefly with a hypotonic solution. The hyportonic solution causes the nuclei to swell through osmosis facilitating the spreading of the chromosomes of the metaphase preparation. 
6. The swollen cells are fixed, dropped onto a microscope slide and dried.
7. Slides are stained. 
8. Stained slides are scanned to identify "good" chromosome spreads (chromosomes are not too long or too compact and are not overlapping). Good spreads are then photographed.
9. Karyograms are prepared from developed photos. Chromosomes are cut out and arranged and pasted orderly (from large to small in their different groups) on a prepared form.
10. Today, karyograms are prepared from digital images using a computer. 
11. Karyogram is analysed.
12. Because it is not uncommon for single spreads to have a chromosomes missing or additional chromosomes, several metaphases are processed. This is of particular importance when diagnosing abnormalities.

Description of a karyotype

1. First, the total number of chromosomes is given, followed by a comma and the sex chromosome constitution:           Normal male cat: 38, XY 
2 This general description is then followed by any autosomal abnormalities. 
                For example:
                Horse with three X chromosomes (trisomy X): 65, XXX 
                Female dog with increased length of the short (p) arm of chromosome 2: 78, XX, 2p+ 
                Male pig with a deletion from the long arm (q) of chromosome 10: 38, XY, 10q- 
3. If an abnormality is identified in peripheral blood, it is sometimes desirable to determine whether that abnormality is present throughout the individual, and further studies with tissues other than blood can be performed.