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Preparing a Karyotype
phase cells are required to prepare a standard karyotype, and virtually any population of dividing cells could be used. Blood is easily the most frequently sampled tissue, but at times, karyotypes are prepared from cultured skin fibroblasts or bone marrow cells. None of the leukocytes in blood normally divide, but lymphocytes can readily be induced to proliferate, providing a very accessible source of me taphase cells.
There are many protocols for preparing a karyotype from peripheral blood lymphocytes, but a rather standard series of steps is involved:
- A sample of blood is drawn and coagulation prevented by addition of heparin.
- Mononuclear cells are purified from the blood by centrifugation through a dense medium that allows red cells and granulocytes to pellet, but retards the mononuclear cells (lymphocytes and monocytes).
Q: Why we select mononuclear rather than granulocyte
The mononuclear cells are cultured for 3-4 days in the presence of - a mitogen like phytohemagglutinin, which stimulates the lymphocytes to proliferate madly.
- At the end of the culture period, when there is a large population of dividing cells, the culture is treated with a drug such as colcemid, which disrupts mitotic spindles and prevents completion of mitosis. This greatly enriches the population of m etaphase cells.
- The lymphocytes are harvested and treated briefly with a hypotonic solution. This makes the nuclei swell osmotically and greatly aids in getting preparations in which the chromosomes don't lie on top of one another.
- The swollen cells are fixed, dropped onto a microscope slide and dried.
- Slides are stained after treatment to induce a banding pattern as described above.
Once stained slides are prepared, they are scanned to identify "good" chromosome spreads (i.e. the chromosomes are not too long or too compact and are not overlapping), which are photographed. The photos then are given to kindergarten children, who cut out the images of each chromosome and paste them to a backing sheet in an orderly manner. Alternatively, a digital image of the chromosomes can be cut and pasted using a computer. If standard staining was used, the orderly arrangement is limited to grouping like-sized chromosomes together in pairs, whereas if the chromosomes were banded, they can be unambiguously paired and numbered.
The image below shows chromosomes as they are seen on the slide (left panel) and after arrangment (right panel).
The image below shows chromosomes as they are seen on the slide (left panel) and after arrangment (right panel).
Karyotypes are presented in a standard form.
First, the total number of chromsomes is given, followed by a comma and the sex chromosome constitution. This shorthand description is followed by coding of any autosomal abnormalities. A few (simple) examples of this format are:
- A normal male cat: 38, XY
- 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-
Generally, several met a phases are processed because its not uncommon for a single spread to artifactually have extra chromosomes or be missing chromosomes. This is particularly important if one is to diagnose an abnormality in an individual. It also allows one to diagnose cases of mosaicism, in which an individual has multiple, cytogenetically-distinct populations of cells.
One final point. The discussion above has focused on initial evaluation of an individual's cytogenetic status. If abnormalities are found 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. Also, analysis of diseased tissues can often provide useful information.
One final point. The discussion above has focused on initial evaluation of an individual's cytogenetic status. If abnormalities are found 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. Also, analysis of diseased tissues can often provide useful information.
A prime example of this is the cytogenetic evaluation of cancers, which is not only used diagnostically, but has provided valuable understanding of the pathogenesis of certain types of neoplasia.
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[web]http://learn.genetics.utah.edu/units/disorders/karyotype/flash/karyotype_try_it.swf[/web]
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Next Topic for Cytogenetics: Aneuploidy and Deletions
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