Preparing a Karyotype

Metaphase 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 metaphase 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).


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 metaphase 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.


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).



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 metaphases 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. 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

for more you can see these vedioes

[ame]http://www.youtube.com/watch?v=E0WkZr819UU&feature=related[/ame]


[ame]http://www.youtube.com/watch?v=F7trv8c3Vlo&feature=related[/ame]

finally my question is

Why might a laboratory worker attempting to diagnose a genetic
disorder prefer to work with photographs of chromosomes rather
than the chromosomes themselves?