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Cytogenetics is the study of the structure of chromosome material. It includes routine analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH).



Early years

Cytogenetics is generally said to have begun in 1956 with the discovery that normal human cells contain 46 chromosomes by Tjio and Levan. This discovery was aided by a new technique of slide preparation utilizing a hypotonic solution discovered by TC Hsu in 1952. A hypotonic solution is a salt solution less concentrated than that inside human cells, when added to a cell solution this causes the cells to swell. When the cells are added to the slide after treatment with hypotonic the chromosomes spread much better allowing for much easier enumeration. Previously humans were thought to have 48 chromosomes.

It is notable that while Flemming and Arnold first observed human chromosomes in the 1880s, the number of human chromosomes remained unknown for over 70 years. The causes for this are numerous. For many valid scientific reasons, many geneticists at the time relied heavily on animal models for their research, and thus research in human genetics did not keep up with species like the fruit fly Drosophila. This was also the period of the eugenics movement and many geneticists wanted to distance themselves from human genetics.

Progress in numerical abnormalities

With that advent of harvest procedures which allowed easy enumeration of chromosomes, discoveries were quickly made in abnormalities arising from nondysjunction events which cause cells with aneusomy (additions or deletions of entire chromosomes). In 1959 Lejeune2 discovered patients with Down syndrome had an extra copy of chromosome 21. Down syndrome is also referred to as trisomy 21. In 1960 Nowell3 discovered a small chromosome, dubbed the Philadelphia chromosome which was shown to be the cause of Chronic myelogenous leukemia. 13 years later this was shown to be a translocation or chromosomes 9 and 22 by Janet Rowley

Other numerical abnormalities discovered include sex chromosome abnormalilities. An individual with only one sex chromosome (the X) has Turner syndrome, an additional X chromosome in a male, resulting in 47 total chromosomes has Klinefelter's Syndrome. Many other sex chromosome combinations are compatible with live birth including XXX, XYY, and XXXX. (The ability for mammals to tolerate aneusomies in the sex chromosomes arises from our ability to inactivate it, which is required in normal females to compensate for the dosage difference in having two copies of the chromsome.) Trisomy 13 was also associated with Patau's Syndrome and trisomy 18 was associated with Edward's Syndrome.

Advent of banding techniques

Human female karyotype, giemsa banded
Human female karyotype, giemsa banded

In the late 1960's Casperson developed banding techniques which differentially stain chromosomes. This allows chromosomes of otherwise equal size to be differentiated as well as to elucidate the breakpoints and constituent chromosomes involved in chromosome translocations. Deletions within one chromosome could also now be more specifically named and understood. Deletion syndromes such as DiGeorge syndrome, Prader-Willi syndrome and others were discovered to be caused by deletions in chromosome material.

These combined discoveries in both prenatal and oncological feilds quickly moved cytogenetics into the clinical lab. Techniques were expanded to allow for culture of free amniocytes recovered from amniotic fluid, and elongation techniques for all culture types that allow for higher resolution banding.

Beginnings of molecular cytogenetics

In the 1980s advances were made in molecular cytogenetics. While radioisotope-labeled probes had been hybridized with DNA since 1969 movement was now made in using fluorescently labeled probes, and hybridizing them to chromosomes preparations made using existing techniques, this technique is called fluorescent insitu hybridization (FISH). This change significantly increased the usage of probing techniques as fluorescently labeled probes are safer and can be used almost indefinitely.


In some forms of cancer, especially hematological malignancies, cytogenetics can determine which chromosomal translocations are present in the malignant cells, facilitating diagnosis and susceptibility to treatment (e.g. imatinib mesylate in the presence of the Philadelphia chromosome).

In congenital disorders, such as Down's syndrome, cytogenetics can determine the nature of the chromosomal defect - a "simple" trisomy, a mosaic, "balanced" translocation, a deletion, or an insertion in one - or both - of the parents, or in the fetus.


Routine analysis

Routine chromosome analysis refers to analysis of metaphase chromosomes which have been banded using trypsin followed by Giemsa. This created unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it likely related to replication timing and chromatin packing.

Slide preparation

Cells from bone marrow, blood, amniotic fluid, cord blood, tumor, and tissues (including skin, unbilical cord, liver, and many other organs) can be cultured using standard cell culture techniques in order to increase their number. A mitotic inhibitor (colchicine, colcemid) is then added to the culture. This stops cell division at mitosis which allows an increased yield of mitotic cells for analysis. The cells are then centrifuged and media and mitotic inhibitor is removed, and replaced with a hypotonic solution. This causes the cells to swell so that the chromosomes will spread when added to a slide. After the cells have been allowed to sit in hypotonic, Carnoy's fixative (3:1 methanol to glacial acetic acid) is added. This kills the cells, lyses the red blood cells, and hardens the nuclei of the remaining white blood cells. The cells are generally fixed repeatedly to remove any debris or remaining red blood cells. The cell suspension is then dropped onto specimen slides. After aging the slides in an oven or waiting a few days they are ready for banding and analysis.


Analysis of banded chromosomes is done at a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Generally 20 cells are analyzed which is enough to rule out mosacism to an acceptable level. The results are summarized and given to a board-certified medical geneticist and a pathologist for review, and to write an interpretation taking into account the patients previous history and other clinical findings. The results are then given out reported in an International System for Human Cytogenetic Nomenclature 1995(ISCN1995).

Fluorescent in situ hybridization

Fluorescent in situ hybridization refers to using fluorenscently labeled probe to hybridize to cytogenetic cell preparations.

In addition to standard preparations FISH can also be performed on:

  • bone marrow smears
  • blood smears
  • paraffin embedded tissue preparations
  • uncultured bone marrow
  • uncultured amniocytes
  • cytospin preparations

Slide preparation

This section refers to preparation of standard cytogenetic preparations

The slide is aged using a salt solution ususally consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. The sample DNA and the probe DNA are the co-denatured using a heated plate and allowed to re-anneal for at least 4 hours. The slides are then washed to remove excess unbound probe, and counterstained with 4',6-Diamidino-2-phenylindole (DAPI) or propidium iodide.


Analysis of FISH specimens is done in fluorescence microscopy by a clinical laboratory specialist in cytogenetics (CLSp(CG)). For oncology generally a large number of interphase cells are scored in order to rule out low level residual disease, generally between 200 and 1000 cells are counted and scored. For congenital problems usually 20 metaphase cells are scored.

Future of cytogenetics

Advances now focus on comparative genomic hybridization arrays, and automated systems for counting the results of standard FISH preparations.


  • Tjio HJ, Levan A. The chromosome numbers of man. Hereditas 1956;42:1-6.
  • Note 2: Lejeune J, Gautier M, Turpin MR. Etude des chromosomes somatiques de neuf enfants mongoliens. C R Acad Sci (Paris) 1959;248:1721-2.
  • Note 3: Nowell PC, Hungerford DA. A minute chromosome in human chronic granulocytic leukemia. Science 1960;132:1497-1501.

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Last updated: 08-17-2005 20:16:21