Cytogenetics is essentially a branch of genetics , but it is also a part of cell biology/cytology (a subdivision of human anatomy) that is concerned with chromosomal cell behavior, particularly their interactions during mitosis and meiosis. related to behaviour. [1] Techniques used include karyotyping , 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).



Chromosomes were first observed in plant cells by Carl Wilhelm von Ngeli in 1842. Their behavior in animal ( salamander ) cells was described by Walther Fleming , the inventor of mitosis , in 1882 the name was coined by another German anatomy, von Waldeyer in 1888.

The next stage followed the development of genetics in the early 20th century, when it was appreciated that the set of chromosomes ( karyotype ) was the carrier of genes. It seems that Levitsky was the first to define karyotype as the phenotypic appearance of somatic chromosomes , as opposed to their gene content. [2] [3] The investigation of human karyotype took many years to settle the most fundamental question: how many chromosomes does a normal diploid human cell have? [4] In 1912, Hans von Winiwarter reported 47 chromosomes in spermatogonia and 48 chromosomes in oogonia , an XX/XO sex determination .The conclusion of the system. [5] In 1922 the painter was not sure whether the diploid number of humans was 46 or 48, at first in favor of 46. [6] He later revised his opinion from 46 to 48, and he correctly emphasized that humans have an XX/XY system. of sex determination. [7] Considering their techniques, these results were quite remarkable. In the science books, the number of human chromosomes stood at 48 for more than thirty years. New techniques were needed to correct this error. Jo Hin Tjio working in Albert Levan ‘s laboratory [8] [9] was responsible for discovering this approach:

  1. Using cells in culture
  2. pre-treating cells in a hypotonic solution, which causes them to swell and stretch the chromosomes
  3. A solution of metaphase arrest in mitosis by colchicine
  4. Crush the preparation on the slide to force the chromosomes into a single plane.
  5. Cutting a photomicrograph and arranging the result into an undistorted workogram.

It took until 1956 for the general acceptance that the human karyotype consisted of only 46 chromosomes. The great apes have 48 chromosomes. Human chromosome 2 was formed by the merger of paternal chromosomes, reducing the number.

Applications in Cytogenetics

McClintock’s work on Maize

Barbara McClintock began her career as a maize cytogeneticist. In 1931, McClintock and Harriet Creighton demonstrated that cytological recombination of marked chromosomes is related to the recombination of genetic traits (genes). McClintock, while at the Carnegie Institution, continued previous studies on the mechanisms of chromosome breaks and fusion flares in maize. He identified a particular chromosomal breakage event that always occurs at the same locus on maize chromosome 9, which he named the ” D” or “dissociation” locus. [14]McClintock continued his career in cytogenetics, studying the mechanics and inheritance of broken and ring (circular) chromosomes in maize. During his cytogenetic work, McClintock discovered the transposon, a discovery that eventually earned him the Nobel Prize in 1983.

natural population of drosophila

In the 1930s, Dobzhansky and colleagues collected Drosophila pseudoobscura and D. persimilis from wild populations in California and neighboring states. Using Painter’s technique [15] he studied polytene chromosomes and found that the wild population was polymorphic for the chromosomal inversion. All flies look the same, regardless of the type of inversion: this is an example of a latent polymorphism.

The evidence rapidly accumulated to show that natural selection was responsible. Using a method invented by L’Héritier and Teissier, Dobzhansky bred the population in population cages , which enabled feeding, breeding, and sampling while preventing migration. This had the advantage of eliminating migration as a possible interpretation of the results. Stocks with inversions at a known initial frequency can be maintained under controlled conditions. It was found that the different chromosome types do not fluctuate randomly, as they are selectively neutral, but accommodate certain frequencies at which they become stable. 1951 [16]By the time Dobzhansky published the third edition of his book in 2006, he was persuaded that, like most polymorphisms, chromosomes were being retained in a population by the selective advantage of heterozygosity.

lily and mouse

Lily is a preferred organism for cytological examination of meiosis because the chromosomes are large and each morphological stage of meiosis can be easily identified microscopically. Hotta et al. [19] presented evidence for a general pattern of DNA nicking and repair synthesis in male meiotic cells of lilies and rodents during the zygotene–pachytene stages of meiosis, when crossing over was predicted to occur. The presence of a common pattern among organisms as phylogenetically as far away as lily and mouse the authors concluded that the organization for meiotic crossing-over, at least in higher eukaryotes, is probably universal in distribution.

Human abnormalities and medical applications

After the advent of procedures allowing easy counting of chromosomes, discoveries relating to abnormal chromosomes or chromosome numbers were quickly made. In some congenital disorders such as Down syndrome, cytogenetics reveals the nature of the chromosomal defect: a “simple” trisomy. Abnormalities arising from nondisjunction events can cause cells with aneuploidy (addition or deletion of entire chromosomes) in one of the parents or embryos. In 1959, Lejeune [20] found that patients with Down syndrome had an extra copy of chromosome 21. Down syndrome is also known as trisomy 21.

Other numerical abnormalities discovered include sex chromosome abnormalities. A female with only one X chromosome has Turner syndrome, while a male with an extra X chromosome, resulting in a total of 47 chromosomes, has Klinefelter syndrome. Several other sex chromosome combinations are compatible with live birth, including XXX, XYY and XXXX. The ability for mammals to tolerate aneuploidies in sex chromosomes stems from their ability to deactivate them, which in normal females is required to compensate for two copies of chromosomes. Not all genes on the X chromosome are inactivated, which is why a phenotypic effect is seen in individuals with an extra X chromosome. Trisomy 13 was associated with Patau syndrome and trisomy 18 with Edwards syndrome.

In 1960, Peter Nowell and David Hungerford [21] discovered a small chromosome in the white blood cells of patients with chronic myelogenous leukemia (CML). This abnormal chromosome was called the Philadelphia chromosome – because both scientists were doing their research in Philadelphia, Pennsylvania. Thirteen years later, with the development of more advanced techniques, the abnormal chromosome was shown by Janet Rowley to be the result of a translocation of chromosomes 9 and 22. Identification of the Philadelphia chromosome by cytogenetics is the diagnosis for CML.

The advent of banding techniques


In the late 1960s, Torbjörn Caspersson developed a quinacrine fluorescent staining technique (Q-banding), which revealed the unique banding pattern for each chromosome pair. This allowed the otherwise identical sized chromosome pairs to be differentiated by distinct horizontal banding patterns. Banding patterns are now used to elucidate breakpoints and component chromosomes involved in chromosome translocations. Deletions and inversions within an individual chromosome can be more accurately identified and described using standardized banding nomenclature. G-banding (using trypsin and Giemsa/Wright stain) was developed concurrently in the early 1970s and allows visualization of banding patterns using a bright-field microscope.

Diagrams that identify chromosomes on the basis of banding patterns are known as idiograms . These maps became the basis for quickly transferring cytogenetics to the clinical lab for both the prenatal and oncological fields, where karyotyping allowed scientists to look for chromosomal changes. Techniques were expanded to allow the culture of free amniocytes recovered from the amniotic fluid, and elongation techniques for all types of culture allowed high-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, there was a movement to now use fluorescently labeled probes. Hybridizing them to chromosomal preparations using existing techniques became known as fluorescence in situ hybridization (FISH). [22] This change significantly increased the use of testing techniques because fluorescently labeled probes are safer. Further advances in micromanipulation and investigation of chromosomes led to the technique of chromosome microdissection whereby aberrations in chromosomal structure could be isolated, cloned and studied in ever greater detail.



Routine chromosome analysis (karyotyping) refers to the analysis of metaphase chromosomes that have been banded using trypsin, followed by Giemsa, Leishman, or a mixture of both. This creates unique banding patterns on the chromosomes. The molecular mechanism and reason for these patterns is unknown, although it is probably related to replication timing and chromatin packing.

Several chromosome-banding techniques are used in cytogenetics laboratories. Quinacrine banding (Q-banding) was the first staining method used to produce distinctive banding patterns. This method requires a fluorescence microscope and is no longer as widely used as Giemsa banding (G-banding). Reverse banding, or R-banding, requires heat treatment and reverses the common black-and-white patterns seen in the G-band and Ku-band. This method is particularly helpful for staining the distal ends of chromosomes. Other staining techniques include C-banding and nuclear organizing area stain (NOR stain). These latter methods specifically stain parts of the chromosome. C-banding stains constitutive heterochromatin, usually near the centromere,

High-resolution banding involves the staining of chromosomes during prophase or early metaphase, (prometaphase) before they reach maximal condensation. Because prophase and prometaphase chromosomes are stretched more than metaphase chromosomes, the number of observable bands for all chromosomes increases from approximately 300 to 450 to as many as 800 to detect this less obvious abnormalities. Allows not normally seen with traditional banding.

slide preparation

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


Bonded chromosomes are analyzed under a microscope by a clinical laboratory specialist in cytogenetics (CLSp(CG)). Typically 20 cells are analyzed which is sufficient to rule out mosaicism to an acceptable level. The results are summarized and given to a board-certified cytogeneticist for review, and to write an interpretation taking into account the patient’s past history and other clinical findings. The results are then reported in an International System for Human Cytogenetic Nomenclature 2009 (ISCN2009) .

Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) refers to the use of fluorescently labeled probes to hybridize for cytogenetic cell preparations.

In addition to standard preparations, fish can also be performed:

  • bone marrow smear
  • blood stains
  • Paraffin Embedded Tissue Preparation
  • enzymatically isolated tissue samples
  • Uncultivated bone marrow
  • intractable amniocytes
  • Preparation of cytospin

slide preparation

This section refers to the preparation of standard cytogenetic preparations.

Slides are typically aged using a salt solution consisting of 2X SSC (salt, sodium citrate). The slides are then dehydrated in ethanol, and the probe mixture is added. Sample DNA and probe DNA are co-denatured using a hot plate and allowed to re-anneal for at least 4 h. The slides are then washed to remove excess unbound probe, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide.


FISH samples are analyzed by fluorescence microscopy by a clinical laboratory specialist in cytogenetics. For oncology a large number of interphase cells are typically scored, usually between 200 and 1,000 cells, to rule out low-level residual disease. For congenital problems usually 20 metaphase cells are made.

future of cytogenetics

Advances now focus on molecular cytogenetics, including automated systems for computing the results of standard FISH preparation and techniques for virtual karyotyping, such as comparative genomic hybridization arrays, CGH and single nucleotide polymorphism arrays.