Karyotyping, FISH, CISH, in-situ hybridization, aCGH, LOH, microarrays, mutation analysis – Melbourne Victoria Australia

Cytogenetics

What is Cytogenetics?

Cytogenetics is the study of chromosomes. Chromosomes are found in all cells of your body, except some blood cells. Chromosomes consist of strands of DNA ("deoxynucleic acid") and protective proteins, which microscopically appear as rod-shaped structures during cell division.

Traditional cytogenetics requires a highly experienced scientist meticulously check the chromosomes microscopically to ensure the number, the structure and the banding pattern of all the chromosomes are normal. This process is called karyotyping and the resulting product is a karyotype. Analysis at this level is a relatively broad-brush approach which detects visible chromosome abnormalities.

The limitations of this approach are determined by the quality of the chromosomes produced by the laboratory, the magnification of the microscope and the limitations of light microscopy. Addressing these limitations, newer techniques allow for smaller chromosome abnormalities to be detected.

These newer techniques include:

FISH and CISH (In-situ Hybridization)
aCGH (array Comparative Genomic Hybridization)
LOH (Loss of Heterozygosity)
Microarrays
Mutation analysis

Why is Cytogenetics Important?

There are 46 chromosomes in each cell of your body, 23 from your mother and 23 from your father. Two of these are sex chromosomes (X and Y); the remaining 22 pairs (chromosomes 1-22) are "somatic" chromosomes. All chromosomes have strings of genes on them which are made from DNA.

Chromosomal DNA (our genes) is the encyclopedia of instructions which determines most of our basic qualities, male or female, blond or brunette, and so on. The X and Y chromosomes contain genes chiefly determining gender-related characteristics, while the 22 pairs of somatic chromosomes contain genes important for general body functions. Thousands of diseases are now recognized as being caused by abnormalities in DNA structure which alter how genes function; and susceptibility to hundreds of other diseases (including common illnesses such as high blood pressure, obesity, diabetes, cancer and heart disease) are also strongly influenced by our genes.

We know that when some genes and/or chromosomes show a change from normal, that it can result in syndromes or diseases.

These changes include:

Changes to the number of chromosomes (for eg. Down Syndrome)
Chromosomal or gene deletions (i.e. where some DNA is missing)
Chromosomal or gene duplications (i.e. where there is extra DNA)
Structural abnormalities of the chromosomes (where the genes have been rearranged in a different order)

TissuPath scientists who work in cytogenetics are skilled at identifying these changes and knowing which changes relate to particular syndromes or diseases. They are also aware of the limitations of different tests and can recommend whether karyotyping or FISH is more appropriate to detect a suspected abnormality.

Changes to your genes and/or chromosomes can occur at different times during development.

Prior to conception

as a result of your father or mother carrying the change in their chromosomes/genes. In this instance the change has been inherited and may therefore affect other family members.
as a result of the change occurring during formation of either egg or sperm. In this instance the change has technically also been inherited; however, as it only affected the particular egg or sperm that led to that particular conceptus, other family members are unlikely to be affected.

During early cell division of the embryo

resulting in the presence of two populations of cells, one normal and one abnormal. This is called mosaicism. The two populations of cells can be confined to particular tissues including the placenta, which can lead to a situation called confined placental mosaicism. Cryptic mosaicism, where one cell line cannot be detected, always remains a possibility where clinical findings do not match the cytogenetic findings.

During cancerous growth of tissues

resulting in chromosome/gene abnormalities that are restricted to the tissue which is affected (so-called "somatic genetic" mutations). These changes can be useful for diagnosis and for establishing prognosis, and are also useful for assessing whether the cancer will respond to specific treatments. One well known example of this is an increase in the number of Her-2 genes in breast cancers, which predicts response to trastuzumab (Herceptin®).

Who should have chromosome testing and when?

Your GP or specialist will recommend when it is relevant to have a karyotype and/or FISH test.

Some situations when this may occur include –

During a pregnancy, when it is possible to sample the baby’s cells (either chorionic villi sampling (“CVS”) or amniocentesis (“amnio”) to determine if the baby has a chromosome abnormality. There are various screening tests which your doctor can recommend to determine if your pregnancy is at a high risk of Down Syndrome (trisomy 21) or Edward Syndrome (trisomy 18).
If you have had more than 3 miscarriages, to determine whether either you or your partner have a change to your chromosomes which may be contributing to your miscarriages.
As part of a testing regime if you are having trouble getting pregnant.
If your doctor suspects that either you or your child has a particular syndrome or disease that can be detected by either karyotyping or FISH testing, for example:
- Adolescents who fail to go through puberty (fail to develop secondary sexual characteristics such as pubic and axillary hair)
- Children with delayed developmental milestones such as learning to speak
To assist with diagnosis, prognosis, and/or treatment options if you have cancer or are suspected of having cancer. In this case, Cytogenetic Services requires a sample of the suspect cancer for testing.

Cytogenetic Services provides karyotyping and FISH on all specimen types. Both FISH and CISH are available on specimens which have been prepared for histological examination (Formalin-Fixed and Paraffin Embedded or "FFPE" specimens).

What type of specimen do I need to provide?
Some specimens that are required for karyotyping and/or FISH need to be taken by a specialist doctor. These specimens include:

Amniotic fluid and chorionic villi (taken by a specialist obstetrician trained in ultrasonography)
Products of conception (taken by an obstetrician)
Tumor samples (either whole tumours or biopsies (taken by a specialist surgeon or physician)

Other specimens are more easily obtained by a nurse in a pathology collection centre. These specimens include:

Blood specimens
Buccal smears (from inside the cheek)

The specimen required depends on the reason your doctor wants karyotyping and/or FISH performed.

How long does it take to get a result?

Karyotyping Results
To examine the chromosomes in a particular tissue requires first growing the tissue cells in the laboratory. This step is required because the chromosomes are only visible when the cells are actively dividing. The length of time it takes to grow sufficient cells for “harvesting” depends on the tissue type. This can vary from 3 days for blood samples to 8-14 days for prenatal samples (CVS or amnio).

Once there are sufficient cells dividing a chemical is added to the cultures which stops the cell division at an optimal stage for chromosome analysis. Before microscopic analysis, slides are stained to create an internationally-accepted chromosome banding pattern.

At least 15 cells are examined, with at least five cells having each chromosome thoroughly analysed band by band. In addition, two of the cells are then checked by a senior scientist. Using image analysis software two cells are photographed and a karyotype is prepared.

It takes a minimum of three hours for the analysis, checking, and karyotype to be completed on each specimen, and a conclusion reached about the test result. The actual time taken depends on the quality of the preparation and how many of the original cultures are required for analysis.

Examining all 23 pairs of chromosomes for any abnormality is like looking for a needle in a haystack. Sometimes your doctor may have specific information which indicates that a particular chromosome should be examined in more detail than others. This might be because there is a known chromosome/gene abnormality in the family or it might be because your doctor suspects a specific syndrome or disease. This information is invaluable to the laboratory and will inevitably provide a greater degree of reassurance about the result.

FISH and CISH Results
If your doctor is specific about the syndromes/diseases that they want us to test for, it may be more appropriate for us to do a FISH test, either in conjunction with full chromosome analysis, or as a stand-alone test for chromosomal abnormalities too small to be detected by microscopy alone.

FISH results on uncultured cells can take as little as 24-48 hrs, whilst FISH on cultured cells depends on the length of time it takes to culture the cells and it can take 7-14 days. FISH or CISH testing on formalin-fixed, paraffin-embedded speciments (those used for histopathology) usually takes 2-4 days.