Background Information

In Situ Hybridisation

Advantages of the ISH technology

Localisation of infectious agents in tissues/cells and quantitative measurement in solution are used as complementary methods in diagnostics. For example, ELISA's, Immunohistochemistry, PCR and in situ hybridisation with DNA probes are commonly used in laboratories worlwide. The additional value of ISH over conventional diagnostics lies in the fact that it enables the direct detection of specific DNA or RNA molecules in histological and cytological specimens (cervical smears, samples of blister, nose and other fluids, tissue sections, etc.), without losing the often very essential morphological details.

In contrast to PCR
- ISH can provide molecular information
within the context of histomorphological details.

In contrast to immunohistochemistry
- ISH is a reliable and sensitive method in formalin-fixed and paraffin-embedded tissue sections and additionally offering the possibility for retrospective studies on archival material;
- ISH does not depend on antigen-expression;
ISH is a highly specific technique thanks to
the direct detection of specific DNA/RNA sequences (antibodies are always a second line product).


Current and future applications

The ISH technique is applied in the fields of pathology, cytology, genetics and molecular biology. The ongoing developments in the field of molecular biology, the increase of the number of diagnostic applications of nucleic acid hybridisation techniques and the availability of more specific probes targeting specific diseases will allow better differentiation resulting in individualized patient care.

Viruses
A number of viruses are very difficult to propagate in culture, or in vitro propagation may not be possible at all. Serological detection methods for some viruses are rather complicated, and since virus particles are very unstable outside the body, they bear a risk of false negative testing for infection. With some viruses, a proper diagnosis can only be made when differentiation between an acute phase of the disease, carriers of the disease, or insertion (integration) of the viral genome into the host cell DNA, is possible. ISH offers the solution here.

Another advantage of ISH is that the patient material (cervical smears, samples of blister, nose and other fluids; tissue sections etc.) can directly be examined for the presence of viral DNA and various virus strains and subtypes can be distinguished in a single assay. Retrospective studies with several forms of DNA technology have provided important information revealing viral causes related to various disease processes.

Bacteria
ISH is capable of demonstrating the presence of bacterial nucleic acids by means of DNA probe technology. Genes of bacterial toxins can be demonstrated as well.

Parasites
ISH can be applied for the diagnosis of parasites on routinely obtained patient material, provided the DNA probe is made available.

Genetic defects
The ISH technology is capable of extending prenatal diagnosis of congenital defects, e.g. in amniotic fluid samples and chorionic biopsies. It is quite possible to visualise the absence or presence of a(n) (extra) chromosome (e.g. trisomy 21 in Down's Syndrome) in tissue sections. Also postnatal detection of chromosomal defects in e.g. malignancies and changes (translocations, deletions, inversions, duplications) can be demonstrated by ISH. Recent applications of ISH in tissue sections is the demonstration of numerical chromosomal aberrations in (suspected) human malignancies; i.e haematological disorders, mamma carcinoma and many others.

Gene expression
Immunohistochemistry may be used to test for the presence of cellular antigen but gives no indication whether the gene involved is being actively expressed at that moment, nor whether the protein product is of intracellular or extracellular origin. However, detection of mRNA within a cell by the ISH technique presents direct information about the status of the gene.