In Situ Hybridization: Treatment, Effects & Risks

In situ hybridization is a method for detecting chromosomal aberrations. It involves labeling specific chromosomes with flouroscent dyes and binding them to a DNA probe. This technique is used for prenatal diagnosis of genetic mutations.

What is in situ hybridization?

In situ hybridization involves labeling specific chromosomes with flouroscent dyes and binding them to a DNA probe. This technique is used for prenatal diagnosis of gene mutations. In situ hybridization or fluorescence in situ hybridization involves molecular genetic detection of nucleic acids from RNA or DNA in specific tissues or a cell. Normally, this type of diagnostics is used to detect a structural or numerical chromosomal abnormality in pregnancy. For this purpose, an artificially produced probe is used, which itself consists of nucleic acid. It then binds to the nucleic acids in the organism by base pairing. This binding is referred to by the term hybridization. The detection is carried out on the living structure of the patient and therefore corresponds to in situ detection. To be distinguished from this are in vitro methods, in which the detection takes place in the test tube. The method was developed in the 20th century by scientists Joe Gall and Mary Lou Pardue. The technique has evolved since then. For example, while radioactive probes were used then, flouroscence-labeled probes with a covalent bond to the labeling molecules are used today.

Function, effect, and targets

In situ hybridization is usually used to detect chromosomal aberrations, chromosomal abnormalities that cannot be detected in a karyogram. Thus, the method is always used when hereditary diseases are to be determined during pregnancy. Since chromosomal aberrations are a problem that should not be underestimated today, the application of the method has increased over time. Hybridization is performed using native cells from the mother’s amniotic fluid. The basis of the technique is the binding of the color-labeled probe to DNA fragments. Thanks to the binding, a microscope can later be used to evaluate the number of copies, as the individual copies emit a light signal and can thus be made visible under the microscope. There are different procedures for this. Either the analysis takes place immediately after binding. In this case, a flouroscence dye such as biotin is used, which is bound directly to the DNA probe. In the indirect method of in situ hybridization, the analysis cannot be performed immediately after hybridization because fluorescent substances can bind to the probe only after hybridization. This indirect method is more commonly used than the direct method because it is considered more sensitive. Techniques include chromosome-specific centrometer DNA probes, locus-specific DNA probes, chromosome-specific DNA library probes, and comparative genome hybridizations. Chromosome-specific centromere DNA probes can be used to detect chromosomal numerical abnormalities. That is, they are primarily used when duplicated or deleted chromosomes are suspected. The locus-specific DNA probes are mainly suitable for the detection of minimal mutations that cannot be detected in the karyogram. A chromosome-specific DNA library probe is used in particular to detect insertions and translocations. Comparative genome hybridization, on the other hand, is a comprehensive analysis of losses and gains in chromosomal material. Today, in situ hybridization is of great importance within the diagnostics of various chromosomal mutations. In the diagnostics of Down syndrome, for example, probes bind to chromosome 21. For this purpose, chromosome-specific probes are usually used, which can be applied in cases of suspicion of this disease. A suspicion may arise, for example, if the parents have previously given birth to a child with the disease and the ultrasound image is conspicuous. If there is a triple rather than a double tie, resulting in a triple color signal, the diagnosis is considered confirmed.

Risks, side effects, and hazards

Unlike PCR, for example, in situ hybridization is much less susceptible to contamination. In addition, the time required for the procedure is immensely less.However, because embryos in particular form chromosomal patterns, any pattern present cannot be used to infer with certainty the rest of the chromosomal distribution and thus the genetic status of other cells. Color signals may also overlap or remain invisible for other reasons. Thus, in situ hybridization as a diagnostic tool during pregnancy is relatively prone to error. Misdiagnoses can occur and parents may decide against a healthy embryo. To reduce the error-proneness of in situ hybridization, at least two embryonic cells should be examined simultaneously. By examining two cells in parallel, there is now only a negligible risk of misdiagnosis. Parents can therefore rely on the diagnosis in such a case. In situ hybridization is not offered to every pregnant woman, but only to women from a risk group. Nevertheless, pregnant women are not denied this type of diagnosis at their own request. Abnormal ultrasound findings or an abnormal serum may prompt a physician to offer the diagnostic procedure. Today, in situ hybridization can be used to diagnose a large proportion of chromosomal aberrations, but by no means all of them. Therefore, in situ hybridization must never be performed alone, but must always be used in conjunction with a conventional chromosome test. Care of the pregnant woman plays a major role in this procedure. Therefore, before the analysis, a thorough discussion on the diagnostic method takes place with the expectant mother, informing her about the risks, the possibilities, and the limitations of the technique.