# Invasive Prenatal Diagnostics: A Cornerstone of Perinatal Management
## 2 Techniques of Invasive Diagnostics  
### Amniocentesis  
#### 2.1.1 Genetic Analysis  
Upon early detection of a foetal abnormality, invasive diagnostics should be offered to the patient. Foetal tissue can be obtained either by amniocentesis or chorionic villus sampling (CVS). Aminocentesis is a safe procedure that is usually carried out in an outpatient setting. It is performed by the insertion of a needle inside an amniotic cavity with constant ultrasound guidance. Its safety has been proven numerous times [66], but both maternal and foetal complications can occur. Therefore, patients should always be properly consulted and give written consent. After removing a sample of amniotic fluid, it undergoes a genetic analysis. Currently, a variety of tests are available, as described below. _Karyotyping_--Historically, amniotic fluid routinely underwent a conventional cell culture. After 2-3 weeks, microscope visualisation of chromosomes was feasible. This method was relatively time-consuming and allowed for only a diagnosis of chromosomal number aberrations and significant structural aberrations (resolution limited to 5-10 Mb), neglecting a substantial group of structural defects. Therefore, quality alternatives were needed. _Quantitative Fluorescence Polymerase Chain Reaction (QF-PCR)_--QF-PCR is a rapid method for the quantitative analysis of abnormalities at the chromosomal level. Amplification of repeat sequences of polymorphic loci by PCR with fluorochromic primers allows for the visualisation and quantification of the product by an automated DNA scanner [67]. The results are seen as \"peaks\" corresponding to the number of alleles of amplified genes present in the examined sample. Individuals with a trisomic karyotype will demonstrate three peaks, suggesting the presence of three copies of the same gene, thus enabling the diagnosis of trisomy. This method was initially introduced as a valuable alternative to a standard cell culture. Despite providing results for abnormalities of the chromosomal copies overnight, it was not accepted as a substitute for a classic cytogenetic analysis. It is unlikely to detect some abnormalities of high clinical significance, such as balanced translocations or chromosomal mosaicism [68]. Nevertheless, it is extremely useful in excluding the contamination of the sample with maternal tissue and is still valuable in this indication. _In-situ Hybridisation (ISH)_--This method has been used in prenatal diagnostics since the beginning of the 2000s and is often referred to as \"chromosome painting\" [69]. After obtaining a sample, the genetic material is denatured to a single-stranded state and hybridised to fluorescent probes specific to certain chromosomal regions [70]. This allows for the detection and visualisation of precisely defined sequences, thus making them countable. Therefore, this genetic method provides a quantitative approach rather than a qualitative one. However, the results are usually available in 48 h, which makes it valuable for urgent clinical situations. _Comparative Genomic Hybridisation (CGH)_--This method was first described in 1992 by [PERSON] et al. [71] and was initially useful in oncology, allowing for the detection of discreet gene rearrangements. Eventually, it found a broad application in genetic and prenatal diagnostics. It is based on a standard _FISH_ technique; however, its conceptual difference is crucial. A sample of DNA extracted from the examined material is labelled with green and red fluorescent markers. Subsequently, the test DNA is hybridised on a slide with reference DNA. The target DNA consists of previously prepared metaphase chromosomes derived from the cells of a karyotypically normal individual [72]. Finally, the imaging software compares the metaphase spreads and calculates the green/red ratio along the length of each chromosome. The changes in the green/red ratio correspond to the areas of genomic gains or losses. Therefore, it is possible to distinguish copy number variations (CNVs) with high sensitivity.