Fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) is a combination of cytogenetic and molecular genetic methods. Principle of FISH method is hybridization – linking DNA probe to chromosomal DNA of examined sample of the patient. Probe represents small fragment of DNA labelled with fluorescent colour, which is linked to specific point on the chromosome.

Today there is a wide scale of commercial probes for all chromosomes or its segments, centromeres and many genes. Thanks to those we can closer determine numeric and structural changes of chromosomes in various types of cells and tissues, on chromosomes even in non-splitting cells. FISH method moved forward cytogenetic diagnosis, as it enabled view into sub-microscopic level of chromosomes – that is visualize small chromosomal reconstructions, which are not visible nor valuable under microscope during routine cytogenetic analyses.

FISH method helps to uncover or elaborate small deletions, duplications, translocations, determine original mark of chromosomes (pic. 1A + 1B, pic 2A + 2B) and diagnose cell lines, which are present only in small percentage in tissues or blood, for example small mosaics of sex chromosomes X and Y in persons with reproductive disorders (pic.3). Fluorescent in situ hybridization is used also to set frequency of numeric changes of chromosomes in sperm of men with severe spermatogenesis disorder (evolution of gametes) (pic. 4). From this year pre-implentation genetic diagnosis of chromosomal anomaly has been established, where FISH method is also used (pic. 5, see block PGD).

(Pic. 1A + 1B )) Small excessive marker chromosome of unknown origin has been detected in female with long lasting primary sterility (1A). Origin of marker chromosome has been assessed using FISH method with centromeric probe for chromosome 15 (green signal) and probes specific for two genes on long limb of the same chromosome. There is one green signal (centromere 15) and both genes (red signals) present on normal chromosome 15, marker chromosome contains under FISH findings only centromere and does not carry any genetically active chromatin. Its presence in karyotype may be related to infertility of examined female.

(pic. 2A + 2B ) Inverted duplication of Y chromosome, which has also deletion of significant part of long limb diagnosed in male with azoospermia (pic. 2A). Break point on long limb of Y chromosome happened under centromere in AZF area, which is mostly missing. Chromosomal anomaly has been accurately determined using FISH method – hybridization with SRY gene for evolution of testes has found inverted duplication of broken chromosome (2 red signals labeling presence of 2 copies of SRY gene) – pic. 2B. Molecular genetic methods closely determined dimension of deletion in AZF area.ti.

(Pic. 3) Mosaic of chromosome X diagnosed in female patient with irregular menstruation. Red label – signal representing centromere of X chromosome; one red signal states presence of one X chromosome in line 45,X and two red signals stand for presence of two X chromosomes in line 46,XX.

(Pic. 4) Examination of number of chromosomes X, Y and 18 in sperm of males with severe form of oligospermia. Normal findings represent presence of one X chromosome (green signal) and one chromosome 18 (blue signal), or one Y chromosome (red signal) and one chromosome 18 (blue signal). Presence of two chromosomes 18 and one chromosome Y is a pathological finding.

(Pic. 5) Blastomere from embryo from which foetus affected by Edward’s syndrome could be born (nucleus is under-coloured grey, colour marks are signals for examined chromosomes: X-2 purple signals, 13-2 red signals, 18-3 light blue signals, 21-2 green signals).