Structural and numerical chromosomal alterations aberrations are the hallmarks of malignant diseases. Routine cytogenetic analysis based on G-protein couple receptor GPCR techniques provides important information of diagnostic and prognostic relevance both in hematological malignancies and solid tumors. However, detection of chromosomal alterations by this method is complicated by the difficulty in routinely preparing metaphase spreads of sufficient quality and quantity, the clonal heterogeneity of the tumors, and the complexity of the many c Skip to main content Skip to table of contents.
This service is more advanced with JavaScript available. Five pure spectrally distinct dyes are used either singly or in combination to create a chromosome cocktail of probes, each with a unique spectral signature for each chromosome 1.
Its strength lies in its ability to define translocations, marker chromosomes, and complex rearrangements, and to reveal cryptic change; it cannot, however, detect intrachromosomal rearrangements, such as duplications, very small deletions, or small paracentric inversions 1. Abnormal spectral karyotype of a four-way translocation. Characterization FAQs. Quality Control Testing. A discrete color was assigned to all pixels with identical spectra according to a spectrum classification system, and the pixels were then displayed in the assigned colors.
Images were captured and analyzed according to the manufacturer's instructions. Usually, five to eight metaphase chromosome spreads from each patient were analyzed by SKY. The DAPI images and G-banding results were used in the identification of the chromosome bands and the assignments of the breakpoints.
SKY has been shown to be a useful molecular-based method that can identify chromosomal rearrangements that are not visible by routine light microscopy. To assess the utility of SKY in identifying cryptic chromosomal rearrangements in pediatric patients with ALL, we analyzed a select group of leukemic samples. This group of 20 patients lacked chromosomal abnormalities by conventional cytogenetics.
SKY analysis found no chromosomal aberrations in leukemic cells of 18 of 20 patients. However, FISH using a 19p subtelomeric probe confirmed the presence of the reciprocal t 17;19 data not shown. The two translocations t 7;8 and t 13;17 have not been previously identified in patients with ALL, probably because of the subtle nature of these changes. G-banding a and SKY results b. Display colors are shown on the left; spectral classification colors are shown on the right.
The probe for chromosome 7 was labeled with digoxigenin, and the probe for chromosome 8 was labeled with biotin. Normal chromosomes 7 and 8 are shown to the left of the translocated chromosomes. G banding a and abnormal chromosomes 13, 17 and 19 identified by SKY b. The signal from chromosome 19 was not observed on the other homolog of chromosome 17 by SKY.
Display colors are shown on the left, and the spectral classification colors are shown on the right.
A combination of digoxigenin- and biotin-labeled chromosome 13 probe, biotin-labeled chromosome 17 probe and digoxigenin-labeled chromo-some 19 probe was used to confirm the t 13;17 and der 19 t 17; Display and spectral colors for the abnormal chromosomes are shown on the left and right side respectively. However, FISH with the 19p subtelomeric probe confirmed the balanced t 17;19 data not shown. Display colors are on the left, and spectral classification colors are shown on the right.
Probes for chromosomes X and 5 were labeled with digoxigenin and biotin, respectively. For detection of chromosome 21, we used a combi-nation of digoxigenin- and biotin-labeled probes. The display colors are shown on the left, and the spectral classification colors are shown on the right. The probe for chromosome 9 was labeled with digoxigenin, and the identity of the X chromosome was determined by the inverted DAPI images. Normal chromosome 21 is to the left of the quadruplicated 21q chromosome.
To extend our study, we examined the ability of SKY to help in defining the derivative or marker chromosomes observed by conventional cytogenetics. Ten cases were selected that had either structural or numerical abnormalities. In these patients, conventional cytogenetics detected 29 acquired clonal abnormalities, which included seven unidentified chromosomal material and four deletions.
The redefined karyotype was as follows: 47,X,der X t X;5 p In patient No. The amplification represented the duplication of the 21q22 segment. The significance of AML1 amplification and of extra copies of 21q in the pathogenesis of leukemia is not known. In addition, FISH using an 11q subtelomeric probe did not identify any translocation to any other chromosome; this finding confirmed the del 11 q23 data not shown. Conventional cytogenetic analysis identified an add 4 p16 and a t 8;14 q Furthermore, SKY has been applied to a research field within comparative cytogenetics, which studies chromosomal rearrangements during evolution 4.
Using SKY technique, our laboratory becomes the first to identify chromosome abnormalities in mice and humans with polycystic kidney disease Figure 2. There is no doubt that SKY will be beneficial in many other chromosomal associated diseases. In fact, we propose that SKY would also be useful in many cilia-related pathogenesis, where cilia have been shown to regulate cell division Because mouse and rat are important animal models to study the mechanism of a disease and to perform a functional assessment in vivo , the development of mouse and rat probes for SKY analysis has allowed routine karyotyping of mouse and rat chromosomes.
At present, SKY is therefore limited to chromosomal studies in human, mouse and rat. However, as more research laboratories use other species to study genomic compositions, it is foreseeable that more chromosome painting probes for different organisms will soon be commercially available.
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Treat the cells with colcemid solution at 0. Collect the medium containing any floating cells in ml sterile falcon centrifuge tubes. Rinse the cells on the plate with sterile1X-PBS. After incubating with sterile trypsin for min, harvest and collect the remaining cells into the same tube. Spin the tubes at r. Depending on the pellet size, add ml of hypotonic solution of 0. Centrifuge at r. This procedure is critical so that the metaphase spreads will not be trapped in the cells' clumps which would compromise the experiment.
Centrifuge again at r. Clean slides in absolute ethanol, then dip the slide in dH 2 O for approximately10X in order to form a sheath of water on the surface of the slide.
Check the slide under a light microscope using 10X and 40X dry objectives making sure there is metaphase chromosomes and the spreads are evenly spaced.
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