| When marker chromosomes or unbalanced translocations are encountered during a pre- and post-natal examination, rapid identification of the origin of these chromosomes, or portions of these chromosomes is required. Previously, G-banding has been the method of choice to discern these complex rearrangements. Fluorescence in situ hybridization (FISH) has revolutionized clinical chromosomal identifications, but it can be time consuming and costly, requiring a series of combined analyses if the origin of the marker chromosome is not known. Rapid identification of the origin of complex markers can be accomplished with a whole genome painting approach such as spectral karyotyping (SKY) , It is a 24-color, multi-chromosomal painting assay that allows the visualization of all human chromosomes in one experiment. The ability for SKY analysis to detect equivocal or complex chromosomal rearrangements, as well as to identify the chromosomal origins of marker chromosomes and other extra-chromosomal structures, makes this a highly sensitive and valuable tool for identifying recurrent chromosomal aberrations. In this study, we established a technical system of spectral karyotyping (include investigating the effects of a number of variables, i.e., dropping height of cell suspension, wetness and angle of the slide, drying time, fixative ratio on metaphase chromosome spreading and the underlying mechanism. ) , to meet the needs of the laboratories in China . then, analyzed lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirmed that an algorithm which selectively uses SKY will provide an efficient andimproved method for pre- and post-natal chromosomal analysis.The emphasis of research is the implementation of SKY in clinical.Objective1 , To investigate the effects of a number of variables, i.e., dropping height of cell suspension, wetness and angle of the slide, drying time, fixative ratio on metaphase chromosome spreading and the underlying mechanism.2, Establishing the technical system of spectral karyotyping to meet the needs of thelaboratory in china.3, To evaluate case-study archives involving marker chromosome identification using the protocol of G-band screening, SKY identification and FISH confirmation, to demonstrate the utility of this procedure in a clinical setting.Methods1, To study the effects of a number of variables, i.e., dropping height of cell suspension, relative humidity, slide placed angle condition , fixative ratio, and ets.The quality of metaphase chromosome spreads was assessed with the following quantitative parameters: (a) the metaphase area, (b) the number of chromosome overlaps per metaphase, and (c) the frequency of broken metaphases.2, With a better understanding of fundamental factors influencing chromosome spreading, we developed a new and simple method with which consistent improvement in the quality of metaphase chromosome spreads was obtained.3, To establish and demonstrate the effectiveness of the algorithm, we analyzed lymphocyte slides, using a combination of G-banding, SKY, and FISH.ResultsPart I The effects of a number of variables on SKY metaphase chromosomespreading.and possiable mechanisms1, Measurements showed that the quality of chromosome spreads, judged by themetaphase area, number of chromosome overlaps per metaphase, and percentage ofbroken metaphases, was not significantly affected by the dropping height of cellsuspension.2, The effects of different ratios of methanohacetic acid (from 2:1 to 4:1) were foundthat metaphases fixed with higher ratios of the fixative were more resistant to cellbreaking but had smaller metaphase areas.3, The new and simple method consistently improves chromosome spreading in allcase.4, We found that centrifuge speed influence quality of metaphase in some degree.Compared with 600Xg, centrifuge at 240Xg remains less residuum from cytoplasmaand other impurity, and gives more clear setting.Part II The implemetation of SKY in clincal. Combined with G banding and FISH1, We established the technical system of spectral... |
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