应用双色荧光原位杂交技术检测一例49,XXXXY性染色体异常

目的:探讨用双色荧光原位杂交技术(dual-color fluorescence in situ hybridization,D-FISH)检测性染色体数目异常的实验方法及其应用价值。方法:以Biotin标记的X染色体α-卫星DNA(pBamX7)探针和以Digoxigenin标记的Y染色体长臂末端重复顺序(pY3.4)探针与经处理的标本同时进行外周血染色体及间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体和间期细胞核的杂交信号颗粒数。结果:在显微镜下可见以Biotin标记的pBamX7探针显示4个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或细胞质背景经DAPI复染显示兰色;统计350个中期染色体和间期细胞核,X染色体杂交信号阳性率分别为91.43％和92.57％;Y染色体杂交信号阳性率分别为99.5％和99.8％。结论:双色荧光原位杂交技术是检测49,XXXXY性染色体异常以及其他性染色体数目异常患者的一种十分有价值的技术,具有高效、灵敏、可靠等特点,可为临床提供良好的辅助诊断。

The Application of Dual-color Fluorescence in situ Hybridization to the Diagnosis of a 49,XXXXY Chromosomal Abnormality

Objective:To study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application in the diagnosis of a 49.XXXXY chromosomal anormality. Methods: Biotin labeled alpha satellite X chromosome DNA(pBamX7) probe and Digoxigenin labeled Y chromosome long arm terminal repetitive sequence (pY3. 4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and in-terphase nucleus. After being washed, the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hybridization signals, chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescence microscope Olympus AX-70 and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results:It was seen under the microscope that the Biotin labeled pBamX7 probe showed 4 green hybridization signal and that the Digoxigenin labeled pY3. 4 probe showed 1 red hybridization signal. Chromosomal or cytoplasm counter-stained with DAPI showed blue. The positivity rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91,43% and 92. 57% respectively while that of the Y chromosome hybridization signal was 99. 5% and 99. 8%. Conclusion:Dual-color fluorescence in situ hybridization is a fairly valuable technique in diagnosing 49,XXXXY chromosomal abnormality and other sex chromosomal abnormalities. Being efficient, sensitive and reliable, it can be applied in clinic as a good diagnostic aid and serves as an important complement to conventional cytogenetics.