Bischoff F Z, Lewis D E, Nguyen D D, Murrell S, Schober W, Scott J, Simpson J L, Elias S
Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030, USA.
Am J Obstet Gynecol. 1998 Jul;179(1):203-9. doi: 10.1016/s0002-9378(98)70273-x.
Currently, prenatal diagnosis of chromosome abnormalities requires invasive techniques such as amniocentesis and chorionic villus sampling that carry small but finite risks of fetal loss. A noninvasive approach is to isolate fetal cells from maternal blood by flow sorting followed by genetic interphase analysis with fluorescence in situ hybridization. Because the ratio of fetal to maternal cells is relatively low after flow sorting and to detect 90% to 95% of fetal aneuploidies associated with serious birth defects, a 5-color fluorescent in situ hybridization strategy is necessary for simultaneous detection of chromosomes X, Y, 13, 18, and 21 in all flow-sorted nuclei recovered from a specimen.
Fetal nucleated red blood cells were isolated from maternal blood in 40 cases (10.4 to 27.0 weeks' gestation) by flow cytometry on the basis of positive selection of CD71+ (transferrin receptor), CD45-, and LDS751 staining. Each case was evaluated for 5-color fluorescent in situ hybridization efficiency by determining the percentage of flow-sorted nuclei containing 8 hybridization signals for chromosomes X, Y, 13, 18, and 21.
A total of 42,312 flow-sorted nuclei from maternal blood samples were analyzed. In 5 of 16 (31%) cases with a male fetus, 0.16% of nuclei scored were identified as fetal by the presence of 1 signal each for chromosomes X and Y. Fetal trisomy 21 nuclei were accurately detected in 2 cases with a female fetus, each of which was subsequently confirmed.
Five-color interphase fluorescent in situ hybridization analysis can be used to effectively analyze rare fetal aneuploid nuclei in enriched flow-sorted cells isolated from maternal blood.
目前，染色体异常的产前诊断需要采用侵入性技术，如羊膜穿刺术和绒毛取样术，这些技术存在导致胎儿丢失的小但有限的风险。一种非侵入性方法是通过流式分选从母血中分离胎儿细胞，随后采用荧光原位杂交进行遗传间期分析。由于流式分选后胎儿与母体细胞的比例相对较低，为了检测与严重出生缺陷相关的90%至95%的胎儿非整倍体，需要采用5色荧光原位杂交策略，以便在从标本中回收的所有流式分选核中同时检测X、Y、13、18和21号染色体。
基于CD71 +（转铁蛋白受体）、CD45 - 和LDS751染色的阳性选择，通过流式细胞术从40例（妊娠10.4至27.0周）孕妇的母血中分离胎儿有核红细胞。通过确定含有X、Y、13、18和21号染色体8个杂交信号的流式分选核的百分比，对每例进行5色荧光原位杂交效率评估。
共分析了来自母血样本的42312个流式分选核。在16例男性胎儿病例中的5例（31%）中，通过X和Y染色体各有1个信号，将0.16%的计分核鉴定为胎儿核。在2例女性胎儿病例中准确检测到胎儿21三体核，随后每例均得到证实。
5色间期荧光原位杂交分析可用于有效分析从母血中分离的富集流式分选细胞中的罕见胎儿非整倍体核。
