Davis B H, Olsen S, Bigelow N C, Chen J C
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan, USA.
Transfusion. 1998 Aug;38(8):749-56. doi: 10.1046/j.1537-2995.1998.38898375514.x.
The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples.
Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method.
The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent.
The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.
实验室测定母血循环中胎儿细胞水平，对于疑似子宫创伤女性的产科管理以及正确给予抗-D预防新生儿Rh溶血病的剂量，仍然是一项重要的辅助手段。广泛使用的手工Kleihauer-Betke试验在敏感性和精确度上存在局限性，这促使流式细胞术方法在母血样本中胎儿细胞检测的应用增加。
开发了针对胎儿血红蛋白（HbF）的鼠单克隆抗体，将其与异硫氰酸荧光素偶联，并用于一种为定量胎儿红细胞而开发的多参数流式细胞术检测。开发了一种快速细胞内染色方法，即在单克隆抗体孵育前使用短暂的戊二醛固定和Triton X-100通透处理，同时优化了用于分析50,000个细胞的流式细胞术分析方案。评估了该检测方法的线性和精确度，并与Kleihauer-Betke酸洗脱法进行了相关性分析。
抗-HbF流式细胞术方法与Kleihauer-Betke方法显示出良好的相关性（r2 = 0.86），对于胎儿细胞>0.1%的血样，其精确度更高，变异系数<15%。对150份非妊娠成年人的血样进行分析，包括因血红蛋白病和胎儿血红蛋白遗传性持续存在而HbF升高的个体，胎儿细胞的平均值为0.02%，所有结果均低于0.
