应用聚凝胺筛选-D-表型红细胞的研究

-D- phenotype screening by polybrene method

目的 -D-表型是Rh血型系统中较为特殊的血型表型,该表型仅表达RhD蛋白而不表达RhCE蛋白,其发生频率小于十万分之一。由于目前尚缺乏简单有效的筛选方法,该表型及其基因在中国地区的频率目前还未见报道,本研究拟应用该表型D抗原表达量与正常表型D抗原表达量的差异,开发一种新型筛选试剂对该稀有血型进行初步筛选。方法利用流式细胞术对-D-表型及表型基因携带者D抗原表达量进行检测;再利用抗原表达差异,通过调整筛选体系内聚凝胺浓度,使得RhD抗原表达量较高的红细胞与筛选试剂发生凝集,达到对-D-表型及其基因携带者的初步筛选目的。结果从流式细胞术免疫荧光值定量结果(MFI)分析,-D-表型细胞RhD抗原表达量(284 360±16 698,n=3)为正常RhD阳性细胞抗原表达量(98 642±35 908,n=9)的约3倍(P<0.01),RHD--杂合子RhD抗原表达量(181 109±39 455,n=4)为正常RhD阳性细胞抗原表达量的约2倍(P<0.01);15μL 3%新鲜红细胞悬液与筛选用体系35μL(1μL IgG抗-D,29μL凝聚胺聚凝胺,及5μL低离子强度溶液)的反应体系筛选出的阳性细胞经流式检测RhD抗原表达量(144 538±227 445,n=7)为正常RhD阳性细胞抗原表达量的约1.5倍。结论聚凝胺初筛体系可用于-D-表型高通量筛选,为该表型的人群频率等研究提供可靠的的技术手段。

Objective To develop a novel screening reagent for-D-phenotype preliminary screening based on the difference in RhD antigen expression level of-D-phenotype and normal RhD phenotype. Methods RhD antigen expression of-D-phenotype and Rh D--gene carrier were detected by flow cytometry. By adjusting the concentration of polybrene in the screening system, the red blood cells with high RhD antigen expression level agglutinated, and the preliminary screening of the-D-phenotype and its gene carriers was realized. Results According to the quantitative results of immunofluorescence intensity(MFI) analysis by flow cytometry, the expression level of RhD antigen in-D-phenotype cells(284 360±16 698, n=3) was about 3 times normal RhD positive cells(98 642±35 908, n=9)(P<0.01), while RhD antigen expression level of RhD--gene carrier(181 109±39 455, n=4) was about 2 times normal RhD positive cells(P<0.01). RhD antigen expression(144 538±227 445, n=7) of the positive cells screened by 15 μL 3% fresh red blood cell suspension and screening system 35 μL(1 μL IgG anti-D, 29 μL polybrene polybrene, and 5 μL low ionic strength solution) was about 1.5 times normal RhD positive cells. Conclusion The polybrene preliminary screening system, which can be used for high-throughput screening of-D-phenotype, is a reliable technical method for frequency study of this phenotype.