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目的寻找具有回收率高且适于RhD抗原荧光检测的红细胞固定透化方法。方法分别采用4%甲醛、4%多聚甲醛、2%戊二醛对红细胞进行固定,同时采用4%甲醛-Triton X-100、4%多聚甲醛-Triton X-100、2%戊二醛-Triton X-100对红细胞进行透化固定,未处理红细胞作为对照。冰上固定20 min,洗涤后计算红细胞回收率。在各组红细胞中加入1∶128的RhD Ig G单克隆抗体,同时设组内空白对照,37℃孵育30 min。PBS洗涤3次后加入1∶200的FITC荧光抗体,37℃孵育30 min,PBS洗涤3次后流式细胞仪检测。结果 2%戊二醛及2%戊二醛-Triton X-100 2组的回收率分别为72.83%与72.80%,高于对照组(58.32%),甲醛及多聚甲醛2组的回收率在4.00%左右,远低于对照组。流式检测结果显示,不同固定剂的荧光本底均高于未处理红细胞。与对照组相比甲醛与多聚甲醛会减弱RhD抗原荧光强度,而戊二醛不影响RhD荧光强度,戊二醛-Triton X-100可增强RhD抗原检测的灵敏度。结论2%戊二醛-Triton X-100固定透化法不仅可得到较高的红细胞回收率,且RhD荧光检测的灵敏度也得到提升,是荧光检测RhD抗原较理想的固定透化方法。
Objective To find a red blood cell fixed dialysis method with high recovery rate and suitable for fluorescence detection of RhD antigen. Methods The erythrocytes were fixed with 4% formaldehyde, 4% paraformaldehyde and 2% glutaraldehyde, respectively. The cells were fixed with 4% formaldehyde-Triton X-100, 4% paraformaldehyde-Triton X-100, -Triton X-100 erythrocytes permeabilized fixation, untreated erythrocytes as a control. Fixed on ice for 20 min, and washed to calculate the recovery of erythrocytes. In each group of erythrocytes by adding 1: 128 RhD Ig G monoclonal antibody, while the set within the blank control, 37 ℃ incubated for 30 min. After washing with PBS for 3 times, 1: 200 FITC fluorescent antibody was added and incubated at 37 ° C for 30 min. The cells were washed three times with PBS and detected by flow cytometry. Results The recoveries of 2% glutaraldehyde and 2% glutaraldehyde-Triton X-100 2 were 72.83% and 72.80%, respectively, higher than that of the control group (58.32%). The recoveries of formaldehyde and paraformaldehyde were 4.00%, much lower than the control group. Flow cytometry showed that the fluorescence of different fixatives was higher than that of untreated erythrocytes. Compared with the control group, formaldehyde and paraformaldehyde attenuated the fluorescence intensity of RhD antigen, but glutaraldehyde did not affect the fluorescence intensity of RhD. Glutaraldehyde-Triton X-100 could enhance the sensitivity of RhD antigen detection. Conclusion The 2% glutaraldehyde-Triton X-100 fixed permeabilization method can not only obtain high recovery of erythrocytes, but also improve the sensitivity of RhD fluorescence detection. It is a more ideal fixed dialysis method for detecting RhD antigen by fluorescence.