用于RhD抗原流式细胞检测的固定透化方法评价

Evaluation of RBC fixation and permeabilization methods for RhD fluorescence detection

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 method for RBC fixation and permeabilization with high recovery rate and suitable for RhD antigen fluorescence detection.Methods 4% formaldehyde,4% paraformaldehyde and 2% glutaraldehyde were used to fix RBCs,and 4% formaldehyde-Triton X-100,4% paraformaldehyde-Triton X-100,2% glutaraldehyde-Triton X-100 were used to fix and permeabilize RBCs,untreated RBCs as a control.Fixed for 20 min on ice,washing,calculate cell recovery rate.1 ∶128 RhD Ig G monoclonal antibody was added to each group of RBCs and incubated at 37℃ for 30 min.The blank control group was set at the same time.After washing with PBS for three times,1 ∶200 FITC was added,incubated at 37℃for 30 min,washed for three times with PBS,and then detected by flow cytometry.Results The recoveries of 2% glutaraldehyde and 2% glutaraldehyde-Triton X-100 were 72.83% and 72.80%,respectively,which were higher than the control group（58.32%）.The recoveries of formaldehyde and paraformaldehyde were 4.00% or so,much lower than the control group.Flow cytometry showed that the fluorescence background of different fixatives was higher than untreated RBCs.Compared with the control group,formaldehyde and paraformaldehyde weakened the fluorescence intensity of RhD antigen,2%glutaraldehyde did not affect the fluorescence intensity of RhD,while 2% glutaraldehyde-Triton X-100 could enhance the sensitivity of RhD antigen detection.Conclusion The 2% glutaraldehyde-Triton X-100 fixation and permeabilization method not only can obtain higher erythrocyte recovery rate,but also improve the sensitivity of RhD fluorescence detection,which is the ideal method for fluorescence detection of RhD antigen.