中国输血杂志2017，Vol.30Issue(9)：984-986,3.DOI:10.13303/j.cjbt.issn.1004-549x.2017.09.004
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
摘要
Abstract
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 20min on ice,washing,calculate cell recovery rate.1 ∶ 128 RhD IgG 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.关键词
甲醛/多聚甲醛/戊二醛/固定/透化/RhD抗原Key words
formaldehyde/paraformaldehyde/glutaraldehyde/fixation/permeabilization/RhD antigen/RBC (red blood cell)分类
医药卫生引用本文复制引用
李桢,徐华,周华友,张印则..用于RhD抗原流式细胞检测的固定透化方法评价[J].中国输血杂志,2017,30(9):984-986,3.基金项目
深圳市科技计划项目(JCYJ20140403092619633) （JCYJ20140403092619633）
