红细胞裂解液对CD34+细胞相对计数的影响及其原因初步探讨

Influence of red blood cell lysing solution on comparative counting of CD34+ cells and its causes

摘要:

目的：研究红细胞（red blood cell，RBC）裂解液对CD34+细胞相对计数的影响。方法：收集101份外周血干细胞采集物，CD34-PE及CD45-FITC双色免疫荧光染色后，分别用FACS裂解液（FACSTM lysing solution，FACS）、Optilyse C裂解液（optilyse C lysing solution，Optilyse C）及自配裂解液溶解红细胞，采用洗涤程序和运用ISHAGE设门策略，检测CD34+细胞、淋巴、粒、单核细胞相对数，CD45+ 细胞占全部收集信号百分比及各类细胞前向散射光（forward scatter，FSC）及侧向散射光（side scatter，SSC）信号。结果：比较3种裂解液处理后样本的CD34+ 细胞百分比，FACS裂解液处理的样本CD34+ 细胞百分比最低，自配裂解液最高（FACS vs. Optilyse C vs. 自配=0.49±0.43 vs. 0.67±0.50 vs. 0.73±0.66，P=0.000）。FACS裂解后样本CD45+细胞百分比远高于Optilyse C及自配裂解液（t=142.500，P=0.000）。3种裂解液对淋巴细胞百分比结果（F=35.340，P=0.000）具有统计学差异；而粒细胞百分比（F=0.610，P=0.540）、单核细胞百分比（F=1.590，P=0.210）差异无统计学意义。FACS裂解液处理后，CD34+ 细胞、淋巴细胞的FSC和SSC信号强度均弱于Optilyse C裂解液及自配的裂解液；而粒、单核细胞的SSC信号，FACS裂解液处理后最强，Optilyse C裂解液处理后最弱。另外，动员后的白细胞数多少与CD34+ 细胞百分比不存在相关性（r=0.108，P=0.312）。结论：红细胞裂解液导致CD34+细胞相对计数差异的原因可能与CD45+细胞比例有关，即获取的有效细胞数；裂解液对细胞形态的影响与造成淋巴细胞比例有显著性差异有关。鉴于不同红细胞裂解液对CD34等抗原阳性细胞的相对计数确实有不良影响，因此在应用流式细胞术检测或计数时应慎重选择。

Abstract:

Objective：To analyze the influence of the red blood cell（RBC） lysing solution on the comparative counting of CD34+ cells. Methods：A total of 101 peripheral blood stem cell collections were collected and stained by CD34-PE and CD45-FITC monoclonal antibodies. Then the RBCs were lysed by two commercially available lysing solutions，FACSTM lysing solution（FACS） and Optilyse C lysing solution（Optilyse C） and one self-prepared lysing solution. The washing procedure and ISHAGE gate strategy were used to de－tect the relative numbers of CD34+ cells，lymphoid cells，granulocytes，and monocytes，the percentage of CD45+ cells，and the various types of cell forward scatter（FSC） and side scatter（SSC） signals. Results：The percentage of CD34+ cells was the lowest in the FACS-treated samples，while the highest in self-prepared lysing solution（0.49±0.43 vs. 0.67±0.50 vs. 0.73±0.66，P=0.000）. The percent－age of CD45+ cells in the samples treated by FACS was much higher than that in the samples treated by Optilyse C and self-prepared lysing solution respectively（t=142.500，P=0.000）. The percentage of lymphocytes（F=35.340，P=0.000） had significant differences in samples treated by the three lysates solutions respectively，however，there was no statistically significant difference for the percentage of granulocytes（F=0.610，P=0.540） and monocytes（F=1.590，P=0.210）. After FACS treatment，the signal intensities of FSC and SSC of CD34+ cells and lymphocytes were weaker than those of the Optilyse C and self-prepared lysing solution treatments，while the SSC signals of granulocytes and monocytes were the strongest after FACS lysate treatment and were the weakest after Opti－clese C treatment. In addition，there was no correlation between the number of leukocytes after mobilization and the percentage of CD34+ cells（r=0.108，P=0.312）. Conclusion：The difference in the relative count of CD34+ cells caused by the RBC lysate may be related to the proportion of CD45+ cells，that is，the acquired effi－cient cell numbers. The effect of lysate on cell morphology is related to the significant difference in lymphocyte ratio. As different RBC lysates do have adverse effects on the relative counts of CD34-positive cells，the choice of RBC lysates reagents for the flow cytometry or counting should be careful.