红细胞裂解液对CD34^+细胞相对计数的影响

Effect of RBC Lysing Solution on CD34^+ Cell Counting

为了研究红细胞裂解液对CD34+细胞相对计数结果的影响,对37份外周血干细胞采集物进行CD34-PE及CD45-FITC双色免疫荧光染色后用FACS裂解液(FACSTM lysing solution,FACS)、IOTest3裂解液(IOTest 3 lysings olution,IOTest)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+细胞相对数,同时记录细胞的前向散射(forward scatter,FSC)、侧向散射(side scatter,SSC)信号及CD45+细胞百分比。结果表明,FACS处理的样本CD34+细胞百分比低于IOTest(0.50±0.42vs0.92±0.59,p=0.004);而IOTest与自配红细胞裂解液处理结果的差异无统计学意义(0.92±0.59vs0.95±0.64,p=0.902)。经IOTest裂解后细胞的FSC、SSC信号均显著高于FACS(p<0.01)。IOTest裂解后的样本CD45+细胞百分比低于FACS。WBC数的多少与CD34+细胞百分比不存在相关关系(rs=0.192,p=0.357)。结论:某些红细胞裂解液对CD34等抗原阳性细胞的相对计数确有不良影响,用流式细胞术检测或计数时应慎重选择红细胞裂解液。

To investigate whether the RBC lysing solution can affect the results of relative enumeration of CD34 ^＋ cells ,37 mobiled peripheral blood apheresis products were stained using CD34-PE and CD45-FITC monoclone antibodies and RBCs were then lysed by two lysing solution commercially available（ one named FACSTM Lysing Solution,FACS; another IOTest 3 Lysing Solution, IOTest） and one lysing solution self-prepared. After being processed by lyse-and- then-washed method, samples were detected by FACSCantoTM flow cytometer. The percentages of CD34 ^＋ cells were determined based on ISHAGE gating strategy, forward and side scatter （FSC and SSC） characteristics , percentage of CD45 ^＋ cells were recorded simultaneously. The results showed that by lyse-and-then-wash method, the percentages of CD34 ^＋ ceils in FACS-treated samples were significantly lower compared with that in IOTest-treated samples（0.50 ± 0. 42 vs 0. 92 ± 0.59 ,p = 0.004 ）, but no statistical difference was observed between IOTest-treated and ourselves-prepared- treated samples. The intensities of FSC and SSC in cells of IOTest-treated sample were significantly higher compared with that in cells of FACS-treated sample （p 〈 0.01 ）. The proportion of CD45 ± cells in IOTest-treated samples was lower than that in FACS-treated samples. The WBC count of samples was not correlated to the amount of CD34 ± cells （rs = 0. 192,p = 0. 357）. It is concluded that the red cell lysing solution shows unexpected effect on detecting and counting CD34^＋ cells, prudence should be taken to select such reagents at FCM performance. Key words RBC lysing solution; FACS lysing solntion; IOTest lysing solution; CD34 ^＋ cell