红细胞裂解液对CD34^＋细胞相对计数的影响

摘要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CD34一PE及CD45一FITCx／．色免疫荧光染色后用FACS裂解液（FACS^TM lysingsolution，FACS）、IOTest3裂解液（IOTest3lysingsolution，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（forwardscatter，FSC）、侧向散射（sidescatter，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±0．42v30．92±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。

Effect of RBC Lysing Solution on CD34 + Cell Counting

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