Universität Erlangen-Nürnberg, Erlangen, Germany.
Medizinische Klinik 5, Universitätsklinikum Erlangen, Erlangen, Germany.
Int J Lab Hematol. 2021 Oct;43(5):939-947. doi: 10.1111/ijlh.13484. Epub 2021 Feb 13.
Standard protocols in flow cytometry (FCM) require lysis of erythrocytes, which may induce an unwanted loss of leukocytes as bystander effect.
In the present study, we investigated the influence of 6 laboratory protocols using 4 different lysing reagents, FACS Lysing Solution (FacsL), QUICKLYSIS (QuickL), IOTest 3 Lysing Solution (NH4Cl), VersaLyse (VersaL), and VersaLyse with added fixative (VersaFix) on the relative quantity of leukocyte subsets identified by CD3, CD4, CD8, CD19, CD14, CD16, CD56, and CD45, applying a no-lyse-no-wash (NoL) protocol as reference. In addition, we compared the efficiency of red blood cell (RBC) lysis.
Peripheral blood samples from 52 individuals were analyzed. NoL was suitable as reference method, but led to less clear-cut gating of lymphocyte and monocyte populations due to a wider distribution of light scatter. Best completeness of RBC lysis with remaining erythrocytes below 10% was achieved using NH4Cl and VersaL. We observed a loss of 11% of monocytes after QuickL. Lymphocyte counts were 19% lower after FacsL. Cell subsets within the lymphocyte compartment were rather similar between the different methods with the exception of lower B-cell counts (-8%) and higher NK-cell counts (+11%) after FacsL. NH4Cl and VersaL were in good accordance with the NoL method and also with the mean values of all methods.
Our data show that the lysing reagents tested lead to specific deviations in the quantitation of leukocyte subsets and show different efficiency of erythrocyte lysis.
流式细胞术（FCM）的标准方案要求裂解红细胞，这可能会导致白细胞作为旁观者效应的不必要损失。
在本研究中，我们研究了使用 4 种不同裂解试剂的 6 种实验室方案对白细胞亚群相对数量的影响，这些试剂包括 FACS Lysing Solution（FacsL）、QUICKLYSIS（QuickL）、IOTest 3 Lysing Solution（NH4Cl）、VersaLyse（VersaL）和添加固定剂的 VersaLyse（VersaFix），并以无裂解无洗涤（NoL）方案作为参考。此外，我们比较了红细胞（RBC）裂解的效率。
分析了来自 52 个人的外周血样本。NoL 作为参考方法是合适的，但由于光散射分布较宽，导致淋巴细胞和单核细胞群体的门控不太清晰。使用 NH4Cl 和 VersaL 可以实现最佳的 RBC 裂解完整性，剩余红细胞<10%。QuickL 后单核细胞损失 11%。FacsL 后淋巴细胞计数降低 19%。除了 FacsL 后 B 细胞计数降低（-8%）和 NK 细胞计数升高（+11%）外，淋巴细胞群内的细胞亚群在不同方法之间相当相似。NH4Cl 和 VersaL 与 NoL 方法以及所有方法的平均值非常一致。
我们的数据表明，所测试的裂解试剂导致白细胞亚群定量存在特定偏差，并显示红细胞裂解效率不同。
