Terstappen L W, Meiners H, Loken M R
Becton Dickinson, Monoclonal Center, Mountain View, CA 94039.
J Immunol Methods. 1989 Sep 29;123(1):103-12. doi: 10.1016/0022-1759(89)90034-3.
A simple and rapid method was developed for immunofluorescence measurements of cells by flow cytometry which does not require washing procedures, permitting absolute enumeration of cell subpopulations. Peripheral blood cells were labeled with fluorescein and phycoerythrin conjugated monoclonal antibodies and the nucleic acid stain LDS-751. Distilled water was added following incubation to induce erythrocyte lysis by hypotonic shock. After lysis for 30 s the tonicity of the sample was increased followed by measurement on the flow cytometer. The leukocyte populations were clearly resolved in the correlation of forward and orthogonal light scattering. The immunofluorescence resolution of the labeled leukocytes was equivalent to NH4Cl and a commercial lysing preparation. Absolute number of leukocytes and percentage of leukocyte subpopulations determined with this procedure correlated well with the results obtained with a clinical hematology analyzer. Cell recovery and preservation of cellular characteristics of three different procedures for lysing the human erythrocytes were compared. The LDS-751 permitted the discrimination of intact cells from residual erythrocyte ghosts, platelets and damaged nucleated cells. A considerable loss of cells was found for both NH4Cl and commercial lysing solution; the samples prepared by NH4Cl lysing had a selective loss of lymphocyte subpopulations as compared with the other two techniques. In contrast to the two procedures in which multiple washing steps are involved, the no wash, hypotonic lysis procedure provided a means of obtaining absolute numbers of leukocyte subpopulations identified by combining light scattering and immunofluorescence characteristics with no centrifugation steps required.
开发了一种简单快速的方法，用于通过流式细胞术对细胞进行免疫荧光测量，该方法无需洗涤步骤，可对细胞亚群进行绝对计数。外周血细胞用荧光素和藻红蛋白偶联的单克隆抗体以及核酸染料LDS-751进行标记。孵育后加入蒸馏水以通过低渗休克诱导红细胞裂解。裂解30秒后，增加样品的张力，然后在流式细胞仪上进行测量。在正向和正交光散射的相关性中，白细胞群体清晰可辨。标记白细胞的免疫荧光分辨率与氯化铵和市售裂解制剂相当。用该方法测定的白细胞绝对数和白细胞亚群百分比与临床血液分析仪获得的结果相关性良好。比较了三种不同的人红细胞裂解方法的细胞回收率和细胞特性保存情况。LDS-751可区分完整细胞与残留的红细胞影、血小板和受损的有核细胞。发现氯化铵和市售裂解液均导致相当数量的细胞损失；与其他两种技术相比，氯化铵裂解制备的样品中淋巴细胞亚群有选择性损失。与涉及多个洗涤步骤的两种方法不同，无需洗涤的低渗裂解方法提供了一种通过结合光散射和免疫荧光特性来获得白细胞亚群绝对数的方法，且无需离心步骤。
