Horvatinovich J M, Sparks S D, Mann K P
Duke University Medical Center, Durham, North Carolina, USA.
Cytometry. 1996 Jun 15;26(2):172-7. doi: 10.1002/(SICI)1097-0320(19960615)26:2<172::AID-CYTO12>3.0.CO;2-K.
Development of a more cost-effective and efficient method of performing lymphocyte subset analysis is of continuing importance in clinical flow cytometry laboratories. Current two-color methods utilize forward and right angle light scatter and multiple tubes per sample and are thereby liable to gate contamination. Methods using three-color analysis with CD45 vs. right angle light scatter (RALS) gating cannot always exclude a contaminating nonlymphoid population. We have established a two tube approach to directly measure total T cells, T suppressor, and T helper subsets, total B cells and total natural killer cells. The technique involves staining of whole blood with a mixture of five monoclonal antibodies conjugated to three fluorochromes: CD4+CD19 fluorescein isothiocyanate (FITC), CD3+CD33 phycoerythrin (PE), CD45 peridin chlorophyll alpha protein (PerCP), CD8+CD16 FITC, CD3+CD33 PE, and CD45 PerCP. Analysis is performed using a single laser flow cytometer. This method has equivalent recovery to and improved purity of the lymphocyte gate when compared to well-established methods. These antibody combinations additionally allow clear separation of lymphocytes from other leukocytes and debris as well as separation of the T cell helper and suppressor subsets, natural killer cells and B lymphocytes. We additionally provide preliminary data that an accurate lymphocyte subset analysis can be performed on a single tube containing five antibodies (CD4+CD19 FITC, CD3+CD33 PE, and CD45 PerCP), although some measurements are performed deductively.
开发一种更具成本效益和高效的淋巴细胞亚群分析方法，在临床流式细胞术实验室中一直具有重要意义。当前的双色方法利用前向和直角光散射，且每个样本需要多个试管，因此容易出现设门污染。使用CD45与直角光散射（RALS）设门的三色分析方法并不总能排除污染的非淋巴细胞群。我们建立了一种双管方法来直接测量总T细胞、抑制性T细胞和辅助性T细胞亚群、总B细胞和总自然杀伤细胞。该技术涉及用与三种荧光染料偶联的五种单克隆抗体混合物对全血进行染色：CD4⁺CD19异硫氰酸荧光素（FITC）、CD3⁺CD33藻红蛋白（PE）、CD45叶绿素蛋白（PerCP）、CD8⁺CD16 FITC、CD3⁺CD33 PE和CD45 PerCP。使用单激光流式细胞仪进行分析。与成熟方法相比，该方法具有同等的回收率，且淋巴细胞设门的纯度有所提高。这些抗体组合还能使淋巴细胞与其他白细胞和碎片清晰分离，以及使辅助性T细胞和抑制性T细胞亚群、自然杀伤细胞和B淋巴细胞分离。我们还提供了初步数据，表明可以在含有五种抗体（CD4⁺CD19 FITC、CD3⁺CD33 PE和CD45 PerCP）的单个试管上进行准确的淋巴细胞亚群分析，尽管有些测量是通过推导进行的。
