Hunter S D, Peters L E, Wotherspoon J S, Crowe S M
Flow Cytometry Unit, Macfarlane Burnet Centre for Medical Research, Fairfield Hospital, Victoria, Australia.
Cytometry. 1994 Mar 1;15(3):258-66. doi: 10.1002/cyto.990150311.
Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.
用于评估白细胞亚群的商用试剂试剂盒涉及使用预混合的异硫氰酸荧光素（FITC）和藻红蛋白（PE）偶联的单克隆抗体组合对多达六管样本进行染色。我们描述了一种快速方法，通过该方法，在单管染色后可以对总CD3⁺ T细胞、CD4⁺ T细胞（CD3⁺ CD4⁺）、CD8⁺ T细胞（CD3⁺ CD8⁺）、假定的γδ受体T细胞（CD3⁺ CD4⁻ CD8⁻）以及CD3⁺ CD4⁺ CD8⁺ T细胞、B淋巴细胞和NK细胞进行计数。全血标本用抗体混合物标记：FITC偶联的抗CD4和抗CD19抗体、PE偶联的抗CD8和抗CD16抗体，以及用于抗CD3抗体的多甲藻叶绿素蛋白（PerCP）或别藻蓝蛋白（APC）标记。记录20,000个事件后，在Consort 32计算机系统和LYSYS-II（Becton Dickinson，加利福尼亚州圣何塞）上对数据进行分析，所有淋巴细胞亚群值通过布尔代数使用我们称为布尔门分析（BGA）的技术来确定。我们的研究表明，BGA在统计学上等同于SimulSET淋巴细胞亚群分析。此外，该程序将所需的管数减少到两管，从而节省了试剂、耗材和时间。
