Lymphocyte subset analysis by boolean algebra: A phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence

Abstract

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 γδ‐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. © 1994 Wiley‐Liss, Inc.

Computational and Experimental Analysis Reveals a Requirement for Eosinophil-Derived IL-13 for the Development of Allergic Airway Responses in C57BL/6 Mice

“…In the study of immunological responses, this approach has been implemented in small networks for the analysis of T cell activation and anergy, for the analysis of lymphocyte subsets, and we have used this approach to analyze the pathogenesis of Bordetellae infections (10,(71)(72)(73). In this study, we assembled the literature on the immune components involved in case of the i.p.…”

Modeling Systems-Level Regulation of Host Immune Responses

“…Qualitative discrete modeling such as ours has been previously successfully implemented in gene regulatory networks and signal transduction networks for predicting the dynamic trajectory of biological circuits and for accessing the reliability of gene regulatory networks in signal processing [31,32,50]. In the study of immunological responses, this approach has been implemented in small networks for the analysis of T cell activation and anergy [51] and for the analysis of lymphocyte subsets [52]. Here, a comprehensive network was constructed to study the immunological responses at the systems level, and the dynamic model of this network was successfully validated.…”

Cytokine profiles of BAL T cells and T‐cell clones obtained from human asthmatic airways after local allergen challenge

“…T‐cell clones were phenotyped with FITC‐conjugated antibodies to CD4 and CD19, PE‐conjugated antibodies to CD8 and CD16, and a PerCP‐conjugated antibody to CD3 (Becton‐Dickinson, Abingdon, UK). The stained cells were analysed on a FACScan cytometer (Becton‐Dickinson) by a Boolean gate analysis technique which enables all markers to be stained in one sample ( 13). The antibodies were diluted in PBS, and 20 μl of each antibody was added to a polypropylene tube containing 1×10 5 cells in 100 μl.…”