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

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+...

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Bibliographic Details
Published in:	Cytometry (New York, N.Y.) Vol. 15; no. 3; pp. 258 - 266
Main Authors:	Hunter, Stephen D., Peters, Lyndsay E., Wotherspoon, John S., Crowe, Suzanne M.
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-03-1994
Subjects:	Antibodies, Monoclonal - analysis Antigens, CD - analysis Antigens, CD19 Antigens, Differentiation, B-Lymphocyte - analysis B-Lymphocyte Subsets - chemistry B-Lymphocyte Subsets - cytology B-Lymphocyte Subsets - immunology CD4 Antigens - analysis CD8 Antigens - analysis Flow Cytometry - methods Fluorescein-5-isothiocyanate Fluorescent Antibody Technique Humans Immunocytometry Immunophenotyping Mathematics Receptors, IgG - analysis T-Lymphocyte Subsets - chemistry T-Lymphocyte Subsets - cytology T-Lymphocyte Subsets - immunology three color flow cytometry
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Summary:	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.
Bibliography:	Stephen Hunter and the Flow Cytometry Unit at the Macfarlane Burnet Centre for Medical Research are supported by a grant from the Victorian Health Promotion Foundation, and by the Research Fund of the MBCMR. Suzanne Crowe is a Wellcome Trust Senior Research Fellow. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0196-4763 1097-0320
DOI:	10.1002/cyto.990150311