Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome.
10.3343/alm.2012.32.3.171
- Author:
Joonhong PARK
1
;
Kyungja HAN
Author Information
1. Department of Laboratory Medicine, School of Medicine, The Catholic University of Korea, Seoul, Korea. hankja@catholic.ac.kr
- Publication Type:Original Article
- Keywords:
Monoclonal antibody cocktail;
Lymphocyte subset;
Single-color multitarget flow cytometry
- MeSH:
Antibodies, Monoclonal/chemistry/*immunology;
Antigens, CD19/chemistry/metabolism;
Antigens, CD3/chemistry/metabolism;
Antigens, CD4/chemistry/metabolism;
Antigens, CD8/chemistry/metabolism;
B-Lymphocyte Subsets/immunology/metabolism;
Color;
Flow Cytometry/*methods;
Fluorescein-5-isothiocyanate/*chemistry;
Humans;
T-Lymphocyte Subsets/immunology/metabolism
- From:Annals of Laboratory Medicine
2012;32(3):171-176
- CountryRepublic of Korea
- Language:English
-
Abstract:
BACKGROUND: We developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay. METHODS: We selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples. RESULTS: CV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein alpha-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2> or =0.98, 0.99, 0.99, and 0.99, respectively; P<0.05). CONCLUSIONS: The multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.
