Ann Lab Med.  2012 May;32(3):171-176. 10.3343/alm.2012.32.3.171.

Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome

• Park J
• Han K

• KMID: 1381680
• DOI: http://doi.org/10.3343/alm.2012.32.3.171

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.

Keyword

Monoclonal antibody cocktail; Lymphocyte subset; Single-color multitarget flow cytometry

MeSH Terms

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

Figure

• 

  Fig. 1 Representative dot plots and histograms of single-color multitarget flow cytometry with CD19-FITCdim+CD3-FITCbright and with CD3-FITCdim+CD4-FITCbright. (A) B cells (CD19-FITCdim, brown) and T cells (CD3-FITCbright, green). (B) Non-Thelper cells (CD3-FITCdim, pink) and Thelper cells (CD3-FITCdim+CD4-FTTCbright, blue).Abbreviations: SM-FC, single-color multitarget flow cytometry; PerCP, peridinin chlorophyll protein complex; FITC, fluorescein α-isothyocyanate.

• 

  Fig. 2 Representative dot plots of single-color multitarget flow cytometry with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright. B cells (CD19-FITCdim, brown), Tcytotoxic cells (CD3-FITCbright+CD8-PEdim, pink), and Thelper cells (CD3-FITCbright+CD4-PEbright, blue) in a right dot plot.Abbreviations: SM-FC-SAA s/p uPBSCT, single-color multitarget flow cytometry-severe aplastic anemia after unrelated peripheral blood stem cell transplantation; SSC-A, side scatter; PerCP, peridinin chlorophyll protein complex; PE, phycoerythrin; FITC, fluorescein α-isothyocyanate.

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