Single-color multitarget flow cytometry using monoclonal antibodies labeled with different intensities of the same fluorochrome

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 α-isothyocyanate (FITC)(dim)+CD4-FITC(bright) and with CD19-FITC(dim)+CD3-FITC(bright) 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-T(helper) cells, and T(helper) cells; r(2)=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITC(dim)+CD3-FITC(bright) and CD8-PE(dim)+CD4-PE(bright), and MFC, in the 23 patient samples (B cells, T cells, T(cytotoxic) cells, and T(helper) cells; r(2)≥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.

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

Fig. 1

Representative dot plots and histograms of single-color multitarget flow cytometry with CD19-FITC_dim+CD3-FITC_bright and with CD3-FITC_dim+CD4-FITC_bright. (A) B cells (CD19-FITC_dim, brown) and T cells (CD3-FITC_bright, green). (B) Non-T_helper cells (CD3-FITC_dim, pink) and T_helper cells (CD3-FITC_dim+CD4-FTTC_bright, 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-FITC_dim+CD3-FITC_bright and CD8-PE_dim+CD4-PE_bright. B cells (CD19-FITC_dim, brown), T_cytotoxic cells (CD3-FITC_bright+CD8-PE_dim, pink), and T_helper cells (CD3-FITC_bright+CD4-PE_bright, 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.