title: 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.
authors.affiliation: Department of Laboratory Medicine, School of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: Joonhong
authors.initials: J
authors.lastname: Park
authors.forename: Kyungja
authors.initials: K
authors.lastname: Han
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D018941:Antigens, CD19 / Q000737:chemistry / Q000378:metabolism; D016175:B-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D017252:CD3 Complex / Q000737:chemistry / Q000378:metabolism; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism; D016827:CD8 Antigens / Q000737:chemistry / Q000378:metabolism; D003116:Color; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D006801:Humans; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D016650:Fluorescein-5-isothiocyanate