Original Article

Ann Lab Med 2012; 32(3): 171-176

Published online May 1, 2012

Copyright © Korean Society for Laboratory Medicine.

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

Joonhong Park, M.D. and Kyungja Han, M.D.

Department of Laboratory Medicine, School of Medicine, The Catholic University of Korea, Seoul, Korea

Correspondence to: Kyungja Han
Department of Laboratory Medicine, School of Medicine, The Catholic University of Korea, Seoul St. Mary’s Hospital, 505 Banpo-dong, Seocho-gu, Seoul 137-701, Korea
Tel: +82-2-2258-1644
Fax: +82-2-2258-1719

Received: July 12, 2011; Revised: January 2, 2012; Accepted: February 1, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


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-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≥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: Monoclonal antibody cocktail, Lymphocyte subset, Single-color multitarget flow cytometry