Korean J Lab Med.  2011 Jul;31(3):148-153. 10.3343/kjlm.2011.31.3.148.

Comparative Quantitative Analysis of Cluster of Differentiation 45 Antigen Expression on Lymphocyte Subsets

• Im M
• Chae H
• Kim T
• Park HH
• Lim J
• Oh EJ
• Kim Y
• Park YJ
• Han K

• KMID: 1735845
• DOI: http://doi.org/10.3343/kjlm.2011.31.3.148

Abstract

BACKGROUND
Since the recent introduction of radioimmunotherapy (RIT) using antibodies against cluster of differentiation (CD) 45 for the treatment of lymphoma, the clinical significance of the CD45 antigen has been increasing steadily. Here, we analyzed CD45 expression on lymphocyte subsets using flow cytometry in order to predict the susceptibility of normal lymphocytes to RIT.
METHODS
Peripheral blood specimens were collected from 14 healthy individuals aged 25-54 yr. The mean fluorescence intensity (MFI) of the cell surface antigens was measured using a FACSCanto II system (Becton Dickinson Bioscience, USA). MFI values were converted into antibody binding capacity values using a Quantum Simply Cellular microbead kit (Bangs Laboratories, Inc., USA).
RESULTS
Among the lymphocyte subsets, the expression of CD45 was the highest (725,368+/-42,763) on natural killer T (NKT) cells, 674,030+/-48,187 on cytotoxic/suppressor T cells, 588,750+/-48,090 on natural killer (NK) cells, 580,211+/-29,168 on helper T (Th) cells, and 499,436+/-21,737 on B cells. The Th cells and NK cells expressed a similar level of CD45 (P=0.502). Forward scatter was the highest in NKT cells (P<0.05), whereas side scatter differed significantly between each of the lymphocyte subsets (P<0.05). CD3 expression was highest in the Th and NKT cells.
CONCLUSIONS
NKT cells express the highest levels of CD45 antigen. Therefore, this lymphocyte subset would be most profoundly affected by RIT or pretargeted RIT. The monitoring of this lymphocyte subset during and after RIT should prove helpful.

Keyword

Antibody binding capacity; CD45; Lymphocyte subset

MeSH Terms

Adult
Antibodies/immunology
Antigens, CD45/*analysis/immunology
B-Lymphocytes/immunology/metabolism
CD8-Positive T-Lymphocytes/immunology/metabolism
Female
Flow Cytometry/*methods
Fluorescein-5-isothiocyanate/chemistry
Fluorescent Dyes/chemistry
Humans
Killer Cells, Natural/immunology/metabolism
Lymphocytes/immunology/*metabolism
Lymphoma/radiotherapy
Male
Middle Aged
Natural Killer T-Cells/immunology/metabolism
Protein Binding
Radioimmunotherapy
Reagent Kits, Diagnostic
T-Lymphocytes, Helper-Inducer/immunology/metabolism

Figure

• 

  Fig. 1 Calibration curve of CD45 phycoerythrin-cyanine 7.Abbreviations: MESF, molecules of equivalent soluble fluorochromes; ABC, antibody-binding capacity.

• 

  Fig. 2 Peripheral blood leukocytes after staining with anti-CD45 showing discrete granulocyte (P1), monocyte (P2), and lymphocyte (P3) populations according to the level of CD45 expression (A) FSC and SSC on the scattergrams (B) The lymphocyte subsets are separated using antibodies to surface antigens. CD3+CD56- T cells (gray) are plotted in the P4 region. CD3+CD56+ NKT cells (green) and CD3-CD56+ NK cells (blue) are plotted in the P5 and P2 regions, respectively (C) CD19+ B cells (red) are plotted in P3 region (D) Evidence for different CD45 expression (E) and FSC levels (F) CD3+CD56+ NKT cells (green) showed the highest CD45 and FSC expression levels. The CD3-CD56+ NK cells (blue) showed high level of FSC also.Abbreviations: CD, cluster of differentiation; FSC, forward scatter; SSC, side scatter; NKT, natural killer T cell; NK, natural killer cell.

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