Monoclonal antibodies of IKO series in the three-color analysis of sub-populations of peripheral blood T-lymphocytes of oncological patients
Authors: Zakharova E. N. , Zabotina T. N. , Korotkova O. V. , Kopyrulina M. E. , Panichenko I. V. , Tsiklauri V. T. , Zadеrenko I. A. , Blokhin D. Yu. , Ivanov P. K. 
Published: November 2018

Background.Clinical studies have shown that the most oncological patients have impaired immunological reactivity. Monitoring of the immune system status of patients with solid tumors includes a subpopulation analysis of immunocompetent cells. It is based on the study of expression of surface antigens and intracellular structures, and an analysis of the functional activity of immunocompetent cells. Today two, three, and even four-color reagents are used for subpopulation analysis of lymphocytes. Fluorescent conjugate with monoclonal antibodies (MAB) of the ICO series and fluorescent dyes Imd-306, Imd-506 (cyanine) and phycoerythrin (a group of phycobiliproteins) was received and characterized in N.N. Blokhin National Medical Research Center of Oncology. These conjugates have proved themselves in one and two-color analysis of subpopulations of peripheral blood lymphocytes by flow cytometry.

The purposeof this study is to assess the possibility of sharing several immunofluorescent probes (IFP) on the basis of different Mab series of ICO and dyes FITC, Imd306, Imd506 and PE for three-color analysis of lymphocyte subpopulations of donors and patients with oncological diseases by flow cytometry method.

Materials and methods.A panel of immunofluorescent probes (IFP) CD3FITC, CD4PE, CD8-Imd506, CD4-Imd306 was used in the work. Probes are obtained with MAB (ICO series) and fluorophores FITC, PE, Imd-306, Imd-506. The specific activity of the probes was evaluated in the immunofluorescence reaction by flow cytometry. Donor’s peripheral blood was used to optimize the conditions of the immunofluorescence reaction. Clinical approbation of the obtained sets was carried out on 2 groups of patients with oncological diseases before and after surgical treatment. The first group included 64 patients with cancer of the oral mucosa. The second group consists of 35 patients with ovarian cancer.

Results. We have shown that for the three-color analysis of human lymphocytes by flow cytometry, it is recommended to use sets of labeled MAB: CD3-FITC (10 μg/ml) + CD4-Imd306 at a concentration of 5–10 μg/ml; CD3-FITC + CD4-PE at a concentration of 20 μg/ml and CD3-FITC + CD8 Imd506 at a concentration of 10 μg/ml. The final concentration of immunoglobulin in the solution of IPS is indicated. It was shown that even before the beginning of treatment in patients with cancer of the oral mucosa the subpopulation CD3+ CD4+ content was significantly lowered in the structure of T cells, and the total level of CD8+ lymphocytes significantly exceeds the parameters of the donor group. Analysis of linear CD markers of lymphoid cells in patients with ovarian cancer revealed a significant decrease in the total number of CD3+ T-lymphocytes in comparison with donors. There was no evidence of the effect of surgical treatment on the subpopulation structure of lymphoid cells.

Conclusion.Combinations of fluorescent probes CD3-FITC/CD4-Imd306/CD8-Imd506 and CD3-FITC/CD4-PE/CD8-Imd506 can be used for trichromatic analysis by flow cytometry of the lymphocyte subpopulations of both healthy donors and cancer patients. Comparative studies of the specific activity of combinations of IFP based on Mab ICO series and reference commercial test systems (BD Biosciences) on lymphocytes of healthy donors and oncological patients (ovarian cancer, oral mucosa cancer) showed comparable results. The application of the obtained probes made it possible to reveal violations of the subpopulation structure of peripheral blood lymphocytes in patients with ovarian cancer and cancer of the oral mucosa, in particular, CD3 – /CD8+, CD3+/CD4+, CD3+.