The Impact of Camel Leukocytes Fixation on Cell Count and Monoclonal Antibodies Reactivity in Flow Cytometry

Abstract

Immunophenotyping of separated leukocytes is a common technique used to evaluate the changes in cellular immunity during clinical studies. For fixed cells or blood specimens infected with hazardous pathogens, cell fixation is performed before immunofluorescence. The impact of camel leukocytes fixation before staining on the reactivity of cell surface markers with monoclonal antibodies has not been investigated so far. The aim of the present study was, therefore, to compare cell staining of fixed and unfixed camel leukocytes with monoclonal antibodies to several cell surface antigens. Leukocytes were separated from camel blood and were fixed with paraformaldehyde (PFA) or left without fixation. Cells were labeled with monoclonal antibodies to several leukocyte antigens and the expression pattern of the antigens was compared between fixed and non-fixed cells using flow cytometry. The mean fluorescence intensity of each cell marker was calculated and compared between fixed and unfixed cells. Leukocyte fixation with PFA changed the binding activity of the monoclonal antibodies to CD163 and WC1 markedly, making it unable to stain any cell population. Although the cell staining efficacy of other molecules (such as CD14, CD172a, MHCII, CD11a, CD18, CD44, and CD45) was reduced, they were still able to define the target cells. The fixation-induced changes in the expression density of the analyzed monocytic markers may, however, lead to the misinterpretation of immunophenotyping studies of fixed monocytes or macrophages. Collectively, the obtained results indicated significant changes in the staining efficacy of monoclonal antibodies against several cell surface antigens of camel leukocytes, which should be considered when PFA-fixed cellular targets on camel leukocytes are to be analyzed.

How fixation affects the results of lymph node immunophenotyping by flow cytometry

“…Light scattering of all cells after fixation became similar making it harder to discriminate lymphocytes from cell debris. The data on the effect of fixation on light scatter properties of cells is in agreement with previously reported experiments [22,23]. Generally, fixation significantly affected cell distribution on SSC-A/FSC-A graph as well as the antibody binding efficiency for the majority of surface antigens [22,23].…”

“…The data on the effect of fixation on light scatter properties of cells is in agreement with previously reported experiments [22,23]. Generally, fixation significantly affected cell distribution on SSC-A/FSC-A graph as well as the antibody binding efficiency for the majority of surface antigens [22,23]. Although, the data is variable, and fixation using 4% PFA before lysing red blood cells, might preserve whole blood light scattering profile and antigenicity of some leukocyte surface markers [24].…”