The Impact of Anticoagulation Agent on the Composition and Phenotype of Blood Leukocytes in Dromedary Camels

Abstract

For the analysis of several cellular biomarkers, blood samples are anticoagulated using different agents with different modes of action. However, for the most commonly used anticoagulants, EDTA and heparin, varying effects on blood components have been reported in different species. As little is known about the impact of anticoagulants on the immunological evaluation of camel leukocytes, the present study analyzed the leukogram, the immunophenotype, and the cell vitality of camel leukocytes separated from blood samples anticoagulated with EDTA or lithium heparin. Using flow cytometry and staining with monoclonal antibodies to several cell surface markers, the composition and immunophenotype of camel leukocytes separated from blood anticoagulated with EDTA or heparin were analyzed. In comparison to EDTA-anticoagulated blood, using lithium heparin as an anticoagulant resulted in reduced numbers of total leukocytes and reduced numbers of neutrophils, which led to a reduced neutrophil to lymphocyte ratio. The analysis of cell necrosis and apoptosis after the staining of leukocytes with the DNA-sensitive dye propidium iodide and the mitochondrial membrane potential probe JC1 revealed a higher fraction of necrotic neutrophils and higher fractions of apoptotic neutrophils and monocytes in heparin blood than in EDTA blood. In addition, monocytes from heparin blood showed higher expression levels of the cell surface markers CD14, CD163, and MHCII when compared to cells from EDTA blood. Similarly, in heparin blood, CD44 and CD172a were expressed higher on neutrophils, while CD11a was expressed higher on lymphocytes in comparison to cells from EDTA blood. The results of the current study indicate the importance of considering the type of anticoagulant when investigating the composition, vitality, and immunophenotype of camel leukocytes.

Keywords: anticoagulant; cell biomarkers; dromedary camel; flow cytometry; immunophenotyping; leukocytes.

Figure 1

Flow cytometric analysis of the camel leukogram. (A) Gating strategy for the identification of camel leukocyte populations. Singlets were excluded from the analysis based on their side scatter height (SSC-H) and SSC-Aria (SSC-A) signals. Within the mononuclear cell population, lymphocytes (L) and monocytes (M) were identified as CD14-negative and CD14-positive cells, respectively. In a SSC-A/ FL-1 dot plot, eosinophils (E) were distinguished from neutrophils (N) based on the higher green autofluorescence of their eosinophilic granules. (B) The total leukocyte number was counted under microscope using the Neubauer counting chamber after staining with Türk solution. The relative fractions of leukocyte subsets as determined using flow cytometry were used to calculate the absolute cell numbers. For both heparin and EDTA blood samples, the absolute cell numbers of all leukocyte subsets were presented as the mean and standard error of the mean. Differences between the means were calculated using the t-test and were considered significant (*) if p < 0.05.

Figure 2

Lymphocyte composition in camel blood collected in EDTA or heparin tubes. (A) Gating strategy for the identification of lymphocyte subsets. The whole lymphocyte population was identified within the mononuclear cells in a SSC-A/FSC-A dot plot and the percentage of helper T cells and γδ T cells were identified according to their positive staining with CD4 and WC-1 antibodies, respectively. Camel B cells were identified as MHC-II+CD14-cells in a CD14 against MHC-II dot plot. (B) The cell numbers of B cells, helper T cells, and γδ T cells were estimated and presented as mean and standard error of the mean. Differences between the means were calculated using the t-test and were considered significant (*) if p < 0.05.

Figure 3

The effect of an anticoagulant on the expression density of some cell surface markers on camel neutrophils (A) and monocytes (B). Leukocytes were labeled with monoclonal antibodies to CD172a, CD14, MHCII, and CD163, and labeled cells were analyzed by flow cytometry. The expression of each cell marker was evaluated as mean fluorescence intensity (MFI) and the results are presented as mean ± SEM. * indicates a p value < 0.05.

Figure 4

Ex vivo analysis of the impact of an anticoagulant on the expression levels of the cell surface molecules CD11a, CD18, CD44, and CD45 on camel blood neutrophils, lymphocytes, and monocytes. Separated camel leukocytes were labeled with monoclonal antibodies to CD11a, CD18, CD44 and CD45, and labeled cells were analyzed by flow cytometry. After gating on camel neutrophils, lymphocytes, or monocytes, the expression levels of CD11a, CD18, CD44, and CD45 were measured as mean fluorescence intensities (MFI) of the analyzed markers. Data were presented as mean ± SEM. * indicates a significant difference (p value < 0.05) between groups, as analyzed by the t-test.

Figure 5

Impact of the anticoagulant on the percentage of necrotic leukocytes. Separated blood leukocytes were labeled with PI and analyzed by flow cytometry. (A) Within single cells, granulocytes (G), lymphocytes (L), and monocytes (M) were identified according to their FSC and SSC signals. In a FL3 histogram, PI-negative (viable) cells were distinguished from PI-permeable (necrotic) cells. (B) The percentages of necrotic granulocytes, lymphocytes, and monocytes were calculated and presented graphically as mean ± SEM. Differences between the means were calculated using the t-test and were considered significant (*) if p < 0.05.

Figure 6

The impact of the anticoagulant on camel leukocyte apoptosis. (A) Analysis of cell apoptosis by flow cytometry. After labeling the cells with the mitochondrial membrane potential (MMP) probe JC-1, normal viable cells with orange JC-1 aggregates were detected in FL-2, while apoptotic cells with green JC-1 monomers were detected in FL-1. (B) The percentage of apoptotic cells was presented for gated camel granulocytes, lymphocytes, and monocytes as mean ± SEM. (*) if p < 0.05.