IMPACT OF CELL SEPARATION PROCEDURE ON THE IMMUNOPHENOTYPE OF DROMEDARY CAMEL PERIPHERAL BLOOD MONONUCLEAR CELLS AND TOTAL LEUKOCYTES - Jamal Hussen; Hind Althagafi (Journal of Animal and Plant Sciences, 2026)
The present study investigated the impact of the cell separation procedure and whole blood preservation on the viability and immunophenotype of camel peripheral blood mononuclear cells (PBMC) and total leukocytes. Mononuclear cells were separated by density gradient centrifugation, while total leukocytes were separated by hypotonic lysis of red blood cells. Cell apoptosis, necrosis, forward and side scatter properties, and cell staining patterns with monoclonal antibodies to selected immune cell markers were analyzed by flow cytometry. The results showed only low percentages of apoptotic and necrotic lymphocytes and monocytes within cells separated by both methods, indicating no impact of cell separation on viability. Similarly, comparable percentages of positively stained cells with monoclonal antibodies to the surface antigens CD45, CD44, MHC class I, CD11a, CD14, CD163, CD172a, CD4, BAQ44A, WC1, and MHC class II indicate no impact of the used cell separation methods on the immunophenotype of mononuclear cells. Subsequently, the next experiment was performed to see whether cryopreservation of camel whole blood followed by hypotonic lysis of red blood cells to separate total leukocytes would impact the viability or phenotype of PBMC. Although no impact of cryopreservation on total leukocyte count was observed, hypotonic lysis of cryopreserved blood for the separation of total leukocytes resulted in reduced viability of lymphocytes and monocytes with significant changes in their staining pattern with monoclonal antibodies. In conclusion, PBMC separated by density gradient centrifugation or total leukocytes separated by hypotonic lysis of fresh blood can be used for reliable identification of camel immune cell populations. However, using cryopreserved instead of fresh blood results in significant changes in cell viability and immunophenotype.
Keywords: journal article; Dromedary camel; lymphocytes; monocytes; flow cytometry; apoptosis; density gradient cell separation