Flow cytometric detection of intracellular myeloperoxidase in equine polymophonuclear leukocytes

Myeloperoxidase (MPO) is considered to be selectively expressed in germinative cells of granulocytes and monocytes but not lymphocytes. In human diagnostic medicine the intracellular detection of MPO has become a valuable clinical tool for pathologies such as acute myeloid leukemia. Intracellular MPO detection might also be relevant in equine pathological conditions. Therefore, we developed a standardized flow cytometric protocol for the detection of equine intracellular MPO, including the choice of permeabilization technique (Fix and Perm versus Formaldehyde and FACSPerm), of secondary antibody in the indirect approach (Alexa Fluor 488 versus Allophycocyanin (APC) labeled secondary antibody) and of fluorochrome for the primary antibody in the direct labeling alternative approach (DyLight 649 versus Atto633). Titrations of these antibodies and controls for reproducibility were included. The Fix and Perm protocol resulted in the lower background staining and within-horse variation compared to the tests with Formaldehyde and FACSPerm. In the indirect staining technique, the Alexa Fluor 488 labeled antibody was superior to the APC labeled alternative with respect to variability and discrimination potential of the three leukocyte populations. For the direct staining of the primary antibody, the DyLight 649 fluorochrome was the preferred choice being characterized by a stronger fluorescent signal with lower spread and also the ability to discriminate between equine neutrophil, monocyte and lymphocyte populations, in contrast to the Atto633 labeled antibody. Optimal concentrations for all selected antibodies were determined. The good repeatability of the primary antibody titration illustrated the reproducibility of the flow cytometric technique. The defined flow cytometric protocols for indirect and direct intracellular MPO detection will be applied in ongoing research to validate the possible diagnostic properties of MPO in equine infectious versus non-infectious joint disease. Research funded by a Ph.D. grant of the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen