Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research

Myeloperoxidase (MPO) is considered to be selectively expressed in germinative cells of granulocytes and monocytes but not lymphocytes. In human diagnostic medicine the cellular detection of MPO has become a valuable clinical tool for pathologies such as acute myeloid leukemia. Cellular MPO detection might also be relevant in equine pathological conditions. Therefore, we developed a standardized flow cytometric protocol for the detection of equine surface and total cellular (= surface and intracellular) MPO, including the choice of fixation (Fix versus Formaldehyde) and permeabilization technique (Fix and Perm versus Formaldehyde and FACSPerm), of secondary antibody in the indirect labeling approach (Alexa Fluor 488 versus Allophycocyanin (APC) labeled secondary antibody) and of fluorochrome for the primary antibody in the direct labeling approach (DyLight 649 versus Atto633). Titrations of these antibodies were performed on equine white blood cells and controls for reproducibility were included. For surface staining, membrane fixation with Formaldehyde was used, while the Fix and Perm protocol was chosen for membrane permeabilization. Indirect staining was performed with the Alexa Fluor 488 labeled antibody because of its low variability and excellent discrimination potential of the three leukocyte populations. For the direct staining of the primary MPO antibody, the DyLight 649 fluorochrome was the preferred choice being characterized by a strong fluorescent signal with low spread and also the ability to discriminate between equine neutrophil, monocyte and lymphocyte populations. 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 are 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