Comparison of equine PBMC isolation using two protocols: Impact on key cellular parameters

There are diverse uses for equine peripheral blood mononuclear cells (PBMC) in research. Studies have shown that PBMC isolation protocols can affect immune parameters of diagnostic and research assays. We aimed to assess the impact of equine blood handling techniques on cell yield, viability, and proliferation under mitogen stimulation. Whole blood (WB) samples were collected from eight healthy adult horses into heparinized tubes for PBMC isolation by comparing method 1 (M1) where freshly collected WB was layered directly over a density gradient medium for centrifugation, followed by PBMC harvesting and treating them with erythrocyte lysis buffer (ELB); and M2, where WB rested at room temperature for spontaneous erythrocyte sedimentation before collection of the erythrocyte-depleted upper layer, which was then layered onto the density gradient medium for centrifugation and PBMC harvesting, without ELB washing. Isolated PBMC were stimulated with concanavalin A (Con-A) for proliferation and analyzed by flow cytometry using CFSE and propidium iodide staining. Cell yield of PBMC isolated without ELB (M2) resulted in higher counts (1.4 × 10⁷ ± 0.5 cells/mL) than that of PBMC using M1 (0.4 × 10⁷ ± 0.25 cells/mL) (P = 0.0159). Cell viability was similar between PBMC isolated using either M1 or M2. PBMC isolated using M2 had higher proliferation (40.78% ± 12.4) than PBMC using M1 (21.86% ± 13.5) (P = 0.028). PBMC isolation after spontaneous erythrocyte sedimentation and without the erythrocyte lysis treatment showed a superior performance in the evaluated parameters, underscoring that PBMC separation methods may significantly influence subsequent analyses.