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

Overview

• This study compares two methods for isolating equine peripheral blood mononuclear cells (PBMCs) to determine how different handling techniques affect cell yield, viability, and proliferation.
• The research found that allowing blood to rest for erythrocyte sedimentation before isolation and avoiding erythrocyte lysis improves PBMC yield and proliferation without compromising viability.

Background and Importance

• Equine PBMCs are frequently used in veterinary immunology research and diagnostic tests to study immune responses.
• PBMC isolation protocols can influence the quality and characteristics of the isolated cells, which can affect experimental outcomes.
• Optimizing isolation methods is essential to ensure reliable and reproducible results in studies analyzing immune function.

Study Design and Methods

• Blood samples were collected from eight healthy adult horses using heparinized tubes to prevent clotting.
• Two different isolation protocols were compared:
  • Method 1 (M1): Fresh whole blood was directly layered over a density gradient medium for centrifugation. After PBMC isolation, cells were treated with erythrocyte lysis buffer (ELB) to remove remaining red blood cells.
  • Method 2 (M2): Whole blood was allowed to rest at room temperature for spontaneous erythrocyte sedimentation. The upper layer, depleted of erythrocytes, was then layered over the density gradient medium for centrifugation. PBMCs were harvested without ELB treatment.
• After isolation, PBMCs were stimulated with concanavalin A (Con-A), a mitogen that induces T cell proliferation.
• Proliferation and viability were assessed by flow cytometry using CFSE (to track cell division) and propidium iodide staining (to determine live vs. dead cells).

Key Findings

• Cell Yield: M2 (without erythrocyte lysis) yielded significantly higher PBMC counts (1.4 × 10^6 ± 0.5 cells/mL) compared to M1 (0.4 × 10^6 ± 0.25 cells/mL), with a statistically significant difference (P = 0.0159).
• Cell Viability: Both methods showed similar viability, indicating that the erythrocyte lysis step did not significantly impact survival of isolated PBMCs.
• Cell Proliferation: PBMCs isolated using M2 demonstrated higher proliferation rates (40.78% ± 12.4) following mitogen stimulation compared to M1 (21.86% ± 13.5), with significance (P = 0.028).

Interpretation and Implications

• Allowing whole blood to sediment spontaneously to remove erythrocytes prior to density gradient separation (M2) reduces the need for chemical erythrocyte lysis.
• This gentler technique appears to preserve better the functional capacity of PBMCs, resulting in higher cell yields and greater proliferative responses upon stimulation.
• The findings suggest that excessive handling, such as erythrocyte lysis, may damage or reduce functional immune cells, thus affecting downstream analyses.
• Researchers working with equine PBMCs should consider using sedimentation-based methods to improve isolation outcomes and ensure more accurate immunological assessments.

Conclusion

• The study demonstrates that equine PBMC isolation protocols significantly impact essential cellular parameters relevant to immunological research.
• Specifically, spontaneous erythrocyte sedimentation prior to density gradient separation without the erythrocyte lysis step enhances PBMC yield and proliferation responses compared to a conventional direct layering and lysis method.
• This optimized approach can improve the quality and reproducibility of equine immunology experiments involving PBMCs.