Hemolysis Assay

ASTM F756 classifies hemolysis as: Grade A (≤2% hemolysis) = minimal response, Grade B (2–5%) = mild response, Grade C (5–10%) = moderate response, Grade D (10–50%) = marked response, Grade E (>50%) = severe response. Most regulatory agencies require Grade A or B (≤5% hemolysis) for blood-contacting devices.

Fresh blood is strongly preferred because RBC viability and hemoglobin stability decline over time. Most standards recommend using blood within 4 hours of collection, or up to 24 hours if stored at 4°C with appropriate anticoagulation. Outdated blood may show elevated baseline hemolysis and reduce assay sensitivity.

High baseline hemolysis suggests improper RBC preparation (incomplete removal of plasma, lysis during washing), use of hypertonic or hypotonic buffers, or contamination. Check that the saline is isotonic (0.9% w/v), wash RBCs thoroughly to remove plasma, and ensure aseptic technique throughout.

Regulatory standards typically require human blood for testing of devices intended for human use. If human blood is unavailable due to ethical or practical constraints, some standards permit animal blood (e.g., sheep or porcine) with appropriate justification, though results may not fully predict human response.

Standard hemolysis assays are static and do not reproduce in vivo shear stresses or mechanical trauma. To assess hemocompatibility under flow conditions, more complex assays using parallel-plate chambers or closed-loop bioreactors are available, though these are less routine and more expensive.