Cryoprotective strategies for frozen erythrocytes in POCT whole blood reference materials progress and challenges

Point-of-care testing (POCT) is widely applied in emergency and primary healthcare, but persistent detection bias compromises its reliability. The core causes are erythrocyte matrix effects and the global shortage of commutable whole blood matrix reference materials, while erythrocyte hemolysis during cryopreservation further exacerbates measurement errors and metrological traceability failure. This paper reviews key cryoprotective technologies for frozen erythrocytes over the past decade, including optimized glycerol protocols, novel carbohydrate and non-carbohydrate cryoprotective agents (CPAs), and erythrocyte silicification. Optimized glycerol methods control hemolysis below 0.8% but carry risks of residual interference. Carbohydrate CPAs exhibit excellent biocompatibility and potent ice recrystallization inhibition, yet are limited by low transmembrane efficiency. Non-carbohydrate CPAs are easily removable and free of detection interference, but require improvement in human erythrocyte adaptability. Erythrocyte silicification achieves a 96.9% recovery rate, yet faces high costs, scaling difficulties, and unvalidated clinical safety. Current research gaps include the lack of matrix effect verification, ambiguous residual interference mechanisms, and insufficient long-term stability data. Future efforts should develop low-residue, user-friendly, human-erythrocyte-adapted cryoprotective techniques, strengthen commutability validation, and industrialize freeze-dried whole blood reference materials to minimize hemolysis and matrix interference. This will advance the standardization of in vitro diagnostics and accurate POCT quantitation, ultimately enhancing diagnostic accuracy and patient safety in decentralized healthcare.