Effect of the delayed wash (deglycerolisation) on the red blood cell morphology: Comparison of AFM and optical profilometry

The morphological characterisation is crucial for analysing cell states, especially for red blood cells (RBCs), which are used in transfusions. This study compared the applicability of atomic force microscopy (AFM) and confocal optical profilometry in the accurate characterisation of the RBC morphological parameters. The imaging of RBCs thawed after cryopreservation with immediate and delayed washing steps (deglycerolisation) was performed, and the morphological data obtained with AFM and optical profilometry were compared with the clinical laboratory studies. Both techniques provided close data on the morphological parameters, but optical profilometry allowed a faster and more convenient data acquisition. However, the membrane roughness analysis on discocytes and the submembrane cytoskeleton analysis on RBC ghosts was only possible with AFM due to its higher spatial resolution. Both techniques confirmed that delayed washing did not have negative effects on cells compared to immediate washing. Additional 3-day storage of both types of RBCs resulted in increased haemolysis. A decrease in the fraction of area occupied by pores in the submembrane cytoskeleton with the storage time was observed, possibly associated with the cytoskeleton deterioration. The studied conditions model the transportation of thawed RBCs in a cryoprotectant solution to medical facilities that have technical conditions to wash thawed RBCs and confirm its feasibility.