Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice

The present study examined the damaging effect of intracellular ice on plasma membranes of human erythrocytes. Ice crystals of 0.2–2.0 μm in diameter were formed within the cells as the result of rapid freezing of erythrocytes at the cooling rates around 8000 °C/min. Freeze-fracture and etching studies revealed the ultrastructural alterations of membranes caused by the formation of intracellular ice.In the membrane regions which were in direct contact with intracellular ice, depressions resembling “worm-eaten spots” ranging from 400 to 3000 Å in diameter were observed both on the etched protoplasmic fracture faces (PF) and the exoplasmic surfaces (ES); no perforations were detected in the worm-eaten spots as visualized by slight etching, but artificial destructions occurred on these worm-eaten spots following the increase of etching. The most important phenomenon concerning membrane damage was that in the worm-eaten spots the fracture did not occur along the inner hydrophobic plane of membrane.It was suggested that the formation of intracellular ice in direct contact with a membrane brought about molecular disorganization of bilayer membrane. The presence of these altered membrane regions seems to be responsible for the postthawed hemolysis of the intracellularly frozen erythrocytes.