Hemolysis in several animal species after rapid freezing of blood

The effect of various freezing rates on the extent of hemolysis in human, bovine and ovine erythrocytes, which are known to have different cell volumes, water contents and permeabilities, was investigated. Blood in stainless steel capillary tubes was frozen at various rates by abrupt immersion of the capillaries into cooling baths at temperatures ranging from −20° to −130°C. Minimum lysis values were obtained at freezing temperatures of −40°, −50° and −70°C with, respectively, human, bovine and ovine blood. The smallest, highly permeable sheep erythrocytes were the least damaged at the highest freezing rates; the largest human cells with the highest water content, suffered the greatest damage; intermediate values were obtained with ox blood. At the lower freezing rates, the largest, human cells were the least damaged; the highest hemolysis values were obtained with the smallest, highly permeable sheep erythrocytes; ox blood again gave intermediate values. These results are in agreement with current views that, (1) very rapid freezing results in the formation of damaging intracellular ice; (2) injury associated with slow freezing is related to the extent of dehydration or to the increase in electrolyte concentration which accompanies ice formation; (3) minimum hemolysis is obtained under those freezing conditions in which osmotic dehydration has been sufficient to prevent the formation of intracellular ice, but has left enough water in the cells to prevent the damaging effects of dehydration and high electrolyte concentrations.