Influence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival of slowly frozen human erythrocytes.

We have shown previously that the survival of human red blood cells during slow freezing at 2% hematocrit is dependent more on the magnitude of the unfrozen fraction than on the salt concentration in that unfrozen fraction. In parallel, first Nei and more recently Pegg and colleagues have shown that survival is affected by the hematocrit of the suspension. Freezing at hematocrits above 30% becomes increasingly damaging. The present studies were designed to see whether there is a link between the two phenomena. Cells were suspended at nominal hematocrits of 0.4, 2, 8, 40, or 60% in five test solutions of glycerol-NaCl. The test solutions were of such composition that when frozen to a specified temperature, the magnitude of the unfrozen fraction differed but the NaCl concentration (ms) remained constant. At low hematocrits (0.4 to 8%), red cell survival was dependent predominantly on the unfrozen fraction and was relatively independent of the salt concentration in that fraction. This we term the "rheological" effect because injury appears to be related to interaction with the ice walls and perhaps is due to shearing forces or cell deformation. But at high hematocrits (40 or 60%), cell survival became dependent on both the unfrozen fraction and the salt concentration in that fraction. When freezing occurs at high hematocrits, increasing numbers of cells are presumably brought into contact with their neighbors. Furthermore, they are increasingly shrunken cells, for the progressive removal of liquid water, which is responsible for the crowding, also causes a rise in ms and the consequent osmotic shrinkage of cells. Our data suggest that at unfrozen fractions above those producing injurious rheological forces, the tight packing of less shrunken cells (i.e., high hematocrit, low ms) and the extensive shrinking of loosely packed cells (high ms, low hematocrit) are both quite innocuous. Injury becomes substantial only when extensively shrunken cells are brought into close contact (i.e., high ms, high hematocrit). At high hematocrit the cells occupy a substantial fraction of the unfrozen space, and the water that they lose during slow freezing adds substantially to the volume of extracellular ice. Accordingly, we defined other measures of unfrozen fraction that include these perturbations. However, we found that the conclusions on the relation between survival, unfrozen fraction, and hematocrit were not affected by the method of expressing the unfrozen fraction. Freezing at high hematocrit to high ms and low values of unfrozen fraction is one way to produce contact between shrunken cells at low temperatures.(ABSTRACT TRUNCATED AT 400 WORDS)