Measurement of granulocyte water permeability at subfreezing temperatures

ABSTRACTS, 19th ANNUAL or Hanks’ solution survive the 37°C incubationprovided that autologous plasma is present. Protection is achieved with as little as 0.1 to 0.6% plasma. Interposing the 37°C incubation between the experimental treatment and the fluorescence measurements thus makes the FDA procedure a highly sensitive measure of granulocyte injury. In this assay we measure both the total number of cells before and after treatment and the number of fluorescing cells before and after treatment. The ratio in the latter case provides a more accurate measure of damage than does the ratio of fluorescent cells to total cells in given microscope fields. (Research sponsored by the Oflice of Health and Environmental Research, Department of Energy, under Contract W-7405eng-26 with the Union Carbide Corporation. 1Postdoctoral Investigator supported by Subcontract 3322 from the Biology Division, Oak Ridge National Laboratory, to The University of Tennessee. 2 Postdoctoral Investigator supported by Medical Research Council of Canada.) 89. Transient Volumetric Response of Human Granulocytes to Freezing. GARY J. SCHWARTZ, KENNETH R. DILLER, AND CHARLES R. BAXTER (Biomedical Engineering Center, The Uni-

versity of Texas, Austin, Texas 78712, and Department of Surgery, The University of Texas Health Science Center, Dallas, Texas 75235). A series of experiments was performed to determine the response of human granulocytes suspended in autologous plasma to freezing at various cooling rates. Using cryomicroscopic techniques the suspending medium was nucleated at -2°C in each experiment, and subsequent linear cooling rates of 0, 2, 5, 10, 30, and IO”C/min were effected by a microprocessor-based controller. The volumetric behavior of freezing granulocytes was recorded with a 35-mm camera. Cell volumes were measured from the photomicrographs as a function of temperature over the range from -2 to -50°C. It was observed that at more rapid cooling both the rate and magnitude of freeze-induced volume loss were diminished. For cooling rates of 0, 2, and 5°C min, frozen granulocytes exhibited a volume decrease as large as 50% of the initial cell volume. For greater cooling rates the observed volume was less, with a minimum normalized volume of 90% for lO”C/min and undetectable change for rates of 30 and 80”Clmin. Additionally, it was noted that at temperatures below -2O”C, the volume was constant irrespective of cooling rate. Experimental volume curves were evaluated in terms of a numerical model for cell freezing kinetics. (Sponsored by NSF Grant ECS-8021511.) 90. Measurement of Granulocyte Water Permeability at Subfreezing Temperatures. GARY J. SCHWARTZ AND KENNETH R. DILLER (Bio-

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medical Engineering Center, The University of Texas, Austin, Texas 78712). Small volumes of human granulocytes suspended in autologous plasma were frozen on a cryomicroscope to determine their osmotic properties during freezing stress. The apparent water permeability of the cells was estimated indirectly from measured recordings of the transient volume alterations during isothermal exposure to freeze-concentrated extracellular solutions. The temperature of the granulocyte sample was reduced to a preselected supercooled value between -2 and -lo”C, where it was then maintained constant by a microprocessor-based controller. The extracellular medium was nucleated by spraying droplets of liquid nitrogen over the surface of the cryomicroscope stage. Osmotic dehydration occurred in response to exposure to elevated extracellular electrolyte concentrations. Progressive cell shrinkage was recorded sequentially with a 35-mm camera; the cell volumes were measured subsequently from photomicrographs. The resultant transient granulocyte volume trends exhibited a decrease in both the rate and the extent of volume loss as the temperature was reduced. For example, during 2 min of freezing exposure at -lO”C, the apparent cell volume decreased by only about 10%. The water permeability was deduced by statistically fitting a cell freezing model to the recorded volumetric data. The calculated permeabilities varied from 0.10 ~m/atm/min at -2°C to 0.0002 ~rn/atm/min at -10°C. The activation energy for the transport process at freezing temperatures was considerably higher than values reported for above 0°C. (Sponsored by NSF Grant ECS-8021511.) 91. The Detrimental

Effect of Glycerol on Granulocytes. W. J. ARMITAGE,’ PETER MAZUR, AND K. W. COLE (UT-Oak Ridge Graduate School

of Biomedical Sciences and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830). The survival of human granulocytes after exposure to hyperosmotic solutions of glycerol at 0°C and the subsequent return to isotonic conditions were assessed by fluorescein diacetate after 60-90 min incubation at 37°C. The rapid addition of 2 M glycerol, followed 30 min later by dilution that was slow enough to keep the cell volume below 150% of isotonic volume, was damaging. To determine whether the shrinkage of the cells during the rapid addition of glycerol was the cause of injury, the rate of addition was slowed so that the cells should not have shrunk below 80% of their isotonic volume. However, reducing the osmotic stress in this manner during both addition and removal of glycerol did not ameliorate the damage. Granulocytes were then exposed to a range of glycerol concentrations from 0.5 to 2.0 M at 0°C for 30 min before