The granulocyte suspensions of human granulocytes from the peripheral blood were frozen in the presence of 7.5% dimethylsulfoxide (DMSO) at rates of 1 °C/min until temperatures of -30 °C were reached. The cells were then frozen at a rate of -10-(-)60 °C/min. They were then stored in liquid nitrogen refrigerators from 10 min to 1 1 2 years. Thawed granulocytes did not regain motility even on prolonged incubation and developed homogenous large cytoplasmic bubbles. However, the cells were able to selectively incorporate neutral red granules into the cytoplasm. The cells eventually disintegrated when incubated up to 24 hr. Examination of stained cells showed disruption of the nuclear chromatin. Cells stained with toluidine blue at controlled pH observed under water, showed reversal in normal tinctorial properties of the cells. The nucleus failed to stain, and the cytoplasm stained strongly basophilic. The cytoplasm bubbles showed metachromasia. Cells stained with Feulgen's reaction showed the presence of Feulgen-positive material in the cytoplasm. PAS reaction showed PAS-positive granules in the cytoplasm. Staining for protein revealed strong reaction in the cytoplasm. The cytoplasmic bubbles did not stain. These observations suggest that human PMN cannot withstand freezing in the presence of DMSO, and that the primary site of cryoinjury is the nucleus, and not the cytoplasm.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)