The recovery, structure and function of dog granulocytes were determined before and after freeze preservation. Leucocytes were isolated from defibrinated or anticoagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6 percent)-Isopaque (33.9 percent). Granulocytes isolated by these procedures were examined for changes in O2 consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7. 5 percent) and autologous serum or HBSS and 20 percent autologous serum at the rate of -1°C/ min to -80°C and stored in liquid N2 vapor. After freeze preservation, O2 consumption associated with phagocytosis was decreased by 54 and 64 percent for granulocytes isolated from defibrinated or from ACD anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD anticoagulated blood. Chemotactic response after freeze preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods, However, nuclear, cytoplasmic and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze preservation in DMSO to a degree not previously reported. It is concluded that dog granulocytes freeze preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes. The increased efficacy of granulocyte transfusions in conjunction with antibiotic therapy against microbial infection during agranulocytosis or granulocytopenia has been well documented. An effective method for granulocyte preservation would minimize logistical problems (typing, preservation, matching and transportation) in increasing the availability of this therapy to combat casualties. In order to meet such a requirement, this model system was developed for studying granulocyte preservation methods.
