Liquid preservation of canine granulocytes obtained by counterflow centrifugation-elutriation

Granulocytes (PMN) were isolated from 120 ml of canine whole blood by a modification of the counterflow centrifugation-elutriation technique. Isolated cell suspensions of 96% granulocytes and 4% mononuclear leukocytes with a 21:1 PMN/RBC ratio were stored at 4 degrees C in 4:4:2 medium consisting of four parts HBSS minus Ca++ and Mg++, four parts MEM, twp parts autologous plasma, and 20 microgram/ml gentamicin for 15 days. Granulocytes were stored at concentrations of approximately 4 x 10(6) PMN/ml in polypropylene centrifuge tubes. The stored granulocyte suspensions were assayed in vitro 0, 1, 4, 8, and 15 days to monitor chemotaxis, bacterial growth inhibition, O2 consumption associated with phagocytosis, and enzyme activities. Cell volume analysis was used to evaluate cellular integrity of the liquid-stored granulocytes. Canine granulocytes isolated by the modified dilution technique of counterflow centrifugation-elutriation can be preserved for up to 15 days with 77 +/- 6% granulocyte survival with maintenance of morphological and organelle integrity, as well as retention of in vitro functions of recognition, migration, phagocytosis, and killing of gram-negative bacteria.