CHEMOTAXIS OF CANINE POLYMORPHONUCLEAR NEUTROPHIL GRANULOCYTES USING THE UNDER-AGAROSE METHOD APPLIED TO GLASS MICROSCOPE SLIDES

The under-agarose chemotaxis assay method applied to glass microscope slides was optimised for canine polymorphonuclear granulocytes (PMN) w ith gelatin as protein supplement. Optimum chemotactic response occurr ed after approximately 135 min of incubation at 37 degrees C with 5 x 10(5) cells/well and zymosan-activated serum (ZAS) as chemoattractant. Chemotactic (C) and random migration (R) were stated separately for e ach dog. Storage of blood samples for 90 min before isolation of PMN d id not influence the C or R values (p > 0.05). The preanalytical varia tion was investigated and the proportion of variance, expressed as a p ercentage, due to each source for the C and R values, respectively, wa s determined: dog, 92%, 96%; blood sample, 0.4%, 0%; interslide, 3%, 2 % and intraslide, 3%, 3%. The mean chemotactic responsiveness against ZAS and ZAS diluted 1:1 with RPMI 1640 (dZAS) was determined using 15 laboratory beagle dogs: C-ZAS = 0.67 mm (range: 0.25-2.33 mm), coeffic ient of variation (CV) = 24%, C-dZAS = 0.47 mm (range: 0.18-2.12 mm), CV = 22%. Also, the mean random migration was determined: 0.11 mm (ran ge: 0.05-0.34 mm), CV = 25%. Neither age nor sex had any significant i nfluence on chemotactic or random migration (p > 0.05). The effect of heat-inactivated pooled canine serum (HI-serum) incorporated into agar ose was examined and it was found that this had a significant stimulat ory effect on both chemotactic and random migratory responses (p < 0.0 5). Therefore, agarose with HI-serum is not useful when investigating the chemotactic function.