A New and Simple Approach to Determine Proliferation of Feline Lymphocytes

by: Kim Parkhill

Immune function in felines depends on T-lymphocyte responsiveness. Measurement of T-lymphocyte response can be measured in vivo by stimulating T-cells with mitogen. The goal of this experiment was to determine if polydactyl felines are as immunologically responsive to mitogen as domestic felines by developing as assay to test their lymphoproliferation. In this experiment, a feasible, non-radioactive, and time efficient spectrophotometric microassay using Alamar Blue dye and leukocytes from each of five domestic, normal-toed cats, five domestic, polydactyl cats, and two feline leukemia positive cats (FeLV), was developed and used to test the response of T-lymphocytes from each cat to mitogen induced stimulation using Concanavilin A (Con A). Feline leukocytes were isolated from defibrinated peripheral blood by histopaque 1083-gradient centrifugation and incubated at a concentration of 5 X 10⁵ cells per ml for 72 hours. Alamar Blue was added to each culture after 69 hours of incubation as an indicator of cellular proliferation. The degree of proliferation in each culture was determined by the change in color of Alamar Blue after incubation from 69-72 hours. In this study, the uptake of Alamar Blue by feline leukocytes resulted in a change from the oxidized indigo, non-fluorescent state, to the reduced fluorescent pink state. Reduction of Alamar indicated a proliferation of feline cells whereas oxidation was indicative of little or no cell growth. Based on the calculated mean for feline leukemia, domestic, and polydactyl reduction of Alamar Blue (24.3%, 107.49%, and 201.84%, respectively), the assay developed in this study appears to be useful for distinguishing varying degrees of T-cell response in felines. Standard errors for the mean values of domestic and polydactyl felines did not overlap. Due to small sample sizes, however, standard deviations of the domestic and polydactyl felines overlapped, indicating that this assay did not detect significant differences in T-cell response among the cats studied. Further analysis using larger sample sizes of domestic and polydactyl cats will more effectively demonstrate lymphoproliferation response among domestic and polydactyl cats.

Research Advisor: William L. Hallahan, Ph.D., Nazareth College