Comparison of different procedures to isolate feline peripheral blood mononuclear cells (PBMCs) from small volumes of blood

Studies on leukocytes isolated from feline blood do not provide details on the performances of isolation techniques.1 From the few numerical data available, however, it can be assumed that the purity of isolated cell populations is high but their recovery rate is low.2 Therefore, large volumes of blood (difficult to collect from cats with spontaneous diseases) are required to obtain enough leukocytes for in vitro studies. The aim of this study was to assess the performances of isolation techniques on small volumes of feline blood. Blood samples (1 to 5 mls) were drawn from clinically healthy cats and placed in EDTA-coated tubes. Fifteen session of tests (10 using Ficoll, 5 using Percoll) were performed. In 9 cases cells were further separated by adherence on Petri dishes (PD) and in 5 cases using iron-labelled monoclonal antibodies against leukocyte antigens followed by magnetic sorting (MS). Cell purity (i.e. the percentage of each population) and recovery (i.e. the percentage of cells of each population recorded after isolation compared with blood) were then calculated. The purity of lymphocytes was significantly higher (P=0.015) with Ficoll (79.6 ± 3.3) than with Percoll (61.0 ±12.0); the purity of monocytes was low, and significantly higher (P=0.015) with Percoll (32.7 ± 13.5) than with Ficoll (9.0 ± 1.6). The recovery rate of lymphocytes was low, and significantly higher (P<0.001) with Ficoll (54.7 ± 27.5) than with Percoll (12.0 ± 4.2). The recovery rates of monocytes recorded with Ficoll (59.4± 34.7) or Percoll (38.5 ± 9.38) were not significantly different. The purity of cell types in PD was not morphologically determinable. However, assuming that at least 90% of adherent and non adherent cells were monocytes and lymphocytes, respectively, the recovery rate for both the populations was lower than 10% in most cats. The purity after MS was generally high, but the recovery rate was variable and the number of yielded cells very low. In conclusion, none of the techniques applied in this study provides good performances in terms of number of cells, purity and recovery rate, when applied to small volumes of blood. This suggest that the isolated cells could not be representative of the population in blood, and limits the use of these 3 techniques in cats with spontaneous diseases. Therefore, cell functions in spontaneous feline disease should be investigated in whole blood rather than on isolated cell populations.