Density gradient purification of human lymphocytes from contaminating trypomastigotes of Trypanosoma cruzi

Mixtures of normal human lymphocytes and T. cruzi trypomastigotes obtained from infected mice were centrifuged over Ficoll-Hypaque (FH) continuous and discontinuous gradients. Trypomastigotes were confined to the range 1.051–1.057 g/ml while lymphocytes ranged between 1.046 and 1.080 g/ml. Over 80% of the lymphocytes were found at 1.060 g/ml or higher densities. A discontinuous gradient of FH with 2 layers of 1.060 and 1.077 g/ml of density respectively was selected to obtain trypomastigotes-free white blood cells from blood samples. The functional capacity of lymphocytes recovered from the lower interface, where no parasites were found, was assessed. The response to phytohaemagglutinin of these high density lymphocytes was as good as of total lymphocytes, suggesting that low density lymphocytes are not necessary for proliferative responses. It is postulated that high density lymphoid populations, free from T. cruzi forms, may be used to study the presence of T cell-mediated immune response in Chagas' disease patients.