Simultaneous separation and purification of mononuclear and polymorphonuclear cells from the peripheral blood of cats

Peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMNC) play important roles in immunodeficiency diseases and AIDS-like syndromes in cats caused by feline leukemia virus (FeLV) and presumably also when caused by feline immunodeficiency virus (FIV). For comparative or functional studies it is advantageous or necessary to obtain these cells as separate entities from the same sample of an animal. Therefore, we analyzed the technical parameters of obtaining, separating and purifying these cells simultaneously from several blood samples of several cats. Flow cytometric studies with outbred cats indicated that of various cell separation/purification methods, e.g. from lysed whole blood, by Histopaque 1.077 or 1.119 single-density, or by 1.077/1.119 double-density centrifugation, the 1.077/1.119 double-density centrifugation of diluted whole blood is the most consistent, practical and effective method of yielding both highly purified PBMC and PMNC as separate entities from the same sample. The interface between plasma and 1.077 contained an average 86% PBMC vs. 14% PMNC, and the interface between 1.077 and 1.119 an average of 2% PBMC vs. 98% PMNC. Lymphocytes separated by this method had an average CD4/CD8 T-cell ratio of 2.0. These data indicate that Histopaque 1.077/1.119 double-density gradient allows purification and physical separation of lymphocytes and phagocytes from a blood sample, enabling the investigator to examine both cell types from the same sample simultaneously.