Centrifugal elutriation as a method for isolation of large numbers of functionally intact human peripheral blood monocytes

Abstract

Centrifugal elutriation was used further to isolate human peripheral blood monocytes (HPBM) from mononuclear-enriched cells harvested as a secondary component following platelet concentration collection samples. HPBM were recovered in either one or two populations consisting of either total HPBM or small (SM) and large monocytes (LM). The elutriation was carried out at 3,500 ± 5 rpm for the separation of lymphocytes and HPBM in Ca⁺⁺ −and Mg ⁺⁺ -free PBS without EDTA. An average of 5.05 ± 1.50 × 10⁸ HPBM were recovered in the total HPBM with a purity of 95% ± 3%. The SM and LM were obtained by splitting the total HPBM into two equal populations with an HPBM purity of 92% ± 3% and 93% ± 3%, respectively, by nonspecific esterase staining. The elutriation media were shown to have no effect on viability by trypan blue exclusion. All three HPBM populations were shown to be histochemically (lack of reactivity to leu-1 and leu-7) and functionally (depletion of NK cell activity) purified from the lymphocyte population. The HPBM populations were enriched in HLA-Dr, OKM-1, OKM-5, MY-8, and leu M-3 monoclonal antibody marker staining. There were no differences in percent positive cells between SM and LM populations for any of the monocyte-specific monoclonal antibodies. All three monocyte populations mediated antibody-dependent cell-mediated cytotoxicity to human red blood cells, with LM mediating more lysis (27.0% ± 5%) than SM (7% ± 3%). The HPBM produced H₂O₂ and were activatable by bacterial lipopolysaccharide (LPS) and recombinant interferon gamma. HPBM did not have any spontaneous cytostasis to K562; HPBM, could be activated by 24-hour exposure to 1.0 μg/ml of LPS (28.0%). The data obtained shows that mononuclear cell enriched plateletpheresis samples can be used to obtain large numbers of functionally active, highly purified HPBM.