Isolation of human PMN leukocytes and granules: observations on early blood diluion and on heparin.

Abstract

Abstract A reproducible procedure for the preparation of PMN leukocytes in high yield (80 ± 15 per cent) from human venous blood and for the recovery of intact granules from disrupted leukocytes is described. By immediate twentyfold dilution of blood in saline, subsequent leukocyte clumping was completely avoided. The PMN leukocytes obtained were morphologically normal, metabolically active, and avidly phagocytic. Granules were isolated following homogenization of cells in sucrose in the presence of heparin. The essential role of heparin or certain other polyanions and the ineffectiveness of some polycations in inducing leukocyte disruption is documented. Cellular homogenates were fractionated by differential centrifugation into an 800 × g nuclear fraction that contained intact cells and nuclear fragments, a 25,000 × g granular fraction that contained the bulk of the lysosomal enzymes, acid phosphatase, and β-glucuronidase and a 25,000 × g postgranular supernatant that contained soluble cellular constituents, including the bulk of cellular protein. Enzyme specific activities in the granule fraction increased 6.4 fold for acid phosphatase and 8.2 fold for β-glucuronidase relative to that of the cell homogenate. Sixty-three per cent of the total acid phosphatase and β-glucuronidase were recovered in the granule fraction. Through application of the methodology developed, it is hoped that further insights into the role of leukocytes and leukocyte granules in human physiology and pathology may be obtained.