Ultrastructure of Human Leukocytes after Simultaneous Fixation with Glutaraldehyde

Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method. Neutrophilic polymorphonuclear leukocytes are commonly not well preserved by standard methods of fixation for electron microscopy. Cold osmium tetroxide produces in these cells variable destruction in their cytoplasmic matrix and other damage probably attributable to oxidative effects or to inadequate fixation of proteins. Neutrophils fixed with glutaraldehyde followed by osmium tetroxide frequently show mottled granules, occasional myelin figures, and poorly defined membranes, findings which may reflect some degree of autolysis or lipid extraction during the aldehyde exposure. We report here a method for obtaining improved fixation of neutrophils and of other white blood cells. Essential features of the method are the following: (a) the cells are fixed and processed to the stage of dehydration as single cells in suspension or as very small clumps; (b) initial fixation is accomplished with a freshly made mixture of glutaraldehyde and osmium tetroxide, similar to that employed by Trump and Bulger (1); (c) the cells are postfixed' by suspension in uranyl acetate soPostfixation in uranyl acetate may be incorrect terminology since uranyl ions are probably acting lution modified from Kellenberger (2). This "mixed fixative," as we call it, has been applied, with good results, to several other types of cells in suspension or in monolayer cultures, some of which were illustrated in separate reports (3, 4). MATERIALS AND METHOD Collection of Leukocytes Venous blood from healthy adults was collected with minimal trauma in a plastic syringe and immediately mixed with heparin (Connaught Medical Research Lab., Toronto, Canada) to give a final concentration of 0.1 mg/ml. The heparinized blood was mixed with an equal volume of similarly heparinized 2% clinical dextran (Abbott Laboratories, North Chicago, 11.) in physiologic saline. The tube was slanted at 45 and kept at room temperature for 45 min to allow red cell sedimentation. The opalescent celland platelet-rich supernatant plasma layer was then collected and centrifuged at room temperature primarily as a stain rather than as a fixative. The term will nevertheless be used since it signifies clearly the technical procedure and avoids confusion with the separate later step of staining of the sections with uranyl acetate. 615 on O cber 9, 2017 jcb.rress.org D ow nladed fom for 5 min at 200 g to deposit the white cells while leaving most of the platelets in suspension. Sedimented leukocytes were fixed directly or were washed once in heparinized saline and suspended in 10-20% autologous serum-Hanks' solution for short term (1-2 hr) culture in plastic Petri dishes or glass T flasks. The Fixation, Staining, and