Scanning electron microscopy of rheumatoid arthritis peripheral blood polymorphonuclear leucocytes.

Peripheral blood polymorphonuclear leucocytes (PMNs) were isolated from six normal individuals and from 27 patients with rheumatoid arthritis (RA) by the Ficoll-Hypaque rapid single step centrifugation technique, fixed in suspension, and examined by scanning electron microscopy (SEM). In addition, four of the preparations from normal individuals and eight from patients with RA were examined by transmission electron microscopy (TEM). Most PMNs in preparations from normal subjects were spherical, unpolarised, and had their surface membrane elaborated into irregular ridges and small ruffles; they contained few phagocytic vacuoles and large numbers of electron dense primary and secondary granules. A minority of the cells were non-spherical, polarised, and had portions of their surface membrane elaborated into ruffled pseudopodia. In contrast, preparations of RA PMNs frequently contained fewer unpolarised PMNs and a higher number of polarised PMNs than did preparations of normal PMNs. Some preparations of RA PMNs also contained substantial numbers of spherical cells whose surface was covered mainly by bulges and blebs. Concurrent examination by TEM showed that RA PMNs frequently contained more phagocytic vacuoles and fewer electron dense primary and secondary granules than normal PMNs. The morphological and ultrastructural changes seen in RA PMNs resembled those which normal PMNs are known to undergo on exposure to C5a in vitro, during adherence to endothelial cells in vivo, or during phagocytosis in vivo or in vitro. Our observations, therefore, provide a useful morphological correlation to those in vitro studies in which differences in the functional activity of RA and normal PMNs have been shown. The possibility that the difference seen between RA and normal PMNs is artefactual and does not represent the genuine in vivo states of these cells is discussed.

A tannic acid based preparation procedure which enables leucocytes to be examined subsequently by either SEM or TEM.

A modification of the glutaraldehyde-osmium tetroxide-tannic acid-uranyl acetate (GOTU) fixation procedure is described which allows human leucocytes to be examined subsequently by either transmission electron microscopy (TEM) or scanning electron microscopy (SEM).

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.