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.

The production of small IgG aggregates by glutaraldehyde cross-linking.

Reaction conditions have been determined for the production of soluble IgG polymers in the size range 10 S to 30 S by covalent cross-linking with glutaraldehyde. This size range is comparable with that of the immune complexes which are frequently found in the circulation of patients with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. The yield of IgG aggregates in this size range is far greater than has been reported for cross-linking by other bifunctional reagents or for aggregation by heating. Glutaraldehyde cross-linked IgG polymers are stable and biologically reactive. They can also be labelled with fluorescein and freeze-dried with minimal loss of integrity or reactivity.

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).

Intrinsically stable IgG aggregates.

Measurement of the absorbance due to light scattering at 40 nm proved to be a simple and reliable way of assessing the extent of aggregation in heat-treated IgG solutions. Using this technique the rate of aggregation was demonstrated to be markedly temperature dependent with a sharp inflexion in the curve close to 63 degrees C. During heating at 63 degrees C the concentration of unaggregated IgG fell in a manner consistent with a first-order process and the mean size of the IgG aggregates increased with time. IgG aggregates could be selectively removed from heat-treated IgG solutions and concentrated by precipitation with 3.5% polyethylene glycol without altering their size distribution. Furthermore, and in contrast with a previous report, the IgG aggregates examined in this study were remarkably stable in the absence of any other protein such as serum albumin. In consequence, several important practical recommendations concerning the production of heat-aggregated IgG for use in immune complex assays are made.