Complement-fixing activity of rheumatoid synovial fluid.
Authors: A S Townes, R L Marcus
Journal: Annals of the Rheumatic Diseases (1972)

Haemolytic complement activity has been shown to be reduced in synovial fluid of patients with rheuma- toid arthritis as compared with patients with in- flammatory effusions of similar protein content due to other causes (Hedberg, 1963; Pekin and Zvaifler, 1964; Fostiropoulous, Austen, and Bloch, 1965; Peltier, Coste, and Delbarre, 1966; Townes and Sowa, 1970).Anticomplementary activity was not observed, however, except as subsequently reported by Hedberg (1967).Using unheated synovial fluid and a period of incubation with a complement source, we have demonstrated anticomplementary or complementfixing activity in a significant number of rheumatoid synovial effusions.This activity demonstrates a pattern of complement-component fixation identical to that of antigen-antibody complexes or gamma globulin aggregates.In order clearly to differentiate this activity from poorly defined anticomplementary activities associated with contaminated, aged, or extensively manipulated biological fluids, we refer to this activity as complement-fixing.The frequency, specificity, and stability character- istics of this complement-fixing activity, are presented below with preliminary observations on its clinical correlates. Material and methodsCOLLECTION AND PROCESSING OF SYNOVIAL FLUID Synovial fluid was obtained by needle aspiration from patients with synovial effusions due to various causes, allowed to stand at room temperature for 30 min., centrifuged at 15,000 G for 15 min.at 4°C. to remove cells, clots, and debris, quick frozen in small aliquots, and stored at -70°C.until used.Patients with rheumatoid arthritis met the ARA criteria (Ropes, Bennett, Cobb, Jacox, and Jessar, 1959) for definite or classical disease.Diagnosis in other patients was based on clinical findings detailed elsewhere (Townes and Sowa, 1970).COMPLEMENT Fresh frozen guinea-pig serum was obtained from Sub- urban Laboratories, Silver Spring, Maryland.Human serum routinely used for complement was obtained from a pool of blood bank donors, or occasionally from individual donors. TESTS FOR COMPLEMENT-FIXING ACTIVITYComplement was diluted in isotonic veronal-buffered saline pH 7-4 containing 0.1 per cent.gelatin, 0-00015 M calcium chloride, and 0-001 M magnesium chloride (Kabat and Mayer, 1961) (hereafter abbreviated GVB) so that approximately 7 to 8 CH50 units of complement were available (before incubation) in 2-4 ml.Synovial fluid samples were thawed rapidly at 37°C. and 0-1 ml. was added to 2-4 ml.complement dilution and incubated at 37°C. for 60 min.10 ml. of cold GVB was then added to the reaction mixture, which was titrated in the usual manner for residual complement activity with sensitized sheep erythrocytes (EA)' in a total volume of 7-5 ml. with incubation for 1 hr at 37°C.(Kabat and Mayer, 1961).Complement fixation is expressed as the percentage of the available complement inactivated by 0-1 ml.synovial fluid.This volume of synovial fluid was chosen for routine testing since the resultant concentration (1/25 in the reaction mixture) minimized the contribution of complement activity from ' The nomenclature used conforms to that published in the Bulletin of the World Health Organization (1968), 39, 935.Sheep erythrocytes (E), sensitized with rabbit antibody (A), react with complement (C), followed by those components which have interacted designated by number, the entire sequence EAC 1, 4, 2, 3, 5, 6, 7, 8, 9 being necessary for haemolysis.Total haemolytic complement in 50 per cent.haemo- lytic units is designated CHso and individual components Cl, C4, etc.