Use of Serum or Buffer-Changed EDTA-Plasma in a Rapid, Inexpensive, and Easy-To-Perform Hemolytic Complement Assay for Differential Diagnosis of Systemic Lupus Erythematosus and Monitoring of Patients with the Disease

Abstract

We previously described a simplified quantitative hemolytic assay for classical pathway (CP) hemolytic function in serum that has been shown to correlate with the 50% hemolytic complement (CH 50 ) assay. In the present study, we used this assay to compare CP functions; plasma levels of C3, C4, and C3dg; and ratios of C3dg to C3 in healthy individuals and patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) with different degrees of complement activation. A significant depression in CP function and levels of C4 and C3 and increased C3dg levels and C3dg/C3 ratios were observed in the SLE patients. In patients with RA, CP function was normal, whereas C3, C4, and C3dg levels and the C3dg/C3 ratio were elevated. The SLE results are compatible with systemic complement consumption, whereas the RA data suggest an acute-phase reaction with a normal C3 catabolic rate. To facilitate the handling of patient samples, we also developed a method to restore the hemolytic function of EDTA-plasma by transferring it to Veronal-buffered saline containing the thrombin inhibitor lepirudin. This process inhibits coagulation and enables complement activation, allowing a longer time lag between sample harvesting and testing. These results, combined with previous correlation studies, suggest that the CP hemolytic assay can effectively replace the CH 50 assay for routine SLE differential diagnosis and monitoring of disease activity.

Complement Diagnostics: Concepts, Indications, and Practical Guidelines

“…C3d,g is continuously generated in vivo under normal conditions, presumably as part of the physiological turnover of C3. Therefore, it is useful to determine the ratio between the C3d,g level and the total level of C3 in order to monitor ongoing complement activation (C3d,g/C3), for example, during a flare in SLE [34]. …”

“…Serum or plasma anticoagulated with a specific thrombin inhibitor, such as lepirudin, is used for the quantification of complement function and autoantibodies. Alternatively, EDTA-plasma can be used for the functional assays, provided that the samples are transferred to Veronal buffer with Ca 2+ and Mg 2+ (to enable complement activation) and lepirudin (to inhibit coagulation) [34]. Plasma and serum should be separated within 2 hr of collection and frozen at −70°C.…”

Prompt reversal of a severe complement activation by eculizumab in a patient undergoing intentional ABO-incompatible pancreas and kidney transplantation

“…C3 was assessed using nephelometry (Beckman Coulter Immage, Ramsey, MA, USA). The C3d,g/C3 ratio was calculated as an indicator of complement activation [4,5]. A modified single‐tube assay to detect classical complement pathway (CPP)‐related hemolytic function was performed as previously described [6].…”

Association of Blood Concentrations of Complement Split Product iC3b and Serum C3 With Systemic Lupus Erythematosus Disease Activity

“…However, 2 issues limit the clinical utility of CB‐CAPs: 1) results are not rapidly available, because detection requires flow cytometry, and 2) erythrocyte measurements are a reflection of complement activation and SLE disease activity over the 120‐day lifespan of the erythrocyte rather than the immediate clinical situation. Efforts to measure C3d on other cell types with shorter lifespans (e.g., reticulocytes [R‐C4d]) have demonstrated better correlation with indices of disease activity, including scores on the Safety of Estrogens in Lupus Erythematosus National Assessment version of the SLE Disease Activity Index (SLEDAI) , while C3dg:C3 ratios were found to be correlated with disease activity in juvenile arthritis , rheumatoid arthritis, and SLE .…”