Analyses of Plasma Split Products
The above-described limitations of the complement proteins C3 and C4 as biomarkers for the diagnosis and monitoring of SLE disease activity have prompted the development of assays to measure the proteolytic fragments of complement proteins. As these fragments are formed upon activation of the complement cascade, complement split products reflect complement activation more accurately than the levels of the individual intact proteins.
Assays for several complement split products—either soluble or cell-bound—have been developed to evaluate whether these fragments can serve as biomarkers of SLE either to aid in the diagnosis of the disease or to monitor disease activity or as prognostic markers for flares of nephritis. There are technical issues related to detection of these split products in plasma. Many have a very short half-life. Also, some complement activation can occur in vitro at room temperature, even when collected in EDTA plasma tubes. Therefore, the split products must be measured within a short time on fresh plasma or the sample must be snap frozen until studied. These challenges have made these tests impractical for general clinical use outside of research laboratories [
7]. However, the information gleaned from the recent studies described below adds to the accumulating evidence that complement activation in vivo in SLE is better detected by the presence of split products than by low complement protein levels.
Complement fragments derived from the classical pathway (C4d) or from the convergence of the three pathways of complement activation (C3dg, iC3b) hold promise as biomarkers of SLE.
C3dg has a half-life of 4 h and can be measured in EDTA plasma frozen within a few hours of collection. Samples appear stable at −80 °C for a year, and values are not significantly affected by up to four freeze-thaw cycles. The assay for C3dg requires a step for the precipitation of larger C3 fragments. The supernatant can then be tested with the time-resolved immunofluorimetric assay or ELISA [
18•]. Because the formation of split products in SLE happens in parallel with the degradation and synthesis of C3 during complement activation, the ratio C3dg/C3 has also been evaluated. The performance characteristics of C3dg and the C3dg/C3 ratio have been compared with C3 levels in SLE and normal healthy subjects. Areas under the curve (AUC) of the receiver operating characteristics (ROC) are high at 0.96 and 0.89, respectively, and much better than those of C3 levels; however, comparison of SLE versus other rheumatic diseases has not been performed [
18•].
iC3b has a half-life of 90 min. Samples of blood, plasma, or serum are placed within 30 min of venipuncture in a buffer that prevents spontaneous complement activation and are frozen immediately. The levels of iC3b and the iC3b/C3 ratio are higher in SLE patients than in normal healthy subjects, and the SLE patients with active disease have iC3b and iC3b/C3 ratio higher than patients with inactive disease. The AUC of the ROC curve of iC3b/C3 is higher than that of iC3b in discriminating active and inactive SLE and flaring vs. nonflaring patients, and it outperformed C3 and C4 levels. In fact, the majority of patients with active SLE had high iC3b and iC3b/C3 ratio whereas only 37% had low C3 levels [
19•]. The quantitative lateral flow assay used in this study mitigates some of the handling issues with split product determination which can lead to in vitro complement activation [
20].
C4d is a promising marker of disease activity in SLE as the delta change in plasma C4d values between low and high disease activities in a certain patient was higher for C4d than for C3 and C4 and the AUC of the ROC curve for high disease activity was higher for C4d than for C3 and C4 [
21•]. In addition, C4d was higher in patients with nephritis than in patients without renal involvement. Of note, although the odds ratios of C4d were higher than those of C3 and C4, they were lower than those of dsDNA. The combination of anti-dsDNA and C4d was significantly associated with nephritis. In addition, data in a small number of subjects also indicate that C4d may be able to predict a lupus nephritis flare in patients who had already an episode of nephritis and, thus, are at increased risk. This study was conducted with plasma samples isolated from blood within 1 h of venipuncture and frozen within 2 h. The ELISA was performed with an anti-C4d antibody that recognizes a neoepitope formed after C4b cleavage to C4d. The same group also analyzed the C4d/C4 ratio in a different patient population and found that both C4d and the C4d/C4 ratio were higher in SLE patients—and especially in lupus nephritis—than in healthy controls [
22••]. The AUC of the ROC curve of the C4d/C4 ratio for lupus nephritis was 0.76, higher than that of C4d and C4 alone (both 0.71). In addition, odds ratio analysis showed that high C4d, low C4, and low C3 were associated significantly with nephritis with the C4d/C4 ratio having the highest relative odds. Importantly, C4d and the C4d/C4 ratio decreased in lupus nephritis patients who responded to therapy—while they did not change in nonresponders—and are associated with histopathological changes. The association with both clinical and histopathological responses in lupus nephritis suggests that the determination of C4d in plasma may at least partially replace invasive biopsies to evaluate active nephritis.
Limited data exists on soluble complement fragments derived from the lectin pathway. Some proteins of the lectin pathway are lower in SLE patients than in controls, while others have the opposite trend. In addition, some display a negative correlation with disease activity and a positive correlation with C3, indicating that consumption of some of the proteins of the lectin pathway occurs in SLE. In some studies, significant differences in concentrations between patients with SLE and controls have been observed, and in one study, there was a correlation of MASP-2 levels with SLE disease activity [
23]. However, the wide range of the concentrations of these proteins in patients and controls makes their use as biomarkers of SLE uncertain at this time. Genetic deficiencies of mannose-binding lectin (MBL) have been associated with SLE [
24].
Cell-Bound Complement Activation Products (CB-CAPs) as Markers in SLE
C4d as well as C3d is not only present in solution but also bound to the membrane of blood cells, such as erythrocytes, reticulocytes, B and T lymphocytes, and platelets. These complement fragments which are covalently bound to cell membranes are collectively called cell-bound complement activation products (CB-CAPs) [
7]. They can be measured in EDTA-anticoagulated blood by flow cytometry, and their stability allows for samples to be shipped refrigerated to a central laboratory with overnight delivery. Although flow cytometry is labor intensive, sample processing in the clinic is minimal and does not require centrifugation or storage and transportation at low temperatures.
Extensive research has demonstrated the value of CB-CAPs to aid in the diagnosis of SLE as well as to monitor disease activity, and a recent review on CB-CAPs in SLE has been published [
25•]. In essence, CB-CAPs are more sensitive than plasma/serum levels of C3 and C4 for the diagnosis of SLE. A more recent study showed that CB-CAPs (BC4d, EC4d) are more prevalent than low complement levels in patients with probable SLE [
11••] and when used in a multiparameter assay panel are better than other biomarkers at predicting transition to SLE.
We will review here recent data on C4d bound to erythrocytes (EC4d) and platelets (PC4d).
An analysis of three independent SLE populations showed that EC4d is associated with disease activity measured with the SELENA-SLEDAI or with the physician global assessment. Association is increased when EC4d is combined with low complement C3/C4, indicating that both biomarkers have value in monitoring disease activity in SLE. Interestingly, in the majority of patients, serum complement levels did not change over time and were chronically low or chronically normal. In this subset, EC4d remained associated with disease activity, suggesting that EC4d is superior to C3/C4 and can give information on disease activity in patients whose C3/C4 does not reach abnormally low levels during high disease activity [
14•].
Because of the increased risk of thrombosis in SLE, recent studies have evaluated whether CB-CAPs are associated with history of thrombotic events. EC4d has weak association, and BC4d is not associated with thrombosis. PC4d, however, has strong association with history of thrombosis (odds ratio = 8.4). The association is particularly strong with venous thrombosis (odds ratio = 19.2) and weaker with arterial thrombosis (odds ratio = 4.0) [
26••]. Multivariate logistic regression revealed that PC4d, low C3, and lupus anticoagulant are all significantly and independently associated with thrombosis, indicating additive utility, especially for venous thrombosis.
Association of PC4d with history of thrombosis was recently found also by another group that analyzed PC4d by flow cytometry utilizing frozen platelet-rich plasma [
27•] instead of fresh blood. Comparison of PC4d in patients with SLE to controls in the general population showed that PC4d was associated with venous thromboembolism and ischemic stroke, but not with ischemic heart disease or subclinical atherosclerosis. Interestingly, this study found not only a negative correlation between PC4d and C3 or C4 but also a positive correlation between PC4d and C3dg and, to a lesser extent, with soluble C5a-9 [
27•]. This suggests that both soluble and cell-bound complement split products may be useful biomarkers in SLE and may be associated with thrombosis. Complement-mediated thrombosis has been found in many complementopathies, even though the mechanisms remain unclear [
28].
The ability of PC4d to predict the occurrence of cardiovascular events has yet to be demonstrated. In fact, prospective studies on thrombosis in SLE are particularly challenging owing to the relatively low number of events over a long period of time.