Despite the advancement of novel immunosuppressive agents, antibody-mediated rejection (AMR) continues to be the principal cause of premature kidney allograft loss.1-3 In both acute and chronic forms of AMR, development of antibodies against foreign HLA (donor-specific antibody [DSA]) initiates a cascade of immune injury that culminates in the eventual destruction of kidney vasculature and glomeruli. In kidney recipients, the presence of DSA before4 and after transplantation correlates with poor graft survival.5 Recently, the advent of single-antigen bead (SAB) assay on the Luminex platform allows HLA laboratories to detect even low-level HLA antibodies and more precisely define their specificities, augmenting the accuracy of recipient humoral immune risk assessment. It is recognized, however, that not all DSA as identified by SAB are associated with AMR or other inferior graft outcomes; thus, the need for improved assays that can better discriminate the clinical relevance of preformed and de novo DSA is an unmet challenge in transplant diagnostics.6
Various biologic properties are used to infer the destructive potential of antibodies on the allograft, including number, strength as (crudely) estimated by SAB mean fluorescence intensity (MFI) values,7-10 epitope pattern,11,12 titer,13,14 duration/persistence,15 HLA class specificity,5,15 and isotype.16 Since Patel and Terasaki’s17 landmark study in 1969, the classical complement pathway has been widely regarded as the principal mechanism by which DSA mediate graft injury. On this basis, modifications of the SAB assay (C4d, C1q, C3d) have been designed to interrogate the complement-activating potential of anti-HLA antibodies in the solid phase system,18-20 with the premise that complement-fixing antibodies are more detrimental to the graft compared with their nonactivating counterparts, although complement independent pathways can also be deleterious.21,22 Starting with the solid phase C4d test, we review the evolution of 3 complement dependent assays, focusing primarily on 2 commercially available products: SAB-C1q (C1qScreen, One Lambda, Inc., Canoga Park, CA) and C3d (Lifecodes C3d detection, Immucor Transplant Diagnostics, Inc., Stamford, CT), which evaluate activation of different components of the complement cascade. Here, we highlight the technical features of, and differences between these assays, including their respective strengths and limitations. We summarize key clinical studies and emerging mechanistic evidence, with an emphasis on where data and conclusions diverge. Gaps in knowledge and assay limitations are discussed; within this context, we provide our perspective on the current utility of these tests in clinical transplantation.
EVOLUTION AND TECHNICAL ASPECTS OF COMPLEMENT DEPENDENT ASSAYS
The C4d Assay
The earliest solid phase complement dependent assay was developed by Wahrmann et al18,23 in Vienna, through modification of a flow cytometric based assay which detects binding of IgG antibodies to purified HLA antigens immobilized on inert beads (FlowPRA Screening, One Lambda, Inc., Canoga Park, CA). After incubation of patient sera with HLA coated microparticles, a human complement source (normal human serum obtained from healthy nontransfused male volunteers) is added to allow functional assessment of complement activation. A positive signal is indicated by the detection of complement split product C4d, generated through alloantibody-mediated activation of the classical complement pathway. Early solid phase C4d studies correlated [C4d]FlowPRA+ sera with both intragraft C4d deposition and inferior kidney allograft survival at 3 years posttransplant.24,25 However, when the C4d assay was adapted to the more sensitive SAB platform, the ability of C4d+ DSA to predict outcomes was demonstrated in some,26,27 but not all studies.28,29 It was also pointed out that the dynamic range of signal output on C4d-SAB was rather limited compared with the later developed C1q test.6,30 Although early in the development and understanding of complement tests, Mizutani and Gotoh31 provided key evidence that C4d fixation only occurred in the presence of high titer antibodies, and that the ability to trigger complement activation was lost with dilution of test sera. The significance of this finding would be confirmed and realized in subsequent studies. Because the C4d assay was never broadly commercialized, detailed examination of this assay has remained limited to date and will not be further discussed.
The C1q Assay
In 2011, Chen et al19 published a detailed technical report describing their in-house development and validation of the C1q test. In the standard IgG-SAB Luminex assay, a fluorescent-labeled anti-human IgG antibody is used to detect binding of all IgG antibodies to HLA coated beads. Building on this platform, Chen et al, developed a C1q test that substitutes the IgG reporter antibody with an anti-C1q reagent to uncover a subset of microsphere-bound HLA antibodies that possess the functional capacity to fix C1q, the first component of the classical complement pathway. Given the amount of C1q binding is used as assay readout, standardization of complement concentration in the test serum is achieved by removing endogenous complement through heat treatment followed by spiking with a uniform concentration of purified human C1q.
In the initial study, 96 sera containing 2118 antibody specificities were tested in parallel using complement-dependent cytotoxicity (CDC), IgG-SAB, and C1q. All CDC+ antibodies screened positive when tested using C1q, whereas only a minority (19%) of C1q+ antibodies elicited CDC positivity. On this basis, the authors declared C1q to be a more sensitive and clinically relevant tool compared with CDC, although sera in this study were not correlated with biologic evidence of complement fixation in vivo. The study also suggested C1q positivity was independent of IgG-SAB MFI strength. Antibodies with IgG MFI as low as 1000 to 3000 were found to be complement-activating; conversely, many with MFI > 10 000 demonstrated no cytotoxic potential. Despite inadequate statistical power and resultant limitation in appropriate analyses to draw this conclusion, C1q was quickly heralded as a novel diagnostic assay that unlocked a new dimension to the risk assessment of HLA antibodies.
The C3d Assay
After C1q, the next complement dependent assay involves detection of complement split product C3d positioned downstream in the cascade (Figure 1). Unlike C1q which only measures the potential of antibodies to initiate the classical pathway, detection of C3d may be a more valid reflection of physiologic complement activation and perhaps more indicative of complement-mediated injury in the allograft. Additionally, given that the complement cascade amplifies as it moves downstream, measuring C3d may yield higher sensitivity as a readout for complement-fixation compared with C1q.
ABILITY OF C1Q TO PREDICT CLINICAL OUTCOMES IN KIDNEY TRANSPLANTATION
C1q in the Risk Assessment of Preformed DSA
The growing concern that IgG-SAB may inadvertently reduce access to transplantation through identification of less relevant antibodies was a major impetus driving the development of the C1q test. The potential for reduced donor access is of concern in highly sensitized recipients where the donor pool is already restricted, and in thoracic organ transplantation where pretransplant crossmatches are not always possible due to time limitation, and adjudication of organ acceptance is based on antibodies defined using IgG-SAB. In kidney transplantation, 2 studies specifically examined the utility of C1q in preformed DSA. The largest experience was reported by Otten et al,32 who compared the 10-year death-censored graft survival in 21 patients with pretransplant C1q+ DSA versus 318 patients with C1q− DSA. Acknowledging the small proportion of patients with C1q+ DSA in this cohort, the authors found no significant differences in graft survival between the 2 groups at any point after transplantation. A subsequent study by Crespo et al33 further verified this finding; additionally, they found that C1q positivity in preformed DSA did not correlate with intragraft C4d deposition, AMR, or T cell–mediated rejection (TCR). In a large multicenter study, Loupy et al34 found that the presence of any pretransplant DSA (hazard ratio, 3.53) performed as well as C1q+ DSA (hazard ratio, 2.95) in predicting posttransplant kidney allograft failure. In contrast, a significantly higher risk of graft loss was observed in DSA that converted from C1q− at day 0 to C1q+ posttransplant compared with those that switched from C1q-binding at day 0 to C1q− posttransplant. Together, these studies suggest that the C1q test cannot accurately discriminate preformed antibodies for future posttransplant outcomes distant to the test. It is also important to note that unlike some of the earlier C1q studies that used an in-house preparation, both Otten and Crespo using the later commercialized C1qScreen (One Lambda, Inc.), described a strong relationship between the MFI of antibodies and their C1q status.
C1q in the Risk Assessment of De Novo DSA
Early studies evaluating the performance of in-house C1q19,30,35 demonstrated the association of de novo C1q+ DSA with poor clinical and histopathologic outcomes. In a pediatric cohort of 193 kidney recipients, Sutherland et al35 found C1q+ DSA to be significantly correlated with biopsy C4d staining and acute rejection compared with noncomplement-binding DSA. Furthermore, patients with C1q+ DSA were almost 6 times more likely to suffer graft loss compared with their C1q− counterparts. Another study by Yabu et al30 found the presence of de novo C1q+ DSA to be specific for the development of transplant glomerulopathy (TG), although the assay did not predict death-censored graft survival.
After commercialization of the C1q test by One Lambda Inc. (C1qScreen), the assay was investigated under various study designs to predict outcomes in kidney transplantation. In the largest C1q study to date, Loupy et al34 analyzed sera obtained from 1016 kidney recipients at day 0, 1 year posttransplant, and during acute rejection. Among those with circulating anti-donor HLA antibodies, 77 patients (24%) had C1q-binding DSA while 239 (76%) had non–C1q-binding DSA. Overall, the ability to fix complement was significantly associated with a lower estimated glomerular filtration rate at 1 year (42 ± 22 vs 51 ± 20 ml/min/1.73 m2), and inferior graft survival at 5 years posttransplant (54% vs 93%). Compared with noncomplement-binding antibodies, C1q+ DSA had a higher risk for AMR, which was correlated with a more severe histological phenotype defined by increased Banff scores for microvascular inflammation, biopsy C4d deposition, and TG. An important finding here was that C1q-binding DSA were associated with poor outcomes independent of antibody strength using an arbitrary MFI cutoff of 6000, although it was later pointed out that patient sera in this study were not treated to eliminate complement interference (prozone effect), which has subsequently been shown to affect the true depiction of antibody strength that is not accurately reflected by MFI values.13,14
Findings from Loupy's work were further corroborated by others in more recent studies. Focusing on the most prevalent de novo DSA posttransplant, Freitas et al36 observed that patients with C1q-binding DQ DSA had a 30% lower allograft survival at 5 years posttransplant. In a large Japanese study, Yamamoto et al37 found C1q positivity to be a major risk factor for the development of subclinical AMR, although they also emphasized that C1q activity was significantly associated with IgG MFI values. In pediatric renal transplantation, Fichtner et al38 aimed to determine the prognostic value of C1q positivity in patients undergoing late (≥1 year posttransplant) indication biopsy. Although the assay did not predict TG on biopsy, C1q positivity was strongly associated with chronic active AMR and inferior graft survival at 4 years postbiopsy. Like other studies, the authors also found a significant association between IgG MFI strength and C1q activity, with both the MFI of the immunodominant donor-specific-bead and the sum-MFI of all DSAs yielding a strong predictive power for C1q positivity.
Not all studies confirmed the benefit of C1q in risk-stratifying de novo DSA. In a select cohort of kidney recipients with TG, Messina et al39 found that the majority of TG-associated DSA were in fact non-C1q fixing. In a pediatric kidney cohort of 82 patients, de novo complement-binding DSA were not predictive of humoral rejection, graft dysfunction or failure.40 In a more recent and comprehensive evaluation, Wiebe et al41 discovered that although C1q positivity was associated with a nonsignificant trend toward death-censored graft survival, the C1q status of de novo DSA did not predict graft survival after adjustment for nonadherence and clinical dysfunction, highlighting the critical importance of considering laboratory data in the multifactorial complexity of the patient condition. Importantly, C1q positivity was found to have poor sensitivity and specificity for predicting cellular rejection (0.35 and 0.80, respectively) and AMR (0.31 and 0.82, respectively). These findings suggest that the assay is imperfect in the prognostication of graft survival and cannot be used reliably to inform the selection of patients to undergo kidney biopsy at the time DSA detection.
C3d in the Risk Assessment of DSA
As previously discussed, C3d may offer several technical advantages including greater physiologic validity, over the C1q assay in evaluating complement-fixation in the solid phase system. In an adult kidney cohort of 69 patients, detection of circulating C3d-binding DSA at the time of AMR diagnosis was better correlated with allograft loss compared with C1q.20 In a pediatric study, the C3d-binding status of de novo DSA also produced superior test performance compared with C1q in predicting graft failure.42 In keeping with most C1q publications, recent C3d studies all demonstrated a tight correlation between the IgG MFI of DSA and their ability to trigger C3d deposition.20,42 Despite this, Lan et al43 found that even when restricting analysis to DSA with MFI < 10 000 and adjusting for the prozone effect, C3d-binding DSA were still associated with inferior graft survival compared with non–C3d-binding DSA. Interestingly, almost 30% of C3d− DSA were unexpectedly positive for biopsy C4d staining, which appeared to be related to the number of DSA detected. Although this observation could simply be the result of variability in test sensitivity and specificity between C3d and intragraft C4d staining, the possibility of adjacent noncomplement-fixing DSA synergizing to elicit complement activation on graft endothelium requires further evaluation. In previous experimental models, binding of 2 poly-specific antibodies to distinct epitopes on the same HLA molecule produced synergistic complement activity beyond the effect attributed to the amount of antibody binding alone.44,45 This cooperative mechanism is poorly defined in complement dependent assays, and may in part explain why certain low MFI HLA antibodies have the capacity to induce complement fixation in solid phase tests. Whether a similar process could occur between antibodies binding to adjacent HLA antigens located in steric proximity in the physiologic environment warrants further attention, as this information may shed light on the ability (or lack of) of solid phase diagnostics to predict complement fixation in vivo (Figure 2).
EVALUATING THE DETERMINANTS OF COMPLEMENT ACTIVATION
After critically reviewing the existing body of literature, a detectable, but imperfect relationship is found between solid phase complement activity and the ability to predict outcomes (Table 1). Although heterogeneity in study design, patient population, definition of assay threshold for positivity, and outcome measures of interest undoubtedly play an important role in diverging study conclusions, we postulate that most these inconsistencies may be better explained by taking a closer look at the biological mechanisms which dictate complement activation.
The literature suggests that factors both intrinsic to the serum (antibody titer, avidity, subclass, glycosylation) and extrinsic in the host (antigen/epitope expression and density, local inflammatory environment, complement concentration) regulate complement activation.46 Although many elements including antibody titer and IgG subclass are presently being evaluated as independent predictors of outcomes and are discussed in greater detail in other sections, they will be briefly mentioned here to enhance our understanding of what complement diagnostics measure in the solid phase system.
Most C1q and C3d studies show that a positive signal in both assays is strongly dependent on antibodies’ strength as estimated by IgG MFI. This is especially true when analyzed sera are pretreated with EDTA or serial dilution to eliminate complement interference, which complicates accurate assessment of antibodies’ quantitative strength.9,13,14,41,47,48 In a study by Wiebe et al,41 an EDTA-treated IgG MFI threshold of 10,126 predicted C1q positivity with impressive sensitivity and specificity (100% and 99%, respectively). Supporting this finding, mechanistic evidence revealed that effective complement activation requires a threshold density of IgG molecules to be reached on the cell surface, thereby initiating formation of a stable IgG-hexameric arrangement to permit high affinity interaction with C1q.49 In an elegant series of experiments, Yell et al50 showed that the C1q status of antibodies could be manipulated by changing the concentration of DSA found in sensitized patients, confirming and extending the earliest solid phase strength versus complement activation data.31 Specifically, C1q+ DSA were rendered C1q negative when antibodies were diluted to MFI values comparable to those found in C1q− DSA. Conversely, by concentrating C1q− DSA to achieve an MFI level similar to that observed in C1q+ DSA, noncomplement-fixing sera became C1q positive. Together, these findings suggest that antibody strength is the principal determinant of complement activation (at least in solid phase), and that results of complement assays depend chiefly on the quantity of antibody binding to antigen-coated microspheres, rather than true functional differences between unique antibody molecules. Indeed, for all antibodies of unique specificity detected in a serum, the occurrence of isolated weak/noncomplement-binding HLA DSA is rare, estimated to be in the range of 1% to 5% per 4 clinical studies.51-54 Another study also revealed that complement-activating IgG1 and IgG3 subclasses were the major IgG subtypes found in sensitized patients.51 In fact, 93% of C1q− DSA were found to contain strong complement-fixing IgG1/3 subclasses. Besides complement activity, inferences regarding the pathogenicity of IgG subclasses should also consider their differential affinity for Fcγ receptors (FcγR) expressed on immune cells. Compared with IgG1/3, IgG2/4 exhibit significantly weaker affinity for activating FcγRs that mediate leukocyte recruitment, phagocytosis and cell-mediated cytotoxicity, and IgG4 has enhanced affinity for the inhibitory FcγRIIB receptor relative to other subclasses.46,55
Current Gaps and Limitations of Complement Dependent Assays
In an effort to elucidate the mechanisms which underlie the CYNAP (CDC negative, adsorptive positive) phenomenon and the antiglobulin-augmented lymphocytotoxicity procedure, Rodey’s group44 developed the first complement analytical assay which quantitated C1q interaction with HLA antibodies bound to lymphocyte surface via flow cytometry. In contrast to early work which sought to dissect and tease out the major factors which govern complement activation by using cellular cytotoxicity as the readout,44,45 modern solid phase complement fixation tests have largely relied on clinical endpoints, such as graft loss and rejection as the outcome of interest, which are clearly influenced by a myriad of external elements (ie, immunosuppression, nonadherence, viral infection, donor-recipient epitope compatibility) that are unrelated to the physiologic characteristic that the assays supposedly measure: the level of complement activation. Within this context, the biologic significance of complement-fixing antibodies as identified by these tests remains unknown; instead, these tests are best viewed as biomarkers which predict certain graft outcomes. Accordingly, it is essential that they go through rigorous assay sensitivity/specificity evaluation, test standardization, internal validation, and external multi-center cohort validation before adoption into routine practice.56 Importantly, clinical studies that evaluate these tests must also consider and adjust for patient data and serum factors (MFI, complement interference) which may confound the interpretation of assay results with respect to outcomes. Until recently, adequately powered studies with appropriate statistical modeling of covariates have been lacking, further limiting robust conclusions to be drawn regarding the incremental benefit of these tests in clinical transplantation.
Whether in vivo complement activity could be truly represented and detected in the solid phase system will require correlation of assay data with either cell-based or intragraft evidence of complement activation. In previous experimental work, antigen-antibody interaction and synergy between poly-specific antibodies binding in steric proximity emerged as the most important determinants of complement-mediated cell lysis.44,45 Although these complex interactions being orchestrated on cell surface cannot be fully mimicked on antigen-coated beads, similar experimental approaches may be applied to solid phase assays, with the goal of defining the functional limitations of these tests and providing insights to rationalize discrepant results in the literature.
Perspective on the Clinical Utility of Complement Dependent Assays
The development of novel complement assessment assays has significantly advanced our understanding of the critical factors that govern complement activation. Although most studies associate complement-binding de novo DSA with clinically relevant endpoints, emerging data suggest that antibody quantity is the dominant force driving the observed correlations, and that this information may be available through careful interpretation of the standard IgG-SAB platform. The clinical utility of C1q/C3d in posttransplant antibody monitoring, especially after factoring in the added reagent/labor cost and time-around-time, must take into consideration the ability to infer the same conclusion from other data available. Nevertheless, it is important to point out that complement dependent assays seem to discriminate the pathogenicity of certain low-moderate range MFI antibodies, which appear to depend on factors other than antibody strength (at least to the extent that antibody strength can be reliably estimated on current SAB platforms) in mediating complement activation. However, the occurrence of these antibodies is relatively infrequent; whether the added cost of complement assays justifies this indication will depend on the specific clinical scenario and communication between the treating physician and the HLA laboratory.
Perhaps, the more relevant clinical question is whether different treatment options should be offered based on results of complement diagnostic assays. An argument could be made that irrespective of antibody strength, complement-fixing DSA are associated with poor outcomes and therefore should be treated with more aggressive immunomodulation compared with noncomplement-fixing antibodies. Before succumbing to this line of reasoning, it is important to remember that, as with all antibody or immune system testing, even the most sophisticated method of evaluation can only provide a snapshot appraisal of a patient’s immunological risk at a single time point. When interpreting results of complement dependent assays, it is especially critical to consider the history and longitudinal trend of an antibody’s reactivity. Given the strong dependency of complement activation on antibody strength, weak noncomplement-binding DSA may quickly become complement-activating in the span of even only a few days if a strong memory response is mounted. Indeed, a recent study that examined the kinetics of DSA found that progressive acquisition of DSA’s ability to activate complement was associated with poor outcomes.42 Although outside the scope of this review, a body of literature also supports complement-independent mechanisms of chronic allograft injury.21 Notably, a recent study involving nonsensitized kidney recipients found that the presence of C1q+ DSA was predictive of a rapid progression to graft loss; however, the persistence of C1q− DSA over time also led to inferior graft survival.57
Importantly, when considering the broad spectrum of antibody-mediated paths to adverse outcome, we must remember that complement activation is not the sole pathway of injury. In a series of important experimental studies, Reed’s group and others demonstrated ligation of Class I HLA molecules with antibodies can lead to: (1) partnering with integrin β4 to trigger an intricate intracellular cascade involving focal adhesion kinase, mammalian target of rapamycin, S6 ribosomal protein, and extracellular regulated kinases (ERK1/2), resulting in proliferation and migration of endothelial cells21,22,46,58; (2) release of von Willebrand factor and expression of p-selectin to recruit leukocytes59; and (3) activation of NK cells,60 all of which culminate in endothelial injury in the absence of requirement for complement. Given this mechanistic potential, it would be premature to assume that the detection of DSA, even those of IgG 2/453 that do not bind complement cannot be deleterious, and we posit that the over simplicity of these dichotomous associations is a limitation of our current ability to detect, define and classify the true clinicopathologic spectrum of antibody-mediated injury.
In conclusion, antibody strength appears to be the principal determinant of HLA DSA’s complement binding capacity as detected by any of the assays currently available. Accurate risk assessment of HLA antibodies should continue to integrate different antibody characteristics, the clinical history, and results of complementary assays, while paying special attention to the limitations of the methods utilized and the importance of repeated testing over time. Overreliance on complement dependent assays at a single time point may underestimate the pathogenic potential of DSA in both complement and non–complement-mediated pathways and miss the important window of opportunity for treatment.
The authors thank Dr. Nicole Valenzuela for her artistic contributions to the figures used in this article.
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