Soluble C5b-9 as a Biomarker for Complement Activation in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a rare renal disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. It is caused by abnormal activation of the complement pathway at the cell-surface level.1Legendre C.M. Licht C. Muus P. et al.Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome.N Engl J Med. 2013; 368: 2169-2181Crossref PubMed Scopus (1019) Google Scholar A soluble C5b-9 (sC5b-9) ELISA can be used to monitor activity of the terminal pathway of complement, although discrepant results in 3 recent publications call into question the clinical value of this assay in aHUS. Noris et al2Noris M. Galbusera M. Gastoldi S. et al.Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.Blood. 2014; 124: 1715-1726Crossref PubMed Scopus (226) Google Scholar reported that sC5b-9 is unsuitable as a biomarker of complement activation in aHUS because they found: (1) sC5b-9 levels were elevated in only 10 of 19 patients in the acute phase of disease, (2) 23 of 36 patients in remission continued to have elevated sC5b-9 levels, and (3) sC5b-9 levels did not change in 8 patients receiving eculizumab. By contrast, Volokhina et al3Volokhina EB, Westra D, van der Velden TJ, van de Kar NC, Mollnes TE, van den Heuvel LP. Complement activation patterns in atypical hemolytic uremic syndrome during acute phase and in remission [published online ahead of print July 31, 2014]. Clin Exp Immunol. http://dx.doi.org/10.1111/cei.12426.Google Scholar reported that all patients in their cohort in the acute phase of disease (n = 6) had significantly elevated sC5b-9 levels, while all patients in remission (n = 11) had sC5b-9 levels comparable to healthy controls. Similarly, Cataland et al4Cataland S.R. Holers V.M. Geyer S. Yang S. Wu H.M. Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP.Blood. 2014; 123: 3733-3738Crossref PubMed Scopus (113) Google Scholar found elevated sC5b-9 in all patients with acute disease (n = 19). Both Volokhina and Cataland advocated sC5b-9 as a helpful biomarker to confirm the clinical diagnosis of aHUS. These discrepant conclusions prompted us to evaluate our aHUS cohort. Patients were included if they had a clinical diagnosis of aHUS, which consisted of the presence of nonimmune hemolysis, elevated lactic acid dehydrogenase, and shistocytes on peripheral smear (n = 86). Two patients were then excluded: one with glucose-6-phosphate dehydrogenase deficiency and another given eculizumab prior to sC5b-9 testing. We differentiated active (n = 55) from inactive (n = 29) disease by the presence of both hemolytic anemia (hemoglobin < 10 g/L) and thrombocytopenia (platelets < 150,000/μL) in the former; ADAMST-13 activity was >10% in all patients. Demographic data are shown in Table 1. Like Noris et al2Noris M. Galbusera M. Gastoldi S. et al.Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.Blood. 2014; 124: 1715-1726Crossref PubMed Scopus (226) Google Scholar and Cataland et al,4Cataland S.R. Holers V.M. Geyer S. Yang S. Wu H.M. Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP.Blood. 2014; 123: 3733-3738Crossref PubMed Scopus (113) Google Scholar we measured sC5b-9 plasma using the commercially available ELISA kit from Quidel Corp (MicroVue SC5b-9 Plus).Table 1Summary of Enrolled PatientsActive (n = 55)Inactive (n = 29)M:F21:3414:15Mean age (y)23.115.9Mean platelet count (/μL)82,360276,652Low C3 cases53%33%Low C4 cases21%0%Genetic screen: positive/tested casesaGenetic screen of CFH, CD46, C3, CFI, CFB, and THBD. No significant difference in positive rates between the 2 groups (Fisher exact P=0.3).14/5211/29Homozygous deletion in CFHR3-CFHR1/tested cases4/514/28CFH autoantibody: positive/tested casesbNo significant difference between active and inactive groups (Fisher exact P=0.9). There is no overlap between genetic-positive cases and autoantibody-positive cases.3/543/27a Genetic screen of CFH, CD46, C3, CFI, CFB, and THBD. No significant difference in positive rates between the 2 groups (Fisher exact P = 0.3).b No significant difference between active and inactive groups (Fisher exact P = 0.9). There is no overlap between genetic-positive cases and autoantibody-positive cases. Open table in a new tab In our series, 24 of 55 active cases and 3 of 29 inactive cases had elevated sC5b-9 levels (P = 0.003 by Fisher exact test; Fig 1). Mean sC5b-9 values were 289 ± 120 (SD) and 191 ± 112 ng/mL (active and inactive, respectively; P < 0.001 by Wilcoxon-Mann-Whitney test). Our data suggest that elevated sC5b-9 levels are indicative of active disease (positive predictive value, 89%) but should be used in the context of other complement assays (negative predictive value, 46%). Figure 1 also highlights the inconsistent readouts of sC5b-9 across the studies, which likely arise for multiple reasons. First (and likely key) are the enrollment criteria for aHUS, which had varied inclusion and exclusion criteria. For instance, to identify acute cases, Cataland et al4Cataland S.R. Holers V.M. Geyer S. Yang S. Wu H.M. Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP.Blood. 2014; 123: 3733-3738Crossref PubMed Scopus (113) Google Scholar used platelets < 100,000/μL, serum creatinine > 2.25 mg/dL, and ADAMTS-13 activity > 10%, while Noris et al2Noris M. Galbusera M. Gastoldi S. et al.Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.Blood. 2014; 124: 1715-1726Crossref PubMed Scopus (226) Google Scholar used platelets < 150,000/μL, serum creatinine > 1.3 mg/dL (for adults) or >0.5 to 0.8 mg/dL (for children), and ADAMTS-13 activity of 50% to 150%. Volokhina et al3Volokhina EB, Westra D, van der Velden TJ, van de Kar NC, Mollnes TE, van den Heuvel LP. Complement activation patterns in atypical hemolytic uremic syndrome during acute phase and in remission [published online ahead of print July 31, 2014]. Clin Exp Immunol. http://dx.doi.org/10.1111/cei.12426.Google Scholar defined acute-phase disease as the presence of hemolytic anemia, thrombocytopenia, and acute kidney injury, whereas we used hemolytic anemia, platelets < 150,000/μL, and ADAMST-13 activity > 10%. Second, specimen collection and processing likely affect results. Although sC5b-9 is believed to be relatively stable,3Volokhina EB, Westra D, van der Velden TJ, van de Kar NC, Mollnes TE, van den Heuvel LP. Complement activation patterns in atypical hemolytic uremic syndrome during acute phase and in remission [published online ahead of print July 31, 2014]. Clin Exp Immunol. http://dx.doi.org/10.1111/cei.12426.Google Scholar plasma preparation, long-term storage, and freeze-thaw cycles influence readouts. Third, sC5b-9 detection methods have minor differences. Although Noris et al,2Noris M. Galbusera M. Gastoldi S. et al.Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.Blood. 2014; 124: 1715-1726Crossref PubMed Scopus (226) Google Scholar Cataland et al,4Cataland S.R. Holers V.M. Geyer S. Yang S. Wu H.M. Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP.Blood. 2014; 123: 3733-3738Crossref PubMed Scopus (113) Google Scholar and our laboratory used the same ELISA kit, definitions of normal sC5b-9 levels differed (Noris 127-400 ng/mL; Cataland, 33.9-238.2 ng/mL; our laboratory, 50-300 ng/mL), suggesting protocol and reference sample variation among groups. Volokhina et al3Volokhina EB, Westra D, van der Velden TJ, van de Kar NC, Mollnes TE, van den Heuvel LP. Complement activation patterns in atypical hemolytic uremic syndrome during acute phase and in remission [published online ahead of print July 31, 2014]. Clin Exp Immunol. http://dx.doi.org/10.1111/cei.12426.Google Scholar used a different ELISA, another potential source of variability. For the clinician, the value of sC5b-9 as a biomarker of aHUS disease activity remains unresolved. A collaborative study is needed in which sC5b-9 is measured in a large aHUS cohort in different disease phases by different laboratories. Recruitment protocols (including the ability of sC5b-9 to differentiate aHUS from noncomplement thrombotic microangiopathies), plasma preparation, sC5b-9 measurement, and result interpretation should be optimized and standardized for interlaboratory consistency. In the meantime, the complement evaluation in each patient must be interpreted in the context of clinical history to determine disease status. We appreciate the patients and physicians providing data for this study and the support of the Foundation for Children With Atypical HUS. Support: This study was supported in part by a grant from the Foundation for Children With Atypical HUS. The funder did not play a role in this study. Financial Disclosure: The authors declare that they have no other relevant financial interests. Contributions: Research idea and study design: FB, NGB, RS; data acquisition: FB, NGB, NCM, YZ; data analysis/interpretation: FB, CT, CN, RJS; statistical analysis: FB, YZ; supervision or mentorship: RJS. Each author contributed important intellectual content during manuscript drafting or revision and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. RJS takes responsibility that this study has been reported honestly, accurately, and transparently; that no important aspects of the study have been omitted, and that any discrepancies from the study as planned have been explained.