Clinical Kidney Journal, 2021, vol. 14, no. 4, 1055–1066 doi: 10.1093/ckj/sfaa195 Advance Access Publication Date: 6 November 2020 CKJ Review CKJ R EV IEW Thrombotic microangiopathies assessment: mind the complement Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 Miquel Blasco 1,2,*, Elena Guillén1,*, Luis F. Quintana 1,2, Adriana Garcia-Herrera3, Gastón Pin~ eiro1,2, Esteban Poch 1,2, Enric Carreras4,5, Josep M. Campistol1,2, Maribel Diaz-Ricart5,6 and Marta Palomo4,5,6 1 Department of Nephrology and Kidney Transplantation, Hospital Clı́nic, Centro de Referencia en Enfermedad Glomerular Compleja del Sistema Nacional de Salud (CSUR), University of Barcelona, Barcelona, Spain, 2 Institute of Biomedical Research August Pi i Sunyer (IDIPABS), Malalties Nefro-Urològiques i Trasplantament Renal, Barcelona, Spain, 3Department of Pathology, Hospital Clı́nic, University of Barcelona, Barcelona, Spain, 4 Josep Carreras Leukaemia Research Institute, Hospital Clinic/University of Barcelona Campus, Barcelona, Spain, 5Barcelona Endothelium Team, Barcelona, Spain and 6Department of Pathology, Hematopathology Unit, Hospital Clı́nic of Barcelona, Biomedical Diagnosis Centre (CDB), Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain Correspondence to: Miquel Blasco; E-mail: miblasco@clinic.cat *These authors contributed equally to this work. ABSTRACT When faced with microangiopathic haemolytic anaemia, thrombocytopenia and organ dysfunction, clinicians should suspect thrombotic microangiopathy (TMA). The endothelial damage that leads to this histological lesion can be triggered by several conditions or diseases, hindering an early diagnosis and aetiological treatment. However, due to systemic involvement in TMA and its low incidence, an accurate early diagnosis is often troublesome. In the last few decades, major improvements have been made in the pathophysiological knowledge of TMAs such as thrombotic thrombocytopenic purpura [TTP, caused by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin Type 1 motif, member 13) deficiency] and atypical haemolytic uraemic syndrome (aHUS, associated with dysregulation of the alternative complement pathway), together with enhancements in patient management due to new diagnostic tools and treatments. However, diagnosis of aHUS requires the exclusion of all the other entities that can cause TMA, delaying the introduction of terminal complement blockers, which have shown high efficacy in haemolysis control and especially in avoiding organ damage if used early. Importantly, there is increasing evidence that other forms of TMA could present overactivation of the complement system, worsening their clinical progression. This review addresses the diagnostic and therapeutic approach when there is clinical suspicion of TMA, emphasizing complement evaluation as a potential tool for the inclusive diagnosis of aHUS, as well as for the improvement of current knowledge of its pathophysiological involvement in other TMAs. The development of both new complement activation biomarkers and inhibitory treatments will probably improve the management of TMA patients in the near future, reducing response times and improving patient outcomes. Received: 21.6.2020; Editorial decision: 31.8.2020 C The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1055 1056 | M. Blasco et al. Keywords: complement system, C5b-9 deposits, endothelial cells (ECs), membrane attack complex (C5b-9), soluble C5b-9, thrombotic microangiopathies INTRODUCTION primary TMA forms (Table 1). However, the classification of TMAs is challenging and constantly evolving [3, 13, 15]. On the Thrombotic microangiopathy (TMA) is a histological lesion com- basis of clinical findings, TTP often presents with ischaemia of mon to multiple diseases and with variable clinical presentation the brain or gastrointestinal tract, and rarely causes severe due to its systemic nature [1, 2]. Recently, advances have been acute kidney injury (AKI) [3]. Diagnosis of TTP relies on mea- made in the understanding of the pathophysiological mecha- surement of very low plasma ADAMTS-13 (a disintegrin and nisms involved [3, 4]. However, an accurate diagnosis and early metalloproteinase with a thrombospondin Type 1 motif, mem- start of aetiological treatment able to change the natural history ber 13) activity (<10%) [3]. However, when ADAMTS-13 testing is of these serious conditions may be challenging. In addition, the unavailable and urgent therapy is needed, the PLASMIC score development of new-generation functional and genetic studies can be used, which is a valuable clinical tool based on clinical has revealed an emerging role of the complement system—at Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 and laboratory findings [16]. If the predominant feature at pre- different levels and degrees—in the pathogenesis of many of sentation is severe AKI, then the disorder will most probably be the entities that can produce TMA [5–10]. classified as HUS. The most common form (90% of HUS)—typical Given the high complexity of the clinical management of HUS—is associated with bloody diarrhoea due to different patients with TMA, this review attempts to shed light on the di- pathogens, especially Escherichia coli. Atypical HUS (aHUS) is an agnostic process when there is clinical suspicion and/or histo- ultra-rare disease (incidence 0.5–2 per million per year), and it is logical confirmation of TMA, and stresses the need for complete based on a clinical diagnosis after the exclusion of other causes and comprehensive assessment of complement activation. of TMA. aHUS is caused by dysregulation of the alternative com- Complement status evaluation will not only improve our patho- plement pathway, of either acquired or genetic origin [13, 17, 18]. physiological knowledge of the different TMA forms but could TMAs classified as secondary forms can occur in multiple also have diagnostic and therapeutic implications. Due to the clinical settings (Table 1), among which the most important are: current development of multiple complement blockers [11, 12], autoimmune diseases; cancer and its treatment; pregnancy- these data could eventually allow inclusive diagnosis and per- related TMA; solid organ transplantation (SOT) and haemato- sonalized medicine. poietic cell transplantation (HCT); and multiple infections [13, 19]. CLINICAL SUSPICION Thrombotic microangiopathies are a group of disorders charac- HISTOLOGY terized by non-immune intravascular haemolysis and ischae- Because the histological findings of TMA are not pathogno- mic organ dysfunction. The classical laboratory findings are monic, histological confirmation is not mandatory to establish thrombocytopenia (or >25% decrease in platelet count), result- a diagnosis. Therefore, an aetiological diagnostic approach to ing from platelet aggregation and consumption, and microan- TMA can be initiated once suspicion is established. However, as giopathic haemolytic anaemia (MAHA), resulting from TMA can manifest as a diverse range symptoms and conditions, erythrocyte fragmentation in the microcirculation due to partial histological confirmation is useful, especially when the clinical occlusion by platelet aggregates [1, 13]. MAHA is defined by presentation is uncommon or incomplete, as well as to deter- the presence of schistocytes (fragmented erythrocytes) that can mine prognosis [2]. Several organs may be affected by TMA, and be found on peripheral blood smear, reticulocytosis and a nega- histopathological findings in all of them have been described in tive direct antiglobulin (Coombs) test. Other less specific labora- the literature. Classic findings of TMA consist of endothelial cell tory findings for MAHA are: increased serum lactate (EC) swelling, often associated with luminal fibrin thrombi, and dehydrogenase (LDH), as a result of tissue ischaemia and cell minimal or absent inflammation. Heart, lung and brain biopsies lysis; decreased haptoglobin levels, after its binding to circulat- have only been described in autopsies, whereas the skin, gastro- ing haemoglobin (Hb) realeased by haemolysis; and unconju- intestinal tract and gingiva are the suggested sites for sampling, gated hyperbilirubinaemia. The severity of all these findings whether or not there is a visible lesion [20–22]. Kidney tissue reflects the extent of microvascular platelet aggregation [14]. can also be sampled and remains the gold standard for kidney From a clinical point of view, most patients present with non- TMA diagnosis and to exclude other potential causes of kidney specific symptoms that are a result of microvasculature injury. TMA patients often present with high blood pressure and, in some Table 1. Primary and secondary TMA forms cases, with hypertensive retinopathy [2]. The results of coagulation tests [prothrombin time (PT) and activated partial thromboplastin Primary TMA Secondary TMA time (aPTT)] are usually normal [13]. However, patients with very TTP Autoimmune diseases severe disease associated with disseminated intravascular coagula- Hereditary TTP, acquired/ Infections tion (DIC) are the exception, as they frequently present with septic immune-mediated TTP Pregnancy shock and prolongation of PT and aPTT [13]. The most commonly HUS SOT affected targets in TMA are the brain, kidneys, heart, skin and gas- Acquired HUS: infectious causes HCT trointestinal system, although the clinical presentation can differ (STEC-HUS, S. pneumoniae, Shigella, Methylmalonic acidaemia depending on the underlying aetiology of the disease [2]. etc.); antibody against CFH (aHUS) Malignancy Classically, thrombotic thrombocytopenic purpura (TTP) and Hereditary HUS: complement gene Medications haemolytic uraemic syndrome (HUS), both entities occurring mutations (aHUS) Malignant hypertension with no associated underlying cause, have been classified as Thrombotic microangiopathies assessment | 1057 injury. However, the decision to perform a kidney biopsy must resulting in changes in different structures that progress over be individualized on a case-by-case basis, given that in patients time [23]. These changes can be categorized as below. with suspected TMA there is a high bleeding risk, due to the presence of high blood pressure, thrombocytopaenia and urae- Glomerular changes mia, among other factors. The histopathological findings in kid- ney biopsy reflect tissue response to endothelial injury, In the acute phases of TMA, ECs may appear swollen and fibrin- platelet thrombi are identified within glomerular capillaries Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 FIGURE 1: Pathological findings of renal biopsy in acute and chronic TMA. (A) A thrombus is identified within glomerular capillaries (arrow head) (Masson’s trichrome, 60). (B) Segmental mesangiolysis is indicative of active lesion (arrow head) (silver, 40). (C) Chronic lesions show reduplication of GBM (arrow head) (silver, 60). (D) Necrosis of the arterial wall (arrow head) and glomeruli of shrunken appearance (Masson’s trichrome, 20). (E) Artery show intimal thickening with concentric appearance (‘onion-skin’) (haematoxylin and eosin, 60) (F) Acute tubular necrosis (arrow head) is a finding resulting of ischemic changes (haematoxylin and eosin, 20). 1058 | M. Blasco et al. (Figure 1A). Other findings such us detachment of the ECs from administering early specific treatment. With the aim of improv- glomerular basement membranes (GBMs), mesangiolysis or ne- ing TMA management, we propose an algorithm describing the crosis can be observed (Figure 1B). If TMA persists, then ECs con- sequence of all the complementary tests that should be carried tinue to produce basement membrane material, leading to GBM out according to patient characteristics and clinical settings remodelling with double contours and mesangial expansion, (Figure 2). resulting in a membranoproliferative glomerulonephritis-like pattern indicative of chronic phases of TMA [24–26] (Figure 1C). TTP The cause of TPP is an ADAMTS-13 deficiency, which can be Vascular changes congenital or acquired. This deficiency results in unusually When the arterioles and arteries are involved, intimal swelling, large von Willebrand factor (VWF) multimers and, therefore, a mucoid change and proliferation, and necrosis of the arterial high risk of platelet thrombi in small vessels. Measurement of wall or thrombi in the lumen are observed (Figure 1D). In this ADAMTS-13 activity and inhibitors is crucial to confirm the di- context, glomeruli appear ischaemic and shrunken. In the agnosis of hereditary and autoimmune TTP, but there are still chronic phase, these changes may lead to concentric lamination uncertainties about the interpretation of these data. Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 of intimal fibrosis causing ‘onion-skin’ appearance [24, 26] Importantly, ADAMTS-13 activity is usually normal or modestly (Figures 1E). reduced (>20%) in pregnancy [27] and other forms of TMA asso- ciated with severe sepsis, HCT, systemic lupus erythematosus Tubulointerstitial changes (SLE), disseminated malignancy, recent surgery, severe liver im- Acute tubular necrosis is a finding resulting from ischaemic pairment and DIC [28–30]. Partial ADAMTS-13 deficiency (be- changes. Severe TMA cases can show cortical necrosis, which is tween 10% and 60% of activity) was found in 50% of aHUS predictive of a poor outcome [25, 26] (Figure 1F). patients and Shiga toxin (Stx)-producing Escherichia coli HUS (Stx E. coli HUS; STEC-HUS) [31]. The commercially available techni- ques to measure ADAMTS-13 activity, autoantibodies and anti- DIFFERENTIAL DIAGNOSIS gen (Ag) differ, depending on the laboratories. Regarding When there is clinical suspicion and/or histological confirma- activity, fluorogenic assays based on the use of a peptide con- tion of TMA, a broad and rapid differential diagnosis must be taining 73 amino acid residues from the central A2 domain of made to achieve an aetiological diagnosis and reduce delay in VWF (FRETS-VWF73) as a substrate, conjugated with FIGURE 2: TMA management algorithm. When there are TMA signs, clinical and laboratory evaluation is needed to achieve an aetiological diagnosis and specific treat- ment. ADAMTS-13 activity assessment is urgent, and if <10%, a TTP should be diagnosed, followed by specific treatment. If Stx is detected, then diagnosis of STEC-HUS should be followed by supportive therapy. This supportive therapy, among early start of aetiological treatment, follows the differential diagnosis of all secondary TMA forms. When aHUS is suspected (by exclusion of other TMA forms), a complete evaluation of complement should be performed, eculizumab being the first-line treat- ment. Complement assessment though both in vitro evaluation of C5b-9 deposits on ECs and soluble C5b-9 levels is strongly encouraged in all cases of TMA, when pos- sible. Especially in secondary forms, where if the patient shows complement overactivation and no response to the treatment of choice, a second-line treatment consisting of administration of complement terminal blockers may be contemplated. If complement assessment is not available—in secondary TMA forms—a thera- peutic approach with eculizumab may be also considered (in the absence of response to aetiological treatment).ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin Type 1 motif, member 13; AF-ab, Anti-phospholipid antibodies; HAV, hepatitis A virus; HBV, hepatitis B virus; HCV, hepatitis C virus; CMV, cytomeg- alovirus; EBV, Epstein–Barr virus; HIV, human immunodeficiency virus. Thrombotic microangiopathies assessment | 1059 fluorescence energy resonance transfer (FRETS), offers quick on the clinical suspicion, different diagnostic tests should be and reproducible results [32]. The International Society of performed (serological tests, qPCR, cultures, etc.) [41]. Thrombosis and Haemostasis guidelines establish that when plasma ADAMTS-13 activity is <10 IU/dL (often referred to as Pregnancy-associated TMA 10% of normal ADAMTS-13 activity), the diagnosis of TTP is There are different circumstances in which TMA can occur dur- more likely [33]. Autoantibodies against ADAMTS-13, usually ing pregnancy. Haemolysis, Elevated Liver enzymes and Low immunoglobulin G, are the cause of acquired TTP, a rare auto- Platelets (HELLP) syndrome, which usually occurs after the 20th immune disease [34]. These antibodies show inhibitory activity week of pregnancy, is defined by the coexistence of thrombocy- primarily mediated by their binding to ADAMTS-13 spacer do- topenia, MAHA and liver damage. It can be associated with pre- main, critical for VWF recognition. However, there are also anti- eclampsia, defined by new-onset hypertension and proteinuria, bodies that may cause rapid clearance of ADAMTS-13. Regarding ADAMTS-13 Ag, the concentrations of this enzyme in with or without end-organ dysfunction. If seizures occur in a human plasma range from 0.7 to 1.4 lg/mL (3.5–7.0 nM) [35] and patient with preeclampsia, then the diagnosis changes to can be severely reduced or even non-existent in congenital TTP. eclampsia [42]. Moreover, pregnancy may trigger new-onset TTP This very rare condition, with a prevalence of 0.05–0.4/100 000, or a relapse, especially in the first trimester [8]. Additionally, in Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 is transmitted by autosomal recessive inheritance [36]. up to 20% of women, onset of aHUS occurs during the post- partum period [43–45]. In this case, it is hypothesized that there are different complement system regulatory proteins on the STEC-HUS placental surface, and that their loss in conjunction with com- The most common cause of HUS, particularly in children, is Stx- plement overactivation during delivery may be a perfect trigger producing bacteria, especially E. coli serotype O157:H7 [37]. [46]. STEC-HUS is usually initiated a few days after clinical gastroen- teritis (typically bloody diarrhoea) caused by STEC. Stx, a potent SOT and HCT cytotoxin, apparently initiates the disease process, playing a central pathophysiological role damaging ECs from the vascula- Aetiological diagnosis is challenging because TMA may be asso- ture [6], among red cells and platelets [18]. This damage could ciated with several triggers, such as ischaemia–reperfusion in- constitute triggers leading to enhanced complement activation. jury and antibody-mediated rejection in the case of SOT, or The clinical manifestations of Stx-induced HUS overlap with induction treatment and graft-versus-host prevention regimens those of other related syndromes, but the molecular mecha- in HCT. Opportunistic infections and immunosuppressive treat- nisms differ considerably. Accurate diagnosis of STEC infections ment, mainly related to calcineurin and mammalian Target of is important for the appropriate management of infected Rapamycin (m-TOR) inhibitors, could be the cause of TMA in patients. Candidates for the detection of all STEC serotypes in- both transplantation modalities. TMA associated with HCT is clude chromogenic agars, enzyme immunoassays, quantitative especially recognized to be associated with excessive comple- real-time polymerase chain reaction (qPCR) and, most recently, ment activation, likely triggered by endothelial injury, and new methods of whole-genome analysis [38]. evidence suggests neutrophil extracellular trap (NETs) forma- tion as the mechanistic link between these two entities [47]. NETs are deposits of antimicrobial proteins on a single DNA Autoimmune diseases framework released by activated neutrophils [48], and can acti- The main systemic disorders related to TMA are systemic vate complement via both alternative and non-alternative path- scleroderma (SSc) and SLE, with or without associated antiphos- ways [49]. Levels of NETs are measured through quantification pholipid syndrome (APS). Their diagnosis requires the perfor- of circulating ds-DNA in a quick and easy assay, and could mance of a complete clinical assessment and autoimmune eventually constitute a new marker to be considered in the di- panel, including: serum antinuclear antibodies (ANA), anti- agnosis of TA-TMA. double-stranded DNA (ds-DNA) antibodies (using Crithidia luci- liae indirect immunofluorescence test) and anti-extractable Methylmalonic acidaemia and/or aciduria nuclear Ag antibodies, including anti-Sm, anti-SSA, anti-SSB and anti-RNP antibodies (the latter associated with overlap con- This is an uncommon and heterogeneous congenital metabolic nective tissue diseases), as well as serum complement factors disorder of cobalamin (vitamin B12), resulting in methylmalonic (C3, C4 and CH50) for SLE diagnosis; antitopoisomerase I (anti- acid and homocysteine accumulation in blood and tissues, with Scl-70) antibody, anticentromere antibody (ACA) and anti-RNA increased urinary excretion of both compounds. In this setting, polymerase III antibodies for SSc diagnosis; and anti-cardiolipin endothelial damage is caused by high homocysteine levels, antibody, anti-b2GP-1 antibody and lupus anticoagulant (LA) for among other factors. Confirmatory genetic testing should be APS diagnosis [39]. performed in patients with positive metabolic screening [50]. Infections Malignancy Many systemic infections have been identified as a TMA- If there is any symptomatic suggestion of malignancy or a his- causing agent [40]. Among them, the most important ones are: tory of cancer in the context of TMA, it is imperative to perform numerous viral infections (outlined in Figure 2); bacterial infec- diagnostic tests (CT scan, bone marrow study, serum tumour tions such as bacterial endocarditis, spotted fever rickettsiosis markers). The evaluation of TMA in patients with cancer fo- or infections caused by Streptococcus pneumoniae (the latter cuses on two principal aetiologies: (i) cancer-induced TMA, shows a positive Coombs test as a differentiating feature); and commonly caused by systemic metastases due to microvascular infections caused by red blood cell parasites (Plasmodium falcipa- obstruction by tumour cells; and (ii) chemotherapy-induced rum, Plasmodium vivax). In each particular case, and depending TMA, caused by dose-dependent toxicity (with gradual onset of 1060 | M. Blasco et al. TMA signs), or by an immune-mediated reaction (drug-depen- treatment required. If, after optimal blood pressure control, dent antibodies), the latter leading to rapid onset of TMA [51]. there is no improvement in platelet count, other causes of TMA must be considered [2]. Medications In most cases, TMA is caused by dose-dependent direct en- dothelial damage (non-immune-mechanism). The most frequent forms of drug-induced TMA are summarized in Table 2 [52]. aHUS This entity is caused by a dysregulation of alternative comple- Malignant hypertension ment pathway over cell surfaces [presence of complement pro- A direct damage of the endothelium is produced in this entity. teins mutations or antibodies against complement factor Ocular fundus study may show hypertensive retinopathy H (CFH)], damaging ECs through complement terminal phase Grades III–IV. In these patients, optimal blood pressure control activation/membrane attack complex (MAC) formation [19, 53]. Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 is the most critical initial management and may be the only aHUS diagnosis is by exclusion of other TMA forms. Table 2. Main drug-induced TMA Cancer therapies Immunosuppressants Others Cytotoxic agents: Calcineurin inhibitors: Biological agents: gemcitabine, mytomicin C cyclosporine, tacrolimus adalimumab, imatinib VEGF inhibitors: m-TOR inhibitors: Antibiotics: bevacizumab, sunitinib sirolimus, everolimus ciprofloxacin, metronidazole, nitrofurantoin, trimethoprim/sulfamethoxazole Proteasome inhibitors: Commonly used drugs: ibuprofen, ketorolac, clopidogrel, simvastatin, oestrogen/ bortezomib, carfilzomib progesterone FIGURE 3: Complement system. The complement system is the first line of physical defence against pathogens and altered host cells and is composed of a cascade of several plasmatic and membrane proteins expressed on the cell surface. There are three different pathways through which the complement system can be activated: the classical, the lectin and the alternative pathway (upper part of the image). Each of the pathways leads to the activation of C3 convertase that cleaves C3 into C3a and C3b, which binds Factor B and generates active convertases on targeted surfaces. The terminal complement cascade is then initiated by the cleavage of protein C5 by C5 convertase, followed by C5a release (potent anaphylotoxin) and C5b-9 molecule assembly, forming the MAC. This structure becomes inserted into the target cell lipid bilayer and induces cell lysis through proton influx. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, com- plement regulatory proteins or complement deficiencies, whether genetic or acquired. Soluble C5b-9 levels and C5b-9 deposition induced by patient activated plasma on ECs in vitro are two of the most promising complement monitoring tools used to provide insight into the integrity of the entire complement reaction cascade. Thrombotic microangiopathies assessment | 1061 COMPLEMENT ASSESSMENT IN TMA alternative therapeutic process when there is a lack of response to initial treatment in other forms of TMA [10, 61–63] (Figure 2). Complement is a part of the innate immune surveillance sys- tem (Figure 3), its main functions being chemotaxis, opsoniza- tion, clearance of immunocomplexes and cell lysis through Functional analysis MAC formation. Complement dysregulation has been linked to Complement functional analyses provide insight into the integ- several forms of TMA [5–10]. Therefore, we suggest a study of rity of the entire complement reaction cascade. These tests are complement in all TMA patients, immediately after diagnosis suitable for investigating suspected complement deficiencies and before any specific treatment (Figure 2). Among all the labo- and must be interpreted in the context of the complete comple- ratory tests for complement assessment (discussed below), we ment picture. However, there is still an open discussion about strongly recommend, when possible, analysis of C5b-9 deposition the utility of complement functional tests as biomarkers of com- on ECs in culture and quantification of soluble C5b-9 levels as plement activation in both TMA debut and response to therapy. useful markers of terminal complement pathway overactivation Complement assays are performed on serum or plasma samples. [54–60] (Figure 3). Although this assessment is not a quick proce- It is important to maintain the samples on ice in order to limit dure, and is not commercially available, knowledge of comple- the amount of ongoing tick-over. Functional complement activity Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 ment involvement may critically reduce diagnostic times when assays are used as screening tests and are based on the ability of aHUS is strongly suspected (Figure 4), and could offer an complement to induce lysis of erythrocytes. FIGURE 4: Clinical case—complement assessment in TMA for early aetiological treatment. A 52-year-old man, with a past medical history of pancreatic ductal adeno- carcinoma (cT1cN0M0), was treated with surgery and concomitant gemcitabine from March to August 2017. After 2 months, he showed nephrotic syndrome, MAHA, thrombocytopenia and progressive kidney failure. A kidney biopsy confirmed TMA both in the glomerulus and arterioles with negative immunofluorescence. To make a rapid differential diagnosis, we ruled out TTP (ADAMTS-13 activity: 87%), STEC-HUS (negative Stx detection), infections (negative cultures) and autoimmune diseases, among other causes of TMA. Concomitantly, we performed a complement assessment with plasma levels of complement components (C3, C4, CH50 and C5b-9) and also with C5b-9 deposition evaluation on ECs culture [57]. Complement levels were within the normal range, but 5 days after admission, we identified significant depo- sition of C5b-9 on EC (3.2 6 0.2-fold increase, P < 0.05) (Figure 4) (A). Despite these results, we spent 4 weeks in the initial management of TMA and especially ruling out tumour recurrence (tumour markers, PET-CT) and gemcitabine-induced TMA. MAHA, thrombocytopenia, kidney failure and nephrotic syndrome persisted despite sup- portive therapy and plasma exchange (18 sessions). Finally, we reached a clinical diagnosis of aHUS by exclusion. At that point (November 2017), we started eculizumab following the data sheet, with TMA haematological response (normalization of platelet count, LDH, haptoglobin and reticulocytes) after 2 weeks, and normal kidney function with complete remission of nephrotic syndrome after 4 weeks, as well as a negativization of C5b-9 deposits (Figure 4) (B). Three months after admission, ge- netic study revealed a CFI mutation (heterozygous variant in exon 5, c.739T>G [p.Cys247Gly] considered as pathogenic) and risk haplotypes in heterozygosis for CFH and MCP. After 29 months, the patient maintained haematological and kidney remission on eculizumab. In conclusion, complement assessment in all TMA cases (the proposed algorithm in Figure 2) could critically reduce diagnostic times for aHUS (supporting physician suspicion) and establish the therapeutic potential of comple- ment blockade in secondary TMA forms when aetiological treatment—if available—fails. 1062 | M. Blasco et al. C5b-9 deposition on ECs in culture. This technique is used to including aHUS [58]. However, comprehensive knowledge of dif- screen for complement abnormalities. It was set up using aHUS ferent complement components or/and complement activation as the gold standard disease in which complement is dysregu- marker levels could provide hints about possible complement lated over cell surfaces. Furthermore, it has been proven useful to pathway overactivation. The quantification of the majority of monitor eculizumab treatment in aHUS patients, and to assess individual complement components can be performed through complement involvement in severe preeclampsia and HELLP syn- standard immunochemical assays [70]. C3 and C4 plasma levels drome or to exclude complement as a pathophysiological factor have been available in routine diagnostic laboratories for deca- in malignant hypertension [57]. To evaluate complement activa- des. Low levels of these two components are associated with a tion, human dermal microvascular EC line CDC/EU. HMEC-1 number of pathologies such as aHUS (C3 consumption only in (HMEC) [64] is seeded on gelatin-covered glass coverlips. Cells are 30–50% of patients), TTP, STEC-HUS and glomerulonephritis, washed with test medium [54] and are then incubated with acti- among others [67, 70, 71]. The quantification of Factor B levels vated plasma (a-plasma), obtained by mixing patients’ plasma could provide information regarding which pathway is actively with control pooled sera (1:1) and diluting the mix with test me- consuming C3. Properdin quantification is available only in spe- dium. Control samples consist of mixing healthy plasma from cialized laboratories, but knowledge of its levels could be useful donors with pooled serum from controls. Then, cultures are as it is the only positive regulator of the alternative pathway Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 washed and fixed with paraformaldehyde. For C5b-9 detection, and its levels could be reduced in cases of complement activa- immunostaining is performed with an anti-human complement tion. The decay products of C3, C4, C5 and Factor B can be quan- C5b-9 complex antibody followed by a fluorescent conjugated tified in specialized laboratories and offer information about secondary antibody and DAPI. The area covered by C5b-9 depos- the activation state of the complement system. The main draw- its is calculated from fluorescent micrographs and expressed as back to its use in clinical practice is that, in general, decay prod- the average fold increases of each condition versus control. This ucts have a very short decay time or half-life. From all the test is a modification of that developed by Noris et al. [54] but complement activation molecules evaluated, soluble C5b-9 takes advantage of the multiple links between the complement (sC5b-9) may reflect the overall activation of the complement and the clotting system [65] to enhance C5b-9 deposition and re- system as the activation of any of the three initiating comple- duce the coefficient of variation obtained for each sample. ment pathways ultimately leads to the generation of the MAC. However, a laboratory that offers this evaluation needs to have However, there are discrepant conclusions regarding the clinical ECs in culture available constantly. value of this assay, especially in aHUS. Palomo et al. [57] and Total haemolytic complement (CH50) assay. This assay anal- Noris et al. [54] reported that sC5b-9 is not a suitable biomarker yses the functional ability of serum complement components to of complement activation in aHUS as they found no correlation lyse sheep red blood cells (SRBCs) pre-coated with rabbit anti- between levels of this molecule and clinical progression of the sheep antibodies. Therefore, it mainly evaluates the classic disease. Contrarily, Volokhina et al. [59] and Cataland et al. [60] complement activation pathway. In this assay, serial dilutions suggested sC5b-9 as a helpful biomarker to confirm clinical di- of the sample are made and incubated with SRBC and the agnosis of aHUS as levels were elevated in patients at the acute results are reported as the reciprocal of the dilution at which phase of the disease and comparable to healthy controls when 50% haemolysis occurs (CH50) [66]. Low levels of CH50 may oc- patients were in remission. Bu et al. [58] reported data suggest- cur due to congenital complement deficiencies, increased con- ing that increased sC5b-9 levels are indicative of active disease sumption of complement components or insufficient synthesis (positive predictive value, 89%) but should be used in the con- of complement factors, and during infections, disease exacerba- text of other complement assays (negative predictive value, tion in patients with SLE and in patients with immune complex 46%). diseases such as glomerulonephritis. Haemolytic and enzyme- linked immunosorbent assays are currently commercially avail- Genetic analysis able [67]. Complement alternative pathway (AP50) assay. This assay Molecular and genetic complement study is mandatory in incubates serial dilutions of patients’ sera with rabbit or guinea patients with clinical suspicion of aHUS [23], reinforcing the di- pig erythrocytes in conditions that specifically activate the al- agnosis if pathogenic variants or CFH antibodies are identified ternative pathway [68]. A low AP50 result is associated with de- (50–60% aHUS patients), and allowing genetic counselling and ficiencies in FB, FD, FH and properdin, glomerulonephritis, offering genotype–phenotype correlations that predict progno- aHUS, STEC-HUS and consumption of complement proteins sis, treatment response and post-transplant recurrence risk [67]. [72]. Genetic testing must be carried out in reference Modified Ham test. The principle of this technique is that paroxysmal nocturnal haemoglobinuria cells are more sensitive Table 3. Genetic and acquired complement abnormalities in aHUS to alternative pathway of complement due to due to deficiency of complement regulatory proteins. The incubation of cells with Complement Coagulation- this deficiency with sera from aHUS patients is supposed to re- Complement regulatory activation related sult in a significant increase of nonviable cells compared with factors factors factors serum from healthy controls [69]. CFH C3 THBD CFH/CFHR hybrid gene CFB DGKE Quantification of individual complement components CFI PLG and regulators and activation markers MCP Anti-CFHantibodies Unfortunately, the quantification of levels of the different com- plement components in serum is not a straightforward ap- CFHR, complement factor H related; CFI, complement factor I; MCP, membrane proach in TMA diagnosis, as soluble levels are not reliable cofactor protein; C3, complement component C3; CFB, complement factor B; biomarkers of complement activation in any form of TMA, THBD, thrombomodulin; DGKE, diacylglycerol kinase epsilon; PLG, plasminogen. Thrombotic microangiopathies assessment | 1063 complement laboratories, capable of identify genetic variants, STEC-HUS. Supportive therapy based on hydration, fluid and risk haplotypes, complex genomic rearrangements and hybrid electrolyte management, antihypertensive drugs and renal re- genes (Table 3). Its main limitations are its high cost and long placement therapy (if needed), is the treatment of choice for response time; therefore, clinical diagnosis of aHUS, by exclu- STEC-HUS [79]. sion, and initiation of an aetiological treatment should never be aHUS. Treatment with complement blockade (Anti-C5) has delayed. In other forms of TMA, there is increasing evidence of significantly improved clinical outcomes in this ultra-rare dis- the presence of genetic variants in complement proteins that ease, in both paediatric [80, 81] and adult patients [82–84]. In could be related to its pathophysiology [73–76]. However, there this regard, treatment with eculizumab was approved by the are no cost–effectiveness studies that allow a universal recom- FDA in 2011, and actually remains the first-line treatment for mendation to carry out these studies in all TMA cases. aHUS patients. It has to be emphasized that early eculizumab use is associated not only with high haematological response, but also with organ dysfunction recovery (especially kidney TREATMENT OF DIFFERENT TMA FORMS function) [85, 86]. Ravulizumab, a long-acting complement in- Primary TMA forms hibitor, was subsequently developed, thereby requiring less fre- quent infusions than eculizumab, and demonstrating also rapid Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 TTP. The mainstay treatment for hereditary TTP is plasma infu- haematological and renal improvement. It received regulatory sion to raise ADAMTS-13 activity. Regarding acquired TTP, cur- approval for aHUS in 2019 [79]. All patients treated with C5 rent standard management consists in therapeutic plasma inhibitors must be immunized with meningococcal vaccines exchange (TPE). TPE involves removal of the patient’s plasma by (both ACYW135 and serogroup B) at least 2 weeks prior to ad- apheresis and replacement with donor plasma, thus removing ministering the first dose of eculizumab. For patients who must ADAMTS-13 autoantibodies and replacing plasma with normal receive the treatment emergently, prophylactic antibiotics to ADAMTS-13 activity. The duration of TPE and the number of prevent meningitis may be administered until the vaccinations procedures required to achieve remission, defined by the recov- take effect. ery of the platelet count, is highly variable. Immunosuppressive When complement blockers are not available, TPE must be agents are routinely added to TPE for the treatment of acquired early started after aHUS diagnosis, until anti-C5 treatment could TTP; however, there are limited data from high-quality clinical be administered. Also, if aHUS is mediated by auto-antibodies trials to direct the most appropriate use of these treatments [17, against CFH, immunossuppresants and TPE should be consid- 77]. Glucocorticoids are thought to speed up the recovery be- ered, in conjunction with the above-explained therapies. cause they reduce the production of both ADAMTS-13 inhibitor (auto-antibody) and cytokines production. The administered dose of glucocorticoids varies according to the severity of TTP Secondary TMA forms presentation; for severe cases, high dose of glucocorticoids is In secondary TMA forms, the treatment of choice should aim to given, starting with pulses of intravenous methylprednisolone correct the underlying disease together with supportive care. and tapering the dose to oral prednisone afterwards. Typically, It has been suggested there is a possible benefit of TPE for the regimen is prednisone 1 mg/kg/day orally followed by a some secondary TMA forms, including those related with auto- rapid taper over 3–4 weeks once a normal platelet count has immune conditions, HCT, pregnancy and drug-induced TMAs, been achieved. In combination with the above measures, rituxi- all of them having a potential immune-mediated endothelial mab (anti-CD20 antibody) is also indicated in patients with pre- damage as a common pathologic mechanism. However, limited sumptive and/or confirmed diagnosis of acquired TTP, since published information exists, and in most cases, American available data suggest that it has a beneficial effect in prevent- Society for Apheresis guidelines categorizes the use of TPE as a ing disease relapse, and possibly in hastening the response to Category III indication; that is, the role of TPE is uncertain, and therapy. However, along with the sparse data, the need for par- decision-making should be individualized [87]. enteral administration and drug cost are conditioning factors Importantly, there is increasing evidence that secondary that have to be taken into consideration when rituximab is pre- TMA forms could present complement system dysregulation, scribed [17, 77]. leading to an endothelial damage. Although experience is Recently, caplacizumab, a humanized monoclonal anti-VWF scarce, cases of secondary TMA of distinct aetiology (including antibody with subcutaneous route of administration, has been TMAs secondary to pregnancy, drugs, transplant, humoral re- approved by the Food and Drug Administration (FDA) (February jection and systemic diseases) refractory to the conventional 2019) for acquired TTP treatment. Caplacizumab targets the A1 treatment with good response to eculizumab have been domain of VWF and blocks its interaction with platelet glyco- reported [10, 47, 61, 62, 88–90]. In these settings, demonstration protein 1b, thereby preventing the formation of microvascular of terminal complement overactivation (C5b-9 monitoring) thrombosis in small arterioles and capillaries. In clinical prac- could be a powerful tool in the decision-making process regard- tice, caplacizumab is reserved for patients who present with se- ing the potential use of complement inhibitors (Figure 2). vere features of TTP. Although the benefit of caplacizumab is greatest when given earlier in the course of disease, it should be given under the guidance of an experienced clinician since it is CONCLUSIONS associated with side effects (mainly, risk of bleeding) and signif- Major advances in the pathophysiological knowledge of entities icant cost. However, because it does not affect the underlying such as TTP and aHUS have allowed a clear improvement in pa- cause of acquired TTP, the above-explained co-treatments to re- tient prognosis. However, in many other causes of TMA, the un- move the antibodies are required along with caplacizumab [78]. derlying pathogenic process is still unknown. Furthermore, In this regard, it has to be noted that the sequencing of adjunc- despite progress achieved in aHUS, its diagnosis continues to be tive therapies are yet to be characterized, since there are limited by exclusion, sometimes delaying the start of high-impact treat- data from prospective, randomized trials to direct the most ap- ment. To improve the management of patients with TMA, we propriate use of these treatments. recommend a clinical algorithm, with special emphasis on 1064 | M. Blasco et al. terminal complement pathway monitoring at the beginning of 10. Kello N, Khoury L, El Marder G et al. Secondary thrombotic the diagnostic process. This review—as a practical guide in the microangiopathy in systemic lupus erythematosus and anti- diagnostic process of TMA—will evolve with the development phospholipid syndrome, the role of complement and use of of new biomarkers and treatments, but its application will allow eculizumab: case series and review of literature. Semin a better understanding of the role of complement in the patho- Arthritis Rheum 2019; 49: 74–83 genesis of these serious systemic processes with potential ther- 11. Frazer-Abel A. The effect on the immunology laboratory of apeutic interventions. the expansion in complement therapeutics. J Immunol Methods 2018; 461: 30–36 12. Mastellos DC, Ricklin D, Lambris JD. Clinical promise of next- ACKNOWLEDGEMENTS generation complement therapeutics. Nat Rev Drug Discov Fundació Miarnau (Spain); Shook Studio for figures design; 2019; 18: 707–729 Jazz Pharmaceuticals Plc (IST-16-10355, to M.D.R. and E.C.); 13. Arnold DM, Patriquin CJ, Nazy I. Thrombotic microangiopa- and the German José Carreras Leukaemia Foundation (03R/ thies: a general approach to diagnosis and management. 2019 to M.D.R. and E.C.), grant PI19/00888 (to M.D.-R.) CMAJ 2017; 189: E153–E159 14. Moake JL. Thrombotic microangiopathies. N Engl J Med 2002; Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 347: 589–600 FUNDING 15. Scully M, Cataland S, Coppo P et al.; the International This study has been partially funded by Fundació Miarnau Working Group for Thrombotic Thrombocytopenic (Spain), Jazz Pharmaceuticals Plc (IST-16-10355, E.C. and Purpura. Consensus on the standardization of terminology M.D.R.); and the German Josè Carreras Leukaemia in thrombotic thrombocytopenic purpura and related Foundation (03R/ 2019 to E.C. and M.D.R.), Instituto de Salud thrombotic microangiopathies. J Thromb Haemost 2017; 15: 312–322 Carlos III through the Project PIE15/00027 ã´ and Generalitat 16. Bendapudi PK, Hurwitz S, Fry A et al. Derivation and external de Catalunya (2017-SGR675, CERCA). validation of the PLASMIC score for rapid assessment of adults with thrombotic microangiopathies: a cohort study. CONFLICT OF INTEREST STATEMENT Lancet Haematol 2017; 157–164 17. Scully M, Hunt BJ, Benjamin S et al.; on behalf of British M.B. reports advisory boards and symposium speaker hono- Committee for Standards in Haematology. Guidelines on the raria from Alexion Pharmaceuticals; E.C., and M.D.R. re- diagnosis and management of thrombotic thrombocytope- ceived research grant and symposium speaker fees from nic purpura and other thrombotic microangiopathies. Br J Jazz Pharmaceutical and M.P. received symposium speaker Haematol 2012; 158: 323–335 fees from Jazz Pharmaceutical. 18. Jokiranta TH. Atypical HUS. Blood 2017; 129: 2847–2856 19. Teixeira CM, Silva HT, De Moura LAR et al. Clinical and path- REFERENCES ological features of thrombotic microangiopathy influencing longterm kidney transplant outcomes. PLoS One 2020; 15: 1. Brocklebank V, Wood KM, Kavanagh D. Thrombotic micro- e0227445 angiopathy and the kidney. Clin J Am Soc Nephrol 2018; 13: 20. Magro CM, Momtahen S, Mulvey JJ et al. Role of the skin bi- 300–317 opsy in the diagnosis of atypical hemolytic uremic syn- 2. Campistol JM, Arias M, Ariceta G et al. An update for atypical drome. Am J Dermatopathol 2015; 37: 349–359 haemolytic uraemic syndrome: diagnosis and treatment. A 21. El-Bietar J, Warren M, Dandoy C et al. Histologic features of consensus document. Nefrologia 2015; 35: 421–447 intestinal thrombotic microangiopathy in pediatric and 3. George JN, Nester CM. Syndromes of thrombotic microangi- young adult patients after hematopoietic stem cell trans- opathy. N Engl J Med 2014; 371: 654–666 plantation. Biol Blood Marrow Transplant 2015; 21: 1994–2001 4. Shatzel JJ, Taylor JA. Syndromes of thrombotic microangiop- 22. Nishioka GJ, Chilcoat CC, Aufdemorte TB et al. The gingival athy. Med Clin North Am 2017; 101: 395–415 biopsy in the diagnosis of thrombotic thrombocytopenic 5. Noris M, Mescia F, Remuzzi G. STEC-HUS, atypical HUS and purpura. Oral Surg Oral Med Oral Pathol Oral Radiol 1988; 65: TTP are all diseases of complement activation. Nat Rev 580–585 Nephrol 2012; 8: 622–633 23. Goodship THJ, Cook HT, Fakhouri F et al. Atypical hemolytic 6. Buelli S, Zoja C, Remuzzi G, Morigi M. Complement activa- uremic syndrome and C3 glomerulopathy: conclusions from tion contributes to the pathophysiology of Shiga Toxin- a “Kidney Disease: Improving Global Outcomes” (KDIGO) associated hemolytic uremic syndrome. Microorganisms Controversies Conference. Kidney Int 2017; 91: 539–551 2019; 7: 15 24. Lusco MA, Fogo AB, Najafian B et al. AJKD atlas of renal pa- 7. Dhakal P, Bhatt VR. Is complement blockade an acceptable thology: thrombotic microangiopathy. Am J Kidney Dis 2016; therapeutic strategy for hematopoietic cell transplant- 68: 33–34 associated thrombotic microangiopathy? Bone Marrow 25. Chang A. Thrombotic microangiopathy and the kidney. Transplant 2017; 52: 352–356 Diagnostic Histopathol 2017; 23: 101–108 8. Fakhouri F. Pregnancy-related thrombotic microangiopa- 26. Ernandes Neto M, Soler L, de M, Gallindo Vasconcelos HV et thies: clues from complement biology. Transfus Apher Sci al. Histopathological features of thrombotic microangiopa- 2016; 54: 199–202 thies in renal biopsies. J Nephropathol 2019; 8: 27 9. Zheng L, Zhang D, Cao W et al. Synergistic effects of 27. Sánchez-Luceros A, Farı́as C, Amaral M et al. von Willebrand ADAMTS13 deficiency and complement activation in patho- factor-cleaving protease (ADAMTS13) activity in normal genesis of thrombotic microangiopathy. Blood 2019; 134: non-pregnant women, pregnant and post-delivery women. 1095–1105 Thromb Haemost 2004; 92: 1320–1326 Thrombotic microangiopathies assessment | 1065 28. Ono T, Mimuro J, Madoiwa S et al. Severe secondary defi- thrombotic microangiopathy and GVHD. Blood 2017; 130: ciency of von Willebrand factor–cleaving protease 1259–1266 (ADAMTS13) in patients with sepsis-induced disseminated 48. Ravindran M, Khan MA, Palaniyar N. Neutrophil extracellu- intravascular coagulation: its correlation with development lar trap formation: physiology, pathology, and pharmacol- of renal failure. Blood 2006; 107: 528–534 ogy. Biomolecules 2019; 9: 365 29. Mannucci PM, Canciani MT, Forza I et al. Changes in health 49. Yuen J, Pluthero FG, Douda DN et al. NETosing neutrophils and disease of the metalloprotease that cleaves von activate complement both on their own NETs and bacteria Willebrand factor. Blood 2001; 98: 2730–2735 via alternative and non-alternative pathways. Front Immunol 30. Martin-Rodriguez S, Reverter JC, Tàssies D et al. Reduced 2016; 7: 137 ADAMTS13 activity is associated with thrombotic risk in sys- 50. Baumgartner MR, Hörster F, Dionisi-Vici C et al. Proposed temic lupus erythematosus. Lupus 2015; 24: 1143–1149 guidelines for the diagnosis and management of methylma- 31. Feng S, Eyler SJ, Zhang Y et al. Partial ADAMTS13 deficiency lonic and propionic acidemia. Orphanet J Rare Dis 2014; 9: in atypical hemolytic uremic syndrome. Blood 2013; 122: 1–36 1487–1493 51. George JN. Systemic malignancies as a cause of unexpected 32. Kokame K, Nobe Y, Kokubo Y et al. FRETS-VWF73, a first fluo- microangiopathic hemolytic anemia and thrombocytopenia. Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 rogenic substrate for ADAMTS13 assay. Br J Haematol 2005; Oncology 2011; 25: 908–914 129: 93–10 52. Al-Nouri ZL, Reese JA, Terrell DR et al. Drug-induced throm- 33. Saha M, McDaniel JK, Zheng XL. Thrombotic thrombocytope- botic microangiopathy: a systematic review of published nic purpura: pathogenesis, diagnosis and potential novel reports. Blood 2015; 125: 616–618 therapeutics. J Thromb Haemost 2017; 15: 1889–1900 53. Yoshida Y, Kato H, Ikeda Y et al. Pathogenesis of atypical he- 34. Terrell DR, Williams LA, Vesely SK et al. The incidence of molytic uremic syndrome. J Atheroscler Thromb 2019; 26: thrombotic thrombocytopenic purpura-hemolytic uremic 99–110 syndrome: all patients, idiopathic patients, and patients 54. Noris M, Galbusera M, Gastoldi S et al. Dynamics of comple- with severe ADAMTS-13 deficiency. J Thromb Haemost 2005; ment activation in aHUS and how to monitor eculizumab 3: 1432–1436 therapy. Blood 2014; 124: 1715–1726 35. Righini M, Le Gal GD, Lucia S et al. Relation between 55. Timmermans SAMEG, Abdul-Hamid MA, Potjewijd J et al.; on ADAMTS13 activity and ADAMTS13 antigen levels in healthy behalf of the Limburg Renal Registry. C5b9 Formation on en- donors and patients with thrombotic microangiopathies dothelial cells reflects complement defects among patients (TMA). Thromb Haemost 2006; 95: 715–719 with renal thrombotic microangiopathy and severe hyper- 36. Camilleri RS, Scully M, Thomas M et al. A phenotype- tension. J Am Soc Nephrol 2018; 29: 2234–2243 genotype correlation of ADAMTS13 mutations in congenital 56. Galbusera M, Noris M, Gastoldi S et al. An ex vivo test of com- thrombotic thrombocytopenic purpura patients treated in plement activation on endothelium for individualized eculi- the United Kingdom. J Thromb Haemost 2012; 10: 1792–1801 zumab therapy in hemolytic uremic syndrome. Am J Kidney 37. Mayer CL, Leibowitz CS, Kurosawa S et al. Shiga toxins and Dis 2019; 74: 56–72 the pathophysiology of hemolytic uremic syndrome in 57. Palomo M, Blasco M, Molina P et al. Complement activation humans and animals. Toxins 2012; 4: 1261–1287 and thrombotic microangiopathies. Clin J Am Soc Nephrol 38. Parsons BD, Zelyas N, Berenger BM et al. Detection, charac- 2019; 14: 1719–1732 terization, and typing of Shiga toxin-producing Escherichia 58. Bu F, Meyer NC, Zhang Y et al. Soluble c5b-9 as a biomarker coli. Front Microbiol 2016; 7: 478 for complement activation in atypical hemolytic uremic 39. Song D, Wu L, Wang F et al. The spectrum of renal throm- botic microangiopathy in lupus nephritis. Arthritis Res Ther syndrome. Am J Kidney Dis 2015; 65: 968–969 59. Volokhina EB, Westra D, van der Velden TJAM et al. 2013; 15: R12 40. Nester CM, Barbour T, de Cordoba SR et al. Atypical aHUS: Complement activation patterns in atypical haemolytic state of the art. Mol Immunol 2015; 67: 31–42 uraemic syndrome during acute phase and in remission. 41. Infections S, Thrombotic M, Purpura T. Systemic infections Clin Exp Immunol 2015; 181: 306–313 mimicking thrombotic. Am J Hematol 2012; 86: 743–751 60. Cataland SR, Holers VM, Geyer S et al. Biomarkers of terminal 42. Tranquilli AL, Dekker G, Magee L et al. The classification, di- complement activation confirm the diagnosis of aHUS and agnosis and management of the hypertensive disorders of differentiate aHUS from TTP. Blood 2014; 123: 3733–3738 pregnancy: a revised statement from the ISSHP. Pregnancy 61. Cavero T, Rabasco C, López A et al. Eculizumab in secondary Hypertens 2014; 4: 97–104 atypical haemolytic uraemic syndrome. Nephrol Dial 43. Dashe J. The long-term consequences of thrombotic micro- Transplant 2017; 32: 466–474 angiopathy (thrombotic thrombocytopenic purpura and he- 62. Román E, Mendizábal S, Jarque I et al. Secondary thrombotic molytic uremic syndrome) in pregnancy. Obstet Gynecol 1998; microangiopathy and eculizumab: a reasonable therapeutic 91: 662–668 option. Nefrologia 2017; 37: 478–491 44. Noris M, Remuzzi G. Atypical hemolytic–uremic syndrome. 63. Weitz IC. Thrombotic microangiopathy in cancer. Semin N Engl J Med 2009; 361: 1676–1687 Thromb Hemost 2019; 45: 348–335 45. Fakhouri F, Roumenina L, Provot F et al. Pregnancy-associ- 64. Ades EW, Candal FJ, Swerlick RA et al. HMEC-1: establish- ated hemolytic uremic syndrome revisited in the era of com- ment of an immortalized human microvascular endothelial plement gene mutations. J Am Soc Nephrol 2010; 21: 859–867 cell line. J Invest Dermatol 1992; 99: 683–690 46. Huerta A, Arjona E, Portoles J et al. A retrospective study of 65. Amara U, Flierl MA, Rittirsch D et al. Molecular intercommu- pregnancy-associated atypical hemolytic uremic syndrome. nication between the complement and coagulation systems. Kidney Int 2018; 93: 450–459 J Immunol 2010; 185: 5628–5636 47. Gloude NJ, Khandelwal P, Luebbering N et al. Circulating 66. Costabile M. Measuring the 50% haemolytic complement dsDNA, endothelial injury, and complement activation in (CH50) activity of serum. J Vis Exp 2010; 37: 1923 1066 | M. Blasco et al. 67. Angioi A, Fervenza FC, Sethi S et al. Diagnosis of complement atypical hemolytic uremic syndrome in children. Pediatr alternative pathway disorders. Kidney Int 2016; 89: 278–288 Nephrol 2016; 31: 15–39 68. Servais G, Walmagh J, Duchateau J. Simple quantitative hae- 80. Giordano M, Castellano G, Messina G et al. Preservation of re- molytic microassay for determination of complement alter- nal function in atypical hemolytic uremic syndrome by ecu- native pathway activation (AP50). J Immunol Methods 1991; lizumab: a case report. Pediatrics 2012; 130: e1385–e1388 140: 93–100 81. Greenbaum LA, Fila M, Ardissino G et al. Eculizumab is a safe 69. Gavriilaki E, Yuan X, Ye Z et al. Modified Ham test for atypical and effective treatment in pediatric patients with atypical hemolytic uremic syndrome. Blood 2015; 125: 3637–3646 hemolytic uremic syndrome. Kidney Int 2016; 89: 701–711 70. Mollnes TE, Jokiranta TS, Truedsson L et al. Complement 82. Fakhouri F, Hourmant M, Campistol JM et al. Terminal com- analysis in the 21st century. Mol Immunol 2007; 44: 3838–3384 plement inhibitor eculizumab in adult patients with atypical 71. Shih AR, Murali MR. Laboratory tests for disorders of com- hemolytic uremic syndrome: a single-arm, open-label trial. plement and complement regulatory proteins. Am J Hematol Am J Kidney Dis 2016; 68: 84–93 2015; 90: 1180–1186 83. Legendre CM, Licht C, Muus P et al. Terminal complement in- 72. Rodrı́guez de Córdoba S, Hidalgo M, Pinto S et al. Genetics of hibitor eculizumab in atypical hemolytic-uremic syndrome. atypical hemolytic uremic syndrome (aHUS). Semin Thromb N Engl J Med 2013; 368: 2169–2181 Downloaded from https://academic.oup.com/ckj/article/14/4/1055/5957453 by guest on 26 May 2023 Hemost 2014; 40: 422–430 84. Licht C, Greenbaum LA, Muus P et al. Efficacy and safety of 73. Westra D, Volokhina EB, van der Molen RG et al. Serological eculizumab in atypical hemolytic uremic syndrome from 2- and genetic complement alterations in infection-induced year extensions of phase 2 studies. Kidney Int 2015; 87: and complement-mediated hemolytic uremic syndrome. 1061–1073 Pediatr Nephrol 2017; 32: 297–309 85. Walle J, Vande Delmas Y, Ardissino G et al. Improved renal 74. Jodele S, Licht C, Goebel J et al. Abnormalities in the alterna- recovery in patients with atypical hemolytic uremic syn- tive pathway of complement in children with hematopoietic drome following rapid initiation of eculizumab treatment. stem cell transplant-associated thrombotic microangiop- J Nephrol 2017; 30: 127–134 athy. Blood 2013; 122: 2003–2007 86. Menne J, Delmas Y, Fakhouri F et al. Outcomes in patients 75. Salmon JE, Heuser C, Triebwasser M et al. Mutations in com- with atypical hemolytic uremic syndrome treated with ecu- plement regulatory proteins predispose to preeclampsia: a lizumab in a long-term observational study. BMC Nephrol genetic analysis of the PROMISSE cohort. PLoS Med 2011; 8: 2019; 20: 125 e100103 87. Winters JL. Plasma exchange in thrombotic microangiopa- 76. Vaught AJ, Braunstein EM, Jasem J et al. Germline mutations thies (TMAs) other than thrombotic thrombocytopenic pur- in the alternative pathway of complement predispose to pura (TTP). Hematology 2017; 2017: 632–638 HELLP syndrome. JCI Insight 2018; 3: e99128 88. Ardissino GW, Ossola, M Baffero, GM et al. M. Eculizumab for 77. Zheng XL, Vesely SK, Cataland SR et al. ISTH guidelines for atypical hemolytic uremic syndrome in pregnancy. Obstet treatment of thrombotic thrombocytopenic purpura. J Gynecol 2013; 122: 487–489 Thromb Haemost 2020; 00: 1–7 89. Faguer S, Huart A, Frémeaux-Bacchi V et al. Eculizumab and 78. Chung C. New therapeutic targets and treatment options for drug-induced haemolytic-uraemic syndrome. Clin Kidney J thrombotic microangiopathy: caplacizumab and ravulizu- 2013; 6: 484–485 mab. Ann Pharmacother 2020; 00:1–14 90. Rosenthal J. Hematopoietic cell transplantation-associated 79. Loirat C, Fakhouri F, Ariceta G et al.; for HUS International. thrombotic microangiopathy: a review of pathophysiology, An international consensus approach to the management of diagnosis, and treatment. J Blood Med 2016; 7: 181–186