Diagnostic approach to microangiopathic hemolytic disorders

Transcription

1 Received: 8 January 2017 Accepted: 3 March 2017 DOI: /ijlh REVIEW ARTICLE Diagnostic approach to microangiopathic hemolytic disorders K. Kottke-Marchant Medical Director Hemostasis and Thrombosis Robert J. Tomsich Pathology and Laboratory Medicine Institute Cleveland Clinic 9500 Euclid Avenue, LL3-1 Cleveland, OH Correspondence Kandice Kottke-Marchant marchak@ccf.org Abstract Thrombotic micro- angiopathies (TMA) are a group of related disorders that are characterized by thrombosis of the microvasculature and associated organ dysfunction, and encompass congenital, acquired, and infectious etiologies. A hall mark of TMAs is the fragmentation of erythrocytes by the microvascular thrombi, resulting in a hemolytic anemia. There are several distinct pathophysiologies leading to microangiopathic hemolysis, ranging from decreased degradation of von Willebrand factor as seen in thrombotic thrombocytopenic purpura (TTP) to endothelial damage facilitated by Escherichia coli shiga toxin or complement dysregulation, seen in shiga toxin- related hemolytic- uremic syndrome (Stx- HUS) and complement- mediated TMA (also called atypical hemolytic- uremic syndrome), respectively. Distinguishing these disorders is important, as many TMAs are life- threatening, the treatments are distinct and selecting appropriate therapy can improve patient prognosis. Laboratory testing, including measurement of ADAMTS13, ADAMTS13 inhibitor, shiga toxin, and complement factors, can help establish diagnoses and guide therapy. KEYWORDS Thrombotic microangiopathy, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, ADAMTS13, atypical hemolytic uremic syndrome, complement-mediated, thrombosis, platelets, von Willebrand factor 1 THROMBOTIC MICROANGIOPATHIES Thrombotic microangiopathies (TMA) are a group of related disorders that are characterized by thrombosis of the microvasculature and associated organ dysfunction, and encompass congenital, acquired, and infectious etiologies. 1,2 A hall mark of these disorders is the fragmentation of erythrocytes by the microvascular thrombi, resulting in a nonimmune microangiopathic hemolytic anemia (MAHA). Figure 1. As the pathophysiology of these disorders has become better understood, the classification has been clarified into primary TMA syndromes, both hereditary and acquired. 2 Table I. The characteristic and best studied TMA disorder is thrombotic thrombocytopenic purpura, or thrombotic thrombocytopenia purpura (TTP), also called ADAMTS13 deficiency- mediated TMA, which has both hereditary and acquired forms. 2,3 TTP is associated with ultra- large von Willebrand factor (VWF) multimers causing microvascular platelet thrombi and vascular occlusion. 4 TTP is a medical emergency as it has a very high mortality rate (90%) if not treated promptly. 5 Another hereditary TMA, complement- mediated TMA (CM- TMA), also called atypical HUS, is caused by abnormal activation or regulation of the alternate pathway of complement. 2,6 An acquired TMA, shiga toxinassociated hemolytic- uremic syndrome (Stx- HUS), is due to enterohemorrhagic Escherichia coli strains that produce shiga toxin, leading to endothelial and glomerular damage. 7 2 CLINICAL AND PATHOLOGIC FEATURES Most TMAs have clinical symptoms that include microangiopathic hemolysis, with thrombocytopenia and signs of organ damage ranging from fever and mental status changes to renal dysfunction, but may also have atypical presentations such as cardiac damage. 2,3 TTP classically has been described to have a pentad of symptoms (thrombocytopenia, microangiopathic hemolysis, fever, mental status changes, and renal dysfunction), but the presentation is variable and criteria have recently been streamlined to microangiopathic hemolytic anemia and thrombocytopenia. 8 Complement- mediated and shiga toxin- mediated TMAs (also called hemolytic- uremic syndromes) tend to have Int J Lab Hem. 2017;39(Suppl. 1): wileyonlinelibrary.com/journal/ijlh 2017 John Wiley & Sons Ltd 69

2 70 KOTTKE- MARCHANT cells and tethered to the luminal cell membrane as unusually large VWF (ULVWF) via binding to the transmembrane protein P- selectin. A region in the A2 domain of VWF is responsible for binding to platelet GP Ib/ IX/V, and the larger VWF multimers more avidly bind to platelets due to the presence of multiple binding sites. Tethered VWF is rapidly processed into smaller fragments released into the plasma due to cleavage of Tyr Met bonds by the ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13). 12 In individuals with a deficiency of ADAMTS13, the ULVWF remains attached to endothelial cells, leading to platelet adhesion, aggregation, and microvascular thrombosis. It is thought that these long VWF/platelet aggregates can detach and embolize, further facilitating microvascular thrombosis. FIGURE 1 MAHA smear. Typical peripheral blood smear from a patient with thrombotic microangiopathy, displaying thrombocytopenia and erythrocyte fragmentation (schistocytes). Wright Giemsa Stain, original magnification 400X. [Colour figure can be viewed at wileyonlinelibrary.com] prominent renal dysfunction. 9,10 The differential diagnosis of TMAs includes other thrombotic disorders with thrombocytopenia, such as disseminated intravascular coagulation (DIC), catastrophic antiphospholipid antibody syndrome, and heparin- induced thrombocytopenia. von Willebrand factor is a large, multimeric protein produced by endothelial cells that is involved in platelet adhesion to sites of vascular injury. The general pathophysiology of TMAs involves abnormal binding of VWF to platelets in the microvasculature, either due to endothelial damage with release of ultra- large molecular weight VWF molecules or persistence of abnormally large molecules. 11 VWF typically exists in plasma in a range of molecular sizes, but it is initially released from endothelial 3 THROMBOTIC THROMBOCYTOPENIC PURPURA: HEREDITARY AND ACQUIRED (ALSO CALLED ADAMTS13 DEFICIENCY- MEDIATED THROMBOTIC MICRO- ANGIOPATHIES) Thrombotic thrombocytopenia purpura is the classical TMA that was initially described by Elie Moschcowitz in 1924 in a young woman with hyaline thrombi and a rapidly progressive febrile illness. 13 It is thought to be secondary to deficiency of or an autoimmune response against a VWF- cleaving metalloproteinase, ADAMTS13, in many patients, leading to diffuse thrombus formation in small vessels and a decrease in circulating platelets. These patients will show characteristic clinical symptoms with fever, thrombocytopenia, and microangiopathic hemolytic anemia accompanied by multi- organ failure, often manifest as renal failure and mental status changes. 11 However, MAHA and thrombocytopenia are TABLE I Primary Thrombotic Microangiopathy Syndromes Disorder Cause Laboratory Features Treatment Hereditary ADAMTS13 deficiency- mediated TMA (also called TTP) Complement- mediated TMA (also called atypical HUS) ADAMTS13 mutations leading to persistence of ultra- high MW von Willebrand multimers Mutations in complement regulatory genes leading to activation of alternate pathway of complement ADAMTS13 <5% ADAMTS13 >5%; Mutation analysis Plasma exchange Eculizumab Metabolism- mediated TMA Mutations in MMACHC Mutation analysis Vitamin B12 Coagulation- mediated TMA Mutations in DGKE, PLG, THBD Mutation analysis Plasma infusion Acquired ADAMTS13 deficiency- mediated TMA (also called TTP) Shiga toxin- mediated TTP (also called Stx- HUS) Antibodies to ADAMTS13 Gastrointestinal infection with Escherichia coli 0157:H7 producing Shiga toxin with endothelial damage ADAMTS13 <10% with presence of inhibitor or autoantibody Positive serology for shiga toxin and E. coli 0157:H7 Plasma exchange supportive Drug- mediated TMA (immune) Quinine, etc. Discontinue drug Drug- mediated TMA (toxic) multiple Discontinue drug Complement- mediated TMA Antibodies to complement factor H Detection of factor H inhibitor Plasma exchange, anticomplement drug Adapted from George, et al. 2.

3 KOTTKE- MARCHANT 71 sufficient to suspect TTP. 8,14 The erythrocyte fragmentation likely occurs as red cells are sheared by fluid turbulence in areas of platelet aggregates. Two types of TTP have been described: acquired and hereditary. 11,15 The most common form of TTP is an acquired disorder that is seen in adulthood associated with autoantibodies directed against ADAMTS13. Pathogenic antibodies that have been identified are typically directed against the cysteine- rich/spacer domain of ADAMTS13. Other acquired forms of TMA that resemble TTP have been described in association with pregnancy, malignancy, and transplantation. The hereditary deficiency of ADAMTS13, known as the Upshaw- Shulman syndrome, often presents in infancy or childhood and may recur as chronic relapsing TTP. Over 70 genetic mutations of the ADAMTS13 gene on chromosome 9q34 have been identified. 16 Symptomatic onset in about half of the hereditary TTP patients is before the age of 5, with the remainder presenting later, usually after the age of Laboratory Diagnosis Pathologic features and laboratory studies In patients with TTP, review of the complete blood count (CBC) highlights a normocytic anemia with profound thrombocytopenia; platelet counts are frequently less than /μl. The reticulocyte count, red cell distribution width (RDW), and mean platelet volume (MPV) are often increased, indicating the increased turnover of both erythrocytes and platelets. 17 Morphologic evaluation of the peripheral blood smear reveals erythrocyte polychromasia and anisocytosis with prominent schistocytes (Figure 1). Recent guidelines for assessment and quantification of schistocytes include specific morphologic features (helmet cells, small, irregular triangular, or crescent- shaped cells, pointed projections, and lack of central pallor) and recommendations to perform assessment using an automated cell counter. 18,19 Bone marrow evaluation is typically not necessary in establishing a diagnosis of TTP but may be helpful to exclude other causes of thrombocytopenia. Bone marrow morphology is typically nonspecific and shows normal to increased numbers of megakaryocytes. Small vessels may show platelet thrombi, but these are not always observed. Autopsy reflects the pathophysiology of the disease, with plateletrich thrombi plugging the microvasculature of the brain, heart, pancreas, spleen, adrenal gland, and kidneys but rarely involving the liver or lungs, in distinction from DIC. 20 These thrombi are also rich in VWF, if studied by immunohistochemistry, in contrast to the platelet- fibrin thrombin in DIC. Several different assays for the VWF- cleaving metalloproteinase are now available, including assays of ADAMTS- 13 activity, antigen, neutralizing or nonneutralizing autoantibodies, and genetic characterization of the ADAMTS-13 gene for mutations. 21,22 The functional assays are based on the degradation of VWF or VWF- derived peptides and detection of the cleavage products. The initial assay based on degradation of intact VWF measured the enzyme activity by following a progressive decrease in VWF multimer size, but this relied on a VWF multimer Western blot assay that took as long as 2 days to perform and was not readily available in most laboratories. 23 Further modifications of the assay based on intact VWF measured the metalloprotease- induced decrease in VWF multimer size by either immunoradiometric assay in microtiter plates or tracking the ability of VWF to bind to collagen or ristocetin, an activity that is directly proportional to VWF molecular weight. 21,24 These modifications shortened the assay time to about 24 hours, but had a lower limit of quantification of 5% 10% ADAMTS13 activity. More recently developed functional assays are based on the degradation of VWF peptides with direct detection of ADAMTS13 metalloprotease activity, which cleaves the A2 domain between Y1605 and M1606. These assays have employed recombinant ADAMTS13 substrate peptides from the VWF A2 domain, such as L1591- R1668 or VWF73 (D1596- R1668). 25,26 Peptide cleavage is often facilitated by a denaturing agent (i.e., guanidine HCl or urea). Peptide cleavage products (labeled and/or unlabeled) are detected by various techniques, including ELISA, Western blot, and fluorescence resonance energy transfer. 24 These newer assay are rapid, with results available in hour and have a lower limit of quantification down to 0.5% ADAMTS13 activity. 3,21 ADAMTS13 levels <10% are highly indicative of TTP. Some considerations in the ADAMTS13 activity assays include pre- analytic handling, as long storage at room temperature can lead to leukocyte activation and release of proteases that nonspecifically cleave ADAMTS Samples with high levels of plasmin, such as in DIC, can lead to low ADAMTS13 levels, however, not usually below 10%. High levels of bilirubin (>50 umol/l) or hemolysis (plasma Hb >0.05 ug/l) also may interfere with the assay results. Immunoassays for quantitative measurement of ADAMTS- 13 antigen are available. 21 They may be normal in patients with TTP due to neutralizing autoantibodies, where immune complexes are formed. Alternatively, the antigen levels may be decreased if non- neutralizing autoantibodies lead to ADAMTS- 13 clearance. 27 Neutralizing antibodies can be detected using a mixing study of heat- inactivated patient and normal plasma (typically, 1:1 mix) or quantitative Bethesda inhibitor assay, combined with either an immunoassay or functional assay. Measurement of non- neutralizing antibodies to ADAMTS13 can be taken by specific antibody immunoassay. 14,28 Diagnostic strategies for TTP start with the findings of thrombocytopenia, anemia, and schistocytes on the peripheral smear in the setting of a normal PT (prothrombin time) and APTT (activated partial thromboplastin time). The finding of a decreased ADAMTS13 functional level (<10%) should be followed by a Bethesda inhibitor assay to detect a neutralizing antibody. If no neutralizing antibody is detected, an antibody immunoassay may be useful to detect non- neutralizing antibodies. See the diagnostic algorithm (Figure 2). Other supportive laboratory studies include features of hemolytic anemia (decreased haptoglobin, increased lactate dehydrogenase, with a negative Coombs test) and features of renal dysfunction (elevated creatinine with proteinuria and hematuria) or cardiac dysfunction (elevated troponin T). Genetic studies of ADAMTS13 by sequence analysis can be performed. Published studies have shown the majority of mutations to be missense (59%), with 13% nonsense, 13% deletions, 6% insertions, and 9% splice site mutations. 16 Plasma ADAMTS13 activity in normal individuals typically ranges from 50% to 180%. 29 Patients with acute TTP usually have activity less than 10% and often <5%. 30 Variably decreased ADAMTS13 activity can also be observed with liver disease, disseminated malignancy, inflammatory disorders, and during pregnancy. Infants may also have a lower activity than adults.

4 72 KOTTKE- MARCHANT FIGURE 2 Diagnostic algorithm. This is a suggested laboratory diagnostic algorithm for distinguishing the various thrombotic microangiopathies. Testing for ADAMTS13 activity and inhibitor typically begins with clinical suspicion for TMA in the setting of 1 thrombocytopenia and 2 microangiopathic hemolytic anemia (schistocytes on peripheral smear, anemia, decreased haptogloin, increased lactate dehydrogenase, and negative Coombs). [Colour figure can be viewed at wileyonlinelibrary.com] Traditional screening coagulation studies, such as PT and APTT, are normal in patients with TTP. 2,3 Results of assays for VWF, such as VWF antigen, ristocetin cofactor, and factor VIII, are typically normal. However, VWF multimer analysis may show an increase in ULvWF multimers during the acute disease presentation. 3.2 Differential diagnosis Thrombotic thrombocytopenia purpura can be diagnosed definitively in many patients based on the distinct clinical features and markedly decreased ADAMTS13 activity. However, ADAMTS13 assays are not widely available, so rapid diagnosis often rests on excluding other consumptive thrombocytopenic disorders. Additionally, many patients with clinical symptoms of TTP have only modestly decreased levels of ADAMTS13, while other individuals with markedly decreased ADAMTS13 levels may not have clinical symptoms of TTP. Thrombotic thrombocytopenia purpura and DIC are both thrombotic microangiopathies with schistocytes on the peripheral smear, but these two disorders can be distinguished by the characteristic coagulation test abnormalities found in DIC, such as elevated PT, elevated APTT, decreased fibrinogen, and elevated D- dimer. Shiga toxin- mediated TMA (Stx- HUS) is distinguishable by serology for E. coli 0157:H7, a lack of abnormalities in ADAMTS13, and the more severe renal dysfunction. Patients with ITP typically lack hemolytic anemia and multi- organ failure and do not have schistocytes on the peripheral smear. TTP can be distinguished from post- transfusion purpura due to lack of association with transfusions or platelet HPA- 1a/1b genotype. Distinction of TTP from drug- induced thrombocytopenia may be difficult in patients treated with multiple drugs, but these disorders usually lack microangiopathic thrombosis or organ failure. Heparin-induced thrombocytopenia (HIT) is one drug- induced thrombocytopenia that is complicated by thrombosis, but HIT is preceded by heparin exposure 5 to 14 days prior and is characterized by large- vessel thrombosis and the presence of antiplatelet factor 4 antibodies. Paroxysmal nocturnal hemoglobinuria may occasionally present with thrombocytopenia and microangiopathic hemolysis but is usually distinguishable by a lack of renal dysfunction and a flow cytometric analysis demonstrating decreased expression of glycophosphatidylinositol- linked proteins such as CD55 and CD59 on red cells or neutrophils. TTP should also be distinguished from systemic causes of thrombotic microangiopathy that may present with thrombocytopenia and microangiopathic hemolytic anemia, such as hypertensive disorders of pregnancy, malignant hypertension, rheumatologic disorders, infection, and some malignancies. 3.3 Prognosis and Treatment Untreated TTP is associated with a high mortality due to the multiorgan failure. Therapy with the early initiation of daily plasma exchange using fresh frozen plasma is crucial, and the majority of patients now survive the initial episode. 5 However, relapse of TTP may be seen in 30% to 60% of patients, with relapse most frequent in the first month. Newer therapies based on infusion of recombinant ADAMTS13 or rituximab to suppress the immune response have shown promise. 2,5 Recently, caplacizumab, an anti- von Willebrand factor single- variable domain immunoglobulin that inhibits the interaction between ultralarge VWF multimers and platelets has shown

5 KOTTKE- MARCHANT 73 efficacy in resolution of acute acquired TTP, but at the expense of increased bleeding tendency HEMOLYTIC- UREMIC SYNDROME Hemolytic- uremic syndrome has been classically described as a disorder with thrombocytopenia, microangiopathic hemolytic anemia, and renal failure/uremia. It is now appreciated that HUS is actually two distinct disorders: 1 a complement- mediated TMA (CM- TMA; also known as atypical HUS) that is due to a hereditary or acquired defect in a complement regulatory protein and 2 diarrhea- associated HUS caused by a Shiga toxin- producing strain of E. coli (serotype 0157:H7) known as Shiga toxin- associated hemolytic- uremic syndrome (Stx- HUS). 4.1 Complement- Mediated Thrombotic micro- angiopathies: Hereditary and Acquired (Also known as Atypical HUS) A complement- mediated TMA has been described in both familial and sporadic manifestations. 6 Compared to other forms of TMA, CM- TMA can have a more insidious onset with nonspecific symptoms including malaise, fatigue, and anorexia, but is usually accompanied by acute renal failure. 10 A minority of patients may display neurologic symptoms. Severe forms can be associated with myocardial infarction and multi- organ failure. The etiology of CM- TMA is now known to be due to activation or abnormal regulation of the alternate pathway of complement. 32 Specifically, activation of C3a and C5a can lead to endothelial injury with release of ultra- high molecular weight VWF and slightly low levels of ADAMTS13. There are many reasons for activation of the alternate pathway of complement in CM- TMA, including mutations of complement regulators, such as factor H (CFH), factor I (CFI), membrane cofactor protein as well as gain of function mutation of complement effectors such as C3 (C3) and factor B (CFB). 6,33,34 Approximately 10% of CM- TMA is acquired due to antibodies against complement regulatory factors, such as factor H Laboratory Testing Laboratory diagnosis of CM- TMA includes demonstrating thrombocytopenia with MAHA, together with abnormal creatinine and negative Shiga toxin assay. ADAMTS13 levels may be slightly decreased, but always are over 5%. 35 Assay of complement factors may demonstrate C3a or C5a activation, and mutation studies may show mutations in one or more complement factors, although these assays are only available in reference laboratories and up to 30% of patients with CM- TMA may not have a demonstrable mutation. 36 In patients with acquired CM- TMA, antibodies against factor H may be identified Treatment While rare, CM- TMA is important to diagnose as the treatment is specific and effective. Anticomplement therapy has been shown to be effective in CM- TMA. Eculizumab, an anti- C5 monoclonal antibody, is approved for treatment of CM- TMA. 35 The MAb prevents activation of C5a and the subsequent membrane attack complex, thus preventing endothelial injury and release of VWF. Treatment is associated with a high response rate and effective renal recovery. 35 However, as complement testing is often not available to establish a specific diagnosis, treatment often starts with plasma exchange while awaiting results of ADAMTS13 and Shiga toxin assays; detectable ADAMTS13 and negative Shiga toxin assays would indicate therapy with Eculizumab (Figure 2). Patients with inhibitors to complement factors may benefit from immunosuppressive therapy. 4.2 Shiga Toxin- associated Hemolytic- uremic Syndrome (Stx- HUS) An acquired TMA has been described as a complication of gastrointestinal E. coli infections that produce Shiga toxin (Stx)- 1 or Stx- 2. Originally described in children with a diarrheal illness preceding MAHA and acute renal failure, the disorder acquired the name hemolytic- uremic syndrome. 37 The pathogen with outbreaks of Stx- HUS is E. coli O0157:H7 in Europe and the Americas, while S. dysenteriae type 1 is causative in other countries. 7,38 Rarely, pneumococcal infections can also lead to HUS. 39 The Shiga toxin damages intestinal epithelium, resulting in diarrhea. The Stx also adheres to globotriaosylceramide 3 receptors on endothelial cells in the brain and kidney, leading to endothelial cell damage with release of high molecular weight VWF multimers. 40 The Stx may also have an inhibitory effect on ADAMTS13, leading to persistence of high molecular weight VWF multimers. The clinical scenario in Stx- HUS is an acute onset, often bloody diarrhea and is frequently observed in children. An epidemic form seen in food- related outbreaks is associated with a particular serotype of E. coli, namely 0157:H Laboratory Diagnosis Patients with Stx- HUS often have normal ADAMTS13, with elevated D- dimer and detectable fecal Stx by immunoassay or genetic testing. Serology for E. coli will show serotype 0157:H7 in the majority of patients Treatment Treatment of Stx- HUS is usually supportive, with dialysis and RBC transfusion, as needed. Unlike TTP, plasma exchange is not indicated and the prognosis is usually favorable. 5 DRUG- MEDIATED THROMBOTIC MICROANGIOPATHIES 5.1 Immune Reaction Drug- mediated TMA with acute renal injury has been associated with an immune reaction to quinine. Quinine- dependent antibodies have

6 74 KOTTKE- MARCHANT been described to interact with multiple cell types, including activation of endothelial cells. 41 Laboratory findings may include demonstration of quinine drug- dependent antibodies. Therapy is typically supportive with discontinuation of drug therapy. 5.2 Toxic Dose- Related Many drugs have been associated with an incidence of TMA, with the cause ascribed to time and dose- dependent toxicity. Implicated drugs include calcineurin inhibitors (cyclosporine, tacrolimus), vascular growth factor (VEGF) inhibitors, and thienopyridines (ticlopidine, clopidogrel) METABOLISM- MEDIATED THROMBOTIC MICROANGIOPATHIES A rare cause of TMA is cobalamin C disease, which is a hereditary defect of vitamin B12 metabolism. This has been termed metabolism- mediated TMA (MM- TMA) and is typically seen in infants 43 associated with mutation of methylmalonic aciduria and homocystinuria type C protein (MMACHC). The pathophysiologic link between the mutation and TMA is not fully established, but generation of activated oxygen and endothelial dysfunction has been implicated. 44 Treatment is therapy with vitamin B12. 7 COAGULATION- MEDIATED THROMBOTIC MICROANGIOPATHIES Once considered in the spectrum of atypical HUS, rare mutations of several coagulation- affiliated proteins with associated TMA are now classified as coagulation- mediated TMA. These include mutations of thrombomodulin, plasminogen, and diacylglycerol kinase ε (DGKE). 45,46 The pathophysiology of coagulation- mediated TMA has not been established, but DGKE mutations may lead to protein kinase C activation and up- regulation of prothrombotic factors, such as VWF. 47 SUMMARY There has been rapid progress in understanding the pathophysiology of TMAs so that they can now be classified as separate, but related disorders. 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