In spite of the complicated etiologic, clinical, and pathologic scenario of cryoglobulinemia, physicians can play a key role in its successful management by early recognition of the most common clinical presentations.
“Cryoglobulinemia” refers to the presence of cryoglobulins (immunoglobulins that precipitate at variable temperatures < 37°C [98.6°F]) in serum. Monoclonal cryoglobulinemia (type I) involves a single type of monoclonal immunoglobulin, while mixed cryoglobulinemia involves a mixture either of polyclonal immunoglobulin (Ig) G and monoclonal IgM (type II), or of polyclonal IgG and polyclonal IgM (type III); both monoclonal and polyclonal IgM have rheumatoid factor activity. Cryoglobulinemia is a unique model of human disease for several reasons: (1) cryoglobulins are detected using a simple technical approach that is based on in vitro laboratory observation of cold precipitation in serum; (2) cryoglobulinemic organ damage may be produced by two different etiopathogenic mechanisms (accumulation of cryoglobulins and autoimmune-mediated vasculitic damage); and (3) cryoglobulinemia is associated with a wide range of etiologies, symptoms, and outcomes, and is considered a disease that combines elements of autoimmune and lymphoproliferative diseases. There are three main broad treatment strategies in cryoglobulinemia-conventional immunosuppression, antiviral treatment, and biologic therapy. Some agents, such as corticosteroids and rituximab, have been successfully used in all types of cryoglobulinemia; however, treatment should be modulated according to the underlying associated disease (chronic viral infections, autoimmune diseases, or cancer), the predominant etiopathogenic damage (vasculitis vs hyperviscosity), and the severity of internal organ involvement.
Cryoglobulins are immunoglobulins that precipitate in vitro at temperatures below 37°C (98.6°F) and redissolve after rewarming. Reversible precipitation upon exposure to low temperatures allows laboratory detection of cryoglobulins. These properties also have pathogenic implications, although the molecular mechanisms involved are not entirely understood. Several specific features make cryoglobulinemia a unique model of human disease. First, cryoglobulins are detected using a simple technical approach based on in vitro laboratory observation of cold precipitation in serum. Second, cryoglobulins are associated with a wide range of etiologies, symptoms, and outcomes. Third, cryoglobulinemic organ damage may be produced by two different etiopathogenic mechanisms-the accumulation of cryoglobulins and autoimmune-mediated vasculitis. Fourth, cryoglobulinemia is a unique disease that combines features of autoimmune and lymphoproliferative diseases.
Cryoglobulins are not necessarily a sign of disease; healthy people may have low concentrations of cryoglobulins, while polyclonal cryoglobulins may be transiently detected during infection. The term “cryoglobulinemia” refers to the presence of cryoglobulins in serum, while “cryoglobulinemic disease” is used to describe symptomatic patients, since many patients with cryoglobulinemia remain asymptomatic. In 1974, Brouet et al proposed a classification that is still widely used due to its good correlation with clinical features and associated diseases: a) monoclonal cryoglobulinemia (type I), which consists of a single type of monoclonal immunoglobulin (Ig); and b) mixed cryoglobulinemia, which is a mixture of either polyclonal IgG and monoclonal IgM (type II) or polyclonal IgG and polyclonal IgM (type III); both monoclonal and polyclonal IgM have rheumatoid factor (RF) activity. The frequent confluence of musculoskeletal complaints in the setting of RH positivity often leads to an initial misdiagnosis of patients as having rheumatoid arthritis.
Type I cryoglobulinemia consists of a single monoclonal cryoprecipitable immunoglobulin (mainly IgG or IgM) that is produced by monoclonal expansion of a B-cell clone that may be indolent (monoclonal gammopathy of unknown significance [MGUS]), smoldering (WaldenstrÃ¶m macroglobulinemia), or overtly malignant (multiple myeloma). The two key features that define monoclonal cryoglobulinemia are the overwhelming association with hematologic neoplasia and the pathogenic mechanism of tissue damage. The monoclonality of cryoglobulinemia dictates much of this condition’s pathophysiology and is also associated with hematologic neoplasia.
In the largest reported series of patients with type I cryoglobulinemia, all cases were associated with hematologic neoplasia, including MGUS in 44% of patients and overt hematologic malignancy in the remaining 56% (WaldenstrÃ¶m macroglobulinemia and multiple myeloma accounted for 70% of these cases). Another recent study reported seven cases of type I cryoglobulinemia associated with multiple myeloma, and included seven other reported cases with this association.
Type I cryoglobulinemia mainly leads to tissue damage through the occlusion of blood vessels, leading to ischemia. The proximate cause of the occlusions is the precipitation of cryoglobulins from the serum and their accumulation in the microcirculation. This vascular occlusion is usually accompanied by high cryoglobulin concentrations and may be clinically associated with hyperviscosity syndrome and cold-
induced necrotic acral lesions. However, a recent study demonstrated that monoclonal IgG cryoglobulins from two patients with multiple myeloma formed crystals, even at quite low concentrations. Precipitated cryoglobulins appear in vivo as hialin thrombi occluding small blood vessels, mainly of the endoneural microvessels and the renal glomerular tuft; massive intraluminal cryoprecipitation may lead to rapidly progressive renal failure.
Terrier et al recently studied the main clinical expression of type I cryoglobulinemia in 64 patients with cryoglobulinemic vasculitis (Table 1). The main clinical manifestations at diagnosis included cutaneous features (purpura in 69%, acrocyanosis in 30%, skin necrosis in 28%, skin ulcers in 27%, and livedo reticularis in 13%) (Figure 1) and extracutaneous involvement (peripheral neuropathy in 44%, renal involvement in 30%, and joint involvement in 28%); no patient had central nervous system (CNS), pulmonary, gastrointestinal, or cardiac involvement. Nearly 80% of patients presented with severe systemic vasculitis, defined by extensive cutaneous ulcers and/or necrosis, multiple mononeuropathy, and/or glomerulonephritis. A hyperviscosity syndrome is the main clinical presentation in some patients with type I cryoglobulinemia. These patients, who are likely to have either multiple myeloma or WaldenstrÃ¶m macroglobulinemia, demonstrate high quantities of circulating monoclonal Ig. The key symptoms of a hyperviscosity syndrome involve the brain, eyes, and ears: headache and confusion, blurry vision or vision loss, and hearing loss or epistaxis.
The diagnosis of all types of cryoglobulinemia requires demonstration of cryoglobulins in serum. Appropriate sample collection and handling are critical. Blood should be collected in prewarmed syringes and tubes, transported, clotted, and centrifuged at 37o to 40oC (98.6° to 104°F), ensuring that its temperature never falls below 37oC. The serum should be stored at 4oC (39.2°F) for up to 7 days. Precipitation of type I cryoglobulins usually occurs within hours. Cryoglobulin levels are usually > 5 g/L in type I cryoglobulinemia ; quantitation is important because the cryoglobulin level usually correlates with the severity of hyperviscosity symptoms and may be useful in monitoring the response to treatment. In affected patients, physical examination should include funduscopy to rule out retinal hemorrhages, and measurement of serum viscosity, since patients usually become symptomatic at > 4.0 centipoise, although some patients may develop symptoms below this level.
Terrier et al recently studied the therapeutic management of 64 patients with type I cryoglobulinemic vasculitis, of whom 56 received at least 1 course of treatment. Nearly 90% of the patients were treated with corticosteroids. In patients with underlying MGUS, plasmapheresis (13%), immunosuppressant agents (13%), or rituximab (9%) were also required. In patients with underlying overt hematologic neoplasia, chemotherapy (56%), plasmapheresis (18%), rituximab (17%), and bortezomib (6%) were also used. Response to therapy was higher and relapses were less common in patients with hematologic neoplasia than in those with MGUS, with nearly 30% of MGUS cases being refractory to the first-line therapy. With respect to adverse events, 13% of the entire group of 64 patients developed severe infections, and vasculitis worsened in 3 of the 23 patients (13%) who received rituximab, including 1 patient in whom retreatment was associated with a vasculitic relapse. The 5- and 10-year survival rates were 94% and 87%, respectively, but rates were lower in patients with hematologic malignancies.
Patients who present with symptomatic hyperviscosity require a different, urgent therapeutic approach, especially when serum viscosity is very high. Both plasma exchange and plasmapheresis remove circulating cryoglobulins from the circulation, and are the most useful approaches not only for patients with hyperviscosity syndrome, but also for those with immediately life-threatening vasculitic disease. Nonselective plasma exchange and double-filtration plasma exchange are the most commonly used apheretic procedures. However, apheretic techniques do not alter the underlying disease milieu and can lead to a rebound phenomenon in which cryoglobulin production increases after the cessation of apheresis. Although no controlled trials of the treatment of serum hyperviscosity syndrome have been performed, experience suggests that apheresis of one type or another demonstrates consistent efficacy. Most signs and symptoms are reversible with prompt diagnosis and treatment. Because plasma exchange procedures generally remove larger volumes of plasma than does plasmapheresis, plasma exchange is preferable, particularly in patients with severe hyperviscosity symptoms. Cyclophosphamide therapy for up to 6 weeks may be required to prevent a cryoglobulin rebound. However, only 25% of patients with multiple myeloma–related type I cryoglobulinemia responded to cyclophosphamide, while two-thirds responded to bortezomib. The main therapeutic goal seems to be the cure of the underlying hematologic disease, which is responsible for cryoglobulin synthesis. Good results have been obtained in multiple myeloma with high-dose melphalan therapy. Cryoglobulinemic relapses were observed invariably only in the setting of the return
of the underlying multiple myeloma.
In contrast to type I cryoglobulinemia, mixed cryoglobulinemia (MC) is characterized by a large number of associated etiologic processes, including infections, autoimmune diseases, and cancer; in some patients there is no known etiologic factor. If a known cause of cryoglobulinemia cannot be identified, the disorder is termed “essential cryoglobulinemia.” The discovery of the hepatitis C virus (HCV) radically changed the predominant etiology of MC from essential to HCV-related cryoglobulinemia; this change occurred in the early 1990s. MC may be classified as related or unrelated to infectious processes (Table 1).
The main cause of MC is infection. Soon after the discovery of HCV in 1989, various studies reported a significantly high prevalence of HCV antibodies in patients with essential MC. Although other viruses have also been found to be related to MC, HCV infection is now known to account for more than 90% of all cases of MC.
Etiopathogenic role of HCV. HCV is the most frequent infectious trigger of cryoglobulin production; HCV envelope protein E2 interacts with the major extracellular loop of tetraspanin CD81, a signaling molecule expressed by hepatocytes and lymphocytes that triggers chronic B-cell stimulation. B-cell clones have been demonstrated in the liver, peripheral blood, and bone marrow of patients with HCV infection, particularly in patients with type II cryoglobulinemia. These B-cell clones produce monoclonal IgM, which has a cross-reacting idiotype called WA (so named because it was first isolated from the serum of a patient with WaldenstrÃ¶m macroglobulinemia). The WA idiotype is closely associated with the RF activity intrinsic to the IgM found in mixed cryoglobulins, which allows cryoglobulins to bind polyclonal IgG directed at the HCV core protein. In vivo and in vitro cryoprecipitates contain viral core proteins and RNA, indicating that cryoglobulin formation results from the host immune response to HCV. Immune complex–mediated vasculitis is the key etiopathogenic pathway of damage in MC. The monoclonal IgM component of MC generates large immune complexes containing IgG and complement proteins. Complement fractions such as C1q bind to receptors on endothelial cells, facilitating immune complex deposition and subsequent vascular inflammation.
Clinical manifestations. The percentage of patients with mixed cryoglobulins who develop symptoms varies widely (from 10% to 50%), depending on the underlying associated disease. The most common presentation is the triad of purpura, arthralgia, and weakness, which is reported in 80% of patients at disease onset.[17,18] Cutaneous purpura is the most indicative and frequent clinical feature of cryoglobulinemic syndrome; digital ischemia may be severe and lead to necrosis (Figure 2), while livedo reticularis and cutaneous ulcers usually reflect small artery involvement. Cutaneous ulcers or necrosis signify a high risk of infection, sepsis, and death. Cryoglobulinemic vasculitis flares are often accompanied by general symptoms, such as fever, weakness, myalgia, and arthralgia. Articular involvement consists mainly of joint pain (arthralgias, as opposed to arthritis) in the hands, knees, and wrists, without clinical signs of inflammation. A non-erosive arthritis with palpable synovitis is reported in about 10% of patients and perhaps even fewer. Weakness and fatigue are reported by > 50% of patients. The internal organs affected most frequently by cryoglobulinemia are the kidneys. Approximately 30% of patients have renal disease, and kidney biopsy typically shows membranoproliferative glomerulonephritis. The presentation of cryoglobulinemic glomerulonephritis is generally indolent in nearly half of cases (proteinuria, microscopic hematuria, red blood cell casts, and renal failure), while nephrotic and nephritic syndrome are less frequent presentations. More than 70% of patients with MC and renal involvement present with hypertension, and nearly half have creatinine levels > 1.5 mg/dL. Cryoglobulinemic neuropathy is reported in up to 20% of cases. The most common symptoms are sensory, eg, paresthesias (painful or burning sensations) in the lower limbs. These generally precede clinical evidence of motor nerve involvement. Electromyographic studies usually disclose a confluent polyneuropathy rather than mononeuritis multiplex. The predominant peripheral nerve involvement is in the distributions of the peroneal and sural nerves. Vasculitic neuropathy associated with MC rarely presents as a rapidly progressive sensorimotor involvement with severe functional impairment.
Life-threatening cryoglobulinemia. Infrequent severe manifestations include intestinal ischemia, alveolar hemorrhage, and CNS and myocardial involvement. Less than 5% of cryoglobulinemic patients have gastrointestinal involvement; intestinal ischemia is the main clinical presentation, resulting in acute abdominal pain, general malaise, fever, and bloody stools (Figure 3). Some patients may present with life-threatening intestinal perforation and shock, cholecystitis (with cholecystectomy disclosing cryoglobulinemic involvement of the gallbladder), or vasculitic pancreatitis. Pulmonary involvement also occurs in less than 5% of MC patients, but recognition of MC as the underlying cause poses a significant clinical challenge. Some of these patients may present with interstitial lung fibrosis (Figure 4), and others with hemoptysis and acute respiratory failure, with diffuse pulmonary infiltrates caused by cryoglobulinemic alveolar hemorrhage. The most frequent clinical presentation of CNS involvement is stroke with motor or sensory deficits, aphasia, or dysarthria-although encephalopathy has also been described; isolated cases of myelitis and ischemic spinal cord involvement have been reported as well. Myocardial involvement, ie, ischemic heart disease caused by coronary vasculitis in the absence of cardiovascular risk factors, has been reported in fewer than 10 cases.
A small proportion of cryoglobulinemic patients present with multiorgan vasculitic involvement of the small-to-medium–sized arteries, capillaries, and venules.
Diagnosis. Cryoglobulinemic syndrome is often diagnosed when typical vasculitic organ involvement (mainly of the skin, kidneys, or peripheral nerves) appears in a patient with mixed cryoglobulins. However, expert laboratory interpretation that takes the clinical context into account is essential, since false-negative results may be caused by incorrect collection or transport of blood samples. When there is justified clinical suspicion, serum cryoglobulins should be determined again,[3,20] or more sensitive diagnostic techniques, such as flow cytometry, should be used.Laboratory tests and histopathologic studies are key tools in evaluating patients with MC. Biochemical tests for renal and liver involvement are mandatory, as are immunologic tests, since hypocomplementemia (especially low C4 levels) and raised serum RF levels are characteristic of cryoglobulinemic vasculitis. Vasculitis is the typical pathologic lesion of MC; tissue biopsies show mixed inflammatory cellular infiltrates involving the small and, less frequently, medium-sized vessels. The most common target tissues are the skin, kidneys, and peripheral nerves; fibrinoid necrosis may be observed. Indirect immunofluorescence studies identify deposits of complement and immunoglobulins of the same type as those included in serum cryoglobulins. A multicenter project by de Vita et al has developed preliminary international classification criteria for cryoglobulinemic syndrome (Table 2).
Outcomes. Half of patients with MC have chronic disease with no involvement of vital organs, one-third have moderate-to-severe disease with chronic renal failure or liver cirrhosis, and nearly 15% present with sudden, life-threatening involvement. The prognosis is heavily influenced by cryoglobulinemic damage to vital organs; the mortality rates for intestinal ischemia and alveolar hemorrhage are 40% and 80%, respectively.[23,24] In addition, chronic HCV infection may result in liver cirrhosis and cancer (either B-cell lymphoma or hepatocarcinoma), which worsen the prognosis of patients with HCV-related MC substantially.
Antiviral therapies. Although earlier studies used interferon alfa (IFNÎ±) monotherapy, current evidence suggests that the most effective approach to the treatment of HCV-related cryoglobulinemic vasculitis is the use of combined antiviral therapies (Table 3), since the eradication of HCV (or at least significant lowering of HCV RNA viremia) should be considered the principal therapeutic objective. The combination of pegylated IFNÎ± (peg-IFNÎ±) and ribavirin is currently considered the standard of care therapy, and sustained virologic responses to this approach have been reported in nearly half of patients with HCV genotypes 1 and 4, and in more than 75% of those with genotypes 2 and 3. A recent study has confirmed the usefulness of testing for the rs12979860/rs8099917 IL28B polymorphism as a strong independent predictor of response to IFN-based antiviral therapeutic regimens in HCV patients with MC. Drug intolerance and dose reductions are common with both ribavirin and IFNÎ±, which have recently been found to be associated with retinal toxicity. The usefulness and safety of adding a third antiviral agent to the standard therapy (triple therapy) has recently been evaluated by Terrier et al in an open-label study including 23 patients with HCV-related MC, in whom telaprevir (375 mg 3 times daily, for 12 weeks) and boceprevir (800 mg 3 times daily, for 44 weeks) were added to peg-IFNÎ±/ribavirin in 15 and 8 patients, respectively. At week 24, all patients had responded clinically (nearly 40% showed a partial response), with a rate of virologic response of nearly 70%. Unfortunately, there was a high rate of adverse events, with more than 40% of the 23 patients developing severe cytopenia. Nearly 90% of patients required erythropoietin, and antiviral therapy had to be withdrawn in 1 in 3 patients. These preliminary results suggest that the balance between the benefits and safety of adding a protease inhibitor to standard antiviral therapy should be carefully evaluated in individual patients with HCV-related MC.
B-cell depletion therapy. The most promising non-antiviral therapeutic approach to HCV-related cryoglobulinemia is B-cell depletion with rituximab (Table 3).[29,30] A prospective cohort study of 93 consecutive patients found better results for a combination of rituximab and standard antiviral therapy compared with antiviral therapy alone, while another study found similar results in 37 patients, and Petrarca et al found excellent tolerance of the rituximab + antiviral therapy combination in cirrhotic patients, with improvement in liver cirrhosis. Sneller et al recently conducted a randomized controlled trial comparing rituximab (375 mg/m2/wk for 4 weeks) with standard immunosuppressive therapy in 24 patients whose disease was refractory to antiviral therapy; after 6 months, 10 patients in the rituximab group and 1 in the control group were in remission (83% vs 8%), a result that met the criterion for halting the trial. De Vita et al have published the results of a large controlled trial that included 59 patients with severe, refractory HCV-related MC (skin ulcers, active glomerulonephritis, or refractory peripheral neuropathy) who were randomly assigned to receive either rituximab (2 infusions of 1 g every 2 wks) or conventional treatment (glucocorticoids, azathioprine/cyclophosphamide, or plasmapheresis). The primary end point was the proportion of patients who continued taking their initial therapy; the percentages were 71% vs 3% at 6 months, and 61% vs 3% at 2 years. In the two trials, no significant adverse effects of rituximab, including increased HCV RNA viremia or elevated liver transaminase levels, were reported. One note of caution with regard to the use of rituximab is the potential for the formation of immune complexes that could exacerbate a patient’s vasculitis as a result of the binding of rituximab (a chimeric monoclonal antibody) and cryoglobulinemic IgM with RF activity. The formation of such immune complexes can be a dramatic clinical event, requiring prompt and intensive immunosuppression.
Non-infectious MC is mainly associated with systemic autoimmune diseases (SAD) and lymphoproliferative diseases, although in nearly half of cases no underlying disease is found (essential mixed cryoglobulinemia).[37,38] In patients with primary SjÃ¶gren syndrome, the main SAD associated with cryoglobulinemia, cryoglobulins have a close association with extraglandular involvement, B-cell lymphoma, and poor survival[39,40]; cryoglobulins are also detected in nearly 10% of patients with systemic lupus erythematosus and rheumatoid arthritis. With respect to hematologic neoplasia, MC is predominantly associated with B-cell lymphoma, which often appears as a late manifestation of cryoglobulinemic syndrome; cryoglobulins may also be detected in patients with solid tumors.
The clinical manifestations and outcomes are very similar to those reported in patients with HCV-related MC (Table 1). In the largest reported series, Terrier et al described 242 patients, of whom 83% presented with cutaneous involvement, 52% with peripheral neuropathy, 40% with joint involvement, and 40% with renal involvement. With regard to diagnosis, Quartuccio et al have confirmed that the recently proposed preliminary classification criteria also performed well in noninfectious MC.
The therapeutic approach to noninfectious MC is mainly based on the use of conventional immunosuppression, with the aim of suppressing clonal B-cell expansion and the production of cryoglobulins. The immunosuppressive approaches traditionally used in cryoglobulinemic vasculitis-high-dose glucocorticoids and cyclophosphamide-were derived primarily from strategies employed in the treatment of other systemic vasculitides. Azathioprine and mycophenolate mofetil may be used in lieu of cyclophosphamide or following cyclophosphamide as “remission maintenance” agents, although neither medication has been evaluated for this purpose in formal clinical trials. The efficacy of rituximab in noninfectious MC has recently been evaluated in 23 patients, with rituximab demonstrating a clinical and immunologic response in 80%. Another recent study evaluated 209 patients with noninfectious MC who received corticosteroids (all patients), alkylating agents (44%), rituximab (41%), plasmapheresis (21%), and azathioprine or mycophenolate mofetil (15%). Multivariate analysis showed that the combination of corticosteroids and rituximab was more effective than corticosteroids alone at achieving a complete clinical response; the addition of rituximab was associated with a higher rate of severe infection but not with a higher mortality rate. Patients who received corticosteroids in combination with alkylating agents had severe infections less frequently than those who received rituximab, but the efficacy of the former combination was lower. The main factors associated with the development of severe infection were older age, renal failure, and the concomitant use of high doses of corticosteroids. The authors suggested that corticosteroids plus rituximab could be the first-line therapeutic approach in patients with severe noninfectious MC but that corticosteroids should be rapidly tapered with the aim of decreasing the risk of severe infection.
In spite of the complicated etiologic, clinical, and pathologic scenario of cryoglobulinemia, physicians can play a key role in its successful management by early recognition of the most common clinical presentations. A complete workup to assess the different organs that may be affected by cryoglobulinemic disease requires multidisciplinary collaboration led by a specialized autoimmune disease unit. However, some aspects of cryoglobulinemia remain unresolved, such as the etiologic agents involved in essential cryoglobulinemia or the optimal therapeutic approach to patients with refractory/severe disease.
Treatment should be tailored according to the predominant etiopathogenesis (vasculitis vs hyperviscosity), the severity of internal organ involvement, and especially the underlying associated disease (viral, autoimmune, or hematologic). Corticosteroids remain the cornerstone of therapy for any type of cryoglobulinemia, but the results of the principal studies show that high doses of corticosteroids can have deleterious effects, especially in HCV patients, when given over long periods. Therefore, the dose of corticosteroids required to control cryoglobulinemic disease activity should be reduced to the minimum effective dose as rapidly as possible. Rituximab may also be recommended for the treatment of all types of cryoglobulinemia, especially in patients with severe/refractory disease, but its use is currently limited by the lack of US Food and Drug Administration/European Medicines Agency approval for these indications. With respect to antiviral therapies, the addition of new anti-HCV agents (protease inhibitors) to the classic treatment regimen may increase the virologic response by up to 70%, although there is a very high rate of severe adverse events with these regimens; the future use of new generations of anti-HCV agents (combinations of direct-acting antiviral agents) in HCV-related cryoglobulinemia may lead to better tolerability. The use of other drugs, including thalidomide-, lenalinomide-, and bortezomib-based regimens, may be especially useful in patients with type I cryoglobulinemia; B-cell activating factor (BAFF)-blocking agents, interleukin-2 agonists, or Toll-like receptor agonists may be promising future therapies. While there remain three main broad treatment strategies for cryoglobulinemia (conventional immunosuppression, antiviral treatment, and biologic therapy), the most recent studies suggest a change in the therapeutic approach from monotherapy to combined/sequential regimens, including both etiologic- and pathologic-driven therapies, the aim of which is to block the different etiopathogenic pathways involved.
Acknowledgement:The work of Dr. Brito-ZerÃ³n is supported by “Ajut per a la Recerca Josep Font” from Hospital ClÃnic-Barcelona 2012–2015.
The authors have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.
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