Cryoglobulinaemia and HCV

EULAR on-line course on Rheumatic Diseases
Cryoglobulinaemia and
Hepatitis C Virus
Pauline Baudart, Christian Marcelli, Clodoveo Ferri

A previous version was coauthored by Clodoveo Ferri, Maria Teresa Mascia, David
Saadoun, Patrice Cacoub and Marco Sebastiani
LEARNING OBJECTIVES
 Correctly classify/diagnose cryoglobulinaemia and mixed cryoglobulinaemia (MC).
 Use the correct technical procedures to detect and characterise cryoglobulins.
 Describe and explain the main mechanisms involved in the aetiopathogenesis of MC syndrome
(cryoglobulinaemic vasculitis).
 Outline the epidemiology, prognosis and main clinical manifestations of MC syndrome.
 Make a differential diagnosis between MC syndrome and other autoimmune rheumatic disorders
(Sjögren’s syndrome, rheumatoid arthritis, other systemic vasculitides).
 Define the main organ, systemic autoimmune disorders, and neoplastic complications possibly
triggered by hepatitis C virus (HCV) infection, the HCV syndrome.
 Describe and explain the pathogenetic mechanisms of HCV-related autoimmune and
lymphoproliferative disorders.
 List the main levels of intervention (etiological, pathogenetic, and symptomatic) and targets of HCV-
mixed cryoglobulinaemia therapy: clinical response (organ target manifestations), virological response
(HCV RNA) and immunological response (cryoglobulinaemia, C4 serum level).
 Understand that antiviral therapy is the cornerstone of HCV-mixed cryoglobulinaemia treatment.
 State that B cell depleting therapy (rituximab) is an interesting additional therapeutic option.
 Explain the timing of action and the limitations of rituximab.
 Explain concerns about immunosuppressant agent use in HCV-related mixed cryoglobulinaemia.
 Describe the use of steroids, immunosuppressant agents and plasmapheresis.
Cryoglobulinaemia and Hepatitis C Virus – Module 42a
1 Cryoglobulinaemia
1.1 Definition
The presence in the serum of one (monoclonal cryoimmunoglobulinaemia) or more immunoglobulins (mixed
cryoglobulinaemia, MC), which precipitate at temperatures below 37°C and redissolve on rewarming, is
termed cryoglobulinaemia or cryoimmunoglobulinaemia and is an in vitro phenomenon (figure 1) (Ferri,
2002*; Ferri, 2008*). Various hypotheses have been suggested to explain the cold precipitation of
immunoglobulin(s): it can be secondary to the intrinsic characteristics of both mono- and polyclonal
immunoglobulin (Ig) components or can be caused by interaction among single components of the
cryoprecipitate. However, the intimate mechanism(s) of cryoprecipitation remains obscure.
Figure 1 Comparison of cryoglobulinaemic and normal serum samples. Cryoprecipitate, evaluated after 7
days’ storage at 4°C, is present in a serum sample from a patient with mixed cryoglobulinaemia compared
with normal serum (left).
1.2 Classification
Cryoglobulinaemia is usually classified into three subgroups according to immunoglobulin composition (table
1). Type I cryoglobulinaemia consists of only one isotype or subclass of immunoglobulin. Both type II and type
III mixed cryoglobulins are immune complexes (IC) composed of polyclonal IgGs, autoantigens and mono- or
polyclonal IgMs, respectively; the IgMs are the corresponding autoantibodies with rheumatoid factor (RF)
activity. With more sensitive methodologies (ie, immunoblotting or two-dimensional polyacrylamide gel
electrophoresis), type II mixed cryoglobulins often show a microheterogeneous composition; in particular,
oligoclonal IgM or a mixture of polyclonal and monoclonal IgM can be detected (Ferri, 2002*; Ferri, 2008*).
This particular serological subset, termed type II–III MC, could represent an intermediate, evolutive state from
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type III to type II MC. Moreover, type II–III MC could fit together with the most recent molecular studies
showing the presence of oligoclonal B lymphocyte proliferation in liver and bone marrow biopsy specimens
from patients with MC. In two-thirds of patients with type II MC, a cross-idiotype WA monoclonal RF (first
isolated from the serum of a patient with Waldenström’s macroglobulinaemia) has been demonstrated (Ferri,
2002*; Ferri, 2008*).
Table 1 Classification and clinico-pathological characteristics of different cryoglobulinaemias
Classification Composition Pathological findings Clinical associations

Type I Monoclonal Ig, mainly IgG, Tissue histological Lymphoproliferative disorders:
cryoglobulinaemia or IgM, or IgA self- alterations of MM, WM, CLL, B-NHL
aggregation through Fc underlying disorder
fragment of Ig
Type II mixed Monoclonal IgM (or IgG, Leucocytoclastic Infections (mainly HCV),
cryoglobulinaemia or IgA) with RF activity vasculitis B autoimmune/lymphoproliferative
(often cross-idiotype WA- lymphocyte disorders, rarely ‘essential’
mRF) and polyclonal Ig expansion with tissue
(mainly IgG) infiltrates
Type II–III mixed Oligoclonal IgM RF or Leucocytoclastic Infections (mainly HCV),

cryoglobulinaemia mixture of vasculitis B autoimmune/lymphoproliferative
poly/monoclonal IgM lymphocyte disorders, rarely ‘essential’
(often cross-idiotype WA- expansion with tissue
mRF) infiltrates
Type III mixed Polyclonal mixed Ig (all Leucocytoclastic Infections (mainly HCV), more
cryoglobulinaemia isotypes) with RF activity vasculitis B often autoimmune disorders,
of one polyclonal lymphocyte rarely ‘essential’
component (usually IgM) expansion with tissue
infiltrates
B-NHL, B cell NHL; CLL, chronic lymphocytic leukaemia; HCV, hepatitis C virus; Ig, immunoglobulin; MM,
multiple myeloma; NHL, non-Hodgkin’s lymphoma; RF, rheumatoid factor; WM, Waldenström’s
macroglobulinaemia.
Type I cryoglobulinaemia is usually associated with well-known haematological disorders but is often itself
asymptomatic (Ferri, 2002*; Ferri, 2008*). Similarly, circulating mixed cryoglobulins can be detected in a great
number of infectious or systemic disorders; generally they represent an isolated laboratory finding with no
clinical consequences. In contrast, ‘essential’ MC represents a distinct disorder, which is classified as one of the
systemic vasculitides. Cryoglobulinaemic vasculitis (figure 2) is secondary to vascular deposition of circulating
IC, mainly cryoglobulins and complement, with the possible contribution of both haemorheological and local
factors. According to its clinical and histological features, MC is included in the subgroup of small-vessel
systemic vasculitides, which also include cutaneous leucocytoclastic vasculitis and Henoch–Schönlein purpura
(table 2).
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Table 2 Proposed classification of systemic vasculitides

Vessels Primary Secondary
Large–medium Takayasu’s arteritis, giant cell Infectious arteritis (syphilis), rheumatoid arthritis
arteries arteritis
Medium arteries Kawasaki’s arteritis, RA, SLE, SSc, PM/DM, Behçet’s syndrome, HBV+,
polyarteritis nodosa SS
Medium–small Granulomatosis with Cogan syndrome, recurrent polychondritis, drugs,
arteries polyangiitis (Wegener’s), HBV, HCV, HIV infection
eosinophilic granulomatosis
with polyangiitis (Churg–
Strauss syndrome),
microscopic polyangiitis
Small arteries, ‘Essential’ MC (<10%),* MC (HCV+ 90%; HBV+ <5%),* HIV infection,
capillaries, venules cutaneous leucocytoclastic others, drugs, lymphoproliferative disorders
vasculitis, Henoch–Schönlein
purpura
*The percentages refer to a Italian mixed cryoglobulinaemia series.
HBV, hepatitis B virus; HCV, hepatitis C virus; MC, mixed cryoglobulinaemia; PM/DM,
polymyositis/dermatomyositis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SS, Sjögren’s
syndrome; SSc, systemic sclerosis.
Figure 2 Cutaneous manifestations of mixed cryoglobulinaemia (MC). (A) Recent-onset orthostatic purpura;
at this stage the histopathological evaluation shows (B) the classic necrotising leucocytoclastic vasculitis
characterised by diffuse fibrinoid necrosis and disintegrated neutrophil permeation of the vessel walls. (C)
Symmetrical hyperpigmentation of the skin on the legs after repeated episodes of purpura, (D) severe
vasculitic manifestation and (E) a large skin ulcer, often resistant to treatment. Both orthostatic purpura (A)
and permanent ochraceous lesions (C) are typical skin manifestations of MC.
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Leucocytoclastic vasculitis is the histopathological hallmark of MC (figure 2). It may involve small- and medium-
sized vessels and may be responsible for multiple organ involvement (Ferri, 2002*; Ferri, 2008*). The term
‘cryoglobulinaemic vasculitis’ is often used as a synonym and better describes the typical histopathological
alterations commonly detectable in the biopsy of cutaneous lesions.
2 Mixed cryoglobulinaemia (cryoglobulinaemic vasculitis)
2.1 Epidemiology
MC is considered to be a rare disorder, but no adequate epidemiological studies of its overall prevalence have
been carried out. Numerous cohort studies of series of patients from different countries suggest that the
prevalence of MC is geographically heterogeneous; the disease is more common in southern Europe than in
northern Europe or North America. MC is characterised by clinical polymorphism – a single manifestation (skin
vasculitis, hepatitis, nephritis, peripheral neuropathy, etc) is often the only apparent or clinically predominant
feature – so that patients with MC are often referred to different specialties (figure 3). Consequently, a correct
diagnosis might be delayed or overlooked entirely. Thus, we can understand why the true prevalence of MC
might be underestimated (Ferri, 2002*; Ferri, 2008*; Ferri, 2016).
Figure 3 Distribution of patients with mixed cryoglobulinaemia (MC) among different medical specialties.
Given its clinical polymorphism, MC syndrome may develop with different, often unpredictable symptoms.
Consequently, patients with MC may be referred to different specialties according to the prevalent or
apparently unique clinical manifestations, such as membranoproliferative glomerulonephritis (MPGN) or
purpuric skin lesions. Patients with very mild manifestations, often arthralgias and/or serum rheumatoid
factor (RF) positivity, are generally referred to a rheumatology clinic. phen, phenomenon.
For the above reasons, mainly the frequent dispersion of patients among different specialties, symptom
composition in MC syndrome may vary greatly among patient series from various tertiary care facilities (figure
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3). Overall, MC is more common in women than in men (female to male ratio of 3:1), disease onset is
particularly common in the fourth to fifth decades and in older people, and the disease is rarely seen in young
people.
The epidemiology of MC associated with hepatitis C virus (HCV) infection has been examined in cohort studies
focusing on HCV-infected subjects, which reported low levels of circulating mixed cryoglobulins in over 50% of
cases, while overt cryoglobulinaemic syndrome developed in about 5%. HCV infection is particularly diffuse
worldwide; therefore, an increasing incidence of some late complications of HCV infection, such as MC, could
be observed, especially in underdeveloped countries where HCV is prevalent in the general population.
In contrast, ‘essential’ MC is generally seen in a significantly lower proportion of patients with MC, being quite
rare in some geographical areas, such as southern Europe (figure 4).
Figure 4 Geographical distribution of some hepatitis C virus (HCV)-related and non-HCV-related disorders.
The prevalence of HCV-related disorders, such as mixed cryoglobulinaemia (cryoglobulinaemic vasculitis
(Cryo. vasc.) and B cell non-Hodgkin’s lymphoma (B-NHL)) shows large geographical heterogeneity. For
example, these disorders are more prevalent in southern than in northern Europe, in contrast to the
distribution seen for other diseases, such as some systemic vasculitides (granulomatosis with polyangiitis
(GPA) and polyarteritis nodosa (PAN)).
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2.2 Aetiopathogenesis
Several clinico-epidemiological studies reported that chronic hepatitis was one of the most common
symptoms of MC; its prevalence progressively increases to over two-thirds of patients during the clinical
course of the disease. This observation suggested a possible role for hepatotropic viruses in the pathogenesis
of the disease and so a causative role for hepatitis B virus (HBV) has been sought since the 1970s. The same
virus has been just correlated with another systemic vasculitis, namely, polyarteritis nodosa. However, HBV
viraemia was rarely recorded, while anti-HBV antibodies varied among different groups of patients with MC
(Ferri et al, 2002*; Ferri, 2008*). HBV may be a causative factor of MC in <5% of people (figure 5).
Figure 5 Schematic representation of different clinical and virological subsets of mixed cryoglobulinaemia
(MC) syndrome. (1) 'Essential' MC (EMC); (2,3) EMC and hepatitis C virus (HCV)-associated MC syndrome in
the setting of definite autoimmune-lymphoproliferative disorders (ALD), such as autoimmune hepatitis,
Sjögren’s syndrome or B cell lymphomas; (4) the most common subset of HCV-associated MC syndrome; and
(5) MC associated with other infectious agents such as hepatitis B virus (HBV).
In 1989, the discovery of HCV as the major aetiological agent of non-A-non-B chronic hepatitis, was crucial for
the aetiopathogenetic studies of MC syndrome. A possible role of HCV infection in MC was proposed
independently by two pioneering reports which showed a significantly higher prevalence of serum anti-HCV
antibodies in these patients than in the general population (Ferri, 2015* review). This hypothesis was deeply
confirmed in 1991, when the presence of HCV RNA was detected by polymerase chain reaction (PCR) in 86% of
Italian patients with MC (Ferri, 1991*). Following this, an increasing number of studies including clinico-
epidemiological observations, as well as both histopathological and virological investigations (HCV RNA
detection by PCR and/or in situ hybridisation) established the important role of HCV in the pathogenesis of
MC. The prevalence of serum anti-HCV antibodies and/or HCV RNA in patients with MC ranged from 70% to
almost 100% among different patient populations. Given the striking association between MC and HCV
infection, the term ‘essential’ is now used to refer to a minority of patients with MC (in Italy <10%; figure 5)
(Ferri, 2002*; Ferri, 2008*).
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The clinical development of MC is closely linked to the natural history of chronic HCV infection. MC
phenotypes are also the result of genetic and/or environmental cofactors, which remain largely unknown
(figure 6). HCV has been recognised to be both a hepato- and lymphotropic virus, as suggested by the presence
of active or latent viral replication in the peripheral lymphocytes of patients with type C hepatitis or MC. HCV is
an RNA virus without reverse transcriptase activity; therefore, the viral genome cannot integrate in the host
genome. It is probable that HCV chronically stimulates the immune system through different viral proteins,
such as core protein. Chronic stimulation of the lymphatic system exerted through viral epitopes, autoantigen
production and/or a molecular mimicry mechanism has been suggested. In this respect, particularly interesting
is the presence, in HCV-positive patients, of anti-GOR antibodies, which are cross-reactive autoantibodies
directed both to the HCV core and a nuclear antigen called GOR. Another pathogenetic hypothesis suggested
that HCV, in association with very low-density lipoprotein (LDL), might induce a T-independent primordial B
cell population, producing monoclonal immunoglobulin with WA idiotype. In turn, HCV–very LDL complexes
may trigger RF production as a consequence of somatic mutations of WA clones; the possible evolution to B
cell lymphomas might be the consequence of the accumulation of stochastic genetic aberrations. The chronic
stimulation of B lymphocytes by HCV epitopes may cause expansion of some B cell subpopulations with
favourable and/or dominant genetic characteristics (Ferri et al, 2002*; Ferri, 2008*). This hypothesis recalls the
pathogenetic role of Helicobacter pylori in MALT lymphoma of the stomach, for which different evolutive
phases are required.
The interaction between the HCV E2 envelope protein and CD81 molecule, a ubiquitous tetraspannin present
on the surface of B cells, may represent another important pathogenetic factor in MC. The consequence of this
interaction may be a strong and sustained polyclonal stimulation of the B cell compartment (figure 6). A
following pathogenetic step of HCV-related autoimmune-lymphoproliferative disorders may be the t(14;18)
translocation found in B cells of HCV-infected subjects. Even if not definitely confirmed, the t(14;18)
translocation may lead to increased expression of Bcl-2 protein with consequent inhibition of apoptosis and
abnormally prolonged B cell survival. Interestingly, a significantly high prevalence of t(14;18) translocation is
found in patients with HCV-related MC (85% in type II MC), and also in patients with isolated hepatitis type C
(about 37–38%). It can be hypothesised that during chronic HCV infection, several factors (including the
interaction between the HCV E2 protein and CD81 molecule, the high viral variability and the persistent
infection of both hepatic and lymphatic cells) may favour a sustained and strong B cell activation (figure 6).
This latter may in turn favour the t(14;18) translocation and Bcl-2 overexpression; the consequent B
lymphocyte expansion is responsible for autoantibody production, including the cryoglobulins. In addition, the
prolonged B cell survival may represent a predisposing condition for further genetic aberrations, which may
lead to frank B cell malignancy as a late complication of MC syndrome (Ferri et al, 2002*; Ferri, 2015*).
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Of interest, HCV-driven lymphoproliferation may also explain the pathogenetic role of HCV infection in
‘idiopathic’ B cell lymphomas. This association was first described in unselected Italian patients with idiopathic
B cell lymphomas and later confirmed by different epidemiological and laboratory studies, mainly in the same
geographical areas as those in which HCV-associated MC is commonly found (figure 4).
Given its biological characteristics, HCV may be involved in many autoimmune and lymphoproliferative
disorders. Figure 6 summarises the main causative factors – infectious, toxic, genetic and/or environmental –
that are potentially involved in the pathogenesis of MC. These factors, alone or in combination, may trigger
two multistep pathogenetic processes, not mutually exclusive, responsible for MC and other HCV-related
disorders. The first process produces a ‘benign’ polyoligoclonal B lymphocyte proliferation responsible for
organ- and non-organ-specific autoimmune disorders, including immune-complex-mediated cryoglobulinaemic
vasculitis; the second is characterised by different oncogenetic alterations, which ultimately may lead to
malignant complications. Comparable pathogenetic mechanisms could be also hypothesised for HCV-negative
MC; this intriguing clinical subset might be correlated with other infectious agents or associated with some
well-known autoimmune/rheumatic or lymphoproliferative disorders (figures 5 and 6) (Ferri et al, 2002*; Ferri,
2015*).
MC with or without overt clinical syndrome has been reported in patients with a great number of infectious
agents, usually as anecdotal observations. A significant prevalence of MC has been found in patients with
human immunodeficiency virus (HIV) infection, with and without HCV co-infection. HIV alone may exert a
continuous antigenic stimulation of B lymphocytes; these latter may be responsible for type III MC production
earlier in the course of HIV infection. In some patients, the B cell disorder may evolve into monoclonal MC
with a typical clinical syndrome. As observed for HCV infection, the prevalence of other virus-related MC is
variable among patient series from different geographical areas (figure 4). Moreover, a number of clinico-
epidemiological studies showed a heterogeneous distribution of different HCV-related extrahepatic
manifestations, including some autoimmune disorders such as primary Sjögren’s syndrome (pSS) (Ferri et al,
2002*; Ferri et al, 2007*; Ferri, 2015*).
Cryoglobulinaemic syndrome may share a number of aetiopathogenetic events and clinical features with both
autoimmune diseases such as autoimmune hepatitis (AIH), Sjögren’s syndrome and polyarthritis, and B cell
lymphomas. Therefore, a differential diagnosis should be carefully made in all patients with MC syndrome
(figure 7) (see also section 2.5); correct disease classification may decisively affect the overall clinico-
therapeutic approach and prognosis.
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Figure 6 Aetiopathogenesis of mixed cryoglobulinaemia (MC) and other HCV-related disorders: the HCV
syndrome. The figure summarises the putative mechanisms involved in the aetiopathogenetic cascade of MC
and other HCV-related disorders. This is probably a multifactorial and multistep process: the remote events
include some infectious agents, mainly HCV (see also Fig. 5), predisposing host factors and, possibly,
unknown environmental/toxic triggers. Viral antigens (eg, HCV core, envelope E2, NS3, NS4, NS5A proteins)
may exert a chronic stimulus on the host immune system through specific lymphocyte receptors, such as
CD81, which may interact with the viral E2. Predisposing host factors may include particular HLA alleles, and
metabolic and hormonal conditions. The main consequence is a ‘benign’ B cell proliferation with a variety of
autoantibodies produced, among which are rheumatoid factor (RF), and cryo- and non-cryoprecipitable
immune complexes (IC). These serological alterations may be correlated with different organ- and non-
organ-specific autoimmune disorders, including MC syndrome (or cryoglobulinaemic vasculitis). Moreover,
the activation of Bcl2 proto-oncogene, responsible for prolonged B cell survival, may be a predisposing
condition to other genetic aberrations, which may lead to frank B cell lymphomas and other malignancies.
The appearance of malignant neoplasias can be seen in a small but significant percentage of patients,
usually as a late complication. Both immunological and neoplastic disorders show a clinico-serological and
pathological overlap. Often, autoimmune organ-specific manifestations may evolve to systemic conditions,
such as MC, and less frequently to overt malignancies (right). Conversely, it is not uncommon that patients
with malignancies develop one or more autoimmune manifestations. In this scenario, MC syndrome is at the
junction between autoimmune and neoplastic disorders. HCV, hepatitis C virus; IC, immune complexes; PCT,
porphyria cutanea tarda ; RF, rheumatoid factor; sicca s., sicca syndrome.
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Figure 7 Clinical overlap and differential diagnosis among some possible hepatitis C virus (HCV)-related
rheumatic diseases. Mixed cryoglobulinaemia (MC) syndrome, primary Sjögren’s syndrome (SS) and
rheumatoid arthritis (RA) show a clinico-pathological overlap, including a possible association with HCV
infection. The following information may be useful for a correct differential classification/diagnosis: primary
SS shows a typical histopathological pattern of salivary gland involvement (inv.) and specific autoantibodies
(anti-RoSSA/LaSSB), which are rarely found in patients with MC; conversely, cutaneous leucocytoclastic
vasculitis, visceral organ involvement (glomerulonephritis, hepatitis), low C4 and HCV infection, are typically
found in MC. Moreover, erosive symmetrical polyarthritis and serum anticyclic citrullinated peptide
antibodies (anti-CCP) characterise classic RA. Finally, B cell non-Hodgkin’s lymphoma (B-NHL) may
complicate these diseases, more frequently MC and primary SS. B-NHL may be suspected following careful
clinico-serological monitoring and diagnosed by bone marrow/lymph node biopsies and total body CT scan.
RF, rheumatoid factor.
2.3 Clinical manifestations
MC syndrome, or cryoglobulinaemic vasculitis, is characterised by a clinical triad of purpura, weakness and

arthralgias, and by a variable combination of symptoms, including chronic hepatitis, membranoproliferative
glomerulonephritis (MPGN), peripheral neuropathy, skin ulcers, diffuse vasculitis and, less frequently,
lymphatic and hepatic malignancies. The clinical pattern of cryoglobulinaemic vasculitis is comparable in
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patients with type II or type III MC. Table 3 gives the prevalence of MC manifestations in an Italian patient
population referred to our university-based division of rheumatology. Patient recruitment at different
specialist centres may influence the symptom composition of MC series; for example, patients with MC
recruited at nephrology units naturally show a higher percentage of glomerulonephritis compared with those
seen at rheumatology units (figures 3 and 13). This factor together with the different geographical origins of
patient series reported in the literature may be responsible for the variable prevalence of individual MC
symptoms (Ferri, 2015*).
Table 3 Demographic, clinico-serological and virological features of 250 patients with mixed
cryoglobulinaemia evaluated at the end of patient follow-up
Features Value
Epidemiological features
Age at disease onset (years), mean (SD) (range) 54 (13) (29–72)
Female:male ratio 3
Disease duration (years), mean (SD) (range) 12 (10) (1–40)
Clinical features
Purpura 98
Weakness 98
Arthralgias 91
Arthritis (non-erosive) 8
Raynaud’s phenomenon 32
Sicca syndrome 51
Peripheral neuropathy 81
Renal involvement* 31
Liver involvement 73
B cell non-Hodgkin’s lymphoma 11
Hepatocellular carcinoma 3
Serological and virological features
Cryocrit (%), mean (SD) 4.4 (12)
Type II/type III mixed cryoglobulins 2/1
C3 (mg/dL), mean (SD) (normal 60–130) 93 (30)
C4 (mg/dL), mean (SD) (normal 20–55) 10 (12)
Antinuclear antibodies 30
Antimitochondrial antibodies 9
Antismooth muscle antibodies 18
Antiextractable nuclear antigen antibodies 8
Anti-HCV antibodies ± HCV RNA 92
Anti-HBV antibodies 32
HBsAg 1
HCV-/HBV- 7
Results are shown as percentages unless otherwise stated.
*Invariably membranoproliferative glomerulonephritis.
HBV, hepatitis B virus; HCV, hepatitis C virus.
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At patient anamnesis, the presenting symptoms of MC vary greatly among cryoglobulinaemic patients;
similarly, at the first examination, MC shows different clinico-serological patterns, varying from apparently
isolated serum mixed cryoglobulins, in some cases associated with mild manifestations such as arthralgias
and/or sporadic purpura, to severe cryoglobulinaemic syndrome with multiple organ involvement (figure 8).
The disease shows a combination of serological findings (mixed cryoglobulins with RF activity and frequently
low C4) and clinico-pathological features (purpura and leucocytoclastic vasculitis with multiple organ
involvement). In some chronically HCV-infected subjects, asymptomatic serum mixed cryoglobulins can be
found. This condition may precede the clinical onset of disease by years or decades. On the other hand, some
patients show typical cryoglobulinaemic syndrome without serum cryoglobulins, the hallmark of the disease
(figure 8). MC is characterised by a large amount of cryoprecipitable IC, with the cryoglobulins representing a
variable percentage of them among different patients as well as in the same patient during follow-up.
Therefore, the absence of serum cryoglobulins may be a transient phenomenon owing to this variable
percentage of cryoprecipitable IC; repeated cryoglobulin determinations are necessary for a correct diagnosis
in these subjects (Ferri et al, 2002*; Ferri, 2008*).
Figure 8 Different clinico-serological patterns of mixed cryoglobulinaemia (MC). (1) Definite MC syndrome
(or cryoglobulinaemic vasculitis) is a combination of serological findings (mixed cryoglobulins with
rheumatoid factor (RF) activity and often low C4) and typical clinico-pathological features (purpura,
leucocytoclastic vasculitis and frequent multiple organ involvement) (also see table 3). However, incomplete
MC syndrome can be seen at any time during the natural history of the disease. In particular, (2) isolated
serological alterations may be detected in the early stages of the disease or during clinical remission. In
contrast, (3) the absence of serum cryoglobulins in patients with overt MC syndrome may be a transient
phenomenon due to the wide variability in the percentage of cryoprecipitable immune complexes (IC) during
the natural history of the disease or, less frequently, to switching from ‘benign’ B cell lymphoproliferation to
malignant lymphoma.
The most common manifestations of MC are cutaneous lesions. Orthostatic purpura is generally intermittent,
with the dimensions and diffusion of purpuric lesions varying greatly from sporadic isolated petechiae to
severe vasculitic lesions, often complicated by torpid ulcers of the legs and malleolar areas (figure 2). In a
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significant proportion of patients, repeated episodes of purpura may lead to stable, often confluent, areas of
ochraceous colouration on the legs (figure 2). Cutaneous manifestations, in particular orthostatic purpura and
ulcers, are the direct consequence of vasculitic alterations with the possible contributions of various cofactors,
in particular chronic venous insufficiency and physical stress, mainly prolonged standing and/or muggy
weather. In addition, haemorheological disturbances due to high cryocrit levels may also be a contributing
factor. In this respect, the purpuric outbreaks are often seen late in the afternoon when the highest cryocrit
levels are generally found, often following prolonged standing (Ferri et al, 2002*; Ferri, 2008*).
Patients with MC frequently have arthralgias, while clear signs of arthritis (usually mild, non-erosive
oligoarthritis) are less often seen.
Almost half of the patients with MC complain of mild sicca syndrome, that is, xerostomia and xerophthalmia;
however, only a few cases meet the current criteria for the classification of pSS (see section 2.5).
Peripheral neuropathy may frequently complicate the clinical course of MC, in most cases as mild sensory
neuritis. The common symptoms are paraesthesias with painful and/or burning sensations in the lower limbs,
often with nocturnal exacerbation. The patient’s quality of life may be severely compromised because of the
chronicity of these symptoms together with their poor response to treatment. In a minority of cases, the
peripheral neuropathy may be complicated by severe sensorimotor manifestations, which usually appear
abruptly, often as asymmetric mononeuritis. In some patients, peripheral neuropathy may complicate
interferon (IFN) α treatment, possibly in predisposed subjects, often during the first weeks of antiviral therapy.
Central nervous system involvement, including dysarthria and hemiplegia, is rarely seen; in older patients, it is
often difficult to distinguish these symptoms from the most common atherosclerotic vascular manifestations
(Ferri et al, 2002*; Ferri, 2015*; Ferri, 2016*).
In over two-thirds of patients (table 3), overt chronic hepatitis, generally with a mild to moderate clinical
course, can be seen throughout the natural history of the disease. This manifestation, uncommon in other
systemic vasculitides, is the direct consequence of HCV infection that represents the underlying disorder in
MC. In our experience, chronic hepatitis may evolve to cirrhosis in one-quarter of patients, while only seven
patients (3%, 7/250; see table 3) developed hepatocellular carcinoma. In a few cases, especially in patients
with renal failure due to chronic glomerulonephritis, hepatorenal syndrome develops as a major life-
threatening complication. On the whole, the clinical course and prognostic value of chronic hepatitis seem to
be less severe than for classic type C hepatitis without MC syndrome; similarly, hepatocellular carcinoma less
often complicates MC syndrome compared with the total population of HCV-positive subjects. These
differences are quite intriguing, but difficult to fully explain. It may be that patients with MC characterised by a
relatively low prevalence of HCV genotype 1b, along with a lower median consumption of alcohol, develop a
rather benign clinical course of liver involvement.
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MPGN type 1 is another important complication, which may severely affect the prognosis and survival of
patients with the disease. MC-related nephropathy is a typical IC-mediated glomerulonephritis, although other
immunological mechanisms have also been hypothesised.
Widespread vasculitis involving medium to small arteries, capillaries and venules with multiple organ
involvement may develop in a small proportion of patients. This extremely severe complication may involve
the skin, kidney, lungs, central nervous system and gastrointestinal tract. In rare cases, intestinal vasculitis may
suddenly complicate the disease, often in patients with renal and/or liver disease; pain simulating an acute
abdomen is the presenting symptom of intestinal vasculitis. A timely diagnosis and aggressive treatment are
necessary for this life-threatening complication.
Interstitial lung disease has been anecdotally observed in MC syndrome as well as in patients with isolated
HCV infection. Almost invariably, lung involvement in MC is characterised by subclinical alveolitis, as
demonstrated by bronchoalveolar lavage in an unselected patient series; this condition may predispose to
pulmonary infectious complications and, in rare cases, may lead to clinically evident interstitial lung fibrosis.
The hyperviscosity syndrome due to high levels of serum cryoglobulins is another rare clinical manifestation of
MC, although haemorheological alterations may contribute to some clinical symptoms such as orthostatic
purpura, skin ulcers and renal involvement.
Generally, there are no associations between the severity of clinical symptoms, such as glomerulonephritis,
skin ulcers and/or diffuse vasculitis, and the serum levels of cryoglobulins and/or haemolytic complement
(Ferri et al, 2002*; Ferri, 2008*). Low complement activity is almost invariably detected in MC. It is
characterised by a typical pattern independently of disease activity; namely, low or undetectable C4 with
normal or slightly reduced C3 serum levels (table 3). Moreover, in vitro consumption of complement can also
be seen owing to the anticomplement activity of some cryoimmunoglobulins. In clinical practice, it is
interesting to note sudden variations in C4, rising from very low to abnormally high levels, in some patients
with MC developing a B cell lymphoma. The lack of correlation between circulating cryoglobulin levels and the
severity/activity of MC manifestations might be explained by different hypotheses: the pathogenic role of
other non-cryoprecipitable IC, their intrinsic ability to activate the complement and/or the in situ formation of
IC, with a relative concentration of HCV virions.
Some endocrinological disorders are significantly more common in patients with MC than in the general
population, including thyroid disorders, diabetes and gonadal dysfunction. The most common thyroid
disorders are autoimmune thyroiditis, subclinical hypothyroidism and thyroid cancer; while hyperthyroidism is
less common, it may appear as a reversible complication of IFN treatment. Moreover, a significantly increased
incidence of diabetes mellitus type 2 has been found in HCV-positive patients with and without MC syndrome
compared with the general population. Finally, HCV-positive men with or without cryoglobulinaemic vasculitis
©2007-2018 EULAR 15
may develop erectile dysfunction, attributable to hormonal or neurovascular alterations, or both (Ferri et al,
2002*; Ferri, 2008*).
B cell lymphoma is the most common malignancy found, often as a late manifestation of MC syndrome. This
complication may be related to peripheral B lymphocyte expansion and to lymphoid infiltrates found in the
liver and bone marrow, which represent the pathological substrate of the disease. In particular, these
infiltrates have been regarded by some authors as ‘early lymphomas’, since they are sustained by lymphoid
components indistinguishable from those of B cell chronic lymphocytic leukaemia/small lymphocytic
lymphoma (B-CLL) and immunocytoma. However, unlike frank malignant lymphomas, they tend to remain
unmodified for years or even decades and are followed by overt lymphoid tumours in approximately 10% of
cases. These characteristics justify the proposed term ‘monotypic lymphoproliferative disorder of
undetermined significance (MLDUS)’. Interestingly enough, the incidence of type II MC-related MLDUS is
highest in those geographical areas where about 30% of patients with ‘idiopathic’ B cell lymphomas also
display HCV positivity and where an increased prevalence of HCV genotype 2a/c has been found in both MC
and lymphomas (figure 4). Type II MC-associated MLDUS presents two main pathological patterns, namely, the
B-CLL-like and the immunocytoma-like. In clinical practice, malignant B cell lymphomas are often seen in
patients with MC with a mild clinical course, sometimes unexpectedly during a routine evaluation. It is possible
to observe a sudden decrease or disappearance of serum cryoglobulins and RF, sometimes associated with
abnormally high levels of C4 as the presenting symptom of complicating B cell malignancy (Ferri et al, 2002*;
Ferri, 2008*).
Other neoplastic manifestations, for example, hepatocellular carcinoma or papillary thyroid cancer, are less
often seen. Thus, MC can be regarded as a pre-neoplastic disorder; consequently, careful clinical monitoring is
recommended, even in the presence of mild MC syndrome.
2.4 Diagnosis
To date there are no available diagnostic criteria for MC; in 1989 the Italian Group for the Study of
Cryoglobulinaemias proposed preliminary criteria for MC classification, later revised with the inclusion of
clinico-pathological and virological findings. This classification is mainly based on the serological and clinical
hallmarks of the disease, namely, circulating mixed cryoglobulins, low C4 and orthostatic skin purpura.
Moreover, leucocytoclastic vasculitis, involving medium and, more often, small blood vessels (arterioles,
capillaries and venules) is the typical pathological finding of affected tissues. It is easily detectable by a skin
biopsy of recent vasculitic lesions (within the first 24–48 h).
More recently, classification criteria for cryoglobulinaemic vasculitis have been validated by a cooperative
study using a standardised methodology from a larger number of countries; these criteria may be usefully
©2007-2018 EULAR 16
employed in epidemiological and clinico-pathogenetic studies, as well as in therapeutic trials (Quartuccio,

2014*).
In all cases, the detection of cryoglobulins in the serum is necessary for a definite classification of MC
syndrome (figure 1) and their characterisation as type II (IgG+IgM monoclonal) or type III (IgG+IgM polyclonal)
mixed cryoglobulins (table 3). Unfortunately, there are no universally accepted methodologies for cryoglobulin
measurement, but simple standardised indications are often sufficient for testing for cryoglobulinaemia.
Cryoglobulins are characterised by high thermal instability. For a correct evaluation of serum cryoglobulins, it
is necessary to avoid false-negative results due to immunoglobulin cold precipitation which also occurs at
room temperature. Blood sampling for cryoglobulin detection should be carried out immediately after blood is
drawn or blood should be rapidly transported to the laboratory using a thermostable device (37°C). In general,
to avoid the possible loss of cryoglobulins, the first steps (blood sampling, clotting and serum separation by
centrifugation) should always be carried out at 37°C. In contrast, isolated serum for cryoglobulin
determination and characterisation should be managed at 4°C. The serum with cryoglobulins should be tested
for reversibility of the cryoprecipitate by re-warming an aliquot at 37°C for 24 h. Cryocrit measurement is
usually carried out in a serum sample stored at 4°C for 7 days. The cryocrit corresponds to the percentage of
packed cryoglobulins with reference to the total serum after centrifugation at 4°C (figure 9); it should be
determined on blood samples without anticoagulation to avoid false-positive results due to cryofibrinogen or
heparin-precipitable proteins. Without the above relatively simple precautions, the quantities of cryoglobulins
measured will be incorrect and the test may completely fail to detect cryoglobulins (Ferri et al, 2002*; Ferri,
2008*).
After isolating and washing the cryoprecipitate, the cryoglobulin components can be identified by
immunoelectrophoresis or immunofixation. These analyses must be performed at 37°C to avoid precipitation
and hence loss of the cryoglobulin during the procedures. More sophisticated methodologies, such as
immunoblotting or two-dimensional polyacrylamide gel electrophoresis, may be used for laboratory
investigations. Although the detection of serum cryoglobulins is fundamental for the diagnosis of MC, the
levels of serum cryoglobulins usually do not correlate with the severity and prognosis of the disease. Very low
levels of cryocrit, often difficult to quantify, can be associated with severe and/or active cryoglobulinaemic
syndrome; in contrast, high cryocrit values may characterise a mild or asymptomatic disease course. In rare
cases, very high cryocrit levels, possibly associated with a cryogel phenomenon, may be associated with classic
hyperviscosity syndrome. A sudden decrease or disappearance of serum mixed cryoglobulins, with or without
abnormally high levels of C4, should be regarded as an alarming signal of complicating B cell malignancy (Ferri
et al, 2002*; Ferri et al, 2015*; Ferri, 2016*).
©2007-2018 EULAR 17
Figure 9 Isolation and measurement of serum cryoglobulins. Graduated glass tubes with serum samples from
a cryoglobulinaemic patient at different time intervals: 0, soon after serum separation from the blood
sample (at least 20 mL of whole blood); 7, after 7 days at 4°C; and centrifugation of the serum for cryocrit
measurement, always at 4°C.
Box 1 summarises the clinico-serological investigations at a patient’s first evaluation in order to classify the MC
syndrome correctly and to identify possible overlapping disorders (figure 7) (see section 2.5) or comorbidities,
or both. The prevalence of these latter, in particular atherosclerosis, may be correlated with the disease
duration, and with cumulative side effects of prolonged treatments. Diagnosis and monitoring of the major MC
manifestations is essential for their timely treatment, especially for life-threatening liver, renal and/or
neoplastic complications.
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Box 1 Clinico-diagnostic assessment of mixed cryoglobulinaemia syndrome (see also Ref. Ferri et al, 2016*)
Clinical and serological investigations at a patient’s first evaluation:

• Past clinical history, physical examination
• Chest X-ray examination, ECG, abdominal ultrasonography (US), blood chemistry and urine analysis
• Cryoglobulin detection and characterisation (see table 1)
• Rheumatoid factor, C3 and C4, antinuclear antibodies (abs), antiextractable nuclear antigen abs,
antineutrophil cytoplasmic abs, antismooth muscle abs, antimitochondrial abs, antiliver/kidney
microsome type 1 abs, other abs
• Virological markers: hepatitis C virus (genotyping), hepatitis B virus, others
• Evaluate possible comorbidities (cardiovascular, endocrine/metabolic, etc)
• Mixed cryoglobulinaemia (MC) classification (definite, essential, secondary)
Diagnosis and monitoring of major MC complications
• Chronic hepatitis, cirrhosis, hepatocellular carcinoma:
o Monitoring (every 6–12 months) of alanine aminotransferase, alkaline phosphatase
o Liver US (biopsy, CT scan)
• Glomerulonephritis:
o Monitoring of urine analysis and serum creatinine (kidney US, biopsy)
• Peripheral neuropathy:
o Clinical monitoring
o Electro-neuro-physiological studies
• Skin ulcers:
o Exclusion of vascular comorbidities (arteriovenous Doppler evaluation)
• Sicca syndrome:
o Differential diagnosis with primary Sjögren’s syndrome
• Arthritis:
o Differential diagnosis with rheumatoid arthritis
• Thyroid involvement:
o Hormones
o Autoantibodies
o Neck US
o Fine-needle aspiration
• B cell lymphoma:
o Clinical monitoring
o Bone marrow/lymph node biopsies
o Total body CT scan
2.5 Differential diagnosis
Initially, the term ‘essential’ referred to MC as an autonomous disease once other well-known systemic,
infectious or neoplastic disorders had been ruled out by a wide clinico-serological investigation. However, in
some patients a definite diagnosis may be difficult because of the clinical polymorphism of the MC. Moreover,
the association of MC with HCV infection may further complicate the differential diagnosis as there is frequent
clinico-pathological overlapping among different HCV-related disorders. Cryoglobulinaemic syndrome can
represent a crossroads between some autoimmune diseases (AIH, Sjögren’s syndrome, polyarthritis,
glomerulonephritis, thyroiditis, type 2 diabetes, etc) and malignancies (B cell lymphomas, hepatocellular
carcinoma) (Ferri et al, 2002*; Ferri et al, 2015*; Ferri, 2016*). We can observe in the same patient a slow
©2007-2018 EULAR 19
progression from mild HCV-associated hepatitis to various extrahepatic manifestations (arthralgias, sicca
syndrome, Raynaud’s phenomenon, RF positivity, etc) and, ultimately, to overt MC syndrome with typical
clinico-serological manifestations. In a minority of patients with MC, a malignancy may develop, generally after
a long follow-up period. Therefore, a careful patient evaluation is necessary for a correct diagnosis of MC
syndrome, particularly to differentiate it from other RF-positive, systemic rheumatic disorders such as
rheumatoid arthritis (RA) and pSS (figure 7).
Arthralgias are one of the most common symptoms, while clear signs of synovitis are quite rare. Patients may
develop mild, non-erosive oligoarthritis, often sensitive to low doses of glucocorticoids with or without
hydroxychloroquine. In contrast, rheumatoid-like polyarthritis is more common in patients with HCV-related
hepatitis without MC syndrome. In patients with HCV-associated MC and symmetrical, erosive polyarthritis,
the diagnosis of overlapping MC/RA syndrome can be suspected. In these cases, the detection of serum
anticyclic citrullinated peptide antibodies, markers of classic RA, may be a useful tool for the differential
diagnosis.
Sicca syndrome may be suspected in about half of the patients with MC; however, current criteria for the
classification of pSS are satisfied in only a few cases. MC and pSS may share various symptoms: xerostomia
and/or xerophthalmia, arthralgias, purpura, RF and serum cryoglobulins and possible complication with B cell
lymphoma. However, a careful patient clinical assessment is usually sufficient for a correct diagnosis in the
large majority of cases following consideration of some important findings: histopathological alterations of the
salivary glands and the specific autoantibody pattern (anti-Ro/SSA-La/SSB) of pSS are rarely found in patients
with MC; conversely, HCV infection, cutaneous leucocytoclastic vasculitis and visceral organ involvement
(MPGN type 1, chronic hepatitis) are seldom recorded in pSS (figure 7). In view of the above considerations, it
has been recently proposed that the presence of HCV infection itself should be considered an exclusion
criterion for the classification of pSS. In rare cases in which the differential diagnosis is very difficult,
particularly in HCV-negative patients, it might be correct to classify the disorder as an overlap syndrome.
Patients with MC/pSS overlap syndrome are often characterised by a more severe clinico-prognostic evolution;
they show a significantly low prevalence of anti-Ro/SSA-La/SSB together with a high prevalence of MC,
hypocomplementaemia, systemic autoimmune manifestations and complicating lymphomas. In clinical
practice, this particular condition might be better regarded as a vasculitic syndrome with relevant implications
for patient prognosis and treatment (Ferri, 2008*; Ferri, 2015*).
Finally, AIH, the old ‘lupoid’ hepatitis, may be associated with HCV infection, generally in the same countries in
which HCV-associated MC is more commonly found. AIH may share with MC syndrome a number of
extrahepatic symptoms, including serum mixed cryoglobulins. The differential diagnosis between these two
conditions may be problematic: some typical features of MC (leucocytoclastic vasculitis,
©2007-2018 EULAR 20
hypocomplementaemia, glomerulonephritis), as well as the presence of serum autoantibodies commonly

found in AIH (antismooth muscle antibodies), should be taken into account.
2.6 Prognosis
The natural history of MC is not predictable and strongly depends on concomitant diseases and complications
and response to treatment. Morbidity due specifically to cryoglobulinaemia may also be significant (infections,
cardiovascular diseases, progressive renal failure, advanced neuropathy). The overall prognosis is worse in
patients with renal disease, liver failure, lymphoproliferative disease and malignancies. Mean survival is
estimated to be about 50–60% at 10 years after diagnosis (Ferri et al, 2004*). Careful monitoring of life-
threatening complications (mainly nephropathy, widespread vasculitis and B cell lymphoma or other
malignancies) should be carried out in all patients with MC.
3 HCV-related extrahepatic disorders
HCV chronic infection is diffuse worldwide and may in a low but significant percentage of patients trigger a
large number of extrahepatic manifestations. These include a variety of autoimmune and neoplastic disorders,
among which MC is the prototypic HCV-associated extrahepatic disease (see section 2). It is a complex
immunological disorder characterised by multiple organ involvement. Since MC syndrome, strongly associated
with HCV infection, mimics other immune-mediated and neoplastic disorders, a possible role of HCV in these
conditions has been also investigated. The spectrum of possible HCV-associated diseases includes a wide
number of organ-specific and systemic diseases. Polyoligoclonal B lymphocyte expansion seems to be the
common underlying alteration in a significant percentage of HCV-infected patients, some of whom may
develop a variable combination of both hepatic and extrahepatic manifestations; the term ‘HCV syndrome’
refers to this complex clinical condition.
HCV-related diseases can be grouped into three different categories according to the strength of association
with the viral infection based on clinico-epidemiological, histopathological and molecular biology studies
(figure 10).
©2007-2018 EULAR 21
Figure 10 Possible clinical manifestations of hepatitis C virus (HCV) syndrome showing the strength of
association between HCV and different diseases in the context of HCV syndrome. The spectrum of different
HCV-associated immunological and neoplastic disorders may be classified on the basis of clinico-
epidemiological, histopathological and molecular biology studies, into three different levels: (1) high: the
association with HCV infection characterises the large majority of patients; HCV infection is one of the major
triggering agents of the disease; (2) medium: the disease shows a significantly higher prevalence of HCV
infection compared to controls; the putative pathogenetic role of HCV is also supported by pathogenetic
studies and may identify at least a specific disease subset; and (3) low: the possible association is suggested
by limited clinico-epidemiological observations; a pathogenetic link in at least a specific disease subset from
some geographical areas is probable, but needs to be definitely demonstrated. Red circles indicate the main
rheumatic diseases potentially HCV related. CNS, central nervous system; dis., disease; HCC, hepatocellular
carcinoma; inv., involvement; MC, mixed cryoglobulinaemia; s., syndrome; t., tarda.
3.1 Pathogenesis of HCV-related autoimmune and lymphoproliferative disorders
The main pathogenetic insights into HCV syndrome have been provided by studies of the biological
peculiarities of this virus and its possible interaction with the host immune system. In addition, studies of HCV-
associated MC provided important insights into the pathogenesis of other HCV-related disorders. HCV
lymphotropism is an important step in the pathogenesis of virus-related immunological disorders. A number of
©2007-2018 EULAR 22
epidemiological studies suggested a pathogenetic role for HCV in MC, a disorder characterised by ‘benign’ B
lymphocyte expansion. Interestingly, the same immune-pathological alteration may also develop in a
significant number of HCV-infected subjects, often in association with one or more serum autoantibodies or
mixed cryoglobulins, or both.
Since HCV is a positive, single-stranded RNA virus without a DNA intermediate in its replicative cycle, viral
genomic sequences cannot be integrated into the host genome. One possible hypothesis is that HCV infection
exerts a chronic stimulus on the immune system, which facilitates clonal B lymphocyte expansion (Ferri et al,
2002*; Ferri et al, 2007*; Ferri, 2008*).
The t(14;18) translocation has been demonstrated in a significant percentage of peripheral blood lymphocytes
from HCV-infected subjects, with consequent activation of the Bcl2 proto-oncogene (figure 6). In addition,
identification of the HCV envelope protein E2, which can bind the CD81 molecule expressed on both
hepatocytes and B lymphocytes, seems to be crucial for HCV-driven autoimmunity. CD81 is a cell-surface
protein that, on B cells, is part of a complex with CD21, CD19 and Leu 13. This complex reduces the threshold
for B cell activation by bridging antigen-specific recognition and CD21-mediated complement recognition. The
interaction between HCV-E2 and CD81 might increase the frequency of VDJ rearrangement in antigen-reactive
B cells. VDJ rearrangement could be responsible for the above-mentioned Bcl2 activation. The activated Bcl2
proto-oncogene can inhibit apoptosis; the consequence is an abnormally extended B cell survival. In turn, B
lymphocyte expansion is responsible for the wide autoantibody production found in HCV-infected subjects,
including cryo- and non-cryoprecipitable IC (figure 6). Specific autoantibodies may characterise some
autoimmune disorders, while mixed cryoglobulins are the serological hallmarks of MC syndrome.
Other mechanisms such as molecular mimicry involving particular HCV antigens and host autoantigens could
be responsible for B lymphocyte activation and autoantibody production (figure 6). In all cases, prolonged B
cell survival can expose these cells to other genetic aberrations, which may lead in predisposed subjects to
overt malignant lymphoma (figure 6). The oncogenic potential of HCV has been confirmed in patients with
hepatocellular carcinoma and in a significant percentage of B cell lymphomas. Of interest, the same virus could
be also involved in other malignancies such as thyroid cancer (Ferri et al, 2002*; Ferri et al, 2015*; Ferri,
2016*).
There is great geographical heterogeneity in the prevalence of HCV-related immunological or neoplastic

disorders (figure 4). This epidemiological observation contrasts with the homogeneous diffusion of HCV
infection worldwide. In this respect, we can hypothesise that HCV itself might be insufficient to drive the
different autoimmune-lymphoproliferative disorders seen in infected subjects. The involvement of particular
HCV genotypes, environmental and/or host genetic cofactors (figure 6) may contribute to the pathogenesis of
©2007-2018 EULAR 23
HCV syndrome. However, the actual pathogenetic relevance of the above cofactors still remains to be fully
demonstrated.
It is well known that most HCV-infected subjects remain asymptomatic, some for a long time. In a small but
significant percentage of patients, the virus is responsible for both hepatic and extrahepatic disorders, usually
as late manifestations (figure 6). These clinico-epidemiological observations suggest that HCV syndrome is the
consequence of a multifactorial and multistep pathogenetic process. It is quite common to see in the same
patient a progression from mild, often isolated manifestations, to systemic manifestations and, finally, to overt
malignancies (figure 6).
3.2 HCV and rheumatic diseases
Various inflammatory rheumatic disorders are the most common extrahepatic manifestations of HCV
syndrome. Among these, MC is at the junction between classic rheumatic diseases, such as RA and Sjögren’s
syndrome, and other autoimmune/lymphoproliferative disorders (figures 5 and 10) (Ferri et al, 2002*; Ferri et
al, 2015*; Ferri, 2016*).
Because of its clinical polymorphism, cryoglobulinaemic vasculitis may overlap with other rheumatic disorders
such as Sjögren’s syndrome and RA (figure 7); on the other hand, these rheumatic disorders may be
occasionally associated with HCV infection (figure 7). The possible aetiopathogenetic role of HCV in pSS
remains controversial. Patients with HCV-associated Sjögren’s syndrome show a significantly low rate of anti-
Ro/SSA-La/SSB (23%) together with a high prevalence of MC (50%), hypocomplementaemia (51%) and
systemic vasculitic manifestations (58%) (figure 7). This particular condition cannot be classified as pSS; at least
50% of patients might be better classified as having cryoglobulinaemic vasculitis, which has important clinico-
prognostic and therapeutic implications. The example of overlapping MC/Sjögren’s syndrome suggests that in
genetically predisposed subjects, HCV infection may trigger complex immune system alterations, which may
produce variable phenotypes mimicking different well-known diseases, namely Sjögren’s syndrome, RA,
dermatopolymyositis, etc (figures 6, 7 and 10).
Chronic oligo-polyarthritis can be found in HCV-infected subjects, is often non-erosive and is barely
progressive. In patients with HCV-related cryoglobulinaemic vasculitis, the joint involvement is generally
characterised by mild oligoarthritis, while symmetrical rheumatoid-like polyarthritis may complicate IFN
treatment in patients with type C hepatitis. Finally, given the relatively high prevalence of the two diseases, it
is not uncommon to observe a simple association between classic RA and HCV infection (figure 11). Figure 7
summarises the main clinico-serological parameters for differential diagnosis between MC, pSS and RA in the
setting of HCV syndrome.
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Figure 11 Proposed flow chart for the classification of patients with chronic arthritis and hepatitis C virus
(HCV) infection. Patients referred for either symmetrical polyarthritis or mono-oligoarthritis should undergo
careful clinical and laboratory investigation, including virological evaluation. On the basis of different
immunological tests, namely, rheumatoid factor (RF), anticyclic citrullinated peptides antibodies (anti-CCP),
antinuclear antibodies (ANA), cryoglobulins and complement, it is possible to correctly classify anti-HCV-
positive patients into two main groups: (a) those with simple association rheumatoid arthritis (RA)+HCV
infection, and (b) those with HCV-related arthritis. This latter condition may include patients with arthritis in
the setting of chronic HCV infection and a variable degree of liver involvement and patients with HCV-
related cryoglobulinaemic syndrome. A possible therapeutic approach to different clinical subsets includes:
for HCV-related arthritis, sequential treatment with antiviral agents, that is, direct-acting antiviral agents
(DAAs, ), and immunomodulating treatments (steroids, hydroxychloroquine (HCQ) and other disease-
modifying antirheumatic drugs, rituximab) can be usefully employed (see text). In patients with concomitant
HCV infection and arthritis, the use of tumour necrosis factor α blocking agents (anti-TNFα) seems to be
useful and safe,. auto-Ab, autoantibodies; Cryo, cryoglobulins; DMARDs, disease modifying anti-rheumatic
drugs.
Osteosclerosis is a rare condition described in adults with HCV infection; it can be defined as an acquired,
painful skeletal disorder characterised by a marked increase in bone mass (figure 10). Osteosclerosis is
clinically characterised by non-specific, often diffuse bony pain and tenderness over involved bones due to
periosteal stretching, in the absence of joint swelling or motion limitation. The radiograph examination shows
©2007-2018 EULAR 25
bony sclerosis and thickening of the long-bone cortices, mainly the diaphyseal cortices. Laboratory
investigations frequently reveal abnormally increased markers of bone formation (alkaline phosphatase and
bone-specific alkaline phosphatase, osteocalcin); these alterations were mirrored by a marked increase in
bone mass (bone mineral density, BMD) and enhanced radionuclide uptake at scintigraphy (99mTc-MDP).
Bone biopsy shows an increased number and thickness of trabeculae with a parallel reduction in bone marrow.
Only a very small percentage of infected patients develop osteosclerosis compared to the widespread diffusion
of HCV. The pathogenesis of this rare condition is still unknown; it has been suggested that HCV alone or in
combination with other unknown agent(s) may infect and alter bone cells or their precursors in predisposed
subjects. These alterations might be mediated by the production of bone growth factors, such as insulin-like
growth factors or osteoprotegerin. The pathogenetic role of the latter factor seems to be relevant; an
imbalance in the osteoprotegerin/RANKL system leading to a predominance of osteoprotegerin has been
documented.
To date, only 16 cases of HCV-associated osteosclerosis have been reported in the literature. In some cases, a
partial or complete spontaneous remission of symptoms and/or bone sclerosis was observed during the
follow-up period. Treatment with antiresorptive agents in some patients was ineffective, while symptomatic
therapies may provide some benefit. The beneficial effect of HCV eradication observed in one patient is quite
intriguing, but should be confirmed in larger numbers of patients.
The number of HCV-associated rheumatic diseases, as well as of other extrahepatic disorders, has grown
during the last two decades (figure 10). In addition to the conditions described above, other rheumatic
diseases have been associated with HCV infection, namely, myalgia, fibromyalgia, poly/dermatomyositis,
polyarteritis nodosa, Behçet’s syndrome, systemic lupus erythematosus and antiphospholipid syndrome.
In a large series of HCV-infected individuals, myalgia was mentioned by a significant number (15%) of patients.
The pathogenesis of this symptom remains difficult to explain; the detection of viral genomic sequences within
muscle fibres suggested a direct involvement of HCV in the pathogenesis of diffuse muscle pain. Fibromyalgia
was also reported by some authors in a significant percentage of patients with chronic HCV infection, but
other studies carried out in patients with typical clinical manifestations of fibromyalgia did not confirm this
association. On the other hand, the differential diagnosis between fibromyalgia and muscle pain, frequently
associated with weakness and arthralgias, may be very difficult in the setting of HCV-positive patients.
Similarly to that proposed for sicca syndrome and pSS, some authors suggest considering HCV-associated
myalgia and classic fibromyalgia as distinct entities.
The association of poly/dermatomyositis with HCV infection is described in numerous anecdotal observations,
more often in patients with long-lasting viral infection or during IFNα treatment. Similarly, cases of vasculitis
involving medium arteries suggesting the diagnosis of polyarteritis nodosa have been associated with HCV
©2007-2018 EULAR 26
infection; moreover, HCV seropositivity has been reported in a significant percentage of patients with
polyarteritis nodosa. This latter association is not surprising in light of the well-known relationship between
polyarteritis nodosa and another hepatotropic virus, namely HBV. Polyarteritis nodosa may share numerous
clinical symptoms with cryoglobulinaemic vasculitis; thus, patients with suspected HCV-associated polyarteritis
nodosa should be correctly classified by means of wide clinico-serological and pathological investigation. As
regards other possible HCV-associated disorders, namely Behçet’s syndrome, systemic lupus erythematosus
and antiphospholipid syndrome, the data reported in the literature are generally anecdotal. Since a
pathogenetic link with HCV cannot be totally excluded, the therapeutic approach in these patients is difficult
(Ferri et al, 2002*; Ferri et al, 2015*; Ferri, 2016*).
As regards HCV-associated MC, patients with concomitant HCV infection and autoimmune systemic disorders,
such as poly/dermatomyositis, polyarteritis nodosa and systemic lupus erythematosus, may be usefully
treated with immunosuppressors (cyclophosphamide, rituximab, etc) with some important precautions and
limitations due to viral infection. In all instances, a preliminary clinical evaluation of liver involvement and viral
replication is necessary before any therapeutic decisions are made. The latter may be based on standard
immunosuppressive treatments, possibly integrated into sequential/combined antiviral treatment.
3.3 Other HCV-related autoimmune disorders
A variety of organ- and non-organ-specific, immune-mediated diseases can be correlated with HCV infection.
Cutaneous manifestations are often seen in HCV-infected subjects. Among these, porphyria cutanea tarda
(PCT) is one of the most investigated. It is the commonest type of porphyria and is characterised by reduced
activity of uroporphyrinogen decarboxylase, an enzyme involved in the haem biosynthetic pathway, and by
frequent chronic liver disease. Since uroporphyrinogen decarboxylase deficiency is an essential condition, but
not sufficient, for definite classification/diagnosis of PCT, various triggering factors, including viral infection,
should be taken into account. A role of HCV infection has been investigated in several studies worldwide,
which have reported a variable percentage of associations. The pathogenesis of HCV-related PCT is particularly
intriguing: both metabolic factors – in particular, altered genes involved in iron metabolism – and a cross-
reactivity of host versus HCV antigens have been proposed. Generally, HCV-positive patients without PCT do
not show significant alteration in porphyrin metabolism; therefore, it may be supposed that a genetically
driven reactivity is decisive, while HCV may exert an indirect role, possibly as a triggering factor (Ferri et al,
2002*; Ferri, 2008*; Ferri, 2015*; Ferri, 2016).
HCV-related lichen planus is another important association, in particular, oral lichen planus, which has a
variable geographical prevalence. Moreover, several mucocutaneous manifestations are variably reported in
HCV-infected subjects, generally based on limited or anecdotal observations. HCV-positive patients may
develop acute episodes or chronic manifestations of well-known skin diseases; these symptoms are an
©2007-2018 EULAR 27
expression of immune-mediated cutaneous injury, triggered by HCV antigens and often amplified by IFN
treatment. In many cases these cutaneous manifestations, often with a contribution from peripheral nerve
alterations, severely affect patients’ quality of life. Peripheral neuropathy is a common complication of HCV
infection, mainly in cryoglobulinaemic vasculitis, while central nervous system involvement is less common.
Peripheral neuropathy is more often seen in patients with overt MC syndrome. Vascular manifestations,
including central nervous system involvement, may represent late comorbidities of HCV syndrome, particularly
in patients with more severe extrahepatic manifestations and receiving long-term steroid treatment. Some
cardiovascular manifestations have been reported during HCV infection, mainly in patient series from eastern
Asian countries; if confirmed by further studies, they may support the role of genetic and/or environmental
cofactors in the pathogenesis of HCV-related diseases.
An intriguing, still controversial, aspect is the possible aetiopathogenetic role of HCV in AIH. Patients with AIH
may present serum mixed cryoglobulins, HCV infection and extrahepatic manifestations such as thyroiditis,
sicca syndrome and arthritis; conversely, patients with HCV infection show one or more non-organ-specific
autoantibodies and different organ involvements. In this respect, the antigenic target specificity of HCV-
related autoantibodies shows only quantitative differences compared with those associated with ‘primary’
AIH. Again, the heterogeneous geographical distribution of HCV-associated AIH suggests a possible
involvement of various pathogenetic cofactors; among these HCV might trigger a peculiar AIH clinico-
serological subset which is prevalent in specific geographical areas (figure 4).
Glomerular and tubulointerstitial renal involvement in both native and transplanted kidneys may be associated
with HCV infection (Ferri, 2017 review). HCV-related glomerulonephritis may include various histopathological
types: MPGN with and without MC and, less frequently, membranous nephropathy, fibrillary and
immunotactoid glomerulonephritis, rapidly progressive glomerulonephritis and exudative-proliferative
glomerulonephritis. Cryoglobulinaemic glomerulonephritis – namely, type I MPGN – is more commonly found,
while ‘primary’ MPGN represents less than one-third of HCV-associated MPGN. This latter disease has been
described mainly in the USA and Japan. However, the real prevalence of MPGN without detectable
cryoglobulinaemia is difficult to assess; it may represent a subclinical form of MC, possibly owing to difficulties
and/or inadequate methods of detecting serum cryoglobulins. The possible methodological biases have been
examined in section 2.4.
In some patients, MPGN is the presenting symptom of MC syndrome that may develop later in the course of
the disease. Renal involvement is one of the most harmful complications of HCV-associated MC syndrome, and
may severely affect the patient’s clinical outcome. It is the consequence of cryoprecipitable and non-
cryoprecipitable IC deposition in the glomeruli. However, the exact role of HCV in the aetiology of
glomerulonephritis is not universally accepted. The presence of HCV particles in IC seems to support an
indirect involvement of this virus in the pathogenesis of glomerulonephritis.
©2007-2018 EULAR 28
Thyroid involvement is perhaps the most common and thoroughly investigated endocrine alteration in HCV-
positive patients (Ferri, 2017 review). The prevalence of abnormally high levels of antithyroid antibodies found
in these patients varies markedly, ranging from 2% to 48%, with a heterogeneous geographical distribution
(figure 4). These discrepancies may be correlated with variable genetic predisposition and environmental
cofactors, such as iodine intake or diffusion of other infectious agents. Moreover, subclinical hypothyroidism
has been found in 2–9% of patients with chronic hepatitis C, while miscellaneous thyroid alterations and raised
serum thyroid autoantibodies are generally higher in chronic hepatitis C than in hepatitis B or D. More
recently, the prevalence of thyroid involvement was investigated in a large series of patients with chronic
hepatitis C and compared with that in control groups from the general population from regions with different
iodine intake, as well as that in patients with chronic hepatitis B. Autoimmune thyroid involvement and
hypothyroidism were significantly more common in patients with chronic hepatitis C than in the comparison
groups, whereas the prevalence of hyperthyroidism was similar. Comparable findings were also found in
another study focusing on the thyroid abnormalities of HCV-positive patients with MC (Ferri et al, 2002*; Ferri
et al, 2015*; Ferri, 2016*).
Recent studies have focused on the possible pathogenetic mechanisms responsible for HCV-related thyroid
disorders. HCV thyroid infection may act by upregulating CXCL10 gene expression and secretion in thyrocytes
(as previously shown in human hepatocytes) recruiting Th1 lymphocytes, which secrete IFNγ and tumour
necrosis factor α (TNF-α). In turn, these cytokines may induce CXCL10 secretion by thyrocytes, thus
perpetuating the immune cascade. The consequence may be the appearance of thyroid autoimmune disorders
in genetically predisposed subjects (figure 12). This hypothesis has been recently confirmed by a study that
evaluated CXCL10 serum levels in HCV-positive patients with MC, in the presence or absence of autoimmune
thyroid involvement. Chronic immune-mediated inflammatory thyroid lesions may be responsible for the
papillary thyroid cancer found in a significant percentage of HCV-infected subjects compared with controls.
Analogous pathogenetic mechanisms can be involved as a consequence of HCV infection of pancreatic β cells
responsible for the upregulation of CXCL10 gene expression and secretion. The recruited Th1 lymphocytes,
which secrete IFNγ and TNF-α, amplify CXCL10 secretion by β cells, thus perpetuating the immune cascade.
The final result is the appearance of β cell dysfunction, with the probable contribution of genetic
predisposition.
The abnormalities in thyroid function should be included among the complications of HCV syndrome. These
patients should be periodically screened for thyroid involvement to identify those needing treatment and to
diagnose the rare but harmful neoplastic complication at an early stage.
Type 2 diabetes may be another important manifestation of HCV syndrome, regardless of the presence and
severity of liver damage.
©2007-2018 EULAR 29
Figure 12 Possible aetiopathogenetic mechanisms of hepatitis C virus (HCV)-related thyroid disorders and
diabetes type 2. HCV thyroid infection may act by upregulating CXCL10 gene expression and secretion in
thyrocytes (as previously shown in human hepatocytes) recruiting Th1 lymphocytes, which secrete interferon
γ (IFNγ) and tumour necrosis factor α (TNFα). In turn, these cytokines may induce CXCL10 secretion by
thyrocytes, thus perpetuating the immune cascade. The consequence may be the appearance of thyroid
autoimmune disorders in genetically predisposed subjects.
Initially, clinic-based studies found an excess of type 2 diabetes in non-cirrhotic HCV-positive patients
compared with patients with chronic hepatitis of other origin; however, this was not confirmed by another
large study. An Italian case–control study evaluated 564 non-cirrhotic HCV-positive patients compared with
302 control subjects without a history of alcohol abuse, drug addiction or positivity for markers of viral
hepatitis and 82 non-cirrhotic HBV-positive patients. A significantly high prevalence of type 2 diabetes was
recorded in non-cirrhotic HCV-positive patients compared with control subjects (12.6% vs 4.9% and 7%,
respectively; p = 0.008). Interestingly, the prevalence of type 2 diabetes in non-cirrhotic HBV-positive patients
(7%) was within the range of the reported age-adjusted prevalence rates for the Italian population (4%).
A comparison between the clinical phenotype of hepatic and diabetic patients showed that non-cirrhotic HCV-
positive type 2 diabetes was characterised by slightly older age, higher body mass index (BMI), elevated serum
triglycerides, raised blood pressure levels and lower high-density lipoprotein cholesterol concentrations.
Moreover, type 2 diabetic non-cirrhotic HCV-positive patients had a significantly lower BMI than type 2
©2007-2018 EULAR 30
diabetic control subjects and a slightly but significantly (p < 0.05) higher BMI than non-diabetic non-cirrhotic
HCV-positive patients. Type 2 diabetes itself is characterised by older age, overweight, dyslipidaemia and
higher blood pressure levels, the so-called ‘metabolic syndrome’ phenotype. In contrast, non-diabetic non-
cirrhotic HCV-positive patients were lean and had low LDL cholesterol levels. Low LDL cholesterol levels have
been correlated with HCV-induced hypo-β-lipoproteinaemia due to a binding competition between the virus
and hepatic LDL receptor (Ferri, 2008*).
An immune-mediated mechanism for HCV-associated diabetes has been postulated and a similar pathogenesis
might be involved in the diabetes of HCV-positive patients with MC. This hypothesis is strengthened by the
finding that autoimmune phenomena in patients with type 2 diabetes are more common than previously
thought. Similarly to the above-mentioned pathogenetic mechanisms involved in HCV-related thyroid
disorders, we can hypothesise that HCV infection of β cells may act by upregulating CXCL10 gene expression
and secretion. The recruited Th1 lymphocytes, which secrete IFNγ and TNF-α, amplify CXCL10 secretion by β
cells, thus perpetuating the immune cascade. The final result is the appearance of β cell dysfunction, possibly
in genetically predisposed subjects (figure 12).
Finally, sex hormone alterations have been observed in HCV-positive MC. Erectile dysfunction has been
anecdotally reported in patients with HCV-related chronic hepatitis undergoing treatment with IFNα.
To investigate the possible role of HCV infection in gonadal dysfunction, 207 male patients with HCV infection
(102 with cryoglobulinaemic vasculitis) were compared with 207 age-matched men, randomly selected from a
series of 2010 subjects from the Italian general population previously investigated for erectile dysfunction.
Exclusion criteria were patients aged >55 years, IFNα treatment during the past 12 months, and the presence
of diabetes, renal failure, hypothyroidism, and cardiovascular and psychiatric disorders. Erectile dysfunction
was significantly more common in HCV-infected subjects than in control subjects (p < 0.001). Sex hormone
determinations showed that total and free testosterone plasma levels were abnormally reduced in HCV-
positive patients with erectile dysfunction. Neither erectile dysfunction nor low levels of total and free
testosterone were related to the severity of liver damage. These sex hormone alterations along with the
possible contribution of peripheral neuropathy may be responsible for erectile dysfunction, which should be
confirmed by further investigations. The above findings further support the role of the host hormonal
environment in the pathogenesis of HCV-driven autoimmune disorders. We can hypothesise that reduced
endogenous immunosuppressive activity due to low levels of adrenal-gonadal androgens may amplify the
autoreactive lymphocyte proliferation triggered by HCV infection.
3.4 HCV and malignancies
HCV infection is the major aetiological factor in hepatocellular carcinoma. The oncogenic role of the virus in
hepatocellular carcinoma has been definitely established. Since 1993, a possible role of this virus in the
©2007-2018 EULAR 31
pathogenesis of malignant B cell neoplasias has also been suggested. This hypothesis was initially suspected
because of the striking association between HCV and MC, a condition that may be complicated by B cell
lymphomas, and was further reinforced by the demonstration of HCV lymphotropism. In 1994, a surprisingly
high prevalence of HCV infection in unselected Italian patients with B cell non-Hodgkin’s lymphoma (B-NHL)
was first reported. After this initial observation, an increasing number of epidemiological and laboratory
investigations in patient series from different countries, as well as in animal models, confirmed the
aetiopathogenetic role of HCV in a significant percentage of patients with B-NHL (figure 6). As for HCV-related
MC, this association has a variable geographical distribution. The HCV-induced B cell malignancies have two
main variants: the lymphomas complicating HCV-positive MC and isolated HCV-positive B-NHL. B-NHL
complicating MC syndrome is discussed in section 3.3 (Ferri, 2015*; Ferri, 2016).
A significantly high prevalence of thyroid cancer complicating HCV-related hepatitis and HCV-related MC
compared with controls was first noted in 1999. These data were subsequently confirmed in a case–control
study, which reported a high prevalence of HCV in patients undergoing surgery for papillary thyroid cancer.
Overall, HCV infection was associated with a high risk for liver cancer, multiple myeloma, B-NHL and thyroid
cancer (figure 6). Furthermore, a high prevalence of thyroid cancer in subjects with a history of transfusion
and/or liver disease indirectly supports the role of HCV in this malignancy. A review of the literature shows
discordant results, possibly owing to important epidemiological and methodological bias. The results of a
recent study on a large number of HCV-infected patients seem to confirm the high prevalence of thyroid
papillary cancer, excluding the influence of some possible biases such as gender, age and iodine intake.
In our studies, features of thyroid autoimmunity were more commonly found in patients developing thyroid
papillary cancer irrespective of whether they had type C hepatitis or HCV-related MC syndrome. This
observation suggests that thyroid autoimmunity may be a predisposing condition for this malignancy.
Although a possible association between thyroid cancer and HCV infection is suggested by the above clinico-
epidemiological studies, this needs to be verified by further investigations.
3.5 HCV syndrome
The strength of association as well as the pathogenetic role of HCV varies greatly among different diseases and
for each disease among series of patients from different countries (figures 4,, 10 and 13; Ferri, 2015*; Ferri,
2016). Often, each disease itself represents a clinical syndrome which may comprise different clinico-
serological subsets. These latter are the resulting phenotypes of a variable combination of different – genetic,
environmental, infectious – pathogenetic cofactors. In this scenario, HCV infection, with the contribution of
other pathogenetic agents, may produce distinct autoimmune or neoplastic disease subsets. The complex of
HCV-related disorders is a continuum, as suggested by the clinical history of some patients, which may display
the entire spectrum. It is not uncommon to see HCV-infected subjects with mild, often limited manifestations,
©2007-2018 EULAR 32
which may progress, generally during a long follow-up period, to more severe systemic manifestations,
including malignancies (figure 6).
In general, HCV syndrome (Ferri et al, 2015*) is a multifaceted, clinico-pathological condition (figure 6 and 13);
the challenge for future investigations is to better elucidate the exact boundaries of this syndrome and the
actual pathogenetic role of the virus in different conditions.
Fig. 13. The HCV syndrome.
The figure schematically reproduce the spectrum of Hepatitis C virus (HCV) infection and diseases. HCV is an
hepato- and lymphotropic virus responsible for a wide spectrum of both hepatic and extrahepatic diseases.
The term 'HCV syndrome' refers to the multiform complex of all HCV-related diseases (Ferri, 2015*). Besides
HCV-infected patients without clinical manifestations or isolated liver involvement, HCV-related
extrahepatic manifestations (HCV-EHMs) may include a variety of non-organ and organ-specific
autoimmune/lymphoproliferative and neoplastic disorders (Ferri, 2015*); therefore HCV-positive patients
are commonly referred to different specialists according the prevalent clinical manifestation(s). Mixed
cryoglobulinemia syndrome (MCs), also termed cryoglobulinemic vasculitis, represents the prototype of
extra-hepatic systemic immune-mediated disorder characterized by multiple organ involvement (Ferri,
2015*). Only a minority of MC patients is HCV-negative (see also Fig. 5; Ferri, 2016*; Galli, 2017*)
©2007-2018 EULAR 33
4 Current and future therapeutic strategies for treating mixed cryoglobulinaemia
Cryoglobulinemia are immune complexes that may induce systemic cryoglobulinemic vasculitis (MC), a small
vessel vasculitis involving the skin, the joints, the peripheral nerve system, and the kidneys (Ferri C, 2002,
2008, 2015). With the discovery of hepatitis C virus (HCV) as the etiologic agent for most cases of
cryoglobulinemia vasculitis (CV) new opportunities and problems for crafting therapy of HCV-MC have
emerged. A new and major concern was the potential adverse effects that immunosuppressive therapy with
glucocorticoids and cytotoxic drugs could have on an underlying chronic viral infection. Alternatively the
discovery of HCV provided the opportunity to control HCV-MC with antiviral therapy (AVT) as the underlying
infection drives immune complex formation and resultant vasculitis (Ferri C, 2002, 2008; Saadoun D, 2006,
2013).
Despite the successes with combination antiviral treatment, HCV-MC remains a severe disease. Most series
reporting on the effects of treatment reported a mortality rate of 8-15%. The 1-year, 3-year, 5-year, and 10-
year survival rates are 96%, 86%, 75%, and 63%, respectively. At the time of the diagnosis of HCV-related
systemic vasculitis, severe liver fibrosis and the severity of vasculitis were the main prognostic factors (Terrier
B et al, 2011). Although there is still controversy, the occurrence rate of B-cell NHL remains higher in MC
patients compared with the general population.
The treatment of HCV and consequently the treatment of HCV-CV has evolved over the years. Figure 14
illustrates the therapeutic interventions according to the aetiopathogenesis of cryoglobulinaemic vasculitis.
©2007-2018 EULAR 34
Figure 14 Therapeutic interventions according to the aetiopathogenesis of cryoglobulinaemic vasculitis.

mAb, monoclonal antibody.
Etiologic treatment:
- The historical treatment of HCV cryoglobulinemia vasculitis was Interferon alpha (IFN) in
monotherapy, then in combination with Ribavarine (RBV).
- Then, the treatment was based on the combination of (Pegylated(Peg)-IFN plus RBV, but efficacy was
limited and the side effects were frequent.
• Recently, major therapeutic advances appeared in the treatment of HCV infection, with the possibility
to eradicate HCV following new direct acting antiviral therapies (DAA) (Gragnani L et al 2015 ). The
first generation of HCV NS3 protease inhibitors - also known as “-previrs” - block the catalytic site of
NS3, preventing the poly-protein cleavage and thus HCV replication. Currently approved “-previrs”
include Telaprevir and boceprevir (first wave). The first generation of DAAs needed the combination
of Peg-IFN and RBV and prolonged treatments.
• The second wave of previrs includes simeprevir, paritaprevir and grazoprevir. Two different classes of
second wave DAAs have been introduced : the NS5A and the NS5B inhibitors:
The NS5A inhibitors block the stage of membranous genesis; they are also known as “-asvirs”
(daclatasvir, ledipasvir, ombitasvir, elbasvir, velpatasvir).
The NS5B polymerase inhibitors or “-buvirs”, include nucleos(t)ide analogues (sofosbuvir)
acting as chain terminators within the polymerase catalytic site, and non-nucleotide inhibitors
(dasabuvir), causing conformational changes and making the polymerase ineffective.
©2007-2018 EULAR 35
4.1 Treatment of HCV cryoglobulinemia vasculitis
A. Antiviral agents
a) Interferon alpha (IFN) monotherapy, IFN plus Ribavarin
Treatment of HCV-MC with Interferon alpha (IFN) was associated with a relatively poor response and a high
relapse rate, especially in severe cases. IFN monotherapy was effective in 50 to 100% of patients with purpuric
skin lesions, but did not demonstrate efficacy on nerve or renal involvement. Combination therapy with IFN
plus ribavirin showed a greater antiviral efficacy with sustained virological response in 35-80% of patients with
chronic hepatitis C (Saadoun D, 2006, 2013). Such combination therapy showed much better short and long-
term results in patients with HCV-MC than reported with IFN monotherapy. In three uncontrolled studies,
combination therapy with standard IFN-α and ribavirin demonstrates enhanced efficacy on main HCV-related
vasculitic manifestations (cutaneous, 100% ; renal, 50% ; and neural, 25-75%). Two studies reported a loss of
proteinuria and haematuria in sustained viral responders treated by IFNα plus ribavirin. In a study of 27
patients with chronic hepatitis C complicated by systemic vasculitis, patients received IFN with ribavirin during
20 ± 14 months. After a mean follow-up of 57 months, 25/27 patients (93%) were alive, whereas 2 patients
died secondary to cirrhosis. Most patients (75%) with a negative viraemia at the end of follow up were
complete clinical responders for their HCV-MC vasculitis. Careful monitoring for adverse effects is mandatory
since some manifestations of HCV-MC, such as peripheral neuropathy or skin ulcers, may worsen under IFN-
based therapy. The tolerance of ribavirin is also reasonable with the exception of haemolytic anaemia
requiring dosage reduction in some patients and adjusted dose in case of renal insufficiency.
b) Peg-Interferon alpha plus Ribavirin
The treatment of HCV infection (i.e. in the absence of HCV-MC) has continued to progress. It was based during
the last decade on the standard of pegylated interferon alpha (Peg-IFNα) plus ribavirin leading to a sustained
virological response in nearly 60% of patients (Saadoun D, 2006, 2013).
Mazzaro et al have reported the results of 18 HCV-MC patients treated with Peg-IFNα2b plus ribavirin for 12
months. At the end of follow-up, only eight (44%) patients were still sustained clinical and virological
responders. One major weakness of this study was the lower Peg-IFN dosage used in comparison with that
usually recommended in HCV therapeutic guidelines.
In a monocentric study, 72 consecutive HCV-MC patients received treatment with IFNα-2b (n=32) (3 million IU
x 3/week) or Peg-IFNα-2b (n=40) (1.5 µg/kg/week), both combined with oral ribavirin (600 to 1,200 mg/day)
for at least 6 months. Peg-IFNα plus ribavirin combination achieved a higher rate of complete clinical (67.5% vs
56.2%), virological (62.5% vs 53.1%) and immunological response (57.5% vs 31.2%) as compared with IFNα
©2007-2018 EULAR 36
plus ribavirin, regardless of HCV genotype and viral load. Sub-group analysis of the 40 HCV-MC patients
treated with Peg-IFNα plus ribavirin showed a complete recovery of skin involvement in 21/24 (87.5%),
arthralgia in 18/22 (81.8%), peripheral neuropathy in 20/27 (74%) and nephropathy in 5/10 (50%) cases,
respectively. Compared with standard IFN α-2b/ribavirin, there was a shorter duration of HCV therapy (13.2
vs. 18.3 months), less frequent use of corticosteroids (35 vs. 47%) and a lower rate of death (5 vs. 18.7%) with
Peg-IFNα2b/ribavirin. An early virologic response (i.e. negativation or > 2 log drop viraemia at month +3) (odds
ratio [OR], 3.53; 95% CI 1.18 to 10.59) was independently associated with a complete clinical response of MC.
A glomerular filtration rate lower than 70 ml/min (OR 0.18; 95% CI 0.05 to 0.67) was negatively associated with
a complete clinical response of MC. Epidemiological features, viral load, transaminases or liver damage did not
influence the clinical outcome of MC. The reappearance of HCV RNA was observed in 8 (11.1%) patients and 6
of them experienced a relapse of MC. In 39/70 (56%) patients, side effects included fatigue (47.2%), fever
(37.5%), anaemia (33.3%), myalgia (25%), neutropenia (20%), depression (15.2%), thrombocytopenia (5%),
pruritus (4.1%) and alopecia (2.7%). When compared with IFNα2b/ribavirin, patients who received Peg-
IFNα2b/ribavirin had a similar rate of adverse events (53.1% vs 55%, respectively).
c) Direct acting antiviral drugs (DAA)
c.1) First generation DAAs: Peg-Interferon alpha plus Ribavirin and protease inhibitors
The first generation of DAAs needed the combination of Peg-IFN and RBV and prolonged treatments. Use of
triple therapy with Peg-IFNα, ribavirin, and a specifically targeted antiviral agent (i.e. Boceprevir or Telaprevir)
led to improve sustained virological response rates in patients infected with HCV genotype 1. In a prospective
cohort study, the efficacy of an NS3 protease inhibitor (boceprevir or telaprevir [thrice daily]), in combination
with Peg-INFα2a (180µg) or 2b (1.5 µg/kg) and ribavirin (800 to 1,400 mg/day) has been evaluated in 23 HCV-
MC patients with genotype 1. Thirteen patients (56.5%) were complete clinical responders, and ten (43.5%)
were partial responders at week 24. The virological response (i.e. HCV RNA negativation) was of 69.6% at week
24 (p=0.005). The cryoglobulin level decreased from 0.44 to 0.06 g/l (p=0.0006) and the C4 level increased
from 0.09 to 0.15 g/l (p=0.045). Grade 3 and 4 adverse events (mainly anaemia, neutropenia and
thrombocytopenia) were observed in 10 cases (43.5%). Twenty patients (87%) received erythropoietin, 9
(39.1%) had red cell transfusion and 2 (8.7%) had granulocyte stimulating agents. Antiviral therapy
discontinuation was required in 8 (34.7%) patients for virological non response (n=5), virological relapse (n=2)
and depression (n=1). Four patients received the combination of rituximab plus Peg-IFNa/ribavirin/protease
inhibitor combination with clinical improvement in all cases of whom 2 were complete clinical responders.
Recently, Saadoun et al (2015) reported the efficacy and the safety of this combined therapy in 30 CV patients,
of whom 67% were complete clinical and virological responders while severe side effects occurred in 47%.
Another study (Gragnagni L, 2014)) confirmed the good efficacy of such combination in 22 cryoglobulinemic
©2007-2018 EULAR 37
subjects, with a cryoglobulin disappearance in 86% but a lower sustained virological response (SVR) rate in
cryoglobulinemic patients than in patients without mixed cryoglobulinemia (23.8% vs 70%, p=0.01). Stine et al.
(2014) in a small case series of CV patients treated with triple AVT (Peg-IFN plus RBV plus sofosbuvir or first
generation DAAs) suggested a longer treatment for cirrhotic patients, apparently more refractory to clear
glucocorticosteroids.
c.2) Second DAAs generation:
The second DAAs generation protocols, are IFN-free (sometimes RBV free). Limited, but essentially concordant
data, are available regarding patients with CV. The first study reported the effects of a sofosbuvir/RBV
combination for 24 weeks in 24 CV patients; SVR was scored in 74% patients, with high rate of clinical response
(87%) and low rates of serious adverse events. The rate of SVR was that expected from used AVT protocols.
Sise et al. (2016), using sofosbuvir-based combinations in 12 CV subjects, obtained an improvement in renal
function with or without immunosuppressant and SVR12 in 83%. An interimanalysis, mostly based on the
combination of paritaprevir/ritonavir, ombitasvir, plus dasabuvir, showed a cryocrit decrease and a clinical
response even during treatment. The clinical improvement rate gradually increased from the inhibition of viral
replication, end of treatment and SVR12. A very recent prospective study (Gragnani L, 2016) assessed efficacy
and safety of sofosbuvir-based individually tailored therapy, in a cohort of 44 consecutive CV patients (median
after treatment follow-up: 42 weeks, range 27–53). All patients obtained viral eradication and an overall 100%
rate of clinical (complete or partial) response at week 24 post-treatment. Interestingly, a progressive increase
of complete response rate over time, confirmed previous, preliminary observations. This is also suggested by
another recent study conducted on 64 patients with circulating CGs (35/64 with CV) mostly treated in IFN-free
regimens (Bonacci 2016). Recently, Hegazy et al. (2016) described a cohort of Egyptian and Italian CV subjects
and Kondili et al. (2016) reported results of a nationwide Italian study, showing the disappearance or
improvement of more than 50% of CV symptoms in 31/37 (84%) patients after DAA. Available data suggest
that IFN-free AVT is safe, generally well tolerated and effective in CV patients.
B. Non-etiologic treatment :
a) Rituximab:
Rituximab (RTX) binding to CD20 causes a B-lymphocytes depletion thus justifying the extensive use in B-
lymphoproliferative disorders and severe autoimmune diseases. The safety and efficacy of RTX monotherapy
in CV were clearly shown.
RTX is especially recommended in case of HCV related CV with severe clinical manifestations, preferring it to
more conventional treatments. RTX can improve various manifestations of CV, including skin symptoms
(purpura and ulcers), fatigue, arthralgias/arthritis, glomerulonephritis (GN) and peripheral neuropathy in a
©2007-2018 EULAR 38
relevant number of cases. It has been reported that RTX is able to restore some CV-related immune
abnormalities; it can decrease cryocrit and rheumatoid factor level, increase C4 level, and induce the
disappearance of bone marrow B cell clonal expansion (Saadoun, 2008).
In a literature review, 13 references reported a total number of 57 cases, Patients had a cryoglobulinemia
vasculitis secondary to chronic active HCV infection in 75.4%. Most patients (48/57) received 4 weekly
consecutive i.v. infusions of 375 mg/m2 of rituximab. The mean follow up after rituximab infusions lasted 9.7
months (range, 3 to 24). Rituximab infusions were effective on main vasculitis signs, with a complete clinical
response in 24/33 (73%). Cryoglobulinemic vasculitis relapse was noted in 13/36 (36.1%) patients within few
days to nineteen months (mean 6.7 months) after the last rituximab infusion. This relapse was predictable due
to the absence of viral eradication and therefore to the persistence of the viral antigenic triggering factor
causing vasculitis.
In 2008, Saadoun et al, reported on 16 consecutive HCV-MC patients being treated with rituximab (375 mg/m2
intravenously each week for 4 weeks) combined with Peg-IFNα-2b (1.5 µg/kg/week, subcutaneously) plus
ribavirin (600–1200 mg/day orally) for 12 months. Fifteen patients (93.7%) showed clinical improvement, 10 of
whom (62.5%) were clinical complete responders at 12 months. Rituximab combined with Peg-IFNalpha2b-
ribavirin represents a safe and effective treatment option in severe refractory HCV-MC.
In 2010, Saadoun et al reported, 38 HCV-MC patients who received a combination of rituximab (375 mg/m2)
once a week for 1 month followed by Peg-IFN (2a: 180 µg, or 2b: 1.5 µg/kg) weekly plus ribavirin (600–1200
mg) daily for 48 weeks. They were compared to 55 HCV-MC patients treated with Peg-IFNα/ribavirin with the
same modalities. Compared with Peg-IFNα/ribavirin, patients treated with rituximab plus Peg-IFNα/ribavirin
had a shorter time to clinical remission (5.4±4 vs 8.4±4.7 months; p=0.004), better renal response rates (80.9%
vs 40% complete response; p=0.040) and higher rates of cryoglobulin clearance (68.4% vs 43.6%; p=0.001) and
clonal VH1–69+ B cell suppression (p<0.01). Treatment was well tolerated with no worsening of viraemia
under rituximab and with 11% of discontinuations due to antiviral therapy. Taken together, rituximab
combined with Peg-IFN/ribavirin was well tolerated and more effective than Peg-IFN/ribavirin in HCV-MC.
Rituximab synergises the immunological effect of antiviral therapy.
Dammacco et al reported on 22 patients with HCV-related MC who received Peg-IFNα (2a: 180 µg, or 2b: 1.5
µg/kg) weekly plus ribavirin (1000 or 1200 mg) daily for 48 weeks, and rituximab (375 mg/m2) once a week for
1 month followed by two 5-monthly infusions (termed PIRR) (Dammacco F, 2010). Fifteen additional patients
received Peg-IFNα/ribavirin with the same modalities as the PIRR schedule. Complete response was achieved
in 54.5% (12/22) and in 33.3% (5/15) of the patients who received PIRR and Peg-IFNα/ribavirin, respectively
(p<0.05). Clearance of HCV RNA and conversion of B cell populations from oligoclonal to polyclonal in liver,
bone marrow and peripheral blood was maintained for up to 3 years in 10 of 12 (83.3%) and in two of five
©2007-2018 EULAR 39
(40%) patients receiving PIRR and Peg-IFNα/ribavirin, respectively (p<0.01). Cryoproteins in 22.7% (5/22) of
patients treated with PIRR and in 33.3% (5/15) treated with Peg-IFNα/ribavirin persisted despite sustained
HCV RNA clearance. No response occurred in the remaining five patients in both groups. PIRR therapy was well
tolerated and more effective than the Peg-IFNα/ribavirin combination in HCV-related MC. Its effect may last
for more than 3 years.
Data about combined RTX plus DAA-based therapy are still scarce. However, it is conceivable to figure out
promising successes of combination with IFN-free schedules.
The exact place of RTX in the field of CV has to be more precisely defined. With the approval of recent DAAs
IFN-free combination, which proved very highly and rapidly effective on viremia, we will have to define in the
next future the remaining place of RTX. For example, as a first-line option for severe and life threatening
conditions needing urgent intervention and as a second-line option for those maintaining significant symptoms
after SVR.
Most studies used the standard haematological treatment schedule (375 mg/m2 in four consecutive weekly
infusions). Lower doses and/or shorter treatment have been also reported, i.e., 1g every two weeks for two
infusions or 250 mg/m2 in two administrations.
In most studies, tolerance of Rituximab was good. One potential concern regarding the use of such therapy
reported in initial studies is its suspected propensity to worsen HCV viraemia. Some patients may experience
systemic drug reactions after rituximab infusion. Six cases of HCV-MC patients who developed a severe flare
of vasculitis one or two days after rituximab infusion have been reported. Two patients developed serum
sickness syndrome 7 and 9 days after the first 1,000 mg rituximab infusion. Compared with patients without
drug reactions, those with drug reactions had higher mixed cryoglobulin levels and more of them received
1,000 mg high-dose rituximab protocol.
b) Other treatments
Immunosuppressive agents have been given for many decades to patients with severe MC disease
manifestations such as membranoproliferative glomerulonephritis, severe neuropathy and other life-
threatening complication. A combination of corticosteroids and immunosuppressants, such as
cyclophosphamide and azathioprine, has been used for the control of severe vasculitis lesions.
Mycophenolate mofetil may represent an alternative therapeutic option in patients refractory or intolerant to
these immunosuppressive drugs. In a large retrospective study of 105 patients with renal disease associated
with CV, 80% were administered corticosteroids and/or cytotoxic agents, while 67% underwent
plasmapheresis. Despite this aggressive approach, long lasting remission of the renal disease was achieved in
only 14% of cases, and the 10-year survival rate was only 49%.
©2007-2018 EULAR 40
Corticosteroids, used alone or in addition to IFN, did not favourably affect the response of HCV-CV
manifestations in two controlled studies. In a randomized trial, methylprednisolone alone given for one year
was associated with clinical response in 16.7% of patients, compared with 53.3% and 52.9% in patients
receiving IFNα or IFNα plus methylprednisolone, respectively. GCs are commonly used to control inflammation
and pain. Low-medium doses of GCs (0.1–0.5 mg/kg/day) usually control mild to moderate symptoms,
whereas high-dose pulse therapy (1–10 mg/kg) is commonly indicated to manage severe and acute conditions,
specifically renal failure, neurologic manifestations or hyperviscosity syndrome.
Plasmapheresis offers the advantage of removing the pathogenic cryoglobulins from the circulation of patients
with MC and is particularly effective for rapidly progressive glomerulonephritis and/or severe skin necrosis or
ulcers (Ferri C, 2008). Immunosuppressive therapy is usually needed associated with plasma exchange in order
to avoid the rebound increase in cryoglobulin serum level seen after discontinuation of apheresis. When used
in combination with HCV treatment, plasmapheresis did not modify the virologic response if IFNα was given
after each plasma exchange session.
4.2 Treatment of non HCV cryoglobulinemia vasculitis
In non-HCV cryoglobulinemia vasculitis, the treatment depends of the severity of the vasculitis, of the
associated aetiology (B cell lymphoma, MGUS, myeloma, Sjögren’s syndrome..) and of the type of cryoglobulin
(type 1 vs type 2 and 3). Low dose corticosteroids may help to control minor intermittent inflammatory signs
such arthralgia or purpura, but do not succeed in cases of major organ involvement (i.e. neurologic, renal,
cardiac), or in the long-term control of vasculitis. Plasmapheresis is particularly effective for rapidly progressive
glomerulonephritis, life threatening organ involvement and/or severe skin necrosis or ulcers. Saadoun et al.
(2012) reported the safety and efficacy of fludarabine (40 mg/m2 orally on days 2–4), cyclophosphamide (250
mg/m2 orally on days 2–4), and rituximab (375 mg/m2 on day 1), every 4 weeks, for 3 to 6 cycles in 7
consecutive patients with severe and refractory MC associated with B-NHL . Clinical features of MC included
purpura (n = 7), polyneuropathy (n =6), and kidney (n =4) and cardiac involvement (n =2). Previous treatment
included rituximab (n= 5), corticosteroids (n = 5), antiviral therapy (n= 5), cyclophosphamide (n =3), and
plasmapheresis (n =2). All patients achieved clinical response, with 3 (42.9%) patients achieving a complete
remission and 4 (57.1%) patients a partial remission.
a) Type 1 cryoglobulinemia vasculitis
Type I cryoglobulinemia vasculitis is usually associated with plasma cell hematologic disease while MC is more
likely related to low grade B cell NHL or Sjögren’s syndrome. The treatment of type I cryoglobulinemia
vasculitis is primarily that of the underlying haematological malignancy and the use of thalidomide or
lenalidomide, and bortezomib-based regimens seem to be interesting options. In patients with MGUS-related
type I cryoglobulinemia vasculitis the initiation of therapy is only justified by the severity of the vasculitis. A
©2007-2018 EULAR 41
recent series have reported clinic-biological characteristics and treatment of 64 patients diagnosed with type I
cryoglobulinaemia. Cryoglobulinaemia was IgG in 60% and IgM in 40% of all cases and was asymptomatic in 16
patients (25%). Cold-triggered ischaemic skin manifestations were observed in 33 patients (51%). Neurological
manifestations were observed in 15 patients and renal manifestations in 13. Most of the patients with necrotic
purpura (14/16, P = 0_009) and renal manifestations (11/13, p= 0.057) had IgG cryoglobulinaemia. IgG
cryoglobulinaemia was associated with monoclonal gammopathy of undetermined significance (MGUS),
myeloma, chronic lymphocytic leukaemia and lymphoplasmocytic lymphoma in 18, 13, 5 and 2 patients,
respectively. IgM cryoglobulinaemia was associated with MGUS and Waldenström macroglobulinaemia in 8
and 18 cases, respectively. One third of patients did not receive any specific treatment. Various treatments,
including rituximab, were administered to 25/31 patients with IgG cryoglobulinaemia and 6/25 patients with
IgM cryoglobulinaemia due to cryoglobulinaemia-related symptoms. Rituximab was ineffective in all cases
associated with a predominantly plasmacytic proliferation.
b) Mixed cryoglobulinemia vasculitis
In non HCV-mixed cryoglobulinemia (MC) vasculitis, rituximab plus corticosteroids combination therapy
showed the greater efficacy compared with corticosteroids alone and alkylating agents plus corticosteroids for
clinical, renal, and immunologic responses and corticosteroid-sparing effect. There is no significant difference
in the efficacy of rituximab therapy whether patients presented with HCV-induced or essential
cryoglobulinemia vasculitis. Rituximab plus corticosteroids regimen is associated with frequent severe
infections, particularly when high doses of corticosteroids are used. These infections occur in a particular
subset of patients, characterized by older age, renal failure (GFR < 60 mL/min), and the use of high-dose
corticosteroids. The efficacy of rituximab in type I cryoglobulinemia vasculitis remains controversial The
absence of CD20 expression on plasma cells was supposed to explain its lack of efficacy.
4.3 Treatment of MC vasculitis relapses.
Clinical relapses of HCV-related vasculitis are usually associated with relapsing HCV viraemia. Vasculitis
relapses usually present the same vasculitis manifestations as were noted at presentation. Recurrence of
vasculitis-related symptoms after withdrawal of antiviral therapy with virological relapse (HCV RNA
repositivation) can be successfully treated with another course of combination antiviral therapy with a good
response. Alternatively, repeated infusions of rituximab are effective to control MC vasculitis. However, the
dose, safety and continued efficacy of repeated therapy with rituximab in MC deserve further substantiation.
Authors have reported on 8 patients who presented with cryoglobulinemic vasculitis due to chronic active HCV
infection with no evidence of underlying malignant disease. After a successful treatment of HCV infection,
patients were sustained virological responders as they remained persistently HCV RNA negative. Later on, MC-
related symptoms reappeared, with no HCV infection relapse (as demonstrated by numerous negative
©2007-2018 EULAR 42
viraemia), but a malignant B-NHL was found in two cases. Clinicians should be aware of the possibility of the
presence of underlying malignant lymphoma when patients develop a relapse of cryoglobulinemia vasculitis
without HCV virological relapse.
4.4 Therapeutic guidelines
The therapeutic strategies of cryoglobulinemia vasculitis are summarized in Figure 15. International
therapeutic guidelines for patients with HCV-CV have been published in 2017 in the Autoimmunity Reviews
(Zignego AL, 2017).
Figure 15 Therapeutic strategies in patients with cryoglobulinaemia vasculitis. *The treatment of

cryoglobulinaemic vasculitis (CV) associated with haematological malignancy is primarily that of the
underlying haemopathy. **If failure or contraindication of antiviral therapy or in non-HCV CV, rituximab
may be used alone. CNS, central nervous system; HCV, hepatitis C virus; MPGN, membranoproliferative
glomerulonephritis; RPGN, rapidly progressive glomerulonephritis.
- Aggressive optimal antiviral therapy with new antiviral agents (DAA) should be considered as induction
therapy for HCV-MC patients with mild to moderate disease severity and activity (i.e. without rapidly
progressive nephritis, motor neuropathy or other life threatening complications). The duration of therapy has
not yet rigorously been determined but current treatment duration in HCV-MC patients is 48 weeks for all HCV
genotypes. With this strategy patients with mild or moderate disease (i.e. arthralgia, purpura, and/or sensory
polyneuropathy) may be able to be managed without immunosuppressive agents. Low dose corticosteroids
may also help to control minor intermittent inflammatory signs such arthralgia or purpura.
©2007-2018 EULAR 43
- In patients presenting with more severe MC disease (i.e. worsening of renal function, mononeuritis multiplex,
extensive skin disease including ulcers and distal necrosis), an induction phase with rituximab is often
necessary. In HCV-MC, combination therapy with rituximab plus DAA is recommended as it may target both
the viral trigger and the downstream B cell arm of autoimmunity. The following therapeutic schedule is
recommended: 1) weekly administration of four intravenous infusions of rituximab at 375mg/m² (on days 1, 8,
15 and 22) over a one-month period; 2) antiviral combination starting after the last rituximab infusion for 3 to
6 months duration.
- For patients presenting with the fulminant forms including peripheral necrosis of extremities, rapidly
progressive glomerulonephritis, digestive, cardiac, pulmonary and/or central nervous system involvement and
or signs and symptoms of hyperviscosity, apheresis can have immediate beneficial effects but must be
combined with immunosuppression to avoid post-apheresis rebound of MC. High dose steroids, rituximab, and
sometimes cyclophosphamide combination appeared as an effective salvage treatment for refractory MC. In
such cases, antiviral therapy should be started soon after the critical phase.
The treatment of cryoglobulinemia vasculitis associated with haematological malignancy is primarily that of
the underlying hemopathy. When CG-related symptoms are severe, complete plasmapheresis is indicated,
even though no prospective studies have established their efficacy or optimal use. The key point is to adapt
treatment modalities to the nature of the underlying gammopathy. If it is plasmacytic (i.e. mainly composed of
CD20 negative cells and usually secreting a mIgG), the treatment should be based on anti-myeloma agents
including, in selected young patients with severe disease, autologous stem cell transplantation. If the
underlying B-cell disorder is lympho-plasmacytic, usually associated with a mIgM, the treatment should be as
for Waldenström’s disease.
©2007-2018 EULAR 44
SUMMARY POINTS
 Types II and III mixed cryoglobulinaemia (MC), or cryoglobulinaemic vasculitis, are classified among
the small-vessel systemic vasculitides. The disease is a combination of serological findings (mixed
cryoglobulins with rheumatoid factor (RF) activity and frequent low C4) and clinico-pathological
features (purpura and leukocytoclastic vasculitis with multiple organ involvement).
 There are no diagnostic criteria for MC. The disease can be correctly classified on the basis of typical
orthostatic purpura with the histological pattern of leukocytoclastic vasculitis on skin samples taken
within the first 24–48 h, detection of serum mixed (IgG-IgM) cryoglobulins, low complement C4 and
positive RF.
 MC may be associated with other well-defined immunological, infectious or neoplastic disorders;
when isolated, it is a distinct disease, the so-called ‘essential’ MC. Following the discovery of a
striking association between MC and hepatitis C virus (HCV) infection, the term ‘essential’ is now
used to refer to a minority of patients. In over three-quarters of cases, cryoglobulinaemic vasculitis
is one of the most important extrahepatic complications of HCV chronic infection.
 The main clinical features of cryoglobulinaemic vasculitis are the typical triad of purpura, arthralgias
and weakness. Liver and renal involvement, peripheral neuropathy, skin ulcers and the possible
development of malignancies, mainly B cell lymphomas, generally as a late complication, may also
be seen
 Liver and/or renal involvement, as well as neoplastic complications, may severely affect the overall
prognosis of MC. Such patients, more often women, aged 50–60 years at the time of diagnosis, have
a worse prognosis than the general population.
 HCV is both a hepatotropic and a lymphotropic virus; it may exert a chronic stimulus on the immune
system with both T and B lymphocyte alterations; ‘benign’ B cell lymphoproliferation is responsible
for different autoantibody production, mainly of RF and cryoglobulins. In addition to
cryoglobulinaemic vasculitis, HCV may trigger different immune-mediated extrahepatic disorders
(thyroiditis, diabetes type 2, polyarthritis, glomerulonephritis, porphyria cutanea tarda, sicca
syndrome, etc.), as well as some malignancies, mainly B cell lymphomas.
 The large geographical heterogeneity in the prevalence of HCV-related extrahepatic manifestations
suggests that HCV itself might be insufficient to trigger and maintain these different autoimmune-
lymphoproliferative disorders. A variable combination of HCV with other unknown environmental
and/or host genetic cofactors may lead to the different clinical phenotypes that characterise HCV
syndrome.
 The main targets in the treatment of HCV-related cryoglobulinaemic vasculitis are the clinical
(improvement of organ target manifestations), virological (clearance of HCV RNA) and
immunological response (serum cryoglobulin and C4 levels).
©2007-2018 EULAR 45
SUMMARY POINTS
 Treatment of HCV-mixed cryoglobulinaemia (MC) may target either the viral trigger (HCV) or the
downstream B cell arm of autoimmunity.
 Antiviral therapy should be considered as induction therapy for HCV-MC with mild to moderate
disease severity and activity.
 IFN-free, DAA-based antiviral therapy should be considered a first line therapeutic measure for HCV-
MC that does not need urgent/life threatening measures.
 In patients presenting with severe disease (ie, worsening of renal function, mononeuritis multiplex,
and extensive skin disease including ulcers and distal necrosis), an immunosuppression induction
phase is often necessary while awaiting the generally slow response to antiviral treatments.
 Combination therapy with rituximab and optimal antiviral treatment appears logical as it may target
both the viral trigger (HCV) and cryoglobulin producing B cells.
 An early virological response to antiviral therapy is correlated with a complete clinical response in
MC.
 Clinical relapses in HCV-related vasculitis are usually associated with relapsing HCV viraemia.
 Recovery of B cell count began 6–9 months after rituximab therapy.
 Low-dose corticosteroids may help to control minor intermittent inflammatory signs such arthralgia,
but are not successful in cases of major organ involvement (ie, neurological, renal) in the long-term
control of vasculitis.
 Careful monitoring for adverse effects is mandatory since some manifestations of HCV-related
vasculitis, such as peripheral neuropathy or skin ulcers, may worsen under IFN therapy.
 Clinicians should be aware of the possibility of malignant lymphoma when HCV-positive patients
experience a relapse in cryoglobulinaemia vasculitis without HCV virological relapse.
 The treatment of cryoglobulinemia vasculitis associated with haematological malignancy is primarily
that of the underlying hemopathy.
©2007-2018 EULAR 46
Acknowledgement
Previous editions of this chapter were authored by Clodoveo Ferri, Maria Teresa Mascia, David Saadoun,
Patrice Cacoub, Marco Sebastiani.
Affiliations
Pauline Baudart1, Christian Marcelli1, and Clodoveo Ferri2,
1 Rheumatology Unit, University Hospital, avenue de la Côte de Nacre, Caen, France;
2 Rheumatology Unit, University of Modena and Reggio Emilia, Medical School, Azienda Ospedaliero-
Universitaria, Policlinico di Modena, Modena, Italy
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EULAR on-line course on Rheumatic Diseases

Pauline Baudart, Christian Marcelli, Clodoveo Ferri

Table 1 Classification and clinico-pathological characteristics of different cryoglobulinaemias

Classification Composition Pathological findings Clinical associations

Type II–III mixed Oligoclonal IgM RF or Leucocytoclastic Infections (mainly HCV),

Table 2 Proposed classification of systemic vasculitides

2 Mixed cryoglobulinaemia (cryoglobulinaemic vasculitis)

2.3 Clinical manifestations

MC syndrome, or cryoglobulinaemic vasculitis, is characterised by a clinical triad of purpura, weakness and

employed in epidemiological and clinico-pathogenetic studies, as well as in therapeutic trials (Quartuccio,

Clinical and serological investigations at a patient’s first evaluation:

2.5 Differential diagnosis

hypocomplementaemia, glomerulonephritis), as well as the presence of serum autoantibodies commonly

3 HCV-related extrahepatic disorders

3.1 Pathogenesis of HCV-related autoimmune and lymphoproliferative disorders

There is great geographical heterogeneity in the prevalence of HCV-related immunological or neoplastic

3.2 HCV and rheumatic diseases

3.3 Other HCV-related autoimmune disorders

3.4 HCV and malignancies

3.5 HCV syndrome

Fig. 13. The HCV syndrome.

4 Current and future therapeutic strategies for treating mixed cryoglobulinaemia

Figure 14 Therapeutic interventions according to the aetiopathogenesis of cryoglobulinaemic vasculitis.

4.1 Treatment of HCV cryoglobulinemia vasculitis

a) Interferon alpha (IFN) monotherapy, IFN plus Ribavarin

b) Peg-Interferon alpha plus Ribavirin

c) Direct acting antiviral drugs (DAA)

c.2) Second DAAs generation:

4.2 Treatment of non HCV cryoglobulinemia vasculitis

a) Type 1 cryoglobulinemia vasculitis

b) Mixed cryoglobulinemia vasculitis

4.3 Treatment of MC vasculitis relapses.

4.4 Therapeutic guidelines

Figure 15 Therapeutic strategies in patients with cryoglobulinaemia vasculitis. *The treatment of

Pauline Baudart1, Christian Marcelli1, and Clodoveo Ferri2,

1 Rheumatology Unit, University Hospital, avenue de la Côte de Nacre, Caen, France;

Ferri C. Mixed cryoglobulinemia. Orphanet J Rare Dis 2008; 3 :25.

Antonelli A, Ferri C, Fallahi P, Giuggioli D, Nesti C, Longombardo G, Fadda P, Pampana A, Maccheroni M,

Antonelli A, Ferri C, Ferrari SM, Colaci M, Fallahi P. Immunopathogenesis of HCV-related endocrine

Boonyapisit K, Katirji B. Severe exacerbation of hepatitis C-associated vasculitic neuropathy following

De Vecchi A, Montagnino G, Pozzi C, Tarantino A, Locatelli F, Ponticelli C. Intravenous methylprednisolone

* De Vita S, Quartuccio L, Isola M, Mazzaro C, Scaini P, Lenzi M, Campanini M, Naclerio C, Tavoni A,

* Ferri C, Sebastiani M, Giuggioli D, Cazzato M, Longombardo G, Antonelli A, Puccini R, Michelassi C, Zignego

Ferri C, Baicchi U, La Civita L, Greco F, Longombardo G, Mazzoni A, Careccia G,Bombardieri S, Pasero G,

Ferri C, Marzo E, Longombardo G, Lombardini F, Greco F, Bombardieri S. Alpha-interferon in the treatment of

Ferri C, Sebastiani M, Antonelli A, Colaci M, Manfredi A, Giuggioli D. Current treatment of hepatitis C-

Ishizaka N, Ishizaka Y, Takahashi E, Tooda E, Hashimoto H, Nagai R, Yamakado M. Association between

Manganelli P, Giuliani N, Fietta P, Mancini C, Lazzaretti M, Pollini A, Quaini F, Pedrazzoni M. OPG/RANKL

Mazzaro C, Panarello G, Carniello S, Faelli A, Mazzi G, Crovatto M, et al. Interferon

versus steroids in patients with hepatitis C virus-associated cryoglobulinaemic glomerulonephritis.

Dig Liver Dis 2000;32:708–15.

Monti G, Galli M, Invernizzi F, Pioltelli P, Saccardo F, Monteverde A, Pietrogrande M, Renoldi P, Bombardieri S,

Quartuccio L, Petrarca A, Mansutti E, Pieroni S, Calcabrini L, Avellini C, Zignego A, De Vita S. Efficacy of

Roccatello D, Fornasieri A, Giachino O, Rossi D, Beltrame A, Banfi G, Confalonieri R, Tarantino A, Pasquali S,

Saadoun D, Resche Rigon M, Pol S, Thibault V, Blanc F, Pialoux G, et al. PegIFNalpha/ribavirin/protease

Visentini M, Ludovisi S, Petrarca A, Pulvirenti F, Zaramella M, Monti M, Conti V, Ranieri J, Colantuono S,

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