Introduction
Cryoglobulins are immunoglobulins (Ig) that precipitate when serum is incubated at temperatures lower than 37 °C. The existence of circulating cryoglobulins (cryoglobulinemia) is not always related to the presence of symptomatology, and the term “cryoglobulinemic syndrome” is used when patients present clinical manifestations (44). In 1933, Wintrobe and Buell (55) described cryoprecipitation as a laboratory phenomenon. In 1966, Meltzer and Franklin (27) described the typical clinical symptoms associated with cryoglobulinemia, particularly the triad of purpura, arthralgia, and weakness. In 1974, Brouet et al (7) classified cryoglobulins into 3 types: Type I cryoglobulins were those composed of single monoclonal immunoglobulins, and types II and III were those formed by monoclonal (type II) or polyclonal (type III) IgM with rheumatoid factor activity plus the corresponding antigen (usually polyclonal IgG). For this reason, types II and III are classically referred to as “mixed cryoglobulinemia.” Recently, new types of cryoglobulins have been described (41).
Cryoglobulins have been observed in a wide variety of diseases, including malignancies, infections, and systemic autoimmune diseases (17,40). When there is no demonstrable underlying disease, the condition is called “essential cryoglobulinemia.” Since the initial report (36) in 1990 of the association between cryoglobulinemia and hepatitis C virus (HCV) infection, it has become clear that most cases of so-called essential cryoglobulinemia are in fact associated with HCV infection. This has led to important changes in the etiology, classification, and treatment of cryoglobulinemia in the last 10 years. Several studies have analyzed cryoglobulinemia in patients with HCV infection, but studies performed in groups of patients covering all etiologies of cryoglobulinemia are scarce (7,14,17,19,30). In this study, we analyzed the etiology, clinical manifestations, and immunologic features of a large series of consecutive patients with cryoglobulinemia from a single center (Hospital Clinic, Barcelona, Spain).
Patients and Methods
Patient selection
Sera from 7,043 patients were tested for circulating cryoglobulins at the immunology department of our hospital between 1991 and 1999. A cryocrit of 1% or more was detected in 443 (6.29%) patients. Clinical and serologic characteristics of these patients were retrospectively collected in a protocol form. Salient features included in the protocol were the following: 1) age at onset of the disease, defined as the initial manifestation clearly attributable to cryoglobulinemia; 2) age at diagnosis, defined as the age when circulating cryoglobulins in serum were detected; 3) age at study, defined as the age when the patient entered the protocol study; 4) associated diseases such as hematologic, infectious, and autoimmune processes; 5) clinical manifestations at the diagnosis of cryoglobulinemia; 6) cumulative clinical manifestations during disease evolution (from the onset until the protocol study); 7) laboratory findings at time of entry in study.
Study of etiologic factors
Hepatitis B virus (HBV) surface antigen (HBsAg) was analyzed in all patients by enzyme-linked immunoabsorbent assay (ELISA) and antibodies to HCV by a third generation ELISA (Ortho 3.0 Diagnostic Systems, Neckargemund, Germany). Anti-HIV-1-p24 antibodies were analyzed in 153 patients by ELISA coated with HIV-1-p24 (ETI p24 K Sorin antigen recombinant kit, Saluggia, Italy). For confirmation, Western blots (Epitope, Beaverton, Oregon, USA) with HIV-1gp160, gp120, gp41, p65, p51, p24, and p18 bands were applied. Systemic autoimmune diseases were diagnosed based on the following criteria: 1) primary Sjögren syndrome (SS) according to the preliminary diagnostic criteria for SS proposed by the European Community Study Group (51); 2) systemic lupus erythematosus (SLE) according to the 1982 revised criteria of the American Rheumatism Association (46); 3) rheumatoid arthritis (RA) by American Rheumatism Association criteria (5); 4) systemic sclerosis by American Rheumatism Association preliminary criteria (3); 5) polymyositis-dermatomyositis by Bohan and Peter criteria (6); 6) primary antiphospholipid syndrome by the preliminary classification criteria (53); 7) systemic vasculitis by the Chapel Hill criteria (20). The presence of hematologic malignancies was confirmed by the clinical findings and biopsies of lymph nodes and/or bone marrow. Patients with non-Hodgkin lymphoma were further classified by histology (Working Formulation). Finally, essential cryoglobulinemia was considered in those cases in which no infectious, autoimmune, or hematologic disease was found.
Definition of clinical features
The clinical manifestations evaluated in our protocol were defined as follows:
Articular involvement included arthralgia and/or nonerosive arthritis characterized by tenderness, swelling, or effusion involving 2 or more peripheral joints. Arthralgia and/or arthritis was considered as a manifestation of cryoglobulinemia, when cases with coincidental systemic autoimmune diseases (SLE, SS, or RA) were excluded.
Fever: axillary temperature >37.5 °C.
Lymphadenopathy: enlarged nodes (>0.5 cm) in the cervical region, axilla, or inguinal area, in the absence of hematologic malignancies.
Purpura: recurrent palpable lesions that usually involved the lower extremities. Skin biopsies of purpuric lesions showed leukocytoclastic vasculitis.
Leg ulcers: usually painless, invariably associated with purpura.
Acral cyanosis, ischemia, and/or skin necrosis: affecting the tip of the nose or the ears, fingers, toes, or legs.
Raynaud phenomenon: blanching of the fingers, toes, ears, nose, tongue, induced by exposure to cold, stress, or both.
Livedo reticularis: reddish or cyanotic discoloration of the skin with a reticular pattern.
Peripheral neurologic manifestations included paresthesia, numbness, and/or motor defects of the lower extremities, compatible with multiple mononeuritis or polyneuritis. Neuropathy was classified as sensory, motor, or sensory-motor neuropathy according to the symptoms and was confirmed by electromyography. Sural nerve biopsy, when performed, showed vasculitis involving vasa nervorum.
Central neurologic involvement included cerebral ischemia (in the absence of hypercoagulability or vascular risk factors), spinal cord, or cranial nerve involvement.
Nephropathy: a) persistent proteinuria >0.5 g/day, or b) altered urinalysis (hematuria, pyuria, red blood cell casts), or c) raised serum creatinine >1.5 mg/dL. Glomerular injury diagnosed by renal biopsy included: membranoproliferative glomerulonephritis, mesangial proliferative glomerulonephritis, and segmentary and focal glomerulonephritis. Patients with lupus nephropathy were excluded.
Gastrointestinal involvement: intestinal vasculitis was considered in those patients with severe abdominal pain and intestinal bleeding, and confirmed by biopsy or postmortem.
Lung involvement: a) pulmonary fibrosis (chronic diffuse interstitial infiltrates on X-ray with a restrictive pattern on pulmonary function studies), or b) acute pulmonary infiltrates, fever and cough in the absence of pulmonary edema, adult respiratory distress syndrome, infectious pneumonia, lung malignancy, and granulomatous disease, or c) hemoptysis.
Laboratory studies
Serum cryoglobulins were measured after centrifugation. Blood samples were obtained and kept at 37 °C for 30 min before separation. Serum was prepared by centrifuging at 37 °C for 10 min at 2,500 rpm. The serum obtained (10 mL) was transferred to a 15-mL glass graduated conical tube, and incubated at 4 °C for 7 days. If a precipitate or gel was detected, the tube was centrifuged at 2,500 rpm for 30 min at 4 °C. The cryoprecipitate was visually measured according to the graduated level of the glass tube and referred as a cryocrit (%) to the total volume sample. The sera supernatant was discarded, and the cryoprecipitate was washed twice with cold saline at 2,000 rpm for 5 min at 4 °C. The cryoprecipitate was resuspended with saline in a 37 °C water bath. If the cryoprecipitate became soluble, immunofixation with specific antisera to IgA, IgG, IgM, κ, and λ chains and anti total serum proteins was performed. The immunofixation was performed with a Helena “Immunofixation Agarose Kit” following the manufacturer’s procedure. After immunochemical analysis, cryoglobulins were classified according to Brouet et al (7).
Other immunologic tests included antinuclear antibodies (ANA) (indirect immunofluorescence using mouse liver and Hep-2 cells as substrate) and precipitating antibodies to extractable nuclear antigens, including Ro/SS-A, La/SS-B, U1-snRNP, and Sm (counterimmunoelectrophoresis using calf and rabbit thymus and human spleen extracts). Antimitochondrial antibodies, antismooth muscle antibodies, type 1 anti-liver/kidney microsome antibodies, antiparietal cell antibodies, and antithyroidal antibodies (antithyroglobulin and antimicrosomal antibodies) were investigated by indirect immunofluorescence. Rheumatoid factor was detected by latex fixation and Waaler-Rose tests. Complement factors (C3 and C4) were estimated by nephelometry (Behring BNA nephelometer) and CH50 by Lachmann’s hemolytic technique (21). Antineutrophil cytoplasm antibodies were tested by an indirect immunofluorescence assay using ethanol-fixed normal human neutrophils. To establish the antigenic specificities of antineutrophil cytoplasm antibodies, serum samples were further tested with 2 ELISAs using as substrates myeloperoxidase (Bio-Carb, Lund, Sweden) and proteinase 3 (IBL, Hamburg, Germany).
Statistical analysis
We used conventional chi-square and the Fisher exact test to analyze qualitative differences. For comparison of quantitative parameters, the Student t-test was used in large samples of similar variance, and the nonparametric Mann-Whitney U test was used for small samples. A value of p <0.05 indicated statistical significance. When several independent variables appeared to have statistical significance in the univariate analysis, a logistic regression test was performed for multivariate analysis to rule out possible confounding variables. The odds ratio was calculated for assessing the risk of appearance of each variable, with a confidence interval of 95%. This statistical analysis was performed using the SPSS program.
Results
General characteristics
Of the 443 patients with cryoglobulinemia, 258 (58%) were women and 185 (42%) were men (female:male ratio, 3:2). The mean age at the time of cryoglobulinemia detection was 54 years (range, 14–91 yr), and, at the time of the study, was 56 years (range, 14–91 yr). The mean disease duration from onset until time of the study was 2 years (range, 0–20 yr).
Cryocrit levels were <5% in 374 (84%) patients, 5%–10% in 44 (10%), and 10%–20% in 16 (4%) patients; only 9 (2%) patients had cryocrit levels >20%. Cryoglobulins were further characterized when sufficient cryoprecipitate was available, about 4%–5% of cryocrit (Table 1). Thus, 90 cryoprecipitates were analyzed by immunofixation. Thirty-one were classified as type I cryoglobulins, 50 as type II, and only 2 cases as type III. Type I cryoglobulins consisted of monoclonal IgG κ (15 patients), IgM κ (15 patients) and IgG λ (1 patient). In most patients (47 patients), type II cryoglobulins were composed of monoclonal IgM κ + polyclonal IgG. Finally, 7 cases were unclassifiable according to the classification of Brouet et al (see Table 1).
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TABLE 1: Cryoglobulinemia in 90 patients
We compared the main characteristics of patients with type I cryoglobulinemia and mixed cryoglobulinemia (Table 2). Patients with mixed cryoglobulinemia showed a higher prevalence of cutaneous involvement (53% versus 30%, p = 0.039), peripheral neuropathy (26% versus 6%, p = 0.018), and rheumatoid factor (95% versus 72%, p = 0.024).
TABLE 2: Characteristics of patients with type I cryoglobulinemia and patients with mixed cryoglobulinemia
Etiology
Infectious disease was associated with cryoglobulinemia in 331 (75%) patients, autoimmune diseases in 94 (24%), and hematologic disease in 33 (7%) patients (Table 3). Forty-nine (11%) patients had no identifiable disease associated with cryoglobulinemia and were classified as having essential cryoglobulinemia.
TABLE 3: Etiologic factors in 443 patients with cryoglobulinemia
Infection was the main etiologic factor identified in our cryoglobulinemic patients, present in 331 (75%) patients. HCV antibodies were found in 321 (73%) patients, HBsAg in 15 (3%), and human immunodeficiency virus (HIV) antibodies in 29 (19%) of 153 patients tested. We compared the main characteristics of cryoglobulinemic patients according to the presence or absence of HCV infection (Table 4). Compared with those without HCV infection, patients with cryoglobulinemia related to HCV showed a lower prevalence of associated autoimmune (10% versus 43%, p < 0.001) and hematologic (5% versus 15%, p < 0.001) diseases. In addition, HCV patients showed a lower prevalence of articular involvement (16% versus 33%, p < 0.001), cutaneous involvement (22% versus 33%, p = 0.028), lymphadenopathy (3% versus 11%, p = 0.006), fever (3% versus 10%, p = 0.011), Raynaud phenomenon (3% versus 10%, p = 0.011), ANA (32% versus 61%, p < 0.001), anti-Ro/SS-A (3% versus 28%, p < 0.001), anti-La/SS-B (0 versus 11%, p = 0.003), and anti-dsDNA (6% versus 23%, p = 0.003) autoantibodies. In contrast, HCV patients showed a higher prevalence of low CH50 (76% versus 62%, p = 0.014) and rheumatoid factor (68% versus 44%, p = 0.001).
TABLE 4: Characteristics of cryoglobulinemic patients with and without HCV infection
Ninety-four patients had a systemic autoimmune disease associated with cryoglobulinemia: 40 (9%) patients had primary SS, 30 (7%) SLE, 7 (2%) polyarteritis nodosa, 6 (2%) systemic sclerosis, and 3 (1%) primary antiphospholipid syndrome (see Table 3). The main hematologic diseases included 16 (4%) patients with non-Hodgkin lymphoma, 3 with chronic lymphocytic leukemia, 3 with multiple myeloma, 2 with Hodgkin lymphoma, 2 with chronic myeloid leukemia, and 2 with myelodysplasia. Of the 16 patients with non-Hodgkin lymphoma, 8 (50%) were classified as high grade, 4 (25%) as intermediate grade, and 4 (25%) as low grade.
Finally, we analyzed the amount of overlap among the different etiologies (Figure 1). Specifically, we observed a strong overlap between HCV and some autoimmune (50% of SS, 57% of polyarteritis nodosa) or hematologic (50% of non-Hodgkin lymphoma) diseases. We also found that most patients with HBsAg (80%) or HIV infection (76%) also had HCV infection (see Table 3).
Fig. 1: Analysis of overlap among the different etiologies in 443 patients with cryoglobulinemia.
Clinical manifestations
Clinical manifestations attributable to cryoglobulinemia were present in 206 (47%) patients during the evolution of the disease. In the remaining cases, diagnosis of cryoglobulinemia was established by the detection of circulating cryoglobulins.
The main clinical manifestations at the onset and during the evolution of the disease are summarized in Table 5. At onset, the most frequent manifestations were cutaneous involvement in 105 (24%) patients, articular involvement in 82 (19%), renal involvement in 80 (18%), and peripheral neuropathy in 26 (6%) patients. Other clinical manifestations at onset included weakness in 38 (9%) patients, fever in 19 (4%), lymphadenopathy in 16 (4%), central nervous system involvement in 8 (2%), and pulmonary involvement in 6 (1%) patients.
TABLE 5: Clinical manifestations at onset and during disease evolution in 206 of 443 patients who developed cryoglobulinemic symptomatology
During evolution of the disease, the most common cumulative manifestations were renal involvement in 115 (26%) patients, cutaneous involvement in 112 (25%), articular involvement in 91 (21%), and peripheral neuropathy in 35 (8%) patients. Other clinical manifestations included weakness in 45 (10%) patients, polyadenopathies in 22 (5%), fever in 21 (5%), central nervous system involvement in 11 (3%), pulmonary involvement in 9 (2%), and intestinal infarction in 5 (1%). Palpable purpura was the most frequent cutaneous feature (Table 6), present in 67 (15%) patients, followed by Raynaud phenomenon in 23 (5%), skin rash in 20 (5%) patients, supramalleolar ulcers in 16 (4%), distal ischemia or gangrenous changes in 9 (2%), livedo reticularis in 5 (1%), and acrocyanosis in 3 (1%) patients. Renal involvement is summarized in Table 7 (excluding those cases with lupus nephritis). Renal failure (creatinine >1.5 mg/dL) was present in 115 (26%) patients, proteinuria in 87 (20%), and glomerulonephritis in 32 (7%) patients. Twelve patients had membranoproliferative glomerulonephritis, 12 mesangial glomerulonephritis, and 8 segmental and focal glomerulonephritis. Finally, neurologic features are shown in Table 8. Peripheral neuropathy was diagnosed in 35 (8%) patients, and included mixed polyneuropathy in 20 (5%) patients, multiple mononeuritis in 10 (2%), and pure sensory polyneuropathy in 5 (1%) patients. Moreover, 12 (3%) patients showed central nervous system involvement: cerebral ischemia in 7 (2%), spinal cord involvement in 3 (1%), and cranial nerve affectation in 2 (0.5%) patients.
TABLE 6: Cutaneous manifestations during disease evolution
TABLE 7: Renal involvement during disease evolution (excluding SLE patients)
TABLE 8: Neurologic manifestations during disease evolution
We analyzed the relationship between cryocrit levels and cryoglobulinemic features (Table 9). Patients with a cryocrit higher than 5% showed symptomatic cryoglobulinemia more frequently than those with a cryocrit lower than 5% (66% versus 43%, p < 0.001). Specifically, those patients with a cryocrit >5% showed a higher frequency of cutaneous purpura (40% versus 10%, p < 0.001), peripheral neuropathy (19% versus 6%, p < 0.001), and glomerulonephritis (21% versus 9%, p = 0.008).
TABLE 9: Relationship between cryocrit level (above/below 5%) and main cryoglobulinemic manifestations
Immunologic features
The most frequent immunologic features were low CH50 in 201 of 283 (71%) patients, positive rheumatoid factor in 139 of 232 (60%), low C4 in 142 of 283 (50%), ANA in 143 of 349 (41%), low C3 in 72 of 283 (25%), antismooth muscle antibodies in 68 of 346 (20%), and antiparietal cell antibodies in 35 of 347 (10%) (Table 10). We compared the immunologic profile of patients according to the presence or absence of cryoglobulinemic symptoms (Table 11). Patients with symptomatic cryoglobulinemia showed a higher prevalence of low C4 (56% versus 40%, p = 0.009), rheumatoid factor (69% versus 42%, p < 0.001), ANA (52% versus 30%, p < 0.001), and antismooth muscle antibodies (26% versus 13%, p = 0.002) compared with patients without clinical manifestations.
TABLE 10: Immunologic features in patients with cryoglobulinemia
TABLE 11: Main immunologic features in patients according to the presence or absence of clinical manifestations attributable to cryoglobulins
Table 12 shows the main associations between immunologic and clinical features of cryoglobulinemia. Patients with cutaneous involvement showed a higher frequency of low C4 (63% versus 44%, p = 0.003), positive rheumatoid factor (72% versus 52%, p = 0.004) and anti-Ro/SS-A antibodies (23% versus 8%, p = 0.009). Those with articular involvement showed a higher frequency of rheumatoid factor (73% versus 53%, p = 0.004), ANA (61% versus 35%, p < 0.001), antismooth muscle antibodies (29% versus 17%, p = 0.012), and anti-dsDNA antibodies (23% versus 8%, p = 0.002). Patients with peripheral neuropathy more frequently had rheumatoid factor (77% versus 57%, p = 0.048) and those with glomerulonephritis had a higher prevalence of low CH50 (84% versus 69%, p = 0.046) and low C4 (66% versus 47%, p = 0.032).
TABLE 12: Percentage of immunologic features according to the presence or absence of main cryoglobulinemic manifestations
Discussion
In this study, we analyze the main characteristics of the largest series to date of patients from a single center with cryoglobulinemia. This series includes all patients with positive circulating cryoglobulins tested in a tertiary university hospital between 1991 and 1999, and as such includes data from different medical specialities. In this way, selection bias due to a particular specialization was minimized, and the study covers the complete spectrum of cryoglobulinemia. In previous series, the frequency of etiologic factors and clinical or immunologic features was extremely variable, depending on the specialization of the reporting center. For example, in an Italian multicenter study by Monti et al in 1995 (30), the main etiologic factors were only partially studied (HCV infection was only investigated in 24% of patients). However, the possibility of unintentional bias in the design and carrying out of the present study must not be forgotten, due to the different specialities having different levels of interest in the study of cryoglobulinemia. Some units and departments performed a screening search and others analyzed cryoglobulins only in symptomatic patients. In addition, laboratory parameters were not done in all 443 patients, due to the influence of previously performed tests, the presence of clinical manifestations, and, obviously, the different interests of the different specialists.
Since the discovery of HCV in 1989, several authors have reported HCV infection in 80%-90% of cases of cryoglobulinemia (1,12,28,36). In this study, HCV infection is the main etiologic factor identified, present in 73% of patients with cryoglobulinemia. In contrast, we found a lower prevalence of HBsAg-positive patients. Before the isolation of HCV, the possibility that HBV might play a predominant role in causing “essential” cryoglobulinemia was supported by some authors, who reported the existence of HBsAg and/or HBsAb in a substantial number of patients with cryoglobulinemia (24,45). This fact possibly represents a concomitant HCV infection, and studies (1,13) have confirmed that fewer than 5% of patients have HBV-associated cryoglobulinemia. In addition, we also found HIV-1 infection in 29 of 153 (19%) patients tested for HIV, although most showed an associated HCV infection. Dimitrakopoulos et al (10) found a high prevalence of cryoglobulins in HIV-1 infected patients (27%), with a higher prevalence in those with concomitant HCV infection (55% versus 23% in HCV-negative patients). It has been reported that, in HIV infection, the formation of cryoglobulins is strongly associated with high serum titers of HIV-RNA and represents a continuous antigenic stimulation of B lymphocytes (26).
In comparing patients with and without HCV infection, we found a higher prevalence of hematologic and autoimmune diseases in HCV-negative patients, suggesting a greater weight of the hematologic and autoimmune processes in those cryoglobulinemic patients without HCV infection. Moreover, we have described a clearly differentiated immunologic pattern in cryoglobulinemic patients with and without HCV infection. Patients with cryoglobulinemia related to HCV showed a higher frequency of hypocomplementemia and rheumatoid factor, suggesting that the stimulation of the immune system by continuous antigenic exposure to HCV would drive a predominant physiopathologic mechanism based on immunocomplex deposition (related to the rheumatoid factor activity) and the “in situ” complement activation. In contrast, cryoglobulinemic patients without HCV infection showed a higher prevalence of clinical autoimmune features and autoantibodies (ANA, Ro, La, antiDNA), signalling the predominant presence of a systemic autoimmune disease in these patients, in whom the immunologic stimulation would be more heterogeneous than in HCV patients.
Of the systemic autoimmune diseases, the second etiologic factor identified in our study, primary SS and SLE were the most frequent diseases associated with cryoglobulinemia. Invernizzi et al (19) reported 49 patients (5%) with an associated systemic autoimmune disease (most frequently SS). The prevalence of cryoglobulinemia in primary SS has ranged from 5% to 61% in previous reports (19,31,50,52), but 2 studies that analyzed large series (40,49) have found a prevalence of 16%. We previously reported a higher prevalence of HCV infection in SS and cryoglobulinemia (15,40), suggesting that the cryoglobulinemia observed in some cases of “primary” SS might be due to HCV infection. The second autoimmune disease strongly associated with cryoglobulinemia was SLE. The clinical significance of cryoglobulinemia in SLE has been scarcely studied and has been related to the activity and severity of the disease (particularly nephritis) and decreased serum complement levels (2,14,54). The presence of cryoglobulins in autoimmune diseases other than SLE or primary SS has been anecdotally reported (4,9,22). In our series, we describe some patients with cryoglobulinemia and polyarteritis nodosa (7 patients), systemic sclerosis (6 patients) and primary antiphospholipid syndrome (3 patients).
The third etiologic group in our study comprised patients with hematologic processes, which were detected in 33 (7%) patients. The most frequent hematologic disease was non-Hodgkin lymphoma, present in 16 patients. It has long been known that some patients with cryoglobulinemia may develop lymphomas, usually after a long-term follow-up (16,29). Moreover, a possible role of HCV in the development of hematologic malignancies has been suggested, especially in non-Hodgkin lymphoma (11,25,39,57), where HCV infection has been demonstrated in 22%–50% of patients. In addition, a close relationship among cryoglobulinemia, HCV, and non-Hodgkin lymphoma has been documented (16,34,35). It is noteworthy that in our series we found 8 patients with these 3 conditions. We also studied the overlap among the different etiologies of cryoglobulinemia (see Figure 1). We found a strong overlap among certain etiologic factors such as HCV infection, SS, and non-Hodgkin lymphoma. Tzioufas et al (49) reported that mixed cryoglobulinemia and monoclonal rheumatoid factor can be used as laboratory predictive factors for lymphoma development in SS. It is possible that the coincidence in the same patient of cryoglobulinemia, HCV infection, and SS may favor the development of lymphoproliferative diseases. Thus, the frequent association of SS with cryoglobulinemia and HCV infection (41), as well as the possible evolution of any 1 of these 3 entities into a B-cell non-Hodgkin lymphoma (43), suggests a close relationship among HCV infection, cryoglobulinemia, SS, and B-cell lymphoproliferation (42).
Classification of cryoglobulins into 3 types, based on their immunochemical properties, was proposed by Brouet et al (7) in 1974, and is still used since it offers a good correlation with associated diseases and clinical manifestations. The most frequent type found in our 90 characterized patients was type II mixed cryoglobulinemia (56%); similarly, Monti et al (30) detected type II mixed cryoglobulinemia in 47% of their population. However, we report 7 cases of cryoglobulins that do not fit into the classification of Brouet et al (7), a fact also recently reported in other studies. Some authors have described the presence of oligoclonal IgM with trace amounts of polyclonal immunoglobulins, and have proposed the existence of a new cryoglobulin type, the type II-III variant (oligoclonal IgM and polyclonal IgG) (32,48). In another study, Musset et al (33) reported that 25/210 (12%) cases of mixed cryoglobulinemia exhibited an additional monoclonal IgG on immunoblot. The existence of a biclonal IgMk cryoglobulin has been described in a patient with SS (38). In our study, we report 7 cases with biclonal cryoglobulins (monoclonal IgM + monoclonal IgG), either alone or associated with polyclonal IgG. This may reflect a transitional process from oligoclonal to monoclonal B-cell selection, suggesting that cryoglobulinemia is a dynamic process. However, the existence of these oligoclonal cryoglobulins seems to be an infrequent finding.
In the present series, we found a prevalence of cryoglobulinemic symptoms in 46% of patients. Previous studies have found a prevalence of vasculitic symptoms in 30%–50% of patients with cryoglobulinemia associated with HCV infection (8,23,37), although other authors found lower prevalences (18). The most frequent clinical features in our study were cutaneous, articular, and neuropathic manifestations, in accordance with those observed by Gorevic et al (17). We also found a high frequency of renal involvement in our cryoglobulinemic patients (23%), similar to that found by Monti et al (30) (20%). Why only a certain subset of patients with cryoglobulinemia develops symptomatology is an intriguing question. We studied the role of several factors in the development of cryoglobulinemic symptomatology, such as cryoglobulin levels, etiologic factors, and immunologic markers. The appearance of cryoglobulinemic symptoms was related to a higher amount of cryocrit (>5%), association with a systemic autoimmune disease, and presence of some immunologic markers (hypocomplementemia, rheumatoid factor, and ANA). Specifically, we found a close association among some clinical and immunologic features: cutaneous involvement was linked with hypocomplementemia and rheumatoid factor, articular involvement with rheumatoid factor and ANA, neuropathy with rheumatoid factor, and glomerulonephritis with hypocomplementemia. Few studies have analyzed the association between cryoglobulinemic symptoms and etiologic or immunologic factors. Zaltron et al (56) found an association between peripheral neuropathy and the existence of higher rheumatoid factor reactivity and reduced complement C4 levels, and another study (47) correlated the existence of cryocrit levels >10% and low C3 levels with a poor outcome of cryoglobulinemic glomerulonephritis. Finally, in the multicenter study by Monti et al (30), patients with cryoglobulinemia associated with autoimmune diseases showed Raynaud phenomenon, arthritis, and sicca syndrome more frequently.
Patients with mixed cryoglobulinemia showed a higher frequency of cutaneous involvement and peripheral neuropathy (see Table 2). This high prevalence of vasculitic symptoms in patients with mixed cryoglobulinemia probably indicates a different pathophysiologic pathway of tissular damage. Mixed cryoglobulinemia leads to systemic vasculitis because of an inflammation of the vessel walls induced by the deposition of IgM (with rheumatoid factor activity)-IgG complexes with the subsequent complement activation, in a different pathophysiologic mechanism than that observed in type I cryoglobulinemia (predominantly due to a direct obstruction of the vessels) (44). The pathogenicity of mixed cryoglobulins is probably related to the existence of an IgM with rheumatoid factor activity, and we have, in fact, demonstrated a higher prevalence of rheumatoid factor activity in our patients with mixed cryoglobulinemia compared with those with type I cryoglobulinemia.
In conclusion, we analyzed the main etiologic, clinical, and immunologic features in 443 patients with cryoglobulinemia from a single center. HCV infection was the main etiologic factor identified (73%), followed by primary SS (9%), SLE (7%), HIV (7%) and non-Hodgkin lymphoma (4%). However, after a careful analysis, it was not possible to discover any etiology in 11% of patients leading us to consider this group as having essential cryoglobulinemia. A strong overlap was detected among certain etiologies, such as HCV, primary SS, and non-Hodgkin lymphoma. Furthermore, we found cryoglobulinemic symptomatology in nearly 50% of patients; purpura, renal involvement, arthritis, and neuropathy were the most frequent symptoms. The appearance of cryoglobulinemic symptomatology was associated with higher cryocrit levels, the existence of an underlying autoimmune disease, and the presence of immunologic markers such as hypocomplementemia, rheumatoid factor, and ANA.
Summary
We analyzed the clinical manifestations and immunologic features of 443 consecutive patients (258 women and 185 men; mean age, 56 yr) with cryoglobulinemia from a single center. Infectious diseases were detected in 331 (75%) patients, autoimmune diseases in 94 (24%), hematologic diseases in 33 (7%), and essential cryoglobulinemia in 49 (11%). Hepatitis C virus (HCV) infection was found in 321 (73%) patients, Hepatitis B virus surface antigen in 15 (3%), and human immunodeficiency virus (HIV) antibodies in 29 (7%). Forty (9%) patients had primary Sjögren syndrome, 30 (7%) systemic lupus erythematosus, 7 (2%) polyarteritis nodosa, and 6 (2%) progressive systemic sclerosis. Finally, 16 (4%) patients had non-Hodgkin lymphoma, 3 chronic lymphocytic leukemia, 3 multiple myeloma, and 2 Hodgkin lymphoma. We observed a strong overlap between HCV and some autoimmune (50% in Sjögren syndrome, 67% in polyarteritis nodosa) or hematologic (50% in non-Hodgkin lymphoma) diseases.
Clinical manifestations attributable to cryoglobulinemia were present in 206 (47%) patients. The most common manifestations were cutaneous involvement in 112 (25%) patients, renal in 103 (23%), articular involvement in 91 (21%), and peripheral neuropathy in 35 (8%) patients. Other clinical manifestations included weakness in 45 (10%) patients, fever in 21 (5%), and polyadenopathies in 22 (5%). Patients with cryocrits >5% showed symptomatic cryoglobulinemia more frequently than those with cryocrits <5% (65% versus 43%, p = 0.001). Patients with cryocrits >5% showed a higher frequency of cutaneous features (47% versus 21%, p < 0.001), peripheral neuropathy (20% versus 6%, p < 0.001), and renal involvement (42% versus 30%, p = 0.03). Finally, patients with symptomatic cryoglobulinemia showed a higher prevalence of low C4 (56% versus 40%, p = 0.008), rheumatoid factor (69% versus 42%, p < 0.001), and antinuclear antibodies (52% versus 30%, p < 0.001).
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