Cryoglobulinemic glomerulonephritis: clinical presentation and histological features, diagnostic pitfalls and controversies in the management. State of the art and the experience on a large monocentric cohort treated with B cell depletion therapy.

Cryoglobulinemia is defined by the presence of immunoglobulins having the following characteristics: forming a gel when temperature is <37 °C, precipitate in a reversible manner in the serum, and redissolve after rewarming. The presence of both polyclonal IgG and monoclonal IgM (type II), or of polyclonal IgG and polyclonal IgM (type III) identifies the mixed cryoglobulinemia (MC). The identification of the Hepatitis C virus (HCV) infection in most of the cases previously defined as "essential" represented a cornerstone in the understanding the pathogenesis of this condition. The picture of MC comprehends heterogeneous clinical presentations: from arthralgias, mild palpable purpura, fatigue to severe vasculitis features with skin necrotic pattern, peripheral neuropathy and, less commonly, lungs, central nervous system, gastrointestinal tract, and heart involvement. The kidney represents the most common organ presentation, and the presence of glomerulonephritis is a key element when considering prognosis. We discuss the clinical presentation and histological features, diagnostic pitfalls, and controversies in the management of patients with cryoglobulinemic glomerulonephritis, with a special focus on reporting our experience in treating patients with B cell depletion therapy.

Successful evolution of morphea after hepatitis C virus eradication with direct-acting antiviral agent treatment

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[A Case of Secondary Cryofibrinogenemia with Cholangiocarcinoma and Deep Venous Thrombosis].

Cryofibrinogen (CF) is a type of cryoprotein (CP) that can precipitate in cooled plasma but not in serum, and resolves upon warming. We identified a case of secondary cryofibrinogenemia with cholangiocarcinoma and deep venous thrombosis. The patient's cryocrit measured using a Wintrobe tube was 19% in sodium citrate plasma stored for 7 days at 4 degrees C. We performed quantitative analysis of plasma proteins (fibrinogen, IgG, IgA, IgM, C3, C4, α1-antitrypsin, and C-reactive protein) before and after precipitation for 12 hours at 4 degrees C. The plasma fibrinogen concentration decreased by 16.7% (120 mg/dL --> 100 mg/dL), whereas the others were unaffected by precipitation. The CP purified from the patient's plasma was washed three times with saline and subjected to Western blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analyses. Western blot analysis indicated that the purified CP was composed of not only fibrinogen but also fibronectin, α1-antitrypsin, α2-macroglobulin, coagulation factor VIII, and IgG, IgA, and IgM. Interestingly, SDS-PAGE analysis showed that the molecular weight of the patient's CF differed from that of purified normal fibrinogen (340 KDa) and consisted of several low-molecular-weight bands (50-250 KDa). From these results, we speculated that CF found in this case was a mixture of degradated fibrinogen and some plasma proteins. In summary, cryofibrinogenemia is a rare and under-recognized disease. Sample information in routine clinical practice is valuable to diagnose this disease.

Primoinfección por citomegalovirus como causa de desarrollo de crioaglutininas y crioglobulinas en una paciente sometida a trasplante renal / No disponible

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Cryoglobulinemia.

Cryoglobulinemia refers to the presence in serum of immunoglobulins that precipitate at a cold temperature. Type I cryoglobulins are single monoclonal immunoglobulins usually associated with haematological disorders. Types II and III are mixed cryoglobulins, composed of monoclonal or polyclonal IgM respectively, having rheumatoid factor activity that bind to polyclonal immunoglobulins. Mixed cryoglobulinemia (MC) syndrome is a consequence of immune-complex mediated vasculitis and is characterized by a typical clinical triad: purpura, weakness, arthralgias; many organs particularly kidney and peripheral nervous system may be involved. MC may be associated with infectious and systemic disorders and since 1990 studies have demonstrated that hepatitis C virus (HCV) may be considered the principal trigger of the disease. The relation between MC and HCV infection shows new insights in the interpretation of the link between viral infection, autoimmune phenomena and lymphoproliferative disorders evolution. In fact, the virus chronically stimulates B-cell polyclonal proliferation from which a monoclonal population may emerge. In symptomatic patients with HCV related MC therapeutic strategy should include an attempt at viral eradication. Antiviral therapy may also be effective in determining the regression of B-cell lymphoproliferative disorder. Rituximab could represent a safe and effective alternative to standard immunosuppression and exerts selective B-cell control.

Effect of routine therapy and selective plasmapheresis on electrical properties of cryoglobulins during ischemic stroke.

The charge properties of cryoglobulins were examined during the first 21 days of ischemic stroke (atherothrombotic and cardioembolic). Routine drug therapy produced no effect on the charge of cryoglobulins. Plasmapheresis significantly modified the electrokinetic parameters of cryoglobulins during atherothrombotic stroke and produced less pronounced effect on these proteins during cardioembolic stroke.

A critical role for sialylation in cryoglobulin activity of murine IgG3 monoclonal antibodies.

Cryoprecipitating IgG3 autoantibodies have been shown to play a significant role in the development of murine lupus-like autoimmune syndrome. However, the structural basis of IgG3 cryoprecipitation still remains to be defined. In view of the implication of positively charged amino acid residues present in variable regions in IgG3 cryoglobulin activity, we explored the role of terminal sialic acids in oligosaccharide side chains for the cryogenic activity of IgG3 mAb. Comparative oligosaccharide structural analysis of different cryogenic and non-cryogenic IgG3 mAb showed an inverse correlation between the extent of sialylation and cryogenic activity. The inhibitory role of sialylation was further confirmed by the demonstration of enrichment of less and more sialylated IgG3 in cryoprecipitated and noncryoprecipitated fractions, respectively, separated from four different cryogenic IgG3 mAb. Significantly, the sialic acid contents of the latter fraction became comparable to those of non-cryogenic IgG3 mAb. Finally, we observed that highly sialylated non-cryogenic IgG3 mAb was more potent in the inhibition of cryoprecipitation of cryogenic IgG3 mAb. Our results thus suggest that the content of negatively charged sialic acids in oligosaccharide side chains is one of the critical factors to determine IgG3 cryoglobulin activity, along with amino acid sequences of the IgG3 variable regions.

'Agglutination and flocculation' of stem cells collected by apheresis due to cryofibrinogen.

Collection of peripheral stem cells by apheresis is a well-described process. Here, investigations concerning 'agglutination and flocculation' of stem cells collected from two patients are described. In both cases, cryoproteins were observed and cryofibrinogen was identified using high-resolution two-dimensional electrophoresis. In one case, peripheral stem cells were collected after a second course of mobilization, and the cells were immediately washed at 37 degrees C before being frozen, allowing their use, despite the presence of cryofibrinogen. In the other case, 'agglutination' was reversed by warming the bag, and plasma was removed before freezing.

Heat insoluble cryoglobulin associated with gangrene in multiple myeloma.

Cryoglobulins are immunoglobulins that have tendency to precipitate in temperatures below 37 degrees C and dissolve with rewarming. Monoclonal cryoglobulins are usually associated with a distinct hematological disorder and often are asymptomatic. Heat insoluble cryoglobulin has been described with Sjogren's syndrome and glomerulonephritis but, not with multiple myeloma. Severe sensitivity to cold occurs with high thermal insolubility of the cryoprotein, with dramatic symptoms when exposed to minimal lowering of the temperature. We report a case of a 49 year old man with multiple myeloma and an unusual type I cryoglobulin that caused occlusive gangrene. The cryoglobulin appeared as a milky white precipitate that was resistant to re-suspension and did not dissolve at 37 degrees C. Immunoelectrophoresis of the cryoglobulin, which dissolved at 56 degrees C, showed it to be composed of a monoclonal IgG kappa protein (3.5 g/dl). Unlike most high thermal insoluble cryoglobulin, cold associated symptoms were not seen. In addition to steroids, plasmapheresis was initiated thrice a week with albumin fluid replacement. Plasmapheresis caused a marked decline in cryocrit levels from 21% to less than 0.5% in 9 days after 4 procedures with resolution of the gangrene of the feet and after 6 treatments, vasculitic symptoms improved dramatically. The cryoglobulin test was negative 2 weeks after initiation of treatment. The patient was treated for the myeloma and there was no recurrence of occlusive symptoms. Proper laboratory procedure and careful examination and handling of cryoglobulinemic samples facilitate detection of unusual cryoglobulins. This is a unique report of multiple myeloma with gangrene of lower extremities that has a heat insoluble cryoglobulin.

High resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry.

Cryoglobulins are cold-precipitable serum immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders such as hepatitis C, Waldenström's macroglobulinemia, multiple myeloma, chronic lymphocytic leukemia, and rheumatoid arthritis. The mechanism(s) of cryoprecipitation has remained obscure hitherto, which has prompted recent intensive efforts on the identification of cryoglobulin components. In the present study, two-dimensional gel electrophoresis (2-DE) combined with high resolution Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry has been applied as a powerful approach for the analysis of cryoglobulins. While FT-ICR mass spectrometry has been shown to enable the high resolution identification and structure analysis of biopolymers using both electrospray (ESI) and matrix-assisted laser desorption ionization (MALDI), the recently developed MALDI-FT-ICR source is shown here to provide high (sub-ppm) mass determination accuracy and isotopic fine structure as particular advantages in the identification of proteins. The main protein components in a serum cryoprecipitate from a patient with hepatitis C virus (HCV) infection and presenting type II cryogobulinemia are immunoglobulin (Ig)M and IgG which were identified by MALDI-FT-ICR MS analysis after separation by 2-DE as mu- and gamma-heavy chains, kappa- and lambda-light chains, and J-chains. Furthermore, complementarity determining regions CDR1 and CDR2 from monoclonal IgM-RF variable region (V)L were directly identified using accurate mass determinations by FT-ICR-MS. The presence of Spalpha was ascertained as an IgM-associated protein in the serum cryoprecipitate from a patient with HCV infection.

Human IgG1/IgG3 cryoglobulin suggesting lack of allelic exclusion.

Immunoglobulins undergoing cold-dependent precipitation are known as cryoglobulins. A type I cryoglobulin after Brouet et al. from serum of a patient with severe cutaneous vasculitis and membranoproliferative glomerulonephritis was purified by reversible temperature-dependent precipitation and analyzed using FPLC, Western blotting and peptide sequencing. The isolated cryoglobulin consisted of a single complex of a molecular weight of above 210kDa observed under non-reducing conditions in SDS-polyacrylamide gel electrophoresis (PAGE). Under reducing conditions, this complex resolved into three bands, two of which were reminiscent of Ig heavy (HC) chains and one of Ig-light chains (LC). The FPLC-purified type I cryoglobulin showed reversible precipitation analyzed by spectrophotometry. Delineation of the peptides involved in complex formation by immunoblot analysis and peptide sequencing revealed IgG3-V(H)4/Igkappa-VkappaIII/JkappaII and IgG1/V(H)3 molecules with evidence of somatic mutation. Coomassie blue-staining suggested that molar amounts of the IgG3-heavy chain were much higher than that of the IgG1-heavy chain. Treatment with SDS and boiling did not disrupt the unusually high molecular weight Ig complex. Pre-treatment of the cryoglobulin in 6M guadinium hydrochloride followed by gel filtration chromatography suggested covalent association of the IgG3, IgG1 and Igkappa molecules. Therefore, it might be that the cryoglobulin was produced by a single plasma B cell clone which passed immunological check-points in terms of B cell selection in the bone marrow in the absence of allelic exclusion, class switching and affinity maturation by somatic mutation.

Molecular study of an IgG1kappa cryoglobulin yielding organized microtubular deposits and glomerulonephritis in the course of chronic lymphocytic leukaemia.

Glomerulonephritis with organized microtubular monoclonal immunoglobulin deposits (GOMMID) and glomerulonephritis related to type I cryoglobulin are well-known but rare complications of B cell derived chronic lymphocytic leukaemia. In these disorders, monoclonal Ig have never been studied at the molecular level. We conducted a pathological and molecular analysis in a patient with chronic lymphocytic leukaemia, glomerulonephritis and a single circulating monoclonal Ig. Unusual IgG1kappa kidney deposits were observed. The heavy and light chain variable region sequences of that cryoprecipitating monoclonal Ig were characterized. Light microscopy revealed glomerulonephritis typical of cryoglobulinaemia, with neutrophil and macrophage infiltration, endocapillary hyperplasia and few protein thrombi. Electron microscopic study clearly evidenced numerous subepithelial mixed granular and organized deposits with a unique microtubular organization, reminiscent of the GOMMID. The Ig molecule sequence revealed alterations of charge and hydrophobicity potentially promoting a crystal-like aggregation and the aggregation of microtubules. This description suggests that common mechanisms are involved in various forms of precipitation and/or deposition of complete Ig molecules, with a variable extent of organization and with a possible overlap between pathological patterns of either glomerulonephritis with microtubular deposits or type I cryoglobulinic glomerulonephritis.

Current topics on cryofiltration technologies.

In the last 40 years, therapeutic plasmapheresis techniques have been improving considerably. These include cryofiltration technologies providing novel ways of removing large amounts of cryoproteins from plasma. The concept of cryofiltration involves exposure of plasma to below core (37 degrees C) and room temperatures (25 degrees C) without freezing. It was initially used to treat diseases such as cryoglobulinemia with systemic vasculitis, rheumatoid arthritis, systemic lupus erythematosus, and ABO-incompatible transplants. There are 2 basic types of cryofiltration. The first method removes cryoproteins, namely cryoglobulins that precipitate at 4 degrees C. Several filters have been used for this procedure like the AP06M (Asahi Medical, Tokyo, Japan) with a 0.2 microm pore size, a 0.65 m2 surface area, and a cellulose diacetate (CDA) membrane. It has been used in the United States and Japan for treatment of rheumatoid arthritis and cryoglobulinemia. A major disadvantage was frequent filter plugging, which was cumbersome and it is no longer used in the United States. The G3 cryofilter (Gelman Sciences, Ann Arbor, MI, U.S.A.) with a 3 microm pore size was tried in vitro but proved inadequate by design. Currently in our institution, the cryoglobulin filter (Pall Medical, Ann Arbor, MI, U.S.A.) is used with a 4.3 microm pore size, a 0.135 m2 surface area, and an acrylic co-polymer pleat membrane. We performed over 1,200 procedures in 40 patients in the last 8 years. The second type of cryofiltration removes cryogel, which is an agglutination complex of fibrinogen, fibronectin, fibrin split products, and cold insoluble proteins with a heparin core, at temperatures between 2 and 10 degrees C. The AP06M, the AC1740 (Asahi Medical) with a 0.02 microm pore size, a 1.70 m2 surface area, and a CDA membrane, and the Evaflux-4A (Kuraray Company, Osaka, Japan) with a 0.03 microm pore size, a 2 m2 surface area, and an ethylene vinyl alcohol membrane are used to remove cryogel to treat ABO-incompatible transplants as well as rheumatoid arthritis and other previously mentioned diseases. This article will discuss each cryofiltration treatment modality.

Hepatitis G and hepatitis C RNA viruses coexisting in cryoglobulinemia.

OBJECTIVE: Hepatitis G virus (HGV) has been identified as a new member of the Flaviridae family, which includes the hepatitis C virus (HCV). There is a well established association between HCV and cryoglobulinemia; however, to date HGV has not been linked with various types of cryoglobulinemia. We investigated the association of HGV with cryoglobulinemia. METHODS: We studied 10 patients with cryoglobulinemia. The cryoglobulins were purified and identified by immunofixation electrophoresis. HGV and HCV RNA were studied in the serum and in purified cryoglobulins by reverse transcriptase polymerase chain reaction. RESULTS: Nine of 10 patients were women, aged 37-74 years. These patients had combinations of hepatitis C (6), vasculitis (7), lymphoproliferative (3), and autoimmune and connective tissue diseases (3). Of the 10 patients, 3 were positive for both HGV RNA and HCV RNA. Two of three patients with dual infection of HGV and HCV had malignancies (Waldenstrom's macroglobulinemia, B cell lymphoma). In one of these 3 patients HGV RNA was positive in the cryoglobulin fraction, but not in serum. Three other patients were positive for HCV RNA alone. CONCLUSION: HGV may be associated with cryoglobulins. Our three patients were co-infected with HCV. Since our series is small, the pathogenetic role of hepatitis G and its relationship to malignancy remain to be elucidated.

[Cryoglobulinemia in chronic liver diseases: cause or consequence?]. / Cryoglobulinaemia krónikus májbetegségekben: ok, vagy következmény?

Cryoglobulinemia occurs in a wide spectrum of infectious, autoimmune, neoplastic, renal and liver diseases. In the first part of the publication the authors give a review about laboratory and clinical features of cryoglobulinemia and its associated syndromes. In essential mixed cryoglobulinemia (arthralgia-purpura-weakness) clinical and laboratory evidence of liver involvement are frequently found. In chronic liver diseases of different etiologies mixed cryoglobulins can be detected. In 30-94% of cases of chronic hepatitis C virus infection and chronic liver diseases related to hepatitis C virus laboratory signs (and occasionally clinical symptoms as well) of mixed cryoglobulins have recently been reported. In the cryoprecipitates of these cryoglobulins positive patients markers of hepatitis C virus have been detected. The presence of high prevalence of cryoglobulins in chronic hepatitis C virus infection and chronic liver diseases related to hepatitis C virus suggests a significant role for this hepatotropic virus in the pathogenesis of mixed cryoglobulins. On the other side, the impact of impaired liver function in the clearance of (cryo)immunocomplexes from the serum can be a causative factor in the production of cryoproteins in chronic liver disease of different etiologies.

Cryofiltration apheresis in the treatment of cryoprecipitate induced diseases.

Cryoprecipitates include cryoglobulins, cryofibrinogen, cryoparaproteins, and some cold hemagglutinins. We used 3 different methods: plasma exchange (PE), plasma filter (PF), and cryofilter (CF) to remove cryoproteins. Twenty-four patients received more than 800 CF, PF, and PE treatments. The PF and CF methods have average pore sizes of 0.03 microm and 4.3 microm and surface areas of 2.0 m2 and 0.135 m2, respectively. The plasma was separated by centrifugation, cooled to 4 degrees C, and the cryoprecipitate was removed by CF. Albumin solution 5% was used as a replacement fluid in PE, but no albumin was required when using PF or CF. Our results show that the CF and PF methods do not cause complement activation. Although PE is effective for removal of cryoproteins, it is nonspecific, nonselective, and requires human albumin or fresh frozen plasma. If intensive PE is required, it may cause depletion of immunoglobulins and coagulation factors and other vital plasma components. The PF removes macromolecules such as IgM and IgG effectively and it is semiselective. It removes any protein with a molecular weight > or = 200,000, which makes it semispecific treatment. CF is very effective and selectively removes cryoproteins; therefore it is a specific therapy. In conclusion, cryofiltration apheresis is the best method to remove cryoproteins in the treatment of cryoprecipitate induced diseases.

Cryofiltration apheresis for treatment of cryoglobulinemia associated with hepatitis C.

Cryofiltration apheresis (CA) is a specific therapy for treatment of patients with cryoglobulinemia. We evaluated the safety and efficacy of CA in patients with mixed cryoglobulinemia associated with hepatitis C. As reported previously, the Cryoglobulin Filter comprises a membrane module inside a refrigeration unit on-line with a Spectra Apheresis System (COBE, Denver, CO). The efficacy of cryofiltration was measured by comparing the sieving coefficient of cryoprecipitable proteins (CPP) to that of albumin and comparing the systemic CPP concentration ratio post to pre treatment. Five patients were enrolled in this study, and a minimum of 10 procedures were performed for each patient. The risk for hepatitis C was multiple blood transfusions, intravenous drug abuse, immunosuppressive therapy, or renal transplantation. Four patients had Type II mixed cryoglobulinemia, and one patient had Type III. Four patients had chronic renal failure; one with liver cirrhosis received alpha interferon along with CA. One patient had no response to conventional plasma exchange and immunosuppressive therapy secondary to repeated infections and sepsis; CA was the only viable therapy for this patient. The maximum CPP concentration before therapy ranged from 1,440 to 7,440 micrograms/ml. The plasma CPP sieving coefficient at 1 L filtrate ranged from 0.25 to 0.74 (average +/- SE, 0.51 +/- 0.19; n = 39). The sieving coefficient for albumin was 1 (n = 50). The systemic CPP ratio post to pre treatment ranged from 0.28 to 0.83 (average +/- SE, 0.59 +/- 0.20; n = 37). No adverse effects specific to CA were observed. The CA was safe and effective and possibly the only choice of therapy in patients with cryoglobulinemic hepatitis C who have no response to plasma exchange and immunosuppressive therapy.

Clinical trials of a cryoglobulin filter.

The authors report the results of clinical trials of a high capacity cryoglobulin filter (Cryofilter) in seven patients with cryoglobulinemia unresponsive to high doses of prednisone or immunosuppressive drugs who required plasmapheresis. The objective of this study was to test the safety and efficacy of the cryofilter in a limited patient population according to the investigational Device Exemption guidelines of the FDA. The cryoglobulins were selectively filtered from plasma at 4 degrees C by a cryofilter characterized by a membrane surface area of 0.135 m2 and an average pore size of 4.3 microns. Safety was evaluated by patients vital signs, complement activation, and clinical score of symptoms in the course of 10 treatments. Efficacy of cryofiltration was evaluated by comparing sieving of the cryoglobulins to that of albumin; immunoglobulins G, A, and M; and fibrinogen. All seven patients completed the series of 10 treatments without notable complement activation or any signs of discomfort. The cryofilter was particularly selective in patients with high cryoglobulin concentrations. Improvement in clinical symptoms was observed in all patients.