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Prolonged B cells stimulation due to the Hepatitis C virus (HCV) can result in autoimmunity, stigmatized by rising levels of cryoglobulins (CGs), the rheumatoid factor (RF), and free light chains (FLC) of immunoglobulins (Ig) associated with a range of symptoms, from their absence to severe cryoglobulinemic vasculitis and lymphoma. Here, we aimed to identify an immunological signature for the earliest stages of vasculitis when cryoprecipitate is still not detectable. We firstly analyzed the IgG subclasses, FLC, and RF in 120 HCV-RNA-positive patients divided into four groups according to the type of cryoprecipitate and symptoms: 30 asymptomatic without cryoprecipitate (No Cryo), 30 with vasculitis symptoms but without CGs that we supposed were circulating but still not detectable (Circulating), 30 type II and 30 type III mixed cryoglobulinemia (Cryo II and Cryo III, respectively). Our results revealed that patients with supposed circulating CGs displayed a pattern of serological parameters that closely resembled Cryo II and Cryo III, with a stronger similarity to Cryo II. Accordingly, we analyzed the groups of Circulating and Cryo II for their immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) gene rearrangements, finding a similar mixed distribution of monoclonal, oligoclonal, and polyclonal responses compared to a control group of ten HCV-RNA-negative patients recovered from infection, who displayed a 100% polyclonal response. Our results strengthened the hypothesis that circulating CGs are the origin of symptoms in HCV-RNA-positive patients without cryoprecipitate and demonstrated that an analysis of clonal IGH and TCR rearrangements is the best option for the early diagnosis of extrahepatic complications.

Cryoglobulinemic Vasculitis (CV) is an autoimmune/lymphoproliferative disorder associated with HCV infection that in 5%-10% of cases evolves into a B cell Non-Hodgkin's Lymphoma (NHL). B-cell activating factor (BAFF) is a key regulator in B-cell development and survival. Particular genetic variants are responsible for BAFF signaling impairment in autoimmune and neoplastic diseases. We evaluated BAFF and BAFF-receptor (BAFF-R) polymorphisms in order to determine if they predispose to HCV-related CV and NHL. The analysis was performed on 416 HCV-chronically infected patients: 136 HCV without signs/symptoms of lymphoproliferations/autoimmunity (HCV), 166 HCV with CV (HCV-CV) and 114 HCV with NHL (HCV-NHL). Rs9514828 SNP on BAFF promoter, rs61756766 on BAFF-R and rs12428930 on the BAFF gene were evaluated by Real-Time PCR. Concerning rs9514828, the frequency of C/T genotype was significantly higher in HCV-CV than in HCV. The difference in the distribution of the T/T mutant genotype in HCV-CV compared to HCV was significant as well as the distribution of C/T and T/T genotype in HCV-NHL versus HCV. T minor allele was more frequent in HCV-NHL and HCV-CV than in HCV. The distribution of C/T + T/T (for the dominant model of penetrance C/T + T/T vs. C/C) was significantly higher in HCV-CV and HCV-NHL than in HCV. Genotyping of rs61756766 on BAFF-R coding gene, revealed C/T heterozygosis at a frequency of 11% in HCV-NHL versus 3% in HCV. The T minor allele frequency was higher in HCV-NHL than in HCV. No differences emerged by genotyping rs12428930 SNP on BAFF coding gene. Our results reinforce the hypothesis that BAFF/BAFF-R genetic pattern has a role in the pathogenesis of HCV-related lymphoproliferations. BAFF/BAFF-R variants could identify a risk haplotype for HCV related CV and NHL and a BAFF/BAFF-R genetic profile assessment could potentially contribute to tailoring anti-BAFF therapy by identifying patients with BAFF alterations in which the treatment could be more beneficial., (© 2022 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

Background and Aims: Direct-acting antivirals (DAAs) usually lead to improvement/remission of cryoglobulinemic vasculitis (CV), although symptoms may persist/recur after a sustained virological response (SVR). We evaluated hematological and genetic markers in patients with HCV-SVR vasculitis with and without persisting/recurring symptoms to early predict the CV outcome., Approach and Results: Ninety-eight patients with HCV-CV were prospectively enrolled after a DAA-induced SVR: Group A: 52 with complete clinical response; Group B: 46 with symptom maintenance/recurrence. Monoclonal B-cell lymphocytosis, t(14;18) translocation, and abnormal free light chains κ/λ ratios were detected by flow cytometry or nested-PCR or nephelometry in 4% Group A versus 17% Group B (P = 0.04) patients, 17% Group A versus 40% Group B patients (P = 0.02), and 17% Group A versus 47% Group B (P = 0.003) patients, respectively. At least 1 out of 3 clonality markers was altered/positive in 29% of Group A versus 70% of Group B patients (P < 0.0001). When available, pretherapy samples were also tested for t(14;18) translocation (detected in 12/37 [32%] Group A and 21/38 [55%] Group B) and κ/λ ratios (abnormal in 5/35 [14%] Group A and 20/38 [53%] Group B) (P = 0.0006), whereas at least one clonality marker was detected/altered in 16/37 (43%) Group A and 30/38 (79%) Group B (P = 0.002). CV-associated single-nucleotide polymorphisms were tested by real-time PCR. Among them, notch4 rs2071286 T minor allele and TT genotype showed a higher frequency in Group B versus Group A (46% vs. 29%, P = 0.01, and 17% vs. 2%, P = 0.006, respectively)., Conclusions: Hematological or genetic analyses could be used to foresee the CV clinical response after DAA therapy and could be valuable to assess a rational flowchart to manage CV during follow-up., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Objectives: The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia., Methods: We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs., Results: We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia., Conclusion: The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Hepatitis C virus (HCV) represents the major risk factor for mixed cryoglobulinemia (MC), a small-vessel vasculitis that may evolve into an overt B-cell non-Hodgkin's lymphoma. Here, we aimed to identify a biomarker signature for the early diagnosis of minimal residual disease (MRD). We assessed free light chains (FLCs), IgM k,and IgM λ heavy/light chain (HLC) pairs, and vascular endothelial growth factor (VEGF) in sera from 34 patients with MC vasculitis (32 HCV- and 2 HBV-related), treated with low-dose rituximab (RTX). FLCs and IgM HLCs were measured by turbidimetric assay; VEGF by an enzyme-linked immunosorbent assay. After RTX, the positive (complete + partial) clinical and laboratory responses were of 85.29% and 50%, respectively; in contrast, the mean levels of FLCs, IgM HLCs, and VEGF were substantially unaffected in most patients and still above the normal range. In those achieving a reduction of FLCs and IgM k and λ chains values within the range of normality, we found that post-treatment free λ chains and IgM k values correlated with clinical and laboratory response. Our results suggest that high levels of FLCs, IgM HLCs, and VEGF could represent the signature of "dormant" B cell clones' activity that could be very useful to identify MRD indicative of possible relapse or worsening outcome., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Cryoglobulinaemia consists of circulating monoclonal and/or polyclonal immunoglobulins with rheumatoid factor (RF) activity that precipitate at temperatures <37°C. Cryoglobulinaemic syndrome, characterised by clinical signs of systemic vasculitis, is associated with chronic infection of hepatitis C virus (HCV) and might evolve in B-cell malignancies. In about one third of all HCV infection cases, serum autoantibodies are commonly found. This is probably due directly to the transformation of infected B cells but, also, indirectly, to the viral chronic stimulation of a pool of autoreactive B cells. The pattern of IgG subclasses seems to contribute to the worsening progression of HCV infection into lymphoproliferative and/or autoimmune diseases. Many evidences showed that B cells circulating in patients with HCV-associated mixed cryoglobulinaemia (MC) are profoundly abnormal; moreover, in most of cases, normal B cells are replaced by expanded clonal B cells characterized by the low expression of CD21. After viral eradication, these cells persist in circulation and their occurrence does not correlate with serum cryoglobulins nor with vasculitis response or relapse. It is probably due to the persistence of monoclonal B cells producing RF, that in course of MC can be reactivated by circulating immune complexes, highly produced during infections or tumours. Here, we aimed to review current literature focusing the pathogenesis of MC referring to specificity and immunochemical characteristics of the immunoglobulins involved in cryoprecipitation.

Cryoglobulins consist of serum immunoglobulins that precipitate below 37°C and resolubilize upon warming. The clinical triad of cryoglobulinemia usually includes purpura, weakness, and arthralgia. Cryoglobulinemic syndrome, clinically defined as a systemic vasculitis, is associated with chronic infection with hepatitis C virus (HCV) and autoimmune disorders and can evolve into B-cell malignancies. While the current literature about HCV-associated cryoglobulinemia is not very limited, little is known about the immunologic and serologic profiles of affected patients. Therefore, comprehension of the pathogenetic mechanisms underlying cryoprecipitation could be very helpful. Due to the persistence of viral antigenic stimulation, biomarkers to use after the worsening progression of HCV infection to lymphoproliferative and/or autoimmune diseases are widely needed. Laboratory methods used to detect and characterize low concentrations of cryoprecipitates and immunotyping patterns could improve patient management. The most critical factor affecting cryoglobulin testing is that the pre-analytical phase is not fully completed at 37°C., Competing Interests: Competing interests The authors have no conflicts of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Infections, autoimmunity and cancer play a role as determinants of etiology in Hepatitis C virus (HCV) related mixed cryoglobulinemia (MC). Several factors of risk have been suggested as markers of pathogenesis and progression of HCV-related MC into B cell Non-Hodgkin's Lymphoma (B-NHL). Here, we evaluated IgG subclass distribution, free light chains (FLCs) and vascular endothelial growth factor (VEGF) as a new combination of biomarkers., Methods: We measured IgG1-4 subclasses, FLCs and VEGF levels in sera 53 from HCV-related MC, in comparison with 40 sera from HCV negative patients with rheumatoid arthritis (RA) and 30 from healthy blood donors (HBD)., Results: IgG3 levels were significantly higher in HCV-MC patients with a decrement of IgG2 and IgG4; FLC levels significantly increased in both MC and RA patients' groups; serological VEGF was higher in HCV-MC patients than in HBD in correlation with k and λ levels., Conclusion: Our results suggest that a specific IgG subclasses pattern together with raised levels of FLCs and VEGF could represent the biomarker "signature" of an inflammation multistage of acquired immune system., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Clinical spectrum of hepatitis C virus (HCV)-related cryoglobulinemia varies from an asymptomatic presentation to severe vasculitis and lymphoma. A recent study in HCV-negative patients suggests that low cryoglobulins (CGs) levels are responsible for severe renal and neurological complications. The aim of this study was to identify a panel of serological biomarkers associated with low levels of CGs in HCV-positive patients. We studied a population of 79 untreated patients with chronic HCV infection: 13 naïve patients without CGs; 28 patients with asymptomatic mixed cryoglobulinemia (MC) and low levels of CGs (16/28 with polyclonal type III and 12/28 with microheterogeneous type III CGs); 38 patients with symptomatic MC and high levels of type II CGs. Serum samples were collected and examined for rheumatoid factor (RF) IgG and IgM, free light chains (FLCs) and C3 and C4 complement components. We found that RF-IgG and IgM, free k chains and k+λ were increased while C4 component was reduced, both in symptomatic and asymptomatic patients. Our results suggest that, even in absence of MC symptoms, the low levels of CGs may represent a trigger of activation for immune system in course of HCV infection. The identification of a correlated biomarkers panel could improve the clinical management of these patients and pave the way for target treatment strategies. © 2018 BioFactors, 45(3):318-325, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

Background: Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree., Methods: Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns., Results: After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate., Conclusion: Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC)., Methods: Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures., Results: Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test., Conclusions: Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naïve HCV-patients without symptoms of extrahepatic manifestations., (Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Mixed cryoglobulinaemia (MC) is an HCV-related lymphoproliferative disorder characterized by the presence of circulating immune complexes called cryoglobulins. Treatment with anti-CD20 monoclonal antibody rituximab is proved to be very useful, especially in patients ineligible to interferon-based antiviral therapy. Recently, free light chain (FLC) κ/λ ratio and FLC patterns were associated with MC. The aim of this study was to evaluate changes in FLC-κ, FCL-λ, FLC ratio following rituximab treatment in patients with HCV-related MC and to correlate FLC-κ, FCL-λ and FLC ratio values with therapy response., Patients and Methods: We retrospectively enrolled 46 patients with HCV infection (26 females, 20 males), including 10 patients without signs/symptoms of MC-related vasculitis, 36 with MC vasculitis. Clinical and biological data were recorded at baseline and 6 months after RTX treatment. Nephelometric measurement of serum FLCs was taken., Results: The mean serum FLC-κ level and FLC ratio were significantly higher in patients with MC, compared to HCV patients without MC and to blood donors. An abnormal FLC ratio at baseline correlated with the presence of cryoglobulins, C4 consumption, higher RF level and higher vasculitis rate. To evaluate the predictive value of FLCs, patients with MC were divided into two groups according to RTX therapy outcome (responders and no/partial responders). Abnormal baseline FLC ratio was significantly associated with no/partial response., Conclusions: RTX treatment in HCV-related MC induces a reduction in FLC-κ and RF levels. Moreover, pretreatment FLC ratio, which can be easily assessed by a routine test, may be useful to predict response to this expensive treatment for patients with HCV-related MC ineligible to IFN-based therapy., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: A new rapid, automatic, and sensitive screening test useful to detect cryoglobulins in serum samples is proposed., Design and Methods: The increase of turbidity during the cryoglobulin aggregation was monitored spectrophotometrically in sera from 400 patients with clinical evidence of cryoglobulinemia related disorders and 100 controls. Results were correlated to those obtained by the traditional method., Results: Kinetics of the aggregation curves were described by their maximum turbidity increase, lag time, and slope. Despite a partial correspondence between the traditional and the rapid test, patients with symptomatic cryoglobulinemia showed turbidity values significantly higher than the determined cutoff. Moreover, a functional classification of cryoglobulins is proposed., Conclusions: Due to its high reproducibility, operator independence, low cost, and results obtained within 2 hours, the rapid test can be used as a "real time" monitoring of cryoglobulinemia related diseases and for the evaluation of plasmapheresis efficacy.

Background: Anti-nuclear antibodies are immunoglobulins directed against nuclear antigens. They are associated with many autoimmune disorders, but are frequently found in patients infected with hepatitis C virus, possibly indicating an underlying common origin. Likewise, mixed cryoglobulinemia often accompanies autoimmune diseases and hepatitis C infection., Aim: To compare anti-nuclear antibodies and immunoglobulin content of cryoprecipitates from hepatitis C virus-positive patients in order to assess their predictive value in the onset of hepatitis C virus-driven extrahepatic disorders., Methods: Serum from 40 hepatitis C virus-positive patients and 50 controls with rheumatoid arthritis was processed for cryoglobulin detection: all subjects presented with Type III mixed cryoglobulinemia. Immunoglobulin content and immunoglobulin subclasses of cryoprecipitates were assessed by immunofixation and tested by ELISA for rheumatoid factor. Cryoprecipitates were also analysed for anti-nuclear antibodies by indirect immuno-fluorescence to identify specific patterns typical of each condition., Results: Anti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%)., Conclusions: IgG3 are autoreactive clones unrelated to viral recognition and possibly involved in autoimmune disorders. Altogether, these results may represent useful diagnostic device for early detection of hepatitis C virus-induced autoimmune diseases., (Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Cryoglobulins are immunoglobulins that precipitate in serum at temperatures below 37°C and resolubilize upon warming. The clinical syndrome of cryoglobulinemia usually includes purpura, weakness, and arthralgia, but the underlying disease may also contribute other symptoms. Blood samples for cryoglobulin are collected, transported, clotted and spun at 37°C, before the precipitate is allowed to form when serum is stored at 4°C in a Wintrobe tube for at least seven days. The most critical and confounding factor affecting the cryoglobulin test is when the preanalytical phase is not fully completed at 37°C. The easiest way to quantify cryoglobulins is the cryocrit estimate. However, this approach has low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed of cryoglobulins. The characterization of cryoglobulins requires the precipitate is several times washed, before performing immunofixation, a technique by which cryoglobulins can be classified depending on the characteristics of the detected immunoglobulins. These features imply a pathogenic role of these molecules which are consequently associated with a wide range of symptoms and manifestations. According to the Brouet classification, Cryoglobulins are grouped into three types by the immunochemical properties of immunoglobulins in the cryoprecipitate. The aim of this paper is to review the major aspects of cryoglobulinemia and the laboratory techniques used to detect and characterize cryoglobulins, taking into consideration the presence and consequences of cryoglobulinemia in Hepatitis C Virus (HCV) infection. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the efficacy of the use of sample handling and transport devices in the pre-analytical phase of cryoglobulina and cryoglobulinemic syndrome diagnosis.