In from the cold: M-protein light chain glycosylation is positively associated with cold agglutinin titer levels.

BACKGROUND: Primary cold agglutinin disease (CAD) is a monoclonal antibody (M-protein) and complement-mediated chronic hemolytic disease process. Antibody glycosylation can play a role in both antibody half-life and complement fixation. Recently, M-protein light chain (LC) glycosylation has been shown to be associated with AL amyloidosis. We hypothesized that M-protein LC glycosylation is also associated with cold agglutinin (CA) titers and CA-mediated hemolysis. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing CA titer evaluation underwent mass spectrometric analysis for M-proteins and M-protein LC glycosylation. A subset of serum samples also underwent evaluation for the ability to trigger cold hemolysis in vitro. M-protein and M-protein LC glycosylation rates were compared across CA titer groups, clinical diagnosis, direct antiglobulin testing (DAT) results, and cold in vitro hemolysis rates. RESULTS: Both M-protein and M-protein LC glycosylation rates significantly differed across CA titer groups with the highest rates in those with elevated CA titers. M-protein LC glycosylation occurred almost exclusively on IgM kappa M-proteins and was significantly associated with positive DAT results and a clinical diagnosis of CAD. Cold in vitro hemolysis was demonstrated in two patients who both had a CA titer of more than 512 but there was no significant association with CA titer group or M-protein LC glycosylation status. CONCLUSION: M-protein LC glycosylation is significantly associated with higher CA titer levels. Given the role that antibody glycosylation can play in antibody half-life and complement fixation, further studies are needed to clarify the effects of LC glycosylation within the context of CAD.

Resolution of serologic problems due to cold agglutinin mediated autoimmune hemolytic anemia and its transfusion decision.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is a rare disease characterized by hemolysis caused by autoantibodies against erythrocyte surface antigen. These antibodies can be classified as warm, cold, or mixed types. METHODS: We report two cases of cold agglutinin disease (CAD), which were eventually diagnosed owing to blood group discrepancy. Resolution was achieved after washing the red blood cells (RBCs) with warm saline and absorbing the autoantibodies at 4°C with the washed RBCs. We also assessed the patient's condition and discussed the strategy of blood transfusion. RESULTS: The first case occurred after postoperative chemotherapy for rectal cancer, and the other manifested with anemia from the outset. Direct antiglobulin tests were positive and revealed autoantibodies against C3d only. Cold agglutinin titration was performed, and the titers of both were 1:1024. Eventually, the patient's condition stabilized without blood transfusion. CONCLUSION: The serological discrepancies observed in the blood transfusion department can successfully guide blood transfusion decisions in cases of CAD.

Long-Term Outcomes of Patients With HCV-Associated Cryoglobulinemic Vasculitis After Virologic Cure.

Patients with hepatitis C virus-associated cryoglobulinemic vasculitis (HCV-CV) have high rates of clinical remission after treatment with direct-acting antivirals (DAAs), but circulating cryoglobulins persist, and vascular disorders reappear in some patients shortly after DAA treatment ends. We performed a prospective study to assess the long-term clinical and immune system effects of HCV eradication with DAAs in 46 patients with HCV-CV and 42 asymptomatic patients with circulating cryoglobulins. A median of 24 months after DAA treatment (range, 17-41 months), 66% of patients with HCV-CV and 70% of asymptomatic patients with circulating cryoglobulins had an immunologic response, with comparable reductions in cryocrit from 2.6% to 0% (P < .05). However, 20% of patients still had positive test results for cryoglobulins after DAA therapy. Among patients with HCV-CV, 42 (91%) had a clinical response, in that their Birmingham Vasculitis Activity Score (version 3) decreased from 7 to 0 (P < .01). Nevertheless, within 2 years after a sustained viral response to DAA therapy, 5 patients with HCV-CV (11%, 4 with cirrhosis) had relapses of vasculitis that included severe organ damage and death.

Subclasses of Monoclonal (Type I) Immunoglobulin G Cryoglobulins: Report on Two Distinct Cases with Myeloma.

BACKGROUND: While different clinical manifestations of IgM and IgG monoclonal cryoglobulins have been demonstrated, little is known about the roles of IgG subclasses in the pathophysiology of these conditions. METHODS: In two cases of myeloma-associated monoclonal (type I) cryoglobulinemia with quite distinct clinical and biological features, serum samples were analyzed using an original IgG subclass-specific immunoblotting technique. RESULTS: The first case had painful arthritis of hands and feet, with skin purpura and a sharp decrease of complement C4 level, and the cryoglobulin was of IgG1 subclass. The second case displayed mostly thrombotic lesions of the limb extremities, C3 and C4 serum levels were normal, and the cryoglobulin belonged to the IgG2 subclass. CONCLUSIONS: Type I cryoglobulins of distinct IgG subclasses may result in different syndromes. In both cases, the treatment relies on eradication of the underlying plasma cell dyscrasia.

Using direct antiglobulin test results to reduce unnecessary cold agglutinin testing.

BACKGROUND: Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia mediated by autoantibodies that preferentially react at 4°C. Laboratory testing for cold-reactive autoantibodies is laborious and may not be ordered judiciously, particularly in patients with a negative direct antiglobulin test (DAT). We sought to determine whether a negative DAT using anti-human complement (anti-C3) rules out elevated cold agglutinin (CA) titers and the diagnosis of CAD. STUDY DESIGN AND METHODS: We performed a retrospective study of patients with a CA test performed at three major academic medical centers: Barnes-Jewish Hospital (2003-2014), Vanderbilt University Medical Center (2007-2009), and Massachusetts General Hospital (2009-2014). RESULTS: This study included 801 patients, of whom 51% (n = 410) had a DAT within the 7 days before CA testing. A total of 98% of patients with a negative DAT using anti-C3 had a negative CA titer (<64). Only five subjects had a negative DAT using anti-C3 and an elevated CA titer. CONCLUSIONS: Overutilization of CA testing could be reduced by establishing laboratory acceptance criteria based on a positive DAT using anti-C3. Such acceptance criteria would have reduced CA testing by 68% for those with an available DAT result.

Reduction in the presence of cryoglobulins over time in the hemodialysis treatment.

BACKGROUND: The presence of cryoglobulins in patients with chronic kidney disease (CKD) on hemodialysis is well described. However, the generation of cryoglobulins during the dialysis treatment has yet to be established. The aim of the present study was to determine the presence of serum cryoglobulins over time in the dialysis treatment in patients with CKD not infected with hepatitis C virus (HCV). METHOD: Peripheral blood samples were collected at the beginning of dialysis treatment and at 30, 60, 90 and 120 days afterwards. Cryoglobulins were defined by the presence of immunocomplexes that precipitated in vitro with exposure to cold and resolubilized when rewarmed. The components of the cryoprecipitate were analyzed by radial immunodiffusion. RESULTS: In this study, 14 patients were included: 11 male and three female, aged 28-88 years, with mean time on hemodialysis of 57 ± 36 days at baseline. The presence of cryoglobulin, constituted by IgM, IgA, IgG and the C3 and C4 components of the complement, was observed in the serum of all patients at the beginning of hemodialysis. Sequence analyses showed that the amount of cryoprecipitate decreased during the dialysis treatment. CONCLUSION: There was a high prevalence of mixed cryoglobulins in CKD patients at the beginning of hemodialysis, and the amount of cryoprecipitate decreased during the treatment.

[The optimization of technique of detection of cryoglobulins in conditions of clinical diagnostic laboratory].

The technique is proposed to evaluate cryoclobulins associated with IgM and IgG on the basis of detection of immune complexes circulating in healthy individuals and patients with liver cirrhosis. It is demonstrated that in patients with liver cirrhosis content of cryoclobulins associated with IgM increases almost twice and those associated with IgG more than two and a half times as compared with healthy individuals. It is established that in healthy individuals cryoclobulins are also present in blood in small amount. The proposed approach to detection of cryoclobulins permits to minimize time of implementation of study and also discharge of reagents.

The development of mixed cryoglobulinemia in Capillaria hepatica-infected mice is associated with the capillaria antigen-induced selective proliferation of splenic B-1a cells in response to interleukin-5 stimulation.

Chronic infection by pathogens such as hepatitis C virus induces monoclonal or oligoclonal proliferation of B cells, which produce IgM rheumatoid factor, leading to the development of mixed cryoglobulinemia (MC). Antigen-driven lymphoproliferation is essential to the onset of MC; however, the underlying mechanism is largely unknown. Herein, we show that type II MC is induced by Capillaria hepatica infection through a mechanism in which splenic B-1a cells reacting to C. hepatica-specific antigen selectively proliferate, producing IgM rheumatoid factor under co-stimulation of the specific worm antigen and IL-5. In vitro assays using B-1a cells from infected mice showed that stimulation by C. hepatica soluble fraction promoted the proliferation of B-1a cells and the secretion of IgM, which reacted with the 75-kDa antigen in the soluble fraction. The severity of MC was correlated with the increase in serum IL-5 levels in the infected mice. Furthermore, i.p. injection of the soluble worm fraction caused MC without an inflammatory response in IL-5 transgenic mice, indicating that IL-5 is critical for the development of MC. These results indicate that the selective proliferation of IgM rheumatoid factor-secreting B-1a cells is induced by co-stimulation by the specific pathogen antigen and IL-5 in the development of MC in C. hepatica-infected mice.

Paradoxical findings in direct antiglobulin test and classification of agglutinating autoantibodies using eluates and monospecific anti-human globulin sera.

BACKGROUND AND OBJECTIVES: Agglutinating autoantibodies are rarely the cause of autoimmune haemolytic anaemia (AIHA) of warm type. These antibodies can be difficult to classify, and serological testing may result in confusion. Here, we describe the occurrence of paradoxical results in direct antiglobulin test (DAT) and a simple technique for the characterization of such antibodies. MATERIALS AND METHODS: Seven patients with AIHA were included in this study. Serological testing was performed using standard techniques. Classification of autoantibodies was performed by preincubation of the eluates from patients' red blood cells (RBCs) with monospecific anti-human globulin sera (mAHG). RESULTS: Strong autoagglutination of patients' RBCs was observed in six of seven cases, with the identification of panagglutinating serum antibodies in three patients. Initially, cold agglutinins with high thermal amplitude were suggested in four patients, and IgM warm autoantibodies were suggested in the remaining three patients. However, inhibition of the eluates revealed autoantibodies of IgA class in two patients, of IgM class in three other cases and of IgG class in two patients. The results of DAT were confusing due to paradoxical effects of mAHG and/or strong autoagglutination. CONCLUSION: Strongly agglutinating autoantibodies can be a source of confusion and can be classified by inhibition experiments using eluates and monospecific antibodies.

Primary cold agglutinin-associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow distinct from lymphoplasmacytic lymphoma.

Primary chronic cold agglutinin disease is a rare hemolytic disease mediated by monoclonal IGHV4-34-encoded cold agglutinins with a predominant specificity for the blood group antigen I. Bone marrow from 54 patients was studied to type the underlying lymphoproliferative disorder better. Bone marrow biopsies showed circumscribed intra-parenchymatous nodules with small monotonous monoclonal B cells in 40/54 patients (median infiltration: 10% of marrow cells) with a CD20(+), IgMs(+), IgDs(+), CD27(+), CD5(-/+), CD11c(-), CD23(-), CD38(-) immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors MUM1, XBP1 and BLIMP1 were noted in these B cells. However, a limited number of mature monoclonal IgM(+), IgD(-) plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). However, mutations of BCL6 intron 1 were not demonstrated, except in one patient, suggesting that the lymphoma cells had not matured in the germinal center. In conclusion, cold agglutinin-associated lymphoproliferative disease displays homogeneous histological and immunophenotypic features. The absence of plasmacytoid cells, the presence of plasma cells predominantly outside the nodular lymphoid infiltrates, IGHV4-34 restriction and absence of MYD88 L265P mutation strongly suggest that cold agglutinin-associated lymphoproliferative disease is a distinct entity that is different from lymphoplasmacytic lymphoma.

Utility of immunologic testing in suspected rheumatologic disease.

The use of diagnostic testing in the clinical practice of medicine has been a shifting landscape from the time that the first blood test was utilized. This is no different in the field of immunology and in particular rheumatology. As the field of immunology is relatively young, the clinical tests are not well established and therefore guidelines for use are still under debate. In this review, we seek to look at some of the key autoantibodies, as well as other tests that are available to diagnose suspected rheumatologic disease, and examine how to best use these tests in the clinic. In particular, we will focus on the anti-nuclear antibodies, anti-neutrophil cytoplasmic antibodies, complement, cryoglobulins, rheumatoid factor, and anti-citrullinated protein antibodies.

Cryofibrinogenemia triggered by a monoclonal paraprotein successfully treated with cyclophosphamide.

Cryofibrinogenemia is a rare clinical finding with a yet unknown physiopathogenic mechanism. We describe the case of a woman with cold-induced extensive necrotic lesions that responded poorly to initial corticosteroid and anticoagulant therapies. Serum cryoglobulin determinations were persistently negative. After several years of evolution, she developed severe cold-related skin lesions that required left-leg amputation. Further analysis disclosed the presence of cryofibrinogen and an apparently insignificant serum monoclonal immunoglobulin Gκ peak. We additionally demonstrate that the cold precipitation of fibrinogen was directly related to the monoclonal paraprotein presence. The lesions responded dramatically to a B cell-targeted therapy with intravenous cyclophosphamide and dexamethasone.

Sialylation determines the nephritogenicity of IgG3 cryoglobulins.

Monoclonal 6-19 IgG3 anti-IgG2a rheumatoid factor derived from lupus-prone MRL-Fas(lpr) mice can induce GN and cryoglobulinemia, but the features that confer nephritogenic potential are not completely understood. Asparagine-linked oligosaccharide chains of 6-19 IgG3 mAb are poorly galactosylated and hardly sialylated, possibly contributing to the pathogenic potential of 6-19 IgG3 rheumatoid factors. Here, we used the 6-19 model of cryoglobulin-associated GN to define the relative contributions of galactosylation and sialylation, in relation to cryoglobulin activity, to the nephritogenic potential of IgG3 antibodies. We generated one highly sialylated and two distinct more galactosylated 6-19 IgG3 rheumatoid factor variants. Although the mere extent of galactosylation had no effect on either the cryogenic and nephritogenic activities of 6-19 IgG3 rheumatoid factor, terminal sialylation attenuated the nephritogenic potential of 6-19 IgG3 by limiting its cryoglobulin activity. These data suggest a protective role of IgG sialylation against the development of cryoglobulin-mediated GN, highlighting the anti-inflammatory activity of sialylated IgG antibodies.

Detection of low level cryoglobulins by flow cytometry.

Several patients with cryoglobulin (CG) associated symptoms are seronegative for CG and other potentially causative biomarkers. We analyzed whether it is possible to detect cryoprecipitates by flow cytometry and whether the sensitivity of their demonstration can be increased as compared to visual inspection. Sera from 91 patients with suspected CG associated symptoms and 33 healthy controls were examined for the presence of CG by conventional visual testing and by flow cytometry for small diffracting particles. For calibration purposes we tested lipid micelle dilutions (positive controls) by both methods. The minimum concentrations of lipid micelles to be detected by visual inspection and flow cytometry were 128.5 and 2.0 pg ml(-1), respectively. Among the 91 patients and 33 controls, only 1 patient serum was positive for CG by conventional testing. This sample was also positive on flow cytometry. In the serum of a patient known to be positive for CG, laser diffracting particles were quantified by flow cytometry after keeping serum at 4°C for 3 days. Of the 91 patients, 14 additional samples displayed cold precipitates which redissolved after rewarming during flow cytometry. All 15 (1 + 14) patients positive for CG on flow cytometry suffered from symptoms usually associated with CG. Some precipitates were labeled with anti IgG and IgM antibodies confirming that the particles detected by flow cytometry contained immunoglobulins. No small diffracting particles were detected in the sera of the 33 healthy controls. Flow cytometry is equally specific but much more sensitive in the detection of CG than visual inspection.

Rheumatoid factor, complement, and mixed cryoglobulinemia.

Low serum level of complement component 4 (C4) that occurs in mixed cryoglobulinemia (MC) may be due to in vivo or ex vivo activation of complement by the classical pathway. Potential activators include monoclonal IgM rheumatoid factor (RF), IgG antibodies, and the complexing of the two in the cold, perhaps modulated by the rheology and stoichiometry of cryocomplexes in specific microcirculations. There is also the potential for activation of complement by the alternative and lectin pathways, particularly in the setting of chronic infection and immune stimulation caused by hepatitis C virus (HCV). Engagement of C1q and interaction with specific cell surface receptors serve to localize immune complexes (ICs) to the sites of pathology, notably the cutaneous and glomerular microcirculations. Defective or saturated clearance of ICs by CR1and/or Fc receptors may explain persistence in the circulation. The phlogistic potential of cryoprecipitable ICs depends upon the cleavage of complement components to generate fragments with anaphylatoxin or leukocyte mobilizing activity, and the assembly of the membrane attack complex (C5b-9) on cell surfaces. A research agenda would include further characterization of the effector arm of complement activation in MC, and elucidation of activation mechanisms due to virus and viral antigens in HCV infection.

Changes of some humoral immunologic indicators and clinical manifestations of cryoglobulinemia in heroin addicts.

UNLABELLED: Different autoantibodies and immunologic abnormalities have been described in heroin addicts. AIMS: dpending on the route of heroin application in heroin addicts to determine: 1) immunoglobulins: IgA, IgG, IgM; 2) complement (C3, C4); 3) some other autoantibodies RF, anti ß2GP1 fractions: IgA, IgG, IgM, ANA; 4) CIC; 5)monitoring the cryoglobulin presence; 6) clinical manifestations in cryoglobulin positive heroin addicts. A total of 363 heroin addicts were analyzed after previously completed questionnaire; biochemical analyses of blood and urine; creatinine clearance (eC(Cr)) by Cockcroft-Gault formula; proteinuria; 24-hour proteinuria (Uprot/Ucreat); ECG; toxicological analyses; complement (C3, C4); immunoglobulins IgA, IgG, IgM; rheumatoid factor; cryoglobulins; circulating immune complexes; antiphospholipid antibodies (anti ß2GP1: IgA, IgG, IgM); antinuclear antibodies. Male patients were predominating (82.09%). Of them 161 were using intravenous heroin (45.4%). IgA was statistically significantly lower in intravenous heroin addicts. Intravenous heroin addicts contrary to those who inhaled heroin had highly significant levels of IgG, IgM, IgG, antiß2GP1 cryoglobulins; significantly higher mean values of: RF, anti ß2GP1 IgA and IgM. Cryoglobulin positive (CP) heroin addicts compared to cryoglobulin negative (CN) presented significantly more frequently with clinical signs of arthralgia, vasculitis, hematuria; whereas highly significantly were manifested respiratory difficulties, neurological disorders, Raynaud phenomenon, proteinuria, 24-hour proteinuria, highly significantly lower mean values of renal clearance. Intravenous heroin addicts compared to the non-parenteral heroin addicts have shown greater changes in certain parameters of humoral immunity. CP heroin addicts have presented with more frequent clinical manifestations than CN heroin addicts.

Cold agglutinins revealed by abnormalities to the cell blood count: a case report.

Cold agglutinin are erythrocyte antibodies which possess the property of agglutinating red blood cells at temperatures of below 37°C, this phenomenon is reversible after heating. This is usually immunoglobulin M (IgM) class. Their pathogenicity is much more related to their temperature range of activity than their title. As we report in this observation, cold hemagglutination makes it difficult to interpret certain immunological tests such as ABO Rh blood grouping or searching for irregular antibodies (SAI). The discovery of cold agglutinins can be fortuitous revealing itself by disturbances and aberrations in the results of blood count or as part of a suggestive clinical or laboratory table cold hemagglutinin disease. The search for a lymphoid hematological at their diagnosis should be systematic.