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.

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.

Multifocal thrombosis with peripheral gangrene in a young boy: Mycoplasma infection triggered cold agglutinin disease.

Cold agglutinin disease (CAD) is extremely rare in children. We report an 8-year-old boy who presented with gangrene of right foot with hypertension and absent lower limb pulses. Blood peripheral smear evidence of autoagglutination and falsely elevated red blood cell indices were suggestive of CAD and on subsequent investigations he was found to have high titres of cold agglutinin antibodies. He also had evidence of pneumonia on chest X-ray and serology for mycoplasma was positive. Computed tomography angiography showed multifocal thrombotic occlusion in bilateral popliteal arteries. He was effectively managed using antimicrobials, warm clothing, aspirin, anticoagulation and corticosteroids. He remains clinically well on follow-up and had no recurrence. CAD presenting with peripheral gangrene is extremely unusual. A careful look at peripheral blood smear gives an initial diagnostic clue. CAD triggered by infection is often self-limiting and requires supportive care.

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.

Rheumatologic manifestations of Hepatitis C Virus.

INTRODUCTION: Hepatitis C Virus (HCV) is a well-known worldwide infection, responsible for hepatic and extrahepatic complications. Among extrahepatic manifestation, the rheumatologic are the most common ones. With the arrival of Direct Antiviral Agents (DAA), the treatment and the clinical perspective have rapidly changed, permitting to achieve a sustained virological response (SVR) and preventing complications of chronic infection. EVIDENCE ACQUISITION: We performed on PubMed a literature search for the articles published by using the search terms "HCV infection," "HCV syndrome," "HCV-related rheumatologic disorders," "cryoglobulinemia," "cryoglobulinemic vasculitis" and "mixed cryoglobulinemia." EVIDENCE SYNTHESIS: Mixed cryoglobulinemia (MC) is the prototype of HCV-associated rheumatologic disorder. HCV-related MC is typically considered by physicians as a human model disease to linking infection with autoimmune diseases. Chronic HCV infection can lead to a multistep process from a simple serological alteration (presence of circulating serum cryoglobulins) to frank systemic vasculitis (cryoglobulinemic vasculitis [CV]) and ultimately to overt malignant B lymphoproliferation (such as non-Hodgkin lymphoma [NHL]). Antiviral therapy is indicated to eradicate the HCV infection and to prevent the complications of chronic infection. Immunosuppressive therapy is reserved in case of organ threatening manifestations of CV. In this review, we discuss the main clinical presentation, diagnostic approach and treatment of rheumatologic manifestations of HCV infection. CONCLUSIONS: Chronic HCV infection is responsible for complex clinical condition, ranging from hepatic to extra-hepatic disorders. Cryoglobulins are the result of this prolonged immune system stimulation, caused by tropism of HCV for B-lymphocyte.

Contribution of Hepatitis C Infection to a Large Cohort of Cryoglobulin-Positive Patients: Detection and Characteristics.

Cryoglobulins (CGs) are cold precipitating immunoglobulins, and hepatitis C virus (HCV) infection is its most common cause. The purpose of the study was to determine the contribution of HCV in a large cohort of CG. Biological characteristics and specificity of CGs in HCV patients were compared to non-HCV subjects. Cryoglobulin analysis included isotype, clonality, concentration, and rheumatoid factor (RF) in cryoprecipitate and serum complement and RF. This study is an extension of the study carried out on a cohort of 13,439 patients tested for CGs from all medical units, in which 1,675/13,439 (12.5%) patients had a CG, and 680/1,675 (40.6%) had HCV serology or viral load determination (HCV RNA). Among these 680 CG patients tested for HCV, 325 of 680 (47.8%) HCV patients (272 HCV RNA+ and 45 HCV RNA- patients) were compared to 355/680 (52.2%) non-HCV subjects. After a positive detection of CG, HCV status was determined only for 37.7% (256/680) of patients, allowing the diagnosis of a previously unknown HCV infection for 39.8% (102/256). Concentration of HCV RNA+ CGs (median = 80.5 mg/L) was significantly higher than that of HCV RNA- CG (median = 50.5 mg/L, p = 0.001) and HCV- CG (median = 32 mg/L, p < 0.0001). There was no difference of median CG concentration between HCV RNA- patients and non-HCV subjects. Rheumatoid factor titer was significantly higher in type II CG compared to type III CG in HCV RNA+ patients (254 ± 720 vs. 15 ± 21 IU/mL, p < 0.0001) and non-HCV subjects (333 ± 968 vs. 16.8 ± 26 IU/mL, p = 0.0004). Complement functional activity CH50 was lower in HCV RNA+ patients (36 ± 24 U/mL) and in HCV RNA- patients (32 ± 21 U/mL) than in non-HCV subjects (50 ± 25 U/mL, p = 0.001 and p = 0.004). In conclusion, HCV infection and treatment influence CG characteristics. It is essential, and far from always tested, to determine the HCV status of patients with mixed CG, and conversely to search for CG in patients with HCV infection.

New Insights in the Pathogenesis and Therapy of Cold Agglutinin-Mediated Autoimmune Hemolytic Anemia.

Autoimmune hemolytic anemias mediated by cold agglutinins can be divided into cold agglutinin disease (CAD), which is a well-defined clinicopathologic entity and a clonal lymphoproliferative disorder, and secondary cold agglutinin syndrome (CAS), in which a similar picture of cold-hemolytic anemia occurs secondary to another distinct clinical disease. Thus, the pathogenesis in CAD is quite different from that of polyclonal autoimmune diseases such as warm-antibody AIHA. In both CAD and CAS, hemolysis is mediated by the classical complement pathway and therefore can result in generation of anaphylotoxins, such as complement split product 3a (C3a) and, to some extent, C5a. On the other hand, infection and inflammation can act as triggers and drivers of hemolysis, exemplified by exacerbation of CAD in situations with acute phase reaction and the role of specific infections (particularly Mycoplasma pneumoniae and Epstein-Barr virus) as causes of CAS. In this review, the putative mechanisms behind these phenomena will be explained along with other recent achievements in the understanding of pathogenesis in these disorders. Therapeutic approaches have been directed against the clonal lymphoproliferation in CAD or the underlying disease in CAS. Currently, novel targeted treatments, in particular complement-directed therapies, are also being rapidly developed and will be reviewed.

Cryoglobulins Today: Detection and Immunologic Characteristics of 1,675 Positive Samples From 13,439 Patients Obtained Over Six Years.

OBJECTIVE: Cryoglobulins are cold-precipitating immunoglobulins. Through progress in techniques, we undertook this study to update information on the biologic characteristics of cryoglobulins in a very large population. METHODS: A cohort of 13,439 patients was tested for cryoglobulins from January 2010 to December 2016. The analysis included cryoglobulin isotype, clonality, concentration, and IgM rheumatoid factor (IgM-RF) in cryoprecipitate, as well as serum complement and RF. Markers of gammopathy, viral infection, and autoimmunity were also investigated. RESULTS: Of the 13,439 patients, 1,675 (12.5%) tested positive for cryoglobulins: 155 patients (9.3%) with type I, 788 (47%) with type II, and 732 (43.7%) with type III cryoglobulins. Nine percent of patients who were retested after initially testing negative for cryoglobulins showed a positive result on a follow-up test (196 of the 2,213 retested patients). In type I cryoglobulins, IgM was more frequent but occurred at lower concentrations than IgG. Mixed cryoglobulins were found in 34.8% of the tested patients who were positive for hepatitis C virus and <5% of those who were positive for hepatitis B virus or HIV. Of the patients with anti-double-stranded DNA, anti-SSA, or anti-cyclic citrullinated peptide autoantibodies, 25.4% tested positive for mixed cryoglobulins, with type III occurring more frequently than type II. Both cryoprecipitate and serum were RF-positive in 21.6% of type II and 10.1% of type III cryoglobulins. A decrease of C4, with or without accompanying decreases of C3 and CH50, was found in 23.6% of cryoglobulin samples. CONCLUSION: Obtained with the use of modern assays, our findings from this very large collection of cryoglobulins provide an update on cryoglobulin distribution and characteristics, with minimal selection bias. Despite strict preanalytical conditions, a negative finding for the presence of cryoglobulin must be confirmed in a second sample. RF activity and complement decreases were rarely detected.

Features of peripheral CD8+CD57+ lymphocytes in patients with autoimmune hemolytic anemia.

Autoimmune hemolytic anemia (AIHA) is an acquired condition characterized by the presence of autoantibodies recognizing erythrocyte-related antigens. Several components of the immune system are involved in disease pathogenesis. Among them, as for other autoimmune disorders, a role for specific CD8+CD57+ regulatory cells subset could be hypothesized. We evaluated this lymphocyte subset by flow cytometry in 18 AIHA patients randomly selected in a retrospective population of 29 cases. Secondary forms were observed in 65.5% of cases, whereas frequencies of warm, cold, mixed, and atypical forms were similar. Cold agglutinins and cryoglobulins tested positive in 44.8% and 10.3% of cases, respectively. These patients exhibited a higher frequency of peripheral vascular symptoms (odds ratio = 8.2, p = .04) and complement consumption (odds ratio = 7.2, p = .02). Frequency of CD8+CD57+ cells resulted significantly higher in AIHA patients than in control group (17.0 ± 15.8% vs 8.2 ± 5.0%, p = .04). Regardless of therapeutic schedule, patients with partial or no response to therapy (8/18) showed higher frequencies of CD8+CD57+ cells as compared with controls (23.6 ± 21.3% vs 8.9 ± 4.9%, p = .01), whereas 10/18 complete responders (CR) showed lower levels of CD8+CD57+ cells (11.7 ± 6.9%, p = .11). CR and controls showed similar values (p = .24). This study suggests that monitoring this lymphocyte subset before and after treatment administration might have a prognostic value. Moreover, CD8+CD57+ cells may represent a possible therapeutic target to restore the normal balance between lymphocyte populations.

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.

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.

Cryoglobulins: An update on detection, mechanisms and clinical contribution.

Cryoglobulins are immunoglobulins precipitating in cold condition. They are classified in 3 types according to the Brouet classification and may lead to vasculitis of small and medium size vessels. Vasculitis is related to vessel obstruction by monoclonal cryoglobulin aggregates in type I cryoglobulins and immune complex deposition in type II and III mixed cryoglobulins. This phenomenon is favored by low temperature, especially in skin, joints, and peripheral nerves, or increased cryoglobulin concentration in kidneys. For their detection, collection and clotting at 37°C are critical pre-analytical conditions. Cryoglobulin characterization and quantification are important to identify the underlying disease. Since detection and identification of cryoglobulins lack standardization, a protocol for such detection, characterization and quantification is proposed.

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.

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.

[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.