Cryofibrinogen-Characteristics and Association with Cryoglobulin: A Retrospective Study Out of a Series of 1,712 Samples over 7 Years.

OBJECTIVE: Cryofibrinogens (CFs) and cryoglobulins (CGs) are cryoproteins responsible for obstructive vasculopathy and vasculitis. The aim of this study was to compare the characteristics of CF and CG, and to define the conditions of their association. METHODS AND RESULTS: This retrospective study was conducted at the Lyon University Hospitals, and included patients with at least one sample tested for CF and/or CG between September 2013 and April 2021. Serum and plasma samples were analyzed in very strict conditions of temperature. After cold precipitation, CF and CG were characterized and quantified in the cryoprecipitates. CRP and plasma fibrinogen levels were also investigated. Over this 7-year period, 1,712 samples for CF detection and 25,650 samples for CG detection were sent to the laboratory. Simultaneous testing of CF and CG was performed in 1,453/1,712 samples (85%). CF was less often positive than CG (8.3 vs. 13.5%, p < 0.0001). In positive CF samples, CG was associated in 28.9% of cases. In CF, fibrinogen was associated with fibronectin in 98/142 (69%) samples, especially in highly concentrated CF. CF concentration was independent of C-reactive protein and plasma fibrinogen concentrations. CONCLUSION: The simultaneous detection of CF and CG is essential for the diagnosis of vasculitis or thromboembolic events and their treatment.

[Cryoglobulinemic vasculitis due to hyperviscosity and cryogel formation as the onset of multiple myeloma. Report of one case]. / Vasculitis crioglobulinémica por hiperviscosidad y formación de criogel como debut de mieloma múltiple. Caso clínico.

Multiple Myeloma (MM) is characterized by a clonal expansion of plasma cells in the bone marrow. These cells typically produce a monoclonal immunoglobulin, and its symptoms arise either from plasma cell infiltration in several organs, or secondary to the presence of a monoclonal protein peak. Symptoms can be summarized by the acronym CRAB (hypercalcemia, renal failure, anemia and bone lesions). Sometimes, in the setting of a protein secreting monoclonal gammopathy, formation of cryoglobulins develops. Cryoglobulins are plasma proteins that precipitate at low temperatures, forming a cold - induced precipitate at small vessels, causing a wide range of clinical manifestations. We report a female consulting for ulcers lasting 2 months in the left foot associated with purpuric lesions in both lower limbs. Protein electrophoresis showed a monoclonal peak in the gamma region. Bone marrow aspirate showed 27% of plasma cells with kappa chain restriction by cytometry. The presence of cryoglobulins was confirmed. The patient was treated with dexamethasone and bortezomib, with a progressive healing of lower limb lesions and disappearance of cryoglobulins. She was discharged in good conditions.

Cutaneous reactive angiomatosis associated with intravascular cryoprotein deposition as the presenting finding in a patient with underlying lymphoplasmacytic lymphoma: A case report and review of the literature.

Cutaneous reactive angiomatosis, a group of disorders defined by benign vascular proliferation, is associated with a number of systemic processes, including intravascular occlusion by cryoproteins. We report a case of a 64-year-old female patient who presented with a 1-year history of nontender petechiae of the bilateral arms and lower legs. Dermoscopic evaluation showed increased vascularity with a globular pattern. Over a period of months, her findings progressed to erythematous to violaceous plaques with admixed hypopigmented stellate scarring of the bilateral lower extremities, forearms, and lateral neck. Biopsy showed increased thin-walled, small dermal blood vessels with focal inter-anastamosis. Some vessels were occluded by eosinophilic globules suspicious for cryoprotein. Subsequent laboratory studies confirmed a diagnosis of type 1 cryoglobulinemia, prompting a bone marrow biopsy that revealed lymphoplasmacytic lymphoma. Herein, we report the fourth case of angiomatosis secondary to intravascular cryoproteins as the initial presentation of an underlying hematologic malignancy. We also present a review of the literature and emphasize the need for thorough initial workup and close and prolonged clinical monitoring for underlying systemic disease in these patients.

Prospective validation of an automatic reflex test for identifying spurious elevations of mean corpuscular haemoglobin concentration due to the presence of cold agglutinins.

Cold agglutinins (CA) in blood may cause false reduction in red blood cell (RBC) count and false increases of RBC indices, such as mean corpuscular haemoglobin concentration (MCHC). Preheating at 37 °C for 2 h is used to overcome this problem. We previously proposed the integration in a total laboratory automation (TLA) setting of a customized reflex test in the presence of MCHC >385 g/L for identifying spurious elevations due to CA. Here, we prospectively evaluate this approach after its introduction in our clinical practice. We evaluated 73 consecutive blood samples from 34 adult patients. Short heating (<1 min) at 41 °C using the reticulocyte channel of Sysmex XN-9000 platform was followed by calculation of optical parameters by the instrument software to ensure quick solution of the CA-dependent problems. After the reflex test in the reticulocyte channel, MCHC dropped below 385 g/L in 50 samples. The reflex markedly corrected the RBC number in eight samples obtained from three patients with CA condition. Two samples from markedly anaemic patients had low blood haemoglobin and RBC count before and after reflex. The remaining 13 samples were obtained from 12 patients, most of whom were on antiretroviral therapy or suffered severe electrolyte disorders, known conditions associated to increased MCHC. The implementation of the proposed automatic reflex by reticulocyte channel on the Sysmex XN-9000 platform in a TLA setting may solve the problem of spuriously high MCHC due to RBC agglutination for CA in a few minutes instead of waiting hours for sample preheating.

Cryofibrinogen-associated glomerulonephritis accompanied by advanced gastric cancer.

We had a 72-year-old man with advanced gastric cancer, poorly differentiated adenocarcinoma, receiving chemotherapy with S-1 (tegafur, gimeracil, and oteracil potassium) plus oxaliplatin. Ascites developed despite remission of gastric cancer and metastasis. Given no malignant cells in ascites, leg edema, renal impairment, hypoalbuminemia, and massive proteinuria, we diagnosed as nephrotic syndrome with microscopic hematuria. Renal biopsy showed membranoproliferative glomerulonephritis with no deposition of immunoglobulins and complements. Of note, electronic microscopy found organized deposits with microtubular structures in the glomerular capillary lumens and subendothelial spaces. The liquid chromatography-tandem mass spectrometry method detected fibrinogen alpha chain, beta chain, gamma chain, and fibronectin, and we eventually diagnosed cryofibrinogen-associated glomerulonephritis. Cryofibrinogen was not detected in plasma. He was expired at 5 months following renal biopsy due to the progression of refractory nephrotic syndrome. In addition to the detailed assessment of specifically organized deposits, the analysis using liquid chromatography-tandem mass spectrometry method is useful to diagnose cryofibrinogen-associated glomerulonephritis. We should consider cryofibrinogen-associated glomerulonephritis as a differential diagnosis when the patients with malignancy showed abnormal urinalysis and renal impairment, though it is a rare disease.

It Is Just a Rash They Said! Acute Skin Manifestation in a Patient With Vasculitis in Rural Hospitals.

A 76-year-old Caucasian male with a history of rheumatoid arthritis, Raynaud's phenomenon, pulmonary embolism on warfarin, and a previous amputation of his left partial ring and fifth finger presented with acute onset of rash in bilateral lower extremities. He was recently started on trimethoprim-sulfamethoxazole due to concern for cellulitis. Differential diagnosis for acute-onset rash with the patient's history presented as a challenge to the internist, as the differential is broad. Our case goes through the differential diagnosis to contrast the different presentations of rash in a patient with vasculitis. Ultimately skin biopsy in conjunction with a past positive cryoglobulinemic level helped confirm the diagnosis of cutaneous vasculitis, following which he was started on appropriate treatment and recovered.

Cold agglutinin disease: where do we stand, and where are we going?

Primary cold agglutinin disease (CAD) is characterized by a very indolent bone marrow clonal B-cell lymphoproliferative disorder that initiates an autoimmune hemolytic anemia. The clonal B cells produce a monoclonal autoantibody termed cold agglutinin, most often of the immunoglobulin (Ig) Mκ class. After binding to its antigen, the IgM initiates a complement classical pathway-driven erythrocyte destruction, predominantly mediated by opsonization with complement protein C3b and extravascular hemolysis in the liver. We review the molecular biology, histopathology, clinical features, and diagnostic procedures in CAD. Some patients are only slightly anemic and do not require treatment, but moderate or severe anemia frequently occurs, and the disease burden has been underestimated. CAD should not be treated with corticosteroids. Several B-cell-directed treatment options are available, and complement-directed approaches are being rapidly developed. Current and possible future therapies are reviewed.

Characteristic electron-microscopic features of cryofibrinogen-associated glomerulonephritis: a case report.

BACKGROUND: Cryofibrinogenemia is a rare disorder that mainly affects the skin and occasionally the kidney. However, there are few published reports of cryofibrinogenemia-associated renal pathology. We therefore report a patient with cryofibrinogen-associated glomerulonephritis. Samples from this patient were examined by electron microscopy, laser microdissection, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). CASE PRESENTATION: A 78-year-old Japanese man presented with declining renal function, proteinuria, and gross hematuria. Kidney biopsy showed a membranoproliferative pattern with crescent formation and dominant C3c deposition in which subendothelial deposits with uniquely organized electron-microscopic features were observed. Additional ultrastructural analysis of cryoprecipitates extracted from plasma revealed similar structures of the glomerular subendothelial deposits. LC-MS/MS identified an increase in fibrinogen α, ß, and γ chains, fibronectin, filamin-A, and C3. The glomerular lesions were diagnosed as cryofibrinogen-associated glomerulonephritis on the basis of these findings. CONCLUSIONS: Although there are few reports of cryofibrinogen-associated glomerulonephritis, we believe that accurate diagnosis can be achieved by performing LC-MS/MS and ultrastructural analysis.

Cryoglobulinemic vasculitis and glomerulonephritis: concerns in clinical practice.

OBJECTIVE: Cryoglobulinemia often causes systemic vasculitis, thereby damaging to skin and internal organs including kidneys, even life-threatening. This review aimed to introduce the advances in understanding, detection, and treatment of this disease in recent years, with a particular concern to clinical practice. DATA SOURCES: All the data in this review were from the English or Chinese literature in the PubMed and China National Knowledge Infrastructure databases as of March 2019. STUDY SELECTION: This review selected important original articles, meaningful reviews, and some reports on cryoglobulinemia published in recent years and in history, as well as the guidelines for treatment of underlying diseases which lead to cryoglobulinemia. RESULTS: Diagnosis of cryoglobulinemia relies on serum cryoglobulin test, in which to ensure that the blood sample temperature is not less than 37°C in the entire pre-analysis phase is the key to avoid false negative results. Cryoglobulinemic vasculitis (Cryo Vas), including cryoglobulinemic glomerulonephritis (Cryo GN), usually occurs in types II and III mixed cryoglobulinemia, and can also be seen in type I cryoglobulinemia caused by monoclonal IgG3 or IgG1. Skin purpura, positive serum rheumatoid factor, and decreased serum levels of C4 and C3 are important clues for prompting types II and III Cryo Vas. Renal biopsy is an important means for diagnosis of Cryo GN, while membranous proliferative GN is the most common pathological type of Cryo GN. In recent years, great advances have been made in the treatment of Cryo Vas and its underlying diseases, and this review has briefly introduced these advances. CONCLUSIONS: Laboratory examinations of serum cryoglobulins urgently need standardization. The recent advances in the diagnosis and treatment of Cryo Vas and GN need to be popularized among the clinicians in related disciplines.

Cryoglobulinemia: An update in 2019.

Cryoglobulinemia is defined as the persistent presence in serum of abnormal immunoglobulins (Igs) that precipitate at low temperatures and dissolve again upon warming. Cryoglobulins may be composed only of a monoclonal Ig (simple type I cryoglobulinemia), of a monoclonal Ig bound to the constant domain of polyclonal Ig heavy chains (mixed type II cryoglobulinemia), or only of polyclonal Igs (mixed type III cryoglobulinemia). The manifestations of type I cryoglobulinemia are often related to intravascular obstruction, whereas those seen in the mixed cryoglobulinemias often originate in true immune complex-mediated vasculitis. The main clinical manifestations affect the skin (purpura, necrotic ulcers), joints, peripheral nervous system, and kidneys (membranoproliferative glomerulonephritis). Patients with type I cryoglobulinemia should be investigated for hematological malignancies (myeloma and B-cell lymphoma). Hepatitis C is the main diagnosis to consider in patients with mixed cryoglobulinemia, followed by connective tissue disease and B-cell non-Hodgkin's lymphoma. The treatment depends mainly on the cause of the cryoglobulinemia. For instance, hepatitis C virus (HCV) eradication is in order in patients with HCV-associated cryoglobulinemia vasculitis, and the underlying hematological malignancy must be treated in patients with type I cryoglobulinemia.

Serological profile of asymptomatic HCV positive patients with low level of cryoglobulins.

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.

Increased genomic instability following treatment with direct acting anti-hepatitis C virus drugs.

Mixed Cryoglobulinemic Vasculitis (MCV) is a prominent extra-hepatic manifestation of Hepatitis C virus (HCV) infection. HCV has been reported to cause B-cell disorders and genomic instability. Here, we investigated B-cell activation and genome stability in HCV-MCV patients receiving the direct antiviral agent, Sofosbuvir, at multiple centers in Egypt. Clinical manifestations in HCV-MCV patients were improved at the end of treatment (EOT), such as purpura (100%), articular manifestations (75%) and neuropathy (68%). Eighteen patients (56%) showed vasculitis relapse after EOT. BAFF and APRIL were higher at EOT and continued to increase one year following treatment onset. Chromosomal breaks were elevated at EOT compared to baseline levels and were sustained at 3 and 6 months post treatment. We report increased expression of DNA genome stability transcripts such as topoisomerase 1 and TDP1 in HCV-MCV patients after treatment, which continued to increase at 12 months from treatment onset. This data suggest that B-cell activation and DNA damage are important determinants of HCV-MCV treatment outcomes.

Are direct-acting antivirals safe and effective in hepatitis C virus-cryoglobulinemia? virological, immunological, and clinical data from a real-life experience.

OBJECTIVES: Hepatitis C virus (HCV) is the major cause of cryoglobulinemia. Direct-acting antivirals (DAAs) have markedly changed the therapeutic outcomes in the treatment of patients with HCV. We evaluate the efficacy, safety, immunological, and clinical response of different DAA regimens in HCV-cryoglobulinemia. PATIENTS AND METHODS: Ninety-three cryoglobulinemic patients, divided into symptomatic [symptomatic cryoglobulinemic patients (SCP; n=35)] and asymptomatic [nonsymptomatic cryoglobulinemic patients (NSCP; n=60)], underwent DAAs. Eighty-nine comparable noncryoglobulinemic patients were selected as a control group. We evaluated the sustained virological response (SVR), the adverse effects, and the immune and symptomatic response. RESULTS: Percentages of patients who achieved SVR and experienced adverse effects were not statistically different between the three groups (100, 95, 93.3% and 57.1, 53.3, 48.3%). In 68.5% of SCP and in 76.7% of NSCP, cryoglobulins disappeared at SVR. No risk factor was associated with the persistence of cryoglobulins. An increase was observed both in C4 (P=0.002; P=0.018) and in C3 (P=0.0037; P=0.031) in SCP and NSCP. About 70% of symptomatic patients showed a complete or partial symptomatic remission: persistence of symptoms is correlated to the type of clinical picture. CONCLUSION: DAA regimens are safe and effective in patients with HCV-cryoglobulinemia. The achievement of SVR is necessary, but not sufficient, to achieve a complete immunological and clinical response.

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.

IgG3 subclass: A possible trigger of mixed cryoglobulin cascade in hepatitis C virus chronic infection.

HCV is a hepatotropic and lymphotropic virus and is the most frequent cause of "benign" mono-oligoclonal B-lymphocyte proliferation, observed in mixed cryoglobulinemia (MC). The study aims to investigate the presence, prevalence and characteristics of the subclasses of cryoglobulins in HCV-patients to look for a relationship with MC. Fifty HCV-infected patients with cryoglobulins were enrolled. IgG subclasses were characterized in cryoprecipitate, and serum IgG and IgM Rheumatoid Factor (RF) were determined. Patients were stratified into two subgroups according to the presence of IgG3 subclass. Differences were observed in supernatant IgM, IgG3-positive and IgG3-negative patients with a higher IgM concentration in the IgG3-negative cohort (p=0.03). Higher IgM-RF was detected in the IgG3-negative group (p=0.01). IgG3-positive group showed higher IgG-RF compared to the IgG3-negative group (p<0.0001). IgG3-negative/monoclonal-IgM patients had higher cryocrit compared to IgG3-negative/polyclonal-IgM patients (p<0.01). C4 levels were higher in the polyclonal-IgM group compared to monoclonal-IgM group (p<0.01). We speculate that cryoglobulins are part of a progressive clonal selection process in which, B-cells are stimulated to produce oligoclonal IgG3 with RF activity. The persistence of the antigenic stimulus elicits the production of polyclonal IgM-RF and subsequently the formation of oligoclonal IgG/polyclonal IgM containing cryoglobulins. In the last stage, a monoclonal IgM-RF clone is formed which may coexist with a monoclonal IgG3-RF clone.

Efficacy of Cryofiltration for Treatment of Mixed Cryoglobulinemia: A Report of Four Cases.

Cryoglobulinemia can induce systemic vasculitis affecting various organs such as skin, peripheral nerves, and kidney. The disease can induce chronic organ failure and even be life-threatening. Cryofiltration has been applied for the treatment of cryoglobulinemic vasculitis. We have experienced four cases with mixed cryoglobulinemia showing severe and progressive clinical manifestations, including skin purpura, nephrotic syndrome, acute kidney injury, and peripheral neuropathy. Cryofiltration in conjunction with conventional pharmacological therapies appeared to be safe and effective. After the treatments, plasma cryoglobulins were markedly reduced and the disease was well controlled. Although its efficacy has not yet been well established, this report can be another evidence showing efficacy of cryofiltration for treatment of mixed cryoglobulinemia.

HCV-unrelated cryoglobulinaemic vasculitis: the results of a prospective observational study by the Italian Group for the Study of Cryoglobulinaemias (GISC).

OBJECTIVES: To investigate the clinical and laboratory patterns of HCV-unrelated cryoglobulinaemic vasculitis (CV), and the factors influencing its outcome. METHODS: Prospective study of all anti-HCV and HCV-RNA negative patients with CV who have been observed since January 2004 in 17 centres participating in the Italian Group for the Study of Cryoglobulinaemias (GISC). RESULTS: 175 enrolled were followed up for 677 person-years. The associated conditions were primary Sjögren's syndrome (21.1%), SLE (10.9%), other autoimmune disorders (10.9%), lymphoproliferative diseases (6.8%), solid tumours (2.3%) and HBsAg positivity (8.6%), whereas 69 patients (39.4%) had essential CV. There were significant differences in age (p<0.001), gender (p=0.002), the presence of purpura (p=0.005), arthralgia (p=0.009), liver abnormalities (p<0.001), sicca syndrome (p<0.001), lymphadenopathy (p=0.003), splenomegaly (p=0.002), and rheumatoid factor titres (p<0.001) among these groups. Type II mixed cryoglobulins were present in 96 cases (54.9%) and were independently associated with purpura and fatigue (odds ratio [OR]4.3; 95% confidence interval [CI] 1.8-10.2; p=0.001; and OR2.8; 95%CI 1.3-6.3; p=0.012). Thirty-one patients died during follow-up, a mortality rate of 46/1000 person-years. Older age (for each additional year, adjusted hazard ratio [aHR] 1.13; 95%CI 1.06-1.20; p<0.001), male gender (aHR 3.45; 95%CI 1.27-9.40; p=0.015), type II MCG (aHR 3.31; 95%CI 0.09-1.38; p=0.047) and HBsAg positivity (aHR 7.84; 95%CI 1.20-36.04; p=0.008) were independently associated with greater mortality. CONCLUSIONS: HCV-unrelated CV is a multifaceted and often disabling disorder. The associated conditions influence its clinical severity, giving rise to significantly different clinical and laboratory profiles and outcomes.