Detection Of Cryoglobulins Research Articles

Insights into the immunological description of cryoglobulins with regard to detection and characterization in Slovenian rheumatological patients

The detection of cryoglobulins (CG) used to diagnose cryoglobulinemic vasculitis requires strict adherence to protocol, with emphasis on the preanalytical part. Our main objectives were to introduce a more sensitive and specific protocol for the detection of CG and to characterize CG in Slovenian patients diagnosed with cryoglobulinemic vasculitis, other vasculitides, connective tissue diseases or non-rheumatic diseases examined at the Department of Rheumatology (University Medical Centre Ljubljana). Samples were routinely analyzed for the presence of CG with the protocol using the Folin-Ciocalteu reagent. In the newly introduced protocol, the type of CG was determined by immunofixation on visually observed positive samples and the concentration of CG in the cryoprecipitate and rheumatoid factor (RF) activity were measured by nephelometry. RF, C3c and C4 were measured in patients` serum and a decision tree analysis was performed using all results. The agreement between negative and positive results between the two protocols was 86%. Of the 258 patient samples tested, we found 56 patients (21.7%) with positive CG (37.5% - type II, 62.5% - type III). The RF activity was observed in 21.4% of CG positive subjects. The median concentration of type II CG was significantly higher than that of type III CG (67.4 mg/L vs. 45.0 mg/L, p = 0.037). Patients with type II had lower C4 concentrations and higher RF compared to patients with type III CG. In the decision tree, C4 was the strongest predictor of cryoglobulinemia in patients. With the newly implemented protocol, we were able to improve the detection and quantification of CG in the samples of our rheumatology patients and report the results to adequately support clinicians.

Clinical and clonal characteristics of monoclonal immunoglobulin M-associated type I cryoglobulinaemia.

Monoclonal immunoglobulin M-associated type I cryoglobulinaemia is poorly characterised. We screened 534 patients with monoclonal IgM disorders over a 9-year period and identified 134 patients with IgM type I cryoglobulins. Of these, 76% had Waldenström macroglobulinaemia (WM), 5% had other non-Hodgkin lymphoma (NHL) and 19% had IgM monoclonal gammopathy of undetermined significance (MGUS). Clinically relevant IgM-associated disorders (including cold agglutinin disease [CAD], anti-MAG antibodies, amyloidosis and Schnitzler syndrome) coexisted in 31%, more frequently in MGUS versus WM/NHL (72% vs. 22%/29%, p < 0.001). The majority of those with cryoglobulins and coexistent CAD/syndrome had the molecular characteristics of a CAD clone (wild-type MYD88 in 80%). A half of all patients had active manifestations at cryoglobulin detection: vasomotor (22%), cutaneous (16%), peripheral neuropathy (22%) and hyperviscosity (9%). 16/134 required treatment for cryoglobulin-related symptoms alone at a median of 38 days (range: 6-239) from cryoglobulin detection. At a median follow-up of 3 years (range: 0-10), 3-year cryoglobulinaemia-treatment-free survival was 77% (95% CI: 68%-84%). Age was the only predictor of overall survival. Predictors of cryoglobulinaemia-related treatment/death were hyperviscosity (HR: 73.01; 95% CI: 15.62-341.36, p < 0.0001) and cutaneous involvement (HR: 2.95; 95% CI: 1.13-7.71, p = 0.028). Type I IgM cryoglobulinaemia is more prevalent than previously described in IgM gammopathy and should be actively sought.

#5353 THE IDIOPATIC HYPOCRYOGLOBULINEMIA AS AN EMERGING MEMBRANOPROLIPHERATIVE GLOMERULONEPHRITIS

Abstract Background and Aims A considerable number of patients with high clinical suspicion for cryoglobulinaemic vasculitis either show negative results for the detection of cryoglobulins or show only trace amounts which cannot be characterized for composition. We aimed at establishing whether the failure to detect or the detection of trace amounts of cryoglobulin with conventional methods either identifies a peculiar subset of low level cryoglobulinaemia (from now on hypocryoglobulinaemia) or represents a separate entity. Method Using a modified precipitation technique in hypo-ionic medium, we prospectively identified between 2008 and 2021 237 patients (median age 60.8 years [22-97], 137 females) having &amp;lt;0.5% cryocrit and clinical suspicion of autoimmune disorder Results 54 out of 237 patients (22.7%) had a history of HCV infection. 169 out of 237 patients (71%) had an established underlying disease, while 68 patients (28.6%) (median age 62.9 years [29-93], 35 females) did not show either laboratory markers or clinical symptoms consonant with an underlying aetiology. These 68 cases with only trace amounts of cryoglobulins were defined as having a putatively idiopathic hypocryoglobulinaemia. Nineteen of these 68 patients (27.9%) had a history of HCV infection. Twenty-four patients out of 68 (35.3%) were positive for rheumatoid factor (RF), while 25 (36.7%) patients had signs of complement consumption, and 36 (52.9%) had increased inflammatory indexes. Seven patients only had arthralgia and constitutional symptoms while 61 out of 68 (89.7%) presented with at least one of the three cardinal signs of cryoglobulinaemic vasculitis including skin lesions, peripheral nerve involvement, and glomerulonephritis. Seventy-five percent of the subjects had type III hypocryoglobulins. In patients with hypocryoglobulinaemia the histologic features of glomerulonephritis (also examined by electron microscopy) resembled those of mixed cryoglobulinaemia-associated glomerulonephritis Conclusion In conclusion, hypocryoglobulins are often polyclonal and are mainly unrelated to HCV infection. Patients who present high clinical suspicion for vasculitis, especially glomerulonephritis and yet test negative for cryoglobulinaemia detected by standard techniques, could require deeper investigation even in the absence of HCV infection, RF activity or signs of complement consumption.

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

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. 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. The simultaneous detection of CF and CG is essential for the diagnosis of vasculitis or thromboembolic events and their treatment.

Serum protein electrophoresis and rheumatoid factor analysis is an effective screening strategy for cryoglobulinaemia

Accurate serum cryoglobulin detection is important to allow prompt treatment but laboratory testing requires stringent pre-analytical conditions and has long turnaround times. Serum protein electrophoresis (EPG) for paraproteinaemia and rheumatoid factor (RF) analysis may offer an effective initial screening strategy for the presence of cryoglobulinaemia. We retrospectively assessed the sensitivity of ancillary EPG and RF testing for the presence of serum cryoglobulinaemia in 586 eligible cryoglobulin positive samples received at the Royal Prince Alfred and Liverpool Hospital immunopathology laboratories over an 11-year period. Ninety-one percent of all cryoglobulin positive samples had either a detectable paraprotein or RF activity, with greatest sensitivity for type I and type II cryoglobulins (97% and 98%, respectively). The sensitivity remained high irrespective of whether EPG and RF analysis was performed with the same, or different, pre-analytical collection conditions to the cryoglobulin collection (92% vs 90%, p=0.46). Only two patients with detected cryoglobulins and no associated paraprotein or RF activity had clinical features of cryoglobulinaemia and neither required treatment. This study demonstrates that serum EPG and RF analysis has high sensitivity for the detection of clinically relevant cryoglobulinaemia, even when not collected under ideal pre-analytical conditions, and potentially offers a prompt and effective screening strategy.

Recognizing the new disorder “idiopathic hypocryoglobulinaemia” in patients with previously unidentified clinical conditions

A considerable number of patients with high clinical suspicion for cryoglobulinaemic vasculitis either show negative results for the detection of cryoglobulins or show only trace amounts which cannot be characterized for composition. We aimed at establishing whether the failure to detect or the detection of trace amounts of cryoglobulin with conventional methods either identifies a peculiar subset of low level cryoglobulinaemia (from now on hypocryoglobulinaemia) or represents a separate entity. Using a modified precipitation technique in hypo-ionic medium, we prospectively identified between 2008 and 2021 237 patients (median age 60.8 years [22–97], 137 females) having < 0.5% cryocrit and clinical suspicion of autoimmune disorder. Of these 237 patients, only 54 (22.7%) had a history of HCV infection. One hundred and sixty-nine out of 237 patients (71%) had an established underlying disease, while 68 patients (28.6%) (median age 62.9 years [29–93], 35 females) did not show either laboratory markers or clinical symptoms consonant with an underlying aetiology. These 68 cases with only trace amounts of cryoglobulins were defined as having a putatively idiopathic hypocryoglobulinaemia. Nineteen of these 68 patients (27.9%) had a history of HCV infection. Twenty-four patients out of 68 (35.3%) were positive for rheumatoid factor (RF), while 25 (36.7%) patients had signs of complement consumption (i.e., C4 < 15 mg/dl and/or C3 < 80 mg/dl ), and 36 (52.9%) had increased inflammatory indexes. Seven patients only had arthralgia and constitutional symptoms while 61 out of 68 (89.7%) presented with at least one of the three cardinal signs of cryoglobulinaemic vasculitis including skin lesions, peripheral nerve involvement, and glomerulonephritis. Seventy-five percent of the subjects had type III hypocryoglobulins. In patients with hypocryoglobulinaemia the histologic features of glomerulonephritis (also examined by electron microscopy) resembled those of mixed cryoglobulinaemia-associated glomerulonephritis. In conclusion, hypocryoglobulins are often polyclonal and are mainly unrelated to HCV infection. Patients who present high clinical suspicion for vasculitis, especially glomerulonephritis and yet test negative for cryoglobulinaemia detected by standard techniques, could require deeper investigation even in the absence of HCV infection, RF activity or signs of complement consumption.

Serum protein electrophoresis and rheumatoid factor analysis as a screening strategy for cryoglobulinaemia

Introduction: Serum cryoglobulin detection is central to the diagnosis of cryoglobulinaemia, but laboratory testing has long turnaround times. Serum protein electrophoresis (EPG) and rheumatoid factor (RF) testing may be an effective initial screening strategy for the presence of cryoglobulinaemia.

Practical Details for the Detection and Interpretation of Cryoglobulins.

Cryoglobulins are immunoglobulins that precipitate at low temperature. Strict preanalytical and analytical conditions are critical for the detection of cryoglobulins. This review will focus on practical recommendations for detection and characterization of cryoglobulins and the technical problems that may be encountered. A laboratory report format is proposed for presentation of these results that includes the parameters necessary for an optimal interpretation by clinicians. The first step of detection of cryoglobulins can be performed in any laboratory that has a 37 °C incubator and temperature-controlled centrifuge. The second step is the characterization of cryoglobulins, and this often must be performed in more specialized laboratories. Characterization includes immunoglobulin typing, for the classification of cryoglobulins and potential underlying disease(s); quantification of immunoglobulins and rheumatoid factor in the cryoprecipitate to define the pathogenicity; and quantification of serum complement, which is useful for diagnosis. These practical recommendations will be useful for the accurate detection of cryoglobulins, an essential step for the diagnosis of cryoglobulinemic vasculitis, a rare but severe clinical manifestation of cryoglobulins.

Cryoglobulinemic vasculitis: pathophysiological mechanisms and diagnosis.

Cryoglobulins (CG) are immunoglobulins that precipitate in the cold, and dissolve at 37°C. In vivo, in cold exposed tissues and organs, they can induce vasculitis and occlusive vasculopathy after deposition on vascular endothelium under low temperature and high concentration conditions. Clinical manifestations are cutaneous (purpura, ulcers, vasomotor symptoms, and livedo reticularis), rheumatological (arthralgia and arthritis), and peripheral neuropathy (paresthesia and pain in the lower limbs). In profound organs such as the kidneys, CG deposition is less temperature-dependent, favored by local protein and anion concentrations, and can lead to glomerulonephritis. This review will focus on cryoglobulinemic vasculitis and vascular lesion, and their diagnosis. The mechanisms of vascular lesions of pathogenic CG in function of CG type and their characteristics are better defined. Optimal conditions for CG detection are critical. The importance of looking for underlying diseases, especially hepatitis C virus status in mixed CG, is reminded. A decision diagram for CG vasculitis diagnosis based on clinical and biological parameters is proposed.

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.

Finger Necrosis Due to Cryoglobulinemic Vasculitis in Association with Membranous Nephropathy

Cryoglobulinemic vasculitis is a small vessel vasculitis that has been associated with chronic infections and autoimmune, lymphoproliferative, and neoplastic disorders. When no significant etiological factors are identified, it is called essential mixed cryoglobulinemia. A detailed and thorough laboratory investigation is required to exclude all possible causes of cryoglobulin formation. Although cryoglobulin testing is simple, careful temperature regulation is needed to avoid false-negative results. Consensus diagnosis should be developed and implemented for appropriate cryoglobulin detection and accurate clinical diagnosis for cryoglobulinemic vasculitis. Here we present an interesting, first-ever case report of a 54-year-old Hispanic-American woman with essential mixed cryoglobulinemia presenting with significant digital necrosis in association with membranous nephropathy.

Hematological Complications in chronic Hepatitis C Virus Infection

The course of chronic hepatitis C virus infection (c-HCVI) is often asymptomatic at onset; If becomes symptomatic extrahepatic complications may dominate. Among them, hematological complications occur quite frequently including mainly mixed cryoglobulinemia and thrombocytopenia, less common non-Hodgkin’s lymphomas (NHL) and monoclonal gammopathy of unknown significance (MGUS). The incidence of hematological complications in c-HCVI varies from 36-76% and is geographically dependent. The aim of this retrospective study was to evaluate the frequency, co-occurrence and risk factors for hematological complications in 1237 untreated patients (pts) diagnosed with c-HCVI in Pomerania region of Poland in the years 1998-2010.The data included complete blood count (CBC), proteinogram and results of liver biopsy, performed at the time when patients were evaluated for antiviral treatment. Declines for hemoglobin (HGB), leukocyte count (WBC) and platelet count (PLT) were graded as mild//moderate if HGB was not lower than 10//8g/dl, WBC than 3//2G/l and PLT than 100//50G/l. Liver fibrosis was assessed using Scheuer classification. Detection of cryoglobulins was performed only in patients with symptoms of cryoglobulinemia. Data were analyzed using univariate and multivariate logistic regression and ROC curves.Mixed cryoglobulinemia was confirmed in 5.3% of pts. No cases of MGUS were detected; NHL were diagnosed in 0.3% of pts. Hypoalbuminemia (<3.75 g/dl) was found in 20.9% and hypergammaglobulinemia (>1.69 g/dl) in 29% of pts. CBC abnormalities were found in 33% of patients: the most common was thrombocytopenia (PLT<150 G/l) in 23.8%, leucopenia (WBC<4 G/l) in 8.4% and neutropenia (ANC<1.5 G/l) in 4.5% of patients. Anemia (HGB≤11.5 g/dl), lymphopenia (ALC<1 G/l) were rare (about 3% of patients). The CBC declines were generally mild; Thrombocytopenia, anemia and leucopenia were mild in 63%, 75% and 78% of pts, respectively. Thrombocytopenia more often (66%) occurred as a single CBC change. Leucopenia, conversely, more often coexisted (68%) with other CBC abnormalities, especially with thrombocytopenia (96%). In univariate analysis pts with thrombocytopenia had a significantly (p<0.05) 5.1 times higher risk of co-occurrence of other hematological complication, of which the highest (7.3) risk related to leucopenia (table 1). Moreover, the more severe thrombocytopenia the higher risk of co-existence of leucopenia was observed: in patients with PLT≤100 G/l risk of leucopenia increased 14.6 times. Hypergammaglobulinemia more often (56%) occurred as a solitary abnormality. If not solitary, it co-existed mainly with thrombocytopenia (82%). Additionally, incidence of thrombocytopenia and leucopenia increased with advancement of liver fibrosis: In pts with stage>3 fibrosis the incidence of thrombocytopenia and leukocytopenia was 73% and 34%, respectively. In multivariate analysis the risk of thrombocytopenia and leucopenia increased significantly from stage 2 and 3 of liver fibrosis, respectively compared to pts without fibrosis: patients with stage>3 had 16.5 times higher risk of thrombocytopenia and 8.2 higher risk of leucopenia comparing to pts without liver fibrosis. The risks of hypergammaglobulinemia and hypoalbuminemia increased significantly only in patients with stage>3, 2.3 and 4.4 times, respectively. Among PLT, albumin and gamma- globulin concentration, PLT was the strongest predictor of lack of advanced liver fibrosis (grade ≥3) (area under ROC curve- 0.8122). For patients with PLT≥155 G/l sensitivity was 74.2%, specificity 75.7% (figure 1). We conclude that in patients with c-HCVI, CBC abnormalities mainly CBC declines occur often and usually are mild and isolated. The most often is thrombocytopenia. Normal PLT count is a strong predictor of lack of advanced liver fibrosis. Coexistence of thrombocytopenia, leucopenia and presence of hypergammaglobulinemia and hypoalbuminemia may indicate advanced liver fibrosis.Table 1Odds ratios for additional hematological complications in patients with thrombocytopenia in univariate analysis.complicationodds ratioP>|z|95% CI:additional hematological complication5.580.0003.95- 7.87leucopenia7.520.0004.89- 11.56anemia2.480.0061.31- 4.71mixed cryoglobulinemia2.430.0011.46- 4.06 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-nuclear antibody detection in cryoprecipitates: Distinctive patterns in hepatitis C virus-infected patients

BackgroundAnti-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. AimTo 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. MethodsSerum 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. ResultsAnti-nuclear antibody patterns differed significantly; 26 infected subjects (65%) were IgG3 positive: of these, 25 were also anti-nuclear antibody-positive (96.1%). ConclusionsIgG3 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.

Diagnostics and Treatment of Cryoglobulinaemia: It Takes Two to Tango

Cryoglobulins are immunoglobulins that reversibly precipitate in the cold. They come in different flavours and, as such, are differentially associated with lymphoproliferative diseases (type I), or systemic autoimmune diseases, and/or infectious diseases (type II/III). The clinical manifestations of cryoglobulinaemia result from either hyper-viscosity or small vessel vasculitis. Hepatitis C virus (HCV) is a well-known factor in the aetiology of cryoglobulinaemia, but substantial geographical differences exist in the association between cryoglobulins and HCV. In the absence of any underlying disease, cryoglobulinaemia is referred to as 'idiopathic' or 'essential'. Detection of cryoglobulins in the laboratory is hampered by several pitfalls, in particular in the pre-analytical stage as well as in the quantification. In addition, our personal experience reveals that the detection of rheumatoid factor, most often present in high concentrations in patients with mixed cryoglobulinaemia, relies on the choice of the test system. Hence, interpretation of the laboratory results in relation to the clinical manifestations requires a partnership between the clinician and the laboratory specialist in order to make a correct diagnosis. Treatment options are primarily directed by identification of underlying diseases, i.e. infections or systemic autoimmune diseases. Idiopathic cryoglobulinaemia is treated with corticosteroids and immunosuppression, or B cell depleting anti-CD20 biologicals. In this overview, the recent literature on current laboratory and clinical practice of cryoglobulinaemia is discussed from a personal perspective.

A Critical Appraisal of Current Practice in the Detection, Analysis, and Reporting of Cryoglobulins

To assess current practice in the detection, analysis, and reporting of cryoglobulins, a questionnaire was sent to 140 laboratories. Only 36% of laboratories used standard procedures (tube preheating, transport in container, and sedimentation and/or centrifugation at 37 degrees C) to ensure that the temperature did not drop below 37 degrees C until after serum separation. Time periods allowed for cryoprecipitation at 4 degrees C varied from 12 h to 9 days, with 30% of laboratories allowing precipitation for <3 days. After cryoprecipitation, 81% of laboratories resolubilized the cryoprecipitate at 37 degrees C, and 77% further immunotyped the cryoprecipitate. After analysis, 5% referred the sample for confirmation, 58% provided a nonquantitative report, and 37% reported the cryoglobulin concentration in the cryoprecipitate as cryocrit, total protein concentration, and/or immunoglobulin concentration. Only 3 laboratories (2%) provided cryoprecipitate-specific reference values for total protein content, and none provided reference values for immunoglobulins. We believe standardization is needed for cryoglobulin detection to avoid missed diagnoses and improve the comparability of results. Laboratories should ensure that sample temperature does not drop below 37 degrees C until after serum separation. The serum should cryoprecipitate at 4 degrees C for at least 3 (preferably 7) days. The cryoprecipitate should be washed and resolubilized at 37 degrees C for further analysis.

Quantitative measurement of hepatitis C virus core antigen is affected by the presence of cryoglobulins

Mixed cryoglobulinaemia is associated strikingly with HCV infection. The aim of this study was to assess whether the adherence to proper methods of collecting samples for cryoglobulin detection was critical or not on virological parameters in hepatitis C virus (HCV) patients. We studied 56 consecutive patients. Blood samples were collected using a conventional method and a blood collection method at 37 degrees C adapted to cryoglobulin detection. HCV core antigen and HCV RNA were measured in sera and cryoglobulins issued from both blood collection methods. In cryoglobulin-positive patients, serum concentrations of HCV core antigen, but not that of HCV RNA, were significantly higher when a conventional method was used, compared to a blood collection method at 37 degrees C (P = 0.001). In the cryoprecipitates, concentration of HCV core antigen was optimum when the blood collection method at 37 degrees C, rather than the conventional method, was applied for cryoglobulin detection (P < 10(-4)). The recovery of HCV core antigen in the cryoprecipitate was improved when cryoglobulins were isolated using the blood collection method at 37 degrees C rather than the conventional method (P < 0.001). HCV parameter measurements and cryoglobulin study should not be performed on the same serum samples due to the potential impact of blood collection methods on results.

Cryoglobulinemia in Kidney Transplant Recipients

The aim of this study was to assess the presence of cryoglobulins, the constitution of the cryoprecipitate, as well as the possible etiology and clinical features in kidney transplant recipients. We excluded patients with clinical or laboratory evidence of autoimmune, liver or neoplasm disease, infections, blood transfusions or immunizations in the previous 3 months. Detection of cryoglobulins was obtained from the peripheral venous blood. In cases of cryoprecipitate formation it was analyzed using anti-IgG, anti-IgM, anti-IgA, anti-C3, and anti-C4 antibodies. The hepatitis C virus (HCV) was detected by the polymerase chain reaction. Thirty-nine patients were selected, of whom 23 were men and the overall mean age was 40.6 ± 12.7 years. Cryoprecipitate was detected in 74.4% (29/39) patients. Among patients with or without cryoprecipitate formation, the serum creatinine values, the percentage of patients with proteinuria, and the posttransplantation times were similar. In patients with cryoglobulins, 37.9% (11/29) were HCV positive. The etiology was not determined for the other patients. The IgG, IgM, and IgA immunoglobulins and the complement fractions C3 and C4 were found in the cryoprecipitate. Their compositions were similar among patients with or without HCV. Few clinical features were associated with the presence of cryoglobulins, including deep venous thrombosis, cutaneous purpura and peripheral neuropathy. In conclusion, cryoglobulinemia was prevalent in kidney transplant recipients, but appeared to not affect graft function. HCV infection was the most frequently associated etiology and clinical features were infrequent.

Analysis of the dynamics of cryoaggregation by light-scattering spectrometry.

Cryoglobulins are proteins that precipitate at temperatures below 37 degrees C. Cold-induced precipitation of proteins may occur in vivo secondary to several important diseases, and lead to pathological manifestations involving different organs. Cryoprecipitation may be observed in vitro by exposing serum samples, supposed to contain cryoglobulins, to low temperatures, but this needs several days to occur. Protein-protein interactions leading to cryoprecipitation are still poorly understood and the knowledge of the underlying mechanism may be relevant to the understanding of the onset of pathological manifestations. Using light-scattering spectrometry, we studied cryoprecipitation occurring in vitro at different temperatures and cryoglobulin concentrations. We describe the kinetics of the cold-induced precipitation of mixed cryoglobulins, measured as increase in turbidity. The plots obtained demonstrate that the cryoprecipitation did not occur as a single-step reaction, but consisted of four distinct phases where both temperature and cryoglobulin concentration affected the immune complexes formation. Light scatter spectrometry may provide a simple, sensitive and rapid method for the detection of cryoglobulins.

Cryoglobulin detection from a blood smear leading to the diagnosis of multiple myeloma

Cryoglobulin detection from a blood smear leading to the diagnosis of multiple myeloma

Gel Diffusion Procedure for the Detection of Cryoglobulins in Serum

Cryoglobulin is a thermoprotein precipitated at low temperatures in serum (1). It consists mainly of immunoglobulins, which form various complexes. They are classified into three types (I, II, and III) according to their composition (2). In recent years, there have been reports on the relationship between type II cryoglobulinemia and hepatitis C (2)(3)(4), which is being widely studied. The common method of detection of cryoglobulins is as follows. After blood is collected at 37 °C and serum separated, 5 mL of the serum is allowed to stand in a cryocrit tube at 4 °C for 2–7 days, and formation of the precipitate is confirmed visually. When a precipitate is found, the sample is centrifuged at 4 °C; the supernatant is removed; cold physiological saline or phosphate buffer (pH 5.5–7.0) is added; and the mixture is heated at 37 °C. If the precipitate redissolves after heating, …