Short Prewarming at 41°C to Correct the Interference in Samples with the Presence of Cold Agglutination.

BACKGROUND: Cold agglutinins (CAs) in blood samples can cause a reversible agglutination of red blood cell (RBC) which result in an incorrect complete blood count (CBC). So, it is important to explore new simple and feasible treatment conditions for clinical work. METHODS: The CAs group included 32 samples with CAs. The parameters of CBC at room temperature or after prewarming at 37°C or 41°C for different time periods were compared. The consistency and correlation of those parameters were analyzed. The morphology of erythrocytes in the CAs group was observed manually. The control group included 45 samples without CAs and prewarmed at 37°C or 41°C for different time periods. The differences were also analyzed. RESULTS: CAs have a significant effect on CBC. After prewarming at 37°C or 41°C the interferences are all corrected. Consider prewarming at 37°C for 120 minutes as the standard procedure. The consistency and correlation analysis showed there was no statistical difference between the results of each subgroup and standard group, except the MCHC of group 41°C 10 minutes. The correlation of parameters between all subgroups and the standard group is satisfied. Microscopic examination showed no RBC aggregation or fragmentation after prewarming at 41°C or 37°C. According to the maximum bias requirements for expert performance in Validation, Verification, and Quality Assurance of Automated Hematology Analyzers, 2nd Edition (CLSI H26-A2), the differences in overall results in control group are negligible. CONCLUSIONS: The 41°C 2 minutes prewarming method is a rapid and effective way for treating samples with CAs. It is an efficient way to obtain more reliable CBC results, without specific instruments.

A Case of Erythrocyte Condensation Caused by Mycoplasma Pneumoniae Infection.

BACKGROUND: Blood routine testing was the most commonly used laboratory method in clinical practice. The results are often influenced by factors such as instruments, reagents, and samples, among which, the interference of cold agglutinin is a very rare element. In our article, we reported a case of red blood cell agglutination caused by Mycoplasma pneumoniae infection. METHODS: The number of blood cells were detected by blood routine analyzer with or without treatment at 37℃ water bath. The red blood cell agglutination was observed through blood smear staining. The cold agglutination test were performed using O-type red blood cells added into patient's plasma and refrigerated overnight at 4℃. We also used luminescent immunoassay technology to detect the content of MP antibodies in patient's serum. RESULTS: The patient's results were RBC (2.69 x 1012/L), MCH (48.5 pg), MCHC (522 g/L). Through a microscope, we observed red blood cell agglutination. The concentration of MP-igM was 60.37 AU/mL. The cold agglutination test was positive. Following a 37℃ water bath, the patient's results changed: RBC (3.85 x 1012/L), MCH (31.2 pg), MCHC (352 g/L). The phenomenon of massive agglutination of red blood cells has also disappeared. CONCLUSIONS: The cold agglutinin produced by MP infection can alter the results of red blood cell. During the epidemic period of MP infection, it is important to pay attention to the phenomenon of abnormal elevation of MCHC in clinical practice.

Serological and Molecular Characterization of Hepatitis C Virus-Related Cryoglobulinemic Vasculitis in Patients without Cryoprecipitate.

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

CANOMAD: a neurological monoclonal gammopathy of clinical significance that benefits from B-cell-targeted therapies.

CANOMAD (chronic ataxic neuropathy, ophthalmoplegia, immunoglobulin M [IgM] paraprotein, cold agglutinins, and disialosyl antibodies) is a rare syndrome characterized by chronic neuropathy with sensory ataxia, ocular, and/or bulbar motor weakness in the presence of a monoclonal IgM reacting against gangliosides containing disialosyl epitopes. Data regarding associated hematologic malignancies and effective therapies in CANOMAD are scarce. We conducted a French multicenter retrospective study that included 45 patients with serum IgM antibodies reacting against disialosyl epitopes in the context of evocating neurologic symptoms. The main clinical features were sensitive symptoms (ataxia, paresthesia, hypoesthesia; n = 45, 100%), motor weakness (n = 18, 40%), ophthalmoplegia (n = 20, 45%), and bulbar symptoms (n = 6, 13%). Forty-five percent of the cohort had moderate to severe disability (modified Rankin score, 3-5). Cold agglutinins were identified in 15 (34%) patients. Electrophysiologic studies showed a demyelinating or axonal pattern in, respectively, 60% and 27% of cases. All patients had serum monoclonal IgM gammopathy (median, 2.6 g/L; range, 0.1-40 g/L). Overt hematologic malignancies were diagnosed in 16 patients (36%), with the most frequent being Waldenström macroglobulinemia (n = 9, 20%). Forty-one patients (91%) required treatment of CANOMAD. Intravenous immunoglobulins (IVIg) and rituximab-based regimens were the most effective therapies with, respectively, 53% and 52% of partial or better clinical responses. Corticosteroids and immunosuppressive drugs were largely ineffective. Although more studies are warranted to better define the optimal therapeutic sequence, IVIg should be proposed as the standard of care for first-line treatment and rituximab-based regimens for second-line treatment. These compiled data argue for CANOMAD to be included in neurologic monoclonal gammopathy of clinical significance.

Analysis of cryoproteins with a focus on cryofibrinogen: a study on 103 patients.

OBJECTIVES: Cryofibrinogen (CF) is an abnormal protein in plasma that precipitates at 4 °C and dissolves at 37 °C. Whilst serum cryoglobulins (CGs) analysis is common practice, CF investigation is rarely performed. This study aims to describe the testing methodology developed at our laboratory, potential pitfalls for all analytical phases, the distribution among hospital wards and clinical conditions underlying test requests and clinical conditions in which to order CF analysis is useful. METHODS: Retrospective analysis of laboratory samples received between January 2019 and June 2021 with CF testing requests. RESULTS: A complete protocol for CF pre-analytical, analytical and post-analytical phases are supplied. Most test requests were received from the rheumatology department for systemic sclerosis or liver transplant screening. Among the 103 in-patients included, CF+ was confirmed in 68 patients (66%). Of observed CF+ patients (n=68) most cases were CGs- (n=44, 67%). Isolated CF was found in 43% of the cases. Among CF- patients (n=35; 34%) only 2 patients had positive CGs (CGs+). Among rheumatology patients (n=66), isolated CF+ was observed in 45% (n=30/66), whilst among patients with systemic sclerosis with CF+ (n=19), isolated CF+ was detected in 79% (n=15/19). CONCLUSIONS: Described analytical procedures may be used for the creation of harmonized recommendations and indications for CF analysis. Isolated CF positivity among hospitalized patients, predominantly rheumatology and systemic sclerosis patients, appears higher than rates previously reported in literature. We propose CF test recommendations should be included in investigation protocols for diseases where cryofibrinogenemia may occur.

Cryoglobulinemic vasculitis: pathophysiological mechanisms and diagnosis.

PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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. SUMMARY: A decision diagram for CG vasculitis diagnosis based on clinical and biological parameters is proposed.

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.

Delayed diagnosis of Angioimmunoblast T-cell lymphoma presenting with type II Cryoglobulinemia and acute kidney injury: a case report and narrative review of the literature.

BACKGROUND: Angioimmunoblastic T cell lymphoma (AITL) is an infrequent hematological malignancy with variable and often atypical presentations. The presence of dysproteinemia, autoantibodies and systemic involvement in AITL has often led to a delay in diagnosis or even misdiagnosis in practice. We herewith present a case of AITL that primarily presented with acute kidney injury associated with type II Cryoglobulinemia, the underlying cause was only identified 8 months after the emergence of initial symptoms. CASE PRESENTATION: A 67-year old woman presented with 2-month history of intermittent joint pain and a 3-day history of bilateral lower limb edema and acute kidney injury. Initial laboratory investigations showed marked hypocomplementemia with positive autoantibodies of ANA, anti-cardiolipin-IgM and direct antiglobulin. The serum and urinary Immunofixation and serum cryoglobulin tests were negative, while the serum free κ to λ light chain ratio was 0.231. A renal biopsy showed a diffuse proliferative glomerulonephritis with intracapillary pseudothrombi formation. There were orderly arranged microtubular structures of 20-35 nm in diameter in the subendothelial and mesangial area on electron microscopy. Shortly afterwards, the patient developed tingling affecting her finger tips and weak hands and legs. A diagnosis of cryoglobulinemia complicated with cryoglobulinemic glomerulonephritis and polyneuropathy was made. She responded well to methylprednisolone, plasma exchange and rituximab. However, 3 months later, she presented with generalized pruritic rash, weight loss, and inguinal lymphadenopathy. A subsequent inguinal excisional lymph node biopsy at month 8 revealed AITL as the underlying disease. CONCLUSIONS: AITL and its associated B cell dysregulation can give rise to autoimmunity and cryoglobulinemia which may conceal itself as the underlying disorder. In various clinical scenarios of auto-immune diseases, it is advisable that the clinicians should take into consideration the multi-faceted lymphoma.

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.

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.

How immunological profile drives clinical phenotype of primary Sjögren's syndrome at diagnosis: analysis of 10,500 patients (Sjögren Big Data Project).

OBJECTIVES: To evaluate the influence of the main immunological markers on the disease phenotype at diagnosis in a large international cohort of patients with primary Sjögren's syndrome (SjS). METHODS: The Big Data Sjögren Project Consortium is an international, multicentre registry created in 2014. As a first step, baseline clinical information from leading centres on clinical research in SjS of the 5 continents was collected. The centres shared a harmonised data architecture and conducted cooperative online efforts in order to refine collected data under the coordination of a big data statistical team. Inclusion criteria were the fulfillment of the 2002 classification criteria. Immunological tests were carried out using standard commercial assays. RESULTS: By January 2018, the participant centres had included 10,500 valid patients from 22 countries. The cohort included 9,806 (93%) women and 694 (7%) men, with a mean age at diagnosis of primary SjS of 53 years, mainly White (78%) and included from European countries (71%). The frequency of positive immunological markers at diagnosis was 79.3% for ANA, 73.2% for anti-Ro, 48.6% for RF, 45.1% for anti- La, 13.4% for low C3 levels, 14.5% for low C4 levels and 7.3% for cryoglobulins. Positive autoantibodies (ANA, Ro, La) correlated with a positive result in salivary gland biopsy, while hypocomplementaemia and especially cryoglo-bulinaemia correlated with systemic activity (mean ESSDAI score of 17.7 for cryoglobulins, 11.3 for low C3 and 9.2 for low C4, in comparison with 3.8 for negative markers). The immunological markers with a great number of statistically-significant associations (p<0.001) in the organ-by-organ ESS- DAI evaluation were cryoglobulins (9 domains), low C3 (8 domains), anti-La (7 domains) and low C4 (6 domains). CONCLUSIONS: We confirm the strong influence of immunological markers on the phenotype of primary SjS at diagnosis in the largest multi-ethnic international cohort ever analysed, with a greater influence for cryoglobulinaemic-related markers in comparison with Ro/La autoantibodies and ANA. Immunological patterns play a central role in the phenotypic expression of the disease already at the time of diagnosis, and may guide physicians to design a specific personalised management during the follow-up of patients with primary SjS.

Cardiopulmonary Bypass in the Setting of Waldenström's Macroglobulinemia.

Waldenström's Macroglobulinemia (WM) is a rare lymphoma caused by the overproduction of immunoglobulin M (IgM). The elevated level of IgM causes serum hyperviscosity, cold agglutinins, and cryoglobulinemia. Anemia is also present because of impaired production of erythrocytes. For these reasons, placing a patient with WM on cardiopulmonary bypass (CPB) requires careful preparation. In this case, the patient was a 73-year-old male with known Waldenström's disease who required coronary artery bypass graft surgery. This report details the perioperative considerations used for successful CPB on a Waldenström's patient. Critical to this case was the use of plasmapheresis before surgery. Temperature management and acid/base status were carefully controlled. A successful coronary revascularization surgery was performed. Many of the Wadenstrom's disease complications expected on CPB failed to materialize.

[Digital necrosis revealing cold agglutinin disease: Treatment with rituximab]. / Nécroses digitales révélant une maladie des agglutinines froides : traitement par rituximab.

BACKGROUND: Digital necrosis is rarer than lower limb necrosis and constitutes a medical or surgical emergency. Etiological evaluation is required. Cold agglutinin disease is a cause of digital necrosis but diagnosis is difficult. PATIENTS AND METHODS: Herein we report the case of a 57-year-old man presenting recent paroxysmal acrosyndrome of the left hand subsequently complicated by digital necrosis following occupational exposure to cold in his work as a forklift driver. After etiological evaluation, a diagnosis of primary cold agglutinin disease was made. Intravenous rituximab and topical treatment resulted in complete healing. DISCUSSION: Cold agglutinin disease is a rare type of auto-immune hemolytic anemia. Following exposure to cold, paroxysmal cutaneous signs are frequent. The disease may be either primary or secondary with B-cell lymphoproliferative disorder, auto-immune disease or infection. A thorough workup is required. To date, the treatment combining the best positive response rate and good safety is rituximab in weekly perfusions over a 1-month period.

Cryofibrinogen-Associated Glomerulonephritis.

Cryofibrinogen is an under-recognized cryoprotein. Cryofibrinogen is a cryoprecipitate that develops following plasma refrigeration, but does not occur in cold serum. People with cryofibrinogenemia may be asymptomatic, but this cryoprotein can be associated with thromboembolic disease, particularly affecting the skin. Kidney manifestations are relatively uncommon, but are likely underestimated. We describe clinical features and kidney biopsy results in 2 patients with cryofibrinogen-related kidney disease. Both patients presented with proteinuria and hematuria. One had significant cutaneous ulcers and palpable purpura. Kidney biopsy in both cases showed membranoproliferative glomerulonephritis with no immunoglobulin deposition. Weak segmental capillary wall fibrinogen staining was noted in glomeruli. Immunofluorescence studies following pronase digestion failed to reveal masked immunoglobulin deposits. Ultrastructural studies were distinctive and characterized by organized deposits of large-bore with multilayered tubular structures and fine fibrillary structures in a matrix. To confirm the composition of deposits, we extracted the cryoprecipitate from plasma of a patient and performed ultrastructural studies, which showed identical ultrastructural characteristics to those seen on the kidney biopsy. We also performed proteomic analysis of the cryoprecipitate that confirmed the presence of fibrinogen. Subsequent laboratory evaluation was positive for cryofibrinogen in both patients on multiple occasions. Appropriate therapy was instituted in both patients, which included prednisone, immunosuppressive therapy, and avoidance of cold exposure. In summary, we present clinical, kidney biopsy, and laboratory findings and the treatment and follow-up of cryofibrinogen-associated glomerulonephritis. Awareness of this entity will result in accurate diagnoses, appropriate investigation, and 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.

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.