Gene expression analysis revealed downregulation of complement receptor 1 in clonal B cells in cold agglutinin disease.

Cold agglutinin disease (CAD) is a rare B-cell lymphoproliferative disorder of the bone marrow, manifested by autoimmune hemolytic anemia caused by binding of monoclonal IgM autoantibodies to the I antigen. Underlying genetic changes have previously been reported, but their impact on gene expression profile has been unknown. Here, we define differentially expressed genes in CAD B cells. To unravel downstream alteration in cellular pathways, gene expression by RNA sequencing was undertaken. Clonal B-cell samples from 12 CAD patients and IgM-expressing memory B cells from 4 healthy individuals were analyzed. Differential expression analysis and filtering resulted in 93 genes with significant differential expression. Top upregulated genes included SLC4A1, SPTA1, YBX3, TESC, HBD, AHSP, TRAF1, HBA2, RHAG, CA1, SPTB, IL10, UBASH3B, ALAS2, HBA1, CRYM, RGCC, KANK2, and IGHV4-34. They were upregulated at least 8-fold, while complement receptor 1 (CR1/CD35) was downregulated 11-fold in clonal CAD B cells compared to control B cells. Flow cytometry analyses further confirmed reduced CR1 (CD35) protein expression by clonal CAD IgM+ B cells compared to IgM+ memory B cells in controls. CR1 (CD35) is an important negative regulator of B-cell activation and differentiation. Therefore, reduced CR1 (CD35) expression may increase activation, proliferation, and antibody production in CAD-associated clonal B cells.

Hemolytic anemia blunts the cytokine response to transfusion of older red blood cells in mice and dogs.

BACKGROUND: Transfusion of red blood cells (RBCs) stored for longer durations induces hemolysis and inflammatory cytokine production in murine and canine models. Despite immune system activation by stored RBCs, human randomized trials suggest that fresher RBC transfusions do not improve clinical outcomes. We hypothesized that underlying recipient hemolysis may affect cytokine responses to older RBC transfusions. STUDY DESIGN AND METHODS: C57BL/6 mouse cohorts were infused with anti-TER119 antibody to induce hemolysis, rabbit anti-platelet antiserum to induce immune thrombocytopenia (ITP), or appropriate control antibodies. Two days later, mice were transfused with fresh or stored RBCs. Furthermore, in a prospective, randomized, blinded trial, 38 client-owned dogs with primary autoimmune hemolytic anemia (AIHA) and two dogs with ITP, requiring RBC transfusion, were enrolled and randomized to receive fresh (≤7 days) or old (≥21 days) stored RBC transfusions. Monocyte chemoattractant protein (MCP)-1 levels were assessed at defined times after transfusion. RESULTS: Prior immune-mediated hemolysis blunted the MCP-1 response to stored RBC transfusion in mice (361 ± 111 pg/ml vs. 6836 ± 1528 pg/ml in mice with immune hemolysis vs. ITP, respectively; mean ± SD; p < .0001). Although hemolysis markers increased after transfusion of older RBCs, the cytokine response was also muted in dogs with AIHA. No differences in morbidity or mortality were evident comparing dogs randomized to fresh or old RBCs. CONCLUSION: These data suggest that underlying hemolysis blunts inflammatory responses to old RBC transfusions. The canine data support randomized trial results suggesting a lack of clinical benefit with fresh RBC transfusions in subjects with underlying, baseline hemolysis.

Reticulocyte Hemoglobin Equivalent in Patients with Idiopathic Warm Autoimmune Hemolytic Anemia: Implication in the Development of Macrocytosis.

OBJECTIVE: Patients with warm autoimmune hemolytic anemia (WAIHA) present with anemia that is highly heterogeneous, and often have macrocytic anemia with inappropriately elevated mean corpuscular volume (MCV). The goal of this retrospectivecase study is to elucidate the characteristics of anemia in patients with idiopathic WAIHA. PROCEDURES: The hematological parameters were analyzed by automated hematology analyzers in 19 consecutive patients with idiopathic WAIHA. Thecontent of hemoglobin (Hb) in the reticulocytes was assessed as reticulocyte Hb equivalent (RET-He). Relevant laboratory data and medical records were retrospectively studied. RESULTS: The median MCV was 102.7 fL and ten patients had macrocytic anemia with MCV above 100 fL. There was a significant correlation between the percentage of reticulocytes and MCV. The median RET-He value was 35.9 pg, and the reticulocytes of patients with higher MCV had higher RET-He. There was a significant correlation between red cell volume distribution width (RDW) andMCV, while the association between RDW and RET-He was not significant. Red blood cell agglutination was not seen in any of the patients. Relative folate deficiency was implied to contribute to the increased Hb content in the reticulocytes of WAIHA patients. CONCLUSION: Reticulocytes in WAIHA patients often contain more Hb than normal reticulocytes and become inappropriately large, possibly due to relative folate deficiency. Elevated MCV in WAIHA patients is due to the increase in both the number and the Hb content of reticulocytes.

Cold Agglutinin-Mediated Autoimmune Hemolytic Anemia in Association with Antiphospholipid Syndrome.

Antiphospholipid syndrome and cold agglutinin-mediated autoimmune hemolytic anemia are 2 distinct immune-mediated hematologic disorders. While no clear association exists between these 2 entities, complement activation is known to occur in both of them. Herein, we report a unique case of cold agglutinin hemolytic anemia in a patient with a known primary antiphospholipid syndrome.

Rituximab-containing therapy for cold agglutinin disease: a retrospective study of 16 patients.

Cold agglutinin disease (CAD) is a rare form of autoimmune haemolytic anaemia, and because of its rareness, there is no standard treatment for CAD patients. We retrospectively analysed the response to rituximab-containing therapy in CAD patients at our hospital. All patients received rituximab-containing therapy for at least 1 month. A total of 16 patients (11 males and 5 females) were included. The median age at the onset of the disease was 63.5 years (range 41-79). Most patients had manifestations including anaemia (81.3%) or cold-induced circulatory symptoms (75.0%). The median haemoglobin level was 72 g/L (range 29-101), and the median cold agglutinin titre was 1,024 (range 64-2,048). Thirteen of 16 patients (81%) responded to the therapy. Responders achieved a median increase in haemoglobin levels of 45 g/L. Grade 3-4 neutropenia occurred in 3 patients (19%), but only 1 (6%) of them experienced infection. Anaphylaxis related to rituximab occurred in 1 patient. During follow-up, five patients experienced relapse, and two patients died. The estimated median progression-free survival was 36 months, and median overall survival was not yet reached. In conclusion, A rituximab-based therapy in accordance with individual patient characteristics may be a reasonable choice for CAD patients.

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.

Abnormal Biodistribution on 99mTc-Red Blood Cell-Labeled Multigated Acquisition in the Presence of Suspected Cold Agglutinin Disease.

A 99mTc-red blood cell (RBC)-labeled multigated acquisition is a procedure in which the patient's RBCs are radiolabeled and imaged with electrocardiography-gated cardiac scintigraphy to assess the heart's pumping efficiency. Cold agglutinin disease, or cold antibody autoimmune hemolytic anemia, is a rare form of autoimmune hemolytic anemia in which the body's immune system attacks and destroys its own RBCs. This case addresses an altered biodistribution pattern of radiolabeled RBCs in the presence of suspected cold agglutinin disease observed during a multigated acquisition.

Patients with IgG1-anti-red blood cell autoantibodies show aberrant Fc-glycosylation.

Autoimmune hemolytic anemia (AIHA) is a potentially severe disease in which red blood cells (RBC) are destroyed by IgG anti-RBC autoantibodies which can lead to hemolysis. We recently found IgG Fc-glycosylation towards platelet and RBC alloantigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core-fucosylation increases the affinity of the pathogenic alloantibodies to FcγRIIIa/b, and hence RBC destruction. It is known that in autoimmune diseases plasma IgG1 galactosylation and sialylation are lowered, but Fc-glycosylation of RBC-specific autoantibodies has never been thoroughly analyzed. We investigated by mass spectrometry the N-linked RBC autoantibody and plasma IgG1 Fc-glycosylation in relation to occurrence of hemolysis for 103 patients with a positive direct antiglobulin test (DAT). We observed that total IgG1 purified from plasma of patients with RBC-bound antibodies showed significantly decreased galactosylation and sialylation levels compared to healthy controls, similar to what previously has been shown for other autoimmune diseases. The anti-RBC- autoantibodies showed a profile with even lower galactosylation, but higher sialylation and lower bisection levels. In contrast to alloantibodies against RBCs, RBC-bound IgG1 Fc-fucosylation was not different between healthy controls and patients. Analysis of anti-RBC Fc-glycoprofiles suggested that lower bisection and higher galactosylation associate with lower Hb levels.

The diagnosis and management of the haematologic manifestations of lupus.

Haematological manifestations in systemic lupus erythematosus (SLE) are frequently observed. They are diverse and range from mild to severe. Therefore, different treatment approaches are needed from simply keeping vigilant to significant immunosuppression. Most treatment evidence is based on case-reports or small retrospective studies, as few randomized controlled trials have been performed. The development of biological therapy has opened new possible ways to treat the most severe cases but further clinical trials are necessary. In this review we consider the most common and characteristic haematological manifestations of SLE patients, focusing on their pathogenesis and management.

Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.

Neutrophil extracellular traps (NETs) are webs of DNA and protein with both anti-microbial and pro-thrombotic properties which have not been previously reported in dogs. To confirm dog neutrophils can form NETs, neutrophils were isolated from healthy dogs, and stimulated in vitro with 2µM, 8µM, 31µM, and 125µM platelet activating factor (PAF) or 0.03µM, 0.1µM, 0.4µM, 1.6µM and 6.4µM phorbol-12-myristate-13-acetate (PMA). Extracellular DNA was measured using the cell impermeable dye Sytox Green every hour for 4h. At 4h, extracellular DNA was significantly greater than non-stimulated cells at concentrations ≥31µM and ≥0.1µM for PAF and PMA, respectively. Cells stimulated with 31.25µM PAF reached maximal fluorescence by 1h, whereas maximal fluorescence was not achieved until 2h for cells stimulated with 0.1µM PMA. Immunofluorescent imaging using DAPI and anti-elastase antibody confirmed that extracellular DNA is released as NETs. As NETs have been implicated in thrombosis, nucleosomes, a marker correlated with NET formation, were measured in the serum of dogs with the thrombotic disorder primary immune-mediated hemolytic anemia (IMHA) (n=7) and healthy controls (n=20) using a commercially available ELISA. NETs were significantly higher in IMHA cases than controls (median 0.12 and 0.90, respectively, p=0.01), but there were large positive interferences associated with hemolysis and icterus. In summary, the study is the first to describe NET generation by canine neutrophils and provides preliminary evidence that a marker associated with NETs is elevated in IMHA. However, this apparent elevation must be interpreted with caution due to the effect of interference, emphasizing the need for a more specific and robust assay for NETs in clinical samples.

Surface localization of high-mobility group nucleosome-binding protein 2 on leukemic B cells from patients with chronic lymphocytic leukemia is related to secondary autoimmune hemolytic anemia.

Chronic lymphocytic leukemia (CLL) is the main cause of autoimmune hemolytic anemia (AHA). However, the cellular basis underlying this strong association remains unclear. We previously demonstrated that leukemic B cells from patients with CLL recognize the erythrocyte protein Band 3, a prevalent autoantigen in AHA. Here we show that the major binding site of Band 3 on leukemic cells is an extrinsic protein identified as high-mobility group nucleosome binding protein 2 (HMGN2), a nucleosome-interacting factor which has not been previously reported at the cell surface. T lymphocytes do not express HMGN2 or bind Band 3. Removal of HMGN2 from the cell membrane abrogated the capacity of Band 3-pulsed CLL cells to induce CD4 + T cell proliferation. We conclude that surface HMGN2 in leukemic B cells is involved in Band 3 binding, uptake and presentation to CD4 + T lymphocytes, and as such may favor the initiation of AHA secondary to CLL.

Humoral immune mechanism of liver injury in giant cell hepatitis with autoimmune hemolytic anemia.

BACKGROUND AND AIMS: Giant cell hepatitis with autoimmune hemolytic anemia (GCH-AHA) is presumed to be an autoimmune disease, but the mechanism of liver injury is unknown. We proposed that in CGH-AHA, the humoral limb of autoimmunity is the dominant force driving progressive liver injury. METHODS: We studied 6 cases of GCH-AHA and 6 cases of autoimmune hepatitis (AIH) with early childhood onset (3 type 1 and 3 type 2). Liver biopsies were graded for portal and periportal inflammation and for giant cells. Immunohistochemistry characterized cellular inflammation and complement involvement in injury by showing C5b-9 complex in hepatocytes. RESULTS: Clinical and biochemical features at presentation were generally similar; however, the absence of autoantibodies and the presence of Coombs positivity did distinguish GCH-AHA from early-onset AIH. Liver biopsy pathology in CGH-AHA showed giant cells and little inflammation, whereas AIH showed the opposite. C5b-9 staining showed high-grade complement-mediated pan-lobular hepatocyte injury in all of the cases with GCH-AHA, whereas little C5b-9 was seen in hepatocytes in cases with AIH. Inflammation in GCH-AHA comprised mainly lobular macrophages and neutrophils, whereas portal and periportal T-cell and B-cell inflammation characterized cases with AIH. Most cases with AIH responded to therapy with prednisone and azathioprine, whereas most cases with GCH-AHA responded only to rituximab. CONCLUSIONS: Widespread complement-mediated hepatocyte injury and typical C3a and C5a complement-driven liver inflammation along with Coombs-positive hemolytic anemia in GCH-AHA provide convincing evidence that systemic B-cell autoimmunity plays a central pathologic mechanism in the disease. Our findings support B-cell-directed immunotherapy as a first-line treatment of GCH-AHA.