Rumi Clare

Intravenous immunoglobulin as an adjunct to plasma exchange for the treatment of chronic thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening disease. Therapeutic plasma exchange (TPE) is the most effective therapy; however, despite TPE, about one‐third of TTP patients will relapse. A subset of patients with TTP has antibodies to ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) and may become resistant to conventional treatments. We describe a patient with TTP and high‐titre anti‐ADAMTS13 antibodies who developed a chronic, relapsing course of TTP despite frequent TPE. Once adjuvant treatment with intravenous immunoglobulin (IVIG) was added, remission was achieved. Even during remission, anti‐ADAMTS13 antibodies remained elevated. We conclude that IVIG may sustain remission in some patients with chronic, relapsing TTP.

Difficulties in establishing the diagnosis of immune thrombocytopenia: An agreement study

and their cis-acting regulatory elements. Acquired thalassemia has also been described, primarily alpha thalassemia due to somatic point mutations in a trans-acting regulatory factor, ATRX, or, less commonly, due to deletions of the alpha globin gene cluster on chromosome 16 as part of the clonal instability of myeloid neoplasia [1]. Acquired mutations of ATRX, an X-linked chromatin associated factor that has distinct effects on alpha and beta globin gene expression, are observed most commonly in the context of myelodysplastic syndromes (MDS) [1]. In contrast to alpha thalassemia-MDS (ATMDS, Online Mendelian Inheritance in Man #300448), cases of MDS with acquired beta thalassemia are exceedingly rare and poorly characterized [2–4]. Here we describe a patient with previously normal erythrocyte indices who developed microcytic anemia and was subsequently found to have a myeloid neoplasm. Additional evaluation revealed acquired beta thalassemia in association with MDS (BTMDS), resulting from acquired loss of the beta globin gene on chromosome 11p in a neoplastic subclone. A 65-year-old woman with previously normal blood count and erythrocyte indices developed microcytic anemia (hemoglobin 9.7 g/dL, hematocrit 30.7%, mean cell volume 65.7 fL, red cell distribution width [RDW] of 34%) with unremarkable white blood cell and platelet counts. Although the patient was iron deficient at the time of initial evaluation, no bleeding source was identified and anemia failed to improve with parenteral iron repletion (Fig. 1). She subsequently developed worse anemia (hemoglobin of 4.7 g/dL, again with microcytic indices) and new splenomegaly and thrombocytopenia (platelet count of 65 3 10/L); on transfer to our hospital she was noted to have 22% peripheral blasts, leukocyte dysmorphology, and anisopoikilocytosis (Fig. 1B). Hemoglobin electrophoresis (after transfusion) showed hemoglobin A 94.5%, hemoglobin A2 2.4%, and hemoglobin F 3.1%; no hemoglobin variants were detected on isoelectric focusing, and supravital staining with new methylene blue and did not reveal any HbH inclusions. The marrow was hypercellular and left-shifted, with increased reticulin and scattered blast cells with a myeloid phenotype. Next-generation sequencing including 39 genes recurrently mutated in cancer identified single nucleotide variants in TP53 (R175H), APC (E1317Q), and CDH1 (A592T), as well as an insertion/deletion in TP53 at codon Val97 (290delT) resulting in a frame shift mutation. The karyotype was 43 45,XX,add(5)(q31),26,add(7)(q21),28,29,1r[5]/43 45,XX, add(5),26,del(7)(q21),18[2],der(11;17)(q10;q10),del(12)(p11.2),214,add(18)(q21),1r,1mar [15] (Fig. 1C). The derivative chromosome der(11;17)(q10;q10) resulted in loss of chromosomes 11p and 17p, including the locus of beta globin cluster (11p15.5) and the TP53 gene (17p13). Sequencing of the alpha and beta globin genes showed no sequence alterations, but Multiplex Ligation-dependent Probe Amplification (MLPA) demonstrated abnormal copy number of chromosome 11p. Inherited alterations in expression of the beta globin genes are associated with a broad spectrum of clinical presentations, from an asymptomatic beta thalassemia trait that is only of reproductive consequences, to the severe transfusion-dependent phenotype of beta thalassemia major. Acquired clonally restricted deletion of the alpha globin gene cluster or mutation of ATRX is well described in MDS; however, this report is one of only a very small number of descriptions of acquired beta thalassemia. In one such report, two patients with beta thalassemia trait (b) were found to have a somatic mutation that led to a beta thalassemia intermedia phenotype due to somatic deletions of the wildtype b globin gene locus [4]. Other reports of acquired beta thalassemia in MDS identified beta thalassemia using hemoglobin electrophoresis [2,5], including one case of acquired delta beta thalassemia, without genetic analysis [3]. In the present case, cytogenetic and molecular testing revealed beta thalassemia due to a subclone with chromosome 11p loss resulting in HBB haploinsufficiency. Hemoglobin electrophoresis can help distinguish underlying abnormalities in globin synthesis; normal results include 98% HbA, 1–2% HbA2, and <1% HbF. Patients with beta thalassemia have different electrophoresis phenotypes depending on the number of and type of alterations. Beta thalassemia trait has 92–95% HbA, >3,8% HbA2, and 1–4% HbF. Patients with beta thalassemia intermedia have residual variable beta globin synthesis with HbA of 10–30%, HbA2 of 2–5%, and HbF 70–90%. Beta thalassemia major results in no beta globin synthesis and shows 0% HbA, >95% HbF, and 2–5% HbA2. Our patient had 94.5% HbA, 2.4% HbA2, and 3.1% HbF, most consistent with acquired beta thalassemia trait. MDS is commonly associated with ineffective erythropoiesis and normocytic or macrocytic anemia; microcytic red cell indices should prompt assessment for another contributing cause such as iron deficiency, acquired thalassemia, copper deficiency or severe inflammation. Gains and losses in genetic material are characteristic of MDS, including recurrent chromosome 11 abnormalities. Trisomy 11 is observed in 1.5% of cases and deletions in 11q in another 1%, while, in one study of 1084 MDS patients, haploinsufficiency of chromosome 11 or deletions of 11p were present in 2%, including del(11p) in six patients, add(11p) in two, and der(11) in five; all of these patients had complex karyotypes [6]. Therefore, beta thalassemia may be more common than described. These findings underscore the spectrum of phenotypic alterations that may occur in MDS and other hematologic malignancies and highlight another mechanism of ineffective hematopoiesis that may contribute to anemia associated with MDS. Acknowledgment

Producing megakaryocytes from a human peripheral blood source

Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood–derived human hematopoietic stem and progenitor cells (HSPCs).

Misdiagnosis of primary immune thrombocytopenia and frequency of bleeding: lessons from the McMaster ITP Registry

Nonspecific diagnostic criteria and uncertain estimates of severe bleeding events are fundamental gaps in knowledge of primary immune thrombocytopenia (ITP). To address these issues, we created the McMaster ITP Registry. In this report, we describe the methodology of the registry, the process for arriving at the diagnosis, and the frequency of bleeding. Consecutive patients with platelets <150 × 109/L from a tertiary hematology clinic in Canada were eligible. Patients completed a panel of investigations and were managed per clinical need. Two hematologists initially determined the cause of the thrombocytopenia using standard criteria and reevaluated the diagnosis over time, which was adjudicated at regular team meetings. Bleeding was graded from 0 (none) to 2 (severe) prospectively using an ITP-specific tool. Data were validated by duplicate chart review and source verification. Between 2010 and 2016, 614 patients were enrolled. Median follow-up for patients with >1 visit was 1.7 years (interquartile range, 0.8-3.4). At registration, 295 patients were initially diagnosed with primary ITP; of those, 36 (12.2%) were reclassified as having a different diagnosis during follow-up. At registration, 319 patients were initially diagnosed with another thrombocytopenic condition; of those, 10 (3.1%) were ultimately reclassified as having primary ITP. Of 269 patients with a final diagnosis of primary ITP, 56.5% (95% confidence interval [CI], 50.4-62.5] experienced grade 2 bleeding at 1 or more anatomical site, and 2.2% (95% CI, 0.8-4.8) had intracranial hemorrhage. Nearly 1 in 7 patients with primary ITP were misdiagnosed. Grade 2 bleeding was common. Registry data can help improve the clinical and laboratory classification of patients with ITP.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid‐phase to improve binding specificity, the prevalence of anti‐TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non‐immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti‐cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non‐immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti‐TPO or anti‐cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti‐TPO autoantibodies. This suggests that anti‐TPO and anti‐cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia

Essentials The immunogenesis of Heparin‐induced thrombocytopenia (HIT) is not well understood. Immunization to platelet factor 4 (PF4)‐heparin occurs early in life, before any heparin exposure. PF4 and PF4‐heparin complexes induce the proliferation of CD14+ cells. Reduced levels of regulatory cytokines contribute to immune dysregulation in HIT.