Ishac Nazy

Thrombotic microangiopathies: a general approach to diagnosis and management.

Understanding of the pathophysiology of thrombotic microangiopathies — a group of rare yet life-threatening hematologic conditions — has evolved in recent years along with better access to diagnostic testing. Identifying secondary causes requires a thorough diagnostic workup, but appropriate

The effect of rituximab on anti-platelet autoantibody levels in patients with immune thrombocytopenia

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case‐control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet‐bound anti‐glycoprotein (GP) IIbIIIa and anti‐GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti‐GPIIbIIIa levels (P = 0·02) but not anti‐GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.

The unique immunological features of heparin-induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT) is a serious drug reaction that leads to a decrease in platelet count and a high risk of thrombosis. HIT patients produce pathogenic immunoglobulin G (IgG) antibodies that bind to complexes of platelet factor‐4 (PF4) and heparin. HIT immune complexes crosslink Fc‐receptors resulting in platelet and monocyte activation. These events lead to the release of procoagulant chemokines and tissue factor, which together create an intensely prothrombotic state. HIT represents an atypical immune response because it has features of both T cell‐dependent and T cell‐independent mechanisms. The disorder is characterized by newly formed anti‐PF4/heparin IgG antibodies, which are characteristic of a T cell‐dependent mechanism; however, re‐exposure to heparin, months after HIT, does not lead to a memory response, which is consistent with a T cell‐independent mechanism. In this review, we discuss the immunobiological events that can explain these features, including the role for T cell‐dependent and T cell‐independent mechanisms in HIT antibody generation, the immunogenic characteristics of the PF4/heparin antigen, and the concept of a temporary loss in immune regulation contributing to the onset of HIT. We also present a novel immunobiological model to explain the atypical immune response that is characteristic of HIT.

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.

Development of a high-yield expression and purification system for platelet factor 4

Abstract Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure

Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis.

Platelet-Activating Antibodies are Detectable at the Earliest Onset of Heparin-Induced Thrombocytopenia, with Implications for the Operating Characteristics of the Serotonin-Release Assay

Background: Heparin‐induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet‐activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. It is unknown whether platelet‐activating antibodies are detectable at the onset of the HIT‐related platelet count fall. Methods: Available blood samples from 18 patients obtained at onset of HIT were tested using the serotonin‐release assay (SRA), a test for platelet‐activating antibodies, and a PF4‐dependent enzyme‐linked immunosorbent assay (ELISA). Patient samples showing a delay of > 2 days between ELISA and SRA seroconversion were tested for subthreshold levels of platelet‐activating antibodies using two modifications of the SRA that amplify detection of HIT antibodies. We also estimated SRA sensitivity and specificity in two postorthopedic surgery clinical trials (633 samples), including assessing whether a positive SRA influenced platelet count recovery in the absence of thrombocytopenia. Results: Platelet‐activating HIT antibodies were detected in all 18 patients at the beginning of the HIT‐related platelet count fall. Although ELISA seroconversion usually preceded SRA seroconversion by only 1 day (median), subthreshold levels of platelet‐activating antibodies were detected in both patients who exhibited a lag between ELISA and SRA seroconversion. SRA sensitivity was 100% (18/18), and its specificity was 97% (597/615). Nonthrombocytopenic SRA‐positive patients with ongoing heparin treatment exhibited blunted platelet count recovery vs control subjects, suggesting even higher SRA specificity for detecting abnormal platelet count profiles. Conclusions: Platelet‐activating HIT antibodies are detectable at the onset of the HIT‐related platelet count fall. The SRA has high sensitivity and specificity for HIT, and indicates that presence of HIT antibodies can blunt postoperative platelet count recovery.

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.

Megakaryocyte apoptosis in immune thrombocytopenia

Abstract The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.

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.