Thalassemic Red Blood Cells Research Articles

A dataset of digital holograms of normal and thalassemic cells

Digital holographic microscopy (DHM) is an intriguing medical diagnostic tool due to its label-free and quantitative nature, providing high-contrast images of phase samples. By capturing both intensity and phase information, DHM enables the numerical reconstruction of quantitative phase images. However, the lateral resolution is limited by the diffraction limit, which prompted the recent suggestion of microsphere-assisted DHM to enhance the DHM resolution straightforwardly. The use of such a technique as a medical diagnostic tool requires testing and validation of the proposed assays to prove their feasibility and viability. This paper publishes 760 and 609 microsphere-assisted DHM images of normal and thalassemic red blood cells obtained from a normal and thalassemic male individual, respectively.

5613312 PLASMATIC MICROPARTICLES AS POTENTIAL BIOMARKERS IN THE PREVENTIVE DIAGNOSIS OF BETA THALASSEMIA

Background: Plasmatic microparticles (MPs) are submicron (0.1–1 μm) extracellular vesicles that shed during plasma membrane remodeling in response to cell activation and apoptosis. Over the years, microparticles have evolved from their initial status being a cellular discharge mechanism, by which the cell got rid of unwanted materials, they are now considered as functional units produced in a regulated manner and having an important role in the maintenance of homeostasis. However, MPs also participate in the pathophysiology of several diseases such as beta thalassemia. It is a hemoglobinopathy characterized by reduced or absent synthesis of the beta-globin chain resulting in the excessive accumulation of unpaired alpha-globin chains which causes structural and functional alterations of the cytoplasmic membrane of thalassemic red blood cells. These alterations induce membrane budding and the release of MPs, which increases the risk of thrombotic complications observed in beta thalassemia patients. Aims: Therefore, our study suggests the research for plasmatic microparticles as potential cellular biomarkers and the establishment of a new innovative and predictive diagnosis strategy in order to avoid the thrombotic complications of beta thalassemia. Methods: Blood samples were taken from polytransfused beta thalassemia major patients clinically diagnosed at the National Bone Marrow Transplant Center in Tunis and from healthy donors. A cellular study was carried out by flow cytometry in order to quantify the apoptotic MPs derived from erythrocytes, platelets and endothelial cells using different antibodies and specific fluorescent dyes. Results: Our results showed a statistically significant increase in the number of apoptotic MPs, which suggests a high rate of apoptosis in the cells of the patients compared to those of the healthy controls. We also found that MPs derived from erythrocytes and endothelial cells were clearly elevated in beta thalassemia patients, suggesting their potential contribution to thrombotic risk. Conclusion: This discovery makes it possible to consider MPs as potential cellular biomarkers for preventive diagnosis in beta thalassemia.

Mitapivat Improves Markers of Erythropoietic Activity in Long-Term Study of Adults with Alpha- or Beta-Non-Transfusion-Dependent Thalassemia

Background: The thalassemias comprise a group of hemoglobinopathies characterized by ineffective erythropoiesis and hemolysis, which lead to chronic anemia and associated complications. Thalassemic red blood cells (RBCs) do not have sufficient levels of adenosine triphosphate (ATP) to meet their increased energy demands. Mitapivat is an allosteric activator of pyruvate kinase (PK), a key enzyme that regulates the final step of glycolysis responsible for ATP production in RBCs. We previously reported primary results of an open-label phase 2 study (NCT03692052) of mitapivat in pts with α- or β-non-transfusion-dependent thalassemia (NTDT) showing 80.0% (16/20) of pts achieved a hemoglobin (Hb) response of ≥1.0 g/dL increase from baseline at ≥1 assessments between Weeks (Wk) 4-12, inclusive. The increase in Hb was sustained, with a mean Hb (standard deviation) increase of 1.7 g/dL (0.5) at Wk 72; hemolysis markers were also improved. This analysis (data cutoff 27Mar22) evaluated the effect of mitapivat on markers of erythropoietic activity through Wk 72, along with Hb and markers of hemolysis and iron homeostasis. Methods: Eligibility criteria: ≥18 years (y) of age, known medical history of α- or β-thalassemia, Hb concentration ≤10.0 g/dL, and ≤5 RBC units transfused in the prior 24 wk and none in the 8 wk prior to study drug. Pts started mitapivat 50 mg twice daily (BID) and escalated to 100 mg BID based on individual safety and Hb assessments. Pts who completed the 24-wk core period and achieved a Hb response or a delayed Hb response (after Wk 12) were eligible to continue to the extension period (at Wk 24 dose) if they had no ongoing grade ≥3 treatment-emergent adverse events related to study drug. Study visits during the extension occur every 12 wk for up to 3 y. Assessments collected within 8 wk (56 days) after a transfusion were excluded from baseline derivation and analysis. Results: Nineteen of 20 pts completed the core period, 17 entered the extension period; 16 pts (4 with α- and 12 with β-thalassemia) were ongoing at the data cutoff date. At baseline, pts had biomarker levels consistent with ineffective erythropoiesis and hemolysis (Table). Changes from baseline showed a durable increase in Hb with a median (Q1, Q3) of 1.3 g/dL (0.60, 1.90) at Wk 12 and 1.2 g/dL (-0.03, 2.2) at Wk 72 (Table). Concurrently, there were trends for decreases in medians for erythropoietin, erythroferrone, soluble transferrin receptor, indirect bilirubin, and reticulocyte/erythrocyte fraction that were maintained over time. Median hepcidin levels remained relatively stable. Conclusions: These results indicate that mitapivat's mechanism ameliorates multiple aspects of the complex pathophysiology underlying α- and β-NTDT, including ineffective erythropoiesis, hemolysis, and anemia. The sustained long-term effects of mitapivat may offer a novel disease-modifying approach. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LONG-TERM EFFICACY AND SAFETY OF THE ORAL PYRUVATE KINASE ACTIVATOR MITAPIVAT IN ADULTS WITH NON–TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

Thalassemic red blood cells (RBCs) have insufficient levels of adenosine triphosphate (ATP) to meet increased energy demands associated with globin chain precipitation and oxidative stress responses. Mitapivat is a first-in-class, small molecule, oral activator of pyruvate kinase (PK), a key glycolytic enzyme regulating ATP production. In a phase 2, open-label trial of mitapivat in adults with α- or β-non–transfusion-dependent (NTD) thalassemia (NCT03692052), 80% (16/20) of patients (pts) met the primary endpoint of a hemoglobin (Hb) response (≥1.0 g/dL increase from baseline [BL] at ≥1 assessments between Weeks (Wk) 4–12, inclusive). Improvements in markers of hemolysis and ineffective erythropoiesis were also observed and mitapivat was generally well tolerated. Here we report data from the ongoing long-term extension (LTE) period (≤Wk 72; data cutoff 27Mar2021). Pts aged ≥18 years (y) with α- or β-thalassemia, Hb concentration ≤10.0 g/dL, and ≤5 RBC units transfused in prior 24 wk and none in 8 wk prior to study drug were eligible. All pts started mitapivat 50 mg twice daily (BID), escalating to 100 mg BID based on individual safety and Hb assessments. Pts with a Hb response or a delayed Hb response (after Wk 12), with no ongoing study drug-related grade ≥3 treatment-emergent adverse events (AE), could continue on mitapivat in the LTE at the Wk 24 visit dose. LTE study visits occur every 12 wk. Twenty patients started treatment; 19 pts completed the core period, 17 entered the LTE. As of the data cutoff, 1 pt discontinued (pt decision). Median treatment duration for pts in the LTE was 70.9 wk (range 54.7, 105.6); 8 pts received ≥72 wk of treatment as of data cutoff. Baseline values for patients who continued in LTE were: median age 44 y (range 29, 67), mean (standard deviation [SD]) Hb 8.1 (1.2) g/dL, total bilirubin 40.1 (26.2) μmol/L, lactate dehydrogenase (LDH), 272.4 (121.7) U/L, and median erythropoietin (EPO) 70.5 (range 15, 11191) IU/L. Hb improvements achieved in the core period were sustained in the LTE. Mean (SD) Hb increase from BL to Wk 60 (α-thalassemia, n = 4; β-thalassemia, n = 9) and Wk 72 (β-thalassemia, n = 8) were 1.5 (0.4) and 1.7 (0.5) g/dL, respectively. Improvements in markers of hemolysis and ineffective erythropoiesis observed in the core period were maintained in the LTE up to Wk 72 (change in mean [SD] bilirubin and LDH, –15.8 [16.6] μmol/L and –63.6 [216.0] U/L, respectively; change in median [range] EPO, –33.0 [–72.0, –16.0] IU/L). The safety profile was consistent with that observed in the core period. AEs in ≥15% of pts were headache (5/17) and back pain (3/17), 0 were grade ≥3. No treatment-related serious AEs or trends for decreases in bone mineral density were observed. In pts with either α- or β-thalassemia, a favorable efficacy-safety profile was observed with long-term mitapivat treatment. Data show sustained improvements in Hb, hemolysis, and ineffective erythropoiesis across a spectrum of globin genotypes, and no new safety findings. Mitapivat's mechanism of action may represent a novel therapeutic approach for thalassemia. Two phase 3 trials of mitapivat in α- and β-thalassemia for both NTD (ENERGIZE, NCT04770753) and transfusion-dependent (ENERGIZE-T, NCT04770779) pts are enrolling.

An energy booster for thalassaemic red blood cells

Haemoglobinopathies are the most common monogenic disorders with worldwide distribution. Our knowledge on the mechanisms leading to the intramedullary apoptosis of red blood cell precursors and decreased lifespan of red blood cells in thalassaemia has evolved substantially over the past three decades and is guiding the advent of novel therapeutic approaches. Agents targeting different steps of the pathogenesis—namely, the globin chain imbalance, ineffective erythropoiesis, deranged iron homoeostasis, and increased haemolysis—are being developed and studied in the clinical setting.

Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients

Background Hypercoagulability is a known complication of thalassemia, in particular, thalassemia intermedia. Several factors contribute to this hypercoagulability, including chronic platelet activation and the presence of other comorbid conditions. In addition, the oxidation of globin subunits in thalassemic red blood cells (RBCs) triggers the formation of reactive oxygen species. These factors lead to the exposure of negatively charged phospholipids like phosphatidylserine (PS), which ultimately causes increased thrombin generation, particularly in splenectomized patients. Aim This study aimed to assess the risk for hypercoagulability in thalassemic patients using PS expression on RBCs and the thrombin–antithrombin (TAT) complex in plasma. Patients and methods This study included 50 thalassemic patients (19 patients with splenectomy and 31 patients without splenectomy) and 30 apparently healthy individuals as a control group. Patients were subjected to assessment of history for deep venous thrombosis or pulmonary hypertension by echocardiography. Annexin V was used to detect PS expression on RBCs by flow cytometry, while the enzyme-linked immunosorbent assay was used to detect the TAT complex in plasma. Results Erythrocyte PS expression was significantly higher (P<0.001) in thalassemic patients than in the control group. The TAT complex level revealed no significant difference between thalassemia patients and the control group (P=0.468). Patients with pulmonary hypertension showed a statistically significant higher PS expression and TAT complex level. However, there was no significant increase in PS expression or TAT level in patients with a history of deep venous thrombosis only. Conclusion Increased PS expression and TAT complex level may be a risk factor for pulmonary hypertension in thalassemia patients with splenectomy.

P1522: LONG-TERM EFFICACY AND SAFETY OF THE ORAL PYRUVATE KINASE ACTIVATOR MITAPIVAT IN ADULTS WITH NON–TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

Background: Thalassemia is characterized by ineffective erythropoiesis and hemolysis that occur due to imbalanced production and precipitation of globin chains. Thalassemic red blood cells (RBCs) have insufficient levels of adenosine triphosphate (ATP) to meet increased energy demands associated with globin chain imbalance, protein degradation, and cellular oxidative stress responses. Mitapivat is a first-in-class, small molecule, oral activator of RBC pyruvate kinase (PKR), a key glycolytic enzyme regulating ATP production. In a phase 2, open-label trial of mitapivat in adults with α- or β-non–transfusion-dependent (NTD) thalassemia (NCT03692052), 80% (16/20) of patients (pts) met the primary endpoint of a hemoglobin (Hb) response (≥1.0 g/dL increase from baseline [BL] at ≥1 assessments between Weeks (Wks) 4–12, inclusive). Improvements in markers of hemolysis and ineffective erythropoiesis were also observed and mitapivat was generally well tolerated. Aims: To report data from the ongoing long-term extension (LTE) period (≤Wk 72; data cutoff 27Mar2021). Methods: Pts aged ≥18 years (yrs) with a known medical history of α- or β-thalassemia, Hb concentration ≤10.0 g/dL, and ≤5 RBC units transfused in prior 24 wks and none in 8 wks prior to study drug were eligible. All pts started mitapivat 50 mg twice daily (BID), escalating to 100 mg BID based on individual safety and Hb assessments. After completion of the 24-wk core period, pts with a Hb response or a delayed Hb response (after Wk 12), with no ongoing study drug-related grade ≥3 treatment-emergent adverse events (AE), continued on mitapivat in the LTE at the same dose as the Wk 24 visit. Study visits occur every 12 wks for ≤10 yrs. Results: Of 19 pts who completed the core period, 17 entered the LTE (mitapivat 100 mg BID, n=16; 50 mg BID, n=1). As of the data cutoff, 1 pt discontinued (pt decision). Median duration of treatment for pts in the LTE was 70.9 wks (range 54.7, 105.6); 8 pts received ≥72 wks of treatment as of data cutoff. Median pt age in the LTE was 44 yrs (range 29, 67). Mean (standard deviation [SD]) BL Hb, total bilirubin and lactate dehydrogenase (LDH) were 8.1 (1.2) g/dL, 40.1 (26.2) μmol/L, and 272.4 (121.7) U/L, respectively. Median BL erythropoietin (EPO) was 70.5 (range 15, 11191) IU/L. Hb improvements achieved in the core period were sustained in the LTE (Figure). Mean (SD) Hb increase from BL to Wk 60 (α-thalassemia, n=4; β-thalassemia, n=9) and Wk 72 (β-thalassemia, n=8) were 1.5 (0.4) and 1.7 (0.5) g/dL, respectively. Improvements in markers of hemolysis and ineffective erythropoiesis observed in the core period were maintained in the LTE up to Wk 72 (mean [SD] bilirubin and LDH, –15.8 [16.6] μmol/L and –63.6 [216.0] U/L, respectively; median [range] EPO, –33.0 [–72.0, –16.0] IU/L). The safety profile was consistent with that observed in the core period. AEs in ≥15% of pts were headache (5/17) and back pain (3/17), none were grade ≥3. No trends for decreases in bone mineral density were observed. No treatment-related serious AEs occurred. Image:Summary/Conclusion: In pts with either α- or β-thalassemia, a favorable efficacy-safety profile was observed with long-term treatment with mitapivat. Data show sustained improvements in Hb, hemolysis, and ineffective erythropoiesis despite globin genotypic heterogeneity, and no new safety findings. Mitapivat, through its unique mechanism of action, may represent a novel therapeutic approach for this condition. Two phase 3 trials of mitapivat in α- and β-thalassemia for both NTD (ENERGIZE, 2021-000211-23) and transfusion-dependent (ENERGIZE-T, 2021-000212-34) pts are enrolling.

P112: ENERGIZE AND ENERGIZE-T: TWO PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDIES OF MITAPIVAT IN ADULTS WITH NON--TRANSFUSION-DEPENDENT OR TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

Background: Thalassemias are characterized by imbalanced globin-chain production resulting in excess α- or β-globin precipitation, hemolytic anemia, and ineffective erythropoiesis.1,2 ATP levels are reduced in thalassemic red blood cells (RBCs), despite increased energy demands.3,4 Mitapivat is an oral activator of RBC pyruvate kinase (PKR), a glycolytic enzyme that regulates ATP production.5 In a phase 2 study of patients with α- or β-non–transfusion-dependent thalassemia (NTDT), twice-daily (BID) dosing with mitapivat increased hemoglobin (Hb) levels by ≥1.0 g/dL in 80% of patients,6 supporting the broadening of mitapivat’s development in thalassemia. Aims: To report the study designs of ENERGIZE (2021-000211-23) and ENERGIZE-T (2021-000212-34), two phase 3 trials to assess the efficacy and safety of mitapivat in adults with α- or β-NTDT or transfusion-dependent thalassemia (TDT), respectively. Methods: Both studies are phase 3, multicenter, randomized, double-blind, placebo-controlled trials (Figure). In ENERGIZE, approximately 171 eligible adults with NTDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 24 weeks. Upon completion, eligible patients can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (β-thalassemia ± α-globin mutations, Hb E β-thalassemia, or α-thalassemia [Hb H disease]), Hb concentration ≤10.0 g/dL, and NTDT defined as ≤5 RBC units during the 24-week period before randomization and no RBC transfusion ≤8 weeks prior. The primary endpoint is an Hb response defined as a ≥1.0 g/dL increase in average Hb concentration from Week 12 through 24 compared with baseline. Secondary endpoints include patient-reported outcomes, changes in Hb, markers of hemolysis and erythropoiesis, and safety. In ENERGIZE-T, approximately 240 eligible adults with TDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 48 weeks. Upon completion, eligible patients can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (same genotypes as detailed for the ENERGIZE study), and TDT defined as 6–20 RBC units transfused and no transfusion-free period ≥6 weeks during the 24 weeks before randomization. The primary endpoint is a transfusion reduction response, defined as a ≥50% reduction in transfused RBC units with a reduction of ≥2 units of transfused RBCs in any consecutive 12-week period through Week 48 compared with baseline. Secondary endpoints include additional measures of transfusion burden, changes in iron markers, and safety. Results: Not yet available. Conclusions: ENERGIZE and ENERGIZE-T are the first pivotal studies to assess a potential treatment across a broad spectrum of patients with thalassemia (ie, patients with TDT and NTDT; α- and β-thalassemias). ENERGIZE and ENERGIZE-T will evaluate the efficacy and safety of mitapivat, a novel, first-in-class oral activator of PKR. Both studies are actively recruiting.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Cell erythroid maturation approach: a new paradigm in the road map towards a cure for β-thalassaemia syndromes.

Long-Term Efficacy and Safety of the Oral Pyruvate Kinase Activator Mitapivat in Adults with Non-Transfusion-Dependent Alpha- or Beta-Thalassemia

Long-Term Efficacy and Safety of the Oral Pyruvate Kinase Activator Mitapivat in Adults with Non-Transfusion-Dependent Alpha- or Beta-Thalassemia

Luspatercept Redistributes Body Iron to the Liver in Transfusion-Dependent-Thalassemia (TDT) during Erythropoietic Response

Luspatercept Redistributes Body Iron to the Liver in Transfusion-Dependent-Thalassemia (TDT) during Erythropoietic Response

ENERGIZE AND ENERGIZE-T: TWO PHASE 3, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDIES OF MITAPIVAT IN ADULTS WITH NON–TRANSFUSION-DEPENDENT OR TRANSFUSION-DEPENDENT ALPHA- OR BETA-THALASSEMIA

Thalassemias are characterized by imbalanced globin-chain production resulting in excess α- or β-globin precipitation, hemolytic anemia, and ineffective erythropoiesis. Adenosine triphosphate (ATP) levels are reduced in thalassemic red blood cells (RBCs), despite increased energy demands. Mitapivat is an oral activator of RBC pyruvate kinase (PKR), a glycolytic enzyme that regulates ATP production. In a phase 2 study of patients (pts) with α- or β-non–transfusion-dependent thalassemia (NTDT), twice-daily (BID) dosing with mitapivat increased hemoglobin (Hb) levels by ≥1.0 g/dL in 80% of pts, supporting the broadening of mitapivat's development in thalassemia. The study designs of ENERGIZE (2021-000211-23) and ENERGIZE-T (2021-000212-34), two phase 3 trials to assess the efficacy and safety of mitapivat in adults with α- or β-NTDT or transfusion-dependent thalassemia (TDT), respectively, are reported here. Both studies are phase 3, multicenter, randomized, double-blind, placebo-controlled trials. In ENERGIZE, approximately 171 eligible adults with NTDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 24 weeks (wks). Upon completion, eligible pts can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (β-thalassemia ±α-globin mutations, Hb E β-thalassemia, or α-thalassemia [Hb H disease]), Hb concentration ≤10.0 g/dL, and NTDT defined as ≤5 RBC units during the 24-wk period before randomization and no RBC transfusion ≤8 wks prior. The primary endpoint is an Hb response defined as a ≥1.0 g/dL increase in average Hb concentration from Wk 12 through 24 compared with baseline (BL). Secondary endpoints include pt-reported outcomes, changes in Hb, markers of hemolysis and erythropoiesis, and safety. In ENERGIZE-T, approximately 240 eligible adults with TDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 48 wks. Upon completion, eligible pts can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (same genotypes as detailed for the ENERGIZE study), and TDT defined as 6–20 RBC units transfused and no transfusion-free period ≥6 wks during the 24 wks before randomization. The primary endpoint is a transfusion reduction response, defined as a ≥50% reduction in transfused RBC units with a reduction of ≥2 units of transfused RBCs in any consecutive 12-wk period through Wk 48 compared with BL. Secondary endpoints include additional measures of transfusion burden, changes in iron markers, and safety. Not yet available. ENERGIZE and ENERGIZE-T are the first pivotal studies to assess a potential treatment across a broad spectrum of pts with thalassemia (ie, pts with TDT and NTDT; α- and β-thalassemias). ENERGIZE and ENERGIZE-T will evaluate the efficacy and safety of mitapivat, a novel, first-in-class oral activator of PKR. Both studies will start enrollment in 2021.

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Effect of low dose X-ray on membrane fluidity of thalassemic red blood cells

Effect of low dose X-ray on membrane fluidity of thalassemic red blood cells

Automatic detection and characterization of quantitative phase images of thalassemic red blood cells using a mask region-based convolutional neural network.

.Significance: Label-free quantitative phase imaging is a promising technique for the automatic detection of abnormal red blood cells (RBCs) in real time. Although deep-learning techniques can accurately detect abnormal RBCs from quantitative phase images efficiently, their applications in diagnostic testing are limited by the lack of transparency. More interpretable results such as morphological and biochemical characteristics of individual RBCs are highly desirable.Aim: An end-to-end deep-learning model was developed to efficiently discriminate thalassemic RBCs (tRBCs) from healthy RBCs (hRBCs) in quantitative phase images and segment RBCs for single-cell characterization.Approach: Two-dimensional quantitative phase images of hRBCs and tRBCs were acquired using digital holographic microscopy. A mask region-based convolutional neural network (Mask R-CNN) model was trained to discriminate tRBCs and segment individual RBCs. Characterization of tRBCs was achieved utilizing SHapley Additive exPlanation analysis and canonical correlation analysis on automatically segmented RBC phase images.Results: The implemented model achieved 97.8% accuracy in detecting tRBCs. Phase-shift statistics showed the highest influence on the correct classification of tRBCs. Associations between the phase-shift features and three-dimensional morphological features were revealed.Conclusions: The implemented Mask R-CNN model accurately identified tRBCs and segmented RBCs to provide single-RBC characterization, which has the potential to aid clinical decision-making.

Update in Laboratory Diagnosis of Thalassemia.

Alpha- and β-thalassemias and abnormal hemoglobin (Hb) are common in tropical countries. These abnormal globin genes in different combinations lead to many thalassemic diseases including three severe thalassemia diseases, i.e., homozygous β-thalassemia, β-thalassemia/Hb E, and Hb Bart’s hydrops fetalis. Laboratory diagnosis of thalassemia requires a number of tests including red blood cell indices and Hb and DNA analyses. Thalassemic red blood cell analysis with an automated hematology analyzer is a primary screening for thalassemia since microcytosis and decreased Hb content of red blood cells are hallmarks of all thalassemic red blood cells. However, these two red blood cell indices cannot discriminate between thalassemia trait and iron deficiency or between α- and β-thalassemic conditions. Today, Hb analysis may be carried out by either automatic high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CE) system. These two systems give both qualitative and quantitative analysis of Hb components and help to do thalassemia prenatal and postnatal diagnoses within a short period. Both systems have a good correlation, but the interpretation under the CE system should be done with caution because Hb A2 is clearly separated from Hb E. In case of α-thalassemia gene interaction, it can affect the amount of Hb A2/E. Thalassemia genotypes can be characterized by the intensities between alpha-/beta-globin chains or alpha-/beta-mRNA ratios. However, those are presumptive diagnoses. Only DNA analysis can be made for specific thalassemia mutation diagnosis. Various molecular techniques have been used for point mutation detection in β-thalassemia and large-deletion detection in α-thalassemia. All of these techniques have some advantages and disadvantages. Recently, screening for both α- and β-thalassemia genes by next-generation sequencing (NGS) has been introduced. This technique gives an accurate diagnosis of thalassemia that may be misdiagnosed by other conventional techniques. The major limitation for using NGS in the screening of thalassemia is its cost which is still expensive. All service labs highly recommend to select the technique(s) they are most familiar and most economic one for their routine use.

Mitapivat (AG-348), an Oral PK-R Activator, in Adults with Non-Transfusion Dependent Thalassemia: A Phase 2, Open-Label, Multicenter Study in Progress

Background: Thalassemia is a group of inherited blood disorders in which genetic mutation(s) in the α- and/or β-globin locus lead to excess precipitation of β- or α-globin, respectively, and compromised red blood cell (RBC) survival. The condition is characterized by ineffective erythropoiesis and peripheral hemolysis, with resultant anemia. Adenosine triphosphate (ATP) supply appears to be insufficient in thalassemic RBCs to maintain RBC membrane fitness and clearance of globin precipitates. Mitapivat (AG-348) is an oral, small-molecule, allosteric activator of the RBC-specific form of pyruvate kinase (PK-R). PK-R is a key enzyme for maintaining energy homeostasis in RBCs, as they rely almost exclusively on the process of glycolysis to generate ATP. In healthy adults, mitapivat activates wild-type PK-R and increases ATP levels in RBCs. In adults with PK deficiency who were not regularly transfused, oral mitapivat was well tolerated and induced rapid, durable hemoglobin (Hb) increases (NCT02476916). In the Hbbth/3+ mouse model of β-thalassemia, mitapivat increased ATP levels; reduced markers of ineffective erythropoiesis; and improved anemia, RBC survival, and indices of iron overload. These data support the hypothesis that increased ATP synthesis mediated via PK-R activation by mitapivat may improve the survival of thalassemic RBCs in the bone marrow and/or peripheral circulation, and thus represents a novel mechanism to treat patients with thalassemia. Here we present the design of a phase 2, open-label study designed to test this hypothesis and assess the proof of concept of mitapivat in patients with thalassemia. Methods: This phase 2, multicenter, open-label study (NCT03692052) is evaluating the efficacy, safety, pharmacokinetics, and pharmacodynamics of mitapivat in adults with non-transfusion-dependent thalassemia (NTDT). Four sites in North America and the United Kingdom are enrolling patients. The study consists of a 24-week core period followed by a 2-year extension period (Figure). Approximately 17 subjects with NTDT, i.e., β-thalassemia with or without α-globin gene mutations, HbE β-thalassemia, or α-thalassemia (HbH disease), will be enrolled. Non-transfusion dependence is defined as ≤5 units of RBCs transfused in the preceding 24 weeks and no transfusions in the 8 weeks prior to the first day of study drug. The hemoglobin inclusion criterion is ≤10.0 g/dL, which was increased from ≤9.0 g/dL in a recent protocol amendment to better reflect the clinical representation in the NTDT patient population. All eligible patients will receive an initial mitapivat dose of 50 mg twice daily (BID). At the week 6 visit the dose may be increased to 100 mg BID, depending on safety and Hb response. The primary endpoint is the proportion of subjects who achieve an Hb response, defined as an increase in Hb of ≥1.0 g/dL from baseline at any time between week 4 and week 12 (inclusive). Key secondary and exploratory endpoints include changes in Hb and markers of hemolysis; hematopoietic activity; iron metabolism and iron overload; and assessments of safety and pharmacokinetics. The study is currently enrolling. Figure Disclosures Kuo: Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Layton:Cerus Corporation: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Uhlig:Agios: Employment, Equity Ownership. Lynch:Agios: Employment, Equity Ownership. Liu:Agios: Employment, Equity Ownership. Vichinsky:GBT: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Agios: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

PS1569 COMMON SINGLE NUCLEOTIDE POLYMORPHISM OF TMPRSS6 DETERMINES VARIATION OF RED BLOOD CELL INDICES FOUND IN HETEROZYGOUS ALPHA‐THALASSEMIA

Background:Globally, many screening programs for severe thalassemia (thal) syndromes use red blood cell (RBC) indices; mean corpuscular volume (MCV) <80 fl and/or mean corpuscular hemoglobin (MCH) <25 pg as cut‐off to identify thal disease and carriers. These criterion seem to be sensitive for individual with α°‐thal by two α‐globin gene deletions (‐‐/αα) and β‐thal traits. However this approach cannot be used for confidential identification α+‐thal trait (‐α/αα) due to a wide variation of baseline MCV and MCH. Recently, a single nucleotide polymorphism (SNP) of an iron regulation gene; tmprss6 involved in substitution a nucleotide between thymine (T) and cytosine (C) in exon 17 resulted to amino acid change from valine to alanine (V736A) has been described to associate with RBC indices. However its role on thalassemic RBC remains largely unknown.Aims:To determine the effects of common SNP rs855791 of tmprss6 on RBC indices variation in deletional α‐thal carriersMethods:EDTA whole blood samples were collected from Thai healthy volunteers in cohort and measured complete blood count, RBC indices, hemoglobin typing and comprehensive molecular analysis panel covered > 98% of common globin mutations (α‐thal, β‐thal, Hb E) found in Thailand. SNP rs855791 genotypes of tmprss6 were analyzed by PCR‐RFLP with StuI restriction enzyme. Statistical analysis for average (mean), standard deviation (SD) and comparisons was performed by using PASW Statistics 18. The significant difference was determined by p ≤ 0.05.Results:Total 433 cases (age: 23.0 ± 8.7 years old, male: N = 141 (32.6%), female: N = 292 (67.4%)) of normal β‐globin gene (ββ) included three genotypes of α‐globin gene; normal (αα/αα) (N = 357, 82.4%), ‐α/αα (N = 61, 14.1%), and ‐‐/αα (N = 15, 3.5%) (Table 1). The allele frequency of SNP rs855791 of Thai population showed T:C as 0.6 to 0.4 that similar to other Asian population and consistent across different globin genotypes (Table2). The MCV and MCH ranges of two α‐globin genotypes; αα/αα and ‐α/αα showed an overlapping feature. Interestingly, we found no effect of either C or T polymorphism on RBC indices mainly MCV and MCH in individuals with normal globin genes. However the effects of SNP rs855791 genotypes on MCV and MCH were demonstrated in α‐thal traits. Increased MCV and MCH gradually expressed by SNP genotype C/C > C/T > T/T both in α+‐ and α°‐thal trait (Figure 1 and 2). Comparing the level of serum ferritin among different tmprss6 genotypes; the C/C seemed to have higher iron status followed by C/T and T/T, however there was no statistical significance (data not shown). Therefore this epistatic effect of tmprss6 on determining RBC indices in α‐thal carriers may operate through other mechanism.Summary/Conclusion:For the first time, we have identified that tmprss6 polymorphism can determine RBC size and hemoglobinization in carrier of α‐thal. The genetic mechanism of tmprss6 involved in the determination RBC indices in α‐thal mutations would be further studied.image

PB2392 GLOBAL HEMOSTATIC CHANGES AND CIRCULATING MICROPARTICLES IN CHILDREN AND ADOLESCENTS WITH HEMOGLOBIN H AND BETA‐THALASSEMIA/HEMOGLOBIN E DISEASES

Background:Thalassemia is an inherited hemolytic anemia known to be associated with hypercoagulable state. Several factors including the abnormality of thalassemic red blood cell (RBC) membrane, increased platelet count, decreased anticoagulant proteins and increased circulating microparticles (MPs) contribute to the hypercoagulability in thalassemia. Global hemostatic changes as measured by rotatory thromboelastometry (ROTEM®) have been demonstrated in patients with beta‐thalassemia/hemoglobin E disease (beta‐thal/Hb E). However, there is little information in patients with hemoglobin H disease (Hb H) which is a less severe but highly prevalent non‐transfusion dependent thalassemia.Aims:To compare the global hemostatic changes and the levels of circulating MPs among children and adolescents with Hb H, beta‐thal/Hb E and healthy controls.Methods:A cross‐sectional study was conducted in 38 subjects with Hb H, 22 subjects with beta‐thal/Hb E and 12 healthy age‐matched controls. Complete blood count, ROTEM® and quantitation of circulating MPs [annexin V + MPs, red blood cell (RBC) derived MPs (RMPs) and platelet derived MPs (PMPs)] as analyzed by flow cytometry were performed in all subjects.Results:The age (range 5‐21 years) and percentage of male gender (range 50‐58%) among the Hb H, beta‐thal/Hb E and control groups were not statistically different (P = 0.383 and P = 0.798, respectively). The Hb levels were higher and the numbers of nucleated RBC (nRBC) were lower in the Hb H group than in beta‐thal/Hb E group. Overall, the ROTEM® parameters were not statistically different among the thalassemia and control groups. However, within the thalassemia subjects group, the maximum clot firmness (MCF) in splenectomized patients was significantly higher than non‐splenectomized subjects (P < 0.001). The levels of annexin V + MPs in both Hb H and beta‐thal/Hb E groups were significantly higher than the control group (P = 0.001). RMPs in beta‐thal/Hb E group were higher than the controls (P = 0.013) while there was no difference between the Hb H and control groups (P = 0.097). The level of circulating MPs was slightly higher in splenectomized subjects, but the differences were not statistically significant. Only the platelet count and nRBC, but not the circulating MPs, were significantly correlated with MCF in both INTEM (r = 0.620, P < 0.001 and r = 0.322, P = 0.006 respectively) and EXTEM (r = 0.567, P < 0.001 and r = 0.326, P = 0.005, respectively).Summary/Conclusion:The global hemostasis parameters as measured by ROTEM® were not different among the Hb H, beta‐thal/Hb E and control groups in this study. Hypercoagulable state was more commonly found in thalassemia subjects who were splenectomized, regardless of genotypes. The hypercoagulability was associated with higher nRBC and platelet count in the splenectomized group. Patients with Hb H had significantly higher circulating MPs levels than healthy controls although with the lesser extent than those seen in beta‐thal/Hb E.

Role of red blood cells “annexin V” and platelets “P-selectin” in patients with thalassemia

Objective/BackgroundCertain hemostatic anomalies found in patients with thalassemia suggest the existence of a chronic hypercoagulable state. Several etiologic factors may play a role in the pathogenesis of the hypercoagulable state in those patients. One of these factors is abnormal thalassemic red blood cells (RBCs), which may provide a procoagulant. To substantiate these findings, we measured the ability of RBCs from thalassemia patients to bind annexin V with increased fraction of platelets carrying the activation marker CD62P (P-selectin). To study the expression of RBC annexin V and platelets P-selectin in study patients (those with thalassemia major, thalassemia intermedia, thalassemia minor) and control group, four-color flow cytometry was performed and the correlation between these two markers was evaluated. MethodsA case–control study was conducted on 50 β-thalassemia patients (10 patients with thalassemia minor, 30 patients with thalassemia major, and 10 patients with thalassemia intermedia, with 10 normal adult volunteers as a control) from June 2016 to March 2017. Flow cytometry was used to study the expression of anionic phospholipids (Annexin V) on the RBCs and CD62P (P-selectin) on the activated platelet. ResultsThe mean expression of annexin V in patients with thalassemia major and intermedia was significantly higher than that in the control group and patients with thalassemia minor. Although the mean expression was higher in patients with thalassemia intermedia than in those with thalassemia major, it was not statistically significant. ConclusionThe mean expression of platelets P-selectin in patients with thalassemia major and thalassemia intermedia was significantly higher than that in controls and patients with thalassemia minor. However, its expression was significantly higher in patients with thalassemia intermedia than in those with thalassemia major. Annexin V also showed a positive correlation with P-selectin, and both markers positively correlated with regularity of blood transfusion.