A perspective on the added value of red blood cells during cardiac hypothermic oxygenated perfusion.

Hypothermic oxygenated perfusion (HOPE) is an emerging technique for donor heart preservation that is currently being studied in multiple clinical trials with promising results. When compared to HOPE for other organs, cardiac protocols involve red blood cell (RBC) supplementation, despite absence of comparative evidence for its benefits. In this perspective paper, we discuss the pros and cons of the addition of RBCs during cardiac HOPE. Although the current clinical results with RBC supplementation during HOPE seem promising, potential downsides of RBC supplementation cannot be ruled out. The impact of supplemented RBCs during cardiac HOPE requires further investigation to improve HOPE protocols, to optimize heart preservation using this promising technology.

Omega-3 supplementation changes the physical properties of leukocytes but not erythrocytes in healthy individuals: An exploratory trial.

n3-PUFA impact health in several ways, including cardiovascular protection and anti-inflammatory effects, but the underlying mechanisms are not fully understood. In this exploratory study involving 31 healthy subjects, we aimed to investigate the effects of 12 weeks of fish-oil supplementation (1500 mg EPA+DHA/day) on the physical properties of multiple blood cell types. We used deformability cytometry (DC) for all cell types and Laser-assisted Optical Rotational Red Cell Analysis (Lorrca) to assess red blood cell (RBC) deformability. We also investigated the correlation between changes in the physical properties of blood cells and changes in the Omega-3 Index (O3I), defined as the relative content of EPA+DHA in RBCs. Following supplementation, the mean±SD O3I increased from 5.3 %±1.5 % to 8.3 %±1.4 % (p < 0.001). No significant changes in RBC properties were found by both techniques. However, by DC we observed a consistent pattern of physical changes in lymphocytes, neutrophils and monocytes. Among these were significant increases in metrics correlated with the cells' deformability resulting in less stiff cells. The results suggest that leukocytes become softer and have an increased ability to deform under induced short-term physical stress such as hydrodynamic force in the circulation. These changes could impact immune function since softer leukocytes can potentially circulate more easily and could facilitate a more rapid response to systemic inflammation or infection. In conclusion, fish-oil supplementation modulates some physical properties of leukocyte-subfractions, potentially enhancing their biological function. Further studies are warranted to explore the impact of n3-PUFA on blood cell biology, particularly in disease states associated with leukocyte dysregulation.

Osmotic gradient ektacytometry - a novel diagnostic approach for neuroacanthocytosis syndromes.

Introduction: The unique red blood cell (RBC) properties that characterize the rare neuroacanthocytosis syndromes (NAS) have prompted the exploration of osmotic gradient ektacytometry (Osmoscan) as a diagnostic tool for these disorders. In this exploratory study, we assessed if Osmoscans can discriminate NAS from other neurodegenerative diseases. Methods: A comprehensive assessment was conducted using Osmoscan on a diverse group of patients, including healthy controls (n = 9), neuroacanthocytosis syndrome patients (n = 6, 2 VPS13A and 4 XK disease), Parkinson's disease patients (n = 6), Huntington's disease patients (n = 5), and amyotrophic lateral sclerosis patients (n = 4). Concurrently, we collected and analyzed RBC indices and patients' characteristics. Results: Statistically significant changes were observed in NAS patients compared to healthy controls and other conditions, specifically in osmolality at minimal elongation index (Omin), maximal elongation index (EImax), the osmolality at half maximal elongation index in the hyperosmotic part of the curve (Ohyper), and the width of the curve close to the osmolality at maximal elongation index (Omax-width). Discussion: This study represents an initial exploration of RBC properties from NAS patients using osmotic gradient ektacytometry. While specific parameters exhibited differences, only Ohyper and Omax-width yielded 100% specificity for other neurodegenerative diseases. Moreover, unique correlations between Osmoscan parameters and RBC indices in NAS versus controls were identified, such as osmolality at maximal elongation index (Omax) vs. mean cellular hemoglobin content (MCH) and minimal elongation index (EImin) vs. red blood cell distribution width (RDW). Given the limited sample size, further studies are essential to establish diagnostic guidelines based on these findings.

Designing a single-arm phase 2 clinical trial of mitapivat for adult patients with erythrocyte membranopathies (SATISFY): a framework for interventional trials in rare anaemias - pilot study protocol.

INTRODUCTION: Membranopathies encompass haemolytic disorders arising from genetic variants in erythrocyte membrane proteins, including hereditary spherocytosis and stomatocytosis. Congenital dyserythropoietic anaemia type II (CDA II) is associated with the SEC23B gene and can exhibit phenotypic similarities to membranopathies. Current treatment options for these conditions, apart from splenectomy, are primarily supportive. Mitapivat, a novel pyruvate kinase (PK) activator, has demonstrated efficacy in increasing haemoglobin levels and reducing haemolysis in patients with PK deficiency, thalassemia, sickle cell disease and a mouse model of hereditary spherocytosis. METHODS AND ANALYSES: Safety and efficacy of mitapivat sulfate in adult patients with erythrocyte membranopathies (SATISFY) is a prospective, multicentre, single-arm phase two trial involving approximately 25 adult patients (≥18 years) diagnosed with a membranopathy or CDA II. During the 8-week dose escalation period, subjects will receive an initial dose of 50 mg mitapivat two times per day and may increase to 100 mg two times per day at week 4 based on the safety and changes in haemoglobin levels. Patients tolerating mitapivat well may be eligible to continue in two consecutive 24-week fixed dose periods.The primary objective of this study is to evaluate the safety of mitapivat, assessed through the occurrence of treatment-emergent adverse events. Secondary objectives include assessing the effects of mitapivat on haemoglobin levels, haemolysis, erythropoiesis, patient-reported outcome measures and spleen size.SATISFY aims to assess the safety and efficacy of mitapivat in adult patients with red blood cell membranopathies and CDA II, with the aim of establishing proof-of-concept in patients living with these rare conditions. ETHICS AND DISSEMINATION: NCT05935202/CTIS:2023-503271-24-01. Findings will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT05935202. CTIS:2023-503271-24-01. Registered 07-July-2023. Protocol number: 2.1. https://clinicaltrials.gov/study/NCT05935202.

DAHEAN: A Danish nationwide study ensuring quality assurance through real-world data for suspected hereditary anemia patients.

BACKGROUND: Hereditary anemias are a group of genetic diseases prevalent worldwide and pose a significant health burden on patients and societies. The clinical phenotype of hereditary anemias varies from compensated hemolysis to life-threatening anemia. They can be roughly categorized into three broad categories: hemoglobinopathies, membranopathies, and enzymopathies. Traditional therapeutic approaches like blood transfusions, iron chelation, and splenectomy are witnessing a paradigm shift with the advent of targeted treatments. However, access to these treatments remains limited due to lacking or imprecise diagnoses. The primary objective of the study is to establish accurate diagnoses for patients with hereditary anemias, enabling optimal management. As a secondary objective, the study aims to enhance our diagnostic capabilities. RESULTS: The DAHEAN study is a nationwide cohort study that collects advanced phenotypic and genotypic data from patients suspected of having hereditary anemias from all pediatric and hematological departments in Denmark. The study deliberates monthly by a multidisciplinary anemia board involving experts from across Denmark. So far, fifty-seven patients have been thoroughly evaluated, and several have been given diagnoses not before seen in Denmark. CONCLUSIONS: The DAHEAN study and infrastructure harness recent advancements in diagnostic tools to offer precise diagnoses and improved management strategies for patients with hereditary anemias.

Metabolic blood profile and response to treatment with the pyruvate kinase activator mitapivat in patients with sickle cell disease.

Mitapivat is an investigational, oral, small-molecule allosteric activator of pyruvate kinase (PK). PK is a regulatory glycolytic enzyme that is key in providing the red blood cell (RBC) with sufficient amounts of adenosine triphosphate (ATP). In sickle cell disease (SCD), decreased 2,3-DPG levels increase the oxygen affinity of hemoglobin, thereby preventing deoxygenation and polymerization of sickle hemoglobin. The PK activator mitapivat has been shown to decrease levels of 2,3-DPG and increase levels of ATP in RBCs in patients with SCD. In this phase 2, investigator-initiated, open-label study (https://www.clinicaltrialsregister.eu/ NL8517; EudraCT 2019-003438-18), untargeted metabolomics was used to explore the overall metabolic effects of 8-week treatment with mitapivat in the dose-finding period. In total, 1773 unique metabolites were identified in dried blood spots of whole blood from ten patients with SCD and 42 healthy controls (HCs). The metabolic phenotype of patients with SCD revealed alterations in 139/1773 (7.8%) metabolites at baseline when compared to HCs (false discovery rate-adjusted p < 0.05), including increases of (derivatives of) polyamines, purines, and acyl carnitines. Eight-week treatment with mitapivat in nine patients with SCD altered 85/1773 (4.8%) of the total metabolites and 18/139 (12.9%) of the previously identified altered metabolites in SCD (unadjusted p < 0.05). Effects were observed on a broad spectrum of metabolites and were not limited to glycolytic intermediates. Our results show the relevance of metabolic profiling in SCD, not only to unravel potential pathophysiological pathways and biomarkers in multisystem diseases but also to determine the effect of treatment.

The RoxyScan is a novel measurement of red blood cell deformability under oxidative and shear stress.

Exposure to both oxidative and shear stress, a condition that the red blood cell (RBC) continuously experiences in the circulation in vivo can be mimicked in a Couette type viscometer and monitored by ektacytometry. RBCs maintain their deformation and orientation under shear stress and oxidative stress until a threshold is reached at which these conditions appear to overwhelm the elaborate and complex pathways that maintain a proper redox environment in the cell. Oxidative stress under shear alters the ability of the cell to deform, changes cell morphology, its orientation in the shear stress field, and appears to alter intracellular and membrane characteristics. The application of the RoxyScan technology allows the comparison of oxidant effects and the role of antioxidant systems. This provides the opportunity to study the ability of RBC to deal with oxidative stress in various conditions, including RBC disorders such as sickle cell disease (SCD).

Glucose phosphate isomerase deficiency demasked by whole-genome sequencing: a case report.

BACKGROUND: Glucose-6-phosphate isomerase deficiency is a rare genetic disorder causing hereditary nonspherocytic hemolytic anemia. It is the second most common glycolytic enzymopathy in red blood cells. About 90 cases are reported worldwide, with symptoms including chronic hemolytic anemia, jaundice, splenomegaly, gallstones, cholecystitis, and in severe cases, neurological impairments, hydrops fetalis, and neonatal death. CASE PRESENTATION: This paper details the case of the first Danish patient diagnosed with glucose-6-phosphate isomerase deficiency. The patient, a 27-year-old white female, suffered from lifelong anemia of unknown origin for decades. Diagnosis was established through whole-genome sequencing, which identified two GPI missense variants: the previously documented variant p.(Thr224Met) and a newly discovered variant p.(Tyr341Cys). The pathogenicity of these variants was verified enzymatically. CONCLUSIONS: Whole-genome sequencing stands as a potent tool for identifying hereditary anemias, ensuring optimal management strategies.

Biallelic hexokinase 1 (HK1) variants causative of non-spherocytic haemolytic anaemia: A case series with emphasis on the HK1 promoter variant and literature review.

The hexokinase (HK) enzyme plays a key role in red blood cell energy production. Hereditary non-spherocytic haemolytic anaemia (HNSHA) caused by HK deficiency is a rare disorder with only 12 different disease-associated variants identified. Here, we describe the clinical features and genotypes of four previously unreported patients with hexokinase 1 (HK1)-related HNSHA, yielding two novel truncating HK1 variants. The patients' phenotypes varied from mild chronic haemolytic anaemia to severe infantile-onset transfusion-dependent anaemia. Three of the patients had mild haemolytic disease caused by the common HK1 promoter c.-193A>G variant combined with an intragenic HK1 variant, emphasizing the importance of including this promoter variant in the haemolytic disease gene panels. HK activity was normal in a severely affected patient with a homozygous HK1 c.2599C>T, p.(His867Tyr) variant, but the affinity for ATP was reduced, hampering the HK function. In cases of HNSHA, kinetic studies should be considered in the functional studies of HK. We reviewed the literature of previously published patients to provide better insight into this rare disease and add to the understanding of genotype-phenotype correlation.

A novel composition of endogenous metabolic modulators improves red blood cell properties in sickle cell disease.

The most common forms of sickle cell disease (SCD) are sickle cell anemia (SCA; HbSS) and HbSC disease. In both, especially the more dense, dehydrated and adherent red blood cells (RBCs) with reduced deformability are prone to hemolysis and sickling, and thereby vaso-occlusion. Based on plasma amino acid profiling in SCD, a composition of 10 amino acids and derivatives (RCitNacQCarLKHVS; Axcella Therapeutics, USA), referred to as endogenous metabolic modulators (EMMs), was designed to target RBC metabolism. The effects of ex vivo treatment with the EMM composition on different RBC properties were studied in SCD (n = 9 SCA, n = 5 HbSC disease). Dose-dependent improvements were observed in RBC hydration assessed by hemocytometry (MCV, MCHC, dense RBCs) and osmotic gradient ektacytometry (Ohyper). Median (interquartile range [IQR]) increase in Ohyper compared to vehicle was 4.9% (4.0%-5.5%), 7.5% (6.9%-9.4%), and 12.8% (11.5%-14.0%) with increasing 20×, 40×, and 80X concentrations, respectively (all p < 0.0001). RBC deformability (EImax using oxygen gradient ektacytometry) increased by 8.1% (2.2%-12.1%; p = 0.0012), 9.6% (2.9%-15.1%; p = 0.0013), and 13.3% (5.7%-25.5%; p = 0.0007), respectively. Besides, RBC adhesion to subendothelial laminin decreased by 43% (6%-68%; p = 0.4324), 58% (48%-72%; p = 0.0185), and 71% (49%-82%; p = 0.0016), respectively. Together, these results provide a rationale for further studies with the EMM composition targeting multiple RBC properties in SCD.

Diagnosis and management of pyruvate kinase deficiency: international expert guidelines.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.

Oxygen gradient ektacytometry-derived biomarkers are associated with acute complications in sickle cell disease.

ABSTRACT: We investigated the potential of the point of sickling (PoS; the pO2 tension at which red cells start to sickle), determined by oxygen gradient ektacytometry to serve as a biomarker associated with the incidence of acute sickle cell disease-related complications in 177 children and 50 adults. In the pediatric cohort, for every 10 mmHg increase in PoS reflecting a greater likelihood of sickling, the likelihood of an individual experiencing >1 type of acute complication increased; the adjusted odds ratio (aOR) was 1.65. For every 0.1 increase in minimum elongation index (EImin; reflecting improved red blood cell deformability at hypoxia), the aOR was 0.50. In the adult cohort, for every 10 mmHg increase in PoS, we found an aOR of 3.00, although this was not significant after correcting for multiple testing. There was a trend for an association between higher PoS and greater likelihood of vaso-occlusive episodes (VOEs; children aOR, 1.35; adults aOR, 2.22). In children, only EImin was associated with VOEs (aOR, 0.68). When data of both cohorts were pooled, significant associations with PoS and/or EImin were found for all acute complications, independently and when >1 type of acute complication was assessed. These findings indicate that oxygen gradient ektacytometry generates novel biomarkers and provides a rationale for further development of these biomarkers in the assessment of clinical severity, evaluation of novel therapies, and as surrogate clinical trial end points. These biomarkers may be useful in assessing efficacy of novel therapies like pyruvate kinase activators, voxelotor, and L-glutamine.

One-year safety and efficacy of mitapivat in sickle cell disease: follow-up results of a phase 2, open-label study.

Targeting the primary pathogenic event of sickle cell disease (SCD), the polymerization of sickle hemoglobin (HbS), may prevent downstream clinical events. Mitapivat, an oral pyruvate kinase (PK) activator, has therapeutic potential by increasing adenosine triphosphate (ATP) and decreasing 2,3-diphosphoglycerate (2,3-DPG), a glycolytic red blood cell (RBC) intermediate. In the previously reported 8-week dose-finding period of this phase 2, investigator-initiated, open-label study, mitapivat was well tolerated and showed efficacy in SCD. Here, the 1-year fixed-dose extension period is reported in which 9 of 10 included patients (90%) aged ≥16 years with SCD (HbSS, HbS/ß0, or HbS/ß+) continued with mitapivat. Mostly mild treatment-emergent adverse events (AEs) (most commonly, transaminase increase and headache) were still reported. Apart from the reported nontreatment-related serious AE (SAE) of a urinary tract infection in the dose-finding period, 1 nontreatment-related SAE occurred in the fixed-dose extension period in a patient who died of massive pulmonary embolism due to COVID-19. Importantly, sustained improvement in Hb level (mean increase, 1.1 ± 0.7 g/dL; P = .0014) was seen, which was accompanied by decreases in markers of hemolysis. In addition, the annualized rate of vaso-occlusive events reduced significantly from a historic baseline of 1.33 ± 1.32 to 0.64 ± 0.87 (P = .0489) when combining the dose-finding period and fixed-dose extension period. Cellularly, the ATP:2,3-DPG ratio and Hb-oxygen affinity significantly increased and RBC sickling (point of sickling) nonsignificantly reduced. Overall, this study demonstrated 1-year safety and efficacy of treatment with mitapivat in SCD, supporting further evaluation in ongoing phase 2/3 study (RISE UP, NCT05031780). This trial was registered at https://www.clinicaltrialsregister.eu/ as NL8517 and EudraCT 2019-003438-18.

A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia.

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.

Characterization of genetic variants in the EGLN1/PHD2 gene identified in a European collection of patients with erythrocytosis.

Hereditary erythrocytosis is a rare hematologic disorder characterized by an excess of red blood cell production. Here we describe a European collaborative study involving a collection of 2,160 patients with erythrocytosis sequenced in ten different laboratories. We focused our study on the EGLN1 gene and identified 39 germline missense variants including one gene deletion in 47 probands. EGLN1 encodes the PHD2 prolyl 4-hydroxylase, a major inhibitor of hypoxia-inducible factor. We performed a comprehensive study to evaluate the causal role of the identified PHD2 variants: (i) in silico studies of localization, conservation, and deleterious effects; (ii) analysis of hematologic parameters of carriers identified in the UK Biobank; (iii) functional studies of the protein activity and stability; and (iv) a comprehensive study of PHD2 splicing. Altogether, these studies allowed the classification of 16 pathogenic or likely pathogenic mutants in a total of 48 patients and relatives. The in silico studies extended to the variants described in the literature showed that a minority of PHD2 variants can be classified as pathogenic (36/96), without any differences from the variants of unknown significance regarding the severity of the developed disease (hematologic parameters and complications). Here, we demonstrated the great value of federating laboratories working on such rare disorders in order to implement the criteria required for genetic classification, a strategy that should be extended to all hereditary hematologic diseases.

Effect of Cell Age and Membrane Rigidity on Red Blood Cell Shape in Capillary Flow.

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity.

In-vitro and in-silico evidence for oxidative stress as drivers for RDW.

Red blood cell distribution width (RDW) is a biomarker associated with a variety of clinical outcomes. While anemia and subclinical inflammation have been posed as underlying pathophysiology, it is unclear what mechanisms underlie these assocations. Hence, we aimed to unravel the mechanisms in silico using a large clinical dataset and validate our findings in vitro. We retrieved complete blood counts (CBC) from 1,403,663 measurements from the Utrecht Patient Oriented Database, to model RDW using gradient boosting regression. We performed (sex-stratified) analyses in patients with anemia, patients younger/older than 50 and validation across platforms and care settings. We then validated our hypothesis regarding oxidative stress using an in vitro approach. Only percentage microcytic (pMIC) and macrocytic (pMAC) erythrocytes and mean corpuscular volume were most important in modelling RDW (RMSE = 0.40, R2 = 0.96). Subgroup analyses and validation confirmed our findings. In vitro induction of oxidative stress underscored our results, namely increased RDW and decreased erythrocyte volume, yet no vesiculation was observed. We found that erythrocyte size, especially pMIC, is most informative in predicting RDW, but no role for anemia or inflammation. Oxidative stress affecting the size of the erythrocytes may play a role in the association between RDW and clinical outcomes.

Activation of pyruvate kinase as therapeutic option for rare hemolytic anemias: Shedding new light on an old enzyme.

Novel developments in therapies for various hereditary hemolytic anemias reflect the pivotal role of pyruvate kinase (PK), a key enzyme of glycolysis, in red blood cell (RBC) health. Without PK catalyzing one of the final steps of the Embden-Meyerhof pathway, there is no net yield of adenosine triphosphate (ATP) during glycolysis, the sole source of energy production required for proper RBC function and survival. In hereditary hemolytic anemias, RBC health is compromised and therefore lifespan is shortened. Although our knowledge on glycolysis in general and PK function in particular is solid, recent advances in genetic, molecular, biochemical, and metabolic aspects of hereditary anemias have improved our understanding of these diseases. These advances provide a rationale for targeting PK as therapeutic option in hereditary hemolytic anemias other than PK deficiency. This review summarizes the knowledge, rationale, (pre)clinical trials, and future advances of PK activators for this important group of rare diseases.