Iron deficiency anemia is one of the most common diseases worldwide. In the majority of cases, the presence of hypochromic microcytic anemia and biochemical evidence for depletion of body iron stores ...makes the diagnosis relatively straightforward. However, in several clinical conditions, classic biochemical indices such as serum iron, transferrin saturation, and ferritin may not be informative or may not change rapidly enough to reflect transient iron-deficient states (functional iron deficiency), such as the ones that develop during recombinant human erythropoietin (r-HuEPO) therapy. The identification and treatment of iron deficiency in settings such as r-HuEPO therapy, anemia of chronic disease, and iron deficiency of early childhood may be improved by the use of red cell and reticulocyte cellular indices, which reflect in almost real time the development of iron deficiency and the response to iron therapy. In the anemia of chronic disease, measurements of plasma cytokines and iron metabolism regulators such as hepcidin (when available) may be helpful in the characterization of the pathophysiologic basis of this condition. The ratio of serum transferrin receptor (sTfR) to serum ferritin (R/F ratio) has been shown to have excellent performance in estimating body iron stores, but it cannot be used widely because of the lack of standardization for sTfR assays. The combination of hematologic markers such as reticulocyte hemoglobin content, which decreases with iron deficiency, and R/F ratio may allow for a more precise classification of anemias.
Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal ...hemoglobin (HbF, α
γ
)
. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells
. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.
The reticulocyte count reflects the erythropoietic activity of bone marrow and is thus useful in both diagnosing anemias and monitoring bone marrow response to therapy. Automated flow-cytometric ...analysis has led to a significant advance in reticulocyte counting, by simultaneously providing additional parameters and indices such as the reticulocyte immature reticulocyte fraction (IRF), the reticulocyte volume, and the hemoglobin content and concentration. IRF has been proposed as an early marker of engraftment. Reticulocyte hemoglobin content is useful in assessing the functional iron available for erythropoiesis, and reticulocyte volume is a useful indicator when monitoring the therapeutic response of anemias.
The structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of ...Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.
Maintaining a positive iron balance is essential for female athletes to avoid the effects of iron deficiency and anaemia and to maintain or improve performance. A major function of iron is in the ...production of the oxygen and carbon dioxide carrying molecule, haemoglobin, via erythropoiesis. Iron balance is under the control of a number of factors including the peptide hormone hepcidin, dietary iron intake and absorption, environmental stressors (e.g. altitude), exercise, menstrual blood loss and genetics. Menstruating females, particularly those with heavy menstrual bleeding are at an elevated risk of iron deficiency. Haemoglobin concentration Hb and serum ferritin (sFer) are traditionally used to identify iron deficiency, however, in isolation these may have limited value in athletes due to: (1) the effects of fluctuations in plasma volume in response to training or the environment on Hb, (2) the influence of inflammation on sFer and (3) the absence of sport, gender and individually specific normative data. A more detailed and longitudinal examination of haematology, menstrual cycle pattern, biochemistry, exercise physiology, environmental factors and training load can offer a superior characterisation of iron status and help to direct appropriate interventions that will avoid iron deficiency or iron overload. Supplementation is often required in iron deficiency; however, nutritional strategies to increase iron intake, rest and descent from altitude can also be effective and will help to prevent future iron deficient episodes. In severe cases or where there is a time-critical need, such as major championships, iron injections may be appropriate.
Hereditary stomatocytoses (HSts) are a wide spectrum of hemolytic anemias in which the erythrocyte membrane cation permeability is increased. Dehydrated hereditary stomatocytosis is the most frequent ...among HSts. It is caused by missense mutations in PIEZO1 and KCNN4 genes.
We described 123 patients enrolled in our Genetic Unit from 2013 to 2017. Overall HSt subjects exhibit macrocytic mild anemia. We found that PIEZO1 is the most frequent mutated gene within our families (47% of pedigrees). In 59.1% of cases the mutations localized in the nonpore protein domain, while in 40.9% of patients they localized in the central pore region. The genotype‐phenotype correlation analysis on 29 PIEZO1‐patients demonstrated that most of severely affected patients carried mutations in the pore domain, suggesting that the severity of this condition is related to the pore properties and intracellular domain that could be responsible of interactions with intracellular components.
This is the first cohort study on a large set of hereditary stomatocytosis patients, stratified according to their causative gene useful for diagnosis, prognosis, and management of these patients.
Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a ...pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34+ cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.
•Dehydrated hereditary stomatocytosis is characterized by abnormal RBC morphology but may involve pseudohyperkalemia and perinatal edema.•This syndrome is associated with germline mutations in PIEZO1, encoding a transmembrane protein that induces mechanosensitive currents.
Significance Red blood cells with a high density (ρ > 1.120 g/cm ³) are characteristic of sickle cell disease. This paper demonstrates a density-based separation of red blood cells in a system of ...aqueous multiphase polymers that enables a visual test that identifies sickle cell disease, starting from samples of whole blood, in less than 12 min. This low-cost, simple test could provide a means to enable diagnosis of sickle cell disease in low-resource settings and enable life-saving interventions for children with the disease. The method itself provides a demonstration of the use of a biophysical indicator (here, density) rather than a biochemical marker (e.g., proteins separated by gel electrophoresis) as a means to do point-of-care hematology.
Although effective low-cost interventions exist, child mortality attributable to sickle cell disease (SCD) remains high in low-resource areas due, in large part, to the lack of accessible diagnostic methods. The presence of dense (ρ > 1.120 g/cm ³) cells is characteristic of SCD. The fluid, self-assembling step-gradients in density created by aqueous multiphase systems (AMPSs) identifies SCD by detecting dense cells. AMPSs separate different forms of red blood cells by density in a microhematocrit centrifuge and provide a visual means to distinguish individuals with SCD from those with normal hemoglobin or with nondisease, sickle-cell trait in under 12 min. Visual evaluation of a simple two-phase system identified the two main subclasses of SCD homozygous (Hb SS) and heterozygous (Hb SC) with a sensitivity of 90% (73–98%) and a specificity of 97% (86–100%). A three-phase system identified these two types of SCD with a sensitivity of 91% (78–98%) and a specificity of 88% (74–98%). This system could also distinguish between Hb SS and Hb SC. To the authors’ knowledge, this test demonstrates the first separation of cells by density with AMPSs, and the usefulness of AMPSs in point-of-care diagnostic hematology.
FOXP3 deficiency in mice and in patients with immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome results in fatal autoimmunity by altering regulatory T (T
) cells. CD4
T ...cells in patients with IPEX syndrome and Foxp3-deficient mice were analyzed by single-cell cytometry and RNA-sequencing, revealing heterogeneous T
-like cells, some very similar to normal T
cells, others more distant. Conventional T cells showed no widespread activation or helper T cell bias, but a monomorphic disease signature affected all CD4
T cells. This signature proved to be cell extrinsic since it was extinguished in mixed bone marrow chimeric mice and heterozygous mothers of patients with IPEX syndrome. Normal T
cells exerted dominant suppression, quenching the disease signature and revealing in mutant T
-like cells a small cluster of genes regulated cell-intrinsically by FOXP3, including key homeostatic regulators. We propose a two-step pathogenesis model: cell-intrinsic downregulation of core FOXP3-dependent genes destabilizes T
cells, de-repressing systemic mediators that imprint the disease signature on all T cells, furthering T
cell dysfunction. Accordingly, interleukin-2 treatment improved the T
-like compartment and survival.
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