Density, heterogeneity and deformability of red cells as markers of clinical severity in hereditary spherocytosis.

Hereditary spherocytosis (HS) originates from defective anchoring of the cytoskeletal network to the transmembrane protein complexes of the red blood cell (RBC). Red cells in HS are characterized by membrane instability and reduced deformability and there is marked heterogeneity in disease severity among patients. To unravel this variability in disease severity, we analyzed blood samples from 21 HS patients with defects in ankyrin, band 3, α-spectrin or ß-spectrin using red cell indices, eosin-5-maleimide binding, microscopy, the osmotic fragility test, Percoll density gradients, vesiculation and ektacytometry to assess cell membrane stability, cellular density and deformability. Reticulocyte counts, CD71 abundance, band 4.1 a:b ratio, and glycated hemoglobin were used as markers of RBC turnover. We observed that patients with moderate/severe spherocytosis have short-living erythrocytes of low density and abnormally high intercellular heterogeneity. These cells show a prominent decrease in membrane stability and deformability and, as a consequence, are quickly removed from the circulation by the spleen. In contrast, in mild spherocytosis less pronounced reduction in deformability results in prolonged RBC lifespan and, hence, cells are subject to progressive loss of membrane. RBC from patients with mild spherocytosis thus become denser before they are taken up by the spleen. Based on our findings, we conclude that RBC membrane loss, cellular heterogeneity and density are strong markers of clinical severity in spherocytosis.

A Previously Unrecognized Ca2+-inhibited Nonselective Cation Channel in Red Blood Cells.

Supplemental Digital Content is available in the text.

Is Increased Intracellular Calcium in Red Blood Cells a Common Component in the Molecular Mechanism Causing Anemia?

For many hereditary disorders, although the underlying genetic mutation may be known, the molecular mechanism leading to hemolytic anemia is still unclear and needs further investigation. Previous studies revealed an increased intracellular Ca2+ in red blood cells (RBCs) from patients with sickle cell disease, thalassemia, or Gardos channelopathy. Therefore we analyzed RBCs' Ca2+ content from 35 patients with different types of anemia (16 patients with hereditary spherocytosis, 11 patients with hereditary xerocytosis, 5 patients with enzymopathies, and 3 patients with hemolytic anemia of unknown cause). Intracellular Ca2+ in RBCs was measured by fluorescence microscopy using the fluorescent Ca2+ indicator Fluo-4 and subsequent single cell analysis. We found that in RBCs from patients with hereditary spherocytosis and hereditary xerocytosis the intracellular Ca2+ levels were significantly increased compared to healthy control samples. For enzymopathies and hemolytic anemia of unknown cause the intracellular Ca2+ levels in RBCs were not significantly different. These results lead us to the hypothesis that increased Ca2+ levels in RBCs are a shared component in the mechanism causing an accelerated clearance of RBCs from the blood stream in channelopathies such as hereditary xerocytosis and in diseases involving defects of cytoskeletal components like hereditary spherocytosis. Future drug developments should benefit from targeting Ca2+ entry mediating molecular players leading to better therapies for patients.

Déficit de piruvato cinasa eritrocitaria en España: estudio de 15 casos / Red cell pyruvate kinase deficiency in Spain: A study of 15 cases

Antecedentes y objetivo: El déficit de piruvato cinasa (DPK) es una enfermedad hereditaria rara, que cursa con hemólisis crónica y anemia de intensidad variable. Su heterogeneidad genética es elevada, habiéndose descrito unas 240 mutaciones diferentes. Pacientes y metodología: Se han estudiado 15 pacientes con DPK en los que se ha secuenciado la totalidad del gen PKLR, incluyendo las regiones promotora, exónicas, intrónicas flanqueantes y 3’UTR. Resultados: Según la intensidad del cuadro clínico, los pacientes se han clasificado en 3 grandes grupos: I) grave y muy grave (8 pacientes); II) moderado (2 pacientes), y III) leve (5 pacientes). Se han identificado 18 alelos diferentes, de los que 6 son mutaciones nuevas, no descritas con anterioridad, siendo la mutación PKLRc.721G > T la más prevalente (26,67%), seguida de la mutación PKLR c.1456C > T (13,33%). Trece de los 15 pacientes mostraron un genotipo doble heterocigoto y 2 homocigoto. Conclusiones: En España, la heterogeneidad del patrón genético de la PKLR continúa siendo elevada, aunque algo diferente a la observada en un estudio anterior (1998). Se concluye que la secuenciación total del gen PKLR es imprescindible tanto para la caracterización de los pacientes como para la realización del consejo genético (AU)

Background and objective: Pyruvate kinase deficiency (PKD) is a rare, inherited disease causing chronic hemolysis and anemia of varying intensity. The genetic heterogeneity of PKD is high and, to this day, over 240 different mutations have been identified. Patients and methods: 15 unrelated patients affected by PKD have been studied. PKLR gene sequencing was performed by SANGER, including the determination of promoter regions, exonic, intronic flanking and 3’UTR. Results: Patients were classified into 3 groups based on the intensity of their clinical symptoms: I) severe and very severe (8 patients); II) moderate (2 patients), and III) mild (5 patients). Six out of the 18 alleles found were new mutations which had not been described previously, with the PKLR c.721G>T mutation being the most prevalent (26.67%), followed by the PKLR c.1456C>T mutation (13.33%). Conclusions: In Spain, the genetic heterogeneity of PKLR is still high but differs from that observed in the previous study carried out in 1998. Total PKLR gene sequencing is necessary for the characterization of all patients with PKD and for genetic counseling (AU)

Red cell pyruvate kinase deficiency in Spain: A study of 15 cases. / Déficit de piruvato cinasa eritrocitaria en España: estudio de 15 casos.

BACKGROUND AND OBJECTIVE: Pyruvate kinase deficiency (PKD) is a rare, inherited disease causing chronic hemolysis and anemia of varying intensity. The genetic heterogeneity of PKD is high and, to this day, over 240 different mutations have been identified. PATIENTS AND METHODS: 15 unrelated patients affected by PKD have been studied. PKLR gene sequencing was performed by SANGER, including the determination of promoter regions, exonic, intronic flanking and 3'UTR. RESULTS: Patients were classified into 3 groups based on the intensity of their clinical symptoms: I) severe and very severe (8 patients); II) moderate (2 patients), and III) mild (5 patients). Six out of the 18 alleles found were new mutations which had not been described previously, with the PKLR c.721G>T mutation being the most prevalent (26.67%), followed by the PKLR c.1456C>T mutation (13.33%). CONCLUSIONS: In Spain, the genetic heterogeneity of PKLR is still high but differs from that observed in the previous study carried out in 1998. Total PKLR gene sequencing is necessary for the characterization of all patients with PKD and for genetic counseling.

Red Cell Properties after Different Modes of Blood Transportation.

Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca(2+) handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na(+), K(+), Ca(2+)) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca(2+) cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca(2+)-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to the specialized laboratories may be the only option for some groups of patients with highly unstable RBCs.