Vesiculation in irradiated and cation-leaky-stored red blood cells.

BACKGROUND: Extracellular vesicles (EVs) are released by red blood cells (RBCs) throughout their life-span and also during hypothermic storage when they accumulate in the blood bag. We queried whether stored RBCs with increased cation permeability, either from donors with familial pseudohyperkalaemia (FP) or caused by irradiation, vesiculate more readily. STUDY DESIGN AND METHODS: Recent technical advances have revealed at least two sub-populations of MVs in RBC storage units: macrovesicles (2-6 µm) and microvesicles (1-2 µm). Using nanoparticle tracking analysis, imaging flow cytometry, and protein quantification methods, we measured and characterized vesicles released by RBCs from control and FP individuals at three different storage time-points (day 4, day 17, and day 29). The RBCs had either been stored untreated or irradiated on either day 1 or day 14 of storage. RESULTS: We found no difference in the number or size of vesicles released between cation-leaky FP RBCs and non-FP controls. Similarly, irradiated and non-irradiated RBCs showed very similar patterns of vesicle release to during cold-storage. The only significant difference in vesicle release was the increase in accumulated vesicles with length of storage time which has been reported previously. DISCUSSION: EVs in stored blood are potential contributors to adverse transfusion reactions. The number of vesicles released during 35-day hypothermic storage varies between donors and increases with storage duration. However, increased cation permeability and irradiation do not appear to affect vesicle formation during RBC cold-storage.

The compound effect of irradiation and familial pseudohyperkalemia on potassium leak from red blood cells.

BACKGROUND: Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non-FP RBC. STUDY DESIGN: Five FP and 10 non-FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non-FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18-24°C, leucocyte-depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non-irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. RESULTS: As expected, non-irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post-irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post-irradiation). No other parameters studied showed a significant difference between FP and control. DISCUSSION: FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.

Reticulocyte Maturation and Variant Red Blood Cells.

The bone marrow produces billions of reticulocytes daily. These reticulocytes mature into red blood cells by reducing their plasma membrane by 20% and ejecting or degrading residual internal organelles, membranes and proteins not required by the mature cell. This process occurs by autophagy, protein degradation and vesiculation but is not well understood. We previously reported that Southeast Asian Ovalocytic RBCs demonstrate incomplete reticulocyte maturation and we have now extended this study to a number of other variant RBCs. By comparing the profile of a pure reticulocyte preparation of cultured red cells with these variant cells, we show that the largest of these cells, the overhydrated hereditary stomatocytosis cells, are the least mature, they barely reduced their plasma membrane and contain large amounts of proteins that should have been reduced or removed. Intermediate sized variant RBCs appear to be more mature but retain some endoplasmic reticulum and residual membrane proteins. We propose that the size and composition of these variant cell types correlate with the different stages of reticulocyte maturation and provide insight into the reticulocyte maturation process.

Reticulocyte Maturation.

Changes to the membrane proteins and rearrangement of the cytoskeleton must occur for a reticulocyte to mature into a red blood cell (RBC). Different mechanisms of reticulocyte maturation have been proposed to reduce the size and volume of the reticulocyte plasma membrane and to eliminate residual organelles. Lysosomal protein degradation, exosome release, autophagy and the extrusion of large autophagic-endocytic hybrid vesicles have been shown to contribute to reticulocyte maturation. These processes may occur simultaneously or perhaps sequentially. Reticulocyte maturation is incompletely understood and requires further investigation. RBCs with membrane defects or cation leak disorders caused by genetic variants offer an insight into reticulocyte maturation as they present characteristics of incomplete maturation. In this review, we compare the structure of the mature RBC membrane with that of the reticulocyte. We discuss the mechanisms of reticulocyte maturation with a focus on incomplete reticulocyte maturation in red cell variants.

Familial pseudohyperkalemia induces significantly higher levels of extracellular potassium in early storage of red cell concentrates without affecting other standard measures of quality: A case control and allele frequency study.

BACKGROUND: Familial pseudohyperkalemia (FP) is characterized by an increased rate of potassium leakage in refrigerated red cells and is associated with the minor allele of the single nucleotide polymorphism rs148211042 (R723Q) in the ABCB6 gene. The study aims were to obtain the minor allele frequencies of ABCB6 variants and to measure supernatant potassium accumulation, and other red cell storage parameters, in red cell concentrates (RCC) from carriers of variant rs148211042 under standard blood bank conditions. STUDY DESIGN: Whole blood units were collected from 6 FP individuals and 11 controls and processed into RCC in additive solution. RCC were sampled and tested over cold storage for full blood count, extracellular potassium, glucose, lactate, microvesicle release, deformability, hemolysis, pH, adenosine triphosphate, and 2,3-diphosphoglycerate. RESULTS: Screening of genotyped cohorts identified that variant rs148211042 is present in 1 in 394 British citizens of European ancestry. FP RCC had significantly higher supernatant potassium at all time points from day 3 onwards (p < .001) and higher mean cell volume (p = .032) than controls. The initial rate of potassium release was higher in FP RCC; supernatant potassium reached 46.0 (23.8-57.6) mmol/L (mean [range]) by day 5, increasing to 68.9 (58.8-73.7) mmol/L by day 35. Other quality parameters were not significantly different between FP RCC and controls. CONCLUSION: These data suggest that if a blood donor has FP, reducing the RCC shelf-life to 5 days may be insufficient to reduce the risk of hyperkalemia in clinical scenarios such as neonatal large volume transfusion.

Homozygous Southeast Asian Ovalocytosis in five live-born neonates.

Not available.

Expression of South East Asian Ovalocytic Band 3 Disrupts Erythroblast Cytokinesis and Reticulocyte Maturation.

Southeast Asian Ovalocytosis results from a heterozygous deletion of 9 amino acids in the erythrocyte anion exchange protein AE1 (band 3). The report of the first successful birth of an individual homozygous for this mutation showed an association with severe dyserythropoietic anemia. Imaging of the proband's erythrocytes revealed the presence of band 3 at their surface, a reduction in Wr(b) antigen expression, and increases in glycophorin C, CD44, and CD147 immunoreactivity. Immunoblotting of membranes from heterozygous Southeast Asian Ovalocytosis red cells showed a quantitative increase in CD44, CD147, and calreticulin suggesting a defect in reticulocyte maturation, as well as an increase in phosphorylation at residue Tyr359 of band 3, and peroxiredoxin-2 at the membrane, suggesting altered band 3 trafficking and oxidative stress, respectively. In vitro culture of homozygous and heterozygous Southeast Asian Ovalocytosis erythroid progenitor cells produced bi- and multi-nucleated cells. Enucleation was severely impaired in the homozygous cells and reduced in the heterozygous cells. Large internal vesicular accumulations of band 3 formed, which co-localized with other plasma membrane proteins and with the autophagosome marker, LC3, but not with ER, Golgi or recycling endosome markers. Immunoprecipitation of band 3 from erythroblast cell lysates at the orthochromatic stage showed increased interaction of the mutant band 3 with heat shock proteins, ubiquitin and cytoskeleton proteins, ankyrin, spectrin and actin. We also found that the mutant band 3 forms a strong interaction with non-muscle myosins IIA and IIB, while this interaction could not be detected in wild type erythroblasts. Consistent with this, the localization of non-muscle myosin IIA and actin was perturbed in some Southeast Asian Ovalocytosis erythroblasts. These findings provide new insights toward understanding in vivo dyserythropoiesis caused by the expression of mutant membrane proteins.

The Molecular Basis for Altered Cation Permeability in Hereditary Stomatocytic Human Red Blood Cells.

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type.

Band 3 nullVIENNA , a novel homozygous SLC4A1 p.Ser477X variant causing severe hemolytic anemia, dyserythropoiesis and complete distal renal tubular acidosis.

We describe the second patient with anionic exchanger 1/band 3 null phenotype (band 3 nullVIENNA ), which was caused by a novel nonsense mutation c.1430C>A (p.Ser477X) in exon 12 of SLC4A1. We also update on the previous band 3 nullCOIMBRA patient, thereby elucidating the physiological implications of total loss of AE1/band 3. Besides transfusion-dependent severe hemolytic anemia and complete distal renal tubular acidosis, dyserythropoiesis was identified in the band 3 nullVIENNA patient, suggesting a role for band 3 in erythropoiesis. Moreover, we also, for the first time, report that long-term survival is possible in band 3 null patients.

The involvement of cation leaks in the storage lesion of red blood cells.

Stored blood components are a critical life-saving tool provided to patients by health services worldwide. Red cells may be stored for up to 42 days, allowing for efficient blood bank inventory management, but with prolonged storage comes an unwanted side-effect known as the "storage lesion", which has been implicated in poorer patient outcomes. This lesion is comprised of a number of processes that are inter-dependent. Metabolic changes include a reduction in glycolysis and ATP production after the first week of storage. This leads to an accumulation of lactate and drop in pH. Longer term damage may be done by the consequent reduction in anti-oxidant enzymes, which contributes to protein and lipid oxidation via reactive oxygen species. The oxidative damage to the cytoskeleton and membrane is involved in increased vesiculation and loss of cation gradients across the membrane. The irreversible damage caused by extensive membrane loss via vesiculation alongside dehydration is likely to result in immediate splenic sequestration of these dense, spherocytic cells. Although often overlooked in the literature, the loss of the cation gradient in stored cells will be considered in more depth in this review as well as the possible effects it may have on other elements of the storage lesion. It has now become clear that blood donors can exhibit quite large variations in the properties of their red cells, including microvesicle production and the rate of cation leak. The implications for the quality of stored red cells from such donors is discussed.

Familial pseudohyperkalemia in blood donors: a novel mutation with implications for transfusion practice.

BACKGROUND: Familial pseudohyperkalemia (FP) is a dominantly inherited condition in which red blood cells (RBCs) have an increased cold-induced permeability to monovalent cations. Potassium leaks into the supernatant of all stored blood with time, but FP RBCs leak potassium more rapidly. We investigated two unrelated blood donors whose RBC donations demonstrated unexpectedly high potassium after 5 and 6 days' storage. We matched the observed pattern of RBC cation leak to a previously recognized family with FP (FP-Cardiff) and investigated the likely cause with targeted DNA analysis. STUDY DESIGN AND METHODS: Cation leakage from the donor RBCs and from standard donor units was measured. DNA analysis of donors and family members with FP-Cardiff was performed. Allele frequencies were obtained from human variation databases. RESULTS: Both implicated donors were found to have increased cold-induced potassium leak identical in pattern to affected members of the family with FP-Cardiff. We found a heterozygous substitution Arg723Gln in the ATP-binding cassette, Subfamily B, Member 6 protein that segregated with FP in the Cardiff family and was also present in both blood donors. Arg723Gln is listed in human variation databases with an allele frequency of approximately 1:1000. CONCLUSIONS: We describe a novel FP mutation that may affect 1:500 European blood donors and causes rapid loss of potassium from stored RBCs. This finding has implications for neonates and infants receiving large-volume RBC transfusions. Genomic screening of donors could be used to identify donors with this mutation and potentially improve the quality and safety of donor units.

Distal renal tubular acidosis with hereditary spherocytosis.

Hereditary spherocytosis (HS) and distal renal tubular acidosis (dRTA), although distinct entities, share the same protein i.e. the anion exchanger1 (AE1) protein. Despite this, their coexistence has been rarely reported. We hereby describe the largest family to date with co-existence of dRTA and HS and discuss the molecular basis for the co-inheritance of these conditions.

Study of the D-- phenotype reveals erythrocyte membrane alterations in the absence of RHCE.

Red cells with the D-- phenotype do not express the RHCE protein because of mutations in both alleles of the RHCE gene. At present, little is known of the effect this has on the normal function of erythrocytes. In this study a group of five families belonging to a nomadic tribe in Malaysia were identified as carriers of the D-- haplotype. Analysis of homozygous individuals' genomic DNA showed two separate novel mutations. In four of the families, RHCE exons 1, 9 and 10 were present, while the 5th family possessed RHCE exons 1-3 and 10. Analysis of cDNA revealed hybrid transcripts, suggesting a gene conversion event with RHD, consistent with previously reported D-- mutations. Immunoblotting analysis of D-- erythrocyte membrane proteins found that Rh-associated glycoprotein (RHAG) migrates with altered electrophoretic mobility on sodium dodecyl sulphate polyacrylamide gel electrophoresis, consistent with increased glycosylation. Total amounts of Rh polypeptide in D-- membranes were comparable with controls, indicating that the exalted D antigen displayed by D-- red cells may be associated with altered surface epitope presentation. The adhesion molecules CD44 and CD47 are significantly reduced in D--. Together these results suggest that absence of RHCE polypeptide alters the structure and packing of the band 3/Rh macrocomplex.

An infant with pseudohyperkalemia, hemolysis, and seizures: cation-leaky GLUT1-deficiency syndrome due to a SLC2A1 mutation.

CONTEXT: GLUT1 (glucose transporter 1) deficiency syndrome is a well-known presentation in pediatric practice. Very rare mutations not only disable carbohydrate transport but also cause the red cell membrane to be constitutively permeant to monovalent cations, namely sodium and potassium. OBJECTIVE: The aim of this study was to describe the pediatric presentation of a patient with GLUT1 deficiency with such a cation-leaky state. SUBJECT AND METHODS: The infant presented with erratic hyperkalemia, neonatal hyperbilirubinemia, anemia, hepatic dysfunction, and microcephaly. Later, seizures occurred and developmental milestones were delayed. Magnetic resonance imaging and computerized tomography scans of the brain showed multiple abnormalities including periventricular calcification. Visual impairment was present due to the presence of both cataracts and retinal dysfunction. RESULTS: Measurements of red cell cation content showed extremely leaky red cells (causing the hemolysis) and temperature-dependent loss of potassium from red cells (explaining the hyperkalemia as pseudohyperkalemia). A trinucleotide deletion in SLC2A1, coding for the deletion of isoleucine 435 or 436 in GLUT1, was identified in the proband. CONCLUSION: This is the fourth pedigree to be described with this most unusual syndrome. The multisystem pathology probably reflects a combination of glucose transport deficiency at the blood-brain barrier (as in typical GLUT1 deficiency) and the deleterious osmotic effects of a cation-leaky membrane protein in the cells where GLUT1 is expressed, notably the red cell. We hope that this detailed description will facilitate rapid diagnosis of this disease entity.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome.

The hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.