Osmotic Gradient Ektacytometry Research Articles

Next-generation osmotic gradient ektacytometry for the diagnosis of hereditary spherocytosis: interlaboratory method validation and experience.

Osmotic gradient ektacytometry is part of the laboratory diagnosis process of hereditary spherocytosis (HS) and other red blood cell (RBC) membrane disorders. We here present the experience of two independent institutions with a next-generation ektacytometer, the LoRRca MaxSis analyzer, in HS diagnostic settings. Inter- and intra-assay variability and sample stability were analyzed. Samples from patients with HS (n=40), probable HS (n=21), auto-immune hemolytic anemia (n=7), and other pathologies (n=37) were studied. Daily controls were run in parallel with patient samples. Results were expressed as percent of change compared to mean of controls. Analytical performances showed an inter-assay variability between 0.2% and 3%. Samples were stable for 48-72 h depending of temperature storage and anticoagulant used. The following diagnostic cut-offs were established for HS: an increase of more than 21.5% for the osmolality point at the minimal elongation index (O min), a decrease of more than 8.5% for the maximal elongation index (EI max), and a decreased area under the curve (AUC) of more than 18.5% compared to the mean of controls. As the previous instrument, the next-generation ektacytometer is an efficient tool for laboratory diagnosis of HS. Sample stability and standardized reporting of results allow inter-laboratory exchange and comparison. The most useful parameters for HS diagnosis were AUC, EI max, and O min; unfortunately, this method does not differentiate between HS and auto-immune hemolytic anemia (AIHA), but it distinguishes HS from other hereditary membrane pathologies. It can thus be considered as an intermediate step between screening and diagnostic tests.

Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are heterogeneous red blood cell (RBC) membrane disorders that result from mutations in the genes encoding α-spectrin (SPTA1), β-spectrin (SPTB), or protein 4.1R (EPB41). The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. The clinical diagnosis of HE and HPP relies on identifying characteristic RBC morphology on peripheral blood smear and specific membrane biomechanical properties using osmotic gradient ektacytometry. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. Using Next-Generation sequencing, we identified the causative genetic mutations in fifteen patients with clinically suspected HE or HPP and correlated the identified mutations with the clinical phenotype and ektacytometry profile. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP.

The effect of centrifugation at various g force levels on rheological properties of rat, dog, pig and human red blood cells.

Laboratory investigations often require centrifugation of blood samples for various erythrocyte tests. Although there is a lack of data about the effect of centrifugation at various g force levels on erythrocyte rheological properties. We aimed to investigate the effect of a 10-minute centrifugation at 500, 1000 or 1500 g at 15°C of rat, dog, pig and human venous (K3-EDTA, 1.5 mg/ml) blood samples. Hematological parameters, erythrocyte deformability, cell membrane stability, osmotic gradient ektacytometry (osmoscan) and erythrocyte aggregation were determined. Hematological and erythrocyte deformability parameters showed interspecies differences, centrifugation caused no significant alterations. Cell membrane stability for human erythrocytes centrifuged at higher g level showed less decrease in deformability. Osmoscan O min parameter showed slight elevation in dog centrifuged aliquots. Erythrocyte aggregation parameters changed unexpectedly. Rat and dog erythrocyte aggregation indices significantly dropped in centrifuged aliquots. Pig erythrocyte aggregation indices increased significantly after centrifugation. Human erythrocyte aggregation was the most stable one among the investigated species. The used centrifugation protocols caused the largest alterations in erythrocyte aggregation in a controversial way among the investigated species. On the other hand, erythrocyte deformability parameters were stable, cell membrane stability and osmoscan data show minor shifts.

Early hemorheological changes in a porcine model of intravenously given E. coli induced fulminant sepsis.

The pathophysiology of hemorheological and microcirculatory disturbances in septic process -mostly during the early hours- still not clarified in all the details, yet. In anesthetized pigs living E. coli (ATCC 25922 strain) was administered intravenously with an increasing concentration and the animals were observed for 8 hours. Before the intervention and in every 2 hours arterial (cannulated femoral artery) and venous (cannulated external jugular vein) blood samples were collected for hemorheological laboratory tests: blood and plasma viscosity, ESR, leukocyte anti-sedimentation rate, erythrocyte deformability (together with osmoscan parameters) and erythrocyte aggregation (using light-transmission and laser back-scattering methods) Control animals were stable over the 8-hour anesthesia, while septic animals died by the 6th hours in a fulminant sepsis. Over the experimental period, the tendency of impairment in erythrocyte deformability (together with osmotic gradient ektacytometry parameters) and the controversial decreasing of erythrocyte aggregation values (declining all aggregation index values, elongating t1/2) were well detected in this porcine model during the early hours (4- 6) of fulminant sepsis. The in vitro effect of these bacteria on erythrocytes' micro-rheological parameter was similar: decreasing red blood cell deformability and lowering aggregation. Further studies are needed to clarify the early micro-rheological changes of bacteremia and the developing sepsis.

Gardos Channel Mutation Is Associated with Hereditary Dehydrate Stomatocytosis: a Complex Channelopathy

The Gardos channel (KCNN4) is a Ca2+ sensitive, intermediate conductance, K+ selective channel abundant in several tissues including red blood cells (RBC) where it is involved in cell volume regulation (Hoffman et al. 2003, Cahalan et al. 2015).Hereditary dehydrate stomatocytosis (DHSt) is characterized by mild to moderate congenital hemolytic anemia with reticulocytosis and splenomegaly associated with RBC dehydration. Affected RBC are characterized by a nonspecific cation leak, reflected in elevated Na+ content, decreased K+, elevated MCHC and MCV, and decreased osmotic fragility. The definitive diagnosis of DHSt is made by osmotic gradient ektacytometry, which shows a leftward shift of the bell-shaped curve.Heterozygous mutations associated with delayed channel inactivation have been identified for PIEZO1 (Zarychanski et al. 2012), although there are patients lacking a mutation in this gene. Differential diagnosis in hereditary stomatocytosis is important because in DHSt patients, splenectomy is contraindicated due to increased risk of thromboembolic complications.We studied a 40 year-old Italian man, affected since infancy by chronic hemolyitc anemia (Hb 7-9 g/dL, reticulocytes 10%). The unrelated parents and three siblings were healthy with normal hematologic parameters. He was occasionally transfused until splenectomy, performed at the age of 11 years. After splenectomy Hb levels maintained around 10 g/dL, thrombotic events did not occur. Increased ferritin levels (>1000 ng/ml) and moderate iron overload required chelation therapy. Peripheral blood smear showed anysopoikilocytosis with stomatocytes (13%) echinocytes (7%) schistocytes (6%) ellyptocytes (5%) spherocytes (4%). Bone marrow evaluation revealed erythroid hyperplasia with some dyserytrhopoietc changes, in particular binucleated erythroblasts. Eosin 5 maleimide (EMA) binding test was negative; despite the high number of stomatocytes in peripheral blood, the osmotic gradient ektacytometry curve was not suggestive for hereditary xerocytosis.The proband's first daughter, born at term after an uneventful pregnancy, presented with severe anemia (Hb 6.1 g/dL) requiring RBC transfusion at birth and at 4 months; at the age of 1 year she displayed a clinical pattern similar to the one of the father, with median Hb levels of 9 g/dL and no need of transfusions.To uncover the underlying etiologies, all the family members underwent whole exome sequencing. Only one candidate gene, the KCNN4 could be identified: a heterozygous missense mutation (c.1055G>A, p.R352H) was detected as a de novo mutation in the proband, and dominantly transmitted to the daughter. The mutation falling in exon 7 involved in the calmodulin binding domain was predicted to be pathogenic by in silico analysis. Sanger sequencing confirmed the presence of the mutation.Very recently (July 2015), the same mutation in the KCNN4 gene was described by other groups (Rapetti-Mauss et al. 2015; Andolfo et al. 2015). In heterologous expression systems it could be shown that the Ca2+ sensitivity of the mutated Gardos channel was increased.We performed single cell experiments, including patch-clamp recordings in RBCs and precursor cells, Ca2+ fluorescence imaging as well as tracer flux experiments in RBCs of the above described patients and healthy controls to get a mechanistic link between the Gardos channel mutation and the cellular symptoms. Despite the presence of a single mutation in Gardos channel alone, pathological alterations in function of multiple ion transport systems were revealed during these studies.Our findings together with recent reports (Rapetti-Mauss et al. 2015; Andolfo et al. 2015; Glogowska et al. 2015) suggest that the prevalence of mutations in the Gardos channel is higher than previously thought. Therefore it is important to understand the mechanisms implicated by the mutation and to develop reliable diagnostic tools and treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic tool for red blood cell membrane disorders: Assessment of a new generation ektacytometer

Inherited red blood cell (RBC) membrane disorders, such as hereditary spherocytosis, elliptocytosis and hereditary ovalocytosis, result from mutations in genes encoding various RBC membrane and skeletal proteins. The RBC membrane, a composite structure composed of a lipid bilayer linked to a spectrin/actin-based membrane skeleton, confers upon the RBC unique features of deformability and mechanical stability. The disease severity is primarily dependent on the extent of membrane surface area loss. RBC membrane disorders can be readily diagnosed by various laboratory approaches that include RBC cytology, flow cytometry, ektacytometry, electrophoresis of RBC membrane proteins and genetics. The reference technique for diagnosis of RBC membrane disorders is the osmotic gradient ektacytometry. However, in spite of its recognition as the reference technique, this technique is rarely used as a routine diagnosis tool for RBC membrane disorders due to its limited availability. This may soon change as a new generation of ektacytometer has been recently engineered. In this review, we describe the workflow of the samples shipped to our Hematology laboratory for RBC membrane disorder analysis and the data obtained for a large cohort of French patients presenting with RBC membrane disorders using a newly available version of the ektacytomer.

Effect of osmolality on erythrocyte rheology and perfusion of an artificial microvascular network

Plasma sodium concentration is normally held within a narrow range. It may however vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5g/dL), resulting in supernatant osmolalities of 179±4, 213±1, 283±2, 354±3, and 423±5mOsm/kg H2O. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5s−1 and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted toward hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290mOsm/kg H2O and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease and may have significant implications for the management of osmotic homeostasis in clinical practice.

Interpretation of osmotic gradient ektacytometry (osmoscan) data: A comparative study for methodological standards

Osmotic gradient ektacytometry (measuring elongation index in the function of osmolality at a constant shear stress) is a sensitive method to analyze red blood cell (RBC) deformability and investigating the optimal osmolality range for the cells in normal or pathophysiological cellular and micro-environmental conditions. However, the methodological conditions are different, since the results are influenced by the applied shear stress (SS). In this study we investigated rat, dog, pig and human blood samples at SS of 1, 2, 3, 5, 10, 20 and 30 Pa. To describe the range being related to the cell deformability, we introduced new calculated parameters obtained from the raw data of the elongation index (EI)-osmolality (O) curves. Our results showed that: (1) Osmoscan data tested at 20 or 30 Pa do not differ significantly from each other; (2) Under SS of 20 Pa the EImax, the O (EImax), the EI min and the area under curve nearly linearly decrease in the function of SS with different slope in rat, dog, pig and human blood; (3) Measurements under 3 Pa SS become unstable; (4) The differences between minimal and maximal EI and the belonging osmolality values, and their ratios, as new calculated parameters (ΔEI, ΔO, ΔEI/ΔO, EImax/EImin and O (EImax)/Omin) can be suitable for further analysis of the osmoscan curves together with other hemorheological parameters describing RBC deformability; and (5) Decreased erythrocyte deformability (by rigidifying with glutaraldehyde) can be reflected well with the following, calculated osmoscan parameters: ΔO, rO, rEI/rO and ΔEI/ΔO.

Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency.

The cholesterol content in erythrocytes expressing Miltenberger antigen subtype III (Mi.III) has limited effects on membrane deformability (LB161)

The Miltenberger antigen subtype III (Mi.III, GP.Mur), is one of the most important red blood cell (RBC) antigens in Southeast Asia. GP.Mur is a glycophorin B‐A‐B fusion protein evolved from homologous recombination of glycophorin A and glycophorin B. We previously showed that GP.Mur in Mi.III+ cells enhances the expression of band 3, consequently facilitating Cl‐/HCO3‐ transport, CO2 respiration, and membrane resistance to osmotic stress. The aim of this study was to further investigate if the unique expressions of GP.Mur and band 3 in Mi.III also influenced cell membrane deformability. The methods utilized were osmotic gradient ektacytometry, osmotic fragility tests and Amplex Red Cholesterol assay. A total of 114 fresh RBC samples, including 50 Mi.III+ and 64 non‐Mi.III (control), were evaluated. From osmolarity fragility tests, Mi.III+ RBCs consistently exhibited superior resistance to osmotic stress. Mi.III+ RBCs however were less deformable than the control cells. The discordance between the results of membrane deformability and membrane's resistance to osmotic stress suggests that the higher band 3 expression on the Mi.III membrane likely affect the physical properties of the membrane through higher densities of band 3 interactions with adjacent lipids and cytoskeletal proteins. We also only observed the rigidifying effects of cholesterol on membrane deformability in the control cells, and not in Mi.III. We thus conclude that in contrast to the non‐Miltenberger RBCs, the cholesterol content of Mi.III+ cells has relatively limited effects on membrane deformability, presumably due to the higher band 3 levels on the Mi.III membrane.Grant Funding Source: Supported by Taiwan National Health Research Institute, NHRI‐EX102‐10122SI

Comparative osmotic gradient ektacytometry data on inter-species differences of experimental animals

It is known that red blood cell deformability may show colorful inter-species differences, influenced by inner viscosity, cell membrane viscosity, morphology and surface-volume ratio of the erythrocytes. It is also well-known that the cell volume is changing depending on the micro-environmental osmolarity. These changes can be well observable using osmotic gradient ektacytometry (osmoscan). Interestingly, there is a lack of base and comparative osmoscan data regarding the experimental/laboratory animal species. In this study mouse, rat, canine and porcine blood samples were analyzed using a LoRRca MaxSis Osmoscan ektacytometer. The highest elongation index values were found in mouse, typically above shear stress of 1 Pa. Some lower values than these were shown in rat and more lower in canine, while the lowest values were detected in porcine, typically above 3-5 Pa. The optimal osmolarity point value, so the measurable maximal EI osmolarity was in a wide range among the species. While the lowest values were detected in canine, the highest ones in porcine and mouse, the rat values were in between. Further analysis and wider comparison of the osmotic gradient ektacytometry may contribute to the better understanding of the erythrocyte micro-rheological properties, their induced changes and inter-species differences.

A case of hereditary pyropoïkilocytosis with mild expression and delayed onset

We report on a case of hereditary pyropoïkilocytosis fortuitously diagnosed in a 34-year old woman issued from Benin. Laboratory tests indicated a moderate haemolytic anaemia with a marked microcytosis. Blood film examination revealed a striking anisopoikilocytosis characterized by elliptocytes, numerous red blood cells (RBC) fragments and microspherocytes. The histogram of RBC volume distribution showed two populations of RBC: a normocytic and a very microcytic population, this later corresponding to the RBC fragmentation. These features strongly suggested a membrane disorder, particularly an hereditary pyropoïkilocytosis (HPP). The thermal unstability of the cytoskeleton was demonstrated by enhanced red cell fragmentation after in vitro exposure to heat which occurs at a lower temperature as compared to normal red cells. The diagnosis of HPP was confirmed by specialized investigations (osmotic gradient ektacytometry and erythrocytic membrane proteins electrophoresis). HPP is considered as a severe form of hereditary elliptocytosis characterized by jaundice and a severe haemolytic anaemia which usually appears during the neonatal period and the childhood. Our report is intriguing because of the delayed diagnosis of HPP in a patient who presented moderate clinical manifestations.

Asymptomatic elevation of the hyperchromic red blood cell subpopulation is associated with decreased red cell deformability

Hyperchromasia of the red blood cells (RBC), defined as an elevation of the hyperchromic subpopulation, has been described for various medical conditions. However, neither the association of hyperchromasia with an altered RBC membrane nor with other medical conditions has been investigated in a systematic way so far. Since the percentage of hyperchromic RBC is measured on a routine basis by many hematologic laboratories, we evaluated the predictive value of this parameter for the detection of RBC disorders. An extensive workup of all patients undergoing standard hematogram during a period of 6 months at our institution with a fraction of hyperchromic RBC larger than 10 % was collected by reviewing the medical history and performing osmotic gradient ektacytometry on RBC from a part of these patients. Thirty-two thousand two hundred twenty-six individuals were screened; of which, 162 (0.5 %) showed more than 10 % hyperchromic RBC. All of the patients examined by ektacytometry featured abnormal membrane deformability. Hereditary spherocytosis was found in 19 out of these 32 patients, in most cases unknown to the patient and currently asymptomatic. Another 17.9 % of the patients with an elevated subpopulation of hyperchromic RBC suffered from viral infection (human immunodeficiency virus, hepatitis). Our study shows that an elevated proportion of hyperchromic erythrocytes larger than 10 % is associated with both hereditary and acquired RBC membrane disorders and further follow-up should be considered.

A New Case of KLF1 G973A Mutation and Congenital Dyserythropoeitic Anemia (CDA)- Further Definition of Emerging New Syndrome and Possible Association with Gonadal Dysgenesis

Abstract▪2101▪This icon denotes a clinically relevant abstractArnaud et ala recently reported on two children with an as yet unclassified CDA, characterized by hemolytic anemia with high fetal hemoglobin (HbF), presence of embryonic hemoglobin Portland (HbPort) and absence on red blood cells (RBC) of CD44, an adhesion molecule, aquaporin 1 (AQP1) and the linked In(Lu) and Colton blood groups. The underlying molecular defect involves the erythroid transcription factor KLF1 with a mutation in zinc finger 2 at position 973 of Guanine to Adenine. The G973A mutation results in substitution of glutamate to lysine at position 325 in the transcribed protein. The CDA results from a dominant negative effect of KLF1 E325K on the gamma-globin repressor BCL11A, leading to the persistence of fetal erythropoiesis, high Hb F and HbPort. The absence of CD44, AQP1 and deficiency of certain cytoskeletal proteins in the RBC indicate that KLF1 is necessary for the terminal differentiation of RBC. The presence of CD44 on the leucocytes in these cases would appear to suggest an erythroid restrictive effect of the mutation. Of note, the child in whom the E325K mutation was first identified, a 46XY male, was described to have micropenis and hypospadias; the hemolytic anemia was severe and required splenectomy. The original case was that of phenotypic female with 46XX and the clinical course was much milder.We have identified a new case (a caucasian child) with the KLF1 E325K mutation. Comparison with the clinical findings in the two published cases indicates that KLF1 E325K mutation may cause profound developmental disturbances in organs beyond erythroid development, particularly of the gonads. Our patient, now 5 yrs old, was followed with an undiagnosed hemolytic anemia, which started with fetal hydrops at 23 weeks gestation, requiring intrauterine transfusions. Because of the presence of spherocytes on the blood smear and history of renal stones, a mutant band 3 was suspected; however direct nucleotide sequence analysis of the promoter and coding region exons of the band 3 gene from amplified genomic DNA of the proband was normal. Splenectomy was done at 4-years-of-age and the emerging picture was one of extreme normoblastemia, with the presence of both spherocytes and massively enlarged erythrocytes. Osmotic gradient ektacytometry showed reduced erythrocyte deformability with a population of swollen erythrocytes. Hemoglobin analyses revealed a HbF of31% and, HgPort (isoelectric focusing done by James Hoyer at Mayo Laboratories). Flow cytometry demonstrated that CD44 was not expressed on patient's erythrocytes but was expressed on lymphocytes. AQP1 was absent on western blots of RBC ghosts. Nucleotide sequence analysis of the KLF1 gene identified the KLF1 E325K mutation in lymphocyte DNA from the patient, but not in her parents. In addition to the severe anemia, the child who is 46XY, has female external genitalia. Ultrasonography demonstrated a vagina and uterus. Surgically excised streak gonads showed no cellular elements. Array comparative genomic hybridization studies did not reveal any copy number variations in male sex determining genes, but identified polymorphic deletion of estrogen metabolizing UGT2B17.Together these 3 cases suggest that KLF1 E325K may cause more severe anemia and gonadal dysgenesis in 46XY fetuses while the disease is considerably milder in 46XX individuals. Both of the 46 XY cases also had fetal hydrops and were salvaged with transfusions in utero, suggesting that KLF1 mutations may cause early loss of male fetuses. Given that KFL1 interacts with more than 1000 proteins,b,c it is likely that KLF1 is important in the developmental regulation of non erythroid genes including male sex determining genes. Disclosures:No relevant conflicts of interest to declare.

Association between myeloid malignancies and acquired deficit in protein 4.1R: A retrospective analysis of six patients

Constitutional deficit in the erythroid protein 4.1 (4.1R), a structural component of the erythrocyte membrane, is implicated in hereditary elliptocytosis. Acquired deficit in protein 4.1R have been rarely described in myelodysplastic syndromes. Here, we report a series of six patients presenting a myelodysplastic or a myeloproliferative disease in association with an elliptocytosis curve on osmotic gradient ektacytometry and a significant decrease in protein 4.1R level. We confirm that deficit in protein 4.1R is recurrent in myeloid malignancies and should be particularly investigated when deletion del (20 q) is present, since we found this chromosomal abnormality in four out of six patients.

Influence of the retinoid acitretin on erythrocyte microrheology in vitro

To determine in vitro changes in the microrheology of red blood cells (RBCs) treated with acitretin (Ro 10-1670). Venous blood samples were obtained from 27 healthy donors. Washed erythrocytes (5 x 10(6) cells/ml) were incubated for 30 min at 37 degrees C in solutions of low (25 ng/ml) and high (400 ng/ml) acitretin concentration. The whole red cell deformability was measured using osmotic gradient ektacytometry. To assess the contribution of factors influencing changes in red cell deformability, the biophysical state of the phospholipid bilayer was determined using the cation osmotic hemolysis technique (COH) and the internal viscosity using electron paramagnetic resonance (EPR) spectroscopy. Osmotic deformability studies revealed that cells treated with acitretin at low concentration deformed almost normally in isotonic medium, but RBCs incubated with high acitretin concentration showed distinct loss of deformation. However, in hypotonic medium, cell deformability was reduced to abnormally low values and this change was most marked with high acitretin concentration. CHO measurements showed that incubation of red cells at low acitretin concentration caused a significant increase in hemolysis at ionic strengths from 123.2 - 154.0 mmol/1 NaCl in relation to the control (p < 0.001). A high concentration of acitretin caused a significant increase in COH with high ionic strengths in the range 107.8 - 154.0 mmol/l NaCl (p < 0.001) and at low ionic strengths in the range 15.4 mmol/l NaCl (p < 0.05). The measurement of RBC intracellular microviscosity showed no statistically significant differences between control (6.22+/-0.22 cP) and acitretin-treated (6.29+/-0.25 cP) cells. The data show that acitretin, even at low concentration, is capable of inducing the reduction of red cell deformability. A reduced tendency of acitretin-treated RBCs to deform might result from the ability of acitretin to perturb the cell shape and membrane viscoelastic properties.

Mild dehydrated hereditary stomatocytosis revealed by marked hepatosiderosis

We report a patient in whom hepatosiderosis was diagnosed at the age of 55 years and who has since been treated by regular bleeding. The H63D mutation was found in the heterozygous state in the HFE gene. No mutation was recorded in the SLC11A3 gene (ferroportin). Hepatosiderosis did not seem primary, nevertheless its cause long remained elusive. Only 2 years ago did we find the responsible condition, a very mildly expressed form of dehydrated hereditary stomatocytosis (DHS). This genetic disease is a strongly iron-loading condition. Haemolysis was fully compensated. Kalaemia was slightly elevated, suggesting a pseudohyperkalaemia that may be associated with DHS. Osmotic gradient ektacytometry allowed to assess the diagnosis of DHS. The red cell monovalent Na+ and K+ concentrations were moderately elevated and reduced respectively. The temperature dependence of the ouabain + bumetanide-resistant K+ influx produced a shallow slope, above and parallel to the control curve. These features were consistent with the diagnosis of DHS. The pronounced hepatosiderosis contrasted with the mildly expressed DHS, and with the ferritinaemia that was slightly elevated, if at all, prior to bleeding. Bleeding caused ferritinaemia to decrease and hepatosiderosis to recede. The whole picture accounts for a misleading presentation of DHS, in which the primary condition long remained hidden behind one of its remotest complications, hepatosiderosis.

The hereditary stomatocytoses: genetic disorders of the red cell membrane permeability to monovalent cations

The hereditary stomatocytoses are mostly accounted for by genetic disorders of red cell membrane permeability to monovalent cations. These conditions, all very rare, are comprised of a hemolytic anemia, frequently macrocytosis, and the presence of abnormally shaped red blood cells. The key test for diagnosis is osmotic gradient ektacytometry, which measures the osmotic resistance and hydration of the red blood cell; the curve depicting the temperature dependence of the cation leak is also important. Syndromes include familial pseudohyperkalemia (FP), which is devoid of hematological features, dehydrated hereditary stomatocytosis (DHS), and overhydrated hereditary stomatocytosis (OHS). Some forms of DHS may be a pleiotropic, showing pseudohyperkalemia and/or perinatal edema. Perinatal edema, if not properly treated, may be lethal but may also resolve spontaneously prior to or shortly after birth and never reappear. Hereditary cryohydrocytosis, type 1 (CHC 1) is characterized by a dramatic resumption of the leak in vitro as the temperature approaches 0°C; cell hydration seems unaltered. In OHS, stomatin, a membrane protein, is sharply reduced; however, this is a secondary event and the primarily mutated protein remains unknown. Hereditary cryohydrocytosis, type 2 (CHC 2) presents similar to OHS, except that the leak dramatically increases close to 0°C. In addition, hematological manifestations are associated with neurological disorders. Of critical practical importance is that splenectomy in DHS or OHS causes thromboembolic events that may be fatal. The genes involved in hereditary stomatocytoses have yet to be identified. Apart from the 16q24-qter locus, related to subsets of DHS and FP, and a chromosome 2 locus assigned to a single case of FP, gene mapping has been difficult. The eventual discovery of individual genes will clarify complicated classification of the stomatocytoses, now based solely on phenotype.

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid α-spectrin gene

Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

La sphérocytose héréditaire

Physical properties of circulating red cells such as deformability and elasticity are critical for their survival. Such abilities are impaired in hereditary spherocytosis, the most common of heritable hemolytic disorders in Europe. Hereditary spherocytosis can result from various genetic defects leading to decreased production or abnormality of one of the red cell skeleton proteins, mainly ankyrin, band 3, protein 4.2, α or β spectrin. The clinical expression can vary among individuals from a well compensated hemolytic chronic hemolysis to severe anemia. The diagnosis is based on clinical and biological signs of increased hemolysis together with a bundle of criteria such as increase in the percentage of hyperdense cells, increase in osmotic fragility, spherocytes on blood smears. Osmotic gradient ektacytometry provides typical, although not specific, features. Red cell membrane protein electrophoresis and DNA studies can lead to identify the protein and the genetic defect responsible for the disease. The mainstay treatment is total or subtotal splenectomy.