Hemorheological profiles and chronic inflammation markers in transfusion-dependent and non-transfusion- dependent thalassemia.

The rheological properties of blood play an important role in regulating blood flow in micro and macro circulation. In thalassemia syndromes red blood cells exhibit altered hemodynamic properties that facilitate microcirculatory diseases: increased aggregation and reduced deformability, as well as a marked increase in adherence to the vascular endothelial cells. A personalized approach to treating thalassemia patients (transfusions, iron chelation, and splenectomy), has increased patients' life expectancy, however they generally present many complications and several studies have demonstrated the presence of high incidence of thromboembolic events. In this study the hemorheological profiles of thalassemia patients have been characterized to point out new indices of vascular impairment in thalassemia. Plasma viscosity, blood viscosities at low and high shear rates (η1 and η200, respectively), erythrocyte aggregation index (η1/η200), and the erythrocyte viscoelastic profile (elastic modulus G', and viscous modulus G") have been studied in transfusion-dependent and non-transfusion-dependent thalassemia patients. Moreover, the levels of inflammation biomarkers in thalassemia have been evaluated to investigate a relationship between the biomarkers, the disease severity and the rheological parameters. The biomarkers studied are the main components of the immune and endothelial systems or are related to vascular inflammation: cytokines (IL-2, IL-6, IL-10, IL-17A, TNF-alpha), chemokines (IL-8, MIP-1alpha), adipocytokines (leptin and adiponectin), growth factors (VEGF, angiopoietin-1), adhesion molecules (ICAM-1, VCAM-1, E-selectin, L-selectin), and a monocyte/macrophage activation marker (CD163). This study shows that transfusion-dependent thalassemia patients, both major and intermedia, have blood viscosities comparable to those of healthy subjects. Non-transfusion-dependent thalassemia intermedia patients show high blood viscosities at low shear rates (η1), corresponding to the flow conditions of the microcirculation, an increase in erythrocyte aggregation, and high values of the elastic G' and viscous G" modules that reflect a reduced erythrocyte deformability and an increase in blood viscosity. Levels of cytokines, chemokines and adhesion molecules are different in transfusion- and non-transfusion dependent patients and positive correlations between η1 or η1/η200 and the cytokines IL-6 and IL-10 have been observed. The evaluation of the hemorheological profiles in thalassemia can provide new indicators of vascular impairment and disease severity in thalassemia in order to prevent the onset of thromboembolic events.

Innovative screening test for the early detection of sickle cell anemia.

In this study, the capability of thermogravimetry in conjuction with a multivariate statistical analysis, was investigated for the screening of Sickle Cell Anemia (SCA), a hereditary disorder of hemoglobin characterized by severe hemolytic anemia with different severe clinical manifestations. SCA results from a mutation in the sixth codon of the beta globin gene, which results in the substitution of glutamic acid for valine and leads to the production of an altered form of hemoglobin, hemoglobin S (HbS). People with this disorder have atypical hemoglobin molecules which tend to aggregate together and form filaments inside the red blood cells. These deformed red blood cells called half-moon or sickle, are rigid and unable to flow inside the small vessels, creating occlusions of the small circulation. Systematic screening for SCA is not a common practice, and diagnosis is usually made when a severe complication occurs. An early and rapid diagnosis is important for patients in order to prevent and treat the painful episodes that can occur when sickled red blood cells, which are stiff and inflexible, get stuck in small blood vessels. A novel test was developed using whole blood samples from patients with congenital defects and analyzed by the TG7 thermobalance (PerkinElmer) without any pretreatment. The resulting TG and DTG curves of blood samples were compared to those typical of healthy individuals and results demonstrated a different thermal behaviour of the anemic patients with respect to healthy individuals as result of the different amounts of water content and corpuscular fraction. The multivariate statistical analysis performed by chemometrics allowed a quick identification of differences between the two population and provided a model of prediction in patients with heterogeneous congenital hematological disorders. The predictive ability of the model was tested by processing patient affected by SCA and with a confirmed diagnosis obtained by the molecular analysis. The model provided for a sensitivity and an accuracy of a 100% and an error of prediction of about 0.1%.

An Innovative Multilevel Test for Hemoglobinopathies: TGA/Chemometrics Simultaneously Identifies and Classifies Sickle Cell Disease From Thalassemia.

Introduction: Hemoglobinopathies are the most common genetic disorder wordlwide and because of migrations are become an emerging global health problem. Screening programmes for Sickle cell disease and Thalassemia have been implemented in some countries, but are not a common practice, due to a lack in the accuracy of the methods and to the costs of the analyses. Objectives: The objective of this study was the application of the thermogravimetry coupled to chemometrics as new screening method to perform an early diagnosis of thalassemia and sickle cell disease. Methods: Whole blood samples (30 µL) from sickle cell anemia and thalassemia patients were analyzed using the thermobalance TG7 and the resulting curves were compared with those of healthy individuals. A chemometric approach based on Principal Components Analysis (PCA) was exploited to enhance correlation between thermogravimetric profiles and a model of prediction by Partial Least Square Discriminant Analysis (PLS-DA) was developed and validated. Results: The characteristic profile of the blood sample thermal decomposition and the first derivative of the TG curve showed that patients were clearly distinguished from healthy individuals as a result of different amounts of water and corpuscular fraction of blood. The chemometric approach based on PCA allowed a quick identification of differences between healthy subjects and patients and also between thalassemic and sickle cell anemia subjects. Chemometric tools (PLS-DA) were used to validate a model of prediction to process the thermogravimetric curves and to obtain in 1 h an accurate diagnosis. The TGA/Chemometric test permitted to perform first level test for hemoglobinopathies with the same accuracy of confirmatory analyses obtained by the molecular investigation. Conclusions: A screening test based on the coupling of thermogravimetry and chemometrics was optimized for the differential diagnosis of hemoglobinopathies. The novel test is able to simultaneously perform a simple and fast diagnosis of sickle cell anemia or thalassemia, in a single analysis of few microliters of non-pretreated whole blood at low cost, and with high accuracy. Moreover this method results particularly suitable in pediatric patients as it requires small sample volumes and is able to characterize also transfused patients.

Phenotypical and functional abnormalities of circulating neutrophils in patients with ß-thalassemia.

ß-Thalassemia is an inherited single gene disorder related to reduced synthesis of the ß-globin chain of hemoglobin. Patients with ß-thalassemia present variable clinical severity ranging from asymptomatic trait to severe transfusion-dependent anemia and multiple organs complications. Moreover, multiple immune abnormalities are a major concern in ß-thalassemia patients. Aberrant neutrophil effector function plays a pivotal role in infection susceptibility in these patients. In severe and persistent inflammation, immature neutrophils are released from the bone marrow and are functionally different compared with mature ones. Despite some abnormalities reported for thalassemia patient's immune system, few data exist on the characterization of human neutrophils in ß-thalassemia. The aim of this study was to investigate the phenotype and function of circulating neutrophil subsets in patients with ß-thalassemia major and with ß-thalassemia intermedia divided in transfusion-dependent and non-transfusion-dependent. By the use of immunochemical and cytofluorimetric analyses, we observed that patients' CD16+ neutrophils exhibit abnormalities in their phenotype and functions and the abnormalities vary according to the clinical form of the disease and to the neutrophil subset (CD16bright and CD16dim). Abnormalities include altered surface expression of the innate immune receptor CD45, Toll-like receptor 4, and CD32, reduced ability to produce an oxidative burst, and elevated levels of membrane lipid peroxidation, especially in patients with a more severe form of the disease. Overall, our results indicating the occurrence of an immuno-senescent phenotype on circulating neutrophils from thalassemia patients suggest the usefulness of neutrophil feature assessment as a tool for better clinical management of ß-thalassemia.

COVID-19 Sepsis and Microcirculation Dysfunction.

The spreading of Coronavirus (SARS-CoV-2) pandemic, known as COVID-19, has caused a great number of fatalities all around the World. Up to date (2020 May 6) in Italy we had more than 28,000 deaths, while there were more than 205.000 infected. The majority of patients affected by COVID-19 complained only slight symptoms: fatigue, myalgia or cough, but more than 15% of Chinese patients progressed into severe complications, with acute respiratory distress syndrome (ARDS), needing intensive treatment. We tried to summarize data reported in the last months from several Countries, highlighting that COVID-19 was characterized by cytokine storm (CS) and endothelial dysfunction in severely ill patients, where the progression of the disease was fast and fatal. Endothelial dysfunction was the fundamental mechanism triggering a pro-coagulant state, finally evolving into intravascular disseminated coagulation, causing embolization of several organs and consequent multiorgan failure (MOF). The Italian Society of Clinical Hemorheology and Microcirculation was aimed to highlight the role of microcirculatory dysfunction in the pathogenetic mechanisms of COVID-19 during the spreading of the biggest challenges to the World Health.

Differential diagnosis of hereditary hemolytic anemias in a single multiscreening test by TGA/chemometrics.

A multi-screening test based on the coupling of thermogravimetry and chemometrics was optimized for the differential diagnosis of hereditary hemolytic anemias. The novel test is able to simultaneously perform a simple and fast diagnosis of sickle cell anemia, thalassemia, hereditary spherocytosis and hereditary elliptocytosis in a single analysis of a few microliters of non-pretreated whole blood. The thermogravimetric profile of blood from patients affected by such disorders was found to be characteristic of a specific anemic status or a disorder due to membrane defects. In addition, chemometric tools were used to validate a model of prediction to process the thermogravimetric curves and to obtain in 1 hour an accurate diagnosis. The effectiveness of the novel test was evaluated by comparing results with the confirmatory analyses specific for each disorder. The TGA/chemometric test made it possible to perform a first level test of congenital erythrocyte defects, including the hemoglobinopathies and disorders due to membrane defects with the same accuracy of confirmatory analyses obtained by molecular investigation. In addition, the novel test was used for the diagnosis of a number of Italian difficult cases, including neonatal patients for which the conventional screening tests did not manage to obtain a diagnosis confirming the high prediction ability of the single multiscreening test.

Development of a novel test for the identification of hereditary erythrocyte membrane defects by TGA/Chemometrics.

Systematic screening for congenital erythrocyte disorders is not a common practice, due to a lack in the accuracy of the methods and to the costs of the analyses. As a consequence, the diagnosis is usually made when a severe complication occurs. This study introduces an innovative method to perform the screening of a hereditary disease characterized by erythrocyte membrane defects such as hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). Blood samples from healthy donors and patients affected by HS and HE were processed by thermogravimetric analysis (TGA) and the resulting curves were analyzed by chemometrics in order to develop a model of prediction. A novel test was developed and validated by chemometrics and results were found to be in accordance with the genetic diagnosis, confirming the prediction ability of the optimized model that uses few microliters of whole non pretreated blood to perform the diagnosis of HS/HE in less than one hour.

HCV Infection in Thalassemia Syndromes and Hemoglobinopathies: New Perspectives.

Hepatitis C virus (HCV) infection is one of the most serious complications of transfusion therapy in the thalassemia and sickle cell disease (SCD) population before 1990; in fact, since 1990 serological tests were made available to detect infection in blood donors. The iron chelation therapy has improved the life expectancy of these patients and, consequently, a decrease in death due to heart disease may be observed, as well as an increase in liver disease due to the iron overload and HCV infection that lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Until few years ago, the recommended therapy for HCV treatment consisted of pegylated-interferon alpha plus ribavirin, a therapy with important side effects. This treatment has been severely limited to thalassemic and SCD patients due to the hemolytic anemia induced by ribavirin causing an increase in the number of blood transfusions. The development of highly effective Direct-acting Antiviral Agents toward different viral genotypes has led to a real HCV eradication with negative viremia and sustained viral response between 90 and 98%. At the beginning some indications of Direct-acting Antiviral Agents administration were available for those patients exhibiting advanced cirrhosis or needing liver transplantation over time for the high costs of the new drugs. Recently, all treatment regimens can be used for patients with various HCV genotypes, different stages of liver disease, and comorbidities. The HCV eradication has also led to a marked improvement in the parameters of martial accumulation, demonstrating a synergic action also between the effect of antiviral therapy and iron chelation.

Pregnancy in Thalassemia and Sickle Cell Disease: The Experience of an Italian Thalassemia Center.

The life expectancy of thalassemia patients has increased significantly in recent years being the most "elderly" patients approaching or are over 50 years old. Consequently, patients' perspectives have changed, leading them to longer-term planning with a consequent increase in their reproductive potential and desire to have children. Crucial points in the management of pregnancy in thalassemia are the iron chelation therapy before and during pregnancy, the antithrombotic prophylaxis, the management of transfusion therapy according to the modified transfusion requirement, a cardiologic monitoring for hemodynamic changes that expose an increased risk of heart failure. Pregnancy in women with sickle cell disease is still associated with increased rates of maternal and fetal mortality and adverse outcomes. Maternal morbidity may be due to acute sickling crises, thromboembolism, infection, and chronic end-organ dysfunction, while neonatal outcomes may be intrauterine growth retardation, preterm delivery, small infants for gestational age, stillbirth, and neonatal death. The management of pregnancy in thalassemia and sickle cell disease requires to be approached by a multidisciplinary team and followed from the pre-conception phase until the post-partum period with a close monitoring of the maternal and fetal conditions, in order to ensure optimal outcome. This approach requires the application of well-defined protocols that cover all the critical aspects of pregnancies in women affected by these pathologies. We describe our experience of spontaneous and non-spontaneous pregnancies in patients with thalassemia major and intermedia and sickle cell disease followed between 1992 and 2018 at the Thalassemia Unit of S. Eugenio Hospital of Rome.

Hemorheological Alterations and Oxidative Damage in Sickle Cell Anemia.

Sickle cell anemia (SCA) is the most common hereditary disorder of hemoglobin (Hb) characterized by a mutation in the ß globin gene, which leads to synthesis of HbS a hemoglobin which, under hypoxic conditions, gels and leading to the sickling of the red blood cells (RBC). The dehydration of the RBC increases the concentration of the intracellular Hb with an increase in the internal viscosity and consequently a decrease in the erythrocyte deformability. Sickle red blood cells due to their difficulty to flow through the microcirculation cause frequent vaso-occlusive episodes, tissue ischemia, and infarctions. Moreover, the reduced RBC deformability causes cell fragility leading to hemolysis and recently a key role of hemolysis and oxidative stress in the development of vascular dysfunction has been demonstrated. The aim of this study was to evaluate the hemorheological profiles of patients with SCA in order to point out new indices of vascular impairment, and to characterize the membrane oxidative damage of sickled RBC. Blood viscosities, erythrocyte aggregation, and viscoelastic profiles of SCA patients were determined, and the RBC oxidative damage was investigated by comparing metabolic capability and RBC membrane proteins from SCA patients with and without transfusion dependence. The hemorheological profile of SCA subjects demonstrated high blood viscosity, increased RBC aggregation, and decreased RBC deformability. These impaired flow properties were associated with RBC membrane protein oxidation, with degradation of spectrin and increased membrane-bound globin. The comparison between SCA patients with and without transfusion dependence showed metabolic and structural RBC oxidative damage significantly different.

TGA/Chemometric Test Is Able to Detect the Presence of a Rare Hemoglobin Variant Hb Bibba.

In this study the TGA/Chemometric test was applied for diagnosis of a case of congenital hemolytic anemia for which the common first level diagnostic tests were not able to find the erythrocyte congenital defect. A 6 years old girl presented chronic hemolytic anemia characterized by hyperbilirubinemia, increased spleen, negative Coombs tests, normal hemoglobin values, decreased mean corpuscular volume (MCV), increased red cell distribution width (RDW), reticulocytes and lactate dehydrogenase (LDH), and altered erythrocyte morphology (ovalocytes, spherocytes, and rare schizocytes). The diagnostic protocols for differential diagnosis of hereditary hemolytic anemia were carried out by the investigation of the congenital hemolytic anemias due to defects of membrane proteins and the most common erythrocyte enzymes, but no defect was found. The TGA/Chemometric test was applied and the PLS-DA model of prediction was used to process results. The thermogravimetric profile of the patient was very distinct from those of healthy subjects and comparable with those of thalassemia patients. The classification model applied to the patient identified a chronic hemolytic anemia due to a hemoglobin defect and the molecular characterization confirmed the TGA/Chemometrics results, demonstrating the presence of a very rare hemoglobin variant Hb Bibba (α2136(H19)Leu → Proß2). In conclusion the TGA/Chemometric test proved to be a promising tool for the screening of the hemoglobin defects, in a short time and at low cost, of this case of congenital hemolytic anemia of difficult diagnosis. This method results particularly suitable in pediatric patients as it requires small sample volumes and is able to characterize patients subjected to transfusion.

Effect of hemorheological parameters on myocardial injury after primary or elective percutaneous coronary intervention.

BACKGROUND: Abnormal blood viscosity favors atherosclerosis owing to endothelial dysfunction and changes in shear stress. Its effect on coronary microvasculature during percutaneous coronary intervention (PCI) is still unknown. We aimed to investigate the role of hemorheological parameters in the incidence of microvascular obstruction (MVO) and the periprocedural necrosis after primary or elective PCI, and secondarily, we evaluated their prognostic significance. MATERIALS AND METHODS: We enrolled 25 patients with ST-elevation myocardial infarction (STEMI), 30 patients with non-ST-elevation myocardial infarction (NSTEMI), and 30 patients with stable angina (SA) undergoing PCI. MVO in patients with STEMI and periprocedural necrosis in patients with NSTEMI and those with SA were assessed using angiographic/electrocardiographic and laboratory methods, respectively. Hemorheological profile included blood viscosity (η) at shear rates 200 s and 1 s, the erythrocyte aggregation index (η1/η200), and plasma viscosity. Major adverse cardiovascular events occurrence was evaluated at follow-up. RESULTS: Patients with STEMI experiencing angiographic MVO (28%) had higher η200 (5.42±1.28 vs. 3.98±1.22 mPa[BULLET OPERATOR]s; P=0.015). Similarly, patients with STEMI experiencing electrocardiographic MVO (56%) had higher η200 (4.58±0.36 vs. 3.94±0.19 mPa[BULLET OPERATOR]s; P<0.001). Among patients with SA and patients with NSTEMI, those experiencing periprocedural necrosis (23.3%) had higher η200 (5.30±0.86 vs. 4.37±0.88 mPa[BULLET OPERATOR]s; P=0.001), η1 (19.52±9.62 vs. 13.29±7.65 mPa[BULLET OPERATOR]s; P=0.015) and η1/η200 values (3.64±1.50 vs. 2.72±0.92; P=0.007). These significant differences were maintained after adjustment for age, sex, and cardiovascular risk factors. At follow-up (30±6 months), 25 (29.4%) patients presented major adverse cardiovascular events, and they had higher η200 (5.18±1.00 vs. 4.25±1.01 mPa[BULLET OPERATOR]s; P<0.001). CONCLUSION: In patients undergoing either urgent or elective PCI, hemorheological parameters might contribute to myocardial injury and, if furtherly confirmed, to an unfavorable outcome.

Update on thalassemia diagnosis: New insights and methods.

A novel approach based on Thermogravimetric analysis followed by Chemometrics (TGA/Chemometrics) is provided for Thalassemia diagnosis and a comprehensive study consisting of the coupled approach TGA/Chemometrics, the Complete Blood Count (CBC) and Red Blood Cell (RBC) indices is developed and results are compared. A number of 128 subjects were involved in this study included 16 thalassemia intermedia transfusion-dependent (TI-TD) patients, 18 thalassemia intermedia non transfusion-dependent (TI-NTD) patients, and 14 thalassemia major ß (TM-TD) patients. Thalassemic patients were found to be clearly distinct from healthy donors as a function of a different thermal behavior. The chemometric analysis identifies the differences in the composition of blood and a model of prediction for ß-thalassemia was developed and validated to distinguish all patients. TGA/Chemometrics method also permitted to differentiate thalassemic patients according to the severity of anaemia while the evaluation of the indices and the CBC are not able to identify TI-TD, TI-NTD and TM-TD patients at first level test. TGA/Chemometrics was successfully applied for thalassemia diagnosis with 100% of correct classification rate. Chemometric analysis demonstrated that red cell distribution width (RDW), haemoglobin (Hb) and RBC are the diagnostic features in thalassemia compared to mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH). New insights into the significance of the haematological features were provided for an update of the thalassemia classification.

Thermogravimetric analysis coupled with chemometrics as a powerful predictive tool for ß-thalassemia screening.

ß-Thalassemia is a hemoglobin genetic disorder characterized by the absence or reduced ß-globin chain synthesis, one of the constituents of the adult hemoglobin tetramer. In this study the possibility of using thermogravimetric analysis (TGA) followed by chemometrics as a new approach for ß-thalassemia detection is proposed. Blood samples from patients with ß-thalassemia were analyzed by the TG7 thermobalance and the resulting curves were compared to those typical of healthy individuals. Principal Component Analysis (PCA) was used to evaluate the correlation between the hematological parameters and the thermogravimetric results. The thermogravimetric profiles of blood samples from ß-thalassemia patients were clearly distinct from those of healthy individuals as result of the different quantities of water content and corpuscular fraction. The hematological overview showed significant decreases in the values of red blood cell indices and an increase in red cell distribution width value in thalassemia subjects when compared with those of healthy subjects. The implementation of a predictive model based on Partial Least Square Discriminant Analysis (PLS-DA) for ß-thalassemia diagnosis, was performed and validated. This model permitted the discrimination of anemic patients and healthy individuals and was able to detect thalassemia in clinically heterogeneous patients as in the presence of δß-thalassemia and ß-thalassemia combined with Hb Lepore. TGA and Chemometrics are capable of predicting ß-thalassemia syndromes using only a few microliters of blood without any pretreatment and with an hour of analysis time. A fast, rapid and cost-effective diagnostic tool for the ß-thalassemia screening is proposed.

Hereditary spherocytosis and elliptocytosis associated with prosthetic heart valve replacement: rheological study of erythrocyte modifications.

The implantation of a prosthetic heart valve (HVP) in patients with hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) is rare, and the changes in the structure and deformability of erythrocytes that follow implantation in these patients have been poorly described. In the present study, the erythrocytes in HS and HE patients with mechanical HVP were compared to the erythrocytes in patients with only congenital membrane defects, in terms of biochemical modifications and rheological behaviour. Integral and cytoskeletal erythrocyte membrane proteins were studied, and blood viscosity (shear rate/shear stress ratio), aggregation ratio [eta(1 s(-1))/eta(200 s(-1))], and red cell visco-elasticity were determined. Valve replacement with a mechanical prosthesis worsened anaemia and resulted in a change in haemolysis, from sub-clinical to evident. The rheological investigation of erythrocytes from HS patients confirmed the characteristic increased viscosity and aggregation ratio and the decreased deformability. The rheological behaviour of erythrocytes from patients with HVP showed a decrease in viscosity and an increase in elastic modulus. In these patients, the prosthesis seems to have induced traumatic damage to the erythrocyte membrane, leading to fragmentation and lysis, which in turn modified rheological parameters. The biochemical and rheological investigation allowed us to understand the clinical and haematological pictures of the patients and to describe the role played by different factors in haemolytic anaemia.

Sickle cell anaemia: haemorheological aspects.

The maintenance of erythrocyte shape and membrane integrity is bound to the modification of deformability and/or permeability. Usually, this features are not investigated with normal laboratory tests. The membrane stiffness, the cell geometry, and the viscoelasticity components are influencing factors on survival and functionality of the erythrocytes. Only few studies have analyzed the viscoelastic characteristics of red blood cells, even less are the studies on patients affected by sickle cell disease (SCD), a pathology characterized by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle haemoglobin (Hb S) polymers. A critical point of SCD is represented by the rheologic alterations of sickle cells determined by the transition from sol to gel of haemoglobin producing a dramatic change in cell viscosity and viscoelastic properties. We have investigated the behaviour of the blood in SCD, from an original rheological point of view, by evaluating the viscoelastic properties of sickle cells in oscillating harmonic sinusoidal mode. A comparison between patients with different severity of the disease, with transfusion dependence (TD) or without transfusion dependence (NTD), has been carried out. This study has confirmed the rheologic impairment of SC blood. The TD patients showed a minor heterogeneneity of rheologic behaviour in comparison with NTD patients, because of the normalizing effect of transfusion. The analysis of viscoelastic properties might be an additional useful tool for monitoring transfusional and pharmacological treatments.

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein.

BACKGROUND: Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes. CASE PRESENTATION: All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 +/- 0.42) than in controls (4.41 +/- 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced. CONCLUSION: A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.

Prosthetic heart valves' mechanical loading of red blood cells in patients with hereditary membrane defects.

Implantable cardiovascular devices such as prosthetic heart valves (PHVs) are widely applied clinical tools. Upon implantation, the patient can suffer from anemia as a result of red cell destruction and hemolysis can be more relevant whenever the patient is also affected by red cell disorders in which erythrocytes are more susceptible to mechanical stress such as hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). Considering the typical morphological alterations observed in HS and HE, a study of the influence of cell geometry on the distribution of the shear stress on red cells in biological fluids was carried out. A numerical simulation of the loading caused by Reynolds shear stresses on a prolate spheroid was performed, with the ellipticity of the particle as the independent parameter. The average shear stress on a particle in the blood stream was found to depend on the particle's geometry, besides the stress field produced by the prosthetic device. The relevance of an increasing particle ellipticity on the global load is discussed. The model was applied to erythrocytes from implanted patients with HE or HS, enabling to explain the occurrence of moderate or severe anemia, respectively. The clinical data support the relevance of the proposed global parameter as erythrocyte trauma predictor with regard to the fluid dynamics of artificial organs.

Identification of G6PD Mediterranean mutation by amplification refractory mutation system.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X chromosome-linked hereditary enzymopathy in humans. The authors have developed an amplification refractory mutation system (ARMS) to detect the G6PD Mediterranean mutation (nt. 563 C-->T) that is the most frequent among Caucasian population. METHODS: Specific forward polymerase chain reaction (PCR) primers, within exon 6 of the G6PD gene, were designed: ARMS M complementary to the mutated DNA sequence and ARMS N complementary to the wild-type DNA. They were paired with a common reverse primer. The new method was validated using known DNA samples from 72 G6PD-deficient patients carrying the G6PD Mediterranean mutation ascertained by the restriction enzyme analysis. The ARMS test was performed on DNA extracted both from blood or saliva samples. RESULTS: The ARMS test showed an excellent reproducibility and a complete concordance with the endonuclease cleavage reference method. At the same time, it is more rapid and less expensive. CONCLUSIONS: The described molecular test may be a method of choice to identify the G6PD Mediterranean mutation. It could also be helpful to obtain a definite diagnosis of G6PD Mediterranean heterozygotes, which is not feasible by using red blood cell enzyme activity measurements.