Red cell ektacytometry in two patients with chronic hemolytic anemia and three new α-spectrin variants.

Red blood cell (RBC) morphology is, in general, the key diagnostic feature for hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). However, in hereditary pyropoikilocytosis (HPP), the severe clinical form of HE, the morphological diagnosis is difficult due to the presence of a RBC morphological picture characterized by a mixture of elliptocytes, spherocytes, tear-drop cells, and fragmented cells. This difficulty increases in new-borns and/or patients requiring frequent transfusions, making impossible the prediction of the disease course or its severity. Recently, it has been demonstrated that the measurement of osmotic gradient ektacytometry (OGE), using a laser-assisted optical rotational ektacytometer LoRRca (MaxSis, RR Mechatronics), allows a clear differentiation between HS and HE, where the truncated osmoscan curve reflects the inability of the already elliptical cells to deform further under shear stress in the face of hypotonicity. In HPP, however, the RBCs appear to have a significantly decreased ability to maintain deformability in these conditions, and the classical trapezoidal profile of HE is less evident or indistinguishable from HS. Here, two unrelated patients with hereditary hemolytic anemia (HHA) due to HPP and HS, respectively, are described with the joint inheritance of a complex set of five genetic defects. Two of these defects are novel alpha-spectrin gene (SPTA1) variants, one is a microdeletion that removes the entire SPTA1 gene, and two are well-known low-expression polymorphic alleles: α-LELY and α-LEPRA. In the HPP patient (ID1), with many circulating spherocytes, the interactions between the two SPTA1 gene variants may lead, in addition to an elongation defect (elliptocytes), to a loss of membrane stability and vesiculation (spherocytes), and RBCs appear to have a significantly decreased ability to maintain deformability in hypotonic conditions. Due to this, the classical trapezoidal profile of HE may become less evident or indistinguishable from HS. The second patient (ID2) was a classical severe form of HS with the presence of more than 20% of spherocytes and few pincered cells. The severity of clinical manifestation is due to the coinheritance of a microdeletion of chromosome 1 that removes the entire SPTA1 gene with a LEPRA SPTA1 variant in trans. The diagnostic interest of both observations is discussed.

Concomitant Hereditary Spherocytosis and Pyruvate Kinase Deficiency in a Spanish Family with Chronic Hemolytic Anemia: Contribution of Laser Ektacytometry to Clinical Diagnosis.

Background: Hereditary spherocytosis (HS) and pyruvate kinase deficiency (PKD) are the most common causes of hereditary chronic hemolytic anemia. Here, we describe clinical and genetic characteristics of a Spanish family with concomitant ß-spectrin (SPTB) c.647G>A variant and pyruvate kinase (PKLR) c.1706G>A variant. Methods: A family of 11 members was studied. Hematological investigation, hemolysis tests, and specific red cell studies were performed in all family members, according to conventional procedures. An ektacytometric study was performed using the osmoscan module of the Lorca ektacytometer (MaxSis. RR Mechatronics). The presence of the SPTB and PKLR variants was confirmed by t-NGS. Results: The t-NGS genetic characterization of the 11 family members showed the presence of a heterozygous mutation for the ß-spectrin (SPTB; c.647G>A) in seven members with HS, three of them co-inherited the PKLR variant c.1706G>A. In the remaining four members, no gene mutation was found. Ektacytometry allowed a clear diagnostic orientation of HS, independently from the PKLR variant. Conclusions: This family study allows concluding that the SPTB mutation, (c.647G>A) previously described as likely pathogenic (LP), should be classified as pathogenic (P), according to the recommendations for pathogenicity of the American College of Medical Genetics and the Association for Molecular Pathology. In addition, after 6 years of clinical follow-up of the patients with HS, it can be inferred that the chronic hemolytic anemia may be attributable to the SPTB mutation only, without influence of the concomitant PKLR. Moreover, only the family members with the SPTB mutation exhibited an ektacytometric profile characteristic of HS.

Rare anemias in adolescents.

Anemia can be the consequence of a single disease or an expression of external factors mainly nutritional deficiencies. Genetic issues are important in the primary care of adolescents because a genetic diagnosis may not be made until adolescence when the teenager presents with the first signs or symptoms of the condition. This situation is relatively frequent for rare anemias (RA) an important, and relatively heterogeneous group of rare diseases (RD) where anemia is the first and most relevant clinical manifestation of the disease. RA are characterized by their low prevalence (< 1 per 10,000 individuals), and, in some cases, by their complex mechanism. For these reasons, RA are little known, even among health professionals, and patients tend to remain undiagnosed or misdiagnosed for long periods of time, making it impossible to know the prognosis of the disease or to carry out genetic counseling for future pregnancies. Since this situation is an important cause of anxiety for both adolescent patients and their families, the physician's knowledge of the natural history of a genetic disease will be the key factor for the anticipatory guidance for diagnosis and clinical follow-up. RA can be due to three primary causes: 1.Bone marrow erythropoietic defects, 2. Excessive destruction of mature red blood cells (hemolysis), and 3. Blood loss (bleeding). More than 80% of RAs are hereditary, and about 20% remain undiagnosed but when their first clinical manifestations appear during childhood or adolescence, they are frequently misdiagnosed with iron deficiency. For this reason, RA are today an important clinical and social health problem worldwide.

Characterization of hereditary red blood cell membranopathies using combined targeted next-generation sequencing and osmotic gradient ektacytometry.

Hereditary red blood cell (RBC) membranopathies are characterized by mutations in genes encoding skeletal proteins that alter the membrane complex structure. Hereditary spherocytosis (HS) is the most common inherited RBC membranopathy leading to hereditary hemolytic anemia with a worldwide distribution and an estimated prevalence, in Europe, of about 1:2000 individuals. The recent availability of targeted next generation sequencing (t-NGS) and its combination with RBC deformability measured with a laser-assisted optical rotational ektacytometer (LoRRca) has demonstrated to be the most powerful contribution to lower the percentage of hereditary hemolytic anemia undiagnosed cases. In order to know the kind and frequency of RBC membrane mutations in our geographical area (Catalonia) and to better understand their pathophysiology, 42 unrelated, non-transfusion-dependent (NTD) patients with hereditary hemolytic anemia have been studied by combining t-NGS and LoRRca. The osmoscan module of LoRRca provides three rheological profiles that reflect the maximal deformability (EImax), osmotic fragility (Omin), and hydration state (Ohyper) of RBCs and contribute to a better understanding of the contribution RBC rheology to the severity of anemia. From the 42 patients studied, 37 were suspected to be a RBC membrane defect due to phenotypic characteristics and abnormal RBC morphology and, from these, in 31 patients (83.8% of cases) the mutation was identified by t-NGS. No definite diagnosis was achieved in 11 patients (26.2% of cases), including 6 out of 37 cases, with suspected membranopathy, and 5 with unclassifiable HHA. In all these undiagnosed patients, the existence of hemoglobinopathy and/or enzymopathy was ruled out by conventional methods.

Immigration and screening programs for hemoglobinopathies in Italy, Spain and Turkey.

Sickle cell disease (SCD) and thalassemias are the most common monogenic diseases in the world. The number of migrants and refugees in Europe and Turkey, in the past decade, has increased dramatically due to war, violence or prosecutions in their homeland. Prevention and management of haemoglobin disorders is well established and managed in countries where these conditions were traditionally endemic or in countries that have a longstanding tradition of receiving migrants. Therefore, preventive and diagnostic programmes regarding hemoglobinopathies in immigrant populations have been implemented. The purpose of this paper it to report a summary of the experience gained in Italy, Spain and Turkey in migrants, asylum seekers and refugees.

Blood Rheological Characterization of ß-Thalassemia Trait and Iron Deficiency Anemia Using Front Microrheometry.

The purpose of this work is to develop a hematocrit-independent method for the detection of beta-thalassemia trait (ß-TT) and iron deficiency anemia (IDA), through the rheological characterization of whole blood samples from different donors. The results obtained herein are the basis for the development of a front microrheometry point-of-care device for the diagnosis and clinical follow-up of ß-TT patients suffering hematological diseases and alterations in the morphology of the red blood cell (RBC). The viscosity is calculated as a function of the mean front velocity by detecting the sample fluid-air interface advancing through a microfluidic channel. Different viscosity curves are obtained for healthy donors, ß-TT and IDA samples. A mathematical model is introduced to compare samples of distinct hematocrit, classifying the viscosity curve patterns with respect to the health condition of blood. The viscosity of the fluid at certain shear rate values varies depending on several RBC factors such as shape and size, hemoglobin (Hb) content, membrane rigidity and hematocrit concentration. Blood and plasma from healthy donors are used as reference. To validate their potential clinical value as a diagnostic tool, the viscosity results are compared to those obtained by the gold-standard method for RBC deformability evaluation, the Laser-Optical Rotational Red Cell Analyzer (LoRRCA).