Antonella Gambale

Bio: Antonella Gambale is an academic researcher from University of Naples Federico II. The author has contributed to research in topics: Congenital dyserythropoietic anemia type II & Dehydrated hereditary stomatocytosis. The author has an hindex of 16, co-authored 36 publications receiving 1006 citations.

Tomographic flow cytometry by digital holography.

Abstract: High-throughput single-cell analysis is a challenging task. Label-free tomographic phase microscopy is an excellent candidate to perform this task. However, in-line tomography is very difficult to implement in practice because it requires a complex set-up for rotating the sample and examining the cell along several directions. We demonstrate that by exploiting the random rolling of cells while they are flowing along a microfluidic channel, it is possible to obtain in-line phase-contrast tomography, if smart strategies for wavefront analysis are adopted. In fact, surprisingly, a priori knowledge of the three-dimensional position and orientation of rotating cells is no longer needed because this information can be completely retrieved through digital holography wavefront numerical analysis. This approach makes continuous-flow cytotomography suitable for practical operation in real-world, single-cell analysis and with a substantial simplification of the optical system; that is, no mechanical scanning or multi-direction probing is required. A demonstration is given for two completely different classes of biosamples: red blood cells and diatom algae. An accurate characterization of both types of cells is reported, despite their very different nature and material content, thus showing that the proposed method can be extended by adopting two alternate strategies of wavefront analysis to many classes of cells.

New insights on hereditary erythrocyte membrane defects.

Abstract: After the first proposed model of the red blood cell membrane skeleton 36 years ago, several additional proteins have been discovered during the intervening years, and their relationship with the pathogenesis of the related disorders have been somewhat defined. The knowledge of erythrocyte membrane structure is important because it represents the model for spectrin-based membrane skeletons in all cells and because defects in its structure underlie multiple hemolytic anemias. This review summarizes the main features of erythrocyte membrane disorders, dividing them into structural and altered permeability defects, focusing particularly on the most recent advances. New proteins involved in alterations of the red blood cell membrane permeability were recently described. The mechanoreceptor PIEZO1 is the largest ion channel identified to date, the fundamental regulator of erythrocyte volume homeostasis. Missense, gain-of-function mutations in the PIEZO1 gene have been identified in several families as causative of dehydrated hereditary stomatocytosis or xerocytosis. Similarly, the KCNN4 gene, codifying the so called Gardos channel, has been recently identified as a second causative gene of hereditary xerocytosis. Finally, ABCB6 missense mutations were identified in different pedigrees of familial pseudohyperkalemia. New genomic technologies have improved the quality and reduced the time of diagnosis of these diseases. Moreover, they are essential for the identification of the new causative genes. However, many questions remain to solve, and are currently objects of intensive studies.

Recommendations regarding splenectomy in hereditary hemolytic anemias

Abstract: Hereditary hemolytic anemias are a group of disorders with a variety of causes, including red cell membrane defects, red blood cell enzyme disorders, congenital dyserythropoietic anemias, thalassemia syndromes and hemoglobinopathies As damaged red blood cells passing through the red pulp of the spleen are removed by splenic macrophages, splenectomy is one possible therapeutic approach to the management of severely affected patients However, except for hereditary spherocytosis for which the effectiveness of splenectomy has been well documented, the efficacy of splenectomy in other anemias within this group has yet to be determined and there are concerns regarding short- and long-term infectious and thrombotic complications In light of the priorities identified by the European Hematology Association Roadmap we generated specific recommendations for each disorder, except thalassemia syndromes for which there are other, recent guidelines Our recommendations are intended to enable clinicians to achieve better informed decisions on disease management by splenectomy, on the type of splenectomy and the possible consequences As no randomized clinical trials, case control or cohort studies regarding splenectomy in these disorders were found in the literature, recommendations for each disease were based on expert opinion and were subsequently critically revised and modified by the Splenectomy in Rare Anemias Study Group, which includes hematologists caring for both adults and children

Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias.

Abstract: Mutations in more than 70 genes cause hereditary anemias (HA), a highly heterogeneous group of rare/low frequency disorders in which we included: hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA) and Diamond-Blackfan anemia; hemolytic anemias due to erythrocyte membrane defects, as hereditary spherocytosis and stomatocytosis; hemolytic anemias due to enzymatic defects. The study describes the diagnostic workflow for HA, based on the development of two consecutive versions of a targeted-NGS panel, including 34 and 71 genes, respectively. Seventy-four probands from 62 unrelated families were investigated. Our study includes the most comprehensive gene set for these anemias and the largest cohort of patients described so far. We obtained an overall diagnostic yield of 64.9%. Despite 54.2% of cases showed conclusive diagnosis fitting well to the clinical suspicion, the multi-gene analysis modified the original clinical diagnosis in 45.8% of patients (nonmatched phenotype-genotype). Of note, 81.8% of nonmatched patients were clinically suspected to suffer from CDA. Particularly, 45.5% of the probands originally classified as CDA exhibited a conclusive diagnosis of chronic anemia due to enzymatic defects, mainly due to mutations in PKLR gene. Interestingly, we also identified a syndromic CDA patient with mild anemia and epilepsy, showing a homozygous mutation in CAD gene, recently associated to early infantile epileptic encephalopathy-50 and CDA-like anemia. Finally, we described a patient showing marked iron overload due to the coinheritance of PIEZO1 and SEC23B mutations, demonstrating that the multi-gene approach is valuable not only for achieving a correct and definitive diagnosis, but also for guiding treatment.

Novel Gardos channel mutations linked to dehydrated hereditary stomatocytosis (xerocytosis)

Abstract: Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Affected red cells are characterized by a nonspecific cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHSt cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. A recent study has identified two families with DHSt associated with a single mutation in the KCNN4 gene encoding the Gardos channel (KCa3.1), the erythroid Ca(2+) -sensitive K(+) channel of intermediate conductance, also expressed in many other cell types. We present here, in the second report of DHSt associated with KCNN4 mutations, two previously undiagnosed DHSt families. Family NA exhibited the same de novo missense mutation as that recently described, suggesting a hot spot codon for DHSt mutations. Family WO carried a novel, inherited missense mutation in the ion transport domain of the channel. The patients' mild hemolytic anemia did not improve post-splenectomy, but splenectomy led to no serious thromboembolic events. We further characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of CD34+ cells and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development.

Glucose-6-phosphate dehydrogenase deficiency.

Abstract: Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder that occurs almost exclusively in males. This condition mainly affects red blood cells, which carry oxygen from the lungs to tissues throughout the body. In affected individuals, a defect in an enzyme called glucose-6-phosphate dehydrogenase causes red blood cells to break down prematurely. This destruction of red blood cells is called hemolysis.

Quantitative phase imaging in biomedicine

Abstract: Quantitative phase imaging (QPI) has emerged as a valuable method for investigating cells and tissues. QPI operates on unlabelled specimens and, as such, is complementary to established fluorescence microscopy, exhibiting lower phototoxicity and no photobleaching. As the images represent quantitative maps of optical path length delays introduced by the specimen, QPI provides an objective measure of morphology and dynamics, free of variability due to contrast agents. Owing to the tremendous progress witnessed especially in the past 10–15 years, a number of technologies have become sufficiently reliable and translated to biomedical laboratories. Commercialization efforts are under way and, as a result, the QPI field is now transitioning from a technology-development-driven to an application-focused field. In this Review, we aim to provide a critical and objective overview of this dynamic research field by presenting the scientific context, main principles of operation and current biomedical applications. Over the past 10–15 years, quantitative phase imaging has moved from a research-driven to an application-focused field. This Review presents the main principles of operation and representative basic and clinical science applications.

Squeezing for Life - Properties of Red Blood Cell Deformability.

Abstract: Deformability is an essential feature of blood cells (RBCs) that enables them to travel through even the smallest capillaries of the human body. Deformability is a function of (i) structural elements of cytoskeletal proteins, (ii) processes controlling intracellular ion and water handling and (iii) membrane surface-to-volume ratio. All these factors may be altered in various forms of hereditary hemolytic anemia, such as sickle cell disease, thalassemia, hereditary spherocytosis and hereditary xerocytosis. Although mutations are known as the primary causes of these congenital anemias, little is known about the resulting secondary processes that affect RBC deformability (such as secondary changes in RBC hydration, membrane protein phosphorylation, and RBC vesiculation). These secondary processes could, however, play an important role in the premature removal of the aberrant RBCs by the spleen. Altered RBC deformability could contribute to disease pathophysiology in various disorders of the RBC. Here we review the current knowledge on RBC deformability in different forms of hereditary hemolytic anemia and describe secondary mechanisms involved in RBC deformability.

Gradient Light Interference Microscopy for 3D Imaging of Unlabeled Specimens

Abstract: A system and methods for quantitative optical phase imaging of a sample. First second replica field of an image field are generated, characterized by a respective optical phase, cross-polarized and shifted in a shift direction transverse to a normal to the surface of the sample. The replica fields are Fourier transformed, the second replica field is retarded by four successive phase shifts, and, after inverse Fourier transforming, the first and second replica fields pass through an analyzer polarizer and superposing the first and second replica fields on a detector array to create four successive detector signals. The four successive detector signals are solved to derive a gradient of the optical phase of the image field, which may be integrated to obtain a quantitative phase image.

Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia

Abstract: Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions.