Use of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) in the Diagnosis of RBC Membrane Disorders, Enzyme Defects, and Congenital Dyserythropoietic Anemias: A Monocentric Study on 202 Patients

Abstract

Chronic hemolytic anemias are a group of heterogeneous diseases mainly due to abnormalities of red cell (RBC) membrane and metabolism. The more common RBC membrane disorders, classified on the basis of blood smear morphology, are hereditary spherocytosis (HS), elliptocytosis, and hereditary stomatocytoses (HSt). Among RBC enzymopathies, the most frequent is pyruvate kinase (PK) deficiency, followed by glucose-6-phosphate isomerase, pyrimidine 5′ nucleotidase P5′N, and other rare enzymes defects. Because of the rarity and heterogeneity of these diseases, diagnosis may be often challenging despite the availability of a variety of laboratory tests. The ektacytometer laser-assisted optical rotational cell analyser (LoRRca MaxSis), able to assess the RBC deformability in osmotic gradient conditions (Osmoscan analysis), is a useful diagnostic tool for RBC membrane disorders and in particular for the identification of hereditary stomatocytosis. Few data are so far available in other hemolytic anemias. We evaluated the diagnostic power of LoRRca MaxSis in a large series of 140 patients affected by RBC membrane disorders, 37 by enzymopathies, and 16 by congenital diserythropoietic anemia type II. Moreover, nine patients with paroxysmal nocturnal hemoglobinuria (PNH) were also investigated. All the hereditary spherocytoses, regardless the biochemical defect, showed altered Osmoscan curves, with a decreased Elongation Index (EI) max and right shifted Omin; hereditary elliptocytosis (HE) displayed a trapezoidal curve and decreased EImax. Dehydrated hereditary stomatocytosis (DHSt) caused by PIEZO1 mutations was characterized by left-shifted curve, whereas KCNN4 mutations were associated with a normal curve. Congenital diserythropoietic anemia type II and RBC enzymopathies had Osmoscan curve within the normal range except for glucosephosphate isomerase (GPI) deficient cases who displayed an enlarged curve associated with significantly increased Ohyper, offering a new diagnostic tool for this rare enzyme defect. The Osmoscan analysis performed by LoRRca MaxSis represents a useful and feasible first step screening test for specialized centers involved in the diagnosis of hemolytic anemias. However, the results should be interpreted by caution because different factors (i.e., splenectomy or coexistent diseases) may interfere with the analysis; additional tests or molecular investigations are therefore needed to confirm the diagnosis.

Simple Syntheses of New Pegylated Trehalose Derivatives as a Chemical Tool for Potential Evaluation of Cryoprotectant Effects on Cell Membrane

“…Chemical shifts (δ) are given in ppm and J values are given in Hz. 13 C NMR signals were identified by APT experiments. Mass spectra were measured by ESI technique on LCQ Fleet or LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) and MALDI technique on UltrafleXtreme™ MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).…”

“…Our aim was to form a chemical tool for the evaluation of cryoprotectant influences on the cell membrane, which are still not easily predicted during the freezing/thawing process. In [13][14][15], it has been shown that the state of the cell membrane (structure, stiffness, raptures in the membrane) correlates with frozen/thawed cell viability [2,12]. Saccharides are expected to stabilize biological molecules [16,17].…”

Iron overload in congenital haemolytic anaemias: role of hepcidin and cytokines and predictive value of ferritin and transferrin saturation

“…Blood smear was assessed by an expert morphologist. Additional screening tests included RBC osmotic fragility tests, the eosin‐5‐maleimide (EMA)‐binding test, RBC membrane protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and erythrocyte deformability by laser‐assisted optical rotational cell analyser (LoRRca MaxSis; Mechatronics, Zwaag, the Netherlands) (Mariani et al , ; Bianchi et al , ; King et al , ; Zaninoni et al , ). The haemolytic screening included the determination of the following enzymatic activities: pyruvate kinase, glucosephosphate isomerase, pyrimidine‐5′ nucleotidase, glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, adenylate kinase, phosphofructokinase, hexokinase, glyceraldehyde‐P‐dehydrogenase and phosphoglycerate kinase.…”

Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients

“…RBC deformability at normoxic conditions is compromised in patients with SCD due to irreversibly sickled cells, as well as altered hydration causing a decrease in surface/volume ratio . Osmotic gradient ektacytometry, in which deformability is measured as a function of a continuous change in osmolarity, is a well‐established method, in particular in hereditary disorders of the red cell membrane . In SCD, osmotic gradient ektacytometry showed that increasing HbF induces a better surface‐to‐volume ratio thereby improving deformability in untransfused patients with SCD .…”