Experience with eosin-5′-maleimide as a diagnostic tool for red cell membrane cytoskeleton disorders

Abstract

The diagnosis of hereditary spherocytosis (HS) is based on red cell morphology and other conventional tests such as osmotic fragility, autohemolysis and acidified glycerol lysis. However, milder cases are at times difficult to diagnose. Confirmation by red blood cell (RBC) membrane protein analysis is not possible in most laboratories. Recently, a flow cytometric method has been described for quantitating the fluorescence intensity of intact red cells after incubation with the dye eosin-5¢-maleimide (EMA), which binds specifically to the anion transport protein (band-3) at lysine-430. This has been shown to be an effective screening test for red cell membrane disorders. We evaluated the usefulness of this approach for screening membrane protein disorders such as HS and hereditary elliptocytosis (HE) and its value in discriminating this group from other hemolytic anemias, such as glucose-6phosphate dehydrogenase (G6PD) deficiency, b-thalassemia trait, sickle cell anemia and autoimmune hemolytic anemia. Fluorescence intensity, expressed in mean channel fluorescence (MCF) units, was determined using a Becton Dickinson FACS Caliber flow cytometer. Membrane protein analysis was carried out by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE). RBCs from patients with HS and HE gave significantly lower MCF values (P < 0.001) than the normal control group and other patient groups. The diagnosis of HS in four cases was confirmed by RBC membrane protein electrophoresis and all showed a deficiency of spectrin. The advantage of the EMA dye method are its specificity for membrane disorders, as well as being a simple, user-friendly and rapid method which is inexpensive, provided a flow cytometer is available.

Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics

“…4,5,16,17,19,37,38 Among the diagnostic methods considered, the recently proposed EMA-binding test is certainly the most interesting one, and it is being increasingly used by specialized laboratories because of its high sensitivity and specificity. 12,18,[23][24][25][26][27][28][29] This method directly targets the structural lesion of the disease, since the fluorescent probe eosin-5'-maleimide interacts with transmembrane proteins band 3, Rh protein, Rh glycoprotein and CD47 which are reduced in red cells from patients with hereditary spherocytosis; 30 defects of other cytoskeletal proteins, such as spectrin and protein 4.2, also induce a decrease in fluorescence intensity, likely because they create a long-range modulation effect on the dye binding site in band 3 protein. 39 The sensitivity of the EMA-binding test in this series is higher than that recently reported by Crisp et al, 12 and similar to 18 and Girodon et al 25 Interestingly, sensitivity is independent of clinical phenotype, being high also in patients with compensated anemia.…”

“…[13][14][15] More recently, the cryohemolysis test, 16,17 based on the observation that red cells from patients with hereditary spherocytosis are particularly susceptible to cooling at 0°C in hypertonic conditions, and the flow cytometric analysis of eosin-5'-maleimide-labeled intact red blood cells (EMA-binding test) 18 have been proposed as new methods for identifying hereditary spherocytosis. 19 The latter in particular has been proven to be a sensitive and specific diagnostic test for hereditary spherocytosis 12,18,[20][21][22][23][24][25][26][27][28][29] directly targeting the structural lesion of this disease, since the fluorescent probe, eosin-5'-maleimide, interacts with the protein band 3 complex. 30 The performance of the available direct or indirect diagnostic tests has been mostly evaluated individually and on limited number of cases.…”

Flow cytometric osmotic fragility—An effective screening approach for red cell membranopathies

“…Earlier studies showed that storage conditions of the E5 0 M dye were very critical for accurate results. The dye is very unstable in aqueous solution at 4 C. It was also shown that storing the dye in small aliquots at -80 C in the dark maintained its stability over a 4-month period whereas storage of the dye at -20 C resulted in some reduction in MCF over this time (7,8). The E5 0 M-labeled red cells of the two patients of hemolytic anemia and dRTA with Band 3 protein defect gave similar MCF readings (850, 890) to those of HS red cells (831.79 AE 80.70).…”

Erythrocyte Flow Cytometric Analysis in Congenital Dyserythropoietic Anemia Type III-Evaluation of Eosin-5´-Maleimide, CD55, and CD59

“…To a minor extent EMA also interacts with CD47 and Rh-antigen, also reduced in hereditary spherocytosis, contributing to the reduced fluorescence after EMA staining in this disease [4]. The reduced fluorescence in CDA II is probably not due to a quantitative reduction of band 3 in the erythrocyte membrane but rather an affected configuration leading to a decreased affinity to EMA [6]. The increase in EMA fluorescence in CDA III is probably due to the slight increase of erythrocyte volume (MCV) exposing a larger surface per cell to binding of EMA to band 3, Rhesus-antigen and CD47 on the erythrocyte membrane.…”

“…EMA binding appears to be normal in most common anemias such as DAT positive hemolytic anemia and iron deficiency as well as in hemoglobinopathies such as thalassemia [5]. Normal MCF after EMA staining has also been reported in anemias due to enzymopathies such as glucose-6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency [6]. Reduced binding of monoclonal antibodies against the glycosylphosphatidylinositol anchor proteins CD55 and CD59 to erythrocytes and myeloid cells forms the basis of the flow cytometric diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) [7].…”