Evaluation of eosin‐5‐maleimide flow cytometric test in diagnosis of hereditary spherocytosis

Abstract

A flow cytometry-based test using eosin-5-maleimide (EMA) dye was used for diagnosis of hereditary spherocytosis (HS). The mean fluorescence intensiy (MFI) of EMA tagged erythrocytes is lower in HS than that in other hemolytic and nonhemolytic anemias. We enrolled 114 subjects comprising 20 confirmed HS, 20 suspected HS/hemolytic anemia (HA), 20 normal controls, 20 other hemolytic anemias [13 autoimmune hemolytic anemia, three congenital dyserythropoietic anemia (CDA), one pyruvate kinase deficiency, two microangiopathic hemolytic anemia], 18 microcytic anemia and 16 macrocytic anemia cases. All samples were subjected to flow cytometry as per standard protocol. The mean MFI of normal control subjects was 11 861.5 (SD 883.5) and of confirmed HS was 7949.3 (SD 1304.1). Using this test, of 20 patients suspected to be HS/HA but with no confirmatory diagnosis, eight patients were diagnosed as HS. Using logistic regression analysis, the optimum cut-off MFI value between HS and normal controls was 10126. The area under the ROC curve was 0.99. The statistical significance of MFI values was obtained by t-test or Wilcoxon rank sum test as applicable. Compared with normal controls, the MFI values in HS were lower and in megaloblastic anemia were higher which was statistically highly significant (P<0.01), and the MFI values in CDA were lower which was statistically significant (P<0.05). False-positive values were obtained in three cases of AIHA and two cases of CDA. The sensitivity and specificity was 96.4% and 94.2% respectively. The EMA-based flow cytometry test is a highly sensitive and specific method for the diagnosis of HS.

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

“…[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.…”

Identification and Verification of Hereditary Spherocytosis by Means of Laboratory Diagnosis

“…Исследование сохранности цитоскелета эритроцитов методом проточной цитометрии -альтернативная мето-дика с высокой специфичностью и чувствительностью; ее эффективность в верификации наследственного сферо-цитоза подтверждена независимыми исследователями [7][8][9]12]. В нашем исследовании эффективность метода проточной цитометрии с применением эозин-5 малеими-да как диагностического теста для верификации диагноза наследственного сфероцитоза подтверждена для пациен-тов групп взрослого и детского возраста, за исключением пациента, перенесшего трансфузию за несколько дней до проведения исследования.…”

“…Незначительная часть красителя связывается с CD47, RH-ассоциированными белками. Снижение уров-ня экспрессии э5м на эритроцитах у пациентов с наслед-ственным сфероцитозом отражает дефицит мембранных белков, включая белок полосы 3, спектрин и белок поло-сы 4.2 [8,9].…”

Acute Kernicterus in a Neonate With O/B Blood Group Incompatibility and a Mutation in SLC4A1

“…Her parents were hematologically normal, with no spherocytes seen on blood smear. Erythrocyte osmotic fragility testing of the infant was normal, and UGT1A1 promoter genotyping was normal, with homozygosity for TA [6]. A flow cytometry-based test using eosin-5-maleimide dye showed decreased staining, typical of hereditary spherocytosis (HS).…”

“…A decrease in mean fluorescence intensity of eosin-5-maleimide-tagged erythrocytes is associated with band 3 deficiency and red cell cytoskeleton disorders. 5,6 Genetic analysis of erythrocyte membrane proteins was performed by using HaloPlex (Agilent Technologies, Santa Clara, CA) for targeted gene capture and sequencing on a HiSeq 2000 system (Illumina, San Diego, CA). Briefly, DNA was fragmented by using restriction enzymes and then denatured.…”