Facilitating EMA binding test performance using fluorescent beads combined with next‐generation sequencing

Abstract

The eosin‐5′‐maleimide (EMA) binding test is widely used as diagnostic test for hereditary spherocytosis (HS), one of the most common haemolytic disorders in Caucasian populations. We recently described the advantages of replacing the use of healthy control blood samples with fluorescent beads in a modified EMA binding assay. In this study we further explore this novel approach. We performed targeted next‐generation sequencing, modified EMA binding test and osmotic gradient ektacytometry on consecutive individuals referred to our laboratory on the suspicion of HS. In total, 33 of 95 carried a (likely) pathogenic variant, and 24 had variants of uncertain significance (VUS). We identified a total 79 different (likely) pathogenic variants and VUS, including 43 novel mutations. Discarding VUS and recessive mutations in STPA1, we used the occurrence of (likely) pathogenic variants to generate a diagnostic threshold for our modified EMA binding test. Twenty‐one of 23 individuals with non‐SPTA1 (likely) pathogenic variants had EMA ≥ 43.6 AU, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Accuracy was excellent at 93.4% and close to that of osmotic gradient ektacytometry (98.7%). In conclusion, we were able to simplify the EMA‐binding test by using rainbow beads as reference and (likely) pathogenic variants to define an accurate cut‐off value.

Acquired spherocytosis in the setting of myelodysplasia

“…The EMA-binding test was performed on EDTA-stabilized blood within 48 h of sampling [ 8 ]. The labeling of RBCs with EMA, usage of mid-range FL1 Rainbow Fluorescent Particles (BD Biosciences, NJ, USA) and flow cytometry was performed [ 8 , 9 ].…”

DAHEAN: A Danish Nationwide Study Ensuring Quality Assurance through Real-World Data for Suspected Hereditary Anemia Patients

“…In selected cases, other genetic techniques such as array CGH, Sanger sequencing, and targeted sequencing of speci c genes [24] can be employed.…”

“…Membranopathies RBC membrane will be studied by standard diagnostic procedures including osmotic gradient ektacytometry (RR Mechatronics) [24,32,33] and ow cytometry including Eosin-5-maleimide (EMA)binding test [24,28,33].…”

Glucose phosphate isomerase deficiency demasked by whole-genome sequencing: a case report

“…Various tests, including direct anti-globulin test, osmotic gradient ektacytometry [ 7 , 8 ], PKLR sequencing, blood smear, and hemoglobin electrophoresis [ 9 ], ruled out other diagnoses such as hereditary spherocytosis, pyruvate kinase deficiency, autoimmune hemolytic diseases, glucose-6-phosphate dehydrogenase deficiency, thalassemia, and sickle-cell disease. Peripheral blood smear was unremarkable aside from mild stomatocytosis (Fig.…”