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.