Eosin-5'-maleimide Binding Test Research Articles

Novel Biomarkers for Assessing Clinical Severity in Hereditary Spherocytosis - Application of Routine and Advanced Diagnostic Tests

Background: Hereditary spherocytosis (HS) is caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeletal and (trans)membrane proteins. This ultimately leads to instability of the RBC membrane and subsequent membrane loss, resulting in the transformation of the biconcave RBC into a poorly deformable spherocyte, which is susceptible to premature splenic clearance. The diagnosis of HS has substantially improved over the last years and new advanced techniques have been established, including osmotic gradient ektacytometry, eosin-5'-maleimide (EMA) binding test and next-generation sequencing (NGS). However, there is little knowledge on the applicability of these diagnostic outcome parameters as biomarkers for clinical severity in HS. Aim: To explore possible correlations between established and novel diagnostic outcome parameters and clinical severity in a well-defined cohort of HS patients. Methods: This monocenter retrospective cohort study involves patients diagnosed with HS upon referral to our department between 2012 and 2023. Clinical characteristics, routine and advanced diagnostic laboratory parameters, and genotype were evaluated. Clinical severity was classified as previously described, based on parameters such as hemoglobin, reticulocyte percentage and bilirubin 1. The following laboratory tests were included: routine hematological parameters (Cel-Dyn Sapphire or Alinity hq), EMA binding test, osmotic fragility test (OFT) and osmotic gradient ektacytometry (osmoscan, Lorrca MaxSis). The latter technique was also explored for other parameters than the commonly used EI max, O hyper and O min. NGS gene panel analysis was used for molecular diagnosis. Differences between groups were determined using either Chi-squared test, unpaired t-test or Mann-Whitney U test and correlations using point-biserial, Pearson's or Spearman's test via GraphPad Prism. Results: Eighty-two non-splenectomized patients were included. Thirty-five of these (42.7%) were classified as mild, and forty-seven as moderate-severe (57.3%)). Demographic characteristics nor genotype differed significantly, albeit SPTB variants occurred more frequently in the moderate-severe group (15 vs. 4 in the mild group). Expectedly, both reticulocyte count and EI max correlated with clinical severity. RBC distribution width (RDW) correlated moderately, suggesting greater RBC heterogeneity (r=0.606, p<0.0001) is associated with a more severe clinical phenotype. Similarly, EMA binding test results correlated with clinical severity (r=0.390, p<0.001), indicating more pronounced membrane loss in clinically more severe patients. We further evaluated three novel osmoscan parameters ( Figure 1): EI O290, the elongation index (EI) at physiologic isotonicity, which correlated with clinical severity (r=-0.500, p<0.0001), and also with EI max (r=0.856, p<0.0001). The O min-width and O max-width, defined as the difference in osmolality between the intersection points of y=EI min or EI max +/- 0.05 and the osmoscan curve. Thisassesses the effect of changes in osmolality on EI min or EI max, respectively. O min-width correlated with RDW (r-0.439, p<0.0001), OFT results (N=30, r=0.671, p<0.0001) and clinical severity (r=0.390, p<0.001). O max-width correlated with clinical severity (r=-0.375, p<0.001) and strongly with percentage of hyperchromic cells (N=76, r=-0.751, p<0.0001), yet weakly with RDW (-0.273, p<0.05). All significant correlations with clinical severity are displayed in Figure 2. Conclusion: In this study, a number of routine and advanceddiagnostic laboratory parameters were found to correlate with clinical severity in HS. O min-width and O max-width are novel osmoscan-derived biomarkers that correlated with RDW, hyperchromic cells (O max-width), OFT results (O min-width) and clinical severity. EI O290, another novel osmoscan-derived biomarker, also correlated with clinical severity, although not stronger than EI max and AUC. The biomarkers identified in this study, together with patient-reported outcome measures (PROMs), could be used to revise the previously established severity classification system and thereby aid in stratifying clinical severity and subsequent clinical decision-making.

Detection of Red Blood Cell Membrane Proteins in Myelodysplastic Syndromes Using Eosin-5-Maleimide (EMA) Staining by Flow Cytometry.

Background: Eosin-5-Maleimide (EMA)-based flow cytometry binds to red blood cell (RBC) membrane-associated proteins which can be used to detect red blood cell (RBC) membrane disorders. Myelodysplastic syndromes (MDS) are stem cell disorders resulting in ineffective hematopoiesis which is commonly present with anemia and erythroid dysplasia. Objectives: We aimed to study RBC membrane defects in MDS using flow cytometry for EMA staining. Methods: We enrolled anemic patients who were diagnosed with low-risk MDS (R-IPSS score ≤ 3.5), RBC membrane disorders [hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO)], and normal controls. Complete blood count (CBC) and flow cytometry for EMA staining were performed. Results: There were 16 cases of low-risk MDS, 6 cases of RBC membrane disorders, and 15 control cases. Mean fluorescence intensity (MFI) of EMA binding test in the RBC membrane disorders was significantly lower than controls (17.6 vs. 24.3, p < 0.001), but the EMA binding test in the low-risk MDS was not significantly different than the controls (26.5 vs. 24.3, p = 0.08). Conclusion: the RBC membrane defect in low-risk MDS was not demonstrated as having detection ability using EMA binding test with flow cytometry.

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

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.

Improving the EMA Binding Test by Using Commercially Available Fluorescent Beads.

Hereditary spherocytosis (HS) is a common anemia caused by germline mutations in red blood cell cytoskeleton proteins. The flow cytometry-based eosin-5′-maleimide (EMA) binding test is most frequently employed for reliable diagnostics. To perform this test, a number of healthy and ideally also age-matched controls are required, which can be challenging and complicates interlaboratory comparisons. To overcome this limitation, we modified the EMA binding test by replacing healthy controls with commercially available fluorescent beads. Blood samples from 289 individuals with suspected HS were analyzed using the EMA binding test with fluorescent beads and benchmarked against regular EMA binding test using two control samples. Using osmotic gradient ektacytometry as validation, 112 individuals (38.8%) were diagnosed with HS. Performance of the modified EMA binding test was not compromised (accuracy 90.3%) compared to EMA binding test using matched controls (accuracy 88.6%). Based on these findings, we conclude that the modified EMA binding test with fluorescent beads is an attractive alternative, especially in laboratories without easy access to matched controls. Furthermore, as fluorescent beads are stable and easily commutable, they could facilitate both interlaboratory comparisons and quality assessment programs.

Progress on Laboratory Diagnosis of Hereditary Spherocytosis--Review

In recent years, the diagnostic methods of hereditary spherocytosis (HS) have been developed rapidly, including eosin-5'-maleimide (EMA) binding test, flow cytometric osmotic fragility test, osmotic gradient ektacytometry and next-generation sequencing. EMA binding test and flow cytometric osmotic fragility test are recommended as HS screening tests due to their high sensitivity and easy operation. Osmotic gradient ektacytometry has high sensitivity and specificity, thus which can be used to distinguish HS from other hereditary membrane disease, but can not differentiate between HS and auto-immune hemolytic anemia (AIHA) and it is difficult operation, which is used as an intermediate step between screening and diagnostic tests. Next-generation sequencing can detect the molecular defects, identifying the gene encoding defective protein, thus achieving accurate diagnosis. This diagnostic test of HS has become an important diagnostic tool. The development of laboratory diagnosis has reduced misdiagnosis, and significantly improved the level of HS diagnosis.

Usefulness of Eosin-5’-Maleimide (EMA) Dye for the Diagnosis of Hereditary Spherocytosis

Abstract Background the normal biconcave discoid shape of the red blood cell (RBC) is maintained by cytoskeletal proteins underneath the membrane. This protein network and its link with the lipid bilayer are essential for RBCs to remain flexible and carry out their physiological functions. A number of disorders including hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) are caused by decreased expression or mutation of cytoskeletal proteins. Aim of the Work to evaluate the usefulness eosin-5-maleamide (EMA) dye binding flow cytometric test in the diagnosis of HS and assess the sensitivity and specificity of this test to distinguish HS patients from patients having anemia due to other causes. Subjects and Methods this study was conducted on 52 patients with chronic anemia, 21 diagnosed as HS (group I) and 31 patients with anemia due to other causes (group II). The patients were 28 males and 23 females with a male to female ratio 1.2:1, their ages ranged between 3 to 12.5 years old attending at the Pediatric Hematology Clinics, Ain Shams University Hospitals. Normal control group (group III) was included in the study. Results median fluorescence intensity (MFI) had shown significantly lower values in HS patients (group I) (3.78±0.48) than (iron deficiency anemia) IDA patients (group IIa) (5.56±0.35), auto immune hemolytic anemia (AIHA) patients (group IIb) (5.7±0.27), beta thalassemia intermedia (BTI) patients (group IIc) (6.01±0.63) and control group (group III) (5.3±0.43). The % reduction in EMA fluorescence proved to be significantly higher in HS patient (group I) (29.88±8.27) than in IDA patients (group IIa) (1.13±6.18), AIHA patients (-6.14±10.44) and BTI patients (group IIc) (-5.1±10.07). Multi regression analysis had been used to check the most sensitive predictors for diagnosis of HS, both EMA MFI and the % reduction in EMA fluorescence proved to be the best sensitive predictive values for discriminating HS patients, however the higher F-ratio in EMA MFI (F = 25.222) than in % reduction in EMA fluorescence intensity (F = 19.866) showed that the MFI is even better than % reduction in fluorescence in diagnosis of HS. Conclusion the present study highlighted the role of flow cytometric EMA binding test in differentiating HS patients from patients having anemia due to other causes.

PS1198 MOLECULAR DIAGNOSIS OF RARE CONGENITAL ANEMIAS USING TARGETED NEXT-GENERATION SEQUENCING IN INDIAN PATIENTS

Background: Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin-5’-maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next-generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear. Aims: To improve accuracy, robustness, and cost-effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS. Methods: We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA-binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser-assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α-spectrin, β-spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations. Results: A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations. The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry-derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry-derived parameter EImax (<0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin >166 mOsm/kg and EImax <0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty-seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values <40 had mutations only in the SPTA1 gene.Summary/Conclusion: Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and <0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.

PS1197 ASSESSING THE EMA BINDING TEST AND OSMOTIC GRADIENT EKTACYTOMETRY IN THE DIAGNOSIS OF HEREDITARY SPHEROCYTOSIS DEFINED BY THE PRESENCE OF UNDERLYING DIAGNOSTIC MUTATIONS

Background:Hereditary spherocytosis (HS) is one of the most frequent hereditary hemolytic disorders in Caucasian populations. With the emergence of the eosin‐5’‐maleimide (EMA) binding test, osmotic gradient ektacytometry, and targeted next‐generation sequencing (tNGS), the role of single and combined diagnostic analyses remains unclear.Aims:To improve accuracy, robustness, and cost‐effectiveness of the laboratory diagnosis of HS by the EMA binding test and osmotic gradient ektacytometry, individually and in combination, using the occurrence of diagnostic cytoskeleton protein gene mutations determined by tNGS as confirmation of HS.Methods:We included samples from all individuals referred to our laboratory with suspected HS between May 1st, 2017 and July 1st, 2018. In addition to a modified EMA‐binding test using commercial Rainbow Beads as reference, we performed osmotic gradient ektacytometry using the Osmoscan module of the Laser‐assisted Optical Rotational Deformability Cell Analyser (LoRRca MaxSis, RR Mechatronics, The Netherlands), as well as a comprehensive tNGS membranopathy panel (including α‐spectrin, β‐spectrin, ankyrin, band 3, protein 4.1, and protein 4.2). HS was defined as the occurrence of diagnostic cytoskeleton protein gene mutations.Results:A total of 99 individuals were included in the study. Of these, 41 individuals had no proven mutations in red blood cell cytoskeleton protein genes, 34 had one or more diagnostic mutations, and 24 had one or more variants of uncertain significance (VUS). We identified a total 78 different diagnostic mutations and VUS, including 42 novel mutations.The table depicts the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of the EMA binding test as well as the two ektacytometry‐derived parameters Omin (osmotic resistance) and the maximal elongantion index (EImax). Rated individually, the ektacytometry‐derived parameter EImax (&lt;0.579) gave the highest accuracy (91%), when comparing results from individuals with diagnostic mutations and individuals with no mutations. When combining Omin &gt;166 mOsm/kg and EImax &lt;0.6, the highest sensitivity (100%) and accuracy (94.7 %) was reached. Twenty‐seven of 34 individuals with diagnostic mutations had EMA ≥40, which was the optimal threshold in receiver operating characteristic (ROC) analysis. Five of 7 individuals with diagnostic mutations and EMA values &lt;40 had mutations only in the SPTA1 gene.Summary/Conclusion:Individually, osmotic gradient ektacytometry was superior to the EMA binding test in the laboratory diagnosis of HS. In individuals with EMA ≥40 combined with Omin and EImax values of ≥166 mOsm/kg and &lt;0.6, respectively, tNGS provided no additional value in the laboratory diagnosis of HS. In cases of discrepancy between results from the EMA binding test and selected Omin and EImax values, DNA testing may help explain this divergence.image

A Case of Hereditary Spherocytosis Caused by a Novel Homozygous Mutation in the SPTB Gene Misdiagnosed as β-Thalassemia Intermedia Due to a KLF1 Gene Mutation

We report a rare case of hereditary spherocytosis (HS) and hereditary persistence of fetal hemoglobin (Hb) (HPFH) complicated with a β-thalassemia (β-thal) trait and a Krüppel-like factor 1 (KLF1) gene mutation misdiagnosed as β-thal intermedia (β-TI) due to a high percentage of Hb F. The proband presented with pale skin, jaundice and splenomegaly. Analysis of the thalassemia gene indicated βcodon 17/βA (HBB: c.52A>T), while Hb analysis showed significantly increased Hb F levels. The proband was diagnosed to carry β-TI, and a blood transfusion regimen together with iron chelation treatment was recommended. Due to the difference between the phenotype and genotype, next generation sequencing (NGS) was performed and the proband was found to carry a homozygous mutation on the SPTB gene combined with a heterozygous mutation in KLF1. An eosin-5-maleimide binding test (EMA-BT) showed that the mean fluorescence intensity decreased by 47.1%. The proband was finally diagnosed with HS and HPFH complicated with a β-thal trait and the high percentage of Hb F was believed to be ascribed to the KLF1 gene mutation, which is frequent in areas where thalassemia is prevalent. For patients with a β gene mutation accompanying significantly high percentage of Hb F, the diagnosis of β-TI could be warranted, and the influence of the KLF1 gene mutation should be carefully excluded to avoid misdiagnosis of other types of hereditary hemolytic diseases.

Flow Cytometric Eosin-5'-Maleimide Test is a Sensitive Screen for Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is a clinically heterogeneous disease characterized by mild to moderate hemolysis resulting from red cell membrane protein defects. Diagnostic tests include hemogram, reticulocyte count and blood smear evaluation, osmotic fragility, cryohemolysis, SDS-PAGE, flow cytometry using eosin-5'-maleimide (EMA) and genetic studies. We evaluated the flow cytometric EMA-binding test and compared it with osmotic fragility in 51 consecutive cases of suspected HS aged between 10days and 62years. In addition, 4 cases suspected on blood smears underwent EMA testing alone. The 16 EMA-positive cases who were determined to have HS had overlapping hemoglobin levels and reticulocyte counts with the 35 patients with normal EMA results, highlighting the importance of the flow cytometric test in providing a definitive diagnosis. Flow cytometric EMA binding test was thus a simple and relatively faster method to confirm HS in our experience.

Correlation of the degree of band 3 protein absence on erythrocyte membrane by eosin-5'-maleimide binding test and clinical phenotype in hereditary spherocytosis

目的探讨伊红-5′-马来酰亚胺标记的流式细胞术（EMA结合试验）检测红细胞膜骨架带3蛋白缺失程度与遗传性球形红细胞增多症（hereditary spherocytosis，HS）临床表现型的关系。方法分析258例未行脾切除术治疗的HS患者临床和实验室特征，评估EMA结合试验结果与贫血程度、溶血和造血代偿参数的关系。结果258例HS患者中，男128例，女130例，中位年龄23（2~70）岁。代偿性溶血91例、轻度贫血53例、中度贫血78例、重度贫血36例。EMA结合试验荧光强度减低中位数为29.97%（16.09%~47.34%），平均数为（29.70±6.28）%。荧光强度减低程度与红细胞平均体积呈负相关（r=−0.343，P<0.001），与红细胞平均血红蛋白浓度呈正相关（r=0.223，P<0.001），与网织红细胞比例（r=−0.015，P=0.813）和绝对值（r=0.080，P=0.198）均无明显相关性，与血清间接胆红素水平无明显相关（r=−0.009，P=0.902），与HGB水平无明显相关性（r=−0.067，P=0.280）。按EMA标记缺失程度四分位区间分组，不同EMA标记缺失组与HS贫血严重程度分组亦无明显相关性（C=0.150，P=0.746）。结论EMA结合试验结果与HS贫血程度无关。

CD44 as a Potential Screening Marker for Preliminary Differentiation Between Congenital Dyserythropoietic Anemia Type II and Hereditary Spherocytosis.

Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method. Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed. The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells. A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

Towards the harmonization of result presentation for the eosin-5'- maleimide (EMA) binding test in the diagnosis of hereditary spherocytosis.

Background: The EMA Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of 6 normal controls. The latter format has potential for universal reporting. Methods: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. Results:. The FC500 gave a mean ratio of 0.782 (SD = 0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and grey area ratio of 0.868 - 0.918. The Canto II gave a mean ratio of 0.774 (SD = 0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and grey area ratio of 0.859 - 0.925. Conclusion: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of grey-area data requires an assessment of patient's clinical presentation and family history or performing a family study. © 2014 Clinical Cytometry Society.

Screening For Hereditary Spherocytosis: EMA Binding Test and Flow Cytometric Osmotic Fragility Test Are Recommended, But Cryohemolysis Test Is Not Recommended

BackgroundThe hallmark of laboratory test for the diagnosis of hereditary spherocytosis (HS) has been osmotic fragility (OF) test. However, OF test gives false negative results in 10% - 20% of HS patients and false positive results in autoimmune hemolytic anemia (AIHA) patients. Currently, the eosin 5-maleimide (EMA) binding test and hypertonic cryohemolysis (HCH) test have been proposed as screening tests in recently published guidelines for the diagnosis of HS (Br J Haematol 2012;156:37-49). And the flow cytometric OF (FC OF) test is recently developed as an assay that can replace the classical OF test. In this study, we evaluated the performance of 3 screening tests (EMA binding test, FC OF test, and HCH test) for the diagnosis of HS. MethodsA total of 151 patients [32 for HS, 40 for AIHA, 40 for anemia of chronic disease (ACD), 39 for iron deficiency anemia (IDA)] and 140 normal controls (NCs) were enrolled in this study. EMA binding test, FC OF test, and HCH test were performed separately in each patient. Reference ranges (mean ± 1.96SD or 2.5th – 97.5th percentile) for each test were determined from 140 NCs. The cutoff value in EMA binding test (5th percentile) was estimated from 119 anemia patients (AIHA+ACD+IDA), and those in FC OF test (mean -1.65SD) and HCH test (95th percentile) were calculated from 140 NCs. Sensitivity, specificity, negative predictive values (NPV), positive predictive values (PPV), and accuracy of each test were calculated using these cutoff values and compared. Subsequently, receiver operating characteristics (ROC) analysis were performed to determine the best cutoff values of each test for the discrimination of HS from other anemia and area under curve (AUC) of three tests were compared. ResultsThe reference ranges of EMA binding test, FC OF test, and HCH test were determined as 86.9% - 118.68%, 6.6% - 71.6%, and 1.5% - 11.8%, respectively, and estimated cutoff values for the diagnosis of HS were calculated to be< 89.4% (EMA binding test),< 8.0% (FC OF test), and > 9.8% (HCH test). In the EMA binding test, HS patients showed significantly decreased EMA binding to RBC (median 71.3%) than AIHA (median 102.0%, P< 0.001), ACD (median 103.7%, P< 0.001), and IDA (median 100.5%, P< 0.001) patients. In the FC OF test, HS patients also showed significantly decreased residual RBC% (median 3.0%) than AIHA (median 44.6%, P< 0.001), ACD (36.0%, P< 0.001), IDA (median 59.4%, P< 0.001) patients and NCs (median 25.2%, P< 0.001). In the HCH test, HS patients showed significantly increased hemolysis (median 10.2%) than AIHA (median 3.6%, P< 0.001) and ACD (median 4.2%, P< 0.001) patients and NC (median 4.7%, P< 0.001), but IDA patients (median 16.4%) showed significantly increased hemolysis than HS patients (P = 0.027). The sensitivity/specificity/NPV/PPV/accuracy of three tests for the discrimination of HS from other anemia using estimated cutoff values mentioned above were calculated as 96.9%/95.0%/99.1%/83.8%/95.4% for EMA binding test, 84.4%/96.6%/95.8%/87.1%/94.0% for FC OF test, and 50.0%/73.1%/84.5%/33.3%/68.2% for HCH test. When the best cutoff values (≤ 85.0% for EMA binding test, ≤ 10.8% for FC OF test, > 7.1% for HCH test) obtained from ROC analysis are applied, both EMA binding test (AUC 0.996) and FC OF test (AUC 0.986) showed satisfactory performance for the discrimination of HS from other anemia. However, HCH test (AUC 0.703) showed significantly poor performance compared than EMA binding test (P< 0.001) and FC OF test (P< 0.001). ConclusionDespite the current guideline recommends EMA binding test and HCH test as screening tests for the diagnosis of HS, our present results demonstrated that both EMA binding test and FC OF test possess satisfactory performance for the diagnosis of HS but HCH test has relatively poor performance compared to the EMA binding test or FC OF test and failed to discriminate HS patients from IDA patients. These results partly contradict the recommendations from HS guideline and therefore, both EMA binding test and FC OF test is recommended as screening tests for the diagnosis of HS, but HCH test is not recommended. Disclosures:No relevant conflicts of interest to declare.

A new paradigm in the diagnosis of hereditary hemolytic anemia

A new paradigm in the diagnosis of hereditary hemolytic anemia

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis

The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.

Eosin-5-maleimide binding to band 3 and Rh-related proteins forms the basis of a screening test for hereditary spherocytosis.

Flow cytometric analysis of eosin-5-maleimide (EMA) binding to red cells is a screening test for the diagnosis of hereditary spherocytosis (HS). The present study used chemical modifications to determine the integral membrane proteins that react with EMA. The predominant interaction of EMA, contributing c. 80% of fluorescence, was with the epsilon-NH2 group of lysine in band 3 protein, as previously reported. The remainder of the EMA fluorescence was attributable to labelling of accessible sulfhydryl groups on intact red cells. This reaction was heat labile. Three molecules containing sulfhydryl groups were shown to be associated with the Rh blood group protein complex by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These were CD47 and the Rh-associated glycoprotein, both in the Mr 40-60 kD region, and the Rh blood group proteins in the 30-32 kD region. Immunoprecipitation, using specific monoclonal antibodies and antibody binding studies by flow cytometry, showed that the relative content of these three membrane proteins was lower in HS than normal red cells. Thus, the high predictive value of the EMA binding test for HS reflects changes in the relative amounts of the Rh-related integral membrane proteins as well as band 3 in HS red cells.