Eosin-5'-maleimide (EMA) is a fluorescent dye that is used to detect red cell membrane defects by flow cytometry. It is primarily utilized to diagnose hereditary spherocytosis (HS), which may be transfusion-dependent. In addition, reports have indicated the applicability of EMA to hemolytic disorders such as cryohydrocytosis (a type of hereditary stomatocytosis), Southeast Asian ovalocytosis, and congenital dyserythropoietic anemia type II [1]. Studies have also illustrated the use of EMA in the differential diagnosis of HS and hereditary pyropoikilocytosis [2]. The diagnosis of patient susceptibility to hemolytic disorders depends on qualitative parameters such as clinical presentation, family history, peripheral smear profile, and red blood cell (RBC) indices [3].

The RBC membrane is a lipid bilayer consisting of specific protein components such as α and β-spectrin, ankyrin, band 3, protein 4.1, protein 4.2, and actin. These proteins maintain the structural integrity of the RBC membrane and defects in the genes encoding these proteins decrease cell membrane integrity [4]. Red cell membrane disorders are also detected using osmotic fragility (OF) tests, including the NaCl-induced OF test (which measures the resistance of red cells to lysis after spiking the cell sample in NaCl solutions with different concentrations), glycerol lysis test (GLT), acidified GLT, and pink test [5]. The principle of the EMA-RBC binding test is based on the fact that EMA binds covalently with lysine-430 on the first extracellular loop of band 3 protein [6]. Inadequate expression of band 3 protein will result in reduced binding of EMA [6,7].

Patients with HS show reduced mean channel fluorescence (MCF) of EMA based on flow cytometry and reduced mean cell volume (MCV). Hunt et al. [1] described the normalization of EMA-RBC fluorescence data and determined that a cut-off ratio of 0.8 can be used to differentiate normal controls from HS patients. This ratio was calculated as the test MCF normalized by the mean of 6 normal control MCFs. Relative to other tests, the EMA binding test shows the greatest specificity and sensitivity (99.1% and 92.1%, respectively) for HS detection [8,9].

The aim of our study was to determine the fluorescence activity of EMA dye during flow cytometry in normal individuals over a period of one year. A total of 12 healthy volunteers were studied as controls.

The EMA binding test using flow cytometry is a rapid, sensitive, and reliable diagnostic aid [6]. Conventional diagnosis of hereditary RBC membrane disorders is labor-intensive, time-consuming, and has low sensitivity and specificity [10]. In contrast, the EMA test can be performed in less than 2 hours. Previous results have illustrated its high specificity (99.1%) and sensitivity (92.1%), indicating is it a useful diagnostic tool for red cell membrane disorders [8]. This method can be used to detect not only band 3 deficiencies, but also spectrin and protein 4.2 deficiencies [6].

Our study showed minimal changes in the MCF over a 6-month period for EMA. However, the stability of the EMA dye decreased continuously after 6 months, with a significant decrease relative to baseline levels at 8 months. Dye stability, dye concentration, incubation time, storage conditions of blood samples, and delay in flow cytometric analysis of EMA-labeled red cells each play a crucial role in the reproducibility of results. Reports on dye storage conditions have indicated that EMA dye stored at -20℃ remains stable over a period of 4 months, but storage at 4℃ for 1 week results in a rapid decrease in labeling efficiency [6]. However, in this study, the dye was stored at -80℃ over a period of 1 year. Previous studies have also illustrated that EMA binding test results are not influenced by shipping or storage for up to 6 days [8].

EMA is light-sensitive and is hydrolyzed in aqueous buffer. It is recommended that red cells are incubated in the dark for 1 hour at room temperature with intermittent mixing [3]. Although the EMA binding test provides quantitative results that aid in positive and negative discrimination of test results, EMA dye is relatively unstable as a working solution. In a study by Kedar et al., [7] the fluorescence intensity of the working solution remained stable for 4 months when stored at -80℃. However, our results indicate that it remains stable for a period of 6 months. There is a significant decrease in MCF beyond this period of time, after which the same dye should not be used.

In summary, the EMA binding test is a rapid, sensitive, specific, and reliable test for HS. However, despite the specificity of EMA for HS detection, it is important to consider a patient's clinical history, family history, etc. The results of the present study demonstrate high dye stability for up to 6 months, during which the dye can be optimally utilized. We recommend reconstitution of the dye for the test every 6 months and storage at -80℃ in dark conditions.

Box plot summarizing the MCF values for various samples over 12 months, estimated at 2-month intervals. Abbreviation: MCF, mean channel fluorescence.

Table 1 Hematological parameters for all donors.