Mean corpuscular volume of control red blood cells determines the interpretation of eosin-5′-maleimide (EMA) test result in infants aged less than 6 months

Abstract

Eosin-5′-maleimide (EMA) binding test is a flow cytometric test used to detect hereditary spherocytosis (HS). To perform the test sample from patients, 5–6 reference samples of red blood are needed. Our aim was to investigate how the mean corpuscular volume (MCV) of red blood cells influences on the value of fluorescence of bounded EMA dye and how the choice of reference samples affects the test result. EMA test was performed in peripheral blood from 404 individuals, including 31 children suffering from HS. Mean fluorescence channel of EMA-RBCs was measured with Cytomics FC500 flow cytometer. Mean corpuscular volume of RBCs was assessed with LH750 Beckman Coulter. Statistical analysis was performed using Graph Pad Prism. The correlation Spearman coefficient between mean channel of fluorescence of EMA-RBCs and MCV was r = 0.39, p < 0.0001. Interpretation of EMA test depends on MCV of the reference samples. If reference blood samples have lower MCV than the patients MCV, EMA test result might be negative. Due to different MCV values of RBCs in infancy and ca. Three months later, EMA test in neonates might be interpreted falsely negative. Samples from children younger than 3 months old had EMA test result 86.1 ± 11.7 %, whereas same samples that analyzed 4.1 ± 2.1 later had results of 75.4 ± 4.5 %, p < 0.05. Mean fluorescence of EMA-bound RBC depends on RBC’s volume. MCV of reference samples affects EMA test results; thus, we recommend selection of reference samples with MCV in range of ±2 fL compared to MCV of patient RBC’s.

Improved harmonization of eosin‐5‐maleimide binding test across different instruments and age groups

“…However, they did elaborate on different values coming from different instruments. Other studies using a similar metric have cited cutoffs of 81-85% (18,19). From our data, there appears to be a very good correlation between the measurement of the footprint and the MCF ratio/percentage, indicating that the footprint is a valid metric.…”

“…However, the footprint data average was around 99.5% for each group, because the footprint was moved to fit the pattern of the normal and was generated using cord blood samples. Ciepela et al (18) also showed that mean fluorescence of EMA-bound RBC n/a 18.7 n/a n/a, not applicable. Foot print 5 analysis also included examination of a side scatter vs. EMA fluorescence plot also known as "footprint", location of the "footprint" is determined from a group of normal controls.…”

Eosin‐5’‐maleimide binding test—Do we use appropriate reference values to detect hereditary spherocytosis in neonates?

“…Eosin‐5’‐maleimide binding tests were performed as previously described . Briefly, 5 µL of whole blood from each sample was washed with saline and incubated in darkness at room temperature for 1 hour with 25 µL of EMA dye (0.5 mg/mL; Fluka, Gillingham, UK) with intermittent mixing.…”

“…It is more suitable to express EMA test results in percentage of fluorescence of control cells rather than mean fluorescence intensity, since different values of its intensity can be obtained using different flow cytometers or even samples of a dye prepared for staining. The cut‐off value for an EMA test to diagnose HS differs between laboratories and studied populations . Many studies do not include newborns to analyse EMA test specificity and sensitivity.…”

Evaluation of a hereditary spherocytosis screening algorithm by automated blood count using reticulocytes and erythrocytic parameters on the Sysmex XN‐series

“…In this study, EMA was performed by using "MCV matched" controls to reduce the risk of lower sensitivity in neonates. 10 Patients were classified as severe HS (Hb <8 g/dL; n = 1), moderate HS (Hb 8-12 g/dL; n = 5) and mild HS (Hb >12 g/dL; n = 14). In the mild group, three patients were splenectomized.…”