Blood Research  2015;50(2):109-112

doi:10.5045/br.2015.50.2.109

Stability of eosin-5'-maleimide dye used in flow cytometric analysis for red cell membrane disorders.

Simmi MEHRA 1 ; Neetu TYAGI ; Pranav DORWAL ; Amit PANDE ; Dharmendra JAIN ; Ritesh SACHDEV ; Vimarsh RAINA

Affiliations

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Keywords

Flow Cytometry; Red Blood cells; Hereditary spherocytosis

Country

Republic of Korea

Language

English

Abstract

BACKGROUND: The eosin-5'-maleimide (EMA) binding test using flow cytometry is a common method to measure reduced mean channel fluorescence (MCF) of EMA-labeled red blood cells (RBCs) from patients with red cell membrane disorders. The basic principle of the EMA-RBC binding test involves the covalent binding of EMA to lysine-430 on the first extracellular loop of band 3 protein. METHODS: In the present study, the MCF of EMA was analyzed for samples derived from 12 healthy volunteers (controls) to determine the stability (i.e., the percentage decrease in fluorescence) of EMA over a period of 1 year. RESULTS: Comparison of periodical MCF readings over time, that is, at 2-month intervals, showed that there were no significant changes in mean channel fluorescence for up to 6 months; however, there was a significant decrease in MCF at 8 months. CONCLUSION: For optimal dye utilization, EMA remained stable only for up to 6 months. Therefore, we recommend reconstitution of the dye every 6 months when implementing this test and storage at -80degrees C in dark conditions.