Blood Res.  2015 Jun;50(2):109-112. 10.5045/br.2015.50.2.109.

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

• Mehra S
• Tyagi N
• Dorwal P
• Pande A
• Jain D
• Sachdev R
• Raina V

• KMID: 2172775
• DOI: http://doi.org/10.5045/br.2015.50.2.109

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.

Keyword

Flow Cytometry; Red Blood cells; Hereditary spherocytosis

MeSH Terms

Anion Exchange Protein 1, Erythrocyte
Cell Membrane*
Erythrocytes
Flow Cytometry
Fluorescence
Healthy Volunteers
Humans
Reading
Anion Exchange Protein 1, Erythrocyte

Figure

• 

  Fig. 1 Box plot summarizing the MCF values for various samples over 12 months, estimated at 2-month intervals. Abbreviation: MCF, mean channel fluorescence.

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