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

Simmi Mehra; Neetu Tyagi; Pranav Dorwal; Amit Pande; Dharmendra Jain; Ritesh Sachdev; Vimarsh Raina

doi:10.5045/br.2015.50.2.109

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Original Article

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Blood Res 2015; 50(2):

Published online June 25, 2015

https://doi.org/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^*, Neetu Tyagi, Pranav Dorwal, Amit Pande, Dharmendra Jain, Ritesh Sachdev, and Vimarsh Raina

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Department of Pathology, Medanta, The Medicity, Gurgaon, India.

Correspondence to : Correspondence to Simmi Mehra, Ph.D. Department of Pathology, Medanta, The Medicity, Sec-38, Gurgaon, Haryana 122001, India. Tel: +91-8744927108, Fax: +91-124-4834111, mehrasimmi80@gmail.com

Received: April 11, 2015; Revised: May 30, 2015; Accepted: June 4, 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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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 -80℃ in dark conditions.

Keywords Flow Cytometry, Red Blood cells, Hereditary spherocytosis

Article

Original Article

Blood Res 2015; 50(2): 109-112

Published online June 25, 2015 https://doi.org/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^*, Neetu Tyagi, Pranav Dorwal, Amit Pande, Dharmendra Jain, Ritesh Sachdev, and Vimarsh Raina

Department of Pathology, Medanta, The Medicity, Gurgaon, India.

Correspondence to: Correspondence to Simmi Mehra, Ph.D. Department of Pathology, Medanta, The Medicity, Sec-38, Gurgaon, Haryana 122001, India. Tel: +91-8744927108, Fax: +91-124-4834111, mehrasimmi80@gmail.com

Received: April 11, 2015; Revised: May 30, 2015; Accepted: June 4, 2015

Abstract

Background

Methods

Results

Conclusion

Keywords: Flow Cytometry, Red Blood cells, Hereditary spherocytosis

Abstract

Fig 1.

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Figure 1.

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

Blood Research 2015; 50: 109-112https://doi.org/10.5045/br.2015.50.2.109

Table 1 . Hematological parameters for all donors..

Abbreviations: Hb, Hemoglobin; RBC, Red Blood Cell Count; MCV, Mean Cell Volume; MCH, Mean Corpuscular Hemoglobin; RDW, Red Blood Cell Distribution Width; MCHC, Mean Corpuscular Hemoglobin Concentration..