Facilitating EMA binding test performance using fluorescent beads combined with next-generation sequencing.

Area of Science:

• Hematology
• Clinical Diagnostics
• Genetics

Background:

• Hereditary spherocytosis (HS) is a common hemolytic disorder.
• The eosin-5'-maleimide (EMA) binding test is a standard diagnostic tool for HS.
• Current EMA testing relies on healthy blood donors as controls, which can be improved.

Purpose of the Study:

• To evaluate a modified EMA binding test using fluorescent beads instead of healthy controls.
• To establish a diagnostic threshold for the modified EMA test based on genetic variants.
• To assess the accuracy of the modified EMA test compared to osmotic gradient ektacytometry.

Main Methods:

• Targeted next-generation sequencing was performed on individuals suspected of HS.
• A modified EMA binding test using fluorescent rainbow beads as reference was conducted.
• Osmotic gradient ektacytometry was used for comparative analysis.

Main Results:

• A diagnostic threshold of EMA ≥ 43.6 AU was determined using genetic variants, achieving 93.4% accuracy.
• The modified EMA test demonstrated high accuracy, comparable to osmotic gradient ektacytometry (98.7%).
• The study identified 79 different genetic variants, including 43 novel mutations, associated with HS.

Conclusions:

• The modified EMA binding test offers a simplified and accurate diagnostic approach for hereditary spherocytosis.
• Using fluorescent beads and genetic variants as references enhances the reliability of the EMA test.
• This improved diagnostic method aids in identifying HS with high precision.