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Accompanying Hemoglobin Polymerization in Red Blood Cells in Patients with Sickle Cell Disease Using Fluorescence
Fernanda Aparecida Borges da Silva1,2, João Batista Florindo3, Amilcar Castro de Mattos1,4
1Departments of Pathology and Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas 13083-887, Brazil.
International Journal of Molecular Sciences
|November 27, 2024
Summary
Fluorescence lifetime imaging (FLIM) reveals intracellular hemoglobin polymerization in sickle cell disease (SCD) red blood cells (RBCs). This technique accurately distinguishes SCD patients from controls, offering a new tool for studying polymerization inhibitors.
Area of Science:
- Biomedical Optics
- Hematology
- Cellular Imaging
Background:
- Standard staining procedures often miss subtle intracellular changes.
- Fluorescence lifetime imaging (FLIM) shows promise in visualizing unstained cellular structures.
- Sickle cell disease (SCD) involves hemoglobin polymerization within red blood cells (RBCs).
Purpose of the Study:
- To investigate if FLIM can detect intracellular hemoglobin polymerization in RBCs of SCD patients.
- To compare FLIM findings in SCD patients with healthy controls.
- To assess FLIM's potential as a diagnostic and research tool for SCD.
Main Methods:
- Label-free auto-fluorescence FLIM was used on blood films from 45 SCD patients and 27 controls.
- Image analysis involved local binary pattern (LBP) transformation of FLIM data.
- Linear discriminant analysis (LDA) was employed for classification and accuracy assessment.
Main Results:
- Control RBCs showed homogeneous cytoplasm; SCD RBCs exhibited fluorescent foci indicative of polymerization.
- Sickled RBCs displayed larger, irregularly shaped fluorescent areas with increased FLIM values, interpreted as polymerized hemoglobin.
- Texture features from FLIM data distinguished non-sickled SCD RBCs from control cells, achieving 84.7% accuracy in LDA classification.
Conclusions:
- FLIM effectively visualizes the topography of hemoglobin polymerization in SCD.
- The findings support the two-step nucleation theory of polymerization.
- FLIM presents a valuable tool for investigating therapeutic strategies targeting hemoglobin polymerization.
