Comparative Assessment of Hemin-Induced Alterations in Red Blood Cells Using Light Microscopy and Atomic Force Microscopy

Aim of the study : to compare the capabilities of light microscopy and atomic force microscopy (AFM) in assessing the morphological features of red blood cell (RBC) damage. Materials and Methods . The study used blood from three healthy male donors with a mean age of 36 ± 5 years. RBCs were incubated with a hemin solution for 60 minutes. After incubation, blood smears were prepared. Morphological analysis was performed using an Olympus CX41 light microscope. The RBC topography was assessed using AFM on an NTEGRA BIO instrument in semi-contact mode. The distribution of morphological forms, cell diameter, and cell height were analyzed. Results . A predominance of discocytes was observed in the control group, using both methods (light microscopy — 94 ± 2 %, AFM — 94 ± 1 %). After exposure to hemin 96 ± 2 % of the cells lost their central pallor and were classified as planocytes according to light microscopy. However, AFM revealed that these cells were spheroechinocytes (98 ± 1 %), characterized by a decrease in diameter from 7.6 ± 0.6 μm to 5.9 ± 0.4 μm ( p = 0.008), an increase in height from 356 ± 22 nm to 737 ± 148 nm ( p = 0.047), and the presence of multiple protrusions on the membrane. Conclusion . Light microscopy reveals the loss of central pallor but does not allow for verification of the true three-dimensional shape of the damaged cells. AFM is necessary to refine the morphological classification and to quantitatively assess height and diameter. Thus, light microscopy is suitable for initial screening, while AFM is suitable for detailed assessment of RBC morphological alterations.