Evaluation of confocal microscopy as a diagnosis tool on red blood cell diseases

Abstract

In hereditary spherocytosis, mutations in red blood cell membrane proteins result in an overly rigid, misshapen cell whose deformability when traveling through the blood vessels is lost, causing severe anemia and splenomegaly, jaundice, and gallstones. In thalassemia, mutations in the globin genes can cause also severe anemia, skeletal and growth deficits and iron overload. Diagnosing these entities can be difficult due to the coexistence of other causes of anemia and blood transfusions, so complex molecular tests are required. In order to avoid these, we explored the possibility of using spectral confocal microscopy as a diagnostic tool for hereditary spherocytosis and thalassemia in pediatric patients. The red blood cell membrane was stained with different color dyes and immunolabels, to identify possible membrane defects expressed as differences in color and shape under a Leica TCS SP8. Staining the membrane and nuclei with lipophilic fluorescent dyes permitted the precise assessment of cell shape.