Reversible and Irreversible Sickling: a Distinction by Electron Microscopy

Abstract

Abstract Fixatives having either oxidizing or reducing properties were tested in a technic designed to preserve morphologic characteristics of circulating Hb SS erythrocytes. Red cells with cytoplasmic filaments presumably composed of deoxygenated Hb S were found at random by electron microscopy in all preparations. Glutaraldehyde provided the best preservation and was used in subsequent experiments. Venous blood from Hb SS patients was separated by ultracentrifugation into fractions rich in ISC and rich in non-ISC. The two types of erythrocytes, either oxygenated or deoxygenated, were morphologically distinct; and the aberrant appearance of ISC membranes suggested structural damage. Deoxygenation of non-ISC was associated with loose arrangements of filaments demonstrating a tendency to lie parallel within cell protuberances and in cells’ long axes. Cytoplasm of ISC, devoid of detail when oxygenated, acquired a characteristic filamentous fine structure when deoxygenated: the filament pattern implied either i) a molecular organization of hemoglobin within oxygenated ISC invisible in the electron microscope, or ii) filament formation along paths of least resistance in these irreversibly deformed cells. Behavior of ISC in a magnetic field favored the latter explanation. These observations are consistent with at least two sequences possibly associated with reversible sickling: filament formation leads to cell distortion, or cell distortion dictates filament arrangement. Both sequences may operate, each augmenting the other. In contrast, permanent deformation of cell shape known as irreversible sickling appears to be the morphologic contribution of membranes presumably damaged during periods of prolonged sequestration.