(19e) Identification of Rigid Red Blood Cell Population Rigidity in Sickle-Cell Disease Patients Using a Parameterization Model of Ektacytometry

One of the key characteristics of sickle-cell disease (SCD) is the increased rigidity of the red blood cell (RBC) membrane. The decreased flexibility of individual RBCs contributes to the reduction in lifespan of the cell and is well known to be a primary cause of vassocclusion, which lead to painful crisis episodes and other more serious complications in SCD patients. Cell deformability is often overlooked as a critical factor in predicting disease severity. Recent investigations have shown that decreased RBC deformability reduce the ability of leukocytes to effectively bind to the vascular wall under blood flow – hinting that RBC rigidity plays a larger role in hemodynamics and potentially the well-being of SCD patients beyond simply instigating vassocclusion. Ektacytometry is a technique that has developed over the past few decades for measuring RBC deformability. By implementing laser diffraction viscometry, ektacytometry measurements can depict how RBC populations deform under applied shear stress. Deformation in the cell diffraction patterns is translated as an elongation index (EI) ratio. Although a robust technique, it is not perfect, i.e. ektacytometry will only render an average measurement of RBC deformability in a given sample. Diffraction patterns of both rigid RBCs and healthy soft RBCs are mixed together, thus rendering an average measurement of sample deformability and underestimating the bulk rigidity of the rigid RBC population in the whole blood sample.

Here, we present an approach to estimate rigidity of HbS cells, i.e. rigid RBCs, in SCD via the artificial rigidification of healthy RBCs at different degrees of stiffnesses which are then mixed into whole blood samples with different fractions of healthy soft RBCs. Healthy donor, i.e. non-SCD donor, RBCs are incubated with predetermined concentrations of tert-butly hydroperoxide to induce RBC membrane rigidification. Whole blood samples with controlled amounts of rigid RBCs are measured via ektacytometry. We then parameterize these ektacytometry measurements to develop a method that predicts the bulk rigidity of the rigid RBC population in a SCD patient blood sample. Using hemoglobin electrophoresis, we determine rigid fraction of rigid RBCs in SCD patient blood samples. We find that bulk ektactymoetry measurements typically underestimate the stiffness of rigid RBCs. In some SCD patients, the stiffness of rigid RBCs is often underestimated by ~4 times fold when compared to a bulk ektacytometry measurement.

This novel method can estimate bulk rigidity of rigid RBC populations with greater certainty, which can serve as a potential platform for more accurately estimating rigidity of rigid RBC populations in a patient sample and thus better understanding disease severity in a patient and deciding optimal treatment options.