Dynamic fatigue measurement of human erythrocytes using dielectrophoresis.

Erythrocytes must undergo severe deformation to pass through narrow capillaries and submicronic splenic slits for several hundred thousand times in their normal lifespan. Studies of erythrocyte biomechanics have been mainly focused on cell deformability and rheology measured from a single application of stress and mostly under a static or quasi-static state using classical biomechanical techniques, such as optical tweezers and micropipette aspiration. Dynamic behavior of erythrocytes in response to cyclic stresses that contributes to the membrane failure in blood circulation is not fully understood. This paper presents a new experimental method for dynamic fatigue analysis of erythrocytes, using amplitude modulated electrokinetic force field in a microfluidic platform. We demonstrate the capability of this new technique using a low cycle fatigue analysis of normal human erythrocytes and ATP-depleted erythrocytes. Cyclic tensile stresses are generated to induce repeated uniaxial stretching and extensional recovery of single erythrocytes. Results of morphological and biomechanical parameters of individually tracked erythrocytes show strong correlations with the number of the loading cycles. Under a same strength of electric field, after 180 stress cycles, for normal erythrocytes, maximum stretch ratio decreases from 3.80 to 2.86, characteristic time of cellular extensional recovery increases from 0.16s to 0.37s, membrane shear viscosity increases from 1.0(µN/m)s to 1.6(µN/m)s. Membrane deformation in a small number of erythrocytes becomes irreversible after large deformation for about 200 cyclic loads. ATP-depleted cells show similar trends in decreased deformation and increased characteristic time with the loading cycles. These results show proof of concept of the new microfluidics technique for dynamic fatigue analysis of human erythrocytes. STATEMENT OF SIGNIFICANCE: Red blood cells (RBCs) experience a tremendous number of deformation in blood circulation before losing their mechanical deformability and eventually being degraded in the reticuloendothelial system. Prior efforts in RBC biomechanics have mostly focused on a single-application of stress, or quasi-static loading through physical contact to deform cell membranes, thus with limited capabilities in probing cellular dynamic responses to cyclic stresses. We present a unique electrokinetic microfluidic system for the study of dynamic fatigue behavior of RBCs subjected to cyclic loads. Our work shows quantitatively how the cyclic stretching loads cause membrane mechanical degradation and irreversibly deformed cells. This new technique can be useful to identify biomechanical markers for prediction of the mechanical stability and residual lifespan of circulating RBCs.