Tired and stressed: direct holographic quasi-static stretching of aging echinocytes and discocytes in plasma using optical tweezers [Invited].

Red blood cells (RBCs) undergo a progressive morphological transformation from smooth biconcave discocytes into rounder echinocytes with spicules on their surface during cold storage. The echinocytic morphology impacts RBCs' ability to flow through narrow sections of the circulation and therefore transfusion of RBC units with a high echinocytic content are thought to have a reduced efficiency. We use an optical tweezers-based technique where we directly trap and measure linear stiffness of RBCs under stress without the use of attached spherical probe particles or microfluidic flow to induce shear. We study RBC deformability with over 50 days of storage performing multiple stretches in blood plasma (serum with cold agglutinins removed to eliminate clotting). In particular, we find that discocytes and echinocytes do not show significant changes in linear stiffness in the small strain limit (∼20% change in length) up to day 30 of the storage period, but do find differences between repeated stretches. By day 50 the linear stiffness of discocytes had increased to approximately that measured for echinocytes throughout the entire period of measurements. These changes in stiffness corresponded to recorded morphological changes in the discocytes as they underwent storage lesion. We believe our holographic trapping and direct measurement technique has applications to directly control and quantify forces that stretch other types of cells without the use of attached probes.

A comparison of the effect of X and gamma irradiation on red cell storage quality.

BACKGROUND AND OBJECTIVES: Irradiation of red cell components is indicated for recipients at risk of transfusion-associated graft vs. host disease. Current technologies available comprise of a gamma (γ) or an x source of radiation. The benefits of x vs. γ include non-radioactivity and hence no decay of the source. We aimed to compare the effect of the two technologies on red cell component storage quality post-irradiation. MATERIALS AND METHODS: Paired units of red cell concentrates (RCC), neonatal red cell splits (RCS), red cells for intra-uterine transfusion (IUT) or neonatal exchange transfusion (ExTx) were either γ- or x-irradiated. Units were sampled and tested for five storage parameters until the end of shelf life. Equivalence analysis of storage quality parameters was performed for pairs of the same components (RCC, RCS, IUT or ExTx) that were either γ- or x-irradiated. RESULTS: Nearly all component comparisons studied showed equivalence between γ and x irradiation for haemolysis, ATP, 2,3-DPG, potassium release and lactate production. The exceptions found that were deemed non-equivalent were higher haemolysis with x irradiation for ExTx, lower 2,3-DPG with x irradiation for RCS irradiated early and higher ATP with x irradiation for IUT. However, these differences were considered not clinically significant. CONCLUSION: This study has demonstrated that a range of red cell components for use in different age groups are of acceptable quality following x irradiation, with only small differences deemed clinically insignificant in a few of the measured parameters.

A coarse-grained red blood cell membrane model to study stomatocyte-discocyte-echinocyte morphologies.

An improved red blood cell (RBC) membrane model is developed based on the bilayer coupling model (BCM) to accurately predict the complete sequence of stomatocyte-discocyte-echinocyte (SDE) transformation of a RBC. The coarse-grained (CG)-RBC membrane model is proposed to predict the minimum energy configuration of the RBC from the competition between lipid-bilayer bending resistance and cytoskeletal shear resistance under given reference constraints. In addition to the conventional membrane surface area, cell volume and bilayer-leaflet-area-difference constraints, a new constraint: total-membrane-curvature is proposed in the model to better predict RBC shapes in agreement with experimental observations. A quantitative evaluation of several cellular measurements including length, thickness and shape factor, is performed for the first time, between CG-RBC model predicted and three-dimensional (3D) confocal microscopy imaging generated RBC shapes at equivalent reference constraints. The validated CG-RBC membrane model is then employed to investigate the effect of reduced cell volume and elastic length scale on SDE transformation, to evaluate the RBC deformability during SDE transformation, and to identify the most probable RBC cytoskeletal reference state. The CG-RBC membrane model can predict the SDE shape behaviour under diverse shape-transforming scenarios, in-vitro RBC storage, microvascular circulation and flow through microfluidic devices.

Calibration of force detection for arbitrarily shaped particles in optical tweezers.

Force measurement with an optical trap requires calibration of it. With a suitable detector, such as a position-sensitive detector (PSD), it is possible to calibrate the detector so that the force can be measured for arbitrary particles and arbitrary beams without further calibration; such a calibration can be called an "absolute calibration". Here, we present a simple method for the absolute calibration of a PSD. Very often, paired position and force measurements are required, and even if synchronous measurements are possible with the position and force detectors used, knowledge of the force-position curve for the particle in the trap can be highly beneficial. Therefore, we experimentally demonstrate methods for determining the force-position curve with and without synchronous force and position measurements, beyond the Hookean (linear) region of the trap. Unlike the absolute calibration of the force and position detectors, the force-position curve depends on the particle and the trapping beam, and needs to be determined in each individual case. We demonstrate the robustness of our absolute calibration by measuring optical forces on microspheres as commonly trapped in optical tweezers, and other particles such a birefringent vaterite microspheres, red blood cells, and a deformable "blob".

Investigation of red blood cell mechanical properties using AFM indentation and coarse-grained particle method.

BACKGROUND: Red blood cells (RBCs) deform significantly and repeatedly when passing through narrow capillaries and delivering dioxygen throughout the body. Deformability of RBCs is a key characteristic, largely governed by the mechanical properties of the cell membrane. This study investigated RBC mechanical properties using atomic force microscopy (AFM) with the aim to develop a coarse-grained particle method model to study for the first time RBC indentation in both 2D and 3D. This new model has the potential to be applied to further investigate the local deformability of RBCs, with accurate control over adhesion, probe geometry and position of applied force. RESULTS: The model considers the linear stretch capacity of the cytoskeleton, bending resistance and areal incompressibility of the bilayer, and volumetric incompressibility of the internal fluid. The model's performance was validated against force-deformation experiments performed on RBCs under spherical AFM indentation. The model was then used to investigate the mechanisms which absorbed energy through the indentation stroke, and the impact of varying stiffness coefficients on the measured deformability. This study found the membrane's bending stiffness was most influential in controlling RBC physical behaviour for indentations of up to 200 nm. CONCLUSIONS: As the bilayer provides bending resistance, this infers that structural changes within the bilayer are responsible for the deformability changes experienced by deteriorating RBCs. The numerical model presented here established a foundation for future investigations into changes within the membrane that cause differences in stiffness between healthy and deteriorating RBCs, which have already been measured experimentally with AFM.