Impact of the Oscillating Bead Size and Shape on Induced Mechanical Stress on Red Blood Cells and Associated Hemolysis in Bead Milling

Abstract

Background: While in circulation, red blood cells (RBC) need to elastically undergo large deformations without lysing, an ability that may be compromised by cell membrane damage. Such can be tested in vitro by subjecting an RBC sample to external mechanical stress, e.g. through bead milling or oscillation of an object in a sample. In addition to controlling frequency and duration of oscillations, this approach can be further tailored by bead selection/design.Objective: This work studies effects of different beads in creating qualitatively as well as quantitatively different shear stresses when oscillated in a sample containing RBC.Methods: Identical, diluted RBC samples were stressed via bead milling using different beads, with hemolysis profiles developed in each case.Results: Bead dimensions significantly impacted induced stress, both in magnitude as well as type, as reflected by hemolysis under respective conditions. Specifically, cell protection (from lysis) afforded by albumin (when present in the medium) showed a complex dependence on bead length and diameter, with the effect seemingly indicative of multiple combined stresses induced by the bead oscillation. The effect is described as an interplay of stresses generated in bead wake in combination with those generated in the annulus. Conclusion:Bead oscillation based mechanical fragility (MF) profiling of RBC presents a useful tool for exploring effects of different shear stress types for various potential applications for assessment of blood damage, and particularly of sub-hemolytic red cell damage.

Use of Packed Red Blood Cell Mechanical Fragility to Indicate Transfusion Outcomes

“…It is not yet known what flow, cell-cell, and/or cell-wall interactions in circulation may be responsible for pRBC degradation post transfusion, nor their respective potential extents, nor which in-vitro MF testing regimes/conditions may best simulate the relevant physiological stresses that induce hemolysis. Bead milling with cylindrical beads had been shown to generate several different types of mechanical stress in liquid medium, thus allowing testing of RBC MF under substantially different conditions [59, 64]. In this study, MF of each sample was tested using several different approaches.…”

“…Bead milling with cylindrical beads had been shown to generate several different types of mechanical stress in liquid medium, thus allowing testing of RBC MF under substantially different conditions [59,64]. In this study, MF of each sample was tested using several different approaches.…”

“…These two stress application methods (described further below) involve different bead and sample tube geometries, necessitating the use of significantly higher oscillating frequencies in the cam-based system to achieve cumulative hemolysis comparable to that in the electromagnetic setup. Speed of the flow through the annulus of an oscillating cylindrical bead in the tube which is dependent on the speed of the bead and the size of the gap between the bead surface and the wall of the tube, has been shown to be determinatives of generated flow shear stress and resultant hemolysis [59]. Presence of the air with the sample in the cam-based system, as compared to the proprietary electromagnetic approach where no such is present, can potentially add additional stress to cells at the liquid-gas interface.…”

“…"Legacy" cam-based system: This approach utilized a commercial cam-based vertical bead mill (Qiagen GmbH, Redwood City, USA) for stress application, combined with a spectrophotometric analysis of supernatants as described previously [54,55], except with custom-made stainless steel cylindrical beads. Depending on their length and diameter, cylindrical beads were previously shown to generate categorically different types of mechanical stress significantly dependent on erythrocyte environment [59]. Bead dimensions (7 mm in diameter and 18 mm in length) used in this work had been selected so as to provide a substantial difference in induced hemolysis between samples supplemented and nonsupplemented with albumin (SA; Bovine Serum Albumin; RPI Corp, Mt.…”

Red Blood Cell Mechanical Fragility as a Potential Predictor of Long-Term Hemolysis from Ventricular Assist Devices

“…Physiological concentrations of albumin had been shown previously to significantly reduce RBC propensity to hemolyze 34 . Under some stress application regimes, its presence was suggested to alter bead end and annulus flows, resulting in quantitatively and qualitatively altered mechanical stress, with consequently altered RBC response 39 . Thus, while such supplementation was impactful for stress generated with the parameters of regime 2, it had minimal to no impact on stress when generated with the parameters of regime 1.…”