Influence of Standard Laboratory Procedures on Measures of Erythrocyte Damage

Lena Wiegmann,Oliver Speer,Jeroen S Goede,Burkhardt Seifert,Alissa Muller,Vartan Kurtcuoglu,Diane A De Zélicourt

doi:10.3389/fphys.2017.00731

Abstract

The ability to characterize the mechanical properties of erythrocytes is important in clinical and research contexts: to diagnose and monitor hematologic disorders, as well as to optimize the design of cardiovascular implants and blood circulating devices with respect to blood damage. However, investigation of red blood cell (RBC) properties generally involves preparatory and processing steps. Even though these impose mechanical stresses on cells, little is known about their impact on the final measurement results. In this study, we investigated the effect of centrifuging, vortexing, pipetting, and high pressures on several markers of mechanical blood damage and RBC membrane properties. Using human venous blood, we analyzed erythrocyte damage by measuring free hemoglobin, phosphatidylserine exposure by flow cytometry, RBC deformability by ektacytometry and the parameters of a complete blood count. We observed increased levels of free hemoglobin for all tested procedures. The release of hemoglobin into plasma depended significantly on the level of stress. Elevated pressures and centrifuging also altered mean cell volume (MCV) and mean corpuscular hemoglobin (MCH), suggesting changes in erythrocyte population, and membrane properties. Our results show that the effects of blood handling can significantly influence erythrocyte damage metrics. Careful quantification of this influence as well as other unwanted secondary effects should thus be included in experimental protocols and accounted for in clinical laboratories.

Highlights

Erythrocytes, or red blood cells (RBCs), constitute the majority of blood cellular components and are responsible for the vital transport of oxygen and carbon dioxide throughout the body
Our results show that high pressures, centrifuging, vortexing, and pipetting all yield increased levels of free hemoglobin, implying that the forces associated with these methods already translate into hemolytic damage
Since we did not observe an effect on mean cell volume (MCV) or mean corpuscular hemoglobin (MCH), the release of hemoglobin induced by vortexing seems to be governed by complete lysis of the cells, irrespective of their size

Summary

Introduction

Erythrocytes, or red blood cells (RBCs), constitute the majority of blood cellular components and are responsible for the vital transport of oxygen and carbon dioxide throughout the body. The mechanical properties and physical integrity of the erythrocyte plasma membrane are central to this function, allowing RBCs to undergo considerable deformations and travel through the smallest capillaries. Activation of Piezo due to mechanical loading leads to Ca2+ influx, which in turn triggers the dehydration of the cells. This mechanism of volume reduction in response to stress could improve the RBCs’ ability to travel through the smallest capillaries (Cahalan et al, 2015) and was hypothesized to promote oxygen/CO2 exchange in the periphery, which was observed following mechanical stimulation of RBCs (Rao et al, 2009). If the stresses acting on the cell exceed its loading capacity, pore formation (Zhao et al, 2006) or complete membrane rupture (Rand, 1964) occurs, and all cytosolic content including hemoglobin is released into the plasma

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