Sergey S. Shevkoplyas

Washing stored red blood cells in an albumin solution improves their morphologic and hemorheologic properties

Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables.

Effect of osmolality on erythrocyte rheology and perfusion of an artificial microvascular network.

Plasma sodium concentration is normally held within a narrow range. It may however vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5 g/dL), resulting in supernatant osmolalities of 179 ± 4, 213 ± 1, 283 ± 2, 354 ± 3, and 423 ± 5 mOsm/kg H2O. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5s(-1) and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290 mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted toward hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290 mOsm/kg H2O and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease and may have significant implications for the management of osmotic homeostasis in clinical practice.

Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network.

The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN).

A rapid paper-based test for quantifying sickle hemoglobin in blood samples from patients with sickle cell disease

Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per‐test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper‐based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 μL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper‐based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R2u2009=u20090.86) and strong agreement (standard deviation of differenceu2009=u20097%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, nu2009=u20095). The paper‐based assay can be completed in less than 35 min and has a per‐test cost less than

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function.

0.25. The assay is accurate across a wide range of HbS levels (10–97%) and hemoglobin concentrations (5.6–12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper‐based %HbS assay. The paper‐based test could improve clinical care for SCD, particularly in resource‐limited settings, by enabling more rapid and less expensive %HbS monitoring. Am. J. Hematol. 90:478–482, 2015.

Quantifying morphological heterogeneity: a study of more than 1 000 000 individual stored red blood cells

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.1-fold, NADPH-oxidase-dependent increase in intracellular ROS that, in turn, trigger multiple downstream cascades leading to caspase-3 activation, ATP release, and increased band 3 phosphorylation. Functionally, using 2D microchannels to assess membrane deformability, GPS-ligated RBCs travel 33% slower than control RBCs, and lipid mobility was hindered by 10% using fluorescence recovery after photobleaching (FRAP). These outcomes were preventable by pretreating RBCs with cell-permeable ROS scavenger glutathione monoethyl ester (GSH-ME). Our results obtained in vitro using anti-GPA antibodies were validated using complement-altered RBCs isolated from control and septic patients. Our results suggest that during inflammatory conditions, circulating RBCs significantly contribute to capillary flow dysfunctions, and constitute an important but overlooked source of intravascular ROS and ATP, both critical mediators responsible for endothelial cell activation, microcirculation impairment, platelet activation, as well as long-term dysregulated adaptive and innate immune responses.

Influence of red blood cell aggregation on perfusion of an artificial microvascular network

The morphology of red blood cells (RBCs) deteriorates progressively during hypothermic storage. The degree of deterioration varies between individual cells, resulting in a highly heterogeneous population of cells contained within each RBC unit. Current techniques capable of categorizing the morphology of individual stored RBCs are manual, laborious and error‐prone procedures that limit the number of cells that can be studied. Our objective was to create a simple, automated system for high‐throughput RBC morphology classification.

Optimal hematocrit in an artificial microvascular network

RBCs suspended in plasma form multicellular aggregates under low‐flow conditions, increasing apparent blood viscosity at low shear rates. It has previously been unclear, however, if RBC aggregation affects microvascular perfusion. Here, we analyzed the impact of RBC aggregation on perfusion and ‘capillary’ hematocrit in an AMVN at driving pressures ranging from 5 to 60 cm H2O to determine if aggregation could improve tissue oxygenation.

Towards bedside washing of stored red blood cells: a prototype of a simple apparatus based on microscale sedimentation in normal gravity

Higher hematocrit increases the oxygen‐carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist.

Influence of feeding hematocrit and perfusion pressure on hematocrit reduction (Fåhræus effect) in an artificial microvascular network

Infusion of by‐products of red blood cell (RBC) storage‐induced degradation as well as of the residual plasma proteins and the anticoagulant‐preservative solution contained in units of stored blood serve no therapeutic purpose and may be harmful to some patients. Here, we describe a prototype of a gravity‐driven system for bedside washing of stored RBCs.