**1. Introduction**

The circulating cellular elements of blood consist of erythrocytes (red blood cells; RBC), leukocytes (white blood cells; WBC) and platelets. The hemorheology of these blood cells is unique in that these cells exist in a fluid phase subjected to variable, and often extreme, rheological shear stress, viscosity changes and biomechanical obstacles (e.g., capillaries and splenic filtration). Hemodynamically, shear stress is induced by the highly variable flow rate of blood within the ~100,000 kilometers of the human vasculature bed which encompasses both large arteries and veins to the capillary beds (**Figure 1A**) [1]. With an average resting cardiac output of approximately 5 L/min, blood flow in the largest artery (i.e., aorta) is approximately 50 cm/s while flow rates drop to only about 0.03 cm/s in the smallest capillaries and return to about 15–40 cm/s in the largest veins (e.g., superior and inferior vena cava) [1, 2]. In high flow conditions, RBC reside in the fast flowing central axial column of the vessel while WBC (and platelets) are located more peripherally

#### **Figure 1.**

*The physiology and morphology of the vascular bed and blood cells imparts unique rheological stress on circulating blood cells. Panel A: the vascular bed is composed of blood vessels of various sizes which create significant disparity in blood (fluid and cellular) velocity consequent to vessel diameter. The fluid flow induces rheological sheer stress while the vessel size can create biomechanical deformation of cellular elements. Panel B: shown are the general physical parameters of human RBC and WBC. Note that the biconcave RBC is a anuclear while within the WBC, the nucleus:cytoplasm (N:C) ratio of monocytes and lymphocytes are quite divergent. RBC cytoplasmic viscosity is primarily defined by hemoglobin while in WBC, in addition to the nucleus, the presence of granules and vacuoles also impact intracellular viscosity and the aggregate cellular deformability. Panel C: blood cell deformability is crucial during vascular flow due to the size disparity between red blood cells and various leukocytes (e.g., monocytes and lymphocytes) and the capillary (4–58 μm) and splenic interendothelial clefts (0.5 μm). Panels B,C are drawn approximately to scale.*

and prone to mechanical interaction with the endothelial cells lining the blood vessels. WBC also have adhesion molecules on their membrane and, if appropriate signals (e.g., inflammation) are present, they actively roll on the endothelial cells prior to attachment and extravasation (**Figure 1A,B**). Moreover, the viscosity of blood is also variable and is a function of, primarily, red blood cell (RBC) number and flow rate. At high RBC counts and high flow rates, blood is highly viscous while at low RBC counts and low flow rates (capillaries), blood viscosity is greatly reduced. Moreover, as shown in **Figure 1C**, the rheological stress is further exacerbated by the biomechanical stresses induced by the extreme disparity in the size of RBC (~8 μm) and WBC (10–25 μm) to the minimum diameter of the vascular

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transfusion [17–28].

*Assessing the Vascular Deformability of Erythrocytes and Leukocytes: From Micropipettes…*

capillary beds (4–5 μm) and splenic interendothelial clefts (0.5–1.0 μm) [3, 4]. Hence, consequent to both the shear forces, viscosity and biomechanical stresses placed on blood cells, a key biologic/physiologic requirement of both RBC and WBC within the vascular space is rheological deformability. Biomechanically, the intracellular viscosity and membrane rigidity of the RBC and WBC are the key factors in

For the anuclear RBC, intracellular viscosity is primarily determined by hemoglobin content (both absolute content and hemoglobin structure (**Figure 1B**)). RBC membrane deformability/flexibility is primarily imparted by the cytoskeletal structure of the cells and, to a lesser extent, the composition of the bilayer itself (lipid species, protein content, integral versus peripheral membrane proteins, and carbohydrates). For normal RBC the intra- and inter-individual variability of both intracellular viscosity is relatively invariant; however, genetic mutations affecting hemoglobin structure (e.g., HbS, α and β thalassemia, HbE mutations) will dramatically affect both hemoglobin content and the viscosity of the hemoglobin itself. Similarly, the cytoskeletal structure of normal red blood cells is both well characterized and consistent within humans. But, as with hemoglobin variants, mutations in any component of the cytoskeleton can dramatically affect the discoid shape of the RBC and result in size changes and/or altered rigidity or stability of the cytoskeleton and cell itself. Indeed, numerous studies have documented that changes in either the hemoglobin content or structure (the major determinant of viscosity) or mutations to cytoskeletal components (the major determinant of membrane rigidity) can exert significant effects on RBC deformability, biologic function and in vivo circulation. In evidence of this, both biological conditions and pharmacologic agents that affect hemoglobin content and/or viscosity or the RBC cytoskeleton alter cellular deformability and have profound in vivo and in vitro effects on RBC function and survival [5–16]. Indeed, RBC deformability can be a diagnostic indicator of RBC abnormalities and the quality of stored RBC prior to

Intracellular viscosity and membrane structure are similarly key to the rheological deformability of WBC. However, in contrast to RBC, WBC intracellular viscosity is more complex and affected by multiple components including the: nuclear to cytoplasm (N:C) ratio; intracellular granule composition; presence of cytoplasmic vacuoles; as well as the activation state of the immune cell (**Figure 1B**) [28–30]. Similarly, membrane rigidity is also more complex due to: abundance of membrane proteins and protein rafts; changes in protein structure and polymerization consequent to immune activation; and the variability of the membrane and cytoskeletal protein composition of immune cell populations (e.g., monocytes, lymphocytes, granulocytes) and subsets (e.g., T cells versus B cells; CD4+ versus CD8+ T cells; NK cells) [30–35]. Perhaps surprisingly, despite the biologic importance of its rheological deformability within the vasculature, WBC deformability is both poorly defined and much less understood. Indeed, previous studies on WBC have most commonly defined "deformability" as cellular shape change or spreading under extrinsic suction (e.g., micropipette aspiration), compression pressure (e.g., centrifugation and cell poker/probe), or upon activation induced motility [30–32, 34–36]. However, vascular deformability is vastly different from cellular shape change or spreading which are most commonly induced by immune cell activation and, importantly, the actual loss of vascular rheologically-mediated (i.e., fluid motion and spatial confinement) deformability. The paucity of data relating to vascular deformability of WBC has, in large part, been due to the absence of suitable tools for measuring deformability across the broad range of cell types encompassed within leukocyte population. However, the complexity of the leukocyte population and resultant changes in rheological deformability upon activation (e.g., granule

*DOI: http://dx.doi.org/10.5772/intechopen.90131*

imparting their vascular rheological deformability.

#### *Assessing the Vascular Deformability of Erythrocytes and Leukocytes: From Micropipettes… DOI: http://dx.doi.org/10.5772/intechopen.90131*

capillary beds (4–5 μm) and splenic interendothelial clefts (0.5–1.0 μm) [3, 4]. Hence, consequent to both the shear forces, viscosity and biomechanical stresses placed on blood cells, a key biologic/physiologic requirement of both RBC and WBC within the vascular space is rheological deformability. Biomechanically, the intracellular viscosity and membrane rigidity of the RBC and WBC are the key factors in imparting their vascular rheological deformability.

For the anuclear RBC, intracellular viscosity is primarily determined by hemoglobin content (both absolute content and hemoglobin structure (**Figure 1B**)). RBC membrane deformability/flexibility is primarily imparted by the cytoskeletal structure of the cells and, to a lesser extent, the composition of the bilayer itself (lipid species, protein content, integral versus peripheral membrane proteins, and carbohydrates). For normal RBC the intra- and inter-individual variability of both intracellular viscosity is relatively invariant; however, genetic mutations affecting hemoglobin structure (e.g., HbS, α and β thalassemia, HbE mutations) will dramatically affect both hemoglobin content and the viscosity of the hemoglobin itself. Similarly, the cytoskeletal structure of normal red blood cells is both well characterized and consistent within humans. But, as with hemoglobin variants, mutations in any component of the cytoskeleton can dramatically affect the discoid shape of the RBC and result in size changes and/or altered rigidity or stability of the cytoskeleton and cell itself. Indeed, numerous studies have documented that changes in either the hemoglobin content or structure (the major determinant of viscosity) or mutations to cytoskeletal components (the major determinant of membrane rigidity) can exert significant effects on RBC deformability, biologic function and in vivo circulation. In evidence of this, both biological conditions and pharmacologic agents that affect hemoglobin content and/or viscosity or the RBC cytoskeleton alter cellular deformability and have profound in vivo and in vitro effects on RBC function and survival [5–16]. Indeed, RBC deformability can be a diagnostic indicator of RBC abnormalities and the quality of stored RBC prior to transfusion [17–28].

Intracellular viscosity and membrane structure are similarly key to the rheological deformability of WBC. However, in contrast to RBC, WBC intracellular viscosity is more complex and affected by multiple components including the: nuclear to cytoplasm (N:C) ratio; intracellular granule composition; presence of cytoplasmic vacuoles; as well as the activation state of the immune cell (**Figure 1B**) [28–30]. Similarly, membrane rigidity is also more complex due to: abundance of membrane proteins and protein rafts; changes in protein structure and polymerization consequent to immune activation; and the variability of the membrane and cytoskeletal protein composition of immune cell populations (e.g., monocytes, lymphocytes, granulocytes) and subsets (e.g., T cells versus B cells; CD4+ versus CD8+ T cells; NK cells) [30–35]. Perhaps surprisingly, despite the biologic importance of its rheological deformability within the vasculature, WBC deformability is both poorly defined and much less understood. Indeed, previous studies on WBC have most commonly defined "deformability" as cellular shape change or spreading under extrinsic suction (e.g., micropipette aspiration), compression pressure (e.g., centrifugation and cell poker/probe), or upon activation induced motility [30–32, 34–36]. However, vascular deformability is vastly different from cellular shape change or spreading which are most commonly induced by immune cell activation and, importantly, the actual loss of vascular rheologically-mediated (i.e., fluid motion and spatial confinement) deformability. The paucity of data relating to vascular deformability of WBC has, in large part, been due to the absence of suitable tools for measuring deformability across the broad range of cell types encompassed within leukocyte population. However, the complexity of the leukocyte population and resultant changes in rheological deformability upon activation (e.g., granule

*Current and Future Aspects of Nanomedicine*

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**Figure 1.**

and prone to mechanical interaction with the endothelial cells lining the blood vessels. WBC also have adhesion molecules on their membrane and, if appropriate signals (e.g., inflammation) are present, they actively roll on the endothelial cells prior to attachment and extravasation (**Figure 1A,B**). Moreover, the viscosity of blood is also variable and is a function of, primarily, red blood cell (RBC) number and flow rate. At high RBC counts and high flow rates, blood is highly viscous while at low RBC counts and low flow rates (capillaries), blood viscosity is greatly reduced. Moreover, as shown in **Figure 1C**, the rheological stress is further exacerbated by the biomechanical stresses induced by the extreme disparity in the size of RBC (~8 μm) and WBC (10–25 μm) to the minimum diameter of the vascular

*The physiology and morphology of the vascular bed and blood cells imparts unique rheological stress on circulating blood cells. Panel A: the vascular bed is composed of blood vessels of various sizes which create significant disparity in blood (fluid and cellular) velocity consequent to vessel diameter. The fluid flow induces rheological sheer stress while the vessel size can create biomechanical deformation of cellular elements. Panel B: shown are the general physical parameters of human RBC and WBC. Note that the biconcave RBC is a anuclear while within the WBC, the nucleus:cytoplasm (N:C) ratio of monocytes and lymphocytes are quite divergent. RBC cytoplasmic viscosity is primarily defined by hemoglobin while in WBC, in addition to the nucleus, the presence of granules and vacuoles also impact intracellular viscosity and the aggregate cellular deformability. Panel C: blood cell deformability is crucial during vascular flow due to the size disparity between red blood cells and various leukocytes (e.g., monocytes and lymphocytes) and the capillary (4–58 μm) and splenic interendothelial clefts (0.5 μm). Panels B,C are drawn approximately to scale.* release) potentially arising in peripheral blood WBC may be of clinical importance as a biomarker of acute or chronic immune activation.
