**5. Measurement techniques for blood rheology**

#### **5.1. Blood viscometer and ektacytometry**

Blood viscometer measures the viscosity of blood over a wide range of shear rates. Blood viscometer controls either shear stress or shear rate of blood using rational objects. Stresscontrolled blood viscometer applys a constant torque which corresponds to constant rotational speed in a well-designed rotational rheometer. In a rate-controlled system, applied torque is controlled by a stress-sensing device so that a constant rotational speed is achieved. Viscometers can be classified by the cylinder shape: a concentric cylinder, a cone plate, and a parallel plate viscometer (Fig. 9).

Cylinder-type viscometer uses two concentric cylinders: a rotational inner cup and a stationary outer cylinder. Time-independent shear rate can be precisely measured by

concentric cylinder viscometer (Nguyen and Boger,1987). Cone and plate viscometer rotates an inverted cone having very shallow angle (~ 5°); the shear rate under the plate is maintained consistently and independent of a flow curve. Parallel plate viscometer is a simplified version of the cone and plate viscometer and has a advantage of flexible space between two parallel plates. The viscous fluid can confined and rotated in narrow space between two circular parallel plates (Gent,1960).

**Figure 9.** Schematic diagrams of typical viscometers. (a) Concentric cylinder viscometer (or Couette viscometer), (b) cone and plate viscometer, and (c) parallel plate viscometer. (d) Experimental setup of ektacytometer.

Ektacytometer employes a laser diffraction technique with blood viscometer in order to measure RBC deformabiltiy. Conventional blood viscometer applys controlled shear stress to the RBCs in the blood viscometer, and deformability of RBCs can be measured from laser diffraction pattern. Ektacytometer consists of a concentric rotational outer cup and a stationaly inner cylinder; outer cup produces varying shear stress field on blood (Fig. 9d). Through the measurement of diffraction patterns of the laser passing through the blood, RBC deformability can be obtained. The RBC deformation is quantitatively calculated from the scattered laser beam intensity pattern. Under a certain shear rate, isointensity curves in the intensity pattern of the scattered beam will show elliptical shapes, which represent elliptically deformed RBC population (Bessis, Mohandas et al.,1980). From the measured isointensity curves, a deformaion index (DI) of RBCs is calculated as

, *l s DI l s* (9)

Measurement Techniques for Red Blood Cell Deformability: Recent Advances 181

μm. Microfludic device reduced space, labor, and measurement time on numerous experiments, and also enabled precise control and manipulation of the small volume of samples. Microfludic device has been used to study the deformabiltiy of RBCs. Microfluidic channel with a few micrometer diameter mimics micro-capillary structure in blood circulation system. Rheological behaviors of *Pf*-RBCs were studies in microfludic devices (Shelby, White et al.,2003). Microfludic device was used to induce large deformation of

RBCs and its mechanical behavior was studied (Fig. 10) (Li, Lykotrafitis et al.,2007) .

**Figure 10.** (a-d) Snapshot showing the fluidization of a healthy RBC when it passes through a microfluidic channel. Reproduced, with permission, from (Li, Lykotrafitis et al.,2007).

Silva, Park et al.,in press).

**5.3. Filtration test** 

**6.1. Temperature** 

**0 s 0.4 s 0.8 s 1.4 s**

For the study of sickle cell disease, microfluidic device has been used to measure the resistance change rate of blood flow under the sudden change of oxygen concentration (Wang, Ding et al.,2011). Recently, microfludic channels with obstracles have measured the deformabiltiy of malaria infected RBCs in high throughput (Bow, Pivkin et al.,2011; Diez-

Filtration test measures RBC deformabiltiy using a membrane filter with holes of diameter of 3-5 μm (JANDL, SIMMONS et al.,1961). By applying a negative pressure, whole blood is subject to pass through holes in the membrane filter. The deformability of RBCs can affect the speed of flow. RBC deformabiltiy can be calculated from either the flow time or the volume of blood filteres in a certain amount of time (~1 min). Since the filteration test requires for a relatively simple instrument and provides clinically relevant results with high reproducibility, it has been widely used in various studies related to RBC deformability, including the effects of diabetes (Juhan, Buonocore et al.,1982), spesis (Baskurt, Gelmont et al.,1998), sickle cell

disease (JANDL, SIMMONS et al.,1961), and oxygen radical (Srour, Bilto et al.,2000).

Mechanical properties of RBCs is crucial for cell physiology of RBCs. This essential

Temperature plays important roles in RBC deformabilty. The elastic properties of RBC membrane were investigated as function of temperature using the micropipette aspiration

**6. Pathophysiological coditions affecting RBC deformability** 

deformability is in turn affected by various physiological and pathological cues.

where *l* and *s* are distances along the long- and short- axes in the elliptical isointensity curves. DI values are measured at different angular velocities (and thus different shear rate) of the outer cyliner in the ektacytometer. Ektacytometer is a simple and effective technique to measure the deformability of RBC population, and it has been widely used for the study pathophysiology of RBCs. Abnormal deformability in RBCs from patients with hereditary pyropoikilocytosis, hereditary spherocytosis, and Hb CC disease were studies by ektacytometer (Mohandas, Clark et al.,1980).

#### **5.2. Microfluidic device technique**

Microfluidic device has emerged as a promising tool to precisely control fluids with small volumes of fluid containing samples and reagents in channels with dimensions of 10-100 μm. Microfludic device reduced space, labor, and measurement time on numerous experiments, and also enabled precise control and manipulation of the small volume of samples. Microfludic device has been used to study the deformabiltiy of RBCs. Microfluidic channel with a few micrometer diameter mimics micro-capillary structure in blood circulation system. Rheological behaviors of *Pf*-RBCs were studies in microfludic devices (Shelby, White et al.,2003). Microfludic device was used to induce large deformation of RBCs and its mechanical behavior was studied (Fig. 10) (Li, Lykotrafitis et al.,2007) .

**Figure 10.** (a-d) Snapshot showing the fluidization of a healthy RBC when it passes through a microfluidic channel. Reproduced, with permission, from (Li, Lykotrafitis et al.,2007).

For the study of sickle cell disease, microfluidic device has been used to measure the resistance change rate of blood flow under the sudden change of oxygen concentration (Wang, Ding et al.,2011). Recently, microfludic channels with obstracles have measured the deformabiltiy of malaria infected RBCs in high throughput (Bow, Pivkin et al.,2011; Diez-Silva, Park et al.,in press).

## **5.3. Filtration test**

180 Blood Cell – An Overview of Studies in Hematology

ektacytometer.

between two circular parallel plates (Gent,1960).

concentric cylinder viscometer (Nguyen and Boger,1987). Cone and plate viscometer rotates an inverted cone having very shallow angle (~ 5°); the shear rate under the plate is maintained consistently and independent of a flow curve. Parallel plate viscometer is a simplified version of the cone and plate viscometer and has a advantage of flexible space between two parallel plates. The viscous fluid can confined and rotated in narrow space

**Figure 9.** Schematic diagrams of typical viscometers. (a) Concentric cylinder viscometer (or Couette viscometer), (b) cone and plate viscometer, and (c) parallel plate viscometer. (d) Experimental setup of

Ektacytometer employes a laser diffraction technique with blood viscometer in order to measure RBC deformabiltiy. Conventional blood viscometer applys controlled shear stress to the RBCs in the blood viscometer, and deformability of RBCs can be measured from laser diffraction pattern. Ektacytometer consists of a concentric rotational outer cup and a stationaly inner cylinder; outer cup produces varying shear stress field on blood (Fig. 9d). Through the measurement of diffraction patterns of the laser passing through the blood, RBC deformability can be obtained. The RBC deformation is quantitatively calculated from the scattered laser beam intensity pattern. Under a certain shear rate, isointensity curves in the intensity pattern of the scattered beam will show elliptical shapes, which represent elliptically deformed RBC population (Bessis, Mohandas et al.,1980). From the measured

> , *l s DI l s*

where *l* and *s* are distances along the long- and short- axes in the elliptical isointensity curves. DI values are measured at different angular velocities (and thus different shear rate) of the outer cyliner in the ektacytometer. Ektacytometer is a simple and effective technique to measure the deformability of RBC population, and it has been widely used for the study pathophysiology of RBCs. Abnormal deformability in RBCs from patients with hereditary pyropoikilocytosis, hereditary spherocytosis, and Hb CC disease were studies by

Microfluidic device has emerged as a promising tool to precisely control fluids with small volumes of fluid containing samples and reagents in channels with dimensions of 10-100

(9)

isointensity curves, a deformaion index (DI) of RBCs is calculated as

ektacytometer (Mohandas, Clark et al.,1980).

**5.2. Microfluidic device technique** 

Filtration test measures RBC deformabiltiy using a membrane filter with holes of diameter of 3-5 μm (JANDL, SIMMONS et al.,1961). By applying a negative pressure, whole blood is subject to pass through holes in the membrane filter. The deformability of RBCs can affect the speed of flow. RBC deformabiltiy can be calculated from either the flow time or the volume of blood filteres in a certain amount of time (~1 min). Since the filteration test requires for a relatively simple instrument and provides clinically relevant results with high reproducibility, it has been widely used in various studies related to RBC deformability, including the effects of diabetes (Juhan, Buonocore et al.,1982), spesis (Baskurt, Gelmont et al.,1998), sickle cell disease (JANDL, SIMMONS et al.,1961), and oxygen radical (Srour, Bilto et al.,2000).
