Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Summary

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Abstract

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Introduction

Ektacytometry provides a convenient measure of red cell deformability in response to alterations in shear stress (measured in pascals (Pa)) or suspending medium osmolality. Pertinent parameters of red cell deformability include the maximum elongation index (EI Max), a measure of the maximum deformability of a red cell in response to increasing shear stress, and shear stress ½ (SS ½), the shear stress required to achieve half maximal deformability.¹ Osmotic gradient ektacytometry has several informative parameters. These include the elongation index minimum (EI Min), a measure of surface-to-volume ratio and the osmolality at which it o....

Protocol

All subjects in this study gave written informed consent in accordance with the Declaration of Helsinki and the National Institutes of Health Institutional Review Board approved protocols.

1. Turning on the ektacytometer

1. Connect the tubing from the cleaning solution to the low and high osmolar polyvinylpyrrolidone (PVP) solutions. Be careful to connect the 0 osmolar tube to the low osmolar solution and the 500 osmolar tube to the high osmolar solution.
   Note: The low osmolar PVP solution should have an osmolality between 35 and 55 milliosmoles per kilogram (mOsm/kg), a pH of 7.25-7.45 at 25 °C and a viscosit....

Results

The ektacytometry results described in this manuscript can be used to measure red cell deformability in any condition. A schematic of the general set up of an ektacytometer is shown in Figure 1. Homogeneous populations of erythrocytes will produce an elliptical diffraction pattern in response to increasing shear stress that can be used to calculate the elongation index as shown in Figure 2. Diffraction pattern distortion occurs i.......

Discussion

The ektacytometry techniques described are straightforward and well automated, ensuring valid and reproducible results. Nonetheless, some critical steps exist. Proper temperature control of the blood is important. Storage at room temperature for more than eight hours may affect SS ½ values.³⁴ Ensuring that the temperature of the machine is stable at 37 °C is also important, as viscosity of the suspending medium is temperature dependent. Blood should be fully oxygenated to avoid decreased.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LoRRca MaxSis standard version	Mechatronics	LORC109000
LoRRca MaxSis Osmoscan	Mechatronics	LORC109001
Polyvinylpyrrolidone solution (PVP) 0mOsm	Mechatronics	QRR030910
Polyvinylpyrrolidone solution (PVP) 500mOsm	Mechatronics	QRR030930
Polyvinylpyrrolidone solution (PVP) 5mL vials	Mechatronics	QRR030901
X clean	Mechatronics	QRR010946
P1000	MilliporeSigma	Z646555
P200	MilliporeSigma	Z646547
P200 filter tips	MidSci	AV200-H
P1250 filter tips	MidSci	AV1250-H
Kimwipes	MidSci	8091
1.5 mL eppendorf tubes	MidSci	AVSS1700
15 mL conical vial	MidSci	C15R