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Научни трудове на Съюза на учените в България-Пловдив. Серия В. Техника и технологии, естествен ии хуманитарни науки, том XVI., Съюз на учените сесия "Международна конференция на младите учени" 13-15 юни 2013. Scientific research of the Union of Scientists in Bulgaria-Plovdiv, series C. Natural Sciences and Humanities, Vol. XVI, ISSN 1311-9192, Union of Scientists, International Conference of Young Scientists, 13 - 15 June 2013, Plovdiv.
HEMORHEOLOGY IN BIOPHYSICS EDUCATION FOR STUDENTS IN MEDICINE
S. Miteva, S. Alexandrov, N. Hristova-Avakumova, S. Stoeff, L. Traykov,
D. Gerasimova, S. Jovtchev Dept. of medical Physics and Biophysics, medical Faculty, Medical University - Sofia, Bulgaria, 1431 Sofia, Zdrave Street 2, Bulgaria, e-mail: svetla_miteva_nikolova@abv.bg; jovchev@medfac.acad.bg
Abstract
Hemorheology is the science dealing with the deformation and flow properties of cellular and plasmatic components of blood, the interactions between them and their interplay with the vascular system. It investigates the dynamics of the blood flow in the presence of drugs, plasma expanders and prosthetic devices. We offer the basic knowledge on this topic in a lecture for the duration of our biophysics course. During laboratory work students investigate the effect of red blood cell (RBC) concentration and decreased cellular deformability on suspensions viscosity. From the factors involved in RBC aggregation they study the role of chemical nature, concentration and molecular mass of some plasma expanders for this process. The influence of the ionic strength of the medium on RBC aggregation is examined and discussed in respect to the surface electric charge of the cells. The scientific area of hemorheology offers an excellent field for teaching the students the manifold biophysical interrelations between biochemical, electrical, mechanical and rheological properties of blood components as determinants of blood viscosity and circulatory flow.
Introduction
Rheology is a branch of mechanics, which investigates the deformational (rheological) properties of gases, fluids and hard materials, the methods for their determination and description, as well as their physical nature. Hemorheology is the science dealing with the deformation and flow properties of cellular and plasmatic components of blood, the interactions between them and their interplay with the vascular system [1]. Blood performs various essential functions in the body: It delivers oxygen and nutrients to all parts, relays chemical signals to all tissues, removes waste products, defends the organism against infection through the action of antibodies, and maintains constant body temperature. The blood flow in the cardiovascular system depends on the flow conditions: the driving force of the heart and the architecture (geometry) and mechanical properties of the blood vessels. It is also determined through the mechanical (rheological) properties of the blood itself - its viscosity [2]. Whole blood is a concentrated suspension of formed cellular elements including red blood cells (RBC, erythrocytes), white blood cells (WBC, leukocytes) and platelets distributed in blood plasma. Blood viscosity is determined through the volume concentration of blood cells (hematocrit, H), the aggregation and agglutination of
cells at low shear rates, orientation and deformation of cells at high shear rates and viscosity of blood plasma. Within the course of biophysics for students in medicine hemorheology related experimental practices were present since its beginning in 1973-1974 - surface electric charge determination of RBC using microelectrophoresis [3] and determination of hemolytic resistance of erythrocytes [4]. The next step followed within two projects (1984-1990) for exchange of experience in the field of education in medical physics and biophysics for students in medicine and dentistry and investigations on the physical and mechanical properties of cells and membranes - with the Institute of Medical Physics and Biophysics, Charite, Humboldt University, Berlin, Germany. The first lecture with stress on hemorheology was held by Prof. D. Lerche (1987-1988, in Russian). In the next years the lecture was presented by associate professor R. Petrova, after her death in 1989, by assistant professor S. Jovtchev. In the 90-ties three experimental practices were introduced and offered to the students until present. The area of hemorheology was than included in the Biophysics textbook of associated professor M. Marinov [5]. In this paper we present some results obtained from the students within their experimental work under the guidance of the assistant professors for biophysics.
Material and methods
Human whole blood and RBC concentrates are obtained from the National Center for Hematology and Blood Transfusion (NCHBT), Blood Bank Sofia. All the solutions and the stock suspension of washed human RBC (H = 0.60 v/v) in phosphate buffered saline (PBS, pH = 7.4, osmolality = 295 mOsmol/kg) are prepared by our laboratory assistant. The assistant professors discuss the theoretical background with the students. After that they advise the students how to perform the experimental work and control its performance.
Fig. 1. Typical result from students experiment
The specific viscosity increases with the rise in RBC volume concentration. Linear regression according to Einstein's law (line forced through the zero point) gives for the factor a value of 2.00 very close to the expected from the literature - 2.06.
Results and Discussion
Determination of the dependence of specific viscosity on the volume concentration (hematocrit) of red blood cell suspensions
The aim of this experiment is to investigate the validity of Einstein's law (for the specific viscosity (nsp) of diluted suspensions of particles, see below) for the suspension of RBC and from the obtained results to determine the particle shape factor a for the RBC:
nspec =
For hard spherical particles a is equal to 2.5, for hard asymmetric particles it is larger than 2.5 and for native RBC it is around 2.06 due to the deformability of the cells. The experiment starts with RBC suspension in PBS (H ~ 0.1) to be in the range of validity of Einstein's equation. We prepare suspensions of RBC with different hematocrit values diluting the initial suspension. The
students calculate the specific viscosity from the flow times of the suspensions and PBS measured with capillary viscometer of Ostwald type. The hematocrit value is determined with hematocrit centrifuge. The RBC shape factor a is found using linear regression (forcing the line through the zero point) with common statistical tools of Microsoft Excel. Typical result and short comment is shown in Fig 1.
Investigation on the influence of cell deformability on the relative viscosity of erythrocyte suspensions
Aim of the study in this practice is to prove experimentally the role of cell deformability for the relative viscosity of suspensions of RBC. Erythrocyte deformability, i.e. the property of the cells to change their shape passively as a result of applied mechanical stress, is determined through the following cellular factors: geometry - excess of surface area (~30%) compared to the surface needed to encapsulate the volume of the cell cytoplasm in a sphere, internal viscosity - dependent on the biochemical properties (e.g. sickle cell hemoglobin) and concentration of hemoglobin and membrane mechanical properties influenced by membrane skeleton elasticity and lipid bilayer viscosity. Decrease of RBC deformability is achieved by heat treatment (4 or 8 min at 50 °C) or fixation with PBS - glutaraldehyde (GA) solutions (0.3% and 0.5%, for 15 min). The relative viscosity of the suspensions is determined with Ostwald type capillary viscometer.
Fig. 2. The influence of RBC deformability on the relative viscosity of erythrocyte suspensions: left - cells hardened with glutaraldehyde at different concentrations; right - temperature (T) hardened cells.
The RBC hardened with glutaraldehyde (GA) increase the suspensions relative viscosity (Fig. 2., left). Related to the viscosity of control cell suspension - the elevation reaches ~ 30%. The temperature (T) hardened RBC enhance the suspensions relative viscosity in similar manner. Related to control cell suspension viscosity - it reaches ~ 90% for the erythrocytes treated 8 min. It is evident from the graph (Fig. 2, right) that the effect of cell deformability on the relative viscosity is dependent on the hematocrit: ~ 50% at Hct = 0.30 (v/v) vs. 90% at Hct = 60 (v/v). These results are in accordance with experimental data published in the past [6]. They clearly show the important role of RBC deformability for the viscosity of RBC suspensions. The assistant professors discuss these findings with the students in respect to processes in blood in vivo. One example: during in vivo aging of RBC similar effect on cell deformability has the malondialdehyde (MDA) a product of free radical membrane damage. The decreased deformability is one of the reasons responsible
for the removal of the aged RBC in the spleen. In other words GA hardening models the in vivo aging of RBC. In another experiment during our biophysics course we determine the antioxidant capacity (AOC) of blood plasma. We point to the interrelation between RBC deformability and AOC of blood plasma. In this way the students learn to combine knowledge obtained in different experiments. In various disease states this relationship is experimentally verified: sepsis, diabetes, sickle cell disease etc. [7].
Investigations on red blood cell aggregation with the zeta sedimentation method
The biconcave human red blood cells (RBC) form aggregates under low shear stress condition ordering themselves with the flat side to each other, resembling a stack of coins. This type of aggregates are called RBC rouleaux (Fig. 3).
Fig. 3. Microscopic pictures of RBC aggregation in plasma and Dx 500K - 1 g/dl, Dx 70K - 3g/dl, PEG 200K - 0.5 g/dl, PEG 35K - 0.5 g/dl.
These aggregates are easily dispersed in blood flow, but rapidly form again when the fluid forces decrease. Normal RBC aggregation is a reversible process. The factors determining this process are: extracellular and intracellular (aggregability). The extracellular factors involve: flow condition and physicochemical properties ofblood plasma: proteins - fibrinogen, immunoglubulins; plasma expanders (e. g. dextran (Dx), polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), pH - value, ionic strength, osmotic pressure. RBC aggregability, i.e. the complex property of the cell to form aggregates, is influenced by: surface electric charge density - responsible for cell repulsion; cell glycocalyx (as polymer layer on the membrane) thickness and polymer segment surface density - works as a steric barrier, RBC shape and deformability - determines kinetics and extent of the contact area between the cells.
Fig 4. Role of neutral polymer characteristics (chemistry, molecular weight, concentration) on the red blood cell aggregation.
In the experiment the students have to investigate: the role of neutral polymer characteristics (chemistry, molecular weight (MW) and concentration), the influence of ionic strength of suspending medium and the effect of cell deformability on the erythrocyte aggregation. In the years we have used following polymers: dextrans (Dx 70K , Dx 500K), polyethylene glycol (PEG 35K, PEG 200K), polyvinyl pyrrolidone (PVP 40K, PVP 360K) in the concentration range 0 - 3 g/dl. The influence of ionic strength of the medium we tested reducing it with isotonic saccharose solution to about 67 mmol/l NaCl. RBC deformability is modified by the treatments described in the previous chapter. The polymers solved in PBS are added to the stock suspension of washed RBC to obtain final hematocrit ~ 0.40 v/v. The Zeta sedimentaion technique [8, 9] is used to determine the aggregation extent. RBC suspensions are filled in standard hematocrit tubes and sealed at the lower end and spun in an original apparatus (the zetafuge). The spinning axis is essentially vertical. Under the 7-8 g produced by the apparatus, the cells travel to the outer wall of the tube and aggregate. After 45-sec cycle the tubes are rotated through 180o and the process repeated. During each cycle the cells disperse and then aggregate and continuously fall under the downward 1 g. Thus they follow a zigzag course down the tubes. Four spinning cycles take place over three minutes. At the end of the spinning period the apparent packed cell volume (PCV) is measured and referred to as the zetacrit (ZCT). The hematocrit is determined in the same tube after centrifugation at 10 000 g, for 5 min in standard hematocrit centrifuge (HCT). The parameter, which measures the RBC aggregation is the zeta sedimentation ratio (ZSR): HCT/ ZCT. The tremendous advantage of the ZSR technique is that it greatly increases the extent of any possible aggregation overcoming the electrostatic and steric repulsion between the RBC pressing the cells to each other through the applied centrifugal forces [8, 9].
In Fig. 4. we present the results concerning the role of polymer characteristics for the RBC aggregation. The ZSR rises with polymer concentration for all polymers. Within one type of polymer the increase in molecular mass results in higher aggregation parameter values. In the studied concentration range (0 - 3 g/dl) the dependence seems to be linear, since the linear correlation coefficient (R2) is higher than 0.92.
Fig. 5. The aggregation potential (AP) of the neutral polymers evaluated by the slope of the linear regression - it gives the increase in ZSR for 1 g/dl polymer
As measure of the aggregation potential (AP) of the polymers we take the slope of lines from the concentration dependence - it gives the increase in ZSR for 1 g/dl polymer (Fig. 5). For every type of polymer AP increases with MW. For polymers with similar MW the strongest aggregation power has PEG. Hence AP decreases in the order PEG > PVP > Dx. The chemical nature of the polymers is of substantial importance for their aggregation power not only the MW. Decreasing the ionic strength of the suspending medium leads to reduction in RBC aggregation (Fig. 6., left). This is through for 3 g/dl Dx70K (22%) and for PEG 35K 1 g/dl (18%). The reduction in RBC aggregation is discussed with the students to be related to the increased electrostatic repulsion
originating from the enhanced cell zeta potential (Fig. 6., right). This fact is experimentally evaluated in another practice using a microelectrophoresis.
The role of cellular deformability for RBC-RBC interaction is studied for cells treated with glutaraldehyde for 15 min, at room temperature; or temperature hardened (temperature incubation at 48 °C or 50 °C for 5 min). For all aggregating agents used decreasing the RBC deformability lowers the aggregation extent. For the temperature hardened RBC this effect is about 22% (50 °C), in the case of PEG35000 (1 g/dl). For DX 70000 induced aggregation (3 g/dl) the reduction in the ZSR value is similar - 15% (data not shown). The glutaraldehyde hardening of the erythrocytes shows the same effect (data not shown).The results are in agreement with previous reports [11, 12] and already published by us recently [13].
Fig. 6. Influence of ionic strength of the medium on the RBC aggregation (left) and cell zeta potential (right).
Conclusions
The scientific area of hemorheology offers an excellent field for teaching the students the manifold biophysical interrelations between biochemical, electrical, mechanical and rheological properties of blood components as determinants of blood viscosity and circulatory flow. The lecture and the practical experiments aims to help them better understand blood flow disturbances in various pathological states, including cardiovascular diseases - the major cause of death in the human population.
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