Final Project BE 309 Fall 1997
Intrinsic Viscosity of Horse RBC Suspensions
Group M6
Jennifer Russert
Derek Wong
Elizabeth Khaykin
Emily Rothman
Abstract
Intrinsic viscosity is a measure of the interaction of a colloidal particle with the suspending solvent, where there are no interactions between those particles. The main objective of this experiment is to determine the intrinsic viscosity of horse red blood cells (RBCs) in phosphate buffered saline (PBS). This is done by calibrating low concentrations and the testing them with a capillary viscometer at constant temperature of 30° C. The intrinsic viscosity of the red blood cells was found to be 0.0033 ± 0.0024 mPa sec. This experiment outlines the development of the procedures and protocols for measuring the intrinsic viscosity of horse RBCs and identifies the background literature needed for future use as a standard lab experiment.
Background
Blood: a Colloidal Solution
Blood is made up of a suspension of particles in a solution of proteins and electrolytes called plasma. It is a living tissue, which consists of many types of cells. In this experiment we will be concerned primarily with the physical properties of blood, namely its flow behavior.
Erythrocytes, leukocytes, and platelets are the main constituents of blood. The erythrocytes or red blood cells (RBCs) are more than a thousand times more numerous than the leukocytes or white blood cells (WBCs) and much larger than platelets. For this reason the flow properties of blood mainly involve the RBCs. The concentration of RBCs is expressed as the hematocrit, or the percent of RBCs in a sample of blood by volume. Normally an adult human has a hematocrit of 40-45%. Less than that is considered anemia, while more is polycythmia. The normal hematocrit of horses as suggested by literature is around 37% (7).
Rheology is the branch of chemistry and material science that deals with the flow properties of materials. Hemodynamics studies how blood flows in the body and how the hemorheological properties influence flow rates, pressures, etc. The cardiovascular system consists of a complex network of branched vessels, which form a closed loop. Blood relies on its various properties to allow it to pass through tiny capillary passageways and provide all the functions critical to life (8).
Rheological Properties
There are several interrelated factors that regulate the rheological properties of blood. These include the extent of cellular aggregation, the deformability of RBC’s, the composition of plasma, and even the concentration of RBC’s (hematocrit). It is this last factor, hematocrit, that is tested to find the viscosity of red blood cells.
It has been found that many diseases involve changes in various rheological components. For instance, it has been found that patients with myocardial infarction and arterial thrombosis have blood viscosities at low rates of shear, which are elevated above normal values. Patients with anemia or low hematocrit display a lowered value of blood viscosity. On the opposite end, patients with polycythmia or high hematocrit display higher values for viscosity.
Intrinsic viscosity is a property that polymer scientists are often concerned with. It can be an indication of the size and shape of an isolated polymer molecule in a solvent so dilute that there are no interactions between this molecule and any others other than those of the solvent (3).
A normal red blood cell is round and moves through the smallest blood vessels quite easily. But in people who have sickle-cell anemia, the blood cells break down. They lose their round shape and take the form of a sickle-a farmer's implement. These sickled blood cells get stuck in the small veins of the body, blocking other red blood cells from passing. When the flow of blood to a part of the body is reduced, the oxygen supply to that part of the body is cut off and cells begin to die.
Determination of Blood Viscosity
The intrinsic viscosity of RBC’s is a measure of the interaction of colloidal particles such as RBC’s with the suspending solvent. When these are the dominant reactions, particle-particle interactions (e.g. those involving WBC’s or platelets) are negligible. To determine intrinsic viscosity one must first measure the viscosity of RBC suspensions in very dilute systems. Then one extrapolates a suitable function to infinite dilution. The resulting value of intrinsic viscosity is theoretically related to properties of the particles in the particular medium, which in this experiment is PBS.
Since the solutions are dilute, the measured viscosity will be only slightly greater than that of the medium itself, so that very precise methods must be used to determine the viscosity, and great attention must be paid to procedure, temperature control, etc. For our lab the method of choice is the glass capillary viscometer, a very simple system but one requiring careful lab procedures to obtain accurate results.
In order to find the viscosity of the RBC/PBS solutions, the viscosity of PBS alone has to be determined. Using water as a standard, the viscosity of the PBS is found from the ratio (2):
Equation-1
where d represents the density, h the viscosity and t the time for the liquid to flow through the viscometer from the top line of the capillary tube to the lower line. The viscosity of the RBC/PBS samples is found in the same fashion (using water as a standard). Armed with these viscosity values, specific viscosity, hSP, for each sample of RBC/PBS solution can be calculated using the following equation (2):
Equation-2
where hr is the ratio of the viscosity of the solution to that of solvent, hsolution/hsolvent. The specific viscosity is the relative contribution of RBC to the viscosity of the solution. When specific viscosity is divided by concentration, i.e. (hsp/c), the resulting quantity increases with concentration. This relationship is approximately linear, represented by equation-3 (2):
Equation-3
where b is a constant, or the slope. By plotting the quantity (hsp/c) vs. the concentration of the RBC in PBS the intrinsic viscosity [h] can be extrapolated from the y-intercept of the graph which is represented by equation-4 (2):
Literature values for the intrinsic viscosity of human and horse blood can only be related to the known value that can be found. This known value is the viscosity of water, to which all horse red blood cell concentrations relate. The viscosity of water at 30 degrees Celsius is 0.807mPAs (4).
Methods and Procedures
The procedures of this experiment can be split up into three sections: washing of the RBCs and making of PBS solution, blood concentration and concentration calibration, and viscosity measurements
Step one was to wash the red blood cells from the horse donor in order to separate them from the blood plasma and achieve as close to a 100% concentration of the RBCs as possible, also known as a 100% hematocrit. This was done by adding PBS buffer solution to the horse blood then placing the blood in the centrifuge at a speed of 2500 RPM for 10 minutes. The plasma was then suctioned off from the surface of the blood.
This procedure was repeated 4 times, each time bringing the concentration closer to 100%. It was then done again at the beginning of each experimental period in case any blood cells had died during the past week. The PBS solution that was used to wash the cells and later to dilute to varying hematocrits was made using the chemicals in the indicated amounts from Table 1 (1). The solution was mixed in a volumetric flask with an error value of 0.60 mL.
PBS (Phosphate Buffered Saline)
Na2HPO4 (anhydrous) 2.30 g/L
NaH2PO4 (anhydrous) 0.46 g/L
NaCl 8.76 g/L
Table 1
The horse blood was then ready to be diluted into concentrations, which ranged from
0.25 - 3.0 %.
40 ml of each concentration was made using a 100-1000ml micropipette and a graduated cylinder. An amount of blood, amounts are specified in Table 2 for concentration range, was pipetted into a 50mL graduated cylinder. In order to clean all traces of blood from the micropipette tip, it was rinsed with PBS solution, which was then in turn added to the graduated cylinder as well, filling it to 40mL. The concentrations that were created in this manner are as follows
% Concentration / Vol. Blood (mL) / Vol. PBS and Blood (mL)0.25 / 0.100 / 40
1.0 / 0.400 / 40
1.5 / 0.600 / 40
2.0 / 0.800 / 40
2.5 / 1.000 / 40
3.0 / 1.200 / 40
Table 2
Though the concentrations were made with as much care as possible, it was necessary to determine the exact concentrations the solutions turned out to be through a concentration calibration. To do this a Spectrophotometer was used to measure the absorbance of the concentration range. The absorbance was then related through regression to the exact concentration value for each solution.
The trouble that arises with doing this is that the spectrophotometer is not able to read the absorbance of a blood solution that is concentrated to the point were light is not allowed to pass through it. This happens even at the low concentrations of 5%, 4%, or even at 2.5%. The method was still used, and it was found that a concentration of about 0.21% hematocrit was the most concentrated sample that gave an absorbance value directly, without dilution. The other solutions had to be diluted by a specified factor (shown later), then placed into the spectrophotometer.
Several tubes of stock blood samples were given to us initially. These samples contained all components of the blood: red blood cells, plasma, etc. The stock blood samples were washed with PBS and centrifuged in order to remove the plasma component of the blood (the supernatant) as much as possible, isolating red blood cells for our experiment. However, there was still a small amount of plasma and other constituents present in the isolated red-blood cells, therefore, the actual hematocrit of the read blood cells had to be measured. A small amount of the red blood cells were placed in a capillary, and then using microcapillary centrifuge the PBS was separated from the RBCs. After this centrifugation step, the capillary was placed in the hematocrit reader, and the hematocrit of the stock red blood cell sample was found to be 85.5%.
Hematocrit cannot be accurately measured with a hematocrit reader for the small concentrations in our experimental range. Therefore, in order to accurately measure the hematocrit of our samples during our experiment, a spectrophotometer and a calibration curve of absorbance vs. hematocrit were used. The first concentration was made by taking 7.5 mL of the stock red blood cell (85.5%) and adding PBS to make 3000 mL of red blood cell solution. The hematocrit of this solution was 7.5 mL / 3000 mL x 85.5% = 0.21375%. The absorbance reading of this solution was recorded at 577 nm ± 1 nm, one of the absorption peaks of oxygenated blood (BE 209 Final project -Absorbance of Hemoglobin). This solution was then diluted serially, at half the original concentration each time, and the absorbance reading for each concentration was recorded as listed:
Hematocrit (%) / Absorbance Reading0.21 / 1.700
0.11 / 1.040
0.053 / 0.564
0.027 / 0.301
0.013 / 0.160
0.0067 / 0.072
Table 3
A second trial was done with another stock blood in the same fashion, and the calibration is as follows:
Hematocrit (%) / Absorbance Reading0.14 / 1.350
0.068 / 0.774
0.034 / 0.410
0.017 / 0.205
0.0085 / 0.099
0.0042 / 0.049
Table 4
Linear regression was applied to these two trials to obtain a calibration curve.
Graph 1
The slope and the intercept of the calibration curve were found to be 8.418 ± 0.404 and 0.0726 ± 0.0327 respectively. Knowing these values, hematocrit of the samples tested in the experiment could be found by measuring the absorbance of the samples.
However, the concentrations of our samples ranged from approximately 0.25% to 3% while the spectrophotometer was useful only up to 0.25% hematocrit. Therefore, our samples had to be diluted as well. To increase accuracy, each sample of was diluted at two different factors and the corresponding absorbance readings were taken. The results are as follows:
Viscosity measure is the final procedure, which is carried out with a capillary viscometer. The capillary viscometer was contained in a water bath to keep it at a constant temperature of 30 degrees Celsius using a Haake D1 immersion heater/circulator. A thermometer was also clamped to the water bath so that the constant temperature could be further monitored. The heater/circulator was allowed to run at least 10 minutes before any experiments were done.
Water and PBS were first tested, followed by samples of horse blood diluted to the various concentrations with PBS. The lowest concentration was measured first. The sample was dropped down the larger hole of the capillary viscometer (see Picture 1: tube G) and then sucked through the other end with an auto-pipette. It was clamped to the water bath and timed to see just how long the meniscus of the solution would take to fall from point C on the tube to point E (Picture 1). This was repeated three times for every concentration with different samples of blood. Then the viscometer was rinsed twice with the next higher concentration and similar trials were performed.
Picture 1
The water bath not only kept the temperature of the sample constant as it was running through the viscometer, but all of the samples were stored in the same water bath. This kept them at the desired 30 degrees Celsius. Also, the samples were clamped within the water bath making sure nothing bumped into the viscometer disturbing the sample while it was being tested.