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DEVELOPMENT OF THE AGGREGOMETER FOR NEAR-INFRARED
SENSORS THAT STUDY OF THE PLATELET AGGREGATION
Sandoval, G.H.,
Departamento de Engenharia Mecânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, CEP 31270-901, Belo
Horizonte (MG), Brasil
e-mail: geraldo@braile.com.br
Oliveira, M.E.C.,
Departamento de Engenharia Mecânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, CEP 31270-901, Belo
Horizonte (MG), Brasil
e-mail: meneov@ig.com.br
Pinotti, M.
Departamento de Engenharia Mecânica, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, CEP 31270-901, Belo
Horizonte (MG), Brasil
e-mail: pinotti@demec.ufmg.br
Abstract. The cardiovascular disease are the causes one the major of the dead of the Brasil and this has been incentived
the development of artificial devices for implanting in the cardiovascular system that deepen the knowledge process of
thrombus formation, that is, thrombogenesis. This process consists of the aggregates formation of blood cells, of plasma
and of fibrin. Such aggregates (thrombus) can be originated by alterations of the flow conditions and the contact with
the walls of the device, that frequently can leading to the patient death. In this present work, developed three version an
optic device based of the turbidimetric method that uses infrared light sensor capable to analyze the platelet
aggregation, that is, to the measure that platelets agregate ones to the others, the turbidity of the plasma is modified
with increase the area of pass of infrared light produced by the Light Emitting Diode (LED) that can be perceived by
one phototransitor. Thus, the three version of the device is able to perform the analysis of the aggregation for the
variation of the optic density in the plasma sample during the aggregation process. Although similar devices exist in the
market, the devices went developed in the Bioengeneering Laboratories of the Departament of Mechanical Engineering
of the Federal University of Minas Gerais (UFMG) have as main advantage to allow the evaluation of the process of
aggregation in different flow conditions with more an accessible cost. The preliminary results of aggregation obtained
with the three version of the device developed permited quantify to proccess aggregation for the uses of infrared
sensors and went compatible to results similar device existent in the market.
Keywords: platelet aggregation, turbidimetric method, infrared light, hemodinamic, thrombogenesis
1. Introduction
Coronary stenosis is a decrease of the lateral section of arteries surface which propel heart and it is provoked by the
formation of fat plaque below the endothelium. That stenosis decreases the bloodstream toward the cardiac muscle and
provokes severe consequences. These complications are due to the interaction between blood and the lipid plate (known
as atheroma plate) formed in the vessel. When this plate bursts, it reveals sub-endothelial structures (such as collagen)
which activate blood clot. That process forms embolus which causes a blockage in the post lesion and, as a result,
provokes a heart attack. In this case the platelets are activated in two different moments: 1) passing through stenosis and
imposing an unusual flow pattern, 2) when the lipid plaque bursts, reveals the collagen and it develops the process of
adhesion/aggregation of platelets and the formation of fibrin causes thrombus.
Initial studies on the thrombus formation process or thrombogenesis were carried out in 1856 by Virchow and,
since then, it has been known that the phenomenon occurs due to the flow, the blood biochemistry and the surfaces in
which the blood may be in contact (PETSCHEK, 1968). Clot activation and thrombus formation occur when the events
known as Virchow triad are initiated: 1) the activation of platelets; 2) the activation of clot factors; and 3) the activation
of the fibrinolytic system.
By using in vitro studies with blood, the formation of vortex, originated from a stenosis, is characterized by
increasing the shock between the platelets; due to long periods and radial collision toward the wall vessel (KARINO et
al., 1987). This process favours the thrombus development, without adding aggregation agents, or rather, vortex may
cause hemolysis (red globules burst) by liberating the inductor agent of aggregation, Adenosine Diphosphate (ADP),
and activating the platelets. Vortex may conduct to thrombus and thrombosis formation (STEIN and SABBAH, 1974).
One way of studying the aggregation process of in vitro platelets is carried out by the stirring of a suspension of
Platelet Rich Plasma (PRP). Once the platelet aggregation is started (in consequence of its activation) the suspension
speed decreases provoking a change of the infrared light quantity absorbed by the plasma sample. Due to this reason the
stirring method of a PRP suspension is called Method Turbidimetric.
The study on the potential of the in vitro platelet aggregation, or rather, the capability of platelet aggregation when
submitted to either physical or chemical stimulation, as well as the diagnosis method in the area of tissue engineering,
have employed the infrared spectroscopy as sensors (HAZEN et al., 1998); holding the great promise for noninvasive,
nondestructive and therefore less aggressive method (HAZEN et al,1998, FINE and SHVARTSMAN, 2003).
Infrared sensors work according to the absorption frequency of each one of the different blood cells. It makes them
able to detect any blood structural alteration, specially the cellular adhesion and aggregation which alter as the blood is
submitted to different conditions of flow and/or exposed to different kinds of surfaces (BERGER et al., 1997).
Near-infrared spectroscopy is one the three invisible light of the infrared electromagnetic spectrum which has been
used in both tissue and cell stimulation. Near-infrared spectroscopy − NIR − is based on the absorption of
electromagnetic radiation with wavelengths raging from 750 - 2500nm, or rather, it is a radiation close to the visible
light spectrum (HAZEN et al., 1998). The radiation which interacts with a sample may be absorbed, transmitted or
reflected. Reflectance is eliminated so the relation between non-absorbed and transmitted light intensity −from the
sample− can be measured as the transmittance and the absorbance as the quantity of absorbed light.
LEDS (Light Emitting Diode) and the photosensors sensible to emitted radiation appeared as some of the near-
infrared light device, able to analyse blood. It has been verified the development of high intensity LEDS, which have
low spectral rate, with specific wavelength in the near-infrared area and low cost.
Different optical and electrical devices, able to monitor change in blood sample, were developed to the study on
platelet aggregation. One of them was the equipment developed by Feinman et al (1977), which allowed to monitor
both answers given by the stimulated platelets: ATP aggregation and secretion. This device works according to the
already shown turbidimetric method, however, its flow induction speed is steady. This system of aggregation
measurement uses a 55C LED (General Electric) with a 940 nm emission spectrum and a FTP-100 phototransistor
(Fairchild). Another technique used to in vitro analyses consists of detecting the potential difference obtained by the
insertion of electrodes into the sample (CARDINAL and FLOWER, 1982; FREILICH, 1986). There is another
technique of cellular monitoring, of platelet aggregation and of variations in other parameters of platelet aggregation,
which uses a device with a tungsten lamp to create visible light on the filter. For that reason, the method allows to
analyse the electromagnetic radiation at definite wavelengths (RENAUD and RUBEL, 1978). Davis (1968) developed a
measure technique of platelet aggregation and clotting in which both the turbidimetric method and the Chandler tube are
used. The technique consists of adapting a photospectroflowmeter to a 620nm monochromatic filter. Fukuyama et al
.(1989) developed an optical device to promote shear-induced platelet aggregation (SIPA). It was done based on a
turbidimetric technique as employed in commercial platelet aggregation. In this process the optical density of the PRP
sample was detected by a (wavelengths 633 nm) laser light source and a sensor, both guided by an optical fiber.
The degree of platelet aggregation has been used as a sign of some diseases of cardiovascular system or some
blood biochemical disorders in clinical studies. The developed device may be used to: 1) investigate some hemorrhagic
diseases, i. g., Glanzmann’s thrombasthenia, von Willebrand’s disease and Bernard-Soulier disease; 2) monitor the
results of using platelet disaggregates on patients who present platelet hiper-activation; and 3) distinguish those
individuals who present a platelet hiper-activation from the others, pointing out the necessity of prophylactic initiatives
in order to avoid thrombus formation.
The purpose of this study is to developed a Optical Aggregometer which quantifies the platelet aggregation in
equine blood plasma by the Turbidimetric Method, the LED/Phototransistor infrared sensors and variable speed. The
experimental tests were compared to a commercial equipment which works on the same principles.
2. Methods
2.1 Construction of the device
A device was developed due to the need of studying the flow effects on platelet aggregation by the turbidimetric
method as shown in Figure 2.1.
Figure 2.1: Squematic draw of the aggregometer and data acquisition system
used that to study of the Platelet Aggregation process
Speed control, data acquisition system (PC, Multimeter and signal conditioning equipament) are turned on to
activate the stirring system from 300 to 2000 rpm. Due to the necessity of simulating different flow conditions, the
speed can be manipulated. Later, a sample of platelet suspension is placed in the aggregometer channel and closed to
avoid surrounding light.
Turbulence data is read as the optical density difference of the PRP sample is transformed into transmittance sign
by LED/phototransistor optical pair. It occurs as the sample is stirred to quantify this sign as a Platelet Aggregation
percent (% Ag) in terms of time. In this way, platelet aggregation is represented.
Transmittance sign is sent to a conditioned plate to transform this sign into tension sign, which is sent to digital
multimeter. A mobile computer (PC) receives this last sign through a serial port and records that data obtained by
software Masview version 1.1.
The conditioning and amplification system of the sign from LED/photosensor optical pair is done by means of a
micro-controller PIC16F876. There is another micro-controller to monitor the 12 Volts and 3 amperes electric motor
(Tenko Motors). Motor speed control is done by an encoder model PHT18 (Texas Instrumenys).
Comparative tests of platelet aggregation were carried out with a Packs-4 Aggregometer (Helena Laboratories).
2.2 Aggregation measurements
Data was collected using infrared light sensors, spectral scale 880 nm. The transduction system is compound of
LED infrared diode or TIL32 emission (Texas Instruments) and of a phototransistor or receptor model TIL78 (Texas
Instruments) sensible to the emitted radiation.
Magnets with the plasma sample are required to the performing of the magnetic stirring system. The magnets used
in PRP homogenisation from Labbio aggregometer were chosen due to their magnetic intensity, what is fundamental to
the development of the stirring system of Labbio aggregometer. The lower cost of Labbio in comparison to Packs-4
aggregometer was also considered.
2.3 Samples and reagents
Blood samples were collected from 13 adult equines. 20 ml plastic syringes (40 x 16) contained 2 ml of Sodium
Citrate anticoagulant (volume of 3,8%) were used in order to minimize hemolysis. Among those equines, six were
under experimental conditions of induced laminitis. Laminitis is a disease which attaches the animal immune system,
provoking a platelet pre-activation. The other seven animals were healthy.
Blood samples were collected and prepared as stated by Standard ISO 10993-4 (SEYFERT et al., 2002); since red
blood cells may be burst during procedures. After blood collecting, samples were processed to obtain platelet-rich
plasma (PRP) and platelet-poor plasma (PPP).
Some tests were carried out without using ADP, in order to study spontaneous platelet aggregation when it is
submitted to more turbulent flow conditions. Spontaneous aggregation concerns platelet aggregation provoked by the
mechanic shock caused by platelets. The process initiates with platelet membrana bursting what releases ADP in the
dense granules of platelets. ADP activates the liberation of Calcium ions from the dense tubular system to feed the cycle
of platelet activation. However, ADP was used in a majority of the tests to accelerate the activation and aggregation
platelet process.
V A
8.888
1000rpm
LED
Velocity
Control
photosenso
r
Platelets
sus
p
ension
Signals
Conditioner
PC
Multimete
r
Magnetic
agitation
s
y
ste
m
3. Results and Discussion
3.1. Optical model applied for the Aggregometer
The optical model shown in the section (OLIVEIRA,2004) was developed based on the change of optical density in
plasma samples, which occurs as platelets aggregate. This aggregation allows a larger light passage. From the relation
between light quantity emitted by photodiode and perceived by photosensor arises a potential difference called
transmittance.
In the analyses of transmittance data (section 3,2), some conditions were assumed:
1. Radiation absorbed by cells is meant to be proportional to the cell surface and transversal to the direction of
radiation incidence.
2. Spread radiation is absorbed by either other cells or small tube walls.
3. Spread is meant to be proportional to the cell surface and transversal to the direction of radiation incidence.
4. Platelet surface in the suspension can be calculated based on the total number of platelet in the suspension.
5. Transmitted radiation is inversely proportional to the cell number in the sample.
According to these conditions, optical density can be represented by the number of cells in the sample, or rather,
volumetric cellular density. Therefore, the optical density of the sample can be represented cells mean surface by
volume unit; since the transversal surface to the beam is the source of absorption or spread of radiation.
An initial moment of analyses is assumed as a time reference in order to calculate platelet aggregation percentage
of any suspension, at any time. A calculation of the number of platelet is carried out at the initial moment (No). Later,
the same calculation is carried out to greater moments than the initial one (Np). Theoretically, the lowest platelet
number of plasma suspension is the lowest platelet number of a PPP plasma suspension (Nppp). Therefore the greatest
variation of platelet number in any plasma suspension would be (No-Nppp). Any variation between the initial moment
and any other time would correspond to a percent of this number. It is considered that total surface of platelet (Ap) can
be calculated as the surface of one platelet times the platelet number in the suspension.
The relation platelet number in the plasma sample with the Aggregometer data can be obtened for eq. 3.1, where
the platelet number is function tension sign read in the aggregometer of the that form:
⎥
⎦
⎤
⎢
⎣
⎡
⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛−+⋅=
PPP
PRP
PPPp V
V
1
1
1NN
α
3.1
Of the eq. 3.1, verified that number of platelet is carried out at the initial moment can be represent for eq. 3.2:
⎥
⎦
⎤
⎢
⎣
⎡
⎟
⎟
⎠
⎞
⎜
⎜
⎝
⎛−+⋅=
PPP
0
PPP0 V
V
1
1
1NN
α
3.2
Therefore, the percent of aggregation is calculated by the eq. 3.3:
()
()
100%
0
0⋅
−
−
=VV
VV
Ag
PPP
PRP
3.3
3.2 Results of Platelet Aggregation
3.2.1 Results of Platelet Aggregation to Labbio Aggregometer
The first test carried out with Labbio Aggregometer consisted of testing the sensor ability in detect optical density
changes in PRP samples by the formation of platelet aggregation when submitted to different conditions of flow. Or
rather, mechanical aggregation tests were carried out without using ADP (Fig. 3.1). In these tests, PRP samples were
submitted to different conditions of flow by speed variation. It was noticed that, to the three shown curves, there is a
phase of platelet activation which occurs and is characterized by the change of platelet forms. In the aggregation phase,
characterized by the increase of transmittance, the intensity at witch platelets aggregate do not vary. It occurs because
the shear-stress rate imposed is sufficient to burst the platelet membrana and release the Calcium ions.
-30
-20
-10
0
10
20
30
40
50
60
70
80
90
100
0 1000 2000 3000 4000 5000 6000
Time (s)
% Aggregation
1000 RPM
2000 RPM
3000 RPM
Figure 3.1: Mechanical aggregation curve obtened with Labbio
Aggregometer in speed variation
Figure 3.2 shows curves of mechanically induced platelet aggregation when submitted to 3.000 rpm . Aggregation
and activation phase can be observed on both curves. However a disaggregation process can be verified at the end of
each aggregation curve. It can occurs for two reasons: 1) the lack of ADP makes the platelets return to their discoid
form. 2) shear rate may provoke a microaggregate formation.
Tests to obtain curves of platelet aggregation induced by ADP and steady-state velocity of 1000 rpm are shown in
Figure 3.3. The three curves are in the normal rate of aggregation (30 – 70%). Activation phases caused by a quick
decrease in transmittance is noticed after ADP injection, about 60 seconds after the begining of the test. How platelet
aggregation is accelerated with ADP using is verified now.
-20
-10
0
10
20
30
40
50
60
70
80
90
100
0 500 1000 1500
Time (s)
% Aggregation
%agrega1
%agrega3
%agreg2
%agreg4
Figure 3.2: Mechanically Induced Platelet Aggregation with the Labbio Aggregometer
using the same PRP sample in steady-state velocity of 3000 rpm
Activation
p
hases
Aggregation
p
hases
Disaggregation
p
hase
-20
0
20
40
60
80
100
0 100 200 300 400 500 600
Time (s)
% Aggregation
% Agr eg1
% Agr eg2
% Agr eg3
Figure 3.3: Induced Platelet Aggregation curve for ADP obtened with the Labbio
Aggregometer using the same PRP sample and steady speed of 1000 rpm
Figures 3.4 and 3.5 show high levels of fluctuation during aggregation measurements. Figure 3.4 shows high
percent of aggregation due to the fact that the animal had Laminitis, or rather, since the platelets were pre-induced, ADP
adding might have amplified the aggregation process. This fluctuation might have been accentuated by the high number
of platelets in PRP samples.
-10
0
10
20
30
40
50
60
70
80
90
100
110
0 100 200 300 400 500 600
Time (s)
% Aggregation
%Agreg1
%Agreg2
%Agreg3
%Agreg4
%Agreg5
Figure 3.4: Induced agregation curve for ADP obtened with Labbio Aggregometer the steady
speed of 1000 rpm using the same PRP sample of the animal had Laminits
-30
-20
-10
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500 600
Time (s)
%Aggregation
%Agreg1
%Agreg2
%Agreg3
%Agreg4
%Agreg5
Figure 3.5: Induced aggregation curve for ADP with Labbio Aggregometer
using the same PRP sample and steady speed of 1000 rpm
Figure 3.6 shows the comparison between the number of platelet and greatest aggregation of tests which were
carried out with Labbio and Packs-4 aggregometers. It was noted that the preliminary results revealed by the developed
aggregometer show a good correlation with Packs-4. Although the developed system reveals a higher rate of
aggregation. Preliminary results of the prototype of the developed aggregometer reveal the capability of infrared light
sensors − model LED/Phototransistor − to detect Platelet Aggregation. ADP induced aggregation is more accelerated
and amplified than mechanical aggregation. However, ADP stored in platelets is sufficient to provoke their aggregation,
what depends on the flow conditions in which platelets are submitted as time goes by.
0
10
20
30
40
50
60
70
80
90
100
150 170 190 210 230 250 270 290
platelet number*105
% Maximum Aggregation
Max Aggreg Labbio
Max Aggreg Packs-4
Figure 3.6: Comparison between the number of platelet and greatest aggregation
of tests with Labbio and Packs-4 aggregometers
4. Conclusion
An optical aggregometer which uses the Turbidimetric Method was developed by LED/ Phototransistor infrared
sensors to analyse the process of in vitro platelet aggregation. The experimental methodology allowed to obtain needed
values to the determination of platelet aggregation. This process permitted checking the performance of Labbio
aggregometer by comparing Labbio to commercial aggregometer Packs-4, which is on the market.
The measurement system by light sensor with radiation in the near-infrared spectrum model LED/Phototransistor,
wavelength 880 nm, was able to detect the changes in optical density from plasma samples during the aggregation
process.
The system of magnetic stirring of platelet suspension was efficient, since speed could be varied. Therefore, two
facts were verified: 1) the influence of speed on the system of plasma stirring; 2) the preliminary results agree with the
results obtained with the device on the market. An acceptable deviation between the devices was not statistically
established due to the insufficient number of tests.
In relation to preliminary experimental tests, it was observed that the rate of platelet aggregation suffers great
influence from speed and, as a consequence, the degree of turbulence imposed by the flow tends to increase
aggregation. However, from a given speed, the rate of aggregation seems do not alter any longer and the platelet
aggregates begin to be sheared by a turbulent flow.
The time and the magnitude of speed in which platelet aggregates are submitted to a turbulent flow determine the
size of aggregates. These aggregates tend to be great to flows in which the stirring system of platelet suspension is the
least non-invasive one.
Therefore, this paper is a very important step to the study on in vitro aggregation in terms of platelet number and
flow conditions in which platelets are submitted.
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aggregation and test cell for use in such method and apparatus, United States Patent 4,319,194; March 9, 1982.
Davis, R. B., Turbidimetric evaluation of platelet aggregation in the Chandler tube. Am J Clin Pathol. 1968 Jan;49(1):
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measurement of secretion and aggregation by platelets, J Lab Clin Med., 90(1):125-9, 1977.
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