Quantitative Ultrasound System for the Characterization of Animal
Blood Plasma
Leidy T. Molano, Yady M. Jimenez, Julian A.
Villamarìn, Nylho A. Dorado, Muñoz Fabian
G. Muñoz
Facultad Ingeniería Electrónica y Biomédica
Universidad Antonio Nariño
lemolano@uan.edu.co, yadjimenez@uan.edu.co,
jvilla22@uan.edu.co,ndorado@uan.edu.co,
fabian.munoz@uan.edu.co
Abstract
This paper presents the implementation of a quantitative
system of assessment by ultrasound, which allows the
acoustic characterization of animal blood plasma, with
potential applications in hematic biometric techniques.
The system comprises a unidimensional electronic
controller with step size 1 mm which enables the acoustic
inspection of blood samples using an ultrasonic field
incident with central frequency of 5MHz in pulse echo
mode. Additionally, the implemented system incorporates
the development of computer algorithms in Matlab
allowing digital signal processing of ultrasonic
backscattered signals, based on the estimation of acoustic
parameters such as speed of sound and spectral energy
loss, using the logarithmic power spectral density
subtraction in the bandwidth of the transducer (1MHz @ -
3dB). Estimating acoustic parameters from blood samples
centrifuged to 3398 rpm were correlated with density
values, pH and concentration of platelets. The results
showed that samples with densities between 1.040 g/ml -
1.051 g/ml and variations of platelet concentrations
between 45x103 / mm3 - 138x103 / mm3, showed variations
in sound velocity between 1335 m / s - 1572 m / s.
Moreover, measurements showed a sound attenuation
subtle difference variations between 0.21dB/cm to 2.73
dB/cm. Finally quantitative ultrasound system shows that
the estimate of velocity profiles acoustic propagation
enables infer variations of platelet concentration at room
temperature with potential applications in the quality
assessment of blood plasma.
Keywords: Acoustic, Backscattering, Characterization,
Platelts, Ultrasonic.
978-1-5090-3797-1/16/$31.00 ©2016 IEEE
Luis F. Londoño
Politécnico Gran Colombiano Jaime Isaza
Cadavid
lflondono@uan.edu.co
1. Introduction
Plasma rich in platelets (PRP) is a suspension gel
containing high platelet concentrations that allow repair
and enhance tissue injury, such as blood vessels healing to
stop bleeding [1]. The use of platelets in therapeutic
applications involves obtaining autologous PRP, due to
the growth properties of these and their applications in
regenerative medicine [2]. Given that the PRP is defined
as the partial portion of autologous blood plasma and has
higher concentration of platelets than baseline value, it can
be seen that the quality of plasma depends on the amount
of platelets contained [3]. This implies that the quality of
platelets contained in the plasma is directly proportional to
the regeneration capacity. [4].
In this context there is a great scientific and
technological interest in implementing methods to
measure the quantity of plasma and its regenerative
capabilities , as evidenced by some studies in the literature
[5] [6], [7], [8], directed to repair joints, soft tissue and
bone, among others.
The conventional technologies used in clinical
laboratories (hematology equipment) for counting blood
cells, have disadvantages in relation to operating costs,
acquisition and maintenance, in addition to the negative
impacts caused to the environment by the use of chemical
reagents (V-28R, V-28D and others). In this regard, the
use of other methods of counting (Neubauer chamber and
Thom pipette) based on the use of microscope and
techniques are wasteful and susceptible to human error
due to the subjectivity of the observer (bacteriologist) [9].
Considering the disadvantages associated with the above
techniques, evidenced the need to promote the
development of new biomedical technological alternatives
aiming the characterization of biological fluids, according
to the needs stipulated in the national Ten-Year Health
Plan.
In that sense, ultrasound technology are a potential tools
in the assessment non-invasive and nondestructive of
biological tissues, fluids and materials [10], in addition to
presenting optimum cost – benefit relationship. Some
studies related to ultrasonic characterization of blood
coagulation have been reported in [11], [12], showing the
estimation of glucose concentration [13] among others and
reflecting the scope of characterization techniques
ultrasonic.
This paper shows the implementation of a system of
quantitative ultrasonic characterization of blood animal
plasma, which aims to contribute the development of an
acoustic hematological system for estimation of platelets
concentration, through the calculation of acoustic
parameters (speed of the sound and acoustic attenuation
frequency dependent), using spectral analysis and the
measurement of the time of flight of mechanical waves of
high frequency. This way, the intention is to promote the
development of ultrasonic techniques in animal
biotechnology applications considering the potential use
of acoustic techniques used in the calculations of
concentration of substances [1], allowing efficient analysis
noninvasive and nondestructive with a good relation with
the environment in reason of that chemical residues do not
exist.
2. Theoretical Fundament
2.1 Blood Plasma
The blood consists of 55 % of plasma, which is
constituted by water, minerals salts and proteins. 45%
remainder is composed by red globules (erythrocytes),
white globules (Leukocytes) and platelets
(Thrombocytes). The plasma rich in thrombocytes (PRP),
perform a fundamental role in the tissue regeneration and
conventionally it is obtained from processes of
centrifugation. The plasmatic fraction located on the line
basal, it possesses the biggest number of thrombocytes
and factors of growth (PDGF - Factor of Growth Derived
2
from the Thrombocytes, FGF – Factor of growth of
Fibroblast between others), responsible of stimulating the
processes of regeneration and repair tissue.
2.1 Acoustic Parameters
Acoustic parameters as the speed of the sound and the
acoustic attenuation are important in the ultrasonic
characterization of fluids, tissue and materials. The speed
of sound is strictly related with the density and the
acoustic impedance of the sample study. His estimation is
matchless in each material once this one is submitted to
examination by an acoustic field. During the acoustic
characterization of biological tissues the acoustic
propagation velocity provided information about the
features of compressibility, concentration of particles and
soluble solid among others. The measurements were
performed from the time of flight estimates of the
ultrasonic wave. The Equation 1 represents the expression
that allows calculating the speed of sound c in
configuration pulse - echo, measuring the elapsed time in
which, the wave travel through the propagation medium:
c = 2x/t (1)
where 2x it indicates two times the propagation distance
and t the time of flight.
On the other hand, the acoustic attenuation is a
phenomenon in which occurs loss of energy by effects of
dispersion and reflection during the interaction wave-
biological fluid. This loss of energy can be measured by
the calculation of a spectral attenuation coefficient Catt. Its
mathematical model for the experimental estimates is
described by the following equation (2):
Catt= [20 log (Spx/Sp0)] / 2x (2)
Where Spx and Sp0 correspond to power spectral densities
estimated from backscattered ultrasonic signals in a study
sample and a specular reflector respectively.
3. Ultrasonic Characterization System
The experimental setup for the acoustic analysis of the
blood plasma of bovine is illustrated in Figure 1. The
technical characteristics of each component of the device
are described to follow. The Ultrasonic characterization
system has an acoustic tank of 4000 cm3, which contains
the coupling medium to ensure the maximum transfer of
ultrasonic energy to the plasma sample contained in a
vacutainer of 5ml. The system also has a Arduino
microcontroller configured unidirectionally to handle the
movement of the vacutainer, through a step motor ROHS
5V, coupled to a rack rail of 20 cm, which keeps track of
displacement with a step size 1mm .

Figure 2. Blood samples with biological centrifuge process.

SAMPLES DENSITY
(g/mL)
Figure 1. Ultrasonic characterization system a. Computer 1 1.040
equipment; b. Generator of electric pulse excitation; c. 2 1.040
Oscilloscope; d. Electromechanical system of millimetric 3 1.048
positioning e. Acoustic tank; f. Vacutainer; g. Ultrasound 4 1.048
transducer. 5 1.054
6 1.054
Figure 1 also shows a pulse generator Olympus PR
7 1.063
5072, configured in pulse-echo mode to excite an
8 1.063
ultrasonic transducer (Olympus) 5MHz, with bandwidth
of 1 MHz @ -3 dB, using a pulse repetition frequency 9 1.051
(FPR) of 200Hz. Finally the system is composed of an 10 1.051
oscilloscope Gw Instek GDS-1102 AU, used as data 11 1.161
acquisition system with sampling rate of 10M/S and a 12 1.161
computer equipment on which operate computational 13 1.093
procedures in MATLAB (R2014b) for estimating of speed 14 1.093
of sound and the acoustic attenuation. 15 1.073
16 1.073
3.1 Characterization Procedures
Table 1. Physicochemical parameters obtained in blood plasma
samples. pH values were 8 approximately for all samples.
3.1.1 Biological Samples
3.1.1 Ultrasonic Inspection
Biological samples of animal blood (Brown Swiss Cattle
breed) were obtained at the farm of the University
This procedure involves exposing blood plasma samples
Antonio Nariño, collecting 16 samples of 5 ml in
to a pulsatile acoustic field. Initially the vacutainer
vacutainers with heparin to prevent the clotting time.
containing the sample study is positioned by an
Subsequently, centrifugation processes were performed
electromechanical scanning system automated that allow a
with a speed of 3398 rpm for each sample during 10
longitudinal inspection of 3cm at a focusing distance
minutes, aiming the separating of plasma from other blood
(6cm), in which the far field of the transducer is
components as shown in (Fig.2). After the centrifugation
guaranteed, in order to monitor different regions of the
protocol, the physicochemical parameters such as density
study sample. Figure 3 shows the inspection system
and pH (estimated by reactive strip) of biological samples
implemented during acoustic exposure on a plasma blood
were characterized reporting values in Table1.
phantom made with a gel and frost particles.
Immediately to the previous characterization, samples
were exposed to an ultrasonic field for acoustic scanning.

3

Figure 3. Signal Capture System: a. Stepper Motor; b. Sample
(Phantom of biological sample); c. 5MHz; d. Laser for
positioning verification.
3.1.2 Signal Processing
The ultrasonic backscattered signals acquired after
propagation in the study samples were used to estimate the
speed of sound (Fig. 4) and the spectral energy loss
(attenuation).
Figure 4. Speed estimation of the sound.
The Figure 4 presents the algorithm used for the
calculation of the speed of the sound from time of flight
measurements from RF signals (backscattered ultrasonic
signals) stored as files with extension ".CSV". In the first
instance each file is load from a directory and later a
4
region of Interest (RoI) is defined corresponding to the
plasma region inside the vacutainer. This way the time of
flight is estimated from the first wall of the vacutainer up
to the second wall (to see Fig. 5) and finally the speed of
the sound is calculated, considering the propagation
distance to be two times the diameter of the vacutainer,
because the acoustic examination executes in pulse – echo
mode.
Figure 5. Ultrasonic signal in vacutainer and detection of time of
flight.
Otherwise, the Figure 6 presents the algorithm that
allows the calculation of the coefficient of acoustic
attenuation
Figure 6. Estimation of acoustic attenuation coefficient.
by spectral analysis for the estimation of the loss of
acoustic energy using the periodogram method, estimated
as the square module of the Fourier Transform on the
number of samples | (FFT) ^2 |/N. Thus, later to the
lecture of the directory container of backscattered
ultrasonic signals, their power spectral densities (PDS)
estimated on the second wall of the vacutainer during
propagation on blood plasma are compared with the PDS
from an ultrasonic specular reflection (incident pulse).
Finally, by spectral logarithmic difference is measured the
attenuation coefficient by the mathematical model
mentioned previously.

Figure 9. Spectrogram from bovine blood plasma in

vacutainer.

Table II shows the values of the speed of sound,

attenuation and platelets concentration for different study
samples. The results show a linear and proportional
relationship between the increase in concentration of
platelets and increased acoustic parameters.

Figure 7. Power spectral densities from reference medium Considering Table 2, the Figures 10 and 11 show the
power and blood plasma sample.
linear relationship between the platelets concentration in
blood plasma and experimental measurements of the
4. Results speed of sound and spectral energy loss (attenuation).

Considering the ultrasonic backscattered signals

acquired, as shown in Fig.8, signals were analyzed by the
time - frequency duality as shown in Fig. 9, from which it
was possible to estimate velocity values and loss of
spectral energy.

Figure 8. Relationship between the backscattered ultrasonic

signals and the experimental vacutainer inspection.
Fig. 9 presents the ultrasonic energy distribution in the
time – frequency duality showing high energy reflection
regions given by the rigidity of the walls of the
vacutainer.
Figure 10. Comparison of the behavior of speed of sound for the
platelets blood (experimental measurements) and erythrocyte
(reported in the literature).

5
 SAMPLES DENSITY SPEED OF ATTENUATION CONCENTRATION
(g/mL) SOUND (m/s) COEFFICIENT OF PLATELET
(α) dB/cm (x103/mm3)
1 1.040 1669 3.92x10-4 138
2 1.040 1437 3.53x10-4 138
3 1.048 1498 3.17x10-4 78
4 1.048 1401 3.11x10-4 78
5 1.054 1437 3.06x10-5 67
6 1.054 1428 2.85x10-4 67
7 1.063 1413 6.66x10-5 54
8 1.063 1403 2.25x10-4 54
9 1.051 1401 1.82x10-4 45
10 1.051 1379 1.82x10-4 45
11 1.161 1356 1.17x10-4 39
12 1.161 1356 1.11x10-4 39
13 1.093 1350 8.63x10-5 23
14 1.093 1333 1.64x10-4 23
15 1.073 1374 9.10x10-5 20
16 1.073 1333 1.08x10-4 20

Table 2. Acoustic parameters concentration of Thrombocytes

The results obtained were compared with measurements
from blood cells concentration reported in the literature,
indicating a linear proportionality as experimental results.
Moreover, estimates of attenuation showed a linear
relationship according to the increase of platelets, showing
an angular coefficient of 2.46x10-6 dB / cm.

5. Conclusions

This study presents the implementation of an ultrasonic

Figure 11. Linear fit and relationship between the acoustic
attenuation and the thrombocytes concentration.
Figure 10 shows that the speed of sound can be adjusted
by a linear fit with respect to the platelets concentration
increase. The linear regression is described by the
mathematical model y = 4.03x + 1275.60, indicating a
growth rate of 4.03 m/s for each percentage increase in
the value of the platelets concentration.
characterization system of animal blood plasma samples,
based on quantitative estimation of acoustic parameters
(sound speed and attenuation) and its relationship to the
concentration of platelets. The speeds of sound reported a
range of values between 1669 m/s - 1333 m/s, which are
coherent with estimated reported in the literature, for
studies of ultrasound characterization of liquids and
microparticles and values reported by the Laustpederson
database. Experimental results show that there is a linear
and proportional relationship between estimated
parameters and cells concentration, showing the potential
of the quantitative ultrasound characterization techniques
in predicting of platelets concentrations in blood plasma
and the extrapolation to measuring concentrations of
substances in other bioengineering applications.
6. Acknowledgements
The authors of this study are grateful to the Antonio
Nariño University for the financial support of this project

6
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