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Supporting the potential of

quantitative ultrasonic techniques for
the evaluation of platelet
concentration

J. A. Villamarín, Y. M. Jiménez , L. Tatiana Molano, W.

Edgar Gutierrez, L. Fernando Londoño, et al.

J. A. Villamarín, Y. M. Jiménez , L. Tatiana Molano, W. Edgar Gutierrez, L.

Fernando Londoño, D. A. Gutierrez, "Supporting the potential of quantitative
ultrasonic techniques for the evaluation of platelet concentration," Proc. SPIE
10572, 13th International Conference on Medical Information Processing and
Analysis, 1057207 (17 November 2017); doi: 10.1117/12.2288118

Event: 13th International Symposium on Medical Information Processing and

Analysis, 2017, San Andres Island, Colombia

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 Supporting the potential of quantitative ultrasonic techniques for the
evaluation of platelet concentration

J.A. Villamarín, Y. M. Jiménez, L. Tatiana Molano, W. Edgar

Gutierrez, L. Fernando Londoño, D.A. Gutierrez,
Bioengineering Research Group, Antonio Nariño University,
Popayán, Cauca, Colombia.

ABSTRACT

This article describes the results obtained by making use of a non-destructive, non-invasive ultrasonic system for the
acoustic characterization of bovine plasma rich in platelets using digital signal processing techniques. This study includes
computational methods based on acoustic spectrometry estimation and experimental measurements of the speed of sound
in blood plasma from different samples analyzed, using an ultrasonic field with resonance frequency of 5 MHz. The
results showed that the measurements on ultrasonic signals can contribute to the hematological predictions based on the
linear regression model applied to the relationship between experimental ultrasonic parameters calculated and platelet
concentration, indicating a growth rate of 1 m/s for each 0.90 x103 platelet per mm3. On the other hand, the attenuation
coefficient presented changes of 20% in the platelet concentration using a resolution of 0.057 dB/cm MHz.

Keywords: Acoustic, Blood, Plasma, Ultrasound.

1. INTRODUCTION

Platelet rich plasma (PRP) is an important bioactive substance in processes that stimulate the regeneration and healing of
biological tissue, acting as an autologous source of growth factors and stimulates the secretion of proteins involved in the
repair of the pathological tissue [1-3]. Plasma characterization results in great interest in therapeutic applications in
animals [2] and humans [3], since the quality of the platelet concentration present in the PRP contribute to guarantee the
regenerative potential [4]. In the last decade the use of bioactive substances that seek to improve the prognosis in clinical
situations of the stomatognathic system has had an important development. Currently, this technique has been being
applied to patients with dysfunctions in the healing process and more specifically in patients with surgical needs.

Different methods of platelet quantification are based on the observational analysis performed under a conventional
microscope that leads to susceptible measurements by the subjectivity of the operator-observer. Moreover, automated
hematology equipment can identify different cell types through chemical contaminant agents that affect the environment
[6], suggesting the need to research for alternative methodologies that allow to analyze the blood with reliability and
reduce the operative costs required in the hematological systems.

Ultrasound-based technologies are advantageous in the non-invasive characterization of tissues, fluids and materials [7]
having an optimal cost-benefit ratio. Thereby, the ultrasonic characterization of biological fluids has been used recently
at the hematological applications, focusing on processes related to blood coagulation analysis [8], the assessment of
erythrocytes aggregation [9,10] and estimation of glucose level [11], [12], reflecting the scope of ultrasonic
characterization techniques.

Considering the potential of these techniques based on quantitative signal processing of high frequency mechanical
waves, this study shows the estimation of platelet concentration from spectral energy loss estimations and the time of
flight measurements by the systematic wave- biological fluid interaction.

13th International Conference on Medical Information Processing and Analysis, edited by Eduardo Romero,
Natasha Lepore, Jorge Brieva, Juan David García, Proc. of SPIE Vol. 10572, 1057207
© 2017 SPIE · CCC code: 0277-786X/17/$18 · doi: 10.1117/12.2288118

Proc. of SPIE Vol. 10572 1057207-1

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 2. MA
ATERIALS AND
A METHO
ODS

2.1 Hematoological samplles

Twelve sampples of bovine blood each onne with 5 ml werew obtained from the juguular vein in the faculty of veeterinary
medicine of the Antonio Nariño
N A experimenttal tests were carried out with
Univerrsity (UAN). All w the consennt of the
research grouup on Welfaree, Health and Animal
A Producction (UAN). The
T protocol forf obtaining samples of plattelet-rich
plasma consiisted first of the
t provision of o hematologiccal samples inn EDTA tubess at centrifugation rate of 24400 rpm
during 10 miinutes. The sammples were proocessed for hisstological analyysis, mounted on 75 mm x 25
2 mm slides, and then
treated with Masson's trichhrome stainingg. They were analyzed
a undeer an optical microscope
m at 100x, followedd by the
correspondinng quantificatio
on of platelets in
i Neubauer's chamber
c (see Fig.
F 1).

)o.
):°oc' is
;;
04á e . :! 4[ ¡ ¡'
oe
:,..
s,
¡Ñ,.5..
_
='iÌ
,0 .
.
-

Figure 1. Morpphological analy

ysis and platelet count. 100x (Maasson's trichrome staining).

2.2 Ultrason
nic characteriization system
m

The acoustic non-invasive and non-destruuctive characteerization system m comprises: a)a an ultrasonicc field emissionn system
(Olympus 50072PR), b) a positioning
p maachine and c) a data acquissition and proccessing system m. The ultrasonnic field
emission system is configu ured in pulse-eccho mode to provide
p an exciitation energy of 26µJ for ann ultrasound traansducer
(Panametricss A109s) with a central frequuency of 5 MH Hz at bandwiddth of 6 MHz at a -6dB. It is stimulated
s withh a pulse
repetition freequency of 100
0Hz. The posittioning machinne uses a ROH HS stepper motoor (28BYJ-48)) driven by an Arduino
microcontrolller, which allo
ows a control step
s mm by coupling of an endlesss screw. This system also inncludes a
size of 1m
mechanical support
s to hold
d the ultrasoundd transducer annd an acoustic glass tank on which
w a liquidd is attached aim
ming the
coupling betw ween the acoustic wave transsfer and the heematological saample. At last, the data acquiisition system includes
a Gw Instek GDS-1102
G A-U
U oscilloscope,, which stores the ultrasonic backscatter
b siggnals in the interest region (RRoI) with
a sampling raate of 50M/S. The stored siggnal files are suubsequently processed by com mputational prrocedures impllemented
using Matlabb (R2014b). Figgure 2 describees the experimeental set up com
mponents.

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 Figure 2. Ultrrasonic characterrization system implemented. a.a Processing syystem. b. Microccontroller. c. Stepper motor and endless
screw d. Backking. e. Hematoloogical tube. f. Ultrasonic
U transdducer. g. Acousttic glass tank. h. Pulse generatorr. i. Acquisition
n system
(Oscilloscope)).

2.3 Ultrason
nic inspection process

Enlarrgement

6 cm

li Platelet
Rich
Plasma
Ì.
i 11
)ainping
N F

\Zaterial W'all W
Echo Ec:ho Ec

Figure 3. Backkscattering signaal in RoI of plateelet-rich plasma.

The bovine platelet-rich

p plasma samples contained in the
t clinical hemmatology tubess were subjecteed to a high frrequency
and low pow wer acoustic fiield at a focal length of 6 cm, where a more
m uniform wave
w front witth minimizatioon of the
diffraction efffects is guaran
nteed. For eachh sample, twennty signals weree obtained durring the verticaal sweep from the
t basal

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 - line of blood plasma aiming the digital signal processing. An example of the ultrasonic backscattering signal collected
in the RoI is shown in Figure 3.

2.4 Computational procedures

The characterization of the study samples was performed using digital signal processing algorithms, which estimate two
acoustic parameters: speed of sound and acoustic attenuation, being the last based on spectral analysis techniques. The
calculation of the speed of sound is important as it contributes to the understanding about the properties of the medium
through which it propagates such the density and the stiffness of a particle vibrating in an acoustic field. Additionally its
estimate provides information that might be associated with the compressibility characteristics and particles
concentration of biological fluids studied, which allows its characterization.

Experimentally, the speed of sound ( ) was determined by measuring the time of flight (ToF) of a wave travelling
along the propagation distance (x) in the RoI, detecting echoes of high amplitude produced by the high reflection
coefficient of the polypropylene walls of the hematological tube, associated with acoustic impedance change between the
tube wall and the blood plasma. According to the , the speed of sound was estimated based on the expression:

= (1)

Figure 4 shows an example of the echoes selected on the walls of the hematological test tube aiming the calculation of
the speed of sound in the blood plasma. The distance of propagation in the colloidal system is twice the diameter of the
hematological tube (considering that the acoustic inspection was in pulse echo mode). The speed of sound algorithm was
calibrated from experimental measurements in water used as reference liquid.

Sample 1 Sample 2

20

Time (s) x10 -s Time (s)

Semple 3 Sample 4
15 ToF:13.12ps
1
05 05
o 0
-as T. -0.5
-1 ó -1
-1.s
z
2 a 6 8 10 12 14 16 18 20 2 4 6 8 10 21 14 -2 10 20

Time (s) Time (s) x10 ".

Sample 5 Semple 6
15 ToF:13.53ps
1

0.5
II
0 } ^iM'
II
0.5

2
4 6 8 10 12 14 16 18 20 2 t 6 8 10 12 14 16 18 20

Time (s) 110-° Time (s) X10 e

Figure 4. Ultrasonic backscattering signals from platelet-rich plasma.

On the other hand, the acoustic attenuation coefficient is a parameter that describes the loss of acoustic energy caused by
the propagation medium. Its value depends on the medium in question. In soft tissue characterization procedures its
calculation based in spectral method is given by the following equation:

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 = , (2)

where 0 annd are the power spectraal densities estiimated from ulltrasonic echoees obtained durring the propaggating in
water and blood plasma respectively.
r C
Considering thhe power law = , the
t frequency--dependent attenuation
coefficient ( ) can be estim
mated assumingg that experimmentally, the freequency depenndence index for soft tissuees can be
approximatedd linear. Spectral analysis tecchniques are thhe main techniique applied aiiming the attennuation calculaations, as
is displayed in
i Figure 5.

Time (s)
Calcubl#

Linear Fit Spectral

Subtraction

Figure 5. Specctral analysis tech

hnique implemeented.

Computationnally the acousstic attenuationn is measured based on the estimation of the angular cooefficient givenn by the
linear adjustm
ment between the normalizeed power specttral subtractionn obtained froom ultrasound echoes in plattelet-rich
plasma and water
w (used as a reference meedium becausee its acoustic prroperties are known
k and the acoustic energgy loss is
negligible). The
T spectral su ubtraction guarrantees the elimmination of ennergy loss effeccts caused by the
t hematologiical tube
walls and aliignment errorss of the incidennt ultrasonic field.
fi The spectral power dennsities were esstimated by callculating
from the nonnparametric perriodogram metthod. Figure 6 shows an exam mple of the spectral differences between water
w and
samples of thhe bovine bloood plasma. It iss possible to seee an attenuatiion of high frequencies from
m spectra samplles when
they are com mpared with the reference spectrum. Sppectral variatioons corresponnd to a variattion range off platelet
concentrationn of 27.18%.

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 [A]

[B]

Figure 6. A. Spectral differeences between platelet-rich

p plassma samples. B. Spectral compparison betweenn each one of thhe plasma
samples and thhe reference specctrum water.
3. RE
ESULTS

Acoustic paraameters valuess were estimateed for blood plaasma samples. These values are displayed in Table 1.

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 Table 1. Acouustic parameters estimated by ulttrasonic signal processing.
p
Saample Speed of Sounnd Average Attenuation Aveerage (dB/cm-M
MHz) Plateelet Concentratioon (%)
(m/ss)
1 1.6664 3.008 x10-4 100

2 1.617 2.881 x10 -4 98.6

3 1.572 1.663 x10 -4 97.4

4 1.521 1.334 x10 -4 78.3

5 1.488 1.441 x10 -4 78.3

6 1.472 1.110 x10 -4 72.8

A linear relattionship betweeen platelet conncentration andd experimentall estimation off the speed of sound
s and the acoustic
attenuation was
w found (Figu ure 7). The speeed of sound can be adjustedd linearly showwing to the incrrease of plasmaa platelet
concentrationn, indicating a growth rate off 1 m/s for eachh platelets increase of 5.52% per mm3. According to data reported
in the literatuure [13], [14],[15], the measuurements of thhe speed of souund show a lineear proportionaality evidencingg in [15]
a increase off 1 m/s for eaach variation of o blood cells of approximattely 1.52% perr dL. On the other o hand, atttenuation
estimation prresented a lineaar relationship according to thhe increase of platelets,
p evideencing a growtth rate of 0.0577 dB/cm-
MHz for eachh percentage ch hange of platellet concentratioon.

Figure 7. Proffile of the averag

ge of the speed of
o sound propagaation and the atteenuation coefficient average for different percenntages of
platelet concenntrates in plasmaa samples.

4. CONCLUSIO
ONS

The potentiaal of quantitativ

ve ultrasound characterizatioon techniques in non-invasivve and non-desstructive hemooanalysis
was demonsttrated in the present
p article, based on thee estimation of
o acoustic parrameters and their linear reegression
models, whicch showed the relationship between the conncentration of Platelet-Rich Plasma
P and acooustic parametters such
as the veloocity of soun nd and frequuency-dependeent attenuationn. The experrimental proccedures of ultrasonic
characterizatiion evidenced that a variatioon of 53 m/s in
i the speed off sound corresspond approxim mately to variaations of
5.52% of plaatelets per mm3. On the otherr hand, the calcculations assocciated to the atttenuation understood as the acoustic
energy loss inn the study sam
mples allowed to evaluate a variation
v of appproximately 277% of the platellet concentratioon in the

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 analysis samples. Finally, considering the linear regression models obtained from the analysis of 120 ultrasonic signals
from 6 study samples, this study shows a relationship of proportionality of conventional ultrasonic parameters that can be
used to predict platelet concentrations. This approach is expected to stimulate the field of action of acoustic
characterization of biological and industrial fluids.

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