Blood Coagulation Measurements Using Dynamic Speckle Technique

M.M. Patiño-Velasco1, C. Andrade-Eraso1, J. Vásquez-López1, M. Trivi2, and H.J. Rabal2

1
Universidad del Cauca, Popayán, Colombia
2
Centro de Investigaciones Ópticas and Facultad de Ingeniería, Universidad de La Plata, Gonnet- La Plata, Argentina
{mariomilver,ingcarlosandrade,javalop}@gmail.com,
marcelot@ciop.unlp.edu.ar, h_rabal_geb@yahoo.com

Abstract—A series of coagulometers have been developed
with the aim of making easier the measurement of coagulation
times. A phenomenon that emerges as an alternative for the
development of these devices is the use of dynamic laser speck-
les. It is based on the analysis of time variations of a speckle
pattern produced by interference of multiple coherent wavelets
scattered by the sample. We present the results of applying
correlation analysis to dynamic speckle produced by samples
of capillary blood clotting without using any reactive. We also
propose a numerical model which describes the obtained
curves and a method for measuring coagulation time from this
analysis.
Keywords— blood coagulation, optical techniques, image
processing, biospeckle.
I. INTRODUCTION
When highly coherent light illuminates a biological tis-
sue which shows roughness of the order of the wavelength
or higher, the scattered light produces a granular pattern,
called speckle, consisting in bright and dark points. Those
points are produced by the interference of secondary waves
coming from roughness structure. The same phenomenon
occurs when light goes through the sample. If the tissue
presents some type of activity, the dynamics of the scatter-
ing centers modifies the optical path differences, phase
differences between the wavelets are changed and the
speckle pattern intensity changes along time [1]. The activi-
ty of the sample can be inferred from the analysis of the
time space variations of the speckle patterns. Several proce-
dures have been used to describe the activity of dynamic
speckle patterns [2]. From the above mentioned techniques,
it was possible the development of a technique for bio-
speckle activity characterization that can be applied to sev-
eral fields [3-6].
Hemostasis is the control of bleeding through a delicate
balance between procoagulant, anticoagulant, fibrinolytic
and antifibrinolytic activities [7]. It is intended to stop hem-
orrhage though a series of biochemical reactions by convert-
ing blood fibrinogen into fibrin. These reactions are carried
out because of the action of proteins called coagulation
factors [8]. The process is made in three phases: initiation,
amplification and propagation, according to cell based co-
agulation model [9]. As a result of this process, platelets
aggregation occurs, huge amounts of X factors activate and
prothrombinase is generated. The latest converts great vol-
umes of prothrombin in thrombin and, under the action of it,
fibrinogen in fibrin [10]. Besides, series of laboratory
tests to determine abnormalities in hemostasis have been
developed. They are used to identify the deficiency in the
factor that produces it [11]. For control of anticoagulation
therapy in patients prothrombin time (PT) is used [12].
Some coagulometers have been developed to reduce the
variability of the observations based in physical principles
able to detect the changes occurring in blood while it coagu-
lates. In 2004, Piederriere et al. [13,14] proposed a method
for the measurement of platelets aggregation based in the
change of size of speckle grains. They also proposed two
alternative methods to measure blood coagulation time. In
both approaches reagents were included inducing initial
turbulence.
This work is on the same line of the recently published
approaches [15,16] and can be considered as a continuation
of its with some improvements and particular features.
In this work we present a correlation analysis to dynamic
speckle patterns obtained from blood samples that coagulate
spontaneously. Coagulation triggers directly because of the
contact of blood with the tissue factor occurring when the
finger is pricked. We proceed in a similar way to that of
Piederrière et al. [13], and particularly to Faivre et al. [16],
also using correlations between consecutive frames. We
present then a numerical analysis to fit the obtained curves
and propose a method to evaluate the coagulation time. This
results in robust measurements that can be favorably com-
pared with INR classical methods of standardized meas-
urements without the need of reagents and not requiring to
know the precise time of the start of the experiment. This
implies considerable simplicity and avoids possible errors in
composition concentration, initial turbulence of added rea-
gents and added cost. Besides, the results include an analyt-
ical description of the curve with a good fit and a parameter
characteristic of it that could be used in the future as addi-
tional information for diagnosis or following of treatments.

© Springer International Publishing Switzerland 2015 91

A. Braidot and A. Hadad (eds.), VI Latin American Congress on Biomedical Engineering CLAIB 2014, Paraná, Argentina 29, 30 & 31 October 2014,
IFMBE Proceedings 49, DOI: 10.1007/978-3-319-13117-7_24
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II. EXPERIMENTAL METHOD
We used an experimental setup in the so called free
propagation geometry as is shown in Figure 1. A linearly
polarized 5 mW power He-Ne laser (632.8 nm wavelength)
was used for illumination, the speckle patterns were ac-
quired using a lensless CCD camera (SONY® XCD-
SX450CR) with a linear polarizer included in front of it to
control light intensity, a heater with a temperature control
device was specifically designed to maintain the sample
under test at a 37.0 ± 0.5 Celsius without interacting with
the illuminating light, and the blood samples were located
on a removable glass plate on the heater. This experimental
set-up is an improvement of the former one employed in a
previous paper [15].
Fig. 1. Experimental set-up.
Samples corresponding to 14 volunteers were extracted
under general biosecurity protocols [17] and according to
standards of protection of human patients [18] they were
assigned a code and analyzed. Each sample consists of 50
microliters of blood which were extracted from the pad of
the middle finger of the left hand. It was later deposited on
the preheated to 37 Celsius degrees glass plate and a 60
seconds video was immediately recorded to perform the
measurements. Therefore, no reagents such as thromboplas-
tin and anticoagulant were employed.
To detect the time intensity variations in the biologic
dynamic speckle patterns, we used correlation analysis. It
involves low computation time, so that the analysis of the
samples can be performed in real time. The similarity be-
tween consecutive frames was evaluated using the follow-
ing expression:
  I ( x, y)  I  I
1 1 2 ( x , y)  I 2 
r
x y
2 2
  
   I1 ( x , y)  I1      I 2 ( x , y)  I 2  
 x y   x y
Where r is the correlation coefficient, I1 and I2 are the
measured intensities in a pixel located in the same position
(x,y) for two consecutive frames, Ī1 and Ī2 are the mean
M.M. Patiño-Velasco et al.
intensities in each frame. Values of r close to unity indicate
high correlation due to a high similarity between the con-
secutive video frames indicating low activity in the sample.
Conversely, high activity can be inferred if r is close to 0.
Intermediate r values help to follow the time evolution of
the process.
III. RESULTS
Figure 2 shows four correlation index curves obtained
from four volunteers using this procedure. Three regions
can be observed. The first is a high activity one that can be
associated to the start of the hemostatic mechanisms. The
middle one, where the activity diminishes very fast, is due
to the formation of the clot. Finally, in the last region, as the
hemostatic activity is drastically reduced we find a stabiliza-
tion region indicating that the clot has already been formed.
From the numerical analysis of the obtained results, it
was found that the curves could be fitted with good accura-
cy by a sigmoidal curve described by the following equa-
tion:
A1  A2
r  A2  p (2)
t
1   
 t0 
Where r is the correlation coefficient that defines the ac-
tivity estimation, A1 is the lower asymptote, A2 is the upper
asymptote, t0 is the time required for the curve to reach the
inflexion point and p determines the growth speed of the
function. It provides a method to measure the time taken for
clot formation by a simple and predictive function that fits
the experimental data. A very good coincidence between
them the experimental results and the numerical fit can be
observed (R2 = 0.98)
(1)


Fig. 2. Correlation index obtained from four volunteers
IFMBE Proceedings Vol. 49
Blood Coagulation Measurements Using Dynamic Speckle Technique
IV. DISCUSSION
The analysis of the obtained curves shows two processes
that are limited by the inflexion point. The first is the initia-
tion stage that is present since the onset of the activation of
the coagulation up to its first visible manifestations, associ-
ated to fibrin polymerization [19].The second process is the
elongation step, characterized by the evolution of the clot
density which ends with its stabilization [20]. There, the
growth of the curve is associated to the increase in the vis-
cosity of blood, due to the increase in fibrin concentration
[21] and it is this region of the curve that describes the clot
formation.
We propose the time elapsed from the inflexion point un-
til the stabilization of the curve as a descriptor to determine
alterations in the initial coagulation phase. Table 1 shows
the obtained parameters and the measured times using this
this method (in column six (Tclot).
Table 1. Parameters of the clot measurements.
IFMBE Proceedings Vol. 49
93
We established that the clot was formed when the curve
has reached 97% of its upper asymptote. Measurements
were automatically performed within a resolution of 0.1
second, which corresponds to the sampling rate of the regis-
tered video. Furthermore, we used the International Normal-
ized Ratio (INR) obtained for each volunteer using a clini-
cal test of the Prothrombine Time (PT) [12] (measured with
GKV OP-2 coagulometer) as a reference to compare the
obtained results as is shown in the last column in Table 1.
INR is a standardized measure of prothrombine time PT
defined in such way as to be able to compare prothrombine
time measurements obtained using some reagents with the
same magnitude as measured with a different set of rea-
gents. To obtain it the measured PT in seconds is divided by
a value corresponding to a normal control value PTnormal
and the result raised to a power named ISI (International
Sensitivity Index) that accounts for different manufacture
differences.
ISI
 PT 
INR    (3)
 PTnormal 
The International Normalized Ratio (INR) values of
normal coagulation are generally considered between 0.9
and 1.2 [20].
Figure 3 shows the comparison of the times as measured
with the proposed method with the corresponding INR ob-
tained through the clinical test of the Prothrombine Time
(PT) for the same volunteers. As it can be seen, there is a
high level of agreement between both results.
A linear regression of these points shows that the INR
and the measured speckle Tclot are highly correlated (R2 =
0.96), thus indicating that the method of measurement using
biospeckle correlation described here provides important
information that can be used for the estimation of the INR.
Fig. 3. Comparison of the Tclot results with the corresponding INR results
and the least squares linear regression.
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V. CONCLUSIONS
We conclude that there is a strong evidence of the ap-
plicability of the proposed biospeckle method which also
provides a simple, fast and economic observation of the
normal behavior of the human blood coagulation without
using other substances. This facts were already known from
other publications and patents [23,24]. Nevertheless, as the
method that we propose is based on the time elapsed be-
tween the inflexion point and the stabilization of the corre-
lation curve, it does not depend on a precise measurement
of the coagulation start time. Besides, no reagents, such as
thromboplastin and anticoagulant, are required that could
interfere in unknown ways (trough variations in purity
and/or dosage) with the several components of blood and
add turbulence in the early times of coagulation.
The method provides a full curve showing the evolution
of the coagulation process that could be used to screen out
other anomalies. This curve uses many observation points
and least squares fitting, so that it is robust with respect to
start and end only time measurements. Besides of the clot
time estimation provided by the standard clinical methods,
in our proposal is possible to follow the behavior of blood
in all clot process We intend to continue the research on this
line by analyzing a greater number of patients and by in-
cluding samples of people presenting coagulopathies, so
that it could be possible to evaluate the ability of the method
to detect this type of pathologies and evaluate the evolution
of their treatment.
This method also makes possible to ana-
lyze homeostatic response to medical treatment and it could
be tested on animal blood coagulation disorders. Results
will be published in the future.
AKNOWLEDGMENT
This work is supported by CONICET, CIC, ANPCyT
and UNLP (Argentina) and University of Cauca (Colom-
bia).
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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