BLOOD COAGULATION STUDY USING LIGHT-TRANSMISSION
METHOD
Hyunjung Lim1, Jeonghun Nam1, Yongjin Lee1, Shubin Xue1, Seok Chung1 and Sehyun Shin1*
1
Korea University, Korea
ABSTRACT
Blood coagulation is one of the haemostatic processes, which results in a blood clot. It plays a substantial role in preventing the loss of blood. As a result of complex cascade of blood coagulation, fibrin is formed from fibrinogen, which can
be detected using light transmission method. In this study, we introduced a novel microfluidic method to detect blood
coagulation by measurement of the transmitted light intensity. The results indicated that the blood coagulation time is
strongly related to RBC aggregation which was manipulated by adjusting hematocrit. Also, the results were compared
with those measured by INRatio.
KEYWORDS: Blood coagulation, Light-transmission, Microfluidic chip
INTRODUCTION
Blood coagulation is one of the haemostatic process of humans, which consists of a complex, physiological cascade.
When the blood vessel is damaged, the substances released from the destroyed endothelium into blood induce formation
of a platelet aggregation at first. [1, 2] After activation, platelets tend to adhere to the damaged vessel wall and finally, an
aggregated platelet plug is formed to prevent the loss of blood. During this process, plasma clotting also happens. When
the cells those are placed in endothelium are exposed to blood, the plasma clotting, known as blood coagulation, is activated. Blood coagulation process has more complex cascade, which consists of enzymatic reactions in blood plasma [3].
As a result of complicated process, polymerized fibrin is formed from fibrinogen to prevent the loss of blood cells.
It is important to monitor blood coagulation process because disorders in coagulation can lead to higher risk of bleeding. Also, blood coagulation disorders have possibilities to bring pathological complications in increasing thrombosis
and embolism in the vascular system [4]. These are life-threatening, which can induce fatal danger in various clinical
circumstances, such as cardiac surgery [5]. Therefore, it is essential to check the blood coagulation process regularly to
allow the detection of clotting problems.
In this manuscript, we present a new blood coagulation monitoring method using transmitted light intensity, which
can detect the fibrin formation from fibrinogen. The advantages of this method over other methods are simplicity and the
ability to measure blood coagulation with the effect of RBC aggregation. We also report the role of hematocrit on blood
coagulation, followed by deep investigation of RBC aggregation and blood coagulation.
EXPERIMENTAL
The schematic of microchip-based RBC aggregometer is supplied in Figure 1. This system consisted of a microfluidic chip, a light-transmission system, a magnetic-rotation system, and a data acquisition and analysis system.
The microfluidic chip consists of a sample inlet, an air outlet, a test chamber, and a micro-stirrer. The dimensions of a
test chamber, which was validated in a previous study [6] are 4 mm diameter and 0.3 mm height. As the micro-stirrer in
the microchip, made of magnetizable stainless steel (SUS 421), are used after cutting and sterilization. The dimensions
of stirrer were carefully chosen, since a previous study reported that there existed optimal ratios of stirrer to chamber dimensions.
Figure 1: Schematic diagram of experimental apparatus
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14th International Conference on
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The micro-stirrer rotates through the magnetic rotation mechanism. In the magnetic rotation system, a magnet places
on a rotor. As the magnet rotates with the rotor, the stirrer in the microchip rotates at the same speed as the rotor. The rotating speed, which can be controlled by the input voltage of a motor, was found to be optimal at 900 rpm to disperse
RBC aggregates. A mechanical brake is applied to the rotor to abruptly stop the rotating magnet placed on the rotor.
The light-transmission system consists of a laser diode (LM-6305MR, Lanics Co., Ltd., Seoul, Korea) and a photodiode (FDS1010, ThorLab, Boston, USA). Transmitted light signals detected by the photodiode are converted to digital
signals by a DAQ card (NI, USA) and transferred to a computer. The signals are analyzed by the Lab View program to
obtain the indices of RBC aggregation and blood coagulation from the transmitted light intensity over time.
Blood was obtained from a healthy volunteer who was not on any medications and who provided informed consent.
A venous blood sample was drawn from the antecubital vein and collected in Vacutainers(2.7 ml, BD, Franklin Lakes,
NJ, USA) which contained sodium citrate (0.109 M) as the anticoagulant. To initiate blood coagulation cascade, an
aqueous solution of 0.025 M calcium chloride is added to citrated blood samples, which have the final concentration of 4
mmol/L. The measurement starts immediately after recalcifying the blood sample and rapid mixing.
In this study, a series of processes were conducted additionally to adjust hematocrit of blood sample. Whole blood
was centrifuged at 800×g for 12 min. Plasma and buffy coat were then removed. To adjust hematocrit at 20, 40, 60 %,
the RBCs were resuspended in an autologous plasma.
The coagulation time is measured in microchip-based RBC aggregometer, which monitors the transmitted light intensity through the blood sample in the microchip. The procedures of the typical tests we conducted were as follows: first,
the blood sample was placed in the chamber of a microchip after the addition of the calcium solution to blood. Then, the
microchip was mechanically mounted on the jig slightly apart from the magnetic rotating mechanism. In order to mix the
blood sample and disperse the RBC aggregates, a micro-stirrer in the chamber rotated for 10 s and then stopped abruptly.
Despite of the sudden stop of the rotational mechanism, there was a transitional phase due to inertia momentum.
The laser light emitted from the laser diode irradiates the blood sample and part of light is transmitted through the
blood sample. The transmitted light is detected by the photodiode, which is linked to the data acquisition system by a
computer. When the stirring is stopped suddenly, transmitted light intensity increases, because the dispersed RBCs start
to re-aggregate. As shown in Figure 2, the transmitted light intensity keeps increasing asymptotically in case of citrated
blood, while in case of recalcified blood, the intensity of transmitted light starts to decrease at around 250 s. It is affected
by fibrin polymerization, which means that the clot formation with dense polymer network blocks out the light transmission through the blood. The coagulation time is determined as the time when the intensity of transmitted light starts to
decrease.
Figure 2: Syllectogram and definition of clot formation time
RESULTS AND DISCUSSION
The coagulation time was examined by using various blood samples with different hematocrit. Most of aggregation parameters are significantly affected by the hematocrit of the blood sample [7]. Aggregation index showed a strong dependence upon hematocrit, which means that RBCs aggregate more in blood sample with higher hematocrit. On the other hand,
as supported in Figure 3, the coagulation time decreased with the hematocrit. It means that the time needed for fibrin polymerization is decreased when the blood sample has high hematocrit. The results imply that RBC aggregation has an effect on
blood coagulation.
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Figure 3: Effect of hematocrit on blood coagulation time
The results were compared with those done by commercial device (INRatio: HemoSense Inc., San Jose, CA). As supported in Figure 3, the results showed the same tendency with those measured by INRatio and also showed the strong correlation between two instruments.
CONCLUSION
This study introduced a novel microfluidic method to detect the coagulation time by the measurement of the transmitted light intensity, potentially allowing blood coagulation measurements easily in a clinical setting. The feasibility and
accuracy of the new blood coagulation measurement technique have been demonstrated for recalcified blood with various hematocrit. Among the advantages of this method are simplicity, ease of sample control, quick measurement, and the
ability to measure blood coagulation with the effect of RBC aggregation.
ACKNOWLEDGEMENTS
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea
government (MEST) (No. 2009-0086229).
REFERENCES
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U.S.A., pp. 1687, (2007).
[3] James P. Riddel Jr, Bradley E. Aouizerat, Christine Miaskowski, and David P. Lillicrap, Theories of Blood Coagulation, Journal of Pediatric Oncology Nursing, SAGE Publications, U.S.A., pp. 123-131, (2007).
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[6] Sehyun Shin, Yijie Yang and Jang-Soo Suh, Microchip-based cell aggregometer using stirring-disaggregation mechanism, Korea-Australia Rheology Journal, The Korean Society of Rheology, Korea, pp. 109-115, (2007).
[7] Sehyun Shin, Yijie Yang and Jang-Soo Suh, Measurement of erythrocyte aggregation in a microchip stirring system by light transmission, Clinical Hemorheology and Microcirculation, IOS Press, The Netherlands, pp. 197-207,
(2009).
CONTACT
Sehyun Shin, tel: +82-2-3290-3377; [email protected]
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