DEVELOPMENT OF A BIODIAGNOSTIC DEVICE ASSAY FOR

COAGULATION MONITORING

Magdalena M. Dudek and Anthony J. Killard

Biomedical Diagnostics Institute, National Centre for Sensor Research, Dublin City University, Dublin, Ireland

Keywords: Bioassay, Polymer, Warfarin.

Abstract: There is an urgent need for the development of reliable point-of-care devices capable of anticoagulant dose

monitoring due to the increasing number of patients being treated with clotting control therapy. Millions of

patients suffering from cardiovascular-related disorders rely on the anticoagulant therapy. One of the

commonly administered drugs is warfarin. It is effective for primary and secondary prevention of

venous

thromboembolism, for prevention of cardioembolic events

in patients with atrial fibrillation or prosthetic

heart valves,

for prevention of stroke, recurrent infarction or mortality in patients with acute myocardial

infarction and for

the primary prevention of acute myocardial infarction in high-risk

men. Regular

monitoring of warfarin effect is of paramount importance and therefore affords are made to develop novel,

reliable point-of-care devices for drug level determination. Miniaturized microfluidic systems made of

polymers have gained great interest of the diagnostics industry in recent years. Due to the low cost of

manufacturing and processing, they have been employed in the development of several disposable

diagnostic systems. Among the wide selection of different synthetic polymers, thermoplastics have gained

significant popularity. Cyclic polyolefins (COPs) are a relatively new class of thermoplastics with an

excellent combination of optical and electronic properties and are dimensionally stable while being subject

to a range of operational temperatures and pressures. One such COP is marketed by Zeon Corp. under the

brand name Zeonor®. This material has been used as the base for the developed assay. The technology

developed by Åmic B.V. (Sweden) allowed the formation of an ordered array of micropillars which

introduce controlled and highly reproducible capillary filling forces when liquid samples are introduced to

the substrate. Capillary forces play an important role in most of these systems. Assays based on the flow of

a fluid in a device with some form of immobilized reagents are considered as the most commonly used tool

in many detection systems, including diagnostics. Herein, the concept of monitoring blood clotting

properties by measuring a sample distance traveled in a lateral flow system was shown. Substances known

to be strong coagulation activators were employed in the monitoring system. All necessary components

were incorporated into a test strip, so that no pre-treatment steps were required. These were capable of

inducing rapid clot formation and thus arrest of sample flow. The device was shown to be a viable tool for

measuring the clotting status of samples containing different quantities of an anticoagulant. This idea of a

simple assay device could be employed in a point-of-care determination of a drug level.

1 INTRODUCTION

Close to six million people in the world take oral

anticoagulants on a permanent basis, including

patients with artificial heart valves or those affected

by atrial fibrillation or thrombotic disease. There are

also patients who have to rely on them for periods of

several weeks or several months (www.roche.com,

www.argatroban.com). The number of patients

under vitamin K antagonist anticoagulant therapy is

expected to reach 10 million globally by 2010

(www.roche.com). Coagulation monitoring and drug

dosage adjustment are required to maintain the INR

(International Normalized Ratio) within the

therapeutic range (Ansell et al., 2004). INR is a

system that was established by the World Health

Organization (WHO) and the International

Committee on Thrombosis and Hemostasis for

reporting the results of blood coagulation tests. The

WHO procedure is standardized – thromboplastin

reagents are calibrated against a standard by means

of the International Sensitivity Index (ISI). The INR

can be calculated as follows (www.who.int/en):

183

Dudek M. and J. Killard A. (2010).

DEVELOPMENT OF A BIODIAGNOSTIC DEVICE ASSAY FOR COAGULATION MONITORING.

In Proceedings of the Third International Conference on Biomedical Electronics and Devices, pages 183-186

DOI: 10.5220/0002743001830186

 SciTePress

INR = (observed ratio)

ISI

of thromboplastin

Warfarin (Fig. 1) is one of the most commonly

prescribed anticoagulants. It belongs to a group of

coumarins that exert their anticoagulant effect by

interfering with the cyclic interconversion of vitamin

K and its 2,3-epoxide (vitamin K epoxide) (Hart et

al., 2007, Baigent et al., 1998). Warfarin therapeutic

dosage can be affected by several factors and

therefore, it can be difficult to manage (Sick et al.,

2007). The most common complication of warfarin

therapy is bleeding, which occurs in 6 to 39 % of

recipients (Levine et al., 1995). The incidence of

complications varies within this range, depending

upon the clinical indication and the intensity of

anticoagulation. Due to the variability in the

anticoagulant

response to warfarin, regular

monitoring and

dosage adjustment are required to

maintain the INR within the

therapeutic range (Hirsh

et al., 2003).

Figure 1: Chemical structure of warfarin.

The development of microfluidic device platforms is

already an important area for biomedical device

design. Polymer-based microfluidic devices and

their associated materials have gained particular

interest in recent years. Thermoplastics have gained

significant popularity as substrates for the

production of disposable devices for biomedical

applications having low raw material and

manufacturing costs. Their properties which include

thermal stability, ‘mouldability’, precise structural

and morphological control over surface properties,

chemical and biological inertness, good optical and

electrical characteristics and many more are

resulting in the replacement of traditional materials

such as glass, silicon and nitrocellulose as the

foundation of device fabrication.

2 MATERIALS AND METHODS

4Castchips

B2.2 (Fig. 2) were injection molded by

Åmic AB (Uppsala, Sweden) in cyclic olefin

polymer (COP) (Zeonor 1020R

) to form

micropillars (height 65-70 µm, top diameter ca 50

µm, bottom diameter ca 70 µm, the distance between

the centres of the pillars in a row 85 µm, the distance

between the centres of the pillars in a column 185

µm). These facilitated controlled and highly

reproducible capillary filling of liquid samples.

Figure 2: Graphical representation of the B 2.2 micropillar

lateral flow device employed for the detection of

fibrinogen level. The test channel possessed hot-embossed

micropillar structure, as shown in the magnified inset.

The assay platforms were coated with a mixture of

activating reagents by a drop-deposition. 10 μL was

applied and left to dry in under ambient conditions.

Among the active components immobilized on the

assay platform were: activated partial

thromboplastin time (aPTT) reagent, aPTT-SP

(Hemosil), prothrombin time (PT) reagent,

Simplastin HTF (BioMerieux) and Russell’s Viper

Venom (RVV) (Pethapharm). aPTT activator was

ready-to-used solution. PT reagent was reconstituted

in 2- or 4-fold less volume of diluent than suggested

by manufacturer. The activity of RVV solution was

50 U/mL. Positive control was normal clotting,

control plasma (Hemosil), while negative control

consisted of control plasma supplemented with

heparin at a final concentration of 50 U/mL. Such a

high concentration of an anticoagulant was used to

ensure no clotting occurred. The time required for a

test solution to reach each step of a test channel (Fig.

2) was measured. The filling characteristics were

assessed on a basis of obtained filling profiles. In

addition, the device was validated using anonymous

warfarin-treated patient plasma samples with INR

values of 1.1, 2.1 and 9.0.

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3 RESULTS AND DISCUSSION

3.1 Assay Chemistry Formulation

The development of the assay was based on the

monitoring of distance traveled by normal clotting

and non-clotting (heparinized) samples. The aim was

to achieve a significant difference in a distance

traveled between these two variants that would allow

identification of minor clotting disorders (slightly

prolonged CT). Substances known to facilitate rapid

clot formation (aPTT, PT, RVV) were employed in

platform development in order to achieve a flow

cessation. The distances traveled by normal and

heparinized samples on chips coated with a variety

of activator combinations are illustrated in Fig. 3.

0 5 10 15 20 25 30

4xPT:RVV:water (1:1:2)

4xPT:RVV (1:1)

4xPT:RVV (2:1)

4xPT:RVV (3:1)

4xPT:water (1:1)

4xPT:RVV:water (1:1:1)

aP TT:RVV (1:1)

Ratio of immobilized reagents

Distance travelled [ mm]

Figure 3: Distance traveled by normal clotting (plain) and

non-clotting, strongly heparinized (striped) plasma

samples. Test channels were coated with clotting

activators at different ratios.

The difference in the distances traveled by clotting

and non-clotting samples was between 3 and 8.5 mm

depending on a formulation used. The use of the

aPTT reagent did not allow rapid clot formation. The

difference of 3 mm would not allow a precise

differentiation between samples of varying

anticoagulant activity. Similarly, 1:1 mixtures of 4-

fold concentrated PT with water with or without

RVV did not result in good discrimination between

samples of different clotting abilities. Dried mixture

of 4-fold concentrated PT and RVV at ratios of 3:1

and 2:1 yielded a short distances traveled of 12 and

11.5 mm for clotting and 19 and 20 mm for non-

clotting samples. The significant decrease in a

distance traveled was probably not an effect of an

enhancement in clotting, but was more likely due to

high concentration of immobilized PT reagent. The

deposition of high protein concentration (tissue

thromboplastin) and phospholipids could result in a

change of surface properties such as roughness and

wettability. It has been noticed that the distance was

short not only for clotting sample but also for the

negative control sample, for which no clotting

occurred. Therefore, the reduction in the distance

traveled was of no benefit because of the changes

introduced to the surface properties. Formulations

composed of 4-fold concentrated PT and RVV with

or without water dilution at a ratio of 1:1 or 1:1:2

proved to be best at yielding a significant difference

in distance. Fill times were measured at each stage

of the channel coated with these formulations. In

both situations they looked similar as illustrated in

an example in Fig. 4. It has been shown that the

filling profile was very similar for clotting and non-

clotting samples. The flow of a clotting sample was

rapidly arrested at between 15 – 20 mm.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Distance travelled [mm ]

Time [s]

Figure 4: Fill time profiles obtained for normal clotting

(filled symbols) and non-clotting (empty symbols) plasma

samples tested in a channel coated with 4-fold

concentrated PT reagent, RVV and water mixed at a ratio

of 1:1:2 (n=3).

3.2 Validation with Patient Samples

The platforms coated with 4-fold concentrated PT +

RVV (1:1) and 4-fold concentrated PT + RVV +

water (1:1:2) were selected for further validation

using patient samples. Three patient samples with

different INR values were tested: 1.1, 2.1 and 9.0.

Results obtained for normal clotting and heparinized

(50 U/mL) plasma samples and for patient plasma

samples are shown in Fig. 5. The 1:1 PT:RVV

showed good discrimination of INR at the lower

range (1.0 to 2.1) but due to assay variability it was

unable to discriminate higher values (INR 9.0 and

non-clotting controls). However, PT:RVV at 1:1.2

showed poor differentiation between INR 1.0 and

1.1, but was better at distinguishing INR 1.1, 2.1, 9.0

and non-clotting controls. However, variability again

made it difficult to distinguish INR 9.0 from other

values due to the long and variable times which

result.

DEVELOPMENT OF A BIODIAGNOSTIC DEVICE ASSAY FOR COAGULATION MONITORING

185

Non-clotting

sample

INR 9.0

INR 2.1

INR 1.1

INR 1.0

INR value

Distance travelled [mm]

Figure 5: Distances travelled by samples with INR of 1.0

(normal clotting control), 1.1, 2.1, 9.0 and non-clotting.

Chips containing 4-fold concentrated PT:RVV at a 1:1

ratio (empty symbols) and 4-fold concentrated PT : RVV :

water at 1:1:2 ratio (filled symbols) were used for testing

(n=3).

4 CONCLUSIONS

The principle of a point-of-care lateral flow device

for the anticoagulant therapy monitoring has been

shown. The device platform made of cyclic poly

olefin polymer and coated with an optimized

mixture of activating agents has been shown a

reliable tool for an assessment of blood clotting

properties. A mixture of Russell’s Viper Venom and

Prothrombin Time reagents allowed rapid clot

formation which resulted in the cessation of sample

flow. Significant differences in the distance traveled

between a normal clotting and a strongly heparinized

plasma sample were shown. The presence of an

anticoagulant (warfarin) in a patient plasma sample

delayed clot formation and therefore resulted in a

prolonged distance traveled in comparison to a

normal clotting control. The device requires further

optimization in order to obtained better recognition

between samples of different clotting statuses.

However, the idea of an anticoagulant dose

monitoring using the lateral flow device for the

distance traveled measurement was shown to be

viable.

ACKNOWLEDGEMENTS

This material is based upon works supported by the

Science Foundation Ireland under Grant No.

05/CE3/B754.

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www.argatroban.com

www.roche.com

www.who.int/en

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