WO2012025637A1

WO2012025637A1 - An agglutination assay method and device

Info

Publication number: WO2012025637A1
Application number: PCT/EP2011/064841
Authority: WO
Inventors: Anthony Joseph Killard; Nigel Kent; Magdalena Maria Dudek
Original assignee: Dublin City University
Priority date: 2010-08-27
Filing date: 2011-08-29
Publication date: 2012-03-01
Also published as: GB201014316D0; WO2012025637A9

Abstract

The present invention is directed to a method and device for monitoring and measuring agglutination within a sample using a lateral flow assay device. In particular, the present invention is directed to a method for monitoring and measuring the onset of agglutination within a sample on a lateral flow assay device comprising micro-projections to define a lateral flow assay path wherein the method comprises combining the sample with at least one detectable marker and at least one agglutination marker or a combination thereof to form a labelled sample; subjecting the labelled sample to lateral flow; and monitoring the redistribution of the agglutination marker through the lateral flow assay path as agglutination occurs. The invention is also directed to a lateral flow assay device for carrying out the method and a kit comprising the lateral flow assay device and detection means.

Description

AN AGGLUTINATION ASSAY METHOD AND DEVICE FIELD OF THE INVENTION

The present invention is directed to a method and device for monitoring and measuring agglutination within a sample using a lateral flow assay device.

BACKGROUND TO THE INVENTION

There are many different types of agglutination assays commercially available for many different purposes. A significant number of these are immunoassays, utilising antibody/antigen reactions to determine whether a particular target analyte is present in a sample. There are many well know examples of such assays, such as home pregnancy tests, HIV tests, fertility tests etc. These assays utilize many different biological fluids including urine, saliva, blood, or stool samples.

The majority of these assays measure the progress of the reaction in terms of target analyte concentration only. For example, latex agglutination assays detect small qualities of antigen molecules and involve the aggregation of latex particles with surface bound antigenic molecules. Aggregation (or agglutination) occurs when antibody molecules specifically corresponding to the antigen are introduced into the solution of the carrier particles. Mixing antigen-coated latex particles and antibody causes these components to interact and combine. As more antibodies cross-link the antigen coated latex particles and particles, visible clusters are formed.

Other tests involving agglutination include the measurement of blood clotting time. Clotting time tests essentially measure the onset of clot formation, which results from the formation of fibrin fibres in the blood sample. This is important in a range of clinical applications such as assessment of coagulation disorders and controlling the effect of various anticoagulant drug therapies.

Routine tests of blood coagulation , such as the prothrombin time (PT) , activated partial thromboplastin time (aPTT), and thrombin time (TT) are frequently used clinically to assess clotting function in patients. For example, ACT assays are typically performed with addition of contact activator alone (micronized silica, glass, kaolin, celite, ellagic acid, etc). Additionally, aPTT contains such contact activators with add itional phospholi pid mixtu res, referred to as a partial thromboplastin. PT assays possess as their main active ingredient a tissue factor, also known as Factor III. Thrombin Time (TT) also called Thrombin Clotting Time (TCT) is activated by the addition of thrombin reagent. Calcium chloride may also be added in instances where blood samples have been taken into citrated test tubes to prevent coagulation prior to analysis. There are many devices available with which to perform assays relating to the coagulation status of blood. These devices vary as to the types of assay they perform, i.e., whether they use prothrombin time, activated partial thromboplastin time, activated clotting time, thrombin clotting time or a host of other assay techniques. Furthermore, these devices can also employ a range of different transduction methodologies to convert the coagulation status into a quantifiable value. Traditionally, this has been achieved through the formation of a physical blood clot. Alternatively, assays that measure activities of, for example, coagulation factor(s) can also be used, such as the thrombin generation assay or the anti-Factor Xa assay for examples. If the assay results in the formation of a clot, this formation must be sensed in some way. If the assay results in a change in an activity, this can be measured using optical, electrochemical or some other transduction methodology.

However, the clotting time tests presently used in a clinical setting are relatively crude tests. For example, some tests still rely on the observation of fibrin fibre formation in a blood sample tube in a tilting, heated water bath, derived from the classical Lee and White method. Others methods use the principle of aggregometry in which a clotting blood sample occludes and optical or electrochemical pathway. Some others methods use complex arrangements of fluidic channels with pumping processes to effect coagulation. Some assays require multiple assay steps such as pre- mixing with reagents prior to activation, while some others also require sample-processing steps, e.g., plasma formation. Most assays also still require antecubital venipunture for the sample, which adds to patients discomfort, requires trained phlebotomists and requires anti-coagulation prior to testing. Some examples of these are given in US Patent nos. 5908786, 4849340, 4252536, 4963498, 5039617 and 5372946.

These conventional assays for calculating clotting time are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. The principle reason for this is that while most coagulation tests measure a change in the bulk property of blood upon clotting, such bulk measurements do not adequately represent the underlying stochastic nature of the process. That is, in reality, the triggering of coagulation at any point is brought about when there is interplay of appropriate components from the clotting cascade in a complex series of steps. This can occur at multiple locations at slightly different times and is not possible to predict. This is exacerbated by the high levels of variability in the composition of individual donor blood samples. Available clotting tests attempt to reduce such variation through homogenisation and the addition of certain reagents. However, the results of this are still far from satisfactory and clotting time tests continue to suffer from significant analytical errors, more specifically, the precision of the clotting time measurements produced. Commercial devices can still have relative standard deviations of 10% or greater. This significantly reduces their clinical utility. This is a highly competitive field and any improvements in the development of new and more refined assay methods could provide a competitive advantage. Thus, the present invention is directed to an improved method and device for monitoring and measuring agglutination within a sample using a lateral flow assay device. Advantageously, the present invention also provides an improved coagulation method and device.

STATEMENT OF THE INVENTION

According to a general aspect of the invention, there is provided a method for monitoring and measuring agglutination within a sample on a lateral flow assay device comprising micro- projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path wherein the method comprises

combining the sample with at least one detectable marker and at least one agglutination marker or combination thereof to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path; and monitoring the redistribution of the agglutination marker through the lateral flow assay path. According to a first aspect of the invention, there is provided a method for monitoring and measuring agglutination, ideally the onset of agglutination, within a sample on a lateral flow assay device comprising micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path wherein the method comprises

combining the sample with at least one detectable marker and at least one agglutination marker or a combination thereof to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path; and monitoring the redistribution of the at least one or more agglutination markers through the lateral flow assay path as agglutination occurs by observing the enhancement of the detectable marker signal resulting from agglutination in areas around and/or between the micro-projections in contrast to the remaining areas and measuring the change in the standard deviation of the marker signal over time.

According to a second aspect of the invention, there is provided a method for measuring the onset of blood clot formation and determining the associated blood clotting times in a blood or plasma sample wherein the method comprises the following steps combining a blood or plasma sample with at least one detectable marker and at least one agglutination marker or a combination thereof of blood clotting to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path zone; monitoring the formation of a blood clot in the sample and measuring the change in detectable marker signal standard deviation over time resulting from the localised build up of the detectable marker around and/or between the micro-projections, in contrast to the remaining areas without or with reduced observable detectable marker signal. According to one embodiment of this second aspect of the invention, there is provided a method for measuring the onset of blood clot formation and determining the associated blood clotting times in a blood or plasma sample wherein the method comprises the following steps

combining a blood or plasma sample with a fluorescently labelled marker of blood clotting to form a fluorescently labelled sample;

subjecting the fluorescently labelled sample to lateral flow through the lateral flow assay path zone;

monitoring the formation of a blood clot in the sample and measuring the change in fluorescence signal standard deviation over time resulting from the localised build up of the fluorescent label around and/or between the micro-projections in contrast to the remaining areas without or with reduced observable fluorescence.

According to a third aspect of the invention, there is provided a lateral flow assay device for measuring the agglutination, preferably coagulation, within a sample wherein the lateral flow assay device comprises micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path characterised in that the lateral flow assay device comprises a sample receiving zone;

a mixing zone with an agglutination activation zone, wherein the mixing zone comprises at least one deposited detectable marker, agglutination marker and/or agglutination assay reagent;

a lateral flow assay path zone.

According to a fourth aspect of the invention there is provided the use of the lateral flow assay device of the invention in a method for monitoring and measuring the agglutination, preferably coagulation, of a sample. According to a fifth aspect of the invention, there is provide a kit for monitoring and measuring the agglutination, preferably coagulation, of a sample comprising the lateral flow assay devbe of the invention and a detection means for measuring the increase in marker signal standard deviation in the assay zone over time.

DETAILED DESCRIPTION OF THE INVENTION

In the following description the term "lateral flow assay device" and "capillary flow assay device" are understood to be interchangeable.

The term "sample" used herein means a volume of a liquid, solution or suspension of the sample, intended to be subjected to qualitative or quantitative determination of any of its properties. The sample is intended to be any suitable biological liquid which is the subject of the assay, including whole blood, plasma, urine, cerebrospinal fluid, interstitial fluid, lymph fluid, saliva, sputum, sweat, semen, tears and/or faecal matter.

In the following description the term "agglutination" is used in a general context and intended to cover agglutination, aggregation, coagulation and more specifically blood clotting, and the respective agglutination, aggregation, coagulation or clotting of particles of/within a sample. Accordingly, the invention is directed to agglutination assays in general covering but not limited to immunoassays such as latex agglutination immunoassays and more specific coagulation assays and blood clotting assays. Thus, in the following description these terms are interchangeable. Specifically, "clotting" and "coagulation" are understood to be interchangeable. It will be understood that coagulation of any liquid sample effected via the coagulation cascade using fibrinogen or thrombin may be contemplated.

It will be understood that the terms "coagulation activation zone" and "agglutination activation zone" are interchangeable. We have used the term "agglutination activation zone" as the general term encompassing "coagulation".

The terms "zone", "area" and "site" are used in the context of this description, examples and claims to define parts of the fluid passage on a substrate, either in prior art devices or in a device according to an embodiment of the invention. The term "substrate" here means the carrier or matrix to which a sample is added, in this case being the lateral flow assay device. In the following description the term "micropillar" or "micro-projections" or "protruding microstructures" cover a plurality of vertical projections or pillars protruding from the surface of the substrate which define the flow through the substrate. Ideally, such micropillars consist of areas of projections substantially vertical to said surface, and having a height (H), diameter (D) and reciprocal spacing (t1 , t2) such, that lateral capillary flow of the liquid sample in said flow zone is achieved.

In the following description, it will be understood that the term "coating" is interchangeable with the term "layer" or "film". The deposited marker or reagent may be deposited as a thin layer or film on the surface of the lateral flow assay device using available methods.

subjecting the labelled sample to lateral flow through the lateral flow assay path; and monitoring the redistribution of the at least one or more agglutination marker through the lateral flow assay path.

In this method, the monitoring step comprises monitoring the agglutination of the sample by observing the localised build up or signal enhancement of the agglutination marker signal in areas around and/or between the micro-projections in contrast to the remaining areas and measuring the change in the standard deviation of the marker signal over time. In this manner, the change in detectable marker signal standard deviation over time resulting from the localised build up of tie detectable marker around and/or between the micro-projections in contrast to the remaining areas without or with reduced observable detectable marker signal is measured.

Ideally, the agglutination marker is fibrinogen, thrombin, an antibody, a protein, an enzyme, a carbohydrate, a lipid or any other suitable marker of agglutination in a biological fluid and/or a combination thereof. For example, an enzyme may include glucose oxidase, cholesterol oxidase, lactate oxidase, peroxidases (e.g., horseradish peroxidase, catalase) and accompanying substrates including soluble and insoluble substrates. Ideally, the detectable marker is in the form of a fluorescent marker, chromophore, electrochemical marker, enzyme and/or radionuclide and/or a combination thereof.

It will be understood that the detectable marker and agglutination marker may combned with the sample separately. Alternatively, the detectable marker and agglutination marker may be combined together to form a detectable agglutination marker which is subsequently combined with the sample. These steps may take place in situ on the lateral flow assay device itself or prior to application to the lateral flow assay device. Thus, according to one embodiment of the invention, the detectable marker and agglutination marker or combination thereof are combined with the sample prior to use, that is prior to application of the sample to the lateral flow assay device. According to an alternative embodiment, the detectable marker and agglutination marker or combination thereof may be combined with the sample in-situ on the lateral flow assay device. In this embodiment, the lateral flow assay device ideally comprises a sample receiving zone, a mixing zone with a coagulation activation zone and an assay zone. In this manner, the detectable marker and agglutination marker or combination thereof may be pre-deposited in the mixing zone of the lateral flow assay device ready to combine with the sample in the mixing zone. Depending on the specific assay being carried out (including but not limited to ACT, aPPT and PT assays, etc), an agglutination assay reagent may be used and may be selected from one or more of the following: contact or surface activators, such as kaolin, celite, ellagic acid; tissue factors such as tissue thromboplastin; phospholipids; snake venoms; fibrinogen and thrombin; and/or a combinations thereof. Accordingly, thrombin and fibrinogen may be both agglutination markers and agglutination assay reagents.

Optionally, the method may also comprise the step of coating the sample receiving zone and/or mixing zone with a reagent to prevent the irreversible adherence of the agglutination marker to the sample receiving zone and mixing zone surface prior to addition of the sample.

The assay of the invention takes place on a lateral flow assay device, which is advantageously provided with micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path. This specific structure of the lateral flow assay device enables the increase in agglutination marker signal standard deviation resulting from the localised build up of the marker around the micro-projections in contrast to the remaining areas without or with reduced observable detectable marker signal to be measured. With this method agglutination or coagulation takes place throughout the device, and not in a specific location, i.e. to an immobilised binding reagent, as with known assays. We have found that the micro-projections present on the surface of the lateral flow assay device bring about a localised redistribution of the agglutination marker resulting from agglutination occurring within the sample, such that the agglutination marker accumulates and concentrates around the micro-projections during clot formation. Advantageously, these micro-projections form an increased surface area in comparison to a conventional flat substrate surface and enhance agglutination. In this manner, the concentration of the agglutination assay reagent on the pillars contributes to the build up of the agglutination marker on/around the pillars. Accordingly, by measuring the localised redistribution of the marker signal, rather than the increasing magnitude of the marker signal, it is possible to determine the onset of agglutination.

Specifically, the method of the invention requires an area of the sample to be imaged in real time and digitised (pixelated) data collected on the area. Agglutination is measured by measuring the increase in signal standard deviation, which results from this localised build up of the agglutination marker around and between the pillars leaving other areas without any observable marker. This is measured in units of standard deviation where one measure of the standard deviation (s) for a sample is given by:

For use and ease of interpretation in a clinical setting, the test results from the assay of the invention may be correlated to standard methods such as PT-INI (International Normalised Ratio) etc. This is within the scope of the skilled man.

According to one preferred embodiment of the invention, the agglutination marker is labelled with a fluorescent marker and the redistribution of the marker in the lateral flow assay path is measured optically.

It will be understood that the lateral flow assay device may be an open or closed lateral flow assay device. The sample may be any biological fluid as defined above. If for example, whole blood is used, the sample is ideally citrated prior to use and optionally recalcified prior to or upon contact with the lateral flow assay device. In this situation, the device may comprise a recalcification zone. Addition of a buffering agent may also be required and accordingly, the device may comprise a buffering zone. Additionally, a red blood cell removal zone may be required in some circumstances.

According to another embodiment of the invention, the agglutination assay reagent may be deposited on the lateral flow assay path. The reagent may be deposited as a thin layer or film on the lateral flow assay path surface using available methods.

Other agglutination assay reagents include agglutination activating reagents for biomolecular interactions other than blood clotting. For example, the method of the invention may be used in any assay involving the agglutination within a sample. Specifically, the method may be used in any aggregation assay involving a biomolecular interaction process. Such assays involve the use of antibodies and antigens, cell receptor/ligand interactions, nucleic acid interactions, protein/substrate or protein/protein interactions, and/or any combinations thereof.

A particularly preferred embodiment of the invention is the use of the method in a blood clotting time assay and this will be described in further detail later. Accordingly, coagulation assay reagents may also be used and these including contact activators (such as kaolin, celite, ellagic acid), tissue thromboplastin and phospholipid in ACT, aPPT and PT assays, snake venoms, fibrinogen and thrombin. Other coagulation assay reagents including surfactants (such as Triton X- 100) may also be used. Corn trypsin inhibitor may also be added to eliminate spontaneous (non- activated) clotting processes.

According to another embodiment of the invention, the method may be used in an immunoassay, such as a standard latex or particle agglutination assay. According to this alternative embodiment, the method may be used in a particle agglutination assay such as a latex agglutination immunoassay wherein the agglutination marker is an antibody or antigen coated latex bead preparation. We postulate that the present method improves conventional particle agglutination assays by allowing them to be more precisely quantified with regards level and stage of progression of agglutination. Such assays may also contain as yet, unidentified SD response signatures which relate to the kinetics of the process and contains important information relating to concentrations, binding rate constants, and other important measures of the assay. For example, either the antibody antigen, or other ligand may be locally patterned onto the microstructured substrate. The labeled agglutination marker may then interact with the deposited complementary binding partner causing its redistribution from its initial bulk concentration and so result in a visible change in its redistribution which may be associated with some other parameter such as concentration. In this manner, this type of immunoassay may work by the physical capture of the reagents in and around the micropillars.

In this embodiment, latex beads coated with antibodies to a target antigen are mixed with the sample at the mixing zone and travel together along the lateral flow assay path zone. As binding and aggregation occurs, we postulate that the aggregates get physically trapped between the pillars. The higher the concentration of antigen, results in faster and more frequent trapping of aggregates. If the antibody (the agglutination marker) is fluorescently labeled (the detectable marker), the distribution of signal will vary at a time and/ or location and agglutination may be measured.

In this embodiment, the addition of an agglutination assay reagent is not an essential requirement.

It will be understood that the method of the invention may be used in a high throughput screening device.

It will also be understood that the method of the invention may be applied to single analyte/single sample format (as in the case of a Rapid / Point of care (POC) platform). Alternatively, it may be used as an array for multiple measurements and/or multiplexed analytes. In this manner, the invention may relate to the use of an array of multiple lateral flow assay surfaces capable of detecting one or more agglutination marker for use in a high throughput device. Lateral Flow Assay Device

The lateral flow assay device used in the invention ideally comprises a non-porous substrate having a substrate surface and at least one fluid passage or defined fbw path with projections substantially perpendicular to the substrate surface. Ideally, these projections having a height, diameter and a distance or distances between the projections, capable of generating capillary flow, lateral to said substrate surface, of a sample fluid through the fluid passage. It will be understood that the fluid passages define the flow path and support capillary flow.

Thus, the assay device may be any microstructured device comprising an array of micro-pillars or microprojections extending vertically from the substrate surface. The device substrate may be manufactured from a range of materials including glass and thermoplastics, such as polyolefins. The surface of substrate is further structured through, for example, hot embossing, to create uniform and reproducible micro-projections or micro-pillar structures extending vertically from the polymer surface. The regions with the micro-pillars or micro-projections result in the formation of micro-flu id ic channels to enable lateral flow of the sample through the device.

The lateral flow assay device may comprise one or more of the following zones - a sample receiving zone, a cell separation zone, a buffering zone, a recalcification zone, mixing zone(s) with agglutination activation zone, preferably coagulation activation zone and a lateral flow assay path zone. A detection zone may also be present. Such a detection zone is determined by the location of the imaging device, e.g. camera/optical apparatus, needed to monitor the progress of the agglutination/coagulation process.

In the recalcification zone the sample is mixed with calcium chloride as needed. In the mixing zone(s), the sample is ideally mixed with the detectable label and agglutination marker or combination thereof and allowed to mix with the agglutination assay reagents as required. In the assay zone the labelled sample reacts with the agglutination/coagulation reagents along the assay path zone. The agglutination assay reagents may be reacted with the labelled sample in one or both of the mixing zones or assay zones.

In one embodiment, the mixing zone(s) may be coated with a reagent to prevent the detectable agglutination marker (e.g. fluorescently labelled fibrinogen) adhering to the mixing zone prior to application of the blood or plasma sample. The coating reagent may be Teflon®, block copolymers, plasma treatments, derivatives of polyethylene glycols and/or a formulation of supporting bulks proteins, carbohydrates, surfactants, salts, or a composition that prevents or reduces the irreversible adherence of the agglutination marker to the surface of the device. According to one embodiment, the substrate may be glass and the microstructures may be fabricated using conventional photolithography and chemical etching.

In another embodiment, the device may comprise 2 mm wide channels and the micro-pillars may be ellipse shaped as shown in Example 4. It will be understood that other dimensions and shapes may be contemplated. According to another embodiment of the invention, the lateral flow assay device is ideally composed of a polymeric material. Many polymers are suitable due their low cost, are optically transparent and/or amenable to processing through many routes such as hot embossing, injection moulding etc. For example the cyclic polyolefin polymer Zenoor ® may be used. This material is chemically inert, has good optical transparency properties and low intrinsic fluorescence which make it suitable for use in an assay using optical detection.

According to a preferred embodiment of the present invention, the flow path zone of the substrate comprises a plurality of vertical projections protruding from the surface of the substrate which define the flow through the substrate. These are known as "micropillars" or microprojections" or "protruding microstructures". The device with such micropillars may also be known as a "micropatterned device". Ideally, the vertical projections consist of areas of projections substantially vertical to said surface, and having a height (H), diameter (D) and reciprocal spacing such, that lateral capillary flow of the liquid sample in said flow zone is achieved. Such a device is disclosed in WO 03/103835, WO 2005/089082, WO 2006/137785 and related patents. These known devices utilise immobilised binding reagents in which target cells are captured and detected. This differs to the present invention in which agglutination takes place throughout the device and not in a specific location. In one embodiment, the micropillars or projections have a height in the interval of about 15 to about 150 μηη, preferably about 30 to about 100 μηη, a diameter of about 10 to about 160 μηη, preferably 20 to about 80 μηη, and a distance or distances between the projections of about 5 to about 200 μηη, preferably 10 to about 100 μηη from each other. The flow channel may have a length of about 5 to about 500 mm, preferably about 10 to about 100 mm, and a width of about 1 to about 30 mm, preferably about 2 to about 10 mm. It should in this context be noted that a device according to an embodiment of the invention does not necessarily have to have a uniform area of micropillars, but that the dimensions, shape and a distance or distances between the projections of the micropillars may vary in the device. Likewise, the shape and dimensions of the fluid passage may vary. The device may be a disposable assay device or part of such device.

The device of the invention may be used as a point of care device where all the required markers and/or reagents are present/pre-deposited on the surface of the device substrate itself. A particularly preferred embodiment of the invention is the use of the assay method in a clotting time (CT) assay. Alternative assays may be carried out including the prothrombin time (PT) assay, activated clotting time (ACT) assay and activated partial thromboplastin time (aPPT) assay. Clotting Time (CT) Assays

According to a preferred embodiment of the invention, there is provided a method for measuring the onset of blood clot formation and determining the associated blood clotting times in a blood or plasma sample wherein the method comprises the following steps

combining a blood or plasma sample with at least one detectable marker and at least one agglutination marker of blood clotting or combination thereof to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path zone; monitoring the formation of a blood clot in the sample and measuring the change in detectable marker signal standard deviation over time resulting from the localised build up of the detectable marker around and/or between the micro-projections in contrast to the remaining areas without or with reduced observable detectable marker signal.

Ideally, the detectable agglutination marker of blood clotting is fluorescently labelled fibrinogen or thrombin.

Fluorescently labelled fibrinogen is commonly used to study for platelet activation and fibrinogen binding, for instance, in flow cytometry and intravital microscopy. However, these methods merely involve the localization of the fluorescent signal in a sample and are not used to determine clotting times. The present invention does not rely on this simple localization and measurement of the intensity of the fluorescent signal. On the contrary, the method of the present invention relies on the measurement of the fluorescent signal redistribution over time.

We have surprisingly found that this fluorescent signal redistribution is only observed when the clotting process occurs, due to the binding of the conjugate to the activated platelets and incorporation into the forming clots. We have also advantageously found that the use of a micro- structured surface with micro-projections or micropillars in the lateral flow assay device enhances the formation of multiple localised clots and results in the abrupt redistribution of the fluorescent label around the micro-projections at the onset of clot formation. Essentially, the micro-projections bring about a localised redistribution of the fluorescently-labelled fibrinogen resulting from thrombus formation, such that the fluorescence accumulates and concentrates around the micro-projections during clot formation. We have used this abrupt redistribution of the fluorescent label to accurately measure the onset of blood clot formation.

Additionally, the micro-projections or micropillars form an increased surface area in comparison to a conventional flat substrate surface. This results in an enhancement of the action of many adhesive protein factors and enzymes (i.e. fibrinogen and fibrin or thrombin). Additionally, the presence of these micro-projections or micro-pillars may advantageously prevent the nascent fibres from migrating along the test channels. Thus, the method of the present invention measures the increase in fluorescence signal standard deviation resulting from the localised build up of the fluorescent label around the micro-projections in contrast to the remaining areas without or with reduced observable fluorescence.

As expanded on above, we have advantageously found that measuring this localised redistribution of the fluorescence signal, rather than the increasing magnitude of the fluorescence signal provides a far superior determination of the onset of clotting. The method of the invention requires an area of the sample to be imaged in real time and digitised (pixelated) data collected on the area. Thrombus formation is measured by measuring the increase in signal standard deviation which results from this localised build up of label around and between the pillars leaving other areas without any observable fluorescence. As outlined above, this is measured in units of standard deviation where one measure of the standard deviation (s) for a sample is given by:

In this way, the method of the present invention allows a direct optical measurement of thrombus formation by precisely detecting the onset of a fibrin clot formation.

Alternative agents to fluorescently labelled fibrinogen which could be used for clot localisation and the timing of the onset of clotting process, including for example;

• radioisotope-labelled fibrinogen;

• labelled Factor XIII (fluorescently or radiolabeled); • platelet labelling - coagulation activation results in a rapid platelet plug formation which includes platelet activation and aggregation stabilised with cross-linked fibrin mesh. Platelet plug formation could be visualised using labelled agents that have an affinity to any accessible platelet membrane receptor. Although all or majority of available platelets would be labelled, the signal would be amplified in the areas where clot/platelet plug is formed, due to concentration of platelets in that region;

• antibodies reacting with one of the clot components - for example anti-fibrin antibody or antibody directed against the D-dimer region of cross-linked fibrin. This method of the invention can be used in many different clotting time tests including activated partial thromboplastin time (aPTT), activated clotting time (ACT) and prothrombin time (PT) assays, thrombin clotting time, snake venom assays, assays of coagulopathy, or in any assay where a fibrin network is formed as a result of coagulation. Furthermore, this method is also useful for determining the effect of anticoagulant drugs such as heparin on the clotting time. We have found that the method of the present invention allows the accurate determination of the impact of anti-coagulant therapy (e.g., heparin) over a very wide therapeutic range (0-2 U/mL) with exceptional linearity and precision which is not achievable with other systems. We postulate that this precision is achieved by detecting microscopic, rather than bulk, coagulation behaviour. It is well established that, because time is the measured parameter, coagulation assays become increasingly imprecise at increasing clotting times and thus, increasing anti-coagulant drug concentrations. Our assay does not suffer from this increasing imprecision and so has a much extended range of determinable clotting times. This is a significant advantage of the present invention.

Advantageously, the assay has been shown to have good precision and extended dynamic range over conventional techniques, including routine hospital aPTT assays.

General method steps using fluorescently labelled agglutination markers, including fibrinogen

In general terms, the method of the invention involves obtaining a sample and subjecting the sample to conventional pre-incubation steps if necessary. The sample is then mixed with at least one detectable marker and at least one agglutination marker or combination thereof, such as fluorescently labelled fibrinogen; and if the sample was citrated it is then optionally recalcified. The fluorescently labelled sample is applied to the lateral flow assay device with the required agglutination or coagulation assay reagents deposited in-situ on the surface of the device.

The sample advances along the device in a highly controlled, uniform and reproducible fashion, where it dissolves and mixes with the agglutination/coagulation assay reagents in a controlled, uniform and reproducible manner. During this time, the agglutination or coagulation reagents bring about contact activation and the formation of thrombin which is capable of transforming soluble fibrinogen to insoluble fibrin which brings about thrombus formation. This reaction takes place while the sample travels through the device and the concentration and distribution of the fibrinogen and fibrin are changing in a time-dependent manner. In addition, the fluorescently labelled fibrinogen is integrated into the forming thrombus/thrombi, and its concentration and distribution along the channel is changing in a spatial and time-dependent manner.

The redistribution of fluorescence is monitored and measured to enable the correlation with sample clotting time. Specifically, the change in standard deviation of the fluorescent signal is calculated over time to enable the measurement of the clotting time.

Optional Pre-incubation Steps

To perform the assay using blood samples, these samples may need to be stabilised, otherwise, they can begin to coagulate immediately. Typically the blood samples are citrated. Thus, prior to use a citrated sample must generally be recalcified.

For example, the aPTT test typically requires the collection of samples in citrate and also requires their recalcification. This is due to the fact that the contact activation pathway is slow and highly variable and if allowed to proceed without citration leads to high assay variability. Thus, to perform a traditional aPTT test, preincubation with contact activator and phospholipid takes place in the presence of citrate before being reversed with calcium chloride. This is an undesirable step when manufacturing a point of care device. In the present device, the sample may be citrated and recalcified prior to use. This may occur before application of the sample onto the device or on the device itself. I n this case the recalcification may occur in the sample receiving zone which may comprise a recalcification zone. In this situation, no separate pre-incubation with surface activator is required. Alternatively, the present device may be used as a point of care device where freshly obtained samples are used and no citration/recalcification steps are required to perform the assay. This is another major advantage over the current state of the art. Agglutination/Coagulation Assay Reagents

In addition to detectable markers and agglutination markers or combinations thereof, such as fluorescently-labelled fibrinogen, other agglutination or coagulation assay reagents may be required. It will be understood that conventional coagulation (blood clotting) assay reagents may be used used. Conventional coagulation assay reagents used to trigger the intrinsic or extrinsic pathways including contact activators, tissue thromboplastin and phospholipid in ACT, aPPT and PT assays, snake venoms, fibrinogen and thrombin. Contact activators including kaolin, celite, ellagic acid may also be required.

Other coagulation assay reagents including surfactants may also be added to the lateral flow assay surface to reduce surface tension. One exemplary surfactant which may be used is Triton X-100. Other conventional surfactants may be used. I n addition to these assay reagents, corn trypsin inhibitor may also be added to eliminate spontaneous (non-activated) clotting processes.

The agglutination/coagulation assay reagents may be applied to the lateral flow assay device in different ways. In one embodiment of the invention, liquid suspensions of these reagents are applied to the surface of the lateral flow assay device, in the area of the micro-pillar or micro- projections i.e. the lateral flow assay path. There are deposited as a thin film or layer of deposited suspension on the surface of the device. The liquid suspension may then be dried for long term storage. We have found that micro-pillars or micro-projections aid in the deposition and distribution of the assay reagents.

According to an alternative embodiment, the assay reagents may be applied to the surface of the lateral flow assay device in dried form. The assay reagent may be applied to the modified substrate surface using several manual or/and automated techniques including, but not limiting to, pipetting, spray-coating, dip-coating, light-directed patterning, ink-jet printing, screen printing, lithographic techniques (i.e. microcontact printing), electrospray, chemical vapour deposition, atomic force microscope based molecule deposition and others. In this way the assay reagent is immobilized on the substrate surface.

Stability of the assay reagents agents can be further enhanced with the incorporation of supporting reagents including, but not limited to, other proteins, polymers, sugars, surfactants, humectants. Further, stability can be improved by using appropriate drying techniques using techniques such as temperature control, humidity control, air flow and drying rates, e.g., lyophilisation. Further stability can be achieved by using various storage and packaging means such as controlled atmosphere for humidity, temperature, inert gases, vacuum, delivered through various techniques such as pouching and sealing and specified storage conditions.

Optical Apparatus

In order to monitor the redistribution of the detectable agglutination marker on the lateral flow assay, a wide variety of equipment may be used depending on how the type of signal is to be measured.

For example, where the detectable marker is a fluorescently labelled fibrinogen or other coagulation marker, then an optical apparatus comprising a fluorescent image viewer and the lateral flow assay device will be required. The fluorescent image viewer is arranged to focus on the assay zone in this lateral flow assay device. The fluorescent image viewer may be provided above the device.

Any fluorescent image viewer capable of visualizing the formed clot, and optionally recording the viewed image, may be used in the system. For example, the image viewer may comprise a microscope, filter set, light source and/or a camera. When used together, the microscope is connected to the camera such that the fluorescent images visualized and magnified by the microscope are passed to the camera to record. Ideally, the microscope may be a fluorescent microscope.

An alternative optical detection system could comprise a light source, appropriate lenses, filters and a detector. The advantage of this system is that it is simple and more cost effective than using a fluorescent microscope. For example, the light source could be selected from a range of possibilities including lasers, and diodes, including laser diodes, high brightness light emitting diodes (LEDs) and organic LEDs (OLEDs). The system could also comprise an optics housing, wherein the optics housing has means of receiving two filters, an excitation and an emission filter, such that filters are positionable in an excitation and an emission path length. The housing may also contain a lens system to collect fluorescence from the assay zone and direct it to the detector. The detector is a light detector capable of detecting a 2-dimensional array of light emission from the assay zone. This could be either a charge coupled device or CMOS device. The optical detection system would also possess electronics to process the fluorescent signals and a display/readout to convey results to the user.

The system may further comprise at least one platform on which the device of the invention is provided. The platform may be capable of moving the device into a position relative to the fluorescent image viewer such that the image viewer focuses the test material at the test area. Accordingly, the platform may be capable of directional controlled movement in a plurality of axes, e.g. x and/or y and/or z axis.

The system may also further comprise a heater, which is capable of conducting heat to the device. A heater can be incorporated to vary and control the temperature of the assay depending on assay requirements The heater may be provided in or attached to a surface of the platform on which the device is provided, and may therefore be in direct contact with the device. The heater may comprise resistive electrical coils, a printed pattern of resistive ink, or the like. The heatermay be capable of regulating the temperature of sample fluid in the device within the range 37°C to 60°C, preferably around 37°C. Other preferred embodiments of the invention will now be described.

According to one embodiment of the invention, the agglutination assay reagents may be combined with the labelled sample in the agglutination activation zone. Alternatively or additionally, the lateral flow assay path zone may have one or more agglutination assay reagents deposited thereon and the labelled sample (i.e. the sample combined with detectable marker and agglutination marker or combination thereof) is mixed and/or reacts with the deposited assay reagent during passage through the lateral flow assay path zone. According to another embodiment of the invention, the method comprises pre-treating the blood or plasma sample with an anti-coagulant agent. This enables the effect of the anti-coagulant agent to be determined.

According to yet another embodiment of the invention, the detectable agglutination marker, e.g. fluorescently labelled fibrinogen, is combined with the blood or plasma sample prior to use. Alternatively, the detectable marker and agglutination marker or combination thereof, e.g. fluorescently labelled fibrinogen, is combined with the blood or plasma sample in-situ on the lateral flow assay device. In this manner, the lateral flow assay device comprises at least a sample receiving zone, a mixing zone with a coagulation activation zone and a lateral flow assay path zone. The detectable agglutination marker, e.g. fluorescently labelled fibrinogen, is combined with the blood or plasma sample in the mixing zone. Optionally, the sample receiving zone and mixing zone may be coated with a reagent to prevent irreversible adherence of the detectable marker and agglutination marker or combination thereof, e.g. fluorescently labelled fibrinogen, to the sample receiving and mixing zone prior to addition of the blood or plasma sample.

This invention can be used on plasma and whole blood. When whole blood is used, it may be tested directly, or subjected to removal of red blood cells if desired. It will be understood that when the sample is whole blood it is ideally citrated prior to use and/or recalcified prior to, or upon contact with the lateral flow assay device.

Due to the low blood volumes, the invention is suitable for use on blood samples from typical venous sources (antecubital venipuncture), or alternate sites such as finger stick. If collection is into blood tubes using citration for anti-coagulation, re-calcification is required as part of the assay. This could be achieved with external recalcification or in-situ recalcification.

In one embodiment of the invention, the assay can be performed in a single step in-situ on a lateral flow assay device. In this embodiment, the lateral flow assay device comprises a sample receiving zone comprising a red blood cell separation zone (if desired), a mixing zone or zones and an assay path zone. The coagulation assay reagent(s), if employed is pre-applied to the surface of the assay path zone. The blood or plasma sample is first deposited into the red cell removal zone, part of the sample receiving zone, and then passes to the mixing zone of the lateral flow assay device comprising a detectable marker and agglutination marker or combination thereof marker, e.g. fluorescently labelled fibrinogen. No pre-incubation step for the sample is required. The sample mixes with the detectable marker and agglutination marker or combination thereof marker. If recalcification is required, it can be performed in a second mixing zone at this point (a recalcification zone). The labelled sample moves down the assay zone and reacts with the coagulation assay reagent(s). The detectable marker signal is monitored optically. In this embodiment, the detectable marker may be deposited in a dried or liquid form onto the surface of the lateral flow assay device mixing zone. The method of the invention enables assays, for example an aPTT assay, to be performed. Additionally, it has been shown that the assay can be performed at room temperature. Assays are improved at room temperature over 37°C as differences in clotting time are more discernible. Conventional assays are typically carried out at 37°C.

According to a third aspect of the invention, there is provided a lateral flow assay device for measuring the agglutination, preferably coagulation, within a sample wherein the lateral flow assay device comprises micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path characterised in that the lateral flow assay device comprises

a sample receiving zone;

a mixing zone with an agglutination activation zone, wherein the mixing zone comprises at least one detectable marker, agglutination marker and/or agglutination assay reagent; and

a lateral flow assay path.

Additionally or alternatively, the lateral flow assay path zone may comprise a layer of deposited coagulation assay reagent. According to a fourth aspect of the invention there is provided the use of the lateral flow assay device of the invention in a method for monitoring and measuring the agglutination, preferably coagulation, of a sample.

According to a fifth aspect of the invention, there is provided a kit for monitoring and measuring the agglutination of a sample comprising the lateral flow assay device of the invention and a detection means for measuring the increase in marker signal standard deviation in the assay zone over time.

The present invention will now be described with respect to the following non-limiting figures and examples.

Figure 1 (a) is a photographic image showing the results of Comparative Example 1. There is no localised brightness or fluorescence and this can be contrasted with Figs 2c and 2d where the contrast is clearly visible.

Fig. 1 (b) shows the results of photo-bleaching resistance of the green-fluorescent Alexa

Fluor 488 determined by laser-scanning cytometry compared to Oregon Green 488 and fluorescein. Data contributed by Bill Telford, Experimental Transplantation and Immunology Branch, National

Cancer Institute. Fig. 1 (c) is a comparison of pH-dependent fluorescence of the Alexa Fluor 488 (squares), Oregon Green 488 (full circles) and carboxyfluorescein (empty circles) fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm. Sourced from: http://probes.invitrogen.com.

Fig. 2 shows photographic images of recalcified plasma sample supplemented with fluorescently-labelled fibrinogen and tested in aPTT-coated (aPTT-SP) lateral flow platforms captured using (a) brightlight microscopy, 20x, at 5 sec, (b) brightlight microscopy, 20x, at 10 min, (c) fluorescence microscopy, 10x, at 5 sec and (d) fluorescence microscopy, 10x, at 10 min.

Fig. 3 shows the change in mean fluorescence intensity with time measured for fluorescent label-supplemented plasma samples spiked with 0 (circles), 0.25 (squares), 0.5 (triangles) and 1 (crosses) U/mL of heparin tested in aPTT-coated microfluidic channels.

Fig. 4 shows the change in SD over time obtained for plasma sample spiked with 0.25 U/mL heparin. Data analysed using Lab View software at the minimum signal detection set between 0 and 70 f. u.

Fig. 5 shows photographic images of micropillar test channel containing normal clotting plasma sample supplemented with fluorescently-labelled fibrinogen captured using fluorescence microscope with attached video camera at (a) 30 s, (b) 720 s and (c) 1650 s after sample application. Magnification: 10x.

Fig. 6 shows the change in SD of the fluorescence intensity over time for a normal clotting (black) and a non-clotting control (white) plasma sample supplemented with fluorescently-labelled fibrinogen in the micropillar channel modified with aPTT reagents. Arrow indicates the time point for the appearance of highly fluorescent regions. Signal measurement interval: 30 s.

Fig. 7 shows the percentage loss of mass monitored over time as a result of evaporation of the normal clotting plasma sample applied to the open lateral flow channel device.

Fig. 8 includes profiles which illustrate the clotting process in three different areas along the test channel: area 1 , 2 and 3. Addition of heparin at 0.25 U/mL (squares) and at 0.5 U/mL (triangles) was shown to prolong the CT in comparison to a normal sample without anticoagulant (circles).

Fig. 9 shows the change in fluorescence SD over time obtained for 0 U/mL (empty symbols) and 0.5 U/mL or 0.25 U/mL for SynthASIL (filled symbols) heparin in plasma tested on platforms modified with aPTT reagents.

Fig. 10 shows the quality of clotting profiles was influenced by the localisation of a chemistry deposition. Plasma samples without (circles) and with an addition of heparin, 0.5 U/mL (triangles) were tested on platforms with dried aPTT reagent deposited from the sample zone (empty symbols) and the test zone (full symbols). Fig. 11 shows clotting profiles of plasma without (line, no symbols) and with 0.5 U/mL of heparin (circles) were activated by 10 - 50 μΙ_ of an aPTT reagent dried onto the test channel surface.

Fig. 12 shows a barchart comparison of CTs of a normal clotting (white) and a 0.5 U/mL heparin-treated (striped) samples activated by 10 - 50 μί of a dried aPTT reagent.

Fig. 13 shows the change in SD value over time for clotting plasma (white circles) and for whole blood observed at the same settings as plasma (black triangles) and whole blood after adjustment of the brightness adjustment (black circles).

Fig. 14 relates to the incubation of plasma with heparin studies. Comparison of clotting profiles for plasma samples incubated for 0 min (circles), 30 min (squares) and 60 min (triangles) with 0.25 U/mL (empty symbols) and 0.5 U/mL (full symbols) of heparin at RT.

Fig. 15 shows profiles of change in SD over time obtained for plasma samples spiked with heparin at 0 (filled circles), 0.25 (empty circles), 0.5 (filled reversed triangles), 0.75 (empty reversed triangles), 1 (filled squares), 1 .5 (empty squares) and 2 (rhombi) U/mL. Signal measurement interval: 10 sec.

Fig. 16 shows calibration curve of heparin concentration vs. extracted clotting time (CT) in control plasma samples spiked with 0 - 2 U/mL of heparin (n=3). y = 156.29x + 181.28; R² = 0.996.

Fig. 17 shows the correlation between aPTT values for heparin-spiked plasma (0 - 1 U/mL) by the developed aPTT assay device (field method) and coagulometer (n=3). Trend line parameters were as follows: y = 1.0462x + 69.49; R² = 0.9986.

Fig. 18 shows the correlation between aPTT values obtained for plasma samples spiked with heparin at 0 - 0.75 U/mL tested using the fluorescence-based technique (field method, n=3) and using the ACLtop^® (hospital method, n=1). y = 0.8065x + 169.12; R² = 0.9365.

Fig. 19 shows the correlation of patient aPTT values between the hospital method (ACLtop^®, n=1) and using the fluorescence-based assay (field method, n=3). y = 8.7395x + 0.6393; R² = 0.7834.

Fig. 20 shows the correlation between aPTT values by the routine, hospital method (ACLtop^®) and by the fluorescence-based assay (field method) for patient plasma (white) and heparin-spiked control plasma (black). Trend line parameters were as follows: y = 8.7395x + 0.6393; R² = 0.7834 for patient plasma and y = 0.6656x + 137.46; R² = 0.959 for heparin-spiked plasma samples.

Figures 21 to 23 relate to Example 4.

Fig. 21. is an illustrative aerial perspective of an alternative channel consisting of numerous micropillars within the microfluidic lateral flow platform. The fluorescence dye redistribution durhg clotting of a plasma sample supplemented with fluorescently-labelled fibrinogen was monitored using fluorescence microscopy on aPTT-coated chips for 710 s. Representative images captured at 100, 280 and 500 s are shown in Fig. 22. The monitored area was initially dark with evenly distributed fluorescence signal (Fig. 22a). The presence of brightly fluorescing formations could be observed in Fig. 22b. Following that an increase in signal intensity was observed but the signal was mostly confined around the micropillars (Fig. 22c).

Fig. 22 shows photographic images of micropillar test channel containing normal clotting plasma sample supplemented with fluorescently-labelled fibrinogen captured using fluorescence microscope with attached video camera at (a) 100 s, (b) 280 s and (c) 500 s after sample application. Magnification used: 10 x. The insert shows the magnified (20 x) localized redistribution of a fluorescence label during clotting.

Fig. 23. In order to qualitatively determine the onset of clotting images were analysed by calculation of a change in mean fluorescence intensity with time and using SD calculation software. The results are shown in Fig. 23 where the change in the mean fluorescence intensity and the SD of the fluorescence intensity over time for a plasma sample supplemented with fluorescently- labelled fibrinogen in the micropillar channel modified with aPTT reagents. Arrow indicates the time point for the appearance of highly fluorescent regions. Signal measurement interval: 10 s.

Figures 24 to 27 show the results of Example 5.

Fig. 24 are Lab View profiles for normal clotting plasma.

Fig. 25 is a graph of three normal clotting plasma samples. The shaded points illustrated by arrow indicate the CTs.

Fig. 26 are photographic images illustrating sequence of events during plasma clotting in

PT-coated channel; (a) surface background, (b) front of the liquid carrying the clumps of unbound labelled fibrinogen, (c) lag phase with uniformly distributed label, (d) clotting and (e) evaporation.

Fig. 27 is a graph showing the correlation between PT values obtained using the developed system and the INR values of calibrators at the highest (triangles) and the lowest (circles) range.

Figures 28 to 31 show the results of Example 6.

Fig. 28 is a photographic image showing the accumulation of fluorescently-labelled fibrinogen on the surface of particles used as surface activators: kaolin, celite and glass beads.

Fig. 29 shows photographic images showing platelet poor plasma clotting in the channel coated with PT reagent. Images illustrate formation of weak fibrin mesh.

Fig. 30. is a clotting profiles obtained for normal clotting whole blood.

Fig. 31 is a clotting profiles obtained for heparinised whole blood: 1 U/mL (filled symbols) and 100 U/mL, negative control (empty symbols). Comparative Example 1

Blood clotting on flat substrate

Method

Blood clotting was monitored in a capillary channel composed of two laminated sheets of PET polymer coated with a hydrophilic layer, 90368 (Adhesive Research Ireland) to make it suitable for the passage of aqueous samples, but which did not possess and microprojections. Thus, the surface of this channel was a flat surface. The measurement on a flat surface was performed analogously to the previous experiments. The blood sample was supplemented with fluorescently-labelled fibrinogen, externally recalcified with 25 mM CaCI2 at a ratio 1 : 1 and applied to the channel surface. Measurement was carried out in a centre of the channel at the microscope settings used previously for monitoring of the fluorescently- labelled fibrinogen redistribution.

Results & Conclusion

The results are shown in Figure 1 a. We found that after application of the labeled sample to the surface of the channel, the fluorescent label was distributed evenly over the substrate surface. The only possible indication of sample clotting was the cessation of sample movement, however, this was not a conclusive determinant of blood clotting or clotting times.

Thus, these results obtained were not conclusive or helpful in accurately determining the onset of blood clotting or even monitoring blood clotting times.

Example 1

Heparin Dose Monitoring aPTT Assay Development

Materials and Methods

Human fibrinogen

Alexa Fluor 488 (Molecular Probes (F-13191))

Conventional aPPT reagents

4Castchip^® COP (AMIC)

Heparin Method for Making Fluorescently Labelling Fibrinogen

A human fibrinogen conjugate labelled with Alexa Fluor 488, purchased from Molecular Probes (F- 13191) was employed. This fluorescent dye possesses several advantages over traditionally used labels. According to the manufacturer, Alexa Fluor 488 has spectral characteristics similar to fluorescein conjugates. However, it is more photostable (Fig. 1 b) and less pH-dependent (Fig. 1 c) than that of fluorescein-protein conjugates. The conjugate was prepared by attachment of approx. 15 dye molecules per fibrinogen molecule and purified in order to remove any un-reacted dye. Alexa Fluor 488 is a green fluorophore with an absorption and fluorescence emission maxima of 496 nm and 520 nm, respectively. aPTT Assay using Fluorescently Labelled Fibrinogen

The 1.5 mg/mL stock solution of conjugate was prepared in 0.1 M sodium bicarbonate (pH 8.3) and stored at -20°C. 15 - 25 μί of citrated control plasma (Hemosil) or citrated whole blood (3.8 % sodium citrate) were used as test samples. Fluorescently-labelled fibrinogen has been shown to be effective for clot formation localization purposes when used at the concentration of 5 % (v/v) of total fibrinogen content in a sample (Bateman R.M. 2005). Analogously, the same concentration was used here. The normal fibrinogen concentration in human plasma is approx. 2.8 gL (Stief 2007, Boux 1998). Therefore, 1.35 or 2.25 μί of labelled fibrinogen stock solution was added to 15 or 25 μί of a test sample. 15 or 25 μί of 0.025 M CaCb solution (Stago Diagnostica) was then added to reverse the effect of citrate and allow clotting (the exception was the plasma dilution effect studies, where 0.25 M CaCI₂ (Sigma) was used to trigger plasma). Immediately after addition of Ca²⁺ ions, 25 or 50 μί of a test mixture was applied to a test chip and measurement was started. Blank control was prepared by replacing the CaCI₂ with NH₄CI in order to avoid recalcification, prevent from clotting and to keep the dilution factor constant. Positive control was performed using purified thrombin (Sigma) at a concentration of 1 U/mL to trigger clotthg. In heparin sensitivity studies plasma or blood samples were spiked with a series of heparin concentrations (0- 2 U/mL) (Sigma). For consistency between the normal clotting samples with no heparin added and heparinized samples, the former was supplemented with PCR water (Sigma) at a volume equal to the volume of a heparin solution added.

The test chips were prepared by applying 10 - 50 μί of 0.05 % (v/v) Triton X-100 (Sigma) in aPTT reagent to the test channel. Chips were then dried in the open air for 1 h and stored at 4 °C for 1 day to 2 weeks. Five commercially available aPTT reagents were tested: Cephalinex (BioData), C.K.Prest 2 (Stago Diagnostica), aPTT-SP (Hemosil, IL), TriniCLOT aPTT S (Trinity BioTech) and SynthASIL (Hemosil). For an assay optimization purposes TriniCLOT aPTT HS was used. The measurement was performed using a fluorescence microscope at the following settings: magnification: 10 x, green filter, exposure time: 21 ms, minimum brightness: 0, maximum brightness: 594 for plasma and 350 for whole blood, ISO: 200, colour control: red 0.1 , green 0.7 and blue 3.0. The autofocus function was switched off at all times. Experiment was performed in a dark room at 37 °C or RT. Images were taken every 10 or 30 s for up to 1500 s. The saved fluorescence data were then analysed using a LabView programme. The created programme calculated a standard variation/deviation (SD) which reflected the changes in fluorescence signal and a difference between signal generated by formed clots and a background. Where necessary, recorded data was also a subject to visual analysis. SD vs. time profiles were generated (clotting profiles) and the events happening in the monitored area were correlated to the fluctuations in the clotting profile.

The Use of Fluorescently-Labelled Fibrinogen for Clotting Monitoring Purposes

A conjugate of human fibrinogen labelled with Alexa Fluor 488 (described above) was used in the assay. There was no action required to release a fluorescent signal. In the presence of thrombin the soluble fibrinogen was converted into insoluble fluorescently-labelled fibrin. In essence, the labelled fibrinogen competes for unlabelled fibrinogen for incorporation into the forming clot allowing its optical fluorescent localisation. Additionally, binding of the labelled fibrinogen to the GPIIIa-llb receptor on activated platelets could take place which would supplement the process of fibrinogen incorporation into a clot. To assess the potential of the fluorescent label to be used to detect clot formation in vitro, a plasma test sample was externally recalcified and supplemented with the fluorescently-labelled fibrinogen.

A sample was applied to a microfluidic chip that had previously been modified by drying aPTT-SP reagent onto the surface. Passage of the liquid sample would lead to solubilisation and reconstitution of the reagent which would then bring about accelerated clotting.

Results

It was found that the sample advanced along the device channel in a highly controlled and reproducible fashion with an initially uniform distribution of the dye. When analysed by light microscopy, immediately after addition of sample, the sample mixture was homogeneous (Fig. 2a). However, after 10 min, strands which were believed to be fibrin fibres could be seen gathering around and between the micropillars (Fig. 2b). When analysed using fluorescent microscopy, initially, there was an even distribution of fluorophore with relatively low average background intensity (Fig. 2c). However, following clotting, patches of more intense fluorescence in a pattern similar to that seen for the fibrin fibres under light microscopy could be seen (Fig. 2d). It could also be noted that there was a decrease in fluorescence in areas immediately adjacent to the areas of more intense clot formation, which may be due to the concentration of the labelled fibrinogen within the loci of forming clots. The concentration of the fluorescently-labelled fibrinogen and its distribution along the channel appeared to be changing in a time-dependent manner. Appearance of the highly fluorescing formations could be observed after approx. 300 s (Fig. 2d), which corresponded to the time needed for fibrin fibre formation as observed visually by light microscopy (Fig. 2b). Therefore, the timing of the localisation of clot formation as seen with fluorescence could form the basis of a means of defining an assay clotting time.

Plasma samples were spiked with a range of heparin concentrations (0- 1 U/mL) in order to obtain samples with prolonged CTs. The change in the average fluorescence over time was monitored in the test channels (Fig. 3). All samples showed some initial increase in fluorescence intensity, due to the influx of label to the area as it passed down the channel. At some point, however, there was a actual decrease in fluorescence intensity, followed once again by a gradual rise. The point at which this decrease occurred appeared to correlate with the concentration of heparin used, rising from approx. 130 s at 0 U/mL to approx. 220 s for 1 U/mL. However, the inflections generated were not very well defined, particularly at 1 U/mL in which the change in the profile was difficult to identify Thus, alternative methods of correlating clotting with changes in fluorescence were investigated.

Although it did not appear reliable to extract the CT values from the total change in fluorescence intensity, visual observation had suggested that, as well as changes in the average fluorescence, the localised redistribution of the label within a specific area might also be changing in a time- dependent manner. Therefore, the fluorescence standard deviation (SD) of the monitored zone was assessed. Fig. 4 shows the SD change over time obtained for 0.25 U/mL of heparin in plasma with the fluorescence background detection set between 0 and 70 f.u.

The shape of the profiles obtained for 0.25 U/mL heparin-spiked plasma were shown to be highly dependent on the background fluorescence setting. Since all data points in the monitored area were included in the SD measurement even low signal areas like the micropillar surface were included in the SD calculation. Increasing the background cut off resulted in the elimination of the darker/low signal regions within the monitored area (coming most likely from the areas of micropillars) and thus lower SD values were obtained. Thus, below 30 f.u., initial SD values were greater than zero, being between 3 and 4 for 0 f.u. and between 2 and 3 SD for 10 f.u. However, above 30 f.u., little or no change in SD was recorded during the initial profile, which suggests that there was no discrimination between background levels of fluorescent label and the chip substrate.

At approx. 400 s, however, there was a large increase in SD irrespective of background settings, which was most likely due to the onset of coagulation. At this point, samples with background cutoffs of 20 f.u. or greater exhibited sharp, but erratic increases in SD, whereas at 10 and 20 f.u. , the profiles were smooth, but less pronounced in terms of rate of change and peak SD achieved.

The changes in SD for the profiles corrected for background above 30 f.u., combined with the absolute fluorescence values obtained in Fig. 3 suggest that the distribution of fluorescent label within the measured area must be changing, becoming reduced in some areas and increased in others and that this phenomenon appears to be associated with the onset of clotting.

In addition, the change in SD became more significant during clotting for profiles at 20 f.u. or more, while at the same time the signal-to-noise ratio was reduced resulting in noisier profiles which, in turn, made clotting times more difficult to determine. On the other hand, the change in SD obtained with no background rejection (0 f. u.) was slow and gradual and the first point of a change in SD was not well-defined. However, at 10 f.u. cut-off, the initial SD was still quite low, while the change in SD observed during coagulation was quite pronounced and not subject to noise and variability.

LT values were determined for the data shown in Fig 4. In addition, visual examination of the recorded frames was performed. The time points when the creation of the first fibrin fibres could be visually observed were chosen as CTs and compared to the LT values which were taken as the time points when sudden increase in SD was demonstrated on the SD vs. time profiles (Table 1 ).

Table 1 . CT values for plasma samples containing 0 - 2 U/mL of heparin obtained by visual analysis and on a basis of LT calculation for profiles at 0 - 70 f. u. minimum signal detection using the SD calculation program. (-) refers to a meaningless or impossible CT read-out.

Heparin CT [s]

concentration Visual Minimum of signal detection [f. u.]

[U/mL] analysis 0 10 20 30 40 50 60 70

290 290 290 - 260 -

0

290 330 300 350 290

280 280 290

350 350 350 360 370 350 - -

0.25

340 340 340 350

400 410 400 390 420

470 470 470 - -

0.5

460 480 480

470 480 480 480 480 480

500 640 640 - 640 640 - 470

1

500 500 500 510

480 490 490 470

2 560 580 560 480 - 480 500 - -

LTs referred to as CTs were determined for all tested samples at minimum signal detection set to 0 and 10 f.u., while CT determination on the basis of profiles at 20 f.u. or higher detection minimum was very difficult or impossible to perform. The visual analysis was used as a correction method indicating a "real" time point for clotting initiation. Minimum set to 0 and 10 f.u. allowed CT determination in a relatively reliable way. Prolongation in CT value was observed with increased heparin concentration for both settings. However, analysis at 10 f.u. correlated best with the values determined by visual observation for all tested heparin concentrations and reflected the onset of clotting in a more reliable way than for 0 f.u. By setting the detection minimum to 10 f.u., areas of very low fluorescence signal were neglected, which included micropillars as the darkest areas. Excluding these regions from the SD analysis yielded more reliable results. Analysis at the minimum signal detection of 10 f. u. allowed reliable and accurate CT determination and therefore, was chosen as an appropriate method for further CT determination.

Thus, it has been shown that an improved observation of the onset of clotting could be determined from measuring the localised redistribution of the fluorescence signal of a fluorescently-labelled fibrinogen, rather than the simple increase in the magnitude of the fluorescence signal Initial investigation had suggested that the change in SD observed was related to changes in the distribution of fibrinogen and that this was indicative of clot formation and associated CT. However, more detailed studies were undertaken to relate the change in the fluorescent measurement to microscopic changes taking place within the clotting sample. The clotting of plasma samples supplemented with the fluorescently-labelled fibrinogen was monitored using fluorescence microscopy on aPTT-coated chips for 1700 s. Representative images captured at 30, 720 and 1650 s are shown in Fig. 5. The monitored area of micropillar channel covered with plasma and fluorescence label solution was initially dark with evenly distributed fluorophore and low fluorescence signal (Fig. 5a). However, brightly fluorescing formations could be observed at 720 s (Fig. 5b). At 1650 s, the fluorescence intensity in the monitored area increased with the signal mostly confined to the areas around the pillars (Fig. 5c).

Images were then analysed using SD measurements. SD analysis of the citrated plasma triggered with CaCI₂ (clotting) and citrated plasma supplemented with NH₄CI (non-clotting control) is shown in Fig. 6.

For the first 300s only small changes in SD were observed for the clotting sample with values between 3 and 5. This phase corresponded to the period of uniform, low fluorescence signal illustrated in Fig. 5a with the distribution of fluorescence being derived from the difference between the darker areas (pillars) and the brighter fluidic regions between the pillars. This period of minimal change was equated with the lag time of other clotting time assays. After this period, a rapid increase in SD to approx. 14-15 was observed. Following this abrupt change in SD no further change was observed for nearly 500 s. This period was associated with the redistribution of the fluorescent label as illustrated in Fig. 5b and which, we suggest, corresponds to the onset of clotting. During this period, intensely fluorescent areas between the pillars could be seen which conform to patterns of sample flow between the pillars, with adjacent areas with visibly reduced fluorescence. This was supported by visual and white light microscopy which indicated the formation of adherent fibrin fibres at this time. Further intensification is likely as sample continues to flow through and is captured by the forming clot loci. A further significant change in SD was observed at around 810 s followed by a gradual and continuous decrease starting at around 1050 s. Visual analysis revealed that after around 800 s the fluorescent label began to become concentrated around the micropillars (Fig. 5c). Adsorption of the label onto the circle around pillars from the adjacent areas and the dye accumulation resulting in an increased localised signal was most likely an indicator of the onset of evaporation. Part of the liquid evaporated from the free/between-pillars areas, while still significant fraction of the half-liquid solution was relocated to areas adjacent to micropillar walls. The label accumulation and signal enhancement in the circles around the pillars and decreased intensity in the areas between pillars caused significant change in SD, which could be correlated with the appearance of a high peak in SD vs. time profile at approx. 1050 s in Fig. 6. Eventually, evaporation seemed to affect areas adjacent to the pillar as well, which could be observed in a gradually decreasing SD signal after 1050 s. As all liquid evaporated, the fluorescence signal is quenched and decreased. A gradual and steady increase in the SD was observed for the non-clotting sample, where the NH₄CI was added to maintain ion concentration equivalence. No clot formation could be observed visually and the insignificant increase in the SD value may have occurred due to label clumping, accumulation and settling over time rather than clotting.

The rapid increase in SD of the fluorescence signal was observed only for the clotting sample regardless of the fact that the same concentration of fluorescently-labelled fibrinogen was present in both clotting and non-clotting samples. The fibrinogen binding not only made it easy to localise the formed clots, but also allowed recognition of clotting initiation.

Additionally, a further positive control (data not shown) was performed using purified thrombin at 1 U/mL in order to bypass the surface activation phase and induce immediate clotting. As a result, a highly fluorescent clot formed rapidly in the sample application zone. The bulk of the available label was captured and therefore, there was little or no signal measured.

The impact of evaporation on the sample behaviour was assessed over a prolonged period of time in a lateral flow device, which was not equipped with a lid and therefore was exposed to atmospheric conditions. A standard clotting solution consisting of 1 :1 mixture of plasma and CaCb supplemented with fluorescently-labelled fibrinogen (total volume 25 μΙ_) was applied to the microfluidic platform modified with the aPTT reagent. The percentage loss of mass was measured using a standard laboratory scale and was taken as the evaporation rate (Fig. 7).

It was shown that the evaporation process was linear under the given conditions for approx. 1500 s (25 min) at which time it reached a plateau and only approx. 6% of the initial sample mass remained. At approx. 780 - 840 s (13 - 14 min) 50% of the sample mass had evaporated. Previously observed changes of the fluorescence SD signal due to label relocation were attributed to evaporation, especially for times longer than 780 - 840 s. It was also established that the first 600 s (10 min) (in the case of a normal clotting sample) was the period that should be monitored, as although 38% of sample mass had been lost, this did not impact on the measurement of coagulation. However, after approx. 780 s (13 min) the measurement was more significantly influenced by evaporation with nearly 50% loss of sample mass and should not be used for clotting time measurement. Example 2- Assay Platform of Example 1 Optimisation

Area tested

The clotting process began with an addition of Ca²* ions to a citrated sample and accelerated once it came in contact with an aPTT reagent dried onto the surface. The test was performed in an open flow system. The whole length of the test channel was characterised in order to select the best area for the measurement which would be reliable in reflecting the actual clotting process. To show the differences in events occurring in different areas of the channels, three spots along the test channel were selected: area 1 , the sample zone (beginning, labelled with 'S' symbol), area 2, the test zone (middle, labelled with T' symbol) and area 3, the end zone. The behaviour of the normal clotting and heparinized samples (0.25 and 0.5 U/mL of heparin) was monitored in those three areas (Fig. 8).

It was shown that clot formation was not uniform along the test channel. The CTs measured in area 3 were significantly prolonged when compared to areas 1 and 2. The reason for that could be the fact that the clotting process was happening mostly in the earlier parts of the channel. A plasma sample was applied to the sample zone, so before it reached the last stages of its journey most of the fibrinogen and other coagulation factors were probably already consumed in clot formation. Another explanation could be adsorption of the fibrinogen and/or other coagulation factors to the surface, which could affect the efficiency and, subsequently, the rate of clot formation. It was worth noting that the late increase in SD that could be correlated to a clot formation, might have been the result of un-bound, free, labelled fibrinogen accumulation just before the end of the channel. As evaluated by visual observation, the free labelled fibrinogen could form clumps when prevented from further flow by any barrier and monitored over 1200 s or more. Those events resulted in an increase in the overall signal, and therefore, overall SD. That could cause the false positive to occur, by means of a presence of a rapid increase in SD which did not correspond to labelled fibrinogen incorporation into clot, but simply label accumulation. This would be in agreement with what was shown above for area 3 where there was only a slight increase in SD for a normal clotting sample at around 600 s, which suggested that the clot formation took place, even though the signal was very low, and the increase in SD was not significant after 1200 s in comparison to heparinized samples as probably most of the labelled fibrinogen was bound into the quickly forming clots of a non-heparinized sample in the early stages and therefore, not much was left to accumulate in the end of the channel. The CTs for heparinized and non-heparinized plasma samples were similar in areas 1 and 2, with the exception of 0.5 U/mL which affected the clotting process in area 1 so strongly that it actually flattened out the profile making the CT calculation impossible. Normal CT values were similar. The 0.25 U/mL profile looked clearer in the case of area 2, while the area 1 profile was three-steps, which complicated the analysis, as without the exhaustive visual analysis it would be impossible to find out which of those three steps corresponded to the start of clotting process. Therefore, the measurement in area 2 was selected as the most reliable reflection of the clotting process.

Choice of aPTT reagent

Five aPTT reagents were tested in their dried form in the fluorescent clotting assay device. These were Cephalinex, C.K. Prest 2, SynthASIL, aPTT-SP and TriniCLOT aPTT S. Platforms modified with 40 μΙ_ aPTT reagents were used for testing normal clotting and 0.5 U/mL heparin-spiked plasma samples. An exception was SynthASIL for which a heparin concentration of 0.25 U/mL was used because 0.5 U/mL prolonged the plasma CT so drastically that it was impossible to estimate a LT before the sample evaporated. Fig. 9 illustrates the associated clotting profiles obtained for chips modified with these aPTT reagents and Table 2 summarises extracted LTs which were referred to as CTs obtained for the five tested reagents.

Table 2. CT values calculated on the basis of LTs for normal clotting (0 U/mL heparin) and heparinised (0.5 U/mL and 0.25 U/mL for SynthASIL) plasma samples tested on platforms modified with dried aPTT reagents.

It was difficult or impossible to extract the LT/CT values for the samples activated with Cephalinex and C.K. Prest 2 on the basis of profiles shown in Fig. 9. The change in signal SD was gradual throughout the clotting process and therefore the LTs were not wel-defined. However, small inflections in the responses could be observed and so CTs of 360 s and 420 s for Cephalinex and 260 s and 580 s for C.K. Prest 2 were given for normal and heparinised samples, respectively. SynthASIL did show a clear inflection in the SD response for heparinised plasma and yielded a CT of 600 s for normal clotting plasma, which was nearly two-fold longer than CT achieved with any other tested reagent. High baseline CT values would not be suitable for further assay development, therefore the use of SynthASIL was not considered as an optimal strategy for the assay chemistry formulation. The profiles obtained for aPTT-SP and TriniCLOT aPTT S showed very distinctive changes in SD upon clot formation; the profiles were clear, not noisy and thus the CT values were easy to determine for both normal clotting as well as for heparinised samples. However, the delay in clotting due to the addition of heparin was not as significant for TriniCLOT aPTT S as it was for aPTT-SP. The difference in CT between 0 and 0.5 U/mL of heparin was only 80 s for TriniCLOT aPTT S in comparison to 190 s for aPTT-SP. In a case of TriniCLOT aPTT S it would be difficult, if not impossible, to distinguish between 0 and 0.25 and between 0.25 and 0.5 U/mL of heparin in a sample. On the other hand, TriniCLOT aPTT S could be employed in the assays for high heparin dosage monitoring (over 2 U/mL), i.e. during surgery. The CT obtained for non-heparinised sample activated with aPTT-SP was the shortest of all the tested reagents (240 s) and an addition of 0.5 U/mL of heparin resulted in a significant CT prolongation (430 s). aPTT-SP was chosen as the most reliable reagent providing the shortest CT and good differentiation between low heparin dose concentrations and was used for further development of the device for monitoring of therapeutic level of anticoagulant (0 - 2 U/mL).

Localization of aPTT deposition

Test platform preparation, including chemistry deposition, was of importance. aPTT reagent was deposited by drop-deposition and left for drying. The reagents were distributed along the channel in a manner brought about by capillary flow introduced by the micropillars. A study was carried out to determine the difference in strip performance as a function of the area where the chemistry deposition started. 40 μί of an aPTT reagent solution was applied either from a sample zone (beginning of a channel) or from a test zone (middle of a channel). Measurement was always performed in the test zone at Area 2. Fig. 10 shows the clotting profiles of 0 and 0.5 U/mL of heparin samples tested on chips with aPTT reagent deposited either from the sample or the test zone.

It was shown that the deposition process had a significant impact on the test performance and, the resulting profile. Profiles obtained from the test performed on the sample zone-deposited chemistry were noisy and consisted of multiple steps, which complicated the result analysis. It was nearly impossible to calculate the CTs and to distinguish between 0 and 0.5 U/mL of heparin samples. Deposition from the middle of the channel was shown to be much more effective. Both profiles (0 and 0.5 U/mL of heparin) were one-step, not noisy and easy to read. Therefore, this type of chemistry deposition was used further in the preparation of the aPTT assay platform. Volume of the aPTT chemistry to be deposited

For the aPTT determination in a test tube or on a plate, suggested plasma to aPTT reagent ratio is 1 :1. However, the designed configuration did not allow the equivalent ratio calculation, as the whole volume of a test mixture could not be in contact with a whole area of a channel with dried chemistry at the same time. Sample spread along the channel reaching the end in 30 - 60 s. Therefore, studies aiming in a determination of an optimum aPTT reagent volume to be deposited were performed. 10 - 50 μΙ_ of aPTT-SP was deposited in test channels and dried. Normal clotting (no heparin) and heparinized (0.5 U/mL) samples were tested in duplicates or triplicates. Clotting profiles are shown in Fig. 11. Average CTs are compiled in Fig. 12.

The average CTs were between 245 - 300 s and 400 - 420 s for normal clotting and spiked with 0.5 U/mL of heparin plasma samples, respectively. Surprisingly, there was no significant difference in the CTs obtained from different volumes of aPTT reagent. Even a five-fold difference in an aPTT reagent volume did not bring about any change in CT value. It has been proven that even low aPTT reagent to plasma ratios can activate the coagulation cascade effectively. However, the quality of the clotting profiles was not comparable. This was a significant factor in the determination of an optimum volume to be dried. On the basis of the clotting profiles obtained, the conclusion could be made that the optimum aPTT reagent volume to be deposited was 30 or 40 μΙ_. 10 and 20 μΙ_ were shown to result in noisy profiles and CTs were difficult to calculate. 50 μΙ_ was as good as 30 and 40 μΙ_, however, such a high volume was difficult to apply. Therefore, 30 and 40 μΙ_ were selected as an optimum volume of an aPTT reagent for a deposition in a test channel.

Plasma versus whole blood

Fluorescence-based detection systems have been used before in several whole blood monitoring approaches where the application of other methods, such as absorbance, is not possible due to the presence of red blood cells that strongly interfere with the signal [10,1 1]. Clotting of whole blood was assessed using the fluorescence assay described in this work. A normal clotting whole blood sample was applied to an aPTT reagent-coated platform and the change in the fluorescence SD signal was monitored over time.

Fig. 13 illustrates the clotting profiles obtained for a clotting plasma sample and a clotting whole blood sample monitored at equal brightness setting as optimised earlier for plasma and additionally for whole blood after compensating for brightness. The fluorescence emission from the whole blood sample was significantly lower than for plasma. This is likely due to the presence of the dense network of red blood cells which result in absorption and scatter [12]. Whether uncompensated or not, the observed changes in SD were similar for plasma and whole blood. Following brightness adjustment, no significant difference in the SD profiles was evident, with both showing a clotting time for this sample of approx. 190 s. This would suggest that the assay would ha e the potential for clotting time determination in both plasma and whole blood. Plasma samples with heparin pre-incubation

The assay development and optimization studies were performed on plasma or whole blood spiked with heparin. Therefore, it was necessary to find out if the incubation of plasma with heparin had any effect and if there was any difference between freshly prepared and old mixtures. Plasma samples were spiked with 0.25 and 0.5 U/mL of heparin and tested after 0, 30 and 60 min of incubation at RT.

It can be seen in Fig. 14 that the incubation time did not have a significant impact on the final result. As expected, there was a significant shift in lag times between 0.25 and 0.5 U/mL. However, the clotting profiles for the same heparin concentration were alike and calculated CTs were similar regardless of incubation time. CT values were 330 - 340 s and 430 s for 0.25 and 0.5 U/mL respectively. The above study clearly suggests that there was no need for plasma and heparin incubation in this instance and that the experiment can be carried out immediately after mixing.

Heparin dosage response

The fluorescently-labelled fibrinogen assay device was fully optimised for clotting monitoring in plasma and whole blood. Herein, this assay was evaluated for the affect of heparin dosage on clotting time in an aPTT-type assay. Control plasma samples were spiked with concentrations of heparin from 0 to 2 U/mL and these samples were subject to the fluorescent assay in order to assess the effect of heparin dilution on the plasma CTs. Typical SD vs. time profiles obtained for normal clotting (no heparin) and heparinised samples (0.25 - 2 U/mL) are shown in Fig. 15. In order to emphasise the prolongation in CT due to increased heparin concentration, only the initial parts of the profiles are shown.

The change in the detected SD signal was sudden and well-defined. CT values could be easily extracted on the basis of the generated profiles. A significant prolongation in the onset of clotting with increased heparin concentration was obvious. An un-anticoagulated sample (0 U/mL heparin) returned a rapid CT of 170 s. CTs were between 210 - 483 s for 0.25 - 2 U/mL. As illustrated in Fig. 16, the correlation between the extracted CTs and heparin dose was close to linear (R² = 0.996) in a tested range of heparin concentration. A slope value of 156 s.mL.LT¹ and the difference of at least 36 s between increasing heparin doses indicated a relatively high heparin sensitivity of the assay with the %CV < 8.4%. Results obtained for samples at 0.75 U/mL heparin or more were less reproducible than CTs in the lower heparin range. In these tests, the clotting occurs both later and more slowly. This is known to lead to increased variation in the onset of coagulation [13] with resulting increases in CV. Correlation with coagulometry

Coagu lometry is a wel l-established and widely used methodology for CT determination. Coagulometers measure the ability of blood to clot by performing any of several types of tests including PTT, PT and INR, lupus anticoagulant screens, D dimer assays and factor assays. The onset of clotting is usually determined mechanically (rotating or vibrating metal ball) or optically ("by eye" or measurement of the change in the light transmittance). Several other ways of determining a CT have been developed, including surface acoustic wave device measuring changes in a sample viscosity [14] or thickness-shear mode resonator used to characterise static rheological properties of blood [15]. Although coagulometers are relatively expensive, need to be operated by trained personnel and require large volumes of anticoagulated blood samples and activating reagents, they are still widely used in a clinical practice and for research purposes. Several newly developed coagulation monitoring devices are calibrated/validated against traditionally used coagulometers [16,17].

In this study clinical coagulation analyzer, Amelung KC4^® was used as a reference method. Amelung KC4^® is a semi-automated mechanical clot detection system where the manual addition of a plasma sample to an aPTT reagent was followed by pre-incubation step and automated CT determination. This coagulometer allowed aPTT determination for plasma samples spiked with 0 - 1 U/mL of heparin which were tested in parallel using the developed here technique. aPTT reagent deterioration upon drying might have an impact on the final result, therefore, reagent from the same lot was deposited in the developed device channels and in the coagulometer cuvettes and dried for 24 h. According to the recommended coagulometer procedure, test samples were pre-incubated with aPTT reagent for 3 min at 37°C before the CT measurement started, while the developed fluorescence method did not require the pre-incubation. Contact activation was achieved by exposing a test sample to the activator-phospholipid reagent formulation dried to the surface of the test platform. The resulting CT value (test time) was the total time required for the reversal of the citrate effect and the activation of coagulation factors. Therefore, the total CT value obtained using the fluorescence-based method was a sum of all the assay steps which would otherwise be performed separately in a traditional test. Consequently, the absolute CTs obtained by this method were considerably longer than the standard laboratory aPTT. The test was correlated with standard hospital coagulometry using the Amelung KC4^®, Fig. 17. CTs for plasma spiked with 0 - 1 U/mL of heparin were 71 .7 - 196.8 s and 146.7 - 276.7 s for coagulometer and the new technique, respectively. Given that the laboratory method required 3 min incubation, this would extend the total assay time to 251 .7 - 376.8 s. Thus, the total test times were, on average, 1 10 s faster for the new assay. In spite of the differences in the absolute CTs obtained, the correlation between the fluorescence-based method and coagulometer was found to be close to linear with R² = 0.9986.

Correlation with a routine hospital aPTT determination method

CTs obtained from the fluorescence-based method for heparin-spiked plasma were additionally compared and contrasted against another widely used method for clotting assessment in the clinical settings, an automated hospital coagulation analyzer, the ACLtop^® coagulation system (I L). This instrument measures a change in the light transmittance during clotting of a plasma sample activated with an aPTT reagent (clotting method). Plasma samples spiked with a range of heparin concentrations from 0 to 2 U/mL were tested in parallel using the developed fluorescence-based assay and the ACLtop^® coagulation system. The resulting aPTT CT values are listed in Table 2 and correlation showed in Fig. 18.

Table 2.

Table 2 shows APTT of plasma samples supplemented with heparin at 0 - 2 U/mL tested using a routine, hospital method and the developed fluorescence-based assay in parallel.

CT values derived by the hospital method were lower than the fluorescence assay results. Again, this was due to the introduction of plasma and aPTT reagent in a pre-incubation step (as was the case with the coagulometer). Taking this into account, the methods differed by approx. 10 s. Given that the fluorescent assay only takes a reading every 10 s, these could be considered equivalent. In addition, the hospital method was incapable of determining CT values for high heparin concentrations (≥ 1 U/mL). Therefore, correlation was only established on the basis of CT values obtained for 0 - 0.75 U/mL where it was close to linear (R² = 0.9365, n=3). The new method appeared to be a reliable tool for heparin dose monitoring in spiked samples at least. In addition, higher drug doses (up to 2 U/mL) could be detected using the fluorescence-based method, which was not possible with the standard hospital technique. This higher range typically requires use of the ACT assay. The new assay was also more sensitive to low heparin dosage as plasma CT was prolonged by 43.3 s upon an addition of 0.25 U/mL heparin in comparison to only 18.9 s in the ACLtop^®.

Example 3 - Assay validation in patient samples

The viability and reliability of the fluorescence-based lateral flow assay device for heparin determination was further investigated by testing patient samples with known aPTT. A cohort of 32 normal and abnormal plasma samples were obtained from patients with clotting abnormalities, who were receiving combined anticoagulant therapy (exact dosage and type of treatment was unknown). These were examined using the fluorescence-based aPTT method developed here and the ACLtop^® (Table 3 and Fig. 19).

HOS P ITAL M ETHO D N E W AS S AY

APTT [s] APTT [s] S D C V [%]

21.2 170.0 0.0 0.0

24.0 253.3 30.6 12.1

28.3 170.0 14.1 8.3

29.0 276.7 5.8 2.1

30.0 275.0 7.1 2.6

32.0 383.3 37.9 9.9

34.6 320.0 26.5 8.3

34.6 386.7 23.1 6.0

35.2 263.3 23.1 8.8

36.0 335.0 21.2 6.3

36.2 286.7 5.8 2.0

36.9 266.7 5.8 2.2

37.9 280.0 14.1 5.1

38.1 330.0 17.3 5.2

39.8 250.0 0.0 0.0

41.3 285.0 7.1 2.5

42.4 443.3 20.8 4.7

43.4 405.0 35.4 8.7

45.0 303.3 25.2 8.3

46.9 510.0 28.3 5.5

49.1 556.7 63.5 11.4

50.0 500.0 60.8 12.2

50.3 446.7 56.9 12.7

53.2 430.0 28.3 6.6

53.6 410.0 52.0 12.7

54.5 397.5 53.8 13.5

60.9 583.3 55.1 9.4

61.0 613.3 37.9 6.2

63.8 516.7 25.2 4.9

64.4 453.3 15.3 3.4

76.8 730.0 104.4 14.3

78.4 676.7 107.9 15.9 Table 3 shows the aPTT values of 32 patient plasma samples analysed by the routine hospital method and the new fluorescence-based assay (n=3 or 4). SD values for the hospital method, the ACLtop^® were not available.

Total assay times obtained from the fluorescence-based assay were, again predictably greater than the hospital method. However, taking into account the 3 minute incubation, the new method was, on average 1 10 s quicker to return a CT. Again, samples with significantly prolonged CTs showed poorer reproducibility than those of low or normal aPTT. Nevertheless, the intra-test variability was satisfactory. The slowest clotting sample (78.4 s by hospital technique, 676.7 s by fluorescence- based method) yielded the highest %CV of 15.9%, while 24 out of 32 samples returned CVs of less than 10%, which could be considered very reproducible taking into account the inherent variability of this type of assay, particularly when testing heparinised patients where deviations of up to 200% have been documented [18].

Due to the wide variability in aPTT testing and individual patient response, the relationship between the heparin dosage and the CT expressed as aPTT is very difficult to establish. Therefore, new point-of-care methods are correlated with conventional automated aPTT central laboratory coagulometers [19]. Herein, the obtained coefficient correlation of 0.7834 between the newly developed and standard, reference methods was considered good when taking into account the fact that aPTT test is still non-standardised assay and several factors including factor deficiencies can influence the responses of both methods [20]. Results obtained for real patient samples were additionally compared to the heparin-spiked plasma values (Fig. 20). In this instance, a significant disparity could be seen between the calibration curves for spiked and patient samples by the two methods. The correlations between aPTT by the newly developed assay and the routine method for both heparin-spiked and patient samples were found to be close to linear. However, the slope values were significantly different, even though both types of sample were tested using exactly the same protocols. The laboratory method was seen to be far more sensitive to the spiked samples than the fluorescence method. However, the new test was far more sensitive to real patient samples as was also demonstrated in Table 6.4.

The use of heparin-spiked samples for the purpose of the aPTT reference and therapeutic range establishment has been shown to be misleading [21 ,22]. It has previously been shown that calibrations based on in vitro heparin addition to pooled plasmas yield a much lower aPTT therapeutic range in comparison to calibrations based on heparin-treated patient samples [23]. Therefore, the final device calibration ought to be performed using reference samples obtained from patients on heparin therapy. Example 4 - Clotting Time (CT) Monitoring on Alternative Substrate with Microprojections

The method of Example 1 was utilised on an alternative microfluidic structured platform

This microfluidic structured platform was fabricated from glass using conventional photolithography and chemical etching. The device consisted of 2-mm-wide channels and each channel contained numerous ellipse-shaped micropillars (Fig. 21). The major diameter of these pillars was 50 μηη and the minor diameter was 30 μηη with the between-pillar spacing of 50 μηη.

The fluorescence dye redistribution during clotting of a plasma sample supplemented with fluorescently-labelled fibrinogen was monitored using fluorescence microscopy on aPTT-coated chips for 710 s. Representative images captured at 100, 280 and 500 s are shown in Fig. 22.

Results

The monitored area was initially dark with evenly distributed fluorescence signal (Fig. 22a). The presence of brightly fluorescing formations could be observed in Fig. 22b. Following that an increase in signal intensity was observed but the signal was mostly confined around the micropillars (Fig. 22c).

Despite the differences in the platform design, micropillar shape, channel and micropillars dimensions between this platform and the previously used 4Castchip^®, a similar trend of the fluorescence signal redistribution as an effect of clotting was observed.

In order to qualitatively determine the onset of clotting images were analysed by calculation of a change in mean fluorescence intensity with time and using SD calculation software (Fig. 23).

The clotting sample initially showed some short term increase in mean fluorescence intensity. At some point, however, there was an actual decrease in fluorescence intensity, followed once again by a gradual rise. It was not possible to correlate the changes in fluorescence intensity with the clotting process and indicate the onset of clotting on the basis of this result. Thus, SD of the fluorescence intensity was calculated. For the first 250 s only small changes in SD were observed. At this stage the signal was uniformly distributed within the monitored area (Fig. 22a). After this period, an increase in SD was observed. This period was associated with the redistribution of the fluorescent label (Fig. 22b) and which, as previously suggested, corresponds to the onset of clotting. A continuous increase in the SD started at around 350 s. Similarly to the previously tested platform, adsorption of the label onto the circle around pillars from the adjacent areas and the dye accumulation resulting in an increased localised signal was observed, which was most likely an indicator of the onset of evaporation (Fig. 22c). The label accumulation and signal enhancement in the circles around the pillars and decreased intensity in the areas between pillars caused significant change in SD, which could be correlated with the appearance of a high peak in SD vs. time profile at approx. 550 s in Fig. 23. Eventually, evaporation seemed to affect areas adjacent to pillar as well, which could be observed in a gradually decreasing SD signal. As all liquid evaporated, the fluorescence signal is quenched and decreased. It has been shown that the approach based on the redistribution of the fluorescently-labelled fibrinogen during clotting of an activated plasma sample can be successfully utilized to clot formation localization and the determination of the clotting initiation not only on the 4Castchip^® COP device but also using various alternative platforms comprising micropillar/projections, Example 5 - Prothrombin time (PT)

PT assay was developed using the fluorescence-based clot localisation method.

Platform: 5 μΙ_ of PT reagent, Technoplastin HIS^® reagent (Technoclone) containing standardised Ca-thromboplastin from rabbit brain supplemented with 0.1 % Triton X-100 (v/v) was drop-cast deposited from the centre of the channel and left to dry for 1 h.

Alternative PT reagent, Recombiplastin (HemosIL, IL) as well as a range of different amounts of Technoplastin HIS^® reagent were tested and did not allow CT determination, therefore exactly 5 μΙ_ Technoplastin HIS^® reagent is recommended for use.

Testing: 10 μΙ_ of control plasma (Helena Biosciences) was supplemented with 1.5 μΙ_ fluorescently-labelled fibrinogen (Alexa Fluor 488, Invitrogen) and recalcified externally with 10 μΙ_ of 25 mM CaCI₂ (Stago Diagnostica). Even thought the PT reagent contained Ca ions, these did not bring about necessary sample recalcification when used in a dried form, therefore external recalcification was necessary. Measurement: Images were taken every 1 s for 200 s using the Olympus fluorescence microscope with an attached camera and an environment chamber. All experiments were carried out at 37°C. Frames were converted to .avi files and further analysed using LabView software for calculation of the SD change in time. Lag times (LTs) of the retrieved profiles were referred to as clotting times (CTs).

Results:

Example profiles retrieved from the LabView software for normal clotting plasma control are shown in Fig. 24 and an analysis in Excel that allowed CT determination is shown in Fig. 25. First significant increase in SD value indicated the onset of clotting (relocation of the fluorescence label), as confirmed visually.

Fig. 25 is an Excel analysis of three normal clotting plasma samples. Filled symbols indicate the CTs. The initial phase of the profiles was very noisy; this is due to significant amount of unbound and clumped fluorescence label travelling in the front of the liquid, which should be neglected. The label clumping was not noted in aPTT measurements and could be due to the age of the fluorescently-labelled fibrinogen solution used. This could be possibly eliminated if a new bottle was used. However, regardless of this initial noise, CTs could be easily determined. Fig. 26 shows images illustrating sequence of events during plasma clotting in PT-coated channel; (a) surface background, (b) front of the liquid carrying the clumps of unbound labelled fibrinogen, (c) lag phase with uniformly distributed label, (d) clotting and (e) evaporation.

Images in Fig. 26 illustrate the sequence of event during sample clotting in PT-coated channel and allow understanding of the changes in SD versus time profiles in Figs. 24 and 25. Fig. 26a shows the channel measured before the sample covers the monitored area. It can be observed that the background signal from the coated surface is very low. Once the sample fills the channel, the front of the liquid quickly covers the monitored area carrying the big formations/clumps of the unbound fluorescently-labelled fibrinogen (Fig. 26b). Appearance of these formations causes occurrence of noise in the initial parts of the SD versus time profiles as shown in Fig. 25. Once these move further down the channel, image becomes more uniform again with evenly distributed dye, Fig. 26c, which correlates to the lag phase before clotting. The onset of clotting can be easily determined visually, by observing the formation of typical clot structures and fluorescence redistribution in Fig. 26d, which appears as a sharp increase in SD value in Fig. 25. With prolonged monitoring, evaporation could be observed with characteristic accumulation of the signal around the micropillars and decrease in the signal in between, Fig. 26e. CALIBRATION

The assay was calibrated using AMAX Accuclot INR calibrator set (A6718). Assigned INR values were dependent on the reagent and the analyser used. The highest and the lowest INR range were correlated against the obtained PT values (Table 1 and Fig. 27).

Table 1. PT values obtained using the developed system for calibration plasmas with assigned INR values of the highest and the lowest range (n=4).

INR (high) 1.06 1.8 3.11 4.19

INR (low) 0.98 1 .67 2.87 3.87

PT [s] 20.67 28.67 41.80 50.20

SD 1.21 3.51 4.09 3.63

CV [%] 5.86 12.25 9.78 7.24

Additionally alternative PT formulation was tested: TriniCLOT PT HFT (T1 101 , Trinity Biotech). Normal plasma PTs were 62 s and 50 s achieved on platforms coated with 10 and 20 μΙ_ of PT reagent, respectively. PT for INR 1.6-1.8 plasma samples was at around 100 s and no clotting was observed with samples of higher INR. There was an issue with the reagent stability in a dried format. Nevertheless characteristic fluorescence redistribution was observed so the measurement of PT would be possible if the reagent deterioration was reduced by i.e. alternative reagent deposition method or optimised drying conditions. Example 6 - Activated clotting time (ACT)

Platform: A "cocktail" of surface activators: kaolin, celite and glass beads was reconstituted in MAX- ACT tube in water. 10 μΙ_ of this solution was supplemented with 0.1 % Triton X-100 (v/v) and deposited onto the chip. Chips were dried at RT for 1 h or over night.

Testing: 10 μΙ_ of citrated whole blood was supplemented with 1.5 μΙ_ fluorescently-labelled fibrinogen and externally recalcified with 25 mM CaCb.

Measurement: as previously, images taken every 2 sec.

Results:

The presence of large particles like glass beads on the surface caused accumulation of fluorescent- labelled fibrinogen. This sticky protein appeared to stay attached to these particles even before the clotting started, as illustrated in Fig. 28. These areas should be neglected. Alternatively soluble ellagic acid could be used to induce clotting, however the concentration that would cause clotting has not been optimised.

PLATELET POOR PLASMA

Clots induced in platelet poor plasma with surface activators were weak, thin and hardly visible in the areas in between the micropillars in Fig. 29. Initial attempts to visualise clotting in plasma sample confirmed that the use of platelet poor plasma is not appropriate for ACT measurement.

WHOLE BLOOD

Clear and defined label redistribution was observed during clotting of PT-activated whole blood. Figs. 30 and 31 illustrate the SD versus time profiles obtained for normal clotting whole blood, 1 and 100 U/mL heparin-spiked whole blood. As expected the normal clotting (non-heparinised) samples yielded sharp, easy readable profiles with rapid fluorescence label relocalisation and clearly defined CT (Fig. 30). Addition of 1 U/mL heparin caused prolongation in the LT (Fig. 31). Further heparinisation would cause further prolongation in a LT, which would probably overlap with the part of the profile highly influenced by evaporation.

Following CT values were obtained:

• normal whole blood CT = 164 ± 15 s (n=4)

· 1 U heparin whole blood CT = 257 ± 38 s (n=3)Strongly heparinised sample (100 U) was used as a negative control (n=2), no clotting was observed; an increase in SD was a result of evaporation.

The principle of the ACT measurement in the developed system has been proved, however, for testing of strongly heparinised samples (above 1 U/mL), the redesign of the platform would be required, i.e. a lid or some other method to prevent evaporation should be employed. Alternatively, higher volume of applied sample than 20 μί could reduce the effect of evaporation.

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[17] Quehenberger, P., Kapiotis, S., Handler, S., Ruzicka, K., Speiser, W. (1999). Evaluation of the automated coagulation analyzer SYSMEX CA 6000, Thrombosis Research, 96 (1 ): 65. [18] Shojania, A.M., Tetreault, J., Turnbull, G. (1988). The variations between heparin sensitivity of different lots of activated Partial Thromboplastin Time reagent produced by the same manufacturer, American Journal of Clinical Pathology, 89 (1 ): 19.

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Claims

A method for monitoring and measuring the onset of agglutination within a sample on a lateral flow assay device comprising micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path wherein the method comprises:

combining the sample with one or more detectable markers and one or more agglutination markers or a combination thereof to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path; and

monitoring the redistribution of the one or more agglutination markers through the lateral flow assay path as agglutination occurs by observing the en hancement of the detectable marker sig nal resulti ng from agglutination in areas around and/or between the micro-projections in contrast to the remaining areas and measuring the change in the standard deviation of the marker signal over time.

The method according to claim 1 wherein the detectable marker is selected from one or more of the fol lowing a fluorescent marker, chromophore, electrochemical marker, radionuclide and a combination thereof.

The method according to any of the preceding claims wherein the agglutination marker is selected from one or more of the following fibrinogen, thrombin, an antibody, a protein, an enzyme, a carbohydrate, a lipid, any other suitable marker of agglutination in a biological fluid and a combination thereof.

The method according to any of the preceding claims further comprising the step of com bi n i ng the sample or labelled sample with one or more agglutination assay reagents selected from the following:

contact or surface activators, such as kaolin, celite, ellagic acid;

tissue factors, such as tissue thromboplastin;

phospholipids;

snake venoms;

fibrinogen and thrombin; and combinations thereof.

. The method according to any of the preceding claims wherein the onset of coagulation is measured.

. The method according to any of the preceding claims for use in a particle agglutination assay, such as a latex agglutination immunoassay, wherein the agglutination marker is an antibody or antigen coated latex bead preparation.

. The method according to any of the preceding claims wherein the detectable marker is a fluorescent marker and the redistribution of the marker in the lateral flow assay path is measured optically.

. The method according to any of the preceding claims wherein the detectable marker and agglutination marker or combination thereof are combined with the sample prior to use or are combined with the sample in-situ on the lateral flow assay device.

. The method according to any of the preceding claims wherein the lateral flow assay device comprises

a sample receiving zone; a mixing zone with an agglutination activation zone, wherein the detectable marker and agglutination marker or combination thereof are combined with the sample in the mixing zone; and an assay path zone.

0. The method according to claim 9 comprising coating the sample receiving zone and mixing zone with one or more agents to prevent the irreversible adherence of the agglutination marker to the sample receiving zone and mixing zone surface prior to addition of the sample.

1. The method according to any of the preceding claims wherein an agglutination assay reagent is deposited on the lateral flow assay path zone prior to or during use.

2. The method according to any of the preceding claims wherein the sample is any biological fluid, including whole blood, plasma, urine, cerebrospinal fluid, interstitial fluid, lymph fluid, saliva, sputum, sweat, tears and/or faecal matter, preferably a whole blood sample which is citrated prior to use and optionally recalcified prior to or upon contact with the lateral flow assay device.

13. The method according to any of the preceding claims for use in a blood clotting time (CT) assay, prothrombin time (PT) assay, activated clotting time (ACT) assay and activated partial thromboplastin time (aPPT) assay.

14. The method according to any of the preceding claims for measuring the onset of blood clot formation and determining the associated blood clotting times in a blood or plasma sample wherein the method comprises the following steps: combining a blood or plasma sample with at least one detectable marker and at least one agglutination marker of blood clotting or a combination thereof to form a labelled sample;

subjecting the labelled sample to lateral flow through the lateral flow assay path zone;

monitoring the formation of a blood clot in the sample and measuring the change in detectable marker signal standard deviation over time resulting from the localised build up of the detectable marker around and/or between the micro-projections..

15. The method of claim 14 wherein the detectable agglutination marker is a fluorescently labelled marker of blood clotting, preferably fluorescently labelled fibrinogen or thrombin.

16. The method according to claim 14 or 15 wherein the lateral flow assay path zone is provided with one or more agglutination assay reagents deposited thereon and the labelled sample reacts with the one or more deposited assay reagents during passage through the lateral flow assay path.

17. The method according to any of claims 14 to 16 to determine the effect of anti-coagulant drugs on clotting time comprising the additional step of pre- treating the blood or plasma sample with an anti-coagulant agent.

18. The method according to any of claims 14 to 17 wherein the detectable marker and agglutination marker of blood clotting or a combination thereof is combined with the blood or plasma sample prior to use or in-situ on the lateral flow assay device.

19. A lateral flow assay device for measuring agglutination, preferably coagulation, within a sample wherein the lateral flow assay device comprises micro-projections extending from at least part of the surface of the lateral flow assay device to define a lateral flow assay path characterised in that the lateral flow assay device comprises

a sample receiving zone;

a mixing zone with an agglutination activation zone, wherein the mixing zone comprises at least one deposited detectable marker, agglutination marker and/or agglutination assay reagent; and

a lateral flow assay path zone.

20. The lateral flow assay device according to claim 19 optionally comprising a recalcification zone for mixing the sample with calcium chloride, a buffering zone and/or a red blood cell removal zone.

21. Use of the lateral flow assay device according to claims 19 and 20 in a method for monitoring and measuring the agglutination, preferably coagulation, of a sample.

22. A kit for monitoring and measuring the agglutination, preferably coagulation, of a sample comprising the lateral flow assay device according to any of claims 19 to 20 and a detection means for measuring the increase in marker signal standard deviation in the assay path zone over time.