WO1996000395A1

WO1996000395A1 - Evaluation of blood coagulation activity

Info

Publication number: WO1996000395A1
Application number: PCT/US1995/008014
Authority: WO
Inventors: Karim Boulhimez
Original assignee: Chemtrak, Inc.
Priority date: 1994-06-23
Filing date: 1995-06-23
Publication date: 1996-01-04
Also published as: WO1996000390A1; FR2721714A1; AU2889795A; AU2909495A; FR2721714B1

Abstract

Methods and disposable device are disclosed for determining the clotting time of a blood plasma sample. The device has a sample region (59) and an activating region (62) for activating the clotting cascade of the plasma sample. The clotting time is determined by the migration distance of the fluid or clot distance from the sample region (59). The activation region (62) is impregnated with thromplastin to activate the clotting factors and a clotting region (64) is impregnated with excess fibrinogen for enhancing clot formation. Other reagents may also be provided as mobile or immobile reagents.

Description

EVALUATION OF BLOOD COAGULATION ACTIVITY

INTRODUCTION Technical Field

The field of this invention is diagnostics for blood coagulation activity.

Background Blood coagulation (clotting) activity means the capacity of certain blood and tissue factors to act in unison to form a polymerized, clogging substance called a fibrin clot. The formation of the fibrin clot requires a significant number of different factors in a reaction cascade involving enzymes, which activate proteins which may then in turn activate other proteins further down the chain. Ultimately, thro bin cleaves fibrinogen to form fibrin, which can then be cross-linked and involve other proteins and platelets to form the clot. There is substantial interest in determining the clotting activity of blood samples. For the most part, clotting activity is determined for monitoring patients receiving anticoagulant therapy, such as coumarin, fluindione, and warfarin. For these patients it is important to periodically determine the necessary dosage which will prevent blood clot formation (thrombosis) , while at the same time avoid breakthrough bleeding. The current methodology is called prothro bin time (PT) .

There are also other blood tests based on other indications for determining clotting time. For hemophiliacs, one is interested in their ability to form a clot after a treatment or for monitoring their status. In the case of genetic defects, their is a reduced level of a particular factor that is of interest. There are other situations where the time required to produce a clot may also be of diagnostic or therapeutic interest. Each of the presently available assays for coagulation activity related to prothrombin time is dependent upon the activity of one or more constituents in the blood involved with clotting. Most of the methodologies involve the use of thromboplastin (Factor III)or sometimes called tissular factor (^■■TF") , which binds coagulation Factor Vila. The complex between TF and FVIIa promotes blood clotting by proteolyzing coagulation Factors IX and X, as well as VII. As indicated above, thrombin cleaves fibrinogen and coagulation Factor Xllla stabilizes the fibrin clot by covalent cross-linking. A number of the reactions are calcium dependent.

There is also interest in determining the activated partial thromboplastin time (APTT) , where activation of clotting can be obtained with a wide variety of compositions unrelated to the natural factors associated with clotting. Because of the significant number of factors involved in the process, where the factors may be sensitive to various environmental effects, substantial care must be taken in developing methodologies and devices for determining coagulation time accurately. Conventionally, the methodology involves bringing together blood plasma with thromboplastin and calcium. Where thromboplastin is used as a liquid, it has a short shelf life. The reaction is carried out isothermally, conveniently at 37°C. The blood sample is drawn into a container to be treated, e.g., citrated, followed by centrifuging to separate out the red blood cells. Generally at least about 0.1 ml is used and a precise quantity, usually about 0.2 ml of the thromboplastin- calcium solution added to form the clot. The mixture is stirred until coagulation is observed. Quite evidently, the methodology requires substantial technical skill as well as care in insuring that the conditions are closely controlled. There is, therefore, a substantial need to provide alternative methodologies which are simpler to operate, do not require expensive equipment, can be performed by non-technical people, and provide for a reproducible result.

Relevant Literature

Various devices for measuring prothrombin time or other clotting endpoint include Yano et al., Thromb. Res. (1992) 66:583-90; Walenga et al. , Semin. Thromb. Hemost. (1983) 9:346-54; and U.S. Patent Nos. 5,204,525, 5,140,161; 5,164,598, 5,051,231, 4,756,884, and 4,640,896.

SUMMARY OF THE INVENTION Methods and disposable devices are provided for simple determination of clotting time. The device comprises a sample region for applying blood plasma, a bibulous first region, comprising stabilized thromboplastin and calcium at a non-acidic pH, followed by a bibulous second region comprising stabilized fibrinogen at a non-acidic pH. By applying the biological sample to the sample region, the biological sample, by itself or in conjunction with a buffer, is transported through capillary action into the first region where the coagulation activity process is initiated or activated and the sample is carried into the second region where coagulation occurs. The distance to clot formation in the second region is related to the coagulation activity of the blood. Desirably, a control strip is provided which provides for a standard distance for comparison.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of a device according to the subject invention;

FIG. 2 is a plan view of the support and strip portion of the subject device; FIGs. 3, 4 and 5 are cross sectional views of the device according to FIG. 1 at the compartments with three different types of compartments;

FIG. 6 is a plan view of an alternative embodiment of a device according to this invention;

FIG. 7 is a plan view of the support of the device of FIG. 6;

FIG. 8 is an alternative embodiment of the device providing for buffer release.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS In accordance with the subject invention, methods and disposable devices are provided which allow for rapid reproducible non-technical determination of coagulation time of a blood sample. These determinations include, but are not limited to, prothrombin time (P.T.), activated partial thromboplastin time (A. P. T. T.), thrombin clotting time (T. C. T.), assays for particular factors involved with clotting, and the like. The devices employ bibulous or porous elements, e.g. strips, rods, fibers, etc. , providing fluid transport through the elements by capillary action. Since strips are the most convenient for the devices, strips will be used as exemplary of other bibulous supports. In the direction of fluid flow, the device has a sample region, a bibulous region for initiating or activating endogenous agents involved with the coagulation process, followed by a region comprising fibrinogen for clot formation. In one embodiment, a transport region is provided for providing buffer to the sample region for transport of the sample from the sample region to the coagulation time determination region. Also, filtration means may be provided for removing red blood cells and/or platelets from the blood sample to provide plasma to the sample region.

The initiation or activation region will comprise stabilized lyophilized thromboplastin, which will be non- limiting in amount, conveniently with calcium, to provide for initiation and activation of the coagulation cascade. The amount of thromboplastin in the activation region is not critical, so long as it is above a minimum non- limiting amount. Furthermore, when the thromboplastin is diffusively bound to the porous strip, the thromboplastin will accumulate at the migration front, so as to have the thromboplastin concentrated at the migration front.

Conveniently, the bibulous support may be impregnated with a solution of thromboplastin, generally at a concentration in the range of 10 to 100 mg/ml, more usually in the range of 25 to 75 mg/ml, preferably about 50 mg/ml, desirably containing calcium at a concentration in the range of about 1 to 35 mM, preferably in the range of about 5 to 25 mM, at a pH of about 7 to 9, preferably about 7 to 8.5, more preferably about 7.3 to 8.5.

In addition, various'agents may be employed, which may serve in the solubilization of the thromboplastin, aiding transport of proteins, and the like. Particularly saccharides find use, such as glucose, fructose, trehalose, and the like. Generally, the amount of saccharide in the impregnating solution will be in from about 1 to 20 weight percent, more usually in the range of about 5 to 15 weight percent, preferably about 10 weight percent. Various buffers may be employed to maintain the pH, where the buffer should have minimal calcium binding capability. Desirably, nitrogen based buffers may be employed, such as TRIS, HEPES, and the like where the concentration of buffer will generally be in the range of about 1-200 mM, preferably about 20-60 mM. Also stabilizing agents may be employed in minor amount, usually less than about 0.1%, such as sodium azide, antibiotics, etc., antioxidants, such as -tocopherol at from about 0.1 to 0.5%, and the like.

The support is impregnated with the solution by any convenient means, most conveniently, soaking in the solution, generally at a reduced temperature, so as to minimize any degradation of the thromboplastin. One end of the strip may be introduced into the reagent solution and the migration front monitored to define the length of the zone. Usually, where soaking is involved, in order to insure saturation of the support, the soaking will be maintained for at least 6 hours, more usually at least about 12 hours, and usually not more than about 24 hours. The strip may then be dried at a temperature which does not result in significant degradation of thromboplastin activity, generally a temperature in the range of about 0 to 35°C, preferably at a temperature in the range of about 20 to 30°C. Depending on the temperature, pressure, and the like, the time for drying may vary from about 5 min. to about 12 h.

For assays other than PT, activators other than thromboplastin may find use. For example, for APTT, the activator may be kaolin, silica, diato aceous earth, ellagic acid, silica or glass particles, associated or impregnated with a phospholipid, such as cephalin, lecithin, soybean phospholipid extract, or the like. For TCT, one would use thrombin. For individual factors, one would use a combination of normal blood depleted for the factor(s) of interest and thromboplastin or another activator. Factors of particular interest include Factor VIIIc, Factor VII, etc. For hemophilia A, one monitors FVIII activity with an APTT activator. The amounts used for impregnation are not critical, so long as they are not rate limiting, and can be readily optimized.

If desired, the bibulous strip may be chemically activated so as to covalently bond an activator, e.g. thromboplastin. Various reactive groups may be present on the support, depending upon the nature of the support. The support may be activated with cyanogen bromide, chloroacetyl chloride, Ellman's reagent, p- maleimidobenzoyl chloride, where the thromboplastin may be modified with cysteine, or the like. Alternatively, non- covalent complexes may be formed, where non-inactivating anti-thromboplastin antibodies, or streptavidin molecules, where the thromboplastin is modified with biotin, etc. , are covalently bonded to the support. The particular manner in which the activator becomes bound to the support, either covalently or through a non-covalent complex, is not critical to this invention, so long as sufficient and non-limiting thromboplastin activity is reproducibly maintained on the support.

The next region comprises fibrinogen, where the amount of fibrinogen will be in substantial excess, so as not to be rate limiting and affect the sensitivity of the assay. Again, the bibulous support will be impregnated with the fibrinogen solution by any convenient means, e.g., soaking, as described above. To prevent overlap of the two reagent solutions, if desired, two different strips may be used or the migration front of the reagent solution may be monitored. The solution will generally have from about 5 to 15 g/L, more usually from about 7.5 to 10 g/L. The pH will generally be in the range of about 7.5 to 9.5, preferably from about 8 to 9, more preferably from about 8.4 to 8.8, particularly 8.6. The buffers which have been previously described may also be used here at analogous concentrations. Desirably, a small amount of a surfactant, more particularly a non-ionic detergent, is employed, for example, polyethyleneoxy detergents, e.g., Triton X-100. The surfactant is used in small amount, generally not exceeding 0.2 weight %, usually not exceeding 0.15% and generally in excess of about 0.05%. Other non-ionic surfactants may be employed, such as octylglucoside, and the like. The particular surfactant will be selected so as not to interfere with the coagulation process, where polyethylene glycol is found to precipitate fibrinogen.

A solubilizing agent is also added to the mixture, particularly a saccharide, such as described above. The amount of saccharide will generally be in the range of about 5 to 25 weight percent, more usually in the range of about 10 to 20 weight percent, preferably 15 weight percent. Any saccharide which is used will be selected not to precipitate the fibrinogen and be capable of inhibiting crystallization of the fibrinogen. Various supports may be used, which may be the same or different for the different regions. Convenient materials include cellulosic materials, such as paper, e.g., Whatman CHR1, nitrocellulose, etc.; plastic materials, such as polyethylenes, polypropylenes, etc., or other convenient material, which does not interfere with the coagulation process, provides for reasonable transport of the reactive agents, allows for long term storage, and the like. The pore size controls the rate of flow of the fluid along the strip. By employing strips having a graduated pore size, the rate of flow through the strip may be varied in the different regions. Desirably, the mean pore diameter will be in the range of about 0.45 to 150 μm, more usually in the range of about 15 to 150 μm, preferably in the range of about 15 to 100 μm. Generally, the clot initiation or activation strip will be from about 1 to 3 cm, more usually about 1.5 to 2.5 cm, preferably about 2 cm in length. The length of the activation strip will be related to the amount of thromboplastin present on the strip, the rate of flow through the strip, and the rate of activation of FVII. The optimum length can be determined experimentally, by varying the length with samples having known clotting factor times.

The thickness of the strips will generally be in the range of about 0.2 to 1.5 mm, more usually from about 0.5 to 1 mm. The width of the strips will generally be in the range of about 0.5 to 4 mm, more usually about 0.75 to 2 mm, preferably from about 1 to 2 mm.

The length of the coagulation region will be related to the strip porosity, width and thickness and can be any convenient length, so long as it provides for the necessary dynamic range for determining the clotting time. Usually, the coagulation region will be at least about 0.25 times the length of the activation region, more usually 0.5 times the length of the activation region and not more than about 7 times the length of the activation region.

One can provide for enhanced discrimination of the coagulation border, by adding various reagents to the plasma, to the buffer if present, or as mobilizable agents in the sample region or in the activation region. These reagents can take the form of various colorants, latex particles, microvesicles, such as liposomes, carbon particles, colloidal suspensions, e.g. polystyrene particles, latex particles, etc., or silica. Colorants, such as bilirubin, which colorants have protein affinity find particular use as well. The particles will generally be of a diameter in the range of about O.lμ to 50μ.

If desired, a filter can be used which removes red blood cells and/or platelets, so as to deliver plasma sample to the sample region. A wide variety of filters have been reported in the literature, such as U.S. Patent Nos. 5,423,989, 4,816,224, and 4,477,575. For the use of glass fibers, either by themselves or in combination with a membrane which provides for the removal of any red blood cells and/or platelets which escape the glass fiber filter, while avoiding lysis of the red blood cells, the glass fibers should be coated to prevent coagulation.

Where a buffer is used to dilute the sample before introducing the sample into the sample region and/or as an eluent for sample and reagent transport, isotonic saline as diluent may be used or a buffer which will generally have a pH as indicated above for the activation solution, having an analogous concentration of buffer, and such other reagents as may be appropriate, such as calcium ion, generally with a concentration in the range of about the range indicated for the activation solution, nonionic detergents, in from about 0.1 to 1.5%, border enhancers as indicated above, and the like. The buffer may also have an agent which complexes calcium prior to clotting, such as citrate at a concentration of about 0.5 to 5%.

In carrying out the assay, the biological sample is placed in the sample region. When blood, the blood sample, may have been subject to prior treatment to remove the red blood cells and platelets, such as centrifugation, filtration, or the like. Normally, the sample which is applied to the sample region will be a volume from about 5 to 100 μl, where the amount of sample will depend upon whether the sample has been diluted and/or diluent is also employed in the assay or the sample provides the only liquid for transport, the absorption volume of the bibulous strips, and the like. Where the sample is combined with buffer, either prior to or during the assay, the sample will generally be in the range of about 5 to 100 μl, while where the sample is the primary source of fluid, the sample will generally be in the range of about 5 to 50 μl, so that the sample will vary from about 5 to 100 μl.

There may be a single assay pathway or a plurality of pathways. For example, if one wishes to know whether the difference in clotting time between a sample and normal blood is related to a specific factor, one can have a number of pathways, where each of the pathways has a plasma extract depleted for a particular factor. In the pathway where the extract is depleted for the factor in which the blood sample is deficient, the clotting time will be longer, so that the distance along the pathway to the clot will be longer. In this way, with a single sample, one may simultaneously obtain information as to a number of aspects relevant to the patient. If desired, an internal standard may be provided where all the materials necessary for coagulation are available so that a distance is obtained, which may be related to a standard clotting time. For example, by having thrombin in the activation region with or without FXIIIa, the sample comprising the thrombin, upon entering the coagulation region will cause the fibrinogen to clot. Conveniently, one can employ normal blood, plasma, or a plasma extract in substantial excess, so that the blood coagulation time will not be affected by agents in the blood sample. Plasma extracts may include PPSB preparation, prothrombin (FII) , Stuart Factor, Antihemophiliac Factor B, Factor VII, etc. The reagents may be present in the sample region or activation region. For further understanding of the invention, the drawings will now be considered. All of the devices are predicated on single use and being disposable. Furthermore, the devices require very little technical competence for their use and provide for a visual determination of the clotting time, without instrumentation.

The devices depicted in Figures 1 to 5 provide for a control to provide a standardized clotting time, providing for two parallel pathways. A solid housing or platform 4 includes sample well 1. The bottom of sample well 1 has two orofices 2a and 2b in proximity to bibulous migration support strips 3a and 3b. The ends of the strips 3a and 3b are under the orofices 2a and 2b, respectively, which provides that the sample flows from the sample well into the strips 3a and 3b. A slide 5, having transparent window 5a, is attached to platform 4. The window is supplied with reference mark 6 and graduations 7 to evaluate the distance traveled by the fluid on the strips 3a and 3b.

The zone 8 of strip 3a is impregnated with the coagulation activating agents, thromboplastin and calcium, as well as any other appropriate reagents to solubilize the thromboplastin, intiate the coagulation cascade of the components in the sample and stabilize the storage unstable reagents. Zone 9 has fibrinogen, so that prothrombin activated in zone 8 will react with the fibrinogen causing a clot in relation to the clotting activity of the sample. The zone 10 of strip 3b is impregnated with the necessary factors for causing clotting of the fibrinogen in zone 11, e.g. normal plasma, PPSB or thrombin, while zone 11 has the necessary remaining reagents for clotting, e.g., fibrinogen. Conveniently, in zone 10 one may introduce normal blood or plasma in substantial excess to the factors in the blood sample, as well as solubilizing and stabilizing agents. Depending upon the amount of blood introduced in zone 10, the clotting activity or inhibitor in the sample may be overwhelmed so as to have little, if any, effect on the clotting time in the control. Alternatively, one may look to the combination of clotting times from the normal blood and sample blood, so that the distance to clotting observed from the control should be shorter than or equal to the distance to clotting observed with the sample. If desired, one may provide an inert composition which provides for an equalization of the viscosity of the control and the sample to provide for a more accurate comparison. The inert composition will be present in the sample pathway. Among the substances which can be used are water soluble substances, including proteins, such as albumin, sugars, such as dextran and saccharides, and the like. As depicted in Figures 1 to 5, one can employ a single well which has openings 2a corresponding to support 3a and opening 2b which corresponds to support 3b. Alternatively, one may divide the well by wall lc into two compartments la and lb, so that different media (e.g. control and sample) or different reagents may be added to the different compartments. If desired, a filter 16 may be placed over the openings 2a and 2b, which is covered by an absorbent membrane 14. The filter 16 serves to remove red blood cells and/or platelets which might interfere with the assay and feeds plasma to the strips 3a and 3b. Figures 6 and 7 depict an alternative embodiment, comprising a platform 170 having well 100. The well has a single opening 120 in proximity to the strip 110. A slide 172 having a clear window 173 is attached to the support 170 with a graduation scale 174 to evaluate the distance covered by the sample. Zone 130 extends a significant distance away from the sample well 100 and activation zone 160 and is impregnated with fibrinogen. Zone 140 extends away from the sample well 100 in the opposite direction of zone 130 and is also impregnated with fibrinogen.

Activation zone 160 of the strip may be impregnated with thromboplastin and calcium and an inert composition which dissolves with the sample, adjusting the viscosity such that in coming out of zone 160 the fluid is substantially identical in viscosity to the fluid coming out of zone 150. One can determine the amount of the inert composition which is necessary, by adding to one portion of a plasma sample, the reagents and to the other the normal blood and the inert composition and adjusting the amount of inert composition until the two samples have the same viscosity. The inert composition components have been previously described. Zone 150 has a a source of clotting factors to act as the control, such as PPSB and a clotting activation agent (thromboplastin and calcium) , which may be separately placed in zone 150 or other technique used to prevent reaction, while zone 140 is impregnated with the clot formation reagents (fibrinogen and optionally FXIIIa) .

Zones 180 and 190 indicate the presence of clots, so that the distances can be compared for the control and sample, using slide 172 to measure.

The length of zone 140 will be shorter than the length of zone 130, since the sample is expected to have a slower coagulating time than the control.

The devices may be prepared out of any convenient material which is stable, inert, and may be readily handled. The device may be prepared from glass fiber, plastic, treated cardboard or paper, or the like, so long as the material provides for the necessary physical parameters. The particular shape and the dimensions of the material are not critical to this invention, so long as they come within the indicated parameters to provide an observable result.

By appropriate arranging of the calibrated openings in the sample well and the fluid transfer relationships between the openings and bibulous supports, one obtains a constant flow of fluid out of the well to the bibulous supports.

In Fig. 8 a device is depicted which is shown in greater detail in U.S. Patent No. 5,132,086, which disclosure is incorporated herein by reference. Particularly Figs. 2, 3a, 3b and 4 and their decription are incorporated as part of this disclosure. The parts of the device comprise a base plate 40, a slide 42 and a cover plate (not shown) . The base plate consists of a cutout to accept the slide 42, a slot 46 with locating pins 48 into which the assay strip 50 and transport strip 52 are precisely positioned, maintaining about a 2mm gap 54 between them, and a well 56 designed to capture the released transport solution, e.g. wicking buffer. The slide 42 consists of a vented receptor site 58 into which the sample receiving pad 59 is inserted, an arm 60 with shearing action designed to facilitate the release of the transport solution from a pouch which is housed in a well of the cover plate, and a snap 61 to lock the slide in place, once pulled. The strip 50 has two regions, a clotting activation region 62 and a clotting region 64. The clotting activation region 62 is impregnated with thromboplastin and calcium ion, while the clotting region 64 is impregnated with fibrinogen. Initially the slide is at the position indicated in Fig. 8, where the sample receiving pad 59 is out of register with strips 50 and 52. After the sample has been added to the sample receiving pad 59, the slide 42 is pulled to the right to bring the sample receiving pad 59 in register with strips 50 and 52. A ridge in the cover plate squeezes the sample pad to control the volume of the sample in the sample pad. Movement of the slide opens the buffer containing pouch in well 56, which initiates the assay. The buffer is transported to sample receiving pad 59 transporting the sample into activation region 62. The factors associated with clotting in the sample, particularly prothrombin to thrombin, are activated. Movement of the buffer carries the activated factors into clotting region 64, where depending upon the components of the sample controlling clotting, a clot will form some distance into the clotting region. This distance may then be related to prothrombin time. If desired, a second control strip may also be provided, where the sample receiving pad may or may not be involved. The nature of control strips has already been discussed.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Example 1. Polyethylene (Interflo®: Mean Pore Diameter 40μm) of a size of 150 mm x 150 mm was dipped in a thromboplastin-calcium solution (thromboplastin, 50 mg/ml; calcium 20 mM) at one end and the solution allowed to rise until the migration front reached 75% of the height of the polyethylene piece. The piece was then dried at 30°C in vacuo . The piece was then cut into strips of 150 mm x 1.5 mm. These provide the sample strips. For a control strip, in the unimpregnated portion of the strip, 5 μl of a preparation of normal human plasma in a 5% sucrose solution containing sodium deoxycholate (0.01 - 0.04%) and sodium azide (0.4 g/L) was added. A plastic support coated with adhesive is employed. One then fixes the sample strip and control strip by means of the adhesive to the support, so that the two strips are parallel. A sample well is then affixed to the ends of the two strips at the un-impregnated end of the sample strip, but in fluid transfer relationship with both strips.

Water tightness is achieved at the level of the openings in the base by heat welding at 130°C.

Example 2. Following the protocol of Example 1, the reagents are deposited on the strips and lyophilized by dipping the plastic in liquid nitrogen and dehydrating in vacuo .

Example 3. Following the procedure of Example 1, an aliquot of total blood is deposited into the sample well, whereby it migrates along the two strips. The migration front is readily visible in fewer than 5 minutes due to natural coloration of the blood in contrast to the support base. The red blood cells provide for coloration of the fibrin clot.

Example 4. Following the procedure of Example 1, a specific quantity of citrated plasma is mixed with a specific quantity of carbon particles (10v/2v) colloidal solution. A known quantity of the mixture is placed in the sample well, and the sample allowed to migrate along the two strips, where the border is readily detectable due to the carbon particles imprisoned in the fibrin clot. The test can be read in fewer than 5 minutes after addition of the sample.

Example 5. Strips were prepared as follows. Thromboplastin (lyophilized) is regeneratd with 3ml of Tris* HC1 buffer (100 mM, CaCl₂20 mM, glucose 10%, pH 8.4). The strip (porous plastic of pore diameter 25 to 35μ, 0.6 mm thickness),a length of 2 cm, is impregnated with the solution. A second strip of the same material as the first strip is impregnated with a fibrinogen reagent solution comprising human fibrinogen (8.8 g,lL), glucose (15 wt. %) , in Tris* HC1 buffer (100 mM, pH 8.6, Triton X-100, 0.09%).

A temperature response study was performed at 37°C, 23°C and 15°C. At both 37°C and 23°C, the distance was shown to correlate with prothrombin time of the samples over a range of prothrombin times of 100% to 20%. It is evident from the above results, that the subject invention provides for a simple, convenient and rapid method for the determination of clotting time. Disposable devices are provided which may be used without technical competence, simply by adding blood to a device. The device then performs all of the necessary actions and when the assay is completed, the distance may be readily determined and correlated with prothrombin time. Since the migration distance is fixed, one need not observe the assay, but can read the result at a later time. In this way, within a short time, the migration distance can be determined and related to prothrombin time.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED:

1. A disposable device for determining coagulation activity of a blood sample, said device comprising:

a solid support;

a sample assay pathway supported by said support with each component of said pathway in fluid receiving and/or transferring relationship with adjacent components, said assay pathway comprising:

a sample region;

a coagulation activation region comprising a first porous element impregnated with a clotting cascade initiating agent; and

a clotting region comprising a second porous element impregnated with fibrinogen;

wherein the distance traveled of a blood sample introduced into said sample zone is related to said coagulation activity,

2. A disposable device according to Claim 1, further comprising a control assay pathway parallel or in tandem to said sample assay pathway comprising prior to said clotting region one of normal stabilized blood, or thrombin.

3. A disposable device according to Claim 2, wherein said sample region is common to said sample assay pathway and said control assay pathway.

4. A disposable device according to Claim 1, further comprising a filtration device over said sample region capable of removing red blood cells from blood.

5. A disposable device according to Claim 1, wherein said clotting cascade initiating reagent is thromboplastin and calcium ion.

6. A disposable device according to Claim 5, wherein said clotting cascade initiating reagent further comprises a saccharide for stabilizing said thromboplastin.

7. A disposable device according to Claim 5, wherein the protein component of said thromboplastin is covalently bonded to said first porous element.

8. A disposable device for determining coagulation activity of a blood sample, said device comprising:

a solid support;

a sample assay pathway and a control pathway supported by said support with each component of said pathway in fluid receiving and/or transferring relationship with adjacent components, said assay pathway comprising:

a sample region;

a coagulation activation region comprising a first porous strip impregnated with thromboplastin and calcium; and

a clotting region comprising a second porous strip impregnated with fibrinogen;

said control pathway comprising;

a sample region;

parallel or in tandem to said sample assay pathway, comprising prior to a clotting region a third porous strip, one of normal stabilized plasma, an inert composition for modifying the viscosity of a blood sample in said control assay pathway to be equivalent to the blood sample in said sample assay pathway, or thrombin; and

said clotting region comprising a fourth porous strip impregnated with fibrinogen;

wherein the distance traveled of a blood sample introduced into said sample zone in said sample assay pathway as compared to in said control pathway is related to said coagulation activity,

9. A device according to Claim 8, wherein said sample region is common to said sample assay pathway and said control pathway.

10. A device according to Claim 8, wherein said first, second, and fourth strips comprise saccharide in a protein stabilizing amount.

11. A device according to Claim 8, wherein said device comprises means for moving said sample region out of register with said pathways and into register with said pathways.

12. A device according to Claim 11, wherein said device further comprises a buffer source and transport means for transporting buffer from said buffer source to said sample region, and said moving means further comprise buffer releasing means.

13. A device according to Claim 8, further comprising a graduated window over said pathways for measuring the distance traveled by said sample.

14. A device according to Claim 13, wherein said sample region and/or said first and third porous strips comprise a marker for enhancing visualization of the distance traveled by said sample.