WO1996000390A1

WO1996000390A1 - Method and disposable device for determining blood clotting activity

Info

Publication number: WO1996000390A1
Application number: PCT/FR1995/000829
Authority: WO
Inventors: Karim Boulhimez
Original assignee: Karim Boulhimez
Priority date: 1994-06-23
Filing date: 1995-06-22
Publication date: 1996-01-04
Also published as: WO1996000395A1; FR2721714B1; FR2721714A1; AU2889795A; AU2909495A

Abstract

A fully disposable single-use device for determining the blood clotting activity of a biological sample, consisting of a solid holder (4) with a well (1) large enough to hold the sample being tested. Two openings (2a, 2b) at the bottom of the well are in close contact with respective migration substrates (3a, 3b) through which a fluid can flow over a distance corresponding to the viscosity of the fluid. A slide (5) which has a window or is entirely transparent is slidable along the holder (4) and has a reference mark (6) and a scale (7) for determining the distance covered by the fluid on the migration substrates (3a, 3b). Methods for using said device to analyse the blood clotting activity of biological samples are also disclosed.

Description

DISPOSABLE METHOD AND DEVICE FOR EVALUATING BLOOD COAGULATING ACTIVITY

The present invention relates to a device for evaluating the blood coagulant activity of a biological sample, disposable after single use, and the methods of using this device. The term “blood coagulant activity” is understood to mean the ability of certain blood and tissue factors to act in concert in order to form a polymerized and clogging substance called a fibrin clot.

Assessments of blood clotting activity are generally done by measuring the time required for the formation of a fibrin clot when a dose of blood or plasma is put under specific conditions.

The most common evaluation is the one called prothrombin time (TP), also called prothrombin rate or Quick time (TQ.), The main use of which is to monitor patients receiving an anticoagulant medication such as acenocoumarol, fluindione, warfarin. In these patients, it is important to periodically check that the dose administered is sufficient to prevent recurrent clots (thrombosis) but also not too high to prevent phenomena of spontaneous bleeding. The prothrombin time provides information allowing better control of the dose of anticoagulant administered.

Other evaluations of blood clotting activity exist such as for example the activated partial thromboplastin time (TCA), the fibrinogen assay, the thrombin time (TT), the thrombotest of OWREN.

Each of these assessments of blood clotting activity • accounts for the contribution of one or more factors to blood clotting activity. With regard to the prothrombin level, for example, this accounts for the contribution to the coagulant activity of factors whose synthesis is inhibited by anticoagulants of the warfarin type.

Several solutions have been proposed for measuring the time of clot formation when determining the prothrombin time. All have in common the use of thromboplastin as a reagent that triggers the formation of a clot. The differences between the methods lie in the means used to detect the formation of a fibrin clot. The differences also relate to the instrumentation used and the nature of the reagent used.

Conventionally, the prothrombin time (TP) is carried out by bringing a dose of plasma into contact with a dose of liquid calcium thromboplastin. Liquid thromboplastin, which can only be stored for a few hours to a few days, is reconstituted before use by adding an aqueous solution to lyophilized thromboplastin. The evaluation of prothrombin time (PT), like all other evaluations of blood coagulant activity, is done at a constant temperature of 37 ° Celsius. The blood sample is taken from an anticoagulant that captures the calcium in the sample (citrate). Then the sample is centrifuged to separate the red blood cells from the plasma. Then, a non-negligible quantity of plasma (generally 0.1 ml) is pipetted, which is deposited in a cup placed at 37 ° Celsius and a precise quantity (generally 0.2 ml) is added. of reagent triggering the formation of the clot, while ensuring by agitation or by another process, the mixture between the plasma and the reagent triggering coagulation.

Conventional methods suffer from at least one of the following disadvantages: the test must be performed by trained personnel; the reagents used are not stable for a long time once regenerated; there are difficulties in standardizing methods well; the methods require the use of an electric current source, a thermostat or a temperature measurement, a stopwatch, a magnetic field, a photometer.

The object of the invention is to propose a simple and easy-to-use method for evaluating one or more factors contributing to blood coagulant activity.

Another object of the invention is to propose a method which does not require the preparation of the solution containing the reagent triggering the blood coagulant activity. Another object of the invention is to propose a method minimizing the problems of instability of the reagents necessary for the evaluation of blood coagulant activity.

Another object of the invention is to propose a method which allows an intimate mixture of the sample to be evaluated and of the reagents necessary for the evaluation of the blood coagulant activity.

Another object of the invention is to provide a device for measuring the blood coagulant activity on whole blood without the need to separate the plasma or serum from the red blood cells. Another object of the invention is to provide a device allowing the measurement of blood coagulant activity without the need to thermostate the device or to perform a temperature measurement.

Another object of the invention is to provide a device allowing the measurement of blood coagulant activity without the need to use a source of electric current.

Another object of the invention is to provide a device allowing the measurement of the coagulating activity without the need to use a photometer. Another object of the invention is to provide a device for measuring blood coagulant activity and which is entirely disposable after single use. These and other objectives described below have been achieved by the production of devices and methods, the characteristics of which follow. According to the subject of the invention, the device comprises an element which contains all the factors contributing to the blood coagulant activity that the test explores, this element is the witness of a normal • blood coagulant activity, that is to say without a deficiency of factors that are explored by the test, and another item used to assess the factor (s) contributing to blood clotting activity in the test sample.

The device according to the invention, for evaluating the coagulating activity of a biological sample, is essentially characterized by the fact that it is entirely disposable after single use and that it consists of a solid support comprising: a well having a sufficient volume to receive the test sample, at the bottom of which are formed two openings in intimate contact respectively with a test migration support and a control migration support, which allow the flow of a fluid within them on a distance which is a function of the viscosity of the fluid; a cursor, with window or entirely transparent, sliding on the support provided with a reference and a graduation to evaluate the distance traveled by the fluid on the migration supports.

The test migration support of the device of the invention comprises at least one zone for impregnating a reagent which triggers coagulation.

The control migration support comprises an area impregnated with factors necessary for blood clotting activity and an area impregnated with a reagent which triggers coagulation. The test migration support can additionally comprise a zone for impregnating a substance inert with respect to blood coagulant activity, which in contact with the sample, adjusts the viscosity of the fluid so that at the exit from this zone, it is identical to the viscosity of the fluid at the exit from the zone of the control support impregnated with factors necessary for the coagulating activity.

According to another embodiment of the invention, before or after the area of impregnation of a reagent triggering the coagulation of the test support, and before or after the area impregnated with a reagent triggering the coagulation of the control support, is provided an area impregnated with a substrate of coagulating activity. Mention may be made, as substrate of the coagulating activity, of fibrinogen, of human or animal origin.

By coagulation-triggering reagent is meant the factor (s) not belonging to those which are explored by the test but which are necessary for the coagulating activity which the test explores. The composition of the reagent is thus different depending on the factor explored.

By factors necessary for coagulation, is meant the factor (s) which alone suffices to produce a coagulating activity in the presence of the reagent which triggers coagulation. The invention will be further illustrated in more detail by the following description in the light of Figures 1 to 7 in the appendix.

Figure 1 is a top view of a typical device for measuring blood coagulant activity according to the invention.

Figure 2 is a top view of a measurement device as shown in Figure 1, showing the dotted outline, where the movable cursor present in Figure 1 has been removed.

FIG. 3 is a sectional view of a measuring device as shown in FIG. 2.

Figure 4 is a sectional view of a second typical device for measuring blood coagulant activity according to the invention.

Figure 5 is a sectional view of a third device for measuring blood coagulant activity according to the invention.

Figure 6 is a top view of a bidirectional measuring device according to the invention. Figure 7 is a top view of a bidirectional measuring device as shown in Figure 6 where the cursor present in Figure 6 has been removed.

Figure 8 is a top view of another embodiment of the device for measuring blood coagulant activity according to the invention in which the cursor, similar to that of fig 1, has been removed.

According to a particular embodiment of the invention, the device can comprise a well 1 divided into two compartments la and lb at the bottom of which there is respectively the opening 2a opposite the migration support 3a and the opening 2b in look at the migration support 3b. According to another embodiment, the device of the invention can be lined with an absorbent buffer membrane 14 over which a removable filter 16 is affixed.

Another form of device according to the invention and illustrated in FIGS. 6 and 7 consists of a support 170 comprising a well 100, capable of receiving the sample to be tested, at the bottom of which is formed an opening 120 in intimate contact with a migration support 110 in two parts arranged on either side of the well 100, allowing the flow of a fluid over each portion of the migration support 110 over a distance which is a function of the viscosity of the fluid; a cursor 172 with window or entirely transparent, sliding on the support 170 provided with a reference 192 and a graduation 174 to evaluate the distances traveled by the fluid on each portion of the migration support 110. Part of the migration support 110 comprises at least one zone 130 impregnated with a reagent which triggers coagulation and optionally a zone 160 for impregnating a substance inert with regard to blood coagulant activity which, in contact with the sample, adjusts the viscosity of the fluid so that, at the exit from this zone 160, it is identical to that of the fluid at the exit from the zone 150. The other part of the migration support 110 comprises an area 150 for impregnating factors necessary for blood coagulant activity and an area 140 for impregnating a reagent which triggers coagulation. Among the substances which can be used in zone 8 of the migration support 3a or in zone 160 of the migration support 110 of one of the devices defined above, mention may, for example, be made of protein substances such as albumin; dextran, polyethylene glycols; sucrose. The migration supports 3a, 3b and 110 are in the form of strips, rods or tubes. The supports in the form of strips are particularly preferred.

If we refer to Figures 1, 2 and 3, we see a well 1 at the bottom of which are formed two openings 2a and 2b; the opening 2a is located opposite a migration support 3a and the opening 2b is found next to a migration medium 3b. The assembly is arranged on a support 4; a cursor 5 with window or entirely transparent making it possible to see the migration supports, provided with a reference 6 and a graduation 7, slides on the support 4. The well 1 has a sufficient volume to receive the sample to be analyzed and the surface surrounding the opening may be curved and extend downward. The openings 2a and 2b are crimped respectively in the migration supports 3a and 3b in order to ensure sealing during the flow of the fluid. The migration support 3a is impregnated in the zone 9 with a reagent which triggers coagulation and can be impregnated in a zone 8 with a substance inert with respect to blood coagulant activity as indicated above. The migration support 3b is impregnated in zone 10 with factors necessary for blood coagulant activity and in zone 11 with a reagent which triggers coagulation.

In this embodiment, a blood sample for example, is deposited in the well 1 where it flows through the openings 2a and 2b. Through the opening 2a, the sample can mix intimately with the substance that may be present in zone 8 and then migrates to zone 9 where it mixes with the reagent triggering the blood coagulant activity.

In zone 9, a fibrin clot is formed, which has the effect of stopping the migration of the fluid in the migration support 3 all the more quickly the clot has formed. The migration distance is reliable with the coagulating activity of the sample. Through the opening 2b, the sample mixes intimately with the coagulation factors of the zone 10, which makes it possible to have, at the output of zone 10, a fluid having normal coagulating activity; that is to say a coagulating activity which corresponds to that of a biological sample where there is no deficit in factor explored by the test. This activity serves as an internal reference for the device according to the invention. At the exit from zone 10, the fluid meets the substance present in zone 11; a fibrin clot is formed, which causes the migration of the fluid present in zone 11 to stop. When the migration of fluids has stopped in zones 9 and 11, the cursor 5 is moved so as to bring the reference 6 to the end of the migration front of the fluid present in the migration support 3b, then read on the graduation 7 facing the migration front of the fluid present in the migration support 3a, the • value of the blood coagulant activity corresponding to the biological sample tested. In the embodiment shown in Figure 4, we see a well 1 divided into two compartments la and lb at the bottom of which there are respectively the opening 2a opposite the migration support 3a and the opening 2b opposite the migration support 3b. Certain factors necessary for the correct evaluation of blood coagulant activity can be found in compartments la and lb and in the migration supports 3a and 3b. In this embodiment, which is a variant of that presented in FIG. 3, part of the sample to be analyzed is deposited in compartment 1a and another part of the sample in compartment 1b. Thanks to the two compartments, the samples meet substances necessary for the evaluation of the blood coagulant activity in the compartments la and lb and in the migration supports 3a and 3b, without there being any mixing between the fluid present in la and the fluid present in lb. In the embodiment of FIG. 5, the bottom of the well 1 is lined with an absorbent buffer membrane 14 over which is affixed a removable filter 16. The removable filter 16 selectively retains the red blood cells and lets the plasma or the sufficient serum to assess blood clotting activity. At the bottom of the well 1 are the openings 2a and 2b respectively opposite the migration supports 3a and 3b.

In this embodiment, an amount of blood is deposited in well 1 above the filter 16; a sufficient time is required for the red blood cells to be separated, then the filter 16 is removed and a determined quantity of solution containing a marker, such as for example colored particles in colloidal suspension, is added to well 1, above the buffer membrane 14. These particles are sufficiently fine to pass through the membrane 14 and migrate through the openings 2a and 2b in the migration supports 3a and 3b. The fluid-particle mixture meets the substances present in zones 8 and 10 then gains zones 9 and 11 where it meets the reagents triggering coagulation. When fibrin clots are formed in zones 9 and 11, the fluids stop migrating. Reading the migration fronts using cursor 5 is facilitated by the coloring of the fluids by the marker which is taken up in bulk by the fibrin clots.

In the embodiment presented in FIGS. 6 and 7, there is seen a support 170, made of a moisture-resistant material, such as plastic, on which is deposited on part of the surface, a migration support 110 in hydrophilic material, or rendered hydrophilic, having a volume and a porosity sufficient to ensure good migration of fluids. A well 100 capable of receiving the sample to be analyzed is connected to the migration support 110 through the opening 120. The migration support 110 is impregnated with several reagents: in an area 160, there may be an inert substance vis vis-à-vis the blood coagulant activity as indicated above; in zone 150, there are factors necessary for coagulating activity and of the same nature as those which one wants to evaluate in the sample to be tested. Zones 130 and 140 each contain a reagent that triggers blood clotting activity.

In this embodiment, a sample, for example, whole blood, is introduced directly into well 100; the sample enters through the opening 120 in the migration support 110 then migrates bidirectionally. The fluid stops migrating into zone 130 when the fibrin clot is formed. In zone 150, the sample mixes intimately with the substances present, then it migrates to zone 140 where it mixes with the reagent which triggers coagulation. The fluid stops migrating when the fibrin clot is formed.

To evaluate the coagulating activity in the sample to be tested, the reference 192 of the cursor 172 is moved, where a window is provided, facing the migration front of the fluid present in the area 140 and then the pattern is read. of zone 180 facing the fluid migration front and the reference 192 is transferred to the same pattern present in zone 190. Finally, we read on the graduation 174 the value of the activity of the sample to be tested. In the embodiment of FIG. 8, the migration supports 3a and 3b include zones 8, 10, 9 and 11 as in the ^• embodiments previously described, but the zones 9 and 11 are shortened to provide space in the migration supports zones 20 and 21 which are impregnated with fibrinogen. These zones 8, 9, 10, 11, 20 and

21 are in fact produced by separate support portions which are juxtaposed.

According to the present invention, the migration supports of the different types of evaluation devices are made of a certain number of materials which include, but are not limited to, cellulosic materials such as, for example, WHATMAN E T 31 paper, nitrocellulose; plastics such as polypropylene, polyethylenes such as, for example, high molecular weight polyethylenes, polysulfones, polyvinylidene fluorides, polyurethanes, nylons, melamine; mineral materials such as glass fibers. All these materials have an average pore diameter ranging from 0.45 to 130 μm and preferably from 20 to 60 μm. The porosity of the migration band can be controlled and it is well known in the prior art the methods to be used to obtain a porosity gradient from one end to the other of the support of porous material.

As regards the removable filter 16 intended to retain particles such as, for example, the red blood cells of the sample to be evaluated, it is made up of materials which include, but are not limited to, glass fibers, a mixture of glass fibers with plastics and / or cellulosic materials.

The well 1 capable of receiving the sample whose coagulant activity is to be evaluated, is preferably made of plastic material impermeable to liquids. Plastics are particularly useful and convenient to use, but paper treated with hydrophobic substances or metal can also be used.

The absorbent membrane 14 is made of a certain number of porous materials capable of absorbing a sufficient volume of sample after passing through the filter 16. The materials of which the membrane 14 is made up includes, but is not limited to, cellulosic materials such as for example WHATMAN CHR paper, • plastics such as for example high molecular weight polyethylenes, polypropylene, polyurethanes, melamine and have a mean pore diameter in the range of

0.45 to 130 μm.

The whole well assembly, removable filter, absorbent membrane and migration supports is fixed on a support made of solid material 4 or 170 which constitutes the support of the device and ensures its integrity. This support can be made from a sheet of glass, plastic or treated paper cardboard, which is cut to the appropriate dimensions, to hold the entire device for evaluating the coagulating activity, all by ensuring a certain ease of use for the user of the evaluation device. The slider 5 or 172 of the device is made of materials of the same kind as those of the support around which it slides.

As regards the general configuration, it is advantageous that the migration supports have a small thickness and a minimum dead volume, so that the sample volume used is small. The use of migration materials in the form of strips instead of rods or tubes is advantageous because it allows the material to be treated uniformly and then cut to the appropriate dimensions. By providing calibrated openings and making a sealed contact between the well and the migration support, a constant fluid flow is obtained at the outlet of the well in the migration supports. By working with porosity gradient migration supports, the pressure drop due to the tortuosity and friction which sit at the level of the migration supports is avoided and a constant fluid flow is thus ensured. By incorporating a red blood cell filter into the device, it is possible to evaluate the blood coagulant activity on plasma without for this centrifuging and decanting the sample; which is an advantage when the presence of red blood cells interferes with the factors of coagulating activity that we want to evaluate.

The fact of incorporating an internal standard into the device is one of the major advantages of the test and it is known in the prior art that a rigorous evaluation of blood coagulant activity must be made in relation to a standard. In addition, the fact of evaluating under the same conditions the blood coagulating activities of the patient and of the standard makes it possible to overcome the viscosity which can vary from one sample to another as well as the temperature which can vary from one evaluation to another. However, when thermoregulation of the device is provided, the witness support is not necessary.

The devices according to the invention are entirely disposable after single use.

With regard to the reagents incorporated in the migration materials, in all cases, these reagents are dried, lyophilized or bonded by covalent bonding on or in the materials of the test device so that they can be reconstituted following contact with a fluid. Other auxiliary reagents useful for promoting the formation of the fibrin clot and its detection and therefore for increasing the sensitivity of the test device, can also be added to the preceding reagents in the migration materials. These auxiliary reagents include, but are not limited to, buffers, detergents, anticoagulants, colorants, particles of an appropriate diameter to freely penetrate the pores of the migration material, proteins.

Among the proteins, there may be mentioned, in addition to fibrinogen, antibodies directed against the coagulant activity inhibitors, for example antibodies directed against protein C, antibodies directed against thrombomodulin, antibodies directed against protein S, antibodies to plasminogen, antibodies to the plasma tissue factor inhibitor (TFPI).

Certain auxiliary reagents can occupy either the bottom of the wells or the migration supports in the various devices according to the invention. The migration medium (s) may consist of support portions juxtaposed, glued or overlapping to ensure sample migration. The support portions can carry the reagent (s) chosen for the different zones that the samples pass through while migrating. The present invention can be used with both a whole blood sample and a blood sample diluted in isotonic solution or a plasma sample. When working on a plasma sample, it may be necessary to visualize the path of the fluids in the migration bands, in order to make the readings of the distances traveled easy for the user. For this, one can use a marker which accompanies the fluids up to the migration fronts.

This marker can be added beforehand to the plasma sample before depositing the latter in the sample well or this marker can impregnate part of the migration supports.

The markers used are preferably chosen from dyes, latex particles, dyes contained in micro vesicles such as, for example, liposomes, cajbon particles, the latter being in colloidal suspension, polystyrene or silica particles. It appeared advantageous to use carbon particles as a marker because in addition to the effect which they have on the detection of the path of the fluids, they arm the clot in the migration support and therefore favor its formation and its highlighting. When using an affine dye for proteins, it is advantageous to use a very affine marker for albumin such as, for example, bilirubin.

The following examples serve to illustrate the invention without, however, being limiting in nature. EXAMPLES Example 1

• Preparation of the device evaluating the prothrombin rate (TP)

A sheet of high molecular weight polyethylene of the INTERFLO® type is cut having an average pore diameter of 40 μm with dimensions of 150 mm × 150 mm. The plate is immersed in liquid calcium thromboplastin until the migration front reaches 75% of the height of the strips, it is dried at 30 ° C. under vacuum. The plate is then cut into strips of dimensions 150 mm x 1.5 mm. A strip of 150 mm x 1.5 mm is taken on which is deposited in the area not impregnated with thromboplastin, 5 μl of a preparation of human plasma not deficient in coagulation factors in 5% sucrose and containing sodium deoxycholate and sodium azide. This strip serves as a control strip in the device for evaluating the blood coagulant activity according to the invention. On a plastic sheet previously coated with 3M® type adhesive, a strip impregnated solely with calcium thromboplastin of 150 mm × 1.5 mm is deposited and in parallel a control strip as manufactured above. A sample well is deposited on the strips in the areas not impregnated with thromboplastin and the seal is provided at the orifices at the bottom of the well by heat sealing at 130 ° C.

Example 2

The same protocol is followed as in Example 1 except that the reagents deposited in the migration bands are lyophilized as follows.

After impregnating each reagent, the porous plastic is soaked in liquid nitrogen and then dehydrated under vacuum.

Example 3

The device is prepared according to Example 1. When an aliquot of whole blood is placed in the sample well; it migrates in the two bands. Migration stops are clearly visible in less than 5 minutes thanks to the natural coloring of the blood contrasting with the bottom of the support. In fact, the red blood cells are taken up en masse in the fibrin clot.

Example 4

The device is prepared according to Example 1. A determined quantity of citrate plasma is taken which is mixed with a determined quantity of a colloidal solution of carbon particle (10V / 2V). An amount of the mixture (plasma-particle solution) is deposited in the sample well. The migration of fluids is clearly visible thanks to the contrast provided by the carbon particles which are trapped in the fibrin clot. The reading is carried out in less than 5 minutes after depositing the sample.

Example 5

Manufacture of the device for evaluating the prothrombin level (T.P.) Preparation of the fibrinogen-impregnated strips

A plate of high density polyethylene (HDPE) of the POREX® type, 0.5 mm thick and having an average pore diameter of 90 μm, with dimensions of 150 mm × 150 mm, is cut.

The plate is immersed in a solution of human fibrinogen at 11 g / 1 containing glucose at 50 g / 1, Triton X 100 at 0.1% and HEPES buffer at 20 rnM / 1. The whole is stirred overnight.

The plate is removed and dried under vacuum at 35 ° C for 12 hours.

The plate is then cut into strips of 150 mm x 2.5 mm.

Preparation of the bands impregnated with thromboplastin A plate of high density polyethylene of the POREX® type 0.5 mm thick and with an average pore diameter of 30 μm is cut to dimensions of 150 mm × 20 mm. The plate is incubated overnight at 4 ° C. in a solution of Recombinant thromboplastin reconstituted in phospholopid vesicles and containing 20 mM / 1 calcium chloride, lucose at 60 g / 1 and HEPES at 20 mM / 1. The plate is then removed and dried at 35 ° C under vacuum for 12 hours and then cut to the dimensions 20 mm x 2.5 mm.

Preparation of impregnated tape (PPSB)

PPSB (extract of human plasma enriched in prothrombin, proconvertine, Stuart factor, anti-hemophilic factor B) in solution in HEPES 20 rrιM / 1, glucose

50 g / 1 and 0.1% Triton X 100 is prepared.

A plate of high density polyethylene of the POREX® type 0.5 mm thick, with an average pore diameter of 35 μm and dimensions 150 mm × 10 mm is immersed in the PPSB solution prepared as above, for 12 hours at room temperature with stirring. The plate is removed, dried at 35 ° C under vacuum and cut to the dimensions 10 mm x 2 mm.

Preparation of the device: We take two strips of 150 mm x 2 mm impregnated with fibrinogen which are placed in parallel on a rigid plastic support provided with lugs which allow the strips to be wedged without sticking them to the rigid support.

Two strips impregnated with thromboplastin are taken and they are each placed on the support so that each of them slightly overlaps one end of the fibrinogen strips.

A strip impregnated with PPSB of dimensions 10 mm × 2 mm is taken which is placed on the support so that the strip of PPSB overlaps the end of a strip of thromboplastin. On top of the assembly is affixed another plastic plate provided with a sample well with two compartments, each provided with a hole, and two windows in parallel which open over the entire length of the fibrinogen strips. . A fully transparent and suitably graduated cursor slides on the plastic support. Example 6

The device is prepared according to Example 5. 50 μl of whole blood is taken at the fingertip which is diluted in 150 μl of aqueous solution containing NaCl at 140 mM / 1 and sodium citrate at

14 mM / 1. When depositing the diluted sample so as to fill the two compartments of the well; the sample migrates in the two bands. The migration stops are clearly visible in less than 15 minutes. The reading of the result is carried out thanks to the movable cursor whose position is placed opposite the migration front of the control strip and then the test result is read on the graduation opposite the test strip .

Claims

1. Device for evaluating the blood coagulant activity of a biological sample, characterized in that it is entirely disposable after single use and that it consists of a solid support (4) comprising: a well ( 1) having a sufficient volume to receive the sample to be tested, at the bottom of which two openings (2a) and (2b) are formed in intimate contact with respectively a test migration support (3a) and a control migration support (3b ) which allow the flow of a fluid within them over a distance which is a function of the viscosity of the fluid; a cursor (5), with window or entirely transparent, sliding on the support (4), provided with a mark (6) and a graduation (7) to evaluate the distance traveled by the fluid on the migration supports ( 3a) and (3b).

2. Device according to claim 1, characterized in that:

(a) the migration support (3 a) comprises at least one zone (9) for impregnating a reagent triggering coagulation and;

(b) the migration support (3b) comprises a zone (10) for impregnating factors necessary for the coagulant activity and a zone (11) for impregnating a reagent which triggers coagulation.

3. Device according to claim 2, characterized in that the migration support (3a) further comprises a zone (8) for impregnating an inert substance with respect to the coagulating activity which in contact with the sample adjusts the viscosity of the fluid at the exit from this zone, so that it is identical to that of the fluid at the exit from the zone (10) of the migration support (3b).

4. Device according to claim 1 or 2, characterized in that the well (1) is divided into two compartments (la) and (lb) at the bottom of which there is respectively the opening (2a) and the opening ( 2b).

5. Device according to any one of claims 1 to 3, characterized in that the well (1) is lined with a membrane absorbent pad (14) over which a removable filter (16) is affixed.

6. Device for evaluating the blood coagulant activity of a biological sample, characterized in that it consists of a solid support (170) comprising: a well (100) capable of receiving the test sample , at the bottom of which is formed an opening (120) in intimate contact with a migration support (110) in two parts arranged on either side of the well (100), allowing the flow of the fluid on each portion of the support migration, over a distance which is a function of the viscosity of the fluid; a cursor (172) with window or entirely transparent, sliding on the support (170), provided with a marker (192) and a graduation (174) to evaluate the distances traveled by the fluid on each part of the migration support (110).

7. Device according to claim 6, characterized in that a part of the support (110) for migration comprises at least one zone (130) of impregnation of a reagent triggering coagulation and that the other part of the support migration (110) comprises a zone (150) for impregnating factors necessary for blood coagulant activity and a zone (140) for impregnating a reagent which triggers coagulation.

8. Device according to claim 7, characterized in that the part of the migration support (110) comprising the zone (130), further comprises a zone (160) for impregnating an inert substance with respect to of blood coagulant activity which in contact with the sample adjusts the viscosity of the fluid so that when it leaves this zone, it leaves identical to that of the fluid when it leaves the zone (150).

9. Device according to any one of claims 1 to 8, characterized in that the migration supports (3a), (3b) and (110) are made of a porous material in the form of a strip, tube or rod, made of cellulosic, plastic or mineral material, having an average pore diameter of between 0.45 and 130 μm.

10. Device according to claim 9, characterized in that the migration supports (3a), (3b) and (110) are strips of migration.

11. Device according to any one of claims 8 to .10, characterized in that the migration supports (3a), (3b), (110) have a porosity gradient.

12. Device according to any one of claims 1 to

11, characterized in that the well (1) or (100) consists of a plastic material impermeable to liquids, cellulosic material treated with hydrophobic or metallic substances.

13. Device according to any one of claims 1 to 12, characterized in that the support (4) or (170) and the slider (5) or (172) are made from glass sheet, plastic material or from treated paper cardboard, which can be cut to the appropriate dimensions.

14. Device according to claim 5, characterized in that the removable filter (16) consists of a fibrous material chosen from glass fibers, mixtures of glass fibers and plastic material and / or cellulosic materials and that the absorbent membrane (14) is made of a porous cellulosic or plastic material, having an average pore diameter of between 0.45 and 130 μm.

15. Device according to one of claims 1 to 14, characterized in that the migration support (3a) and the migration support (3b) further comprise an area impregnated with a substrate of coagulating activity, disposed before or after the area of impregnation of a reagent triggering coagulation (9) of the test support (3a) and the control support (3b).

16. Device according to claim 15, characterized in that the substrate for the coagulating activity is fibrinogen.

17. Device according to any one of the preceding claims, characterized in that the migration supports consist of juxtaposed or overlapping support portions.

18. Method for evaluating blood coagulant activity of a biological sample, characterized by the fact:

(a) using a device according to any one of claims 1 to 4 and 9 to 14, and placing the biological sample to be tested in the well (1); (b) that the area is impregnated on the migration support (3a)

(9) of a reagent which triggers coagulation and optionally a zone (8) of a substance which is inert with regard to blood coagulant activity, making it possible to adjust the viscosity of the fluid at the outlet of this zone so that that it is identical to that of the fluid at the exit of the zone

(10) the migration medium (3b);

(c) impregnating on the migration support (3b) the area (10) of factors necessary for the coagulating activity and the area (11) of a reagent triggering coagulation; (d) when the migrations of the fluids have ceased in zones (9) and (11), respectively on the migration supports (3a) and (3b), that the cursor (5) is moved so as to bring the mark (6) at the end of the migration front of the fluid present in the migration support (3b) then read on the graduation (7) opposite the migration front of the fluid present in the support migration (3a), the value of the blood coagulant activity of the sample.

19. Evaluation method according to claim 18, characterized in that a device is used according to any one of claims 5 and 9 to 17; that the sample is placed in the well (1); that the red blood cells present in the sample are filtered by means of the removable filter (16); that the filter (16) is then removed; which is added to the well (1), over the buffer membrane (14), a determined quantity of a solution containing a marker.

20. Method for evaluating the blood coagulant activity of a biological sample, characterized by the fact:

(a) using a device according to any one of claims 6 to 17;

(b) impregnating on the migration support (110) the zone (150) and the zones (130) and (140) respectively, of the factors necessary for the coagulating activity and of the reagents triggering the coagulation and possibly a zone (160) of a substance inert with respect to blood coagulant activity making it possible to adjust the viscosity of the fluid at the outlet of this area so that it is identical to that of the fluid at the outlet of this area area (150);

(c) when the migrations of the fluid in the zones (130) and (140) have stopped, as the reference mark (192) of the cursor (172) is moved, facing the migration front of the fluid present in the area (140) to read the pattern of the area (180 ) facing the migration front of the fluid and that the reference (192) is transferred to the same pattern present in the area (190) in the migration area (130); we read on the scale (174) the value of the coagulating activity of the sample.

21. Method according to any one of claims 18 to 20, characterized in that the substance is chosen from a protein substance, dextran, a polyethylene glycol and sucrose.

22. Method according to any one of claims 18 to 21, characterized in that the reagents triggering coagulation are dried, lyophilized or linked by covalent bonding on or in the materials of the device.

23. Method according to any one of claims 18 to

22, characterized in that the migration supports or the well are impregnated with auxiliary reagents chosen from buffers, detergents, anticoagulants, dyes, proteins, substances freely penetrating into the pores of the migration material.

24. Method according to any one of claims 18 to

23, characterized in that the biological sample to be tested is chosen from whole blood and blood plasma.

25. Method according to any one of claims 18 to 24, characterized in that the sample comprises a marker or that the migration supports (3 a) and (3b) or (110) are impregnated with a marker.

26. The method of claim 25, characterized in that the marker is chosen from dyes, latex particles, dyes contained in microvesicles, carbon particles in colloidal suspension, polystyrene particles, silica particles .