MXPA98001050A - Essay for the factor of the san - Google Patents

Abstract

The present invention relates to a method for indicating deficiency of von Willebrand factor in a blood sample or in a sample of platelet-rich plasma which exhibits an abnormal platelet function comprising: (a) adding a von factor preparation Willebrand to the sample, and (b) prove to the sample the platelet function. In this method, the preparation of von Willebrand factor restores normal platelet function and indicates that the sample initially has an absence of von Willebrand factor. The preparation of von Willebrand factor involves purifying von Willebrand factor. The invention also discloses a method which further comprises separating the blood sample into a plasma layer and a cellular component layer and removing the plasma layer, prior to the step of adding a preparation to the sample. In this embodiment, the von Willebrand factor preparation comprises platelet-poor plasma that contains a normal level of von Willebrand factor. The invention also provides a device for indicating von Willebrand factor deficiency in a blood sample which exhibits an abnormal platelet function comprising a preparation of von Willebrand factor, where the preparation is added to the sample and restores the normal function of platelet, and indicates that the initial sample has absence of von Willebrand factor. it also reveals a method which in addition to plasma and a cellular component layer and remove the plasma layer, previous to the step of adding a preparation to the sample. In this embodiment, the von Willebrand factor preparation comprises platelet-poor plasma that contains a normal level of von Willebrand factor. The invention further provides a kit for indicating von Willebrand factor deficiency in a blood sample which exhibits an abnormal platelet function comprising a von Willebrand factor preparation, where the preparation is added to the sample and restoring the normal function of platelet, and indicates that the initial sample has absence of von Willebra factor

Description

TITLE FACTOR ESSAY. OF THE BLOOD.

BACKGROUND PE THE INVENTION. The hemostasis or stoppage of bleeding involves the interaction of two biochemical pathways (the intrinsic and extrinsic pathways) which are controlled by various formed elements and protein factors, by ~ 'I sample platelets. The process by which blood is clotted as currently understood involves a multi-step cascade of activations of the protein factors that culminate in fibrin formation. Interference with any step in this intricate process prevents proper coagulation of the blood and may result in significant bleeding. Several tests have been developed to test -the individual steps of the cascade involved in blood coagulation to determine if a patient's blood can clot properly or if it has a clotting disorder in which it is a REF .: 26698 deficiency of one or more of the factors necessary to coagulate properly. These are a number of coagulation disorders which result in abnormal coagulation including von Willebrand disease, Vlll factor (hemophilia) deficiency, afirogenimia, platelet dysfunction (eg Beard-Soutier syndrome, Glanzman thrombastiaemia) , and the disease of a poor accumulation). Individuals suffering from these disorders suffer a 'potential risk of severe bleeding. Methods to determine the deficiency which is caused by the disorder are desirable for immediate therapeutic or clinical intervention. It is well known that the condition of the function of platelets or blood platelets is an indication of the ability of blood to coagulate properly. Wf deficiency is one of the most common types of platelet dysfunction that occurs in approximately 1% of the population. The v ¥ f is a long multimeric glycoprotein which plays an important role in the adhesion of platelets to the subendatelium that follows the damage of the blood vessel. The interaction of vVf is essential for a normal haemostasis. The most common types of von Willebrand diseases (V ¥ D) include: a deficiency of the normal amount of vWf (severe or not severe deficiency), and WD of type IIA and type IIB (which result from functional deficiency of the vWf protein). WD type IIB is characterized by the lack of high molecular weight multimers of v ¥ f in the plasma. The WD type IIA is characterized by the lack ~ 'of both the high and intermediate molecular weight multimers of v ¥ f. Type IIA is more characterized by a reduced affinity of vWf for glycoprotein Ib (GPIb) platelet receptor, whereas WD type IIB, v ¥ f has an increased affinity of GPIb, see Ribba, et al., J. Biol. Chem., 267 (32): 23209-15 (1992). Type IIA is the most common type II variant of WD. uggeri, et al., J. Clin. Invest., 65: 1318 (1980). The main test that exists in use to test the function of the platelet or the main haemostasis in patients is known as the bleeding time test. "The bleeding time test which has existed for several decades involves making an incision. In the forearm of the patient with a high blood pressure with the fist at 40mmHg.The filter paper was used to absorb the blood from the incision and determine the amount of time from bleeding until it stops.The bleeding usually stops within 10 minutes.The clinical utility of this test is limited by the associated variability with the depth of the incision, the pressure applied fluctuations in the blood pressure of patients - "" "agitated, the method to absorb the blood with the filter paper, the direction of the cuts, among others.

The variables' are difficult to control and cause problems of standardization and interpretation. Therefore, a test was developed for platelet function that does not involve making an incision and is also more accurate. The North American Patents Nos. 4,604,894; 4,780,418; and 5,051,239 disclose an assay system which can be used to develop an in vitro blood test for platelet function, the results of which can be correlated to the in vivo bleeding time test described above, thereby eliminating the patient's involvement. The Trombostat ™ 4000 (Dade International, Inc.), in current use, is a similar system. Platelet function is evaluated in the Trombostat ™ 4000 by aspirating samples of healthy anticaagulated blood at a constant negative pressure through a small opening placed in the center of a separating wall that may be porous or non-porous. In systems where the separating wall is porous, it gets wet before starting the test with an activator that "activates the coagulation of blood platelets A platelet plug is formed in the opening and the time required for the cessation of blood flow to occur is determined The time required to obtain a total occlusion of the opening is called" time of in vitro bleeding. "This time is then related to evaluate the function of the platelet by aggregometry or the bleeding time in vivo, the aforementioned in vitro assay systems which enable the determination of platelet dysfunction, In any case, no distinction is made between differences that can cause abnormal coagulation.A physician who uses such a system does not know if the dysfunction is caused by a deficiency of v ¥ f, or a less common functional platelet disorder, that is why more difficult treatment Current techniques that indicate v ¥ f deficiency include: quantitative tests, eg, immunological detection of v ¥ f; platelet egation, for example, the assay of ristocetin cofactor with fixed platelets; and optical measurements of an aggregation of platelets introduced in 'ristocetin. See Haemostasis and Thrombosis; BASIC Principies and Clinical Practice; 3rd of., Eds. Colman, R.W. , et al., 3. B. Lippincott Co. (1994). These are static tests, which do not simulate in vivo coagulation conditions. These tests are also developed on plasma, instead of healthy blood, which increase steps and time for the development of the trials. In some assays, platelet-rich plasma is used, other assays use platelet-poor plasma with added platelets, others however use fixed platelets or filtered gel. See for example, Miller, et al., J. Clin, Invest., 72: 1532-1542 (1983); Allain, ae al., J. Lab. Clin. Med., Feb .: 318-328 (1975). It may be useful to * have a von Willebrand factor deficiency assay whose tests coagulate in healthy blood samples under conditions that are representative of in vivo conditions. The currently known techniques, described above, are complicated, time-consuming and are only performed in specialized clinical laboratories. The results are usually retired, sometimes require half a day under the best of circumstances, and more typically require 1 to 2 days in usual clinical waiting. • This delay in obtaining the results leads to a delay in identifying the cause of the coagulation disorder and a delay in the treatment. Delayed results are unacceptable in certain emergency surgical procedures.

DESCRIPTION OF THE DRAWINGS Figure 1 shows a cartridge for use in a preferred embodiment of the present method.

Fig. 2 is a cross-section taken along line 2-2 of Fig. 1 where the movable apparatus of Fig. 1 is in a reinforced form and further shows a part of the instrument for use with the apparatus of the present invention. . The figure . is similar to Figure 2, but parts of the instrument are shown which contact and move a component of the apparatus shown in Figure 2 from a first to a second position. Figure 4 is similar to Figure 3 but is "" -: r. '.: 53 r a part that moves through the device.

BRIEF DESCRIPTION OF THE INVENTION The invention provides a method for the rapid indication of VWf deficiency in patients for immediate ur'a clinical and therapeutic intervention. More specifically, the method of the present invention indicates the deficiency of Wf where the deficiency is, for example, an abnormal amount of, in the absence, substantial of the VWf protein, or an absence of the functional protein v ¥ f. The absence of a functional protein is due to an abnormality in the molecular composition of the VWf process (for example, the absence of high and intermediate molecular weight multimers, which cause WD type IIA). The present invention provides a method for indicating the deficiency of vWf in a blood sample or a sample of platelet-rich plasma that exhibits an abnormal platelet function in the test for platelet function where the method comprises adding a vWf preparation to the sample, and test the sample for the platelet function where the vWf preparation restores normal platelet function and indicates that the sample initially has a vWf deficiency. In a preferred embodiment of this method, the VWf preparation consists of purified vWf. Preferably the purified factor is added in an amount to obtain an amount comparable to the VWf activity in normal blood. This amount may vary depending on the specific activity of the purified vWf preparation. Preferably the purified VWf is added in an amount in the range from about 3μg / mL to about 100μg / mL, preferably from about 10μg / mL to about 80μg / mL, more preferably from about 30μg / mL to 5μg. / mL. When commercially available preparations of purified VWf are used (e.g., American Diagnostica), it is preferred that the factor be added at a concentration higher than the vWf concentration in normal plasma which is about 3-12 μg / mL. In another embodiment of the present invention, the method comprises the steps of separating a sample of healthy blood into a plasma layer and a layer of a cellular component, removing the plasma layer, adding a VWf preparation to the layer sample. of the cell-component, and test the sample for the operation of the platelet where the normal function of the platelet indicates that the sample initially has a deficiency of vWf. In a similar embodiment, the vWf preparation comprises platelet-poor plasma containing a normal level of VWf. In a preferred embodiment of the present invention there is provided a method for determining the deficiency of VWf in a blood sample that exhibits, platelet dysfunction where the platelet dysfunction test comprises measuring the closing times in an apparatus for measuring the haemostatic function of the sanqre.

In a preferred embodiment the results are obtained within about 30 minutes and preferably in about 10 minutes. The method of the present invention is useful for indicating the deficiency of vWf in a patient, wherein the VWf deficiency comprises an abnormal amount of the vWf protein, substantial absence of the VWf protein, or absence of a functional VWf protein. The method is especially useful to indicate vWf deficiency where the absence of -VWf protein. functional is - "due to an abnormality in the molecular composition of the VWf protein." The method is preferably useful where the abnormality causes von disease.

Willebrand type IIA. A substantial absence of vWf protein occurs when the sample contains a significantly lower amount of VWf than the amount needed for normal hemostasis, as in WD type III. Examples of the VWf deficiency, where the vWf deficiency is due to an abnormal amount of the W a protein comprising the Y type D. The invention also provides a device for indicating von Willebrand factor deficiency in a blood sample or a sample of platelet-rich plasma which exhibits abnormal platelet function. The equipment comprises a preparation of von willebrand factor, which when added to the sample restores normal platelet function and indicates that the sample initially had absence of von Willebrand factor. In one embodiment of the equipment, the von Willebrand factor preparation comprises the purified von Willebrand factor. Preferably, the von Willebrand factor is added in an amount to obtain activity comparable to the activity of von Willebrand factor in normal blood. In another embodiment of the kit, the van Willebrand factor preparation comprises platelet-poor plasma containing a normal von Willebrand factor level. Preferably, the platelet-poor plasma for use in such equipment comprises fresh platelet-poor plasma in pouch containing normal levels of von Wiilebrand factor, or platelet-poor plasma in normal frozen bag just thawed, containing normal levels of vWf.

DETAILED DESCRIPTION OF THE INVENTION. The present invention provides a rapid method for indicating vWf deficiency in healthy blood samples which exhibit platelet dysfunction, which enables timely treatment of the resulting coagulation disorder. The determination of platelet dysfunction can be performed by any method known in the art, including what was described by Brubaker, US Patent No. 5,089,422 or von der Goltz, US Patent No. 5,051,239. Other similar methods such as conventional aggregometry can also be used. Preferably, the initial determination of platelet dysfunction in blood samples and the subsequent identification of the deficiency of vWf in these blood samples according to the method of the present invention was performed using the PFA-100 ™ (analyzer of the platelet function), described in the pending series Nos. Nos. 08 / 269,184 and 08 / 269,185, incorporated herein by reference. The methods of the present invention can be illustrated using this particular test apparatus. In any case, it is understood that the present invention is not limited to this test equipment.

A preferred embodiment described in the pending application series No: 08 / 269,184, is a test cartridge specifically adapted to be used in an assay to test the blood coagulation function as a measure of platelet function, which includes but is not limited to automated versions of these assays described in US Patents Nos: 4,604,894, 4,780,418 and 5,051,239. This test cartridge comprises: a container, which comprises a receiving chamber for receiving the sample of blood to be tested and a test chamber, where the receiving chamber and the test chamber are separated by a transferable member, a limb for separating disposed within the test chamber, the partition member having an opening through and comprising at least one reagent which activates at least one path for blood coagulation, a movable transfer member mounted on the test chamber so that it moves towards and traverses the transferable member; and a receiving chamber disposed in the test chamber between the partition member and the transfer member for receiving blood from the transfer member. In use, the blood is disposed by a user in the holding chamber and the test cartridge is placed in an instrument for incubation. After incubation, the transfer member moves towards and passes the transferable member to contact the blood and a vacuum is created in the test chamber, blood moves through the transfusion member into the chamber. reception and through the opening in the separation member. In a preferred embodiment, described in "'copending application USSN 08 / 621,821, the transferable member has a cut in. The cut is configured so that the transference member moves easily through the cut .. In any case, the cut is made in order to that the function of the transferable member is not damaged Fig. 1 shows an isometric, schematic view of a preferred apparatus A cross section of the apparatus through line 2-2 of Fig. 1 in assembled form and the sample contained therein it is shown in Figs 2,3 and 4. Figs 2 to 4 also show a component of an instrument which can be used as the apparatus of the present invention.

Referring now to Fig. 1, this preferred apparatus comprises a container 60 in which a retention chamber 61 and a test chamber 63 are defined. The container 60 is provided with a flange 67 and a tongue 69. The geometry of the container 60 which defines a holding chamber 61 and a test chamber is selected to minimize the possibility of an air bubble being trapped in the apparatus and in preferred embodiments the bottom of the holding chamber 61 is tilted to minimize entrapment air when blood is added through the opening 65, to easily insert, for example, the tip of a pipette to release blood into the holding chamber. The geometry of the container is selected to minimize contact of the blood surface to the hot surface of the container, which at the same time minimizes the area of blood exposed to the air to minimize the risk of sample degradation. In the embodiment shown in the figures, the shape-configuration of the container 60 achieves both objectives.

The test chamber 63 is adapted to receive in the sample cup 10. The sample cup 10 supports a partition member 6 with treated reagent having an opening 9 inside and a capillary cube 30 which provides a mechanism for binding the operability from the transfer member 40 to the sample cup 10. The inside of the sample cup 10 is provided with four vacuum shoulder retention ribs 14 for placement, two of which are shown in FIG. 1. The container 60 is adapted to a core with an instrument which can create a vacuum in the test chamber 63. In the embodiment shown, this is done by the edge 12 of the sample cup 10 which comprises a part of the test chamber 63. The instrument has a male instrument which is capable of mating to seal with the rim 12 of the sample cup 10. In the embodiment shown in Figs. 2 to 4, the mating component comprises of vacuum shoulder blades 15 shown. The vacuum shoulder 15 is provided with O-ring 27 which during the test is sealed with the edge 12 and which employs a downward pressure on the sample cup 10 to move the transfer member 40 towards the transferable member. 70, causing it to pierce the transferable member and extend within the sample 11 into the containing chamber. The retention ribs of the vacuum shoulder 14 in the sample cup 10 limit the downward movement of the shoulder to the vacuum 15. FIG. 2 shows a cross-sectional view of the apparatus shown in FIG. 1 throughout of line 2-2 after the shoulder to The vacuum has exerted a downward pressure on the sample cup 10. Fig. 3 shows a cross-sectional view of the apparatus in Fig. 2 after the vacuum shoulder 15 has been moved to contact and move the cup from the sample 10 downwards so that the bottom of the sample cup 10 is in contact with the support member 71 and a transfer member, this capillary 40 has pierced the transferable member 70 and penetrated inside the sample 11. As shown in Fig. 3, the support member 71 contacts the bottom of the sample cup 10 under the downward pressure of the instrument.

The instrument is then able to create a vacuum in the test chamber 63, for example, by the pump movement of the syringe. This vacuum or negative pressure causes the sample 11 to flow from the holding chamber 61 through the capillary transfer member 40 into the receiving chamber 18 and through the opening 11 into the request member 6 as shown in FIG. 4. In the case of the test cartridges for use in the vWf deficiency assay, reagents in the partition member activate the formation of a platelet plug which eventually occludes the opening 9 and the flow of the sample through of the transfer member 4 ceases. The time it takes for the blood flow to cease, called the "closed time", is then compared to the time required for the blood flow to stop when the blood platelet function is normal. A normal range within which the blood flow must stop is obtained by testing normal blood. The opening 9 in the partition member 6 is dimensioned so that under the conditions of the particular test a plug can be formed and the opening closed. If the opening is very small, obstructions related to the test may occur. If it is too large, a stopper can not be formed properly. For the platelet function test, the aperture is preferably between about 100 microns and about 200 microns, more preferably about 140 microns to 160 microns, and most preferably about 150 microns. The dimension of the opening in the partition member 6 does not have a great influence on the initial flow characteristics in the apparatus. The receiving chamber 18 shown in Figs. 2 and 3 are placed in the test chamber 63 between the partition member 6 and the capillary hub 30. The receiving chamber 18 is dimensioned so that the blood entering from the capillary 40 does not get too close to the membrane and disturbs the stopper that is formed. The partition member 6 is a porous or non-porous agglomerated support for one or more reagents capable of initiating platelet aggregation in healthy anticoagulated blood and platelet-rich plasma. For example, in an apparatus specifically adapted to test the function of the platelet, blood enters from the side of the partition member comprising a collagen material as disclosed in US Pat. Mo. 4,604,894 and 5,051,239. When the blood platelets aspirated and anticoagulated come into contact with the collagen in the porous material, the activation and aggregation events take place around the opening, fundamentally forming a platelet plug which occludes the opening and causes the flow to cease of blood. The preferred material for the partition member has absorbency to liquids so that reactants can be applied even to a stable structure so that a precise opening can be made, for example, puncture. Preferred porous partition members for use in the test cartridge and the methods of the present invention include cellulose esters, ceramics, nylon, polypropylene, polyvinylidene fluoride (PVDF), and glass fiber. Particularly a preferred porous partition member is the mixture of cellulose ester (cellulose acetate and nitrate) millipore membrane. In embodiments where the partition member is provided with a collagen cover, a uniform layer of collagen around the opening is highly desirable. The amount of collagen on the membrane is not particularly critical. A range of about l-2μg has been found for a good development in the platelet function test. In a test cartridge of the present invention, the collagen is provided for the porous partition member and then the porous partition member is dried to be stored within the container under an airtight seal. The present invention also provides porous separation members that have incorporated standard platelet aggregation with modulating agents, such as epinephrine or 5'-adenosine diphosphate (ADP). In a preferred test cartridge of the present invention, freeze-dried epinephrine bitartrate (about 10 μg) was incorporated in a separation member. These reagents provide controlled stimulation to the platelets while the blood sample passes through the opening. The collagen surface serves as a binder for depositing and coupling platelets. SI closing time of an opening with a normal blood sample depends in part on the concentration of the biologically active substance incorporated in the membrane. The concentration of the reagents is selected in order to provide a convenient distinction between normal and abnormal coagulation parameters. These can be really determined by someone ordinarily experienced in the art. The concentration ranges of similar reagents reported for use in aggregometry provide a starting point in the determination of the appropriate concentration range. The concentration of the reagents are optimized remembering the desired sensitivity of the assay. For example, it is desired that the concentration of - .epinephrine be sufficient to detect mild platelet dysfunction, but not so low to introduce variable results. "A threshold value of epinephrine is needed for complete activation and aggregation and if mild platelet dysfunction has been studied, when a small amount of reagents is used. It can be seen that a balance between the sensitivity of the test and the reproducible results obtained is desirable. The sensitivity of the test can be controlled by the alteration of the amount of epinephrine bitartrate incorporated within the porous separation member. For example, when using epinephrine ranes, for example, near Sμg, the assay is more sensitive than when using about 20μg. One skilled in the art can easily determine the amount of epinephrine to be used based on the sensitivity desired for the assay. The ranges of closed times for normal blood may be different depending on the amount of epinephrine used in the porous separation member. One skilled in the art can actually determine the standard ranges of closing times for the amount of selected epinephrine. In preferred embodiments, about 10 μg of epinephrine bitartrate are incorporated into the membrane. As shown in Figs. 2 and 3, the test chamber 63 is provided with a double support portion for the sample cup 10, the support comprises the support member 71 and a tightening rib 72. The support member has a central opening dimensioned to allow the separation 31 of the capillary • hub 30 to pass through it As shown in Fig. 2, the rib to tighten (others do not show) keeps the sample cup 10 in a first position in order to that the capillary 40 is near but not in contact with the traversable member 70. As shown in Fig. 3, the sample cup 10 has been moved to the second position by means of which the tightening ribs 72 have been compressed, the sample cup 10 is in contact with and held in position by the support member 71, the separation 31 of the capillary hub 30 has passed through the support member 71, and the capillary 40 has moved towards the through the transferable member 70 to project from nter of the holding chamber 61 and inside the sample 11 arranged therein. The sample is caused to flow from the holding chamber 61 to the test chamber 63 by the / c created by the instrument. The initial velocity of the flow through the apparatus is controlled by the variation of the length and the internal diameter of the capillary. In the platelet function tests, for a sample volume of about 500 to SOOμL it is preferred that the blood flow rate through the apparatus be from 100μL to about 200μL per minute. It is believed that diameters much smaller than 100 microns can have an effect on platelets.

Accordingly, the preferred inner diameter of the capillary 40 is from about 100 to 220 microns. A particularly preferred internal diameter is close to 150-210μ and a capillary length is close to 0.01524 to 0.03048 meters long. In a preferred embodiment especially the inner diameter of the capillary is about 200 ± 10 microns and the length of the capillary is about 0.03048 meters with this configuration and flow, the opening in the membrane can close in about 1 to 3 minutes if the blood It has a normal platelet function. When a test cartridge with epinephrine / collagen with normal blood is used, it is observed that the ranges of the closing times are close to 98-185 seconds. See Mammen, E.F., et., Seminars in thrombosis and Hemostasis-Volume 21, Suppl. 2, pp. 113- 121, 1995. When blood is used from patients with platelet dysfunction (determined by conventional test and clinical indications), closed times greater than the normal range (eg, greater and closer to 185 seconds) are obtained when using the test cartridge with epinephrine / collagen. Therefore, a closing time which exceeds the values within the normal range for the particular apparatus used to perform the assay indicates platelet dysfunction. This test, like others in the art to measure the function of platelets, therefore does not discriminate among the many deficiencies that can cause platelet dysfunction. Requiring to make an additional test which, by currently known methods, consumes time and is complicated. Once it is determined that the patient's blood exhibits platelet dysfunction, the assay of the present invention indicates the deficiency of v ¥ f as the cause of platelet dysfunction by adding a preparation containing vWf to the sample of the healthy blood from the patient and retest the sample for the platelet function A normal closing time indicates a correction in the vWf level in the patient's blood and this indicates the presence of the VWf deficiency in the patient. to indicate the vWf deficiency, for example, an abnormal amount of VWf, a substantial absence of vWf or an absence of the functional Wf protein, an example of the Wf deficiency where the vWf deficiency is due to an abnormal amount of the VWf protein that comprises the WD type I. An absence of the VWf proton occurs when the sample contains a quantity of VWf significantly lower than the amount necessary for a normal hemostasis, as in the WD type III. Finally, certain types of von willebrand diseases result from the absence of the functional vWf protein, as in WD type IIA and IIB. In type 11A, vWf has absence of high and intermediate molecular weight multimers. The method of the present invention is "especially useful for indicating a VWf deficiency due to the absence of the functional VWf protein of the type IIA variety. In the WD of type IIB, the vWx has absence of high molecular weight multimers and vWf has an increase in affinity for the glycoprotein Ib (GPIb) receptor platelet.Thus, platelets tend to become saturated with VWf, and platelet saturation with VWf that occurs in patients affected with type IIB can impact the efficacy of the platelet. If nonconceiving results are obtained and WD type IIB is suspected, another test may be required by other methods.An example of such a method is the addition of small amounts of ristocetin to another aliquot of the blood sample of the patient. patient.

If type IIB WD is present, aggregation of platelets can be seen, for example in an aggregometer.

The blood sample for use in the method of the present invention is prepared in the same way as it is used in the initial test of platelet function, for example, it is treated with an anticoagulant. Any anticoagulant known in the art can be used in this method (e.g., sodium citrate, heparin, hirudin). It is therefore preferred that the anticoagulant does not remove excessive amounts of calcium (e.g. EDTA) or otherwise interferes with platelet aggregation. Any known preparation. n the technique containing Wf is useful in the assay. In a preferred embodiment, the VWf preparation comprises purified VWf. In another preferred embodiment, the preparation comprises platelet-poor plasma containing normal levels of vWf. VWf is present in a natural form as a multi-pandemic glycoprotein. Although the discoverers intend not to be limited by theory, it is hypothesized that the processing involves currently available commercial preparations of purified VWf alters the composition of the VWf, perhaps by breaking its multimeric composition, resulting in a preparation which is different or less active, than that found in normal human plasma. Thus the amount of vWf purified for use in this assay can be optimized to obtain an activity comparable to the activity of VWf in normal blood. Such activity and its optimization are actually carried out by someone with experience in the art. The purified vWf in performing the assay of the present invention can be from an animal or human (eg, bovine) source and obtained from sources known in the art (eg, purified vwf from American Diagnostica). The purified VWf is preferably added to an amount in the range of about 3μg / mL to about 100μg / mL, more preferably from about 10μg / mL to about 30μg / mL, more preferably from 30μg / mL to 50μg / mL. mL. When using available purified vWf preparations (e.g., American Diagnostic), it is preferred that the factor be added at a concentration higher than the VWf concentration in normal plasma which is approximately 3-12 μg / mL. Other sources of VWf are available, such as factor VIII concentrate, which contains VWf levels comparable to normal blood and are used as a therapeutic in hemophilia and in von Willebrand's disease. As disclosed above, preferably the amount of the factor VIII concentrate used is that it requires obtaining an activity (eg, aggregation). ~ and platelet adhesion) comparable to normal blood activity. This amount can be determined and optimized from a particular lot of the factor VIII by someone ordinary experienced in the art. Recycling the VWf can also provide a source of high activity and a pure vWf for use in the present invention. In another preferred embodiment, the VWf preparation useful in performing the present assay comprises platelet poor plasma containing a normal level of vWf (eg, 3-12μg / mL). In this embodiment, the assay further comprises separating the healthy blood sample exhibiting platelet dysfunction in a plasma layer and a cellular component layer, removing the plasma layer, and adding the VWf preparation to the cell layer of the sample. . The vWf preparation comprises platelet poor plasma in normally fresh bag or platelet poor plasma in normally frozen finished defrosting bag containing normal levels of vWf. The plasma can be one or the other human or animal plasma (for example bovine) and can be supplied by a donor or as commercially available frozen plasma. Preferably a volume of this plasma equal to the volume of the plasma removed is added and the sample blended smoothly, as is known in the art, to ensure complete mixing of the plasma and cells and homogenization of the plasma. . sample. The resulting mixture is a reconstituted blood sample. The blood sample mixed with a vWf preparation as taught here is then tested for platelet function. In a preferred embodiment of the present invention the platelet function test comprises measuring the closing times in an apparatus for testing the coagulation of blood function. Preferably this test is done on a PFA-100t test cartridge as described above v shown in Figures 1-4, and more preferably with a test cartridge with collagen / epmephrine. The results of the test prior to the addition of the factor preparation are compared with the results subsequent to the addition. If the addition of a VWf preparation as taught here produces a normal platelet function in the sample, this strongly suggests that the abnormal function previously seen was due to the vWf deficiency. For example, when the test cartridge described above is used, if a correction in the closing time is observed, for example, the closing time is within the normal range, this is a strong indication that the abnormal closing time initially observed in the blood sample was due to the deficiency of vWf in the sample. The clinician may decide to develop additional tests to confirm the diagnosis of W deficiency. Although the PFA-100 ™ is a preferred apparatus, the method of this invention is not limited by the apparatus for testing the blood platelet function. The method is useful in conventional aggregometers and other test apparatus known in the art. For example, the method can be used in the m vitro test system described by Brubaker when developing measures prior to and after the addition of the purified vWf to the sample. If a correction is observed in the time required for the cessation of blood flow, the VWF deficiency in the sample is indicated. A similar method can also be used in the Xylum CSA (Xylum Corp., Y) analyzer and coagulation time techniques in the O'Brien filter. O'Brien, 7. R., Thromb. Res., 76: 103-108 (1994). With some of the test devices as conventional aggregometers, the development of tests on platelet-rich plasma (PP.P), instead of blood, is preferred. The PP.P is obtained by methods known in the art, for example, centrifugation. In this embodiment, the PRP circulates in the test apparatus, with an appropriate agonist to simulate platelet aggregation. The absence of aggregation can indicate the deficiency v ¥ f as the cause. To confirm the V ¥ f deficiency, a preparation v ¥ f as described above, is added to another PRP sample of the patient and the sample is run through the test apparatus. If aggregation is observed, the vWf deficiency is indicated. The VWf preparation useful with PRP is preferred that the von WilLebrand factor or the platelet poor plasma containing a normal level of vWf be purified, as described above. Once the platelet dysfunction has been identified, the method of the present invention enables rapid identification of the vWf deficiency. When the blood samples are tested according to the methods of the present invention and using the PFA-100 ™ apparatus, the test can be developed within 'Approximately 30 minutes in contrast to the prior art tests which requires at least 8-16 hours. Preferably, the results are obtained within 10 minutes when the PFA-ICO ™ system is used. This rapid change eliminates the delay seen in the vWf tests in the prior art. When other types of apparatus are used, such as conventional aggregometers, the test may also be completed within 30 minutes. This test is therefore useful not only because it projects a routine, but for applications where the determination of the function of the platelet can be obtained quickly, as in situations in emergency rooms. Another aspect of this invention is a device for indicating VWf deficiency in a blood sample exhibiting an abnormal platelet function comprising a vWf preparation useful in carrying out the method of the present invention, wherein the preparation is added to the shows previous to the platelet function test. In a preferred embodiment, the kit provides a pre-measured amount of the VWf preparation of the purified von Willebrand factor iofilized in a sterilized vial or container. It is preferred that the preparation is delivered in a dry state. In. In carrying out the assays of the present invention, this preparation is mixed with a quantity of sample, healthy blood or platelet-rich plasma, for example, 1 ml, so that the mixture dissolves the preparation. The sample then circulates in the apparatus that tests the coagulation to determine the presence or absence of the vWf deficiency, as described above. The vwf preparation in the kit is stabilized as is known in the art, for example, by adding buffer salts, glycine bufexl or sodium chloride and antimicrobial agents. The amount of the purified von Wíllebrand factor can depend on the amount of purified factor and can easily be carried out by one skilled in the art. Preferably, the amount of the factor used can provide the equivalent of at least 10g of VWf per mL of sample. This equipment provides sufficient preparation to circulate the samples in duplicate. In another embodiment of a kit of the present invention, the kit delivers a vial for the preparation comprising normal human plasma with normal levels of von Willebrand factor. In use, a sample of blood that is tested is centrifuged to separate the plasma and the cell layer. The plasma layer is removed and an equal volume of VWf preparation provided by the equipment (thawed, if necessary) is added to the sample. The reconstituted sample then circulates in the desired test set to determine the absence or px-essence of the vWf deficiency. A preferred device provides up to about 2 L of plasma, allowing up to 1 mL per sample.

The following examples are provided to more clearly illustrate aspects of the invention and are not intended to limit the scope of the invention.

EXAMPLES I. Determination of platelet dysfunction A healthy blood sample was collected in 3.8% sodium citrate anticoagulant (1 part anticoagulant for 9 parts blood). The function of The sample platelet was tested using the PFA-100 ™ tester as described above, circulated with cartridges with co-gen / epinephrine, approximately 0.8 mL of sample was used for each run in the PFA-100 ™ tester, and the test was developed in duplicate.The time required for closure, for example, cessation of blood flow was observed.If for the test the closed time was higher than the normal reference range, then the results indicate platelet dysfunction. If after evaluation of the patient's history and the -registration of the use of drugs, the clinician suspects von Willebrand's disease, then the sample was again tested according to the methods of the present invention as follows.

II. Determination of the deficiency of VWf-adding plasma. A patient sample was centrifuged at 1500xg or greater to separate cellular components from the plasma. The blood sample used in this test can be taken tube with the blood used in part (a) above, for example, was mixed with an anticoagulant, for example, sodium citrate. A sample of 0.8 mL was used for each circulation in the PFA-100 ™ tester, and the test was developed in duplicate. The platelet-poor plasma collected at high id from the layer of cellular components in the centrifuge tube was removed by methods known in the art, such as aspiration with a pipette. This step was carefully developed so as not to disturb the cell layer. The volume of plasma removed was measured. Preferably at least 85-90% of the plasma collected at the top was removed. A volume, equal to that of the plasma removed, was added to the sample of centrifuged blood of one or the other platelet-poor plasma in a fresh pouch containing nox-mals levels of v ¥ f, or platelet-poor plasma in a freshly frozen freshly thawed pouch. (George ing Bic edical, Overland Park, K "3) The tube was inverted gently, at least six times, to ensure proper mixing of the plasma and cells and homogenize the sample.The PFA-100 ™ test was performed with the reconstituted blood sample using collagen / epinephrine test cartridges as described in pax-te (a) The time required to close, for example, the cessation of blood flow was observed. control measure that the replacement of 100% of the plasma sample with plasma in a normally frozen bag results in closing times similar to normal blood closure times.These results indicate that the plasma in the bag is normally frozen contains an appropriate amount of Wf to obtain closing times comparable to those of normal blood.

TABLE 1 Table 2 shows that blood samples from patients suffering from type IIA WD do not obtain a closed opening, for example, the samples have an average closing time greater than 300 seconds. They did not close due to the absence of normal vWf. Therefore, when the sample plasma of this blood was replaced with an equal volume of fresh normal plasma, the samples have a mean closing time close to those of normal blood. These results indicate that the VWf present in normal fresh plasma was able to correct the deficiency of vWf produces closed times comparable to those of normal blood.

TABLE 2 - 1 D "indicates von Willebrand disease Table 3 shows that when approximately ^, all normal blood plasma is replaced with an equal volume of plasma from a patient suffering from VWf type HA, no closed was obtained. the normal plasma amounts are replaced, the closing times are close to those of normal plasma, indicate normal levels close to vWf.

TABLE 3 III. Determination of the vWf deficiency using the purified v ¥ f preparation. In another embodiment of the method of this invention, 40μg of purified human VWf (American Diagnostica, Inc., Greenwich, CT) was added to approximately ImL of healthy blood sample to obtain a final factor concentration of 40μg / mL. The blood sample was carefully mixed by gently inverting the tube, approximately 5 times to ensure proper mixing and homogenization of the sample. The PFA-100 ™ measurements were then developed using the collagen / epinephrine test cartridges as above. The time required for closure was observed, for example, the cessation of blood flow. The results are shown in Table 4. The addition of 4Cμg of purified vWf per mL of blood results in normal closing times.

TABLE 4 As demonstrated in the examples, a closed time observed after the addition of a VWf preparation delivered within the normal range provides a strong indication that the abnormal closing time observed initially was due to the VWf deficiency in the sample. The clinician can then decide to confirm the diagnosis of the vWf deficiency using other tests.

IV. Determination of the deficiency of v ¥ f using Agregometrla. A blood sample from a patient is centrifuged at 150xg to obtain platelet-rich plasma (PRP): PxRP is aspid and sepad from blood cells with red packaging, by methods known in the art.

The blood sample is then centrifuged at 1500xg to obtain platelet poor plasma (PPP). The platelet account is developed on the PRP. The plate count of the PRP is adjusted to 150,000μL by appropriate mixing of portions of PRP and PPP. An aliquot of the resulting score-adjusted PRP plate is then analyzed in a standard aggregometer (Chrono-Log Corp., riaverts-wn, PA, other devices available from Helena Labory, Beaumont, TX) using a standard group of agonists ( for example collagen, ADP, arachidonic acid, ristocetin). A low platelet aggregation response when using the ristocetin agonist may indicate deficiency of the Willebrand factor vori. The purified VWf is added in another aliquot of the PRP sample, to obtain the equivalent of 40μg of VWf per L of sample, and the aggregometry is repeated with the ristocetin agonist. If a normal aggregation response is observed, the possibility of VWf deficiency in the original sample is confirmed. The clinician can send the patient to develop another and confirmation of this disease. The present invention has been described in detail, it includes the preferred embodiments of this, Therefore, it can be api-eciated that this experience in the art, in consideon of present reveals, can be made modifications and / or improvements of this invention and even it may be within the scope and content of this invention as directed in the following claims.

Claims (23)

What is claimed is:
1. A method for indicating deficiency of von Wiilebrand factor in a blood sample or a sample of platelet-rich plasma which exhibits an abnormal platelet function comprising (a) adding a von Willebrand factor preparation to the sample, and (b) prove to the sample the platelet function, characterized by the fact that the preparation of von Willebrand factor restores normal platelet function.
2. 2. The method according to claim 1, characterized in that the preparation of von illebrand factor comprises the purified von Willebrand factor.
3. 3. The method according to claim 2, characterized in that the purified von Willebrand factor is added to obtain an activity comparable to the activity of von Willebrand factor in normal blood.
4. . The method according to claim 1, characterized in that it further comprises (a) separating the blood sample in a plasma layer and a cellular component layer and (b) removing the plasma layer, prior to the step of adding a von factor preparation. Willebrand to the sample.
5. 5. The method according to claim 4, characterized in that the prepaxation of von Willebrand factor comprises platelet-poor plasma containing a normal level of von Willebrand factor.
6. 6. The method according to claim 5, characterized in that the preparation of the factor see Willebrand comprises platelet-poor plasma in a fresh pouch containing normal levels of von Willebrand factor, or platelet-poor plasma in a normally frozen freshly thawed pouch.
7. 7. The method according to claim 1, characterized in that the test of the platelet function comprises measuring the closing time in an apparatus for testing the haemostatic function of the blood.
8. 8. The method according to claim 7, characterized in that the test of the platelet function comprises measuring the closing time in a test cartridge.
9. 9. The method according to claim 1, characterized in that the von factor deficiency T 'Willebrand comprises an abnormal amount of the vWf protein, a substantial absence of the VWf protein, or the absence of a functional VWf protein.
10. 10. The method of claim 9, characterized in that the absence of a functional vWf protein is due to the abnormality in the molecular composition of the vWf protein.
11. 11. The method according to claim 10, characterized in that the abnormality in the composition of the vWf protein causes von Willebrand disease of type, IIA.
12. 12. The method according to claim 9, characterized in that the abnormal amount of the VWf protein causes von Willebrand disease type I.
13. 13. The method according to claim 9, characterized in that the substantial absence of the VWf protein causes von Willebrand disease type III.
14. 14. A method for indicating the deficiency of von Willebrand factor in a blood sample or a sample of platelet-rich plasma which exhibits an abnormal platelet function comprising a) separating the blood sample in a plasma layer and a cellular component layer b) remove the plasma layer c) add a von Willebrand factor preparation to the sample; and d) p? -ob to the sample platelet function; characterized in that the preparation of von Willebrand factor restores normal platelet function.
15. 15. The method according to claim 14, characterized in that the preparation of von Willebrand factor comprises platelet-poor plasma containing a normal level of von Wiliebrand factor.
16. 16. The method according to claim 15, characterized in that the preparation of von Willebrand factor comprises platelet poor plasma in fresh bag containing normal levels of von Willebrand factor, or platelet poor plasma in freshly thawed normally frozen bag.
17. 17. The method according to claim 14, characterized in that the test of the platelet function comprises measuring the closing time in an apparatus for testing the hemostatic function of the blood.
18. 18. The method according to claim 17, characterized in that the test of the platelet function comprises measuring the closing time in a test cartridge.
19. 19. A kit for indicating deficiency of von Willebrand factor in a blood sample which exhibits an abnormal platelet function comprising a preparation of von Willebrand factor, characterized in that the preparation is added to the sample and re-establishes normal platelet function, and indicates that the initial sample has a von willebrand factor deficiency.
20. 20. The equipment according to claim 19, characterized in that the preparation of von Willebrand factor comprises a purified von Willebrand factor.
21. 21. The kit according to claim 20, characterized in that the purified von Willebrand factor is added to obtain an activity comparable to the activity of von Willebrand factor in normal blood.
22. 22. The kit according to claim 19, characterized in that the preparation of von Willebrand factor comprises platelet-poor plasma containing a normal level of von Willebrand factor.
23. 23. The method according to claim 22, characterized in that the von Willebrand factor preparation comprises platelet poor plasma in fresh bag containing normal levels of von Willebrand factor, or platelet poor plasma in freshly frozen freshly thawed bag. SUMMARY The present invention comprises a method for inactivating von Willebrand factor deficiency in a blood sample or in a platelet-rich plasma sample which exhibits an abnormal platelet function comprising: (a) adding a von Willebrand factor preparation to the sample; and (b) prove to the sample the platelet function. In this method, the preparation of the Willebrand vori factor restores normal function - Platelet and indicates that the sample initially has absence of von Willebrand factor. The von Willebrand factor preparation comprises purifying von factor Willebrand. The invention also discloses a method which further comprises separating the blood sample into a plasma layer and a cellular component layer and removing the plasma layer, prior to the step of adding a preparation to the sample. In this embodiment, the von Willebrand factor preparation comprises platelet-poor plasma that contains a normal level of von Willebrand factor. The invention also provides a device for indicating deficiency of von Willebrand factor in a blood sample which exhibits an abnormal platelet function comprising a von Willebrand factor preparation, where the preparation is added to the sample and restoring the normal function of platelet, and indicates that the initial sample has absence of von Willebrand factor.