MXPA97009953A - Apparatus and method for the preparation of pla - Google Patents

Abstract

The present invention relates to a collection device for the preparation of plasma for diagnostic tests. This device comprises a spray-dried anticoagulant formulation on the interior surface of the device and a thixotropic polymer gel. The device is an improvement over commercially available devices, which contain formulations of liquid anticoagulants, for use in the nucleic acid test, which employs amplification technologies that include, but are not limited to, the polymerase chain reaction (PCR). , Branched DNA (bDNA) and amplification based on nucleic acid sequence (NASB)

Description

APPARATUS AND METHOD FOR THE PREPARATION OF PLASMA BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a device for the preparation of blood plasma for a variety of clinical trials. More particularly, the present invention pertains to a blood collection device, comprising a thixotropic polymeric polyester gel and an anticoagulant formation. The device of the present invention is most preferably used in nucleic acid assays, which use amplification technologies that include, but are not limited to, the polymerase chain reaction (PCR), in branched DNA (bDNA) and the amplification based on nucleic acid sequence (NASBA). 2. Description of the Related Art New amplification technologies, such as polymerase chain reaction (PCR), branched DNA (bDNA) and amplification based on nucleic acid sequence (NASBA), allow researchers to monitor the levels of infectious agents in the plasma. Studies have shown that the number of viral copies of HIV extracellular RNA, or viral load, is a surrogate marker for the progression of HIV infection. Scientific research has shown that HIV replication occurs throughout the life of the infection. After the initial infection, HIV viron enters susceptible cells, doubling rapidly creating billions of copies of HIV viral RNA after infection. Although the viral load of HIV RNA varies throughout the patient's population, the disease follows a specific progressive pattern within each patient. Therefore, monitoring viral load of HIV RNA in patients infected with HIV can be used to manage the disease. In addition, the response of patients to exploited drugs, new drugs and drug combination therapies can be evaluated by monitoring the viral load of HIV RNA. In addition to the HIV virus, there are a number of other infectious diseases that will benefit from monitoring the viral load, such as the Hepatitis C virus. Viral load measurements were determined using the polymerase chain reaction ( PCR), branched DNA (bDNA) and other amplification techniques. The quality and consistency of the sample is critical in obtaining the optimal results of the test using these technologies. There are a number of variables with influence on the quality of the sample, such as the collection method, centrifugation time, sample preparation technique, transport to the test laboratory, contamination with cellular materials, and the like. Numerous sample types have been evaluated for the nucleic acid test, which include whole blood, serum and plasma. Studies have shown that HIV viral load is stable for up to 30 hours in a whole blood sample, using EDTA as the anticoagulant. The coagulation process required to produce the serum can artificially lower the viral load by trapping viral particles in the resulting clot. Although the preferred sample type is plasma, the preparation of a plasma sample can adversely affect the result of the amplification process. For example, if the plasma sample remains in contact with the red blood cells, blood molecules from the hemoglobin, contained within the red blood cells, will interfere with the amplification of the PCR, if hemolysis occurs. In addition, since the half-life of neutrophils is approximately 24 hours in a blood collection tube, and as the neutrophils begin to die, they release granules containing the myeloperoxidase in the sample, and since the myperoperoxidase causes the reduction In viral load, this is also another factor that supports the need to segregate the plasma sample away from blood cells. A further example of the difficulties associated with the current plasma preparation is the fact that the blood collection tubes may contain a liquid anticoagulant to prevent coagulation of the sample. A liquid anticoagulant can dilute the viral load value by volume of the sample. Thus, the viral load value may be below the detection threshold. Commercially available blood collection products, such as (all sold by Becton Dic inson and Company, Franklin Lakes, NJ and all registrations and trademarks are from Becton Dickinson and Company) VACUTAINER brand-name hematology tubes, Nos. Of catalog 367650-1, 367661, 6405, 6385, 6564, 367653, 367665, 367658, 367669, 6450-8, 6535-37, 367662; K2EDTA tubes with trademark VACUTAINER, Nos. of catalog 367841-2, 367856, 367861; PST tubes trademark VACUTAINER, Nos. of catalog 367793-4, 6698, 6595, 6672; CPT tubes, trademark VACUTAINER, Nos. of catalog 362753, 362760-1; SST tubes, trademark VACUTAINER, Nos. of catalog 367782-89, 6509-17, 6590-92; and ACD tubes trademark VACUTAINER, Nos. of catalog 367756, 364012, 4816; they can be used for nucleic acid tests. However, these commercially available products may not consistently deliver a good integrity sample and, therefore, may not provide consistent and adequate amplification results. Therefore, there is a need to provide a standard device designed to collect, process and transport plasma samples for use with amplification technologies. More preferably, the device should be able to assist in standardizing the handling of specimens, provide a closed system, isolate plasma from cellular components, produce minimal plasma dilution and minimal interference with nucleic acid tests. COMPENDIUM OF THE INVENTION The present invention relates to a device for preparing a plasma specimen suitable for diagnostic assays, such as the nucleic acid test. The device comprises a plastic or glass tube, a device for inhibiting the coagulation of blood and a device for separating plasma from whole blood. The device further preferably comprises a recess to close the tube and seal a vacuum within the tube, and to provide easy access into the tube.

Preferably, the remedy for inhibiting blood coagulation is an anticoagulant formulation. Conveniently, this anticoagulant formulation comprises a mixture of water, dihydrate of the dipotassium salt of ethylenediaminetetraacetic acid, also collectively known as K2EDTA or, alternatively, the dihydrate of the tripotassium salt of ethylenediaminetetraacetic acid, also collectively known as K3EDTA. More preferably, the anticoagulant formulation comprises K2EDTA, which has a chemical composition of: 2 (CH2C00K) -C2-N2-H4-2 (CH2C00H) -2H20). More preferably, the K2EDTA formulation is spray-dried over a large surface area of the inner wall of the tube, to substantially reduce local osmolarity and concentration gradients between the anticoagulant and the cells of the blood sample, thereby substantially reducing the the possibility of hemolysis and the rupture of cells within the blood sample. Preferably, the resource for separating the plasma from whole blood is a gel formulation. This gel is conveniently a thixotropic formulation of polymeric gel. The gel conveniently isolates the plasma from the cells of the blood sample in the tube, serving as a means of density separation. As the sample is centrifuged, the gel moves to a point dividing the heavier cellular materials and the lighter fraction of the plasma from the blood sample. In other words, the plasma of the blood sample is divided above the gel and separated from the rest of the blood. More preferably, the tube comprises the gel placed at the bottom end of the tube and the anticoagulant formulation is then spray dried on the inside of the tube, above the gel. The device of the present invention is useful in molecular diagnostic applications, including, but not limited to, nucleic acid testing, detection and quantification of RNA and DNA, with the use of amplification methods. Therefore, the present invention provides an improved method for handling and preparing plasma samples for nucleic acid testing, because the separation of plasma from whole blood can be achieved at the collection point and can minimize any change or degradation of the nucleic acid. The device of the present invention provides a closed one-step system for collecting blood, separating plasma and transporting a specimen for nucleic acid testing. The device maximizes the capabilities of PCR, bDNA, NASBA or other amplification techniques, providing a substantially consistent sample, so that variability from one test to another can be minimized, due to the quality of the sample and variation, and the standard way of handling the sample can be facilitated. In addition, the device of the present invention provides an isolated specimen that is protected when early centrifugation is employed at the collection point and the stability of the specimen is improved during transport. Additional attributes of the device of the present invention are that the spray-dried anticoagulant formulation, which provides a substantially stable ratio of blood to additive over the shelf life of the tube, whereby the device substantially isolates the plasma from the cells and it substantially minimizes degradation due to neutrophils and red blood cells. Most notably, the device of the present invention provides a closed system for collecting a specimen from the blood; resources to anticoagulate the blood without any substantial dilution; resources to facilitate separation of the plasma from the rest of the whole blood by a gel barrier; resources to freeze the plasma inside the device; and resources to transport the specimen to an analytical site, while maintaining the quality and integrity of the sample. Therefore, the device of the present invention provides a means for deriving an undiluted plasma within a closed system configuration with minimal variations of test to test, as compared to commercially available devices. Important attributes of the device of the present invention are that (i) it is compatible with the molecular technologies that are used for nucleic acid tests; (ii) it provides a specimen of substantially pure plasma, with less cellular contamination, compared to devices that do not have a gel barrier and (iii) they allow an undiluted plasma specimen, which intensifies the sensitivity of several molecular technologies, especially for specimens with low viral titer. DESCRIPTION OF THE DRAWINGS Figure 1 is a perspective view of a typical blood collection tube, with a stopper. Figure 2 is a longitudinal sectional view of the tube of Figure 1, taken along line 2-2, comprising the spray-dried anticoagulant formulation and the gel of the present invention. DETAILED DESCRIPTION The present invention can be incorporated into other specific forms and is not limited to any specific modality described in detail, which are merely exemplary. Various other modifications will be apparent and readily obtained by those skilled in the art, without departing from the scope and spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The device of the present invention preferably comprises an anticoagulant formulation, spray dried, and a gel. The device of the present invention more preferably relates to a blood collection device and can be any of an evacuated blood collection device or a non-evacuated blood collection device. This blood collection device is conveniently made of plastic, such as, but not limited to, polyethylene terephthalate, or polypropylene, or glass. Referring to the drawings, in which similar reference characters refer to similar parts throughout the various views, Figure 1 shows a typical blood collection device 10, having an open end 16, a closed end 18, an inner wall 12 and a plug 14, which includes a lower annular portion or flap 15, which extends into and presses against the inner wall 12 of the tube, to hold the plug 14 in place. Figure 2 shows the device 10 with a gel 20 and above the gel, along the inner wall 12, there is an anticoagulant coating 22. A sample of blood specimen of interest can be transferred into the device 10, in which The specimen makes contact with the anticoagulant formulation, so that this anticoagulant formulation dissolves rapidly in the specimen and the coagulation of this specimen is minimized. After the blood is collected within the device of the present invention, a cascade reaction may occur which would cause the blood to clot. Anticoagulants are materials used to prevent blood clotting by blocking the cascade mechanism that would cause clotting. To collect a plasma sample from whole blood, an anticoagulant should be added immediately to preserve the integrity of the sample. There are tubes, commercially available, for plasma collection, which contain numerous types of anticoagulants, such as sodium citrate, heparin, potassium EDTA, and the like. The selection of the type of anticoagulant is important because some additives may interfere with the bDNA, PCT or other amplification techniques used in nucleic acid testing. For example, heparin can interfere with PCR amplification. Preferably, the anticoagulant formulation of the present invention comprises a mixture of water, dihydrate of the dipotassium salt of ethylenediaminetetraacetic acid, also collectively known as K EDTA. The concentration of the anticoagulant formulation is substantially sufficient to minimize the coagulation of a sample of the blood specimen. Conveniently, the concentration of the K2ETA is from about 0.2M to 1M, preferably from about 0.2M to 0.5M and more preferably from about 0.3M to 0.4M. The anticoagulant formulation conveniently has a pH ranging from about 5.6 to 6.2 and preferably about 5.8 to 6.2.

The anticoagulant formulation of the present invention may include additional reagents, in order to provide additional properties to the device. A variety of tube coatings or the addition of other compounds to the anticoagulant formulation may be convenient. They include, but are not limited to, silicone oils and silicone surfactants. Preferably, the gel is a thixotropic polymer gel. The gel preferably has a specific gravity of about 1040 to 1080 g / cm3 and more preferably about 1.043 to 1.050 g / cm3, so, after centrifugation, the plasma of the blood sample is divided above the gel and separates from the rest of the whole blood. The thixotropic polymer gel is substantially insoluble in water and, in substantial form, chemically inert in the blood. The gel can be formulated from dimethyl polysiloxane or polyester and a precipitated methylated silica, in which methylation makes the material partially hydrophobic. The thixotropic polymer gel is first deposited in a tube at the closed end, then the anticoagulant formulation of K2EDTA and the water are applied to the inner wall of the tube above the gel, in the form of fine mist by the spray coating. The applied formulation is then dried by a jet of forced air or air, at elevated temperature, for a period of time. Next, the tube is assembled with a closure and a vacuum is formed inside the tube. The device is then sterilized by gamma irradiation or the like. The main advantages of a tube with a spray-coated anticoagulant formulation on the inner wall are a more precise, stable and uniform anticoagulant filling and an improved anticoagulant solution within the specimen. Due to the fine mist of the anticoagulant formulation, the actual surface area of the anticoagulant formulation exposed to the specimen is maximized. The method for preparing the device of the present invention comprises: (a) depositing a gel at the closed end of a tube; (b) preparing an anticoagulant formulation comprising a mixture of water, dipotassium salt of the ethylenediaminetetraacetic acid salt, at a concentration of about 0.2M to 1M, and a pH of about 5.6 to 6.2; (c) applying the anticoagulant formulation to the surface of the inner wall of the tube with a medium that produces a fine mist of the formulation above the gel; Y (d) drying the applied formulation by applying a jet of air or forced air to the inner wall of the coated tube at an elevated temperature for a period of time. It is preferable that the anticoagulant formulation is dosed and dispensed by a volumetric type device, such as a positive displacement pump. The concentration of the solution (amount of anticoagulant per unit volume of formulation) is adjusted with a volume distributor, so that the desired amount of the anticoagulant is distributed within the device. Other spray techniques include ultrasonic spray. The device of the present invention can be used to collect and prepare a specimen for nucleic acid testing, as follows: (a) collecting a specimen, such as the whole blood sample or the fraction of cells previously treated with the blood in the tube prepared; (b) mix the specimen in the tube with the anticoagulant solution by manual inversion; (c) centrifuge the tube to induce the separation of the plasma from the erythrocytes, leukocytes and platelets, so that the gel is at an intermediate point to the densest erythrocytes and leukocytes and platelets, and the least dense plasma fraction of the sample of blood, thus facilitating the isolation and subsequent removal of the plasma. Various other modifications will be apparent to and can easily be made by those skilled in the art, without departing from the scope and spirit of the invention.

Claims (10)

1. CLAIMS 1. A device for preparing a plasma specimen for diagnostic tests, comprising a plastic or glass tube, a device for inhibiting blood coagulation, including an anticoagulant formulation, and a resource for separating the plasma from whole blood, comprising a thixotropic polymer gel. The device of claim 1, wherein the anticoagulant formulation comprises the water and the dipotassium salt of ethylenediaminetetraacetic acid. 3. The device of claim 2, wherein the anticoagulant formulation is at a concentration of about 0.2M to 1. OM and a pH of about 5.6 to 6.2. 4. A tube for preparing a plasma specimen for diagnostic tests, comprising an upper end, a bottom end, a side wall extending from the upper end to the bottom end and including exterior and interior surfaces, a thixotropic polymer gel at the bottom end of the tube and a spray-coated anticoagulant formulation, comprising a mixture of water and dihydrate of the dipotassium salt of ethylenediaminetetraacetic acid, on the inner surface of the tube. 5. The tube of claim 4, wherein the anticoagulant formulation is at a concentration of about 0.2 to 1. OM and a pH of about 5.6 to 6.2. 6. The device of claim 1, wherein the thixotropic polymer gel has a specific gravity of about 1.040 to 1.080 g / cm3. 7. A method for obtaining a tube for preparing a plasma specimen for diagnostic tests, this method comprises the steps of: a) depositing a gel on the closed end of the tube; b) preparing an anticoagulant formulation comprising a mixture of water and the di-proprate of the dipotassium salt of ethylenediaminetetraacetic acid, at a concentration of about 0.2M to 0.1M, and a pH of about 5.6 to 6.2; c) dispersing the formulation on the inner wall of the tube in a fine mist, above the gel; and d) drying the formulation by applying forced air for a sufficient period of time to dry this formulation, whereby a dry formulation remains. 8. The method of claim 11, wherein the gel is a thixotropic polymer gel. 9. A set for centrifugally separating the plasma from a whole blood sample or its pre-treated cell fraction, this set comprises: a) a container, having an open end, a closed end and an internal and external surface; b) a layer of thixotropic gel, contained within the container in a first position; and c) an anticoagulant solution, to prevent coagulation of the sample, when this sample is introduced into the container, said solution is placed on the internal surface of the container. 10. A method for separating the plasma from a sample of whole blood or its pretreated cell fraction, comprising the steps of: a) supplying a container, having an open end, a closed end, this container further has a layer of thixotropic gel contained therein in a first position, the container also has an anticoagulant solution to prevent coagulation of the sample, when this sample is introduced into the container; b) insert the sample into the container; c) mix the sample inside the container with the anticoagulation solution by manual inversion; d) centrifuge the vessel to induce separation of plasma and erythrocytes and leukocytes, so that the gel migrates to a point dividing the phase of the heaviest erythrocytes and leukocytes and the lightest plasma phase of the blood sample, thus facilitating the isolation and subsequent removal of the plasma.