Abstract:
An assembly device and method for collecting and testing fluid samples more specifically for preparing and stabilizing nucleic acid components in a closed system. The assembly comprises a sample collection container with preloaded testing reagents and a safety separator to contain the testing reagents during sample collection. A fluid sample is delivered to the container and the assembly is subjected to centrifugation whereby the centrifugal load causes the separator to deform so that the separator migrates through the test reagents mixing the sample and reagents, and comes to rest atop the solids at the bottom of the tube.

Description:
This Appln claims benefit of Prov. No. 60/169,166 filed Dec. 6, 1999. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to sample collection tubes provided with a test fluid safety separator, permitting pre-loading of the tubes with potentially toxic testing reagents. More particularly, this invention relates to a closed system for the collection, preparation and stabilization of nucleic acid, comprising a separator device, and method for collecting and transporting fluid samples whereby the separator reduces back flow of potentially toxic testing solutions, reduces the opportunity for contamination and increases amount of target recovered. 
     2. Description of Related Art 
     In blood collection procedures, samples of whole blood are typically collected from a patient by venipuncture through a cannula or needle attached to a syringe or an evacuated collection tube. Typically, the samples are then shipped to a laboratory where personnel experienced in sample preparation add testing reagents such as a lysing solution, and then place the tube in a centrifuge so as to effect mixing of the blood sample with the reagent. Lysing solutions, or other testing reagents, are often toxic, and hence are not included in the sample collection tube because of the possibility of back flow into the veins of the patient during sample collection. Thus, typically, laboratory personnel open the collection tube to add the testing reagent to the collected specimen. This is time consuming and also increases the risk of sample contamination. 
     In another diagnostic area, a patient&#39;s whole blood sample maybe separated into two liquid phases, and separately maintain for subsequent examination of the individual components. A variety of separator devices have been used in collection devices to separate the heavier and lighter phases of a fluid sample. 
     However, to employ a separator device in an evacuated tube for the collection of fluid samples it is desirable that the separator device: (i) is easily and safely used for collecting samples; (ii) is independent of temperature during storage and shipping and stable to radiation sterilization; (iii) permits completion of nucleic acid preparation by centrifugation alone (with no additional step of introducing testing reagents); (iv) minimizes opportunity for cross contamination of samples from introduction of testing solutions before centrifugation; (v) increases the amount of target than can be recovered. Presently known separator devices do not meet all of these requirements. 
     SUMMARY OF THE INVENTION 
     The present invention comprises a closed system for the collection and testing of a fluid sample preferably a blood sample including the preparation and stabilization of nucleic acids. The system includes a method and an assembly for collection and testing. Preferably, the assembly comprises a container and a safety separator. 
     Most preferably, the container is a tube and the separator is arranged to move in the tube under the action of centrifugal force in order to release a testing solution up, into the fluid sample. 
     Most, preferably, the tube includes an open end, a closed end and a sidewall extending between the open end and closed end. The tube further includes a closure with a releasable self-sealing septum disposed to fit in the open end of the tube. A safety separator is positioned atop preloaded testing reagents in the bottom of the tube. Alternatively, both ends of the tube may be open, and both ends of the tube may be sealed by elastomeric closures. At least one of the closures of the tube may include a needle pierceable resealable septum. 
     In one preferred embodiment, the safety separator comprises a toroidal separator and in another preferred embodiment, a bellow separator. 
     Preferably, the safety separator includes an overall specific gravity greater than the specific gravity of the testing reagents (preloaded into the tube) or the mixture of testing reagents and the sample. 
     According to a desired method of the present invention, testing reagents are provided in a typical sample collection evacuated tube. Thereafter, a separator is placed in the tube, above the test reagents. The separator makes physical contact with the tube, presenting a barrier to back flow of the test reagents during sample collection. A resealable closure is placed in the end of the tube so as to create an evacuated space between the closure and the separator. A sample is collected in the evacuated space. Under centrifugal force, the separator is deformed and the barrier with the tube is broken. Because the separator&#39;s density is greater than that of the testing reagents, it begins to migrate toward the closed end of the tube, releasing testing solution to mix with the sample collected. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of the assembly of the present invention. 
     FIG. 2 is a longitudinal sectional view of the assembly of FIG. 1 taken along line  2 — 2  thereof illustrating fluid delivery into the assembly by a needle. 
     FIG. 3 illustrates that assembly under centrifugation and the movement of the separator, and flow of testing reagents into the sample. 
     FIG. 4 illustrates the assembly after centrifugation and the preparation of the nucleic acid within the liquid sample. 
     FIG. 5 is a perspective view of the unassembled elements of the assembly of the present invention. 
     FIG. 6 is a perspective view of an alternate embodiment of the assembly of the present invention. 
     FIG. 7 is a longitudinal sectional view of the assembly of FIG. 6 taken along line  7 — 7  thereof illustrating fluid delivery into the assembly by a needle. 
     FIG. 8 illustrates that assembly under centrifugation and the movement of the separator and flow of testing reagents into the sample. 
     FIG. 9 illustrates the assembly after centrifugation and the preparation of the nucleic acid within the liquid sample. 
     FIG. 10 is a perspective view of the unassembled elements of the assembly of the present invention. 
    
    
     DETAILED DESCRIPTION 
     The present invention provides a fluid collection assembly which allows for the safe and efficient testing of a collected fluid sample with a preloaded testing reagent. More specifically, the present invention provides a closed system which provides for collection as well as preparation and stabilization of nucleic acids. 
     The preferred assembly  20  of the present invention is illustrated in FIGS. 1 to  5 , wherein the assembly comprises a tube, shown generally at  30 , a closure shown generally at  50  and a separator  70 . 
     Tube  30  has an open end  32  that includes a top edge  33 , a closed end  34  and a sidewall  36  extending between the open end and the closed end. Sidewall  36  has an outer surface  38  and an inner surface  40 . Tube  30  defines a receptacle with a central axis “A”. Tube  30  is preferably made from a substantially transparent and rigid material. Suitable materials or the tube include glass, polystyrene, polyethyleneterephthalate, polycarbonate and the like. 
     Closure  50  is disposed to fit over open end  32  of tube  30 . Closure  50  comprises and annular upper portion  52  which extends over top edge  33  of sidewall  36  and a lower annular portion or skirt  54  of lesser diameter than the annular upper portion  52  which extends into the forms an interference fit with inner surface  40  of sidewall  36  for maintaining stopper  50  in place in open end  32 . 
     Annular upper portion  52  includes a top surface area  56 , sidewall  58  that converges from surface area  56  towards upper well area  60 . Well area  60  is most preferably a thin diaphragm or a self-sealing septum for directing and receiving the point of a needle to be inserted into and through the stopper. 
     Lower annular skirt portion  54  defines a lower well  62 , an inner wall surface  64 , an outer wall surface  66  and a bottom surface  68 . Well area  60  and lower well area  62  define a thin diaphragm or self-sealing septum through which a needle may be inserted. The self-sealing septum material allows penetration by a piercing element such as a needle  16  and then reseals when the piercing element is withdrawn. 
     An annular ledge or abutment  57  separates annular upper portion  52  and lower annular portion  54 . Preferably, the closure maybe made of natural rubber elastomer, synthetic thermoplastic and thermoset elastomeric materials. Preferably, the closure is made of a resilient elastomeric material whereby the septum is self-sealing. 
     As shown in FIGS. 1-5, the toroidal separator  70  includes an elastic toroid  72 , a low-density foam float  90  and a high-density sinker  110 . The components of the separator are formed from materials which exhibit a combined density greater than the density of the combined collected fluid sample and the preloaded testing reagents. 
     Toroid  72  includes a top section  86 , a bottom section  88 , and an elastic seal body  91  extending from the top section to the bottom section with a central passageway  98  extending between through the ends and the seal body. 
     Low-density foam float  90  is located at top section  86  and high-density sinker  110  is located at bottom section  88 . High-density sinker  110  surrounds bottom section  88  without obstructing central passageway  98 . Low density foam float  90  is at top section  86  and in direct alignment with central passageway  98 . 
     Low-density foam float  90  may comprise small holes  95  to bleed air out of central passageway  98  when in use. As shown in FIG. 5, the outside diameter “a” of top section  86  and the outside diameter “b” of bottom section  88  are less than the outside diameter “c” of the seal body when the seal body is in its undeformed position. Seal body  91  of toroid  72  and the inner wall of the tube form an interference fit. The low-density foam float and the high-density sinker do not interfere with the inner wall of the tube. 
     Toroid  72  may be assembled by mounting foam  90  over open top section  86  and sinker  110  around the outer circumference of open bottom end  88 . Toroid is then inserted into open end of the tube. Sufficient insertion causes the seal body to sealingly engage the inner tube sidewall, and seal preloaded testing reagents  44 , in the closed end of the tube. Thus, the separator  70  is positioned initially atop the testing reagents  44  and spaced from the closed end of the tube. 
     As shown in FIG. 2, in use, a liquid sample A is delivered to the tube by a needle that penetrates closure  50  in upper well area  60 . For purposes of illustration only, the liquid sample is blood. The liquid sample is introduced into the evacuated space between the closure and the safety separator. The separator  70  effectively blocks movement of the testing reagent  44  into the evacuated space during blood collection. This prevents back flow of the reagents towards the patient. This feature allows blood collection and testing in a closed system, i.e., there is no need to open the tube to introduce the testing reagent after blood collection. The separator&#39;s position atop the testing reagents  44  preloaded in the bottom of the tube  30 , and spaced from the closure, provides easy direct loading of the fluid sample on the separator. Thus, the fluid sample is easily delivered into the tube without exposing the fluid sample needle to the test reagents, reducing back flow to almost zero. After collection, the needle  16  is withdrawn from the tube  30  and the septum of the closure reseals itself. 
     As shown in FIG. 3, in order to effect testing, the assembly  20  is subjected to centrifugation or axial centrifugation force. Seal body  91  of separator  70  deflects, and is thereby released from the inner wall of the tube. The separator  70  descends towards closed end  34  of tube  30 . As the separator descends, seal body  91  of the separator deflects reducing its diameter causing a stretching or elongation and eliminating its interference fit with the inner wall of the tube. The separator  70  is therefore forced to move axially within the tube without any frictional drag. This opens up a path  10  between the tube and the separator, permitting the flow of the testing reagents  44  upwardly past the separator as the separator  70  migrates down the tube. This causes mixing of the testing reagents with the sample so as to permit appropriate testing of the sample. Air will be trapped in the passageway when the bottom section of the toroid contacts the testing reagents. This trapped air could restrict further downward movement of the separator. However, the small holes in the foam defines a path through which trapped air may escape the passageway. Thus, separator  70  is permitted to sink into the closed end of the tube. 
     After centrifugation is terminated, the absence of the centrifugal load will cause the elastic toroid to resiliently return toward an expanded undeformed condition and tightly seal with the inner wall of the tube as shown in FIG.  5 . Thus, separator  70  serves as a divider between the liquid components,  46  and any resultant residue  48  from the test procedure. In nucleic acid preparation for example, gene amplification testing (GAT) blood samples are treated with solutions such as lysing solutions, that break open the cells and release and stabilize the nucleic acid. Nucleic acids are a class of naturally occurring biochemical entities composed of sugar molecules, nitrogenous bases and phosphate groups. Ribonucleic acid (RNA) and deoxynucleic acid (DNA) are prime examples of nucleic acid and may be of viral or genomic origin. The nucleic acid is found in liquid component  46 . Depending on the specific testing reagents, nucleic acid components may be in the liquid component, with the combined residual testing reagents and sera of the sample contained between the separator and the top of the tube, or may be included in the residue or sediment with the cell membrane, cytoplasm and proteins released in the lysing of the sample contained between the separator and the solution of the tube. 
     Tube  30  is compatible with most of the numerous additives used in sample collection tubes such as citrates, silicone, silicates, EDTA and the like that are used to condition the sample either to facilitate or retard clotting, or to preserve the sample for a particular analysis. It is within the purview of this invention that one or more additives may be used in the present invention for particular applications. 
     FIGS. 6-10 represent an alternative embodiment of the present invention. As illustrated in FIGS. 6-10, the alternative embodiment comprises assembly shown generally at  120 , which comprises a tube  30 , a closure  50  as described above, and a separator  170 . 
     As shown in FIGS. 6-10, separator  170  comprises a bellow member  172 , a low-density buoyance or float member  190  and a high-density sinker or ballast member  210 . The components of the separator are formed from materials to exhibit a combined, but greater than the density of the collected fluid sample and the preloaded testing reagents. 
     Buoyance member  190  comprises a top section  211  bottom section  212  and a central passageway  214  extending continuously between the ends. Buoyance member  190  is preferably made of a material which has a component density having the capability to allow it to float in serum of a blood sample. In the present embodiment, buoyance member  190  may be formed of a low density foam. 
     Bellow member  172  comprises a rupturable elastomeric material such as Kraton copolymer, a urethane or PVC. Bellow member  172  includes a bottom  188 , a top  186 , a seal body  191  extending between the top and bottom. Bellow member  172  is made of a material and of a shape which allows deflections caused by opposing forces. 
     Ballast member  210  comprises a cylindrical sidewall  220  extending from a top end  221  to a bottom end  222  and a central passageway  223  extending between the top and bottom ends. The ballast member  210  has a component density whereby it has the capability of sinking in a blood sample. Preferably, the ballast member  210  is made of a high density material such as a substantially rigid moldable thermoplastic material. Such materials include but are not limited to polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester and mixtures thereof that are inert to the fluid sample of interest. 
     The separator is assembled whereby the bottom of bellow member  172  is inserted into the top end of ballast member  210  and then the bottom end of the ballast member is joined with top section  211  of the buoyance member whereby the top section is within central passageway  223  of the ballast member. 
     As shown in FIG. 7, the separator  170  is initially placed atop the testing reagents  44 . A liquid sample A is delivered to the tube by a needle  16  that penetrates closure  50  in upper well area  60  and conical top wall  199  of bellow member  172 . For purposes of illustration only, the liquid sample is blood. The liquid sample is delivered into the evacuated tube above the safety separator. 
     As shown in FIGS. 8 and 9, assembly  120  is subjected to centrifugation or axial centrifugation force. 
     Seal body  191  of the separator deflects reducing its diameter and eliminating its interference fit with the inner wall of the tube. This opens up a path  300  between the tube and the separator, permitting the flow of the test reagents past the separator as the separator migrates down the tube. As the separator descends, the test reagents move upwardly past the separator. Air trapped in the central passageway  223  creates a buoyancy that could prevent further sinking of the separator into the fluid, but venting of air permits further movement of the separator into the fluid. 
     Following immersion of the separator  170  in the fluid sample, the buoyancy member  190  provides a buoyant upward force on the separator due to the displaced fluid. Simultaneously, the ballast member  210  provides an axial force downward on the separator. The combined forces stretch the bellow member  172  axially and pulls it out of contact with the inner wall of the tube so that it is free to move axially without any frictional drag. 
     After centrifugation is terminated, the absence of the centrifugal load will cause the seal body  191  to resiliently return toward an undeformed condition and tightly seal with the inner wall of the tube as shown in FIG. 9 creating a barrier between nucleic acid components  300  and the remainder of the sample fluid and test reagents  310 . 
     In certain applications, the separators of the present invention may be used to trap extracted sediment from the sample plus test reagents. The extracted sediment is trapped below the separator, in the closed end of the tube. If desired, a double ended sample tube may be used, and the extracted sediment removed from the “closed” second end of the tube. 
     With the assembly of the present invention, testing solutions may be preloaded into the sample collection container and an inert barrier added atop the solution to reduce the possibility of back flow. Preloading the testing solutions advances the amount of target that can be recovered, as personnel untrained in sample preparation can collect samples and centrifuge immediately, yielding more non-degraded samples. Lastly, because the safety separator is not intended to come to rest between two solutions of different specific gravity, the manufacturing tolerances of the safety separator are greater. 
     The assembly of the present invention is advantageous over existing separation products that use gel. In particular the assembly of the present invention will not interfere with analytes as compared to gels that may interfere with analytes. Another attribute of the present invention will not interfere with therapeutic drug monitoring analytes. 
     Additionally, the assembly of the present invention does not require any additional steps or treatment by a medical practitioner; and the blood or fluid sample can be drawn in the standard fashion, using standard sampling equipment. 
     The present invention may be embodied in other specific forms and is not limited to any specific embodiments described in detail, which are merely exemplary. Various other modifications will be apparent to and readily made 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.