Abstract:
A mechanically simple, small, hand held device is provided based on filtering and pressure equilibration techniques involving a unique hand-operating sequence that produces air pressure within the collection tube and the device, to enable simple and rapid extraction of blood serum or plasma or other filtrate in milliliter quantities from a collected sample. The device can also provide dilution of the serum, plasma or filtrate, capture of unwanted molecular constituents or dispensing of desired reagents. Pipette extraction of diluted or undiluted blood plasma, serum or filtrate from the device can also be achieved via a septum. The device permits all functions to be performed rapidly and with minimum danger of exposure of the operator or contamination of the sample while enabling standard evacuated collection tubes to be used.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to U.S. Provisional Application Ser. No. 61/103,984, filed on Oct. 9, 2008. 
     
    
     TECHNICAL FIELD 
       [0002]    This invention relates to the separation of blood cells from whole blood to obtain small quantities of plasma or serum that is free of blood cells. It also relates to the optional dilution or treatment of separated fluid (e.g. plasma or serum) and to performance of bio-array assays and other diagnostic procedures using small quantities of the separated fluid at natural or diluted concentrations. (“Plasma” refers to the liquid component of whole blood constituting about one half of the volume of blood, the blood cells constituting the remainder of the volume. “Blood serum” is blood plasma from which fibrinogen or other clotting factors have been removed.) 
         [0003]    The invention also relates more generally to a simple and safe system for transferring liquid, e.g., of volume of a fraction of a milliliter or a few milliliters, from a sealed collection container, and for using that system for producing filtered or treated liquid, for dispensing agents into liquid passing through filter material, and for capturing a molecular constituent of liquid passing through filter material. 
       BACKGROUND 
       [0004]    As traditionally conducted, a set of adult blood tests necessitates collection of whole blood with 3 to 6 evacuated blood collection tubes (Vacutainer™, Becton Dickinson and Company, East Rutherford, N.J.) each with 10 milliliter capacity. Plasma is typically obtained when blood is processed by centrifugal separation or filtering within minutes from being drawn, if unaltered with added substances. Serum is obtained after blood has been kept for a period of time so that fibrinogen forms a clot which sinks to the bottom of the container. Serum is then separated by pipetting, centrifuging or filtering. 
         [0005]    The availability of sensitive biological assays has made it possible to run accurate tests employing much smaller sample volumes than has been traditional. For instance, multiple tests can be preformed employing less than 1 milliliter of plasma or serum using bio-array techniques. No simple and rapidly operable device is presently available for providing serum or plasma extraction at this size volume. 
         [0006]    The need for small volume blood collection itself has been recognized for blood tests for infants and small animals. Evacuated collection tubes have long been available for obtaining a fraction of a milliliter or a few milliliters of blood. 
         [0007]    Extremely small blood volumes have also traditionally been obtained by use of a puncture wound. The finger for instance is pricked with a lancet and then squeezed until a fluid drop of, e.g., 10-20 μ.l, is obtained. 
         [0008]    In most cases of use of small samples for assays, further manipulations have been required once the sample of whole blood has been obtained. The sample may be mixed with a stabilizing agent to permit storage at room temperature prior to separation. Depending on the assay for which the sample is intended, it may also be necessary to add diluents and/or reagents, or it may be necessary to manipulate the sample physically, for example by centrifuging the sample as a means of removing blood cells. 
         [0009]    Current methods of achieving small volumes of blood plasma or serum thus involve numerous steps, employing multiple pieces of equipment and disposable items. Various kits are available for these purposes, examples being Unopette® (Becton Dickinson and Company), Fisherbrand® microhematocrit and capillary tubes (Fisher Scientific Company, Hampton N.H.), and the StatSampler® capillary blood collection kit (StatSpin, Norwood, Mass.). Each relies on multiple separate components for performing the functions of sample collection, processing, and recovery. 
         [0010]    Prior art patents in the general field include U.S. Pat. Nos. 2,460,641; 4,883,068; 4,343,705; 4,477,575; 4,540,492; 4,828,716; 4,906,375; 5,030,341; 5,181,940; 5,308,508; 5,413,246; 5,555,920; 5,681,529; 5,759,866; 5,919,356; 6,261,721; 6,406,671; 6,410,334; 6,465,256; 6,471,069; 6,479,298; 6,497,325; 6,516,953; 6,537,503; 6,755,802; 6,803,022; 6,821,789; 7,070,721 and 7,153,477. 
         [0011]    It is desirable to work efficiently with blood samples of the order of 1 to 5 milliliter. Most protein analyzers for instance necessitate 50 to 100 micro-liters per test and it is common to require 10 tests. Multiplexed biomarker cassettes, e.g. those employing micro arrays, typically run  8  to  12  assays simultaneously and call for 100 to 200 micro-liter of serum or plasma. 
         [0012]    The device made possible by the present disclosure can meet these needs without requiring use of a centrifuge or other inconvenient separation techniques, thus enabling simple and rapid sterile separation at point of collection or point of treatment. 
       SUMMARY 
       [0013]    A mechanically simple, small, hand held device is provided based on pressure equilibration techniques, involving a unique hand-operating sequence that produces compressed air within the collection tube followed by expulsion of liquid from the tube by the air. This is advantageously followed by forced transfer of the liquid through filter medium. The device enables simple and rapid extraction of blood serum or plasma in milliliter quantities from a collected blood sample. The device can also provide dilution of the serum or plasma, or addition of an agent. Pipette extraction of diluted or undiluted blood plasma or serum from the device can also be achieved via a septum. The device permits all functions to be performed rapidly, without exposure of personnel to needles, and with minimum danger of exposure of the operator to the sample or contamination of the sample while enabling standard evacuated collection tubes to be used. 
         [0014]    In preferred implementations, a blood separation device in the form of a cylindrical tubular assembly is provided that employs filtration to produce as much as a milliliter volume of blood plasma or serum, by simple back and forth relative movements of movable parts of the device. The movements produce air flow that pressurizes the previously evacuated collection tube, and forces blood to flow from the collection device and through the filter without exposure to the outside. In certain forms of the device, a preset level of dilution of the sample is achieved within the device. 
         [0015]    The major benefits offered by such devices are:
       Simplicity of operation,   Protection of the operator from exposure,   Freedom of contamination of the sample.       
 
         [0019]    An alternate design simplifies sample dilution. A specific volume of buffer or other fluid is stored in a sealed graduated elongated collection chamber of the device. In this case, a pre-determined volume of filtered sample is introduced into the chamber. 
         [0020]    In some applications the evacuated collection tube (Vacutainer™) is provided with material in the form of a surface coating or as a liquid that prevents blood clotting, or that offers dilution, or that alters the viscosity or other properties of the recovered fluid. 
         [0021]    In its presently preferred implementations, the device comprises a tube-shaped main body closed at one end by a screwed-on small filtrate receptacle. The other end is open, exposing within the main body, a free sliding piston-like member, e.g., a short “poppet,” which is sealed to the inside wall of the main body. The piston is traversed through its center by a fixed, sharp hypodermic needle which protrudes outwardly. The needle is exposed to pierce the end seal of an evacuated collection tube. In the region of the main body of the device, between the poppet and the sample receptacle, is a filter assembly, the “cage”, through which the liquid is forced to pass, e.g., for removing blood cells. 
         [0022]    The device or various of its principles have other potential uses enabling introduction of a sample container to a device, and operating the device to produce a liquid, e.g. a toxic liquid, from which a filterable substance has been accurately removed or to which an agent has been added. 
         [0023]    According to a particular aspect of invention, a device is provided which includes a pump constructed to transfer liquid out of a partially filled, predetermined portable sealed container, the device defining a sleeve, a liquid receptacle communicating with the sleeve, a piston member including at least one seal ring slideably disposed within the sleeve, the piston, sleeve and liquid receptacle forming a closed volume, the piston constructed to couple with the portable container to form a movable assembly within the sleeve, the piston including a passage for enabling fluid communication between the closed volume and the portable container, whereby, forcing the movable assembly in a first direction toward the liquid receptacle can force compressed air captured in the closed volume into the portable container in a first action tending to equilibrate fluid pressures between the closed volume and the sealed container, and releasing the movable assembly enables compressed air captured in the closed volume to move the assembly in pressure-relieving direction opposite to the first direction, so that residual air pressure above liquid within the portable container is effective to force liquid in the portable container to move through the passage into the closed volume in a second action tending to equilibrate fluid pressures between the sealed container and the closed volume. 
         [0024]    Preferred implementations have one or more of the following features: 
         [0025]    The device includes an actuatable pressure relief device associated with the closed volume, constructed, when actuated, to vent the closed volume and enable further movement of liquid from the container in a third action tending to equilibrate fluid pressures between the fluid container and the now-vented closed volume, and enable movement of the piston in the first direction, without air pressure resistance, to force liquid toward the receptacle. 
         [0026]    The device incorporates a filter or filter material to which liquid entering the closed volume is exposed, in preferred cases the device incorporating filter material selected and arranged to filter liquid in the form of blood, or the device incorporating filter material carrying a capture agent selected to remove a constituent of the liquid or the device incorporating filter material carrying an agent exposed to be dispensed into the liquid in which the agent may be a desiccated bio-active substance. 
         [0027]    The device that incorporates a filter or filter material includes an actuatable pressure relief device associated with the closed volume, the pressure release device constructed, when actuated, to vent the closed volume and enable further movement of liquid from the container in a third action tending to equilibrate fluid pressures between the fluid container and the now-vented closed volume. 
         [0028]    Also in the case of the device being provided with a filter or filter material, the device is constructed to enable flow of liquid forced by fluid pressure from the first container to enter into a space preceding the filter or filter material, the device including an actuatable pressure relief device associated with the closed volume, the pressure release device constructed, when actuated, to vent the closed volume and enable movement of the piston, without air pressure resistance, to force liquid through the filter or filter material toward the receptacle. 
         [0029]    In cases employing an actuatable pressure relief device, the relief device comprises a threaded connection capable of being loosened to enable passage of air, in preferred cases the relief device being combined with material selected and positioned to allow passage of air through the threaded connection but to prevent liquid from reaching the threaded connection. 
         [0030]    In cases in which the device with the pressure relief device is also provided with a threaded cover, succeeding clockwise and counter-clockwise screw threads are so associated with the pressure relief device and cover as to ensure that the threaded connections are opened sequentially, for instance a first screw thread enables unsealing and venting a filtrate collecting chamber to permit flow through a filter or filter material, and a second screw thread of opposite hand is associated with the cover that is screwed to close an access port, screwing the cover to close the access port being arranged to force closing of the vent. 
         [0031]    The sleeve of the device is constructed to be hand held and to enable the portable sealed container to be thrust by hand into the sleeve to couple with the piston and produce the movements in the first direction. 
         [0032]    The predetermined portable sealed container is a collection tube terminated in a penetrable end seal, the piston carrying a fixed, hollow penetrating needle having a protruding end exposed to penetrate the end seal during the first movement in the first direction, to enable the coupling of the piston with the predetermined container and to provide the fluid passage between the closed volume and the interior of the container, in certain preferred cases the sleeve is constructed to receive the collection tube in the form of an evacuated blood collection tube. 
         [0033]    The device is constructed to enable filtrate to be pipetted out of a filtrate collection chamber through a septum. 
         [0034]    The device includes pre-stored dilution fluid or reagent positioned to be mixed with liquid removed from the container, as an example dilution liquid is positioned in an end cap isolated from the liquid receptacle by a septum having a burst pressure that enables flow through the septum when the burst pressure is exceeded, the device enabling selective introduction of the liquid from the container to the dilution or reagent liquid by pressure applied to the piston. 
         [0035]    The device in the form of a separation device comprises: (1) a main tubular body having an elongated cylindrical central passage forming the sleeve, the sleeve being open at an upper end to receive the access seal end of a collection tube and closed at its lower end by the liquid receptacle in the form of a sample collection chamber; (2) the piston slideably held in sealed relation within the cylindrical passage, the piston being traversed by a fixed hollow, longitudinally arranged hypodermic tube selected to permit air movement across the piston and having a piercing end directed outwardly, to confront the access seal of the collection tube; (3) and a filter communicating with the main body, a function of which is to permit only liquid to discharge to the collection chamber; the collection chamber arranged to retain filtrate, such as plasma or serum or sample after passing through the filter. In preferred forms the piston is in the form of a poppet element of axial length of the order of the diameter of the sleeve passage. In preferred forms the filter comprises a filter cage element shaped as a cylindrical cup with its closed end formed as a coarse sieve, its cylindrical surface tightly fitted to the inside surface of the tubular main body, the cage holding a mass of glass fiber filter material and having its other end closed with a filter sheet, in certain implementations the collection chamber is attached to the main tubular body via a coarse thread and a seal which hermetically closes the lower end when compressed and permits air movement through the threads when loosened. 
         [0036]    A method is provided of obtaining a filtrate from blood employing the filter device comprising the steps of (a) obtaining a blood sample within an evacuated collection tube having an end sealed with a penetrable seal, (b) holding the filter device vertically, open end up, and introducing the collection tube with sealed end down, and pressing the collection tube down into the sleeve to couple with the slideable piston, then releasing the downward pressure on collection tube, (c) during downward motion some of the compressed captured air beneath the piston entering the collection tube through the passage and bubbles to the top of collection tube, and upon release of the downward pressure, the coupled assembly of collection tube and piston rising due to expansion of air captured in the closed volume, meanwhile, pressure within the collection tube having become higher than that below the assembly, causing blood to be forced out of the collection tube, into the space below, (d) optionally repeating the pressing down step at least once, each cycle causing more air to enter and raise the pressure within the collection tube, then more blood to be forced downwardly, out of the collection tube, (e) subsequently venting the closed space below the piston, (f) repeating the pressing down step once more, with no opposing air pressure, the piston acting to force blood through the filter, and the filtrate (plasma or serum) to enter the collection chamber; also, super atmospheric pressure within the collection tube causing more blood to leave the collection tube and the liquid component to be pushed by the piston through the filter to enter the collection chamber. In certain implementations the closed space is vented by partially unscrewing a bottom collection chamber one or two turns, the threads being coarse to permit air to escape as a cooperating seal formed by an O ring is freed; certain implementations include fully unscrewing a cover of the collection chamber and pipetting a desired volume of filtrate through an exposed septum followed by closing the separation device with the supplied cover and discarding or archiving the unit. 
         [0037]    The method and device are employed in filtering a blood sample followed by conducting an assay with the filtrate. In certain implementations, the assay is conducted by flowing the filtrate or liquid derived from the filtrate over a capture surface having a two dimensional array of spots of protein capture reagents or other array. 
         [0038]    Other features will be understood from the claims, drawings and the following descriptions. 
     
    
     
       DESCRIPTION OF DRAWINGS 
         [0039]      FIG. 1  is a longitudinal cross-section of a filter device assembly and an evacuated collection tube in position to be inserted into the filter device, here the device shown fitted with a filtrate collection assembly having an access septum. 
           [0040]      FIG. 2  is a side view,  FIG. 2A  a detailed axial cross-section,  FIG. 2B  an end view and  FIG. 2C  a detail view of the main body of the filter device of  FIG. 1 . 
           [0041]      FIG. 3A  shows, in axial cross-section, a liquid collection receptacle for the device of  FIG. 1 ;  FIG. 3B  shows, in axial cross-section, a filtrate collection assembly for the device of  FIG. 1  having a septum through which liquid can be withdrawn and  FIG. 3C  shows, in axial cross-section, an alternative liquid collection receptacle for the device comprising a narrow metering tube. 
           [0042]      FIG. 4  is a side view,  FIG. 4A  an end view, and  FIG. 4B  an axial cross section view (the latter with hypodermic needle and O rings installed), of the poppet/piston assembly for the device of  FIG. 1 . 
           [0043]      FIG. 5  shows, in axial cross-section, a filter assembly for the device of  FIG. 1 . 
           [0044]      FIGS. 6A to 6G  show the position of various elements of the assembly as the filtering process takes place. 
           [0045]      FIG. 7  is an exploded cross-section view,  FIG. 7A  an end view and  FIG. 7B  a fragmentary assembled view of a filter assembly which includes a filter cage for holding glass fiber filter and a final filter. 
           [0046]      FIG. 8  is a fragmentary axial cross section of the lower end of a filtrate collection assembly having a closed filtrate collection chamber for serum or plasma, an access septum and a removable cover. 
           [0047]      FIG. 9  is a fragmentary axial cross section of the lower end of the construction of  FIG. 8  having the cover removed to expose the access septum to the filtrate collection chamber and illustrating pipette extraction. 
           [0048]      FIG. 10  is a fragmentary axial cross section of the lower end of an alternative construction having a graduated filtrate collection chamber. 
           [0049]      FIG. 11  is a fragmentary axial cross section of the lower end of an alternative construction having a graduated filtrate collection chamber (e.g. for serum or plasma) partially pre-filled with a defined volume of reagent. 
           [0050]      FIG. 12  is a diagrammatic plan view of the main body of an assay cassette having an array of capture reagents with which filtrate from the filtering device is useful. The Figure is FIG. 4 from provisional U.S. Patent Application 61/030,276, filed Feb. 21, 2008, the entire contents of which are incorporated herein by reference. 
       
    
    
       [0051]    Like reference symbols in the various drawings indicate like elements. 
       DETAILED DESCRIPTION 
       [0052]    Referring to  FIGS. 1 and 6A  to  6 G, in the preferred implementations of the figures a filter device  8  suitable for blood separation is constructed to operate with a standard evacuated blood collection tube  10  (Vacutainer™) which, at its access end, has a needle-pierceable soft rubber seal member  10   a  or other penetrable seal that is capable of self-sealing after being penetrated by a needle. 
         [0053]    The filter device  8  comprises four major components:
       1. A main cylindrical tube-like body  12  which has an elongated central passage that is open at its “upper end” to receive the access end of the collection tube  10  and is constructed to be closed at its “lower end” by structure defining a filtrate collection chamber or receptacle  14 .   2. A cylindrically shaped “poppet” element  16  that is slideably held in sealed relation within the cylindrical passage of main body  12 . In this preferred implementation, poppet element  16  is positioned in axial alignment with the passage by two axially spaced-apart O rings  18   a, b , (or in other embodiments by at least one O ring or equivalent seal and alignment guide), in a piston like manner. Poppet element  16  is traversed centrally by a fixed hollow, longitudinally arranged hypodermic tube needle  20  which permits air movement across the poppet element. A sharp, piercing end of the needle is directed outwardly, to confront the seal member  10   a  of the collection tube  10 .   3. A filter cage element  22  that is shaped as a cylindrical cup with its closed end  22   a  formed as a coarse sieve. Its cylindrical surface is tightly fitted to the inside surface of main body  12 . The cage holds a mass  24  of glass fiber filter material (“glass wool”) and has its other end closed with a film shaped filter  23  a function of which is to permit only fluid to discharge to the collection chamber  14 .   4. A collection chamber  14  in which the filtrate, such as plasma or serum or sample is retained. Collection chamber  14  is typically attached to the main body  12  via a coarse thread  14   a  and seal  14   b  such as an O ring which hermetically closes the lower end when compressed. A number of variations of the collection chamber are suggested below.       
 
         [0058]    The separation process for blood is quite simple and may require about a minute:
   1. Obtain a blood sample within the conventional evacuated collection tube  10 , (Vacutainer™). When inverted with its rubber access seal  10   a  down, blood may reach level L, occupying 70% of the collection space within the tube.   2. (a) Holding the filter device  8  vertically, open end up, introduce the inverted collection tube  10  and press it gently down into the main separator tube body  12 , pushing the poppet element past the commencement of restraint  17  ( FIGS. 2 and 2C ) to a stop. (The first time the poppet element encounters restraint, needle  20  of the restrained poppet  16  penetrates the downward moving rubber seal  10   a  to connect the collection tube  10  and poppet into an assembly that remains together throughout further operation). The downward stroke of the poppet  16  causes air below to be compressed. In a first equilibrating action, some of this compressed air passes from beneath poppet  16  through hypodermic needle tube  20  and bubbles to the top of the space within the collection tube  10 , raising the air pressure within tube  10 . (b) Then release the collection tube  10 , while holding body  12  of device  8 . The connected assembly of collection tube  10  and poppet  16  automatically is forced to rise to a position close to the original position due to expansion of the compressed air captured between the connected assembly and the closed lower end of the main body  12 . With the occurrence of this expansion, air pressure within the collection tube  10  becomes relatively higher than that below the returning assembly. This sets up a second automatic equilibrating action, in which the higher air pressure in the collection tube  10  forces flow of blood out of the collection tube  10 , downwardly through the hypodermic needle  20 , into the space below the poppet  16 , above the filter material  24 .   3. Repeat steps  2 ( a ) and  2 ( b ) one, two or three times depending upon the amount of filtrate desired, each cycle causing ( 2   a ) more air to enter to temporarily raise the pressure within collection tube  10  in the first equilibrating action, then ( 2   b ) more blood to be forced downwardly, out of the collection tube  10 , into the space below, by the second equilibrating action.   4. Partially unscrew the bottom collection chamber  14  one or two turns. The threads are coarse to permit air to escape as the O ring  14   b  is freed and its seal broken.   5. Repeat step  2 ( a ) once more. With no opposing pressure of captured air, poppet  16  acts as a discharge piston to force the below blood through the filter  24 ,  23 , and the filtrate (e.g., plasma or serum) into the collection chamber  14 . Also, superatmospheric pressure within the collection tube  10  causes more blood to leave collection tube  10  and the fluid component to be pushed through the filter to enter the collection chamber  14 . Blood clots, if any, will be retained on top of the filter cage.   6. In the case of use of the filtrate collection assembly of  FIGS. 1 ,  3 B,  8  and  9 , fully unscrew and remove the cover  36  and pipette a desired volume of filtrate from collection chamber through an exposed septum  32 .   7. Close the filter device with the supplied cover  36  and discard or archive the unit.   
 
         [0066]    Blood Collection; Evacuated Collection Tube (Vacutainer™) 
         [0067]    Referring to FIGS.  1  and  6 A- 6 G, in preferred implementations blood is collected from a patient through a vein puncture device into a standard evacuated collection tube  10  such as a Vacutainer™ (Becton Dickinson), preferably container model 10.25×47, 10.25×64 or 10.25×82 with draw capacity of 1.8, 3.0, 3.2 ml respectively, each having a needle-penetrable access seal. The collection tube chosen reflects the volume of plasma or serum required. The tube commonly holds a small volume of material intended to prevent clotting of the blood, occupying as much as 10% of the volume of the blood. 
         [0068]    The air pressure within the evacuated collection tube  10  commences at approximately 30% of sea level atmospheric pressure. When correctly used, tube  10  fills to approximately 70% of its volume with blood, holding air in approximately 30% of the volume, at pressure now close to atmospheric pressure. The evacuated collection tube is then separated from the vein puncture device. 
         [0069]    Main Body  12   
         [0070]    Referring to  FIGS. 1 ,  2  and  2 A, the internal diameter of the main body  12  is slightly larger than the diameter D 3  of the evacuated collection tube (Vacutainer™) such that the collection tube can be installed without difficulty with alignment. In a preferred implementation collection tube  10  is approximately 10.25 mm in outside maximum diameter, D 3 . The main body  12  of the blood separator is approximately 3 inches long, L 1 , made of a transparent plastic with an inside diameter D 2  approximately 0.500 inch and an outside diameter D 4  of ⅝ inch. The upper portion of body  12 , above dimension L 2  may be enlarged to 0.505 inch inside diameter, D 1 . The smaller dimension D 2  in the region below this is intended to create a predetermined holding restraint acting on O rings  18   a  and  18   b  of the poppet for instance of about 2 pound. This is in excess of the resistance force required to cause hypodermic needle  20  to pierce the downwardly moving rubber seal  10   a  of the collection tube, a force less than about 2 pounds in a typical system. The predetermined holding restraint force is sized to be overcome by resilient deformation of the “0” rings. Thus an increased hand force on the collection tube  10  downward propels “poppet”  16 , beyond step  17 , through the main body. 
         [0071]    When commencing use, collection tube  10  is about ⅔ filled with blood. It is inserted in the body  12  of the device and pushed inwardly with sufficient force to impale the septum on the needle and then to proceed downwardly to pressurize captured air, forcing air to pass into the collection tube, thus pressurizing its liquid content and the void space above the liquid. 
         [0072]    The Poppet Element  16   
         [0073]    Referring to  FIGS. 1 ,  4 - 4 B and  FIGS. 6A-6G , the piston, in preferred form the poppet element  16 , with its needle, has 3 functions:
       Pierce the seal  10   a  of the collection tube  10 ,   Pressurize the air in the collection tube to transfer blood or other liquid out,   Force the blood or other liquid through the filter  24 ,  23  and into the filtrate (e.g. plasma or serum) collector  14 .       
 
         [0077]    The poppet  16  is a short rod, its length preferably of the order of its diameter, with two annular grooves ( FIG. 4 ), held in place with two O rings  18   a ,  18   b ,  FIG. 4B , installed in the grooves. The grooves are separated axially by approximately ½ diameter D 2  of the main body internal diameter in order to keep the poppet approximately aligned. The poppet is traversed by a fixed hypodermic needle tubing  20  of approximately 0.036 inch outer diameter with a sharp protruding free length, L 3 , approximately ½ inch, sufficient to pierce through and extend slightly beyond the rubber seal  10   a , into the collection tube  10  (Vacutainer™) 
         [0078]    Prior to use, the poppet  16  with the sharp end of the hypodermic tubing  20  protruding, rests near the entrance of the device but enclosed sufficiently within such that a user would not reach it accidentally. It rests within a slightly enlarged region, typically with diameter of 0.505 inch, such that the force to displace it further downward exceeds the force required to impale the seal  10   a  by the protruding hypodermic tubing  20 . 
         [0079]    The two O rings  18   a ,  18   b  align the poppet and offer a pressure tight seal with main body  12  such that pushing the collection tube  10  (Vacutainer™) further within the main body compresses the air in the device as well as within the collection tube  10 . The volume within the device is preferably defined such that pushing the collection tube  10  to the end of its permitted travel pressurizes the device and collection tube  10  to approximately 3.5 atmospheres. 
         [0080]    The Filter Assembly  22 ,  22 A,  24 ,  23   
         [0081]    Referring to  FIGS. 1 ,  5  and  7  the filter, when adapted to filter, e.g., blood, is preferably built as a subassembly bounded by filter cage  22  that can be tightly fitted within the main body  12 . Cage  22  contains glass wool filter material  24 . The filter cage  22  is preferably shaped as a slightly tapered cylinder closed at its upper end with a very coarse perforated filter  22   a  with as many holes as practical, each of approximately 1 mm opening. This filter  22   a  prevents clots from passing but also retains in place the glass fiber filter material  24  when decompression occurs by the upward movement of poppet element  16 . The filter cage  20  and the coarse filter  22   a  preferably comprise a single molded part of synthetic resin. In a preferred implementation the lower end of the cage is of slightly larger diameter D 5  than the upper end of the cage of diameter D 6 . For instance the cage is formed of plasticized PVC which is malleable, and the cage is press-fit from below into the passage of the main body  12  to form a seal. For instance D 5 =0.503 inch and D 6 =0.495 inch. 
         [0082]    The filter cage  22  may also serve as a stop for the poppet&#39;s travel, but its main function is to block possible clots of red blood cells from entering the glass fiber section and blocking it. 
         [0083]    The middle region of filter cage  22  is approximately 1 diameter long, 0.5 to 0.6 inch long in the preferred implementation. It holds the volume of glass fiber  24  in an approximately uniform distribution 
         [0084]    A finer filter section  23  is provided at the exit end of filter cage  22  to prevent loose fiber elements of the glass fiber filter from escaping into the collection chamber  14 . 
         [0085]    For this purpose, filter cage  22  is closed with a filter material  23  such as Versapor 1200 or Versapor 3000 filter material from VWR international. This is similar to a filter paper with 1.2 or 3 micron porosity. This filter may be bonded to close the filter cage  22  as shown in  FIG. 7B  or placed below it and pushed against a seal such as an O ring or a rubber ring, not shown. 
         [0086]    In some applications, the glass filter or section of the glass filter is coated with a reagent specifically designed to capture some or most of specific molecules that should be excluded from the sample. The high density of fibers and the small cross dimensions and long flow dimensions of the meandering pathways through the filter provide intimate exposure of the filter material to the liquid passing through for such reactions. 
         [0087]    In addition a number of features may be incorporated within the main body in order to retain the filter material located in the filter section. The filter material may include a number of filter media with different properties, some properties being filtering properties and others may have molecular interaction capability with the blood to be processed. For instance, desiccated bio-active reagents having long storage life may be carried by a layer of filter material for release to the liquid or for interaction with designated constituents of the fluid passing through the filter material for labeling, as by fluorescent labels, capture by immobilized capture agents or for other purposes. 
         [0088]    The Filtrate Collection Assembly 
         [0089]    Referring to  FIGS. 1 ,  3 A- 3 C  6 A to  6 G and  8 - 11 , the sample collection assembly of whichever form selected is hermetically sealed on the lower end of the main body  12  so that it can be pressurized. It must be constructed such that at a later stage the seal may be terminated and air can escape and blood can flow through the filter. The air can escape safely to the atmosphere but all liquids must be constrained within the chamber. A micro-porous plug of annular form such as Porex filter material compressed between the lower end of the main body  12  and mating structure of the collection chamber  14  guaranties that no liquid can escape while air can pass through the material. 
         [0090]    An additional function of the collection assembly is to permit easy extraction of the filtrate preferably with a pipette. 
         [0091]    In an alternate construction, see  FIGS. 3C and 11 , the collection assembly may hold, in a sealed manner, a specified volume of buffer or reagent such that a predetermined dilution of the filtrate can take place within the device. 
         [0092]    In preferred implementations the filtrate collection assembly is composed of a chamber that is fastened to the main body  12  via a coarse clockwise thread loosely fitted, such as ½-12 NC. As shown in  FIGS. 1 and 6A , the chamber is spaced with an O ring  14   b  that seals hermetically the two parts when the chamber is fully tightened. As shown in  FIGS. 1  and a micro-porous ring-shaped filter of Porex material  40  for instance is lodged between the two parts to guaranty that no liquid can escape. 
         [0093]    In preferred implementations, see  FIGS. 1 and 8 , the chamber is closed at its outer end with a cover  36  which compresses and seals a septum  32 . The septum is preferably pierced at its center so that when the cover is removed, a pipette (or a syringe) can be entered to extract the filtrate. 
         [0094]    The cover  36  is fastened to the collection chamber  14  with a counterclockwise thread such as ½-20 NF or 7/16-20 NF. The counter clockwise screw thread is employed so that removing the cover  36  causes the chamber  14  to tighten its seal against the main body  12  of the device. 
         [0095]    Referring to  FIGS. 3C ,  10  and  11 , in other implementations, the cover  36 ′ or  38  of transparent material is shaped in an elongated form with volume indications so that a fixed volume of filtrate may be collected by the user. In this condition, after the collection chamber has received all the filtrate, the cover  36 ′ or  38  and the collection chamber are again tightened and filtrate is forced through the perforated septum by pumping movement of the interconnected collection tube  10  and poppet  16  with sufficient force to exceed the fluid “burst” pressure of the perforated, self-sealing septum. The filtrate can than be pipetted out when this elongate cover is removed. Suitable covers may be used to seal both the separated, liquid-filled “cover”  36 ′,  38  and the collection chamber  14  of the device. 
         [0096]    Referring to  FIG. 11 , in another implementation, the elongated cover  36 ′ holds a pre-determined volume of diluent, such as buffer or distilled water. The same process described above can be used to transfer a defined volume of filtrate into the volume by which the filtrate is diluted to the predetermined degree desired. Likewise the pre-stored liquid may contain a reagent for the assay. 
         [0097]    Sample Extraction 
         [0098]    As noted above, in respect of filtering of blood, the blood is drawn from the patient in the conventional manner and the collection tube  10  is inserted vertically, seal  10   a  down, in the appropriate filter device  8 . The open end of the filter device holds poppet element  16  in the main body with the two sealing O rings  18   a  and  18   b , the poppet holding in its center a hollow hypodermic needle  20  that opens the inside of the device, within body  12 , to atmospheric pressure. Pushing the collection tube  10  inside the filter device  8  with a force less than 1000 gram, often under 800 gram, pierces the seal  10   a  which links the inside of the collection tube  10  to the volume of the filter device within body  12  and closes access to atmospheric pressure. The tip of the needle then just protrudes through the seal  10   a , into tube  10 . 
         [0099]    Continuous displacement of the tube  10  downwardly compresses the air within the filter device and forces air within tube  19  until a force of approximately 4 or 5 kilogram is required to reach a stop. The pressure within the device  8  and collection tube  10  reaches a level that is approximately 3.5 the atmospheric pressure and air is forced within the collection tube through the blood to the top of the tube by a first equilibrating action. 
         [0100]    When the force bringing the parts  10  and  8  together is removed, the collection tube  10  is pushed outwardly by the trapped compressed air until the pressure within the device  8  exerts a force equivalent to the friction of the poppet  16  in the tube or about 0.8 kilo. The pressure in the device  8  is reduced to approximately one atmosphere above ambient. In a second equilibrating action, this causes the air trapped in the upper part of the collection tube to expand possibly as much as 3 times, forcing out blood into the body  12  of the device and within or above the filter material. The steps may be repeated until sufficient amount of blood has been pushed within and above the filter material. 
         [0101]    When sufficient blood has been displaced, and the collection tube  10  fully extended outward, unscrewing the filtrate collection chamber  14  from body  12  releases the internal pressure, the captured air escaping through the relieved seal and through the loose-fitting threads. This forces some liquid through the filter and filtrate into the filtrate receptacle  14 . 
         [0102]    If it is necessary that no filtrate should enter the original filtrate receptacle, the device should be turned upside down when the serum receptacle  14  is unscrewed. An alternate receptacle can then be installed and the unit returned to the vertical with the new receptacle at the bottom. 
         [0103]    Pushing collection tube  10  back into device  8  forces more blood through the filter, a process that may be aborted as needed or performed with a different filtrate receptacle. Such receptacle may be graduated so that a specific volume is taken. 
         [0104]    In another implementation, the filtrate chamber may be shaped as a tube to hold a defined volume of buffer or similar dilution fluid required for a later processing of the serum or plasma. Such chamber would preferably be sealed until put in use. 
         [0105]    In another implementation, the filtrate receptacle chamber may be fitted with a septum  32  that can readily be pierced with a pipette or a syringe to meter out a specific volume of serum. 
         [0106]    Filter Description 
         [0107]    Filters are commonly used to separate serum from whole blood. The use of hollow fiber filters are practical if the serum sample is small, typically under 20 microliters (U.S. Pat. Nos. 6,755,802 and 5,919,356). 
         [0108]    The use of filters has been described where the volume and properties of the filter are able to hold the quantity of red cells that need to be separated from the blood sample. 
         [0109]    U.S. Pat. No. 4,477,575, incorporated herein by reference, generously describes such a filter in column 10 line 56-68 and table 2: 
         [0110]    “Separate Recovery of Plasma 
         [0000]    A synthetic resin vessel which downwardly narrows conically (e.g. a synthetic resin tip with a piston pipette, length 5 cm., thickness 0.5 cm.) is loosely filled two thirds full with glass fibers according to the following Table 2, packing densities of 0.1 to 0.4 g./cm 3  being obtained. After the upper free part has been filled with blood, the serum diffuses into the tip of the vessel. From there, an “end-to-end” capillary of 15 μ.l. capacity can be filled by attachment to the opening of the pipette tip. The plasma obtained in this manner can now be used directly for any desired analytical process.” 
         [0111]    The glass fiber filter used in the present devices is generally as described in this patent with the addition of a 1.2 to 3 micron filter downstream that blocks any segment of glass fiber. 
         [0112]    The glass fiber material filter may be purchased from Johns Manville or from PALL/VWR as part 288150-995 and the 0.7 micron filter as part 28149-455 from PALL/VWR. 
         [0113]    Blood serum collection and use is subject to many variables:
       The serum fraction of a blood sample in a normal control subject is similar to that among diabetic patients and ranges from approximately 45% to 70%.   Serum is used undiluted or in a diluted form with dilution ranging from 10% to  2 ×[adding 10% up to an equal amount of diluents]       
 
         [0116]    Some assays demand a filtrate of plasma or serum from which a number of molecules have been removed. This may readily be achieved when appropriate capture agents are imbedded or otherwise immobilized in the filter material that may capture specific molecules such as fibrinogen or minimize the presence of over-expressed proteins the overabundance of which may overwhelm an assay. Amylopectia Sulfate (APS) may be such an agent that can be introduced in a dispersed manner within the glass fiber filter to capture in a distributed, non clogging manner platelets and red cells causing minimum alteration to the serum proper. 
         [0117]    In the event a precise ratio is desirable it may not be practical to incorporate the diluting agent or reagent within the collection tube  10 . The system described here offers a method for accurate dilution or reaction. 
         [0118]    In some assays where the dilution ratio may not be critical, a dilution agent only may be incorporated in the collection tube  10 . 
         [0119]    As described in U.S. patent application 61/030,276 filed Feb. 21, 2008, incorporated by reference, a filter material may be employed to temporarily store a desiccated agent, such as an agent having bio-activity such as a suitably conjugated fluorophore label. Such filter material carrying an agent can be employed as filter  23  as a means to liquefy and dispense the agent into the filtrate. Indeed, it is possible to employ only such filter material, (omitting filters  22   a  and  24 ), and to employ the device simply as a device to dispense an agent into appropriate liquid. 
         [0120]    Blood Protein Assay 
         [0121]      FIG. 12  illustrates the body of a bio-chip cassette for protein. Its plan view is the size of a credit card. Chamber  2  receives the filtrate (plasma or serum) prepared as described above, with or without dilution or additives, depending upon the assay. Chamber  110  holds buffer solution that provides all other liquids for an ELISA-like assay. At chamber  6  is a reaction gap through which the liquids sequentially flow to expose a two dimensional array of spots of capture reagent applied to a solid nitrocellulose coating on a glass substrate, not shown. Spent liquid proceeds to waste chamber  19 . There is a transparent window overlying the array, spaced apart by a small flow gap, not shown. After fluorescent labeling of the captured blood protein and washing by buffer liquid, the array is read by stimulating radiation passing in through the window and exited fluorescent emission passing from the labels out through the window. 
         [0122]    A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. For example, the filter material may be selected for body fluids other than blood, and for other purposes, such as for agent dispensing, instead of for filtering; the pressure relief device may be a valve or other device that can be opened to the atmosphere instead of depending on loosening of a threaded attachment. Accordingly, other embodiments are within the scope of the following claims.