Abstract:
A multi-part device for separating plasma from whole blood is provided, with a sample taking unit for receiving whole blood, a filter unit with a layered filter with multiple layers for extracting plasma, and a pumping unit, typically a plunger pump, for creating a partial vacuum in the filter unit. The filter unit and a plasma collector vessel with a conical tip extending towards the filter unit are contained in a filter cartridge, which may be taken apart after plasma extraction, thus exposing the conical tip of the plasma collector vessel for sample input into an analyzer.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of International Patent Application No. PCT/EP2013/066539, filed 7 Aug. 2013, which claims the benefit of European Patent Application No. 12179900.1 filed 9 Aug. 2012, the disclosures of which are hereby incorporated herein by reference in their entirety. 
    
    
     BACKGROUND 
     The present disclosure relates to a multi-part device for separating plasma from whole blood. 
     Besides centrifuges, which are used mainly in laboratories for separating plasma from whole blood, there are known a number of devices for obtaining very small amounts of plasma at the Point of Care (PoC) by separating plasma from whole blood by means of filtering. 
     In the simplest case plasma separation may be effected by means of a multilayer test strip as described in U.S. Pat. No. 5,262,067 A (BOEHRINGER MANNHEIM), where a transport layer on an inert carrier layer is provided for transporting sample fluid (whole blood) from an input area to a measuring area. The transport layer may for instance be made of glass fibre mat, which in the input area is covered by a plasma separation layer. The procedure is however only suitable for analysers processing test strips. 
     From EP 0 550 950 A2 (SANWA KAGAKU KENKYUSHO) there is known a method and a device for separating blood serum and plasma. This document presents diverse variants of devices for plasma extraction, where for instance in FIGS. 1 to 4 variants are described in which a plasma separating device is integrated in a blood sampler. By means of a partial vacuum blood is first sucked into a collector vessel in which there is disposed a two-layer separating filter. 
     After the blood sample has been taken the collector vessel is connected to an evacuated fluid container, the plasma being separated by the separating filter and collected in the fluid container. In the variant shown in FIGS. 5 and 6 the partial vacuum required for plasma separation is generated by means of a plunger syringe. The variant of FIGS. 9 and 10 furthermore shows a kind of syringe input filter, which may also be used for obtaining plasma. 
     From WO 96/24425 A1 (FIRST MEDICAL INC.), especially from its FIGS. 1 to 3 and 8, a method and device for plasma separation is known. A device called “Blood Separation Device” comprises a filter element, a flexible tube and at its end a needle which is introduced into a “Blood Collection Device”. By means of a motor unit comprising a peristaltic pump acting on the flexible tube whole blood is sucked from the “Blood Collection Device” and pumped through the filter element, whereby plasma is separated and can be obtained for further use at a plasma output opening of the filter unit. The relatively high uncontrolled pressure values met at the filter unit when pressure is applied and the partial vacuum occurring in the collection vessel when whole blood is continuously sucked off are disadvantageous. 
     EP 1 469 068 A1 discloses an apparatus for separating and purifying nucleic acids, which comprises a cylindrical syringe having a leading end part in which a first opening part is formed, and an accommodation part being able to hold liquid therein. A solid phase-holding member is connected to the leading end part of the syringe and a flow hole is formed at the leading end side of the solid phase-holding member. A solid phase comprised of an organic polymer having a hydroxyl group on the surface thereof and being able to adsorb and desorb nucleic acids in a sample solution is accommodated in said solid phase-holding member. In the leading end part of the syringe there is formed a liquid-guiding surface of a shape that increases the diameter of the cross section towards the solid phase element. The apparatus can be used for separating and purifying nucleic acids but does not work as plasma separation device. 
     From U.S. Pat. No. 6,761,855 B1 an improved column for use in solid phase synthesis or purification of complex chemicals, such as biomolecules and more specifically oligonucleotides is known. The column has a top orifice with a sufficient diameter so that a fluid line or a multiple fluid line bundle may dispense fluids into the column with great efficiency. The column has an upper cavity portion configured and sized to render it compatible with dispensing pipettes, so that it can be used as a pipette tip to aspirate the column. The column has a lower cavity portion with a shoulder for ready placement of a lower frit to contain the solid support in a central cavity portion of the column. An upper frit can be conveniently placed in the central cavity portion to seal the solid phase resin. The lower end tip of the column is configured as a Luer-type fitting to provide a male Luer connection. The upper cavity portion is configured to interface with the male Luer of another column, so that two or more columns to be connected in series. 
     U.S. Pat. No. 4,046,145 B1 shows a connector for joining a small dose syringe to a large reservoir syringe for filling the small dose syringe from the reservoir syringe. The connector has a tubular female-to-female coupler and can be furnished with a filter element. Thus, particulate matter that might be within the large reservoir syringe is filtered out prior to its transfer to the small dose syringe. 
     It is an object of the present disclosure to propose a device for separating plasma from whole blood, which should be simple and economical to handle and where plasma samples for subsequent application steps may be obtained even from small blood samples and/or samples with high haematocrit values and where these samples may be fed to the input unit of an analyser in a simple way. 
     SUMMARY 
     It is against the above background that the present disclosure provides certain unobvious advantages and advancements over the prior art. In particular, the inventors have recognized a need for improvements in plasma separation systems and methods for plasma separation. 
     In accordance with one embodiment of the disclosure a multi-part device for separating plasma from whole blood is provided comprising a sample taking unit for receiving whole blood, a filter unit with a layered filter with multiple layers for extracting plasma, and a pumping unit, typically a plunger pump, for creating a partial vacuum in the filter unit. The disclosure further relates to a filter cartridge with a multi-layer filter unit for separating plasma from whole blood. 
     According to the disclosure this object is achieved by proposing that the filter unit and a plasma collector vessel with a conical tip extending towards the filter unit be contained in a filter cartridge, which is separable or may be taken apart after plasma extraction, thus exposing the conical tip of the plasma collector vessel for sample input into an analyser. The plasma collector vessel penetrates with its tip a seal towards the filter unit and is held on its opposite end by a supporting element, typically by a clamping seal, which has a passage leading to the pumping device, e.g., a plunger pump. 
     The seal towards the filter unit and the supporting element or clamping seal in the filter cartridge define a dead volume or compensation volume, which is connected via an air-permeable connection, for instance a compensation opening or a porous membrane, with the pumping device. In this way, the filter unit will not be subjected to pressure in an uncontrolled direct way (dependent on the handling of the plunger of the pumping unit) but slowly and with uniformly diminishing intensity, the pressure situation being determined by the geometry (e.g., the ratio between suction volume of the plunger pump and compensation volume in the filter cartridge) of the individual parts of the separation device and the characteristics of the filter element. 
     Typically the filter cartridge may be taken apart or separated by pulling, screwing or wrenching off the plasma applicator containing the plasma collector vessel with conical tip from the filter housing containing the filter unit. After plasma extraction and detaching of the plasma applicator, the plasma collector vessel with conical tip may by means of this tip be directly docked onto the input opening of an analyser, and the plasma sample obtained may be sucked into the analyser for subsequent analyte determination. 
     According to another embodiment of the disclosure, a filter cartridge with a multi-layer filter unit for extraction of plasma from whole blood is provided, the filter cartridge having in its interior a plasma collector vessel with a conical tip that penetrates a seal towards the filter unit and where the plasma collector vessel is held in the filter cartridge on its opposite end by a clamping seal. The filter cartridge may be disposed within or integrated or set in a syringe or a so-called Monovette. (Sarstedt AG &amp; Co. Kommanditgesellschaft, having a location at Sarstedstraβe 1, 51588 Nuembrecht, Germany, sells a line of blood collection devices under the registered trademark S-Monovette®.) 
     In accordance with one or more other embodiments of the disclosure, the collector vessel containing whole blood can be connected to a filter unit by introducing a suction tube and an aeration tube of the filter unit into the collector vessel. Also, the partial vacuum in the filtering device can be controlled by a control device of the analyser, typically by pressure dependent control of the flow rate of the suction pump. 
     These and other features and advantages of the embodiments of the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following detailed description of the embodiments of the present disclosure can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which: 
         FIG. 1  is a first variant (syringe) of the device according to the disclosure for separating plasma from whole blood in sectional view; 
         FIG. 2  is the first part of the device according to  FIG. 1  after plasma extraction; 
         FIG. 3  is the second part of the device according to  FIG. 1  after plasma extraction; 
         FIG. 4  is a second variant (Monovette) of the device according to the disclosure for separating plasma from whole blood in sectional view; 
         FIG. 5  is a Monovette for plasma extraction according to  FIG. 4 ; 
         FIG. 6  is a filter cartridge according to the disclosure for separating plasma from whole blood, to be inserted into a Monovette according to  FIG. 5 ; and 
         FIG. 7  is the filter cartridge of  FIG. 6  inserted into the Monovette of  FIG. 5  in a sectional view. 
     
    
    
     Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present disclosure. 
     DETAILED DESCRIPTION 
     The various embodiments of the present disclosure have a filter cartridge  13  as a common element, either as an integral component of the sample taking unit (see syringe  11  of  FIG. 1  or  3 ) or as a separate part, which is inserted by the user into a sample taking unit (see Monovette  12  of  FIGS. 4 to 7 ). The filter cartridge  13  comprises a filter unit  10  with a multi-layered filter, and a plasma collector vessel  17  with a conical tip extending towards the filter unit  10  (plasma tip). The filter cartridge  13  may be separated or broken apart after plasma extraction along a plane ε, yielding a plasma applicator  14  containing the plasma collector vessel  17  and a filter housing  8  containing the filter unit  10 . 
     The first variant shown in  FIGS. 1 to 3  has a filter cartridge  13  integrated in a syringe  11  with a Luer cone  22  formed on the filter housing  8 , onto which a needle may be placed for taking a whole blood sample. In the housing of the syringe  11 , in whose plunger housing  20  a plunger  18  is disposed that can be manually moved by means of a rod from an initial position to an end position indicated by a broken line, a filter unit  10  and a plasma collector vessel  17  with a conical tip extending towards the filter unit  10  are provided as the essential parts of the filter cartridge. The plunger housing  20  with the slideable plunger  18  serves as pumping device for creating the partial vacuum required for plasma separation. 
     On activation of the device the whole blood sample  41  is sucked into the filter unit  10  by pulling back the plunger  18 , whereby a plasma front or plasma fraction  40  is generated, which moves into the tip  9  of the plasma collector vessel  17  through the multi-layered filter consisting of a deep-bed filter  3 , a small-pore stop membrane  4  for complete removal of solid blood components (mainly red blood cells, RBCs) and a lateral grid  5 . 
     The plasma collector vessel  17  with its tip  9  penetrates a seal  15  towards the filter unit  10  and is held and sealed on the opposite open end by a supporting element, typically by a clamping seal  27 , which has a passage towards the pumping device (plunger part  20 ). In this passage towards the pumping device a hydrophobic, air-permeable element  16  (liquid stop) is provided. This will prevent the outflow of separated plasma through the passage opposite the tip  9 . 
     The seal  15  towards the filter unit  10  and the clamping seal  27  towards the plunger housing  20  define a compensation volume  21  in the filter cartridge  13 , which is connected to the pumping device of the syringe  11  via an air-permeable passage, for instance a compensation opening  25  in the clamping seal  27  or a porous membrane. 
     In a first variant of the device according to  FIGS. 1 to 3  plasma extraction may be carried out as follows:
         Taking the syringe  11  with integrated filter cartridge  13  from a sterile package.   Placing a needle onto the Luer cone  22 .   Puncturing a selected blood vessel with the needle.   Sucking in a blood sample by pulling back the plunger  18  until it hits the stop.   Locking the plunger rod in a locking position  24 .       

     (Option: the plunger rod may be broken off to avoid reversal of the internal flow direction or pressure fluctuations and any resulting contamination of the obtained plasma at high haematocrit values and/or small sample volume.) 
     The deep-bed filter  3  of the filter unit  10  may for instance be built up from glass fibers without binding agent (typically FV-2, Whatman Inc., resp. DE 40 15 589 A1, or EP 0 239 002 A1 Böhringer-Mannheim) with a retention range of 0.5 μm to 10 μm, typically 1 μm to 5 μm, more typically &lt;3 μm. The red blood cells (RBCs) will collect on the thin glass fibers of the deep bed filter  3  without bursting or unduly influencing the rate of flow.
         Depending on the cross-section of the filter unit  10  and on haematocrit a “plasma front” or “plasma fraction”  40  will form, which can pass the stop membrane  4  unimpededly. Residual RBCs not held back by the deep-bed filter are filtered out by the stop membrane  4 . For this purpose the stop membrane  4  has a pore size significantly smaller than that of the deep-bed filter  3 , i.e., pore diameters of less than 400 nm, typically less than 200 nm. By combining a deep-bed filter  3 , which on account of its pore size already retains the greater part of blood cells but does not impede the flow of the plasma fraction, with a subsequent stop membrane  4 , which due to its smaller pore size will reliably retain remaining blood cells, but would clog swiftly on account of its limited number of pores if the preceding deep-bed filter  4  were absent, a reliable separation of blood cells without clogging of the filter can be achieved, thus making it possible to obtain a sufficiently large volume of plasma sample.   The partial vacuum of not more than 500 mbar, typically 300 mbar, more typically 100 to 150 mbar, established in the filter unit  10 , together with the geometry of the filter unit  10  (ratio of compensation volume  21  to suction volume  23  of the plunger housing  20 ) will determine the flow rate and thus the shear forces acting especially on the RBCs within the stop membrane  4  of the filter unit  10 . Bursting of RBCs (haemolysis) can efficiently be prevented by optimizing the compensation volume  21 .   The lateral grid  5  of the filter unit  10  permits plasma to be collected and sucked off behind the stop membrane  4  towards the plasma collector vessel  17  by efficiently preventing the stop membrane  4  from “sealing off” tightly. Due to its grid structure the lateral grid  5  on the one hand acts as a non-continuous support for the stop membrane  4 , letting plasma flow out on the output side of the stop membrane  4 . By forming channels the grid structure furthermore enables plasma that exits over the area of the stop membrane  4 , to converge towards the plasma collector vessel  17 .       

     (This functionality of the lateral grid  5  may alternatively be provided by structuring the side of the seal  15  facing the stop membrane, e.g., by stamping, or otherwise providing for sufficient roughness of its surface.)
         Plasma extraction is also ended by:
           when the stop membrane  4  is clogged by particulate components of blood, or   especially in the case of haematocrit values &lt;40%, by a hydrophobic, air-permeable element  16  (liquid stop) at the end of the plasma collector vessel  17  or in the passage of the clamping seal  27 , which terminates further plasma extraction when the lumen of the plasma collector vessel  17  bounded by the hydrophobic, air-permeable element  16  is completely filled with plasma.   
           By means of marks on the plasma collector vessel  17  one can optionally ascertain by visual inspection that the desired amount of plasma has been obtained.   The filter housing  8  of the syringe  11  (see arrow  26  in  FIG. 2 ) is unscrewed from or wrenched off the plunger part  20  and thus the filter cartridge  13  is divided along a plane E into a first part containing the filter unit  10  and into a plasma applicator part  14  containing the plasma collector vessel  17  with conical tip, the tip  9  of the plasma collector vessel  17  thus being exposed ( FIG. 3 ).       

     In the variant shown the inherently higher static friction of the clamping seal  27 , as compared with the seal  15  of the plasma collector vessel  17  with its smaller sealing surface, will ensure safe undocking of the tip  9  of the plasma collector vessel  17  prior to the exposure of aeration channels  19  in the housing walls due to further turning or pulling-off of the housing, which channels will permit fast pressure compensation between the suction volume  23  in the plunger housing  20  and the compensation volume  21 , either via the partly porous clamping seal  27  and/or via the compensation opening  25 . 
     Early undocking will also ensure that unforeseen complications and contaminations do not occur at the tip  9  of the plasma collector vessel  17  during the separation procedure. 
     The liquid-stop  16  in the clamping seal  27  at the end of the plasma collector vessel  17  will also prevent fractionating of the plasma sample in the area at the point of tip  9  if pressure compensation between suction volume  23  and compensation volume  21  is retarded. 
     (Alternatively the tip  9  of the plasma collector vessel  17  may be designed as a Luer cone).
         Docking the plasma collector vessel  17  (plus plunger housing  20  acting as a handle), which is at least partly filled with the plasma obtained, onto the input opening of an analyser not further shown here.   Entering the plasma sample into the analyser by sucking it, by analyser means, from the plasma collector vessel  17  that is docked onto the input opening of the analyser. The compensation opening  25  or, alternatively, the porous areas of the clamping seal  27  permit total drainage of the plasma collector vessel  17 .   Analytic determination of the substances contained in the plasma sample obtained according to the present disclosure, for instance the haemoglobin values, in the analyser.       

     In a second variant of the device according to  FIGS. 4 to 7  plasma extraction may be carried out as follows:
         Taking a Monovette  12  as in  FIG. 5  from a sterile package.   Unscrewing the adapter cap  28  with puncturing membrane  29  from the plunger housing  20 .   Placing the filter cartridge  13  between adapter cap  28  and plunger housing  20  according to  FIG. 4 .   Creating a partial vacuum in the Monovette  12  by
           pulling back the plunger  18  until it meets the stop,   locking the plunger rod in locking position  24  shown in broken lines.   
               

     (Optionally: breaking off the plunger rod to avoid reversal of internal flow direction).
         Docking the puncturing membrane  29  of the adapter cap  28  onto a puncturing needle, e.g., butterfly.   Sucking in the blood sample by means of the partial vacuum prevalent in the Monovette  12 .       

     The deep-bed filter  3  of the filter unit  10  may for instance be built up from glass fibers without binding agent (typically FV-2, Whatman Inc., resp. DE 40 15 589 A1, or EP 0 239 002 A1, Böhringer-Mannheim) with a retention range of 0.5 μm to 10 μm, typically 1 μm to 5 μm, more typically &lt;3 μm. The red blood cells (RBCs) will collect on the thin glass fibers of the deep bed filter  3  without bursting or unduly influencing the rate of flow.
         Depending on the cross-section of the filter unit  10  and on haematocrit a “plasma front” or “plasma fraction”  40  will form, which can pass the stop membrane  4  unimpededly. Residual RBCs not held back by the deep-bed filter are filtered out ( FIG. 4 ) by the stop membrane  4 . For this purpose the stop membrane  4  has a pore size significantly smaller than that of the deep-bed filter  3 , i.e., pore diameters of less than 400 nm, typically less than 200 nm. By combining a deep-bed filter  3 , which on account of its pore size already retains the greater part of blood cells, but does not impede the flow of the plasma fraction, with a subsequent stop membrane  4 , which due to its smaller pore size will reliably retain remaining blood cells, but would clog swiftly on account of its limited number of pores if the preceding deep-bed filter  4  were absent, a reliable separation of blood cells without clogging of the filter can be achieved, thus making it possible to obtain a sufficiently large volume of plasma sample.   The partial vacuum of not more than 500 mbar, typically 300 mbar, more typically 100 to 150 mbar, established in the filter unit  10 , together with the geometry of the filter unit  10  (ratio of compensation volume  21  to suction volume  23  of the plunger housing  20 ), will determine the flow rate and thus the shear forces acting especially on the RBCs within the stop membrane  4  of the filter unit  10 . Bursting of RBCs (haemolysis) can efficiently be prevented by optimizing the compensation volume  21 .   The lateral grid  5  of the filter unit  10  permits plasma to be collected and sucked off behind the stop membrane  4  towards the plasma collector vessel  17 , by efficiently preventing the stop membrane  4  from “sealig off” tightly (see also the syringe variant).       

     (As an alternative, this functionality of the lateral grid  5  may be provided by structuring the side of seal  15  facing the stop membrane, e.g., by stamping, or otherwise providing for sufficient roughness of its surface.)
         Plasma extraction is also ended by:
           when the stop membrane  4  is clogged by particulate components of blood, or   especially in the case of haematocrit values &lt;40% by a hydrophobic, air-permeable element  16  (liquid stop) at the end of the plasma collector vessel  17  or in the passage of the clamping seal  27 , which terminates further plasma extraction when the lumen of the plasma collector vessel  17  bounded by the hydrophobic, air-permeable element  16  is completely filled with plasma.   
           By means of marks on the plasma collector vessel  17  one can once again ascertain by visual inspection that the desired amount of plasma has been obtained.   The front part of the Monovette  12  is unscrewed from or wrenched off the plunger part  20  and thus the interposed filter cartridge  13  is divided along a plane ε in a filter housing  8  containing the filter unit  10  and in a plasma applicator part  14  containing the plasma collector vessel  17  with conical tip, the tip  9  of the plasma collector vessel  17  thus being exposed ( FIG. 7 ). The plasma applicator  14  remains connected to the filter housing  20  for ease of handling. After the parts have been separated the device corresponds to that of  FIG. 3 .       

     The further steps of the procedure correspond to those described in the first variant of the device according to the disclosure. 
     It is noted that terms like “preferably”, “commonly” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure. 
     For the purposes of describing and defining the present disclosure it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue. 
     Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present disclosure are identified herein as preferred or particularly advantageous, it is contemplated that the present disclosure is not necessarily limited to these preferred aspects.