EXHIBIT 101 8
`
`US. PATENT NO. 5,064,541 TO JENG ET AL.
`
`Infopia Ex. 1018 pg. 1
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`

`

`United States Patent
`Jeng et al.
`
`[19]
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,064,541
`* Nov. 12, 1991
`
`[541
`
`DEVICES AND METHODS FOR THE
`COLLECTION OF A PREDETERMINED
`VOLUME OF PLASMA OR SERUM
`
`1751
`
`Inventors:
`
`szy-Wen Jeng, Vernon Hills;
`Kristin D. Elmore, Waukegan; Gary
`M. Costa, Gurnee; Terry A. Pry,
`Libertyville, all of Ill
`
`[73]
`
`Assignee: Abbott Laboratories, Abbott Park,
`Ill.
`
`1*]
`
`Notice:
`
`The portion of the term of this patent
`subsequent to Jun. 12, 2007 has been
`disclaimed.
`
`[21]
`
`[22]
`
`[63]
`
`[51]
`[521
`
`[58]
`
`[56]
`
`Appl. No.: 499,864
`
`Filed:
`
`Mar. 27, 1990
`
`Related U.S. Application Data
`
`Continuation-1n—part of Ser. No. 335,064, Apr 7,1989,
`Pat. No 4,.933,092
`
`.......... B01D 37/00; B01D 39/14
`Int. Cl.5 .........
`US. Cl. .................................... 210/767; 210/295;
`210/502.1; 210/510.1; 210/806; 422/100,
`422/101; 436/177
`Field of Search ............... 210/206, 259, 295, 314,
`210/323.1, 335, 502.1, 510.1, 651, 702, 729, 730,
`732, 782, 789, 806, 767; 435/2; 422/101, 56,
`100; 436/177, 178, 808
`References Cited
`U.S. PATENT DOCUMENTS
`
`3,888,629 6/1975 Bagshawe ........................... 436/804
`3,891,553
`6/1975 Ayres .............. 210/789
`
`3,902,964 9/1975 Greenspan
`.. 436/177
`3,960,727 6/ 1976 Hochstrasser ,
`., 210/782
`
`4,464,254
`8/1984 Dojki et a1.
`.. 210/516
`
`4.477.575 10/1984 Vogel et a1.
`.. 210/767
`4,594,327
`6/1986 Zuk .......
`422/56
`4,753,776
`6/1988 Hillman e a.
`.. 422/101
`
`4,820,644 4/1989 Sch‘afer et al.
`.. 422/101
`4,933,092
`6/1990 Aunet et a1.
`........................ 210/729
`
`Primaiy Examiner—W. Gary Jones
`Attorney, Agent, or Firm~—Frank S. Ungemach; Daniel
`R Curry
`
`[57]
`
`ABSTRACT
`
`A device and method for permitting the separation of
`plasma or serum from whole blood. The device com-
`prises a matrix of hydrophilic sintered porous material
`to which at least one red blood cell agglutinating agent
`has been applied. According to a first method of using
`the device, a sample of whole blood is applied to a first
`end of the matrix and the red blood cells within the
`sample come in contact with the agglutinating agents
`present in the matrix. The blood cells agglutinate, and
`are entrapped in the interstices of the matrix, while
`substantially blood-ce11~free serum or plasma accumu-
`lates near the outlet of the device. A filter means in
`liquid receiving relationship with the matrix functions
`to wick the serum of plasma from the matrix.
`According to an alternative aspect of the invention, a
`filter means in liquid receiving relationship with the
`outlet of the matrix functions to retain any blood cells
`, which pass through the matrix as the filter means wicks
`the plasma or serum from the matrix. Additional agglu-
`tinating agents may be incorporated within the filter
`means to facilitate retention of blood cells which pass
`through the matrix.
`Another aspect of the present invention involves a de-
`vice for the measurement of a serum or plasma sample,
`utilizing a matrix of sintered porous material, such as
`sintered glass, sintered steel, sintered ceramics, sintered
`plastics, and equivalents thereof. The matrix is charac-
`terized by a reproducible fluid uptake capacity propor-
`tional to the fixed dimensions of said matrix, a minimal
`reactivity with plasma or serum components, and a
`hydrophilic internal surface which enables the matrix to
`collect and retain a predetermined volume of sample for
`analysis.
`
`20 Claims, 2 Drawing Sheets
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`13\
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`‘E'..A"u”.. u
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`20
`15
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`lnfopia Ex. 1018 pg. 2
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`US. Patent
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`Nov. 12, 1991
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`Sheet 1 of 2
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`~ 5,064,541
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`/38
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`K-.—‘_‘
`-—-‘-“-—‘
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`H”.'.'”. I .
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`42'
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`lnfopia Ex. 1018 pg. 3
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`US. Patent
`
`Nov. 12, 1991
`
`Sheet 2 of 2
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`5,064,541
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`! 104
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`I
`{WI/I2:
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`I
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`lnfopia Ex. 1018 pg. 4
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`‘ 1
`
`5,064,541
`
`DEVICES AND METHODS FOR THE
`COLLECTION OF A PREDETERMINED VOLUME
`OF PLASMA OR SERUM
`
`This application is a continuation in—part of U.S. pa-
`tent application Ser. No.~ 335,064 filed Apr. 7, 1989, now
`U.S. Pat. No. 4,933,092.
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention relates generally to methods
`for separating plasma or serum from whole blood. More
`particularly, the invention relates to devices capable of
`separating plasma or serum from whole blood compris-
`ing a hydrophilic sintered porous material in which at
`least one red blood cell agglutinating agent has been
`incorporated. Agglutinated blood cells are removed
`from whole blood by the sieving action of the matrix of
`the sintered porous material, and optional additional
`filter means.
`invention also relates to devices and
`The present
`methods for collecting a predetermined amount of a
`plasma or serum sample for analysis in a diagnostic
`assay. More particularly, the invention relates to a ma-
`trix of sintered porous material, wherein the matrix
`provides a reproducible fluid uptake capacity.
`2. Background
`Modern clinical diagnostic methods are routinely
`carried out on blood samples. Unfortunately, red blood
`cells present in whole blood scatter and absorb light
`thus interfering with assay methodologies which mea-
`sure either reflected or transmitted light. Other cells
`may interfere with particular determinations; for exam-
`ple, cholesterol determinations can be affected by cho-
`lesterol present
`in cell membranes. For this reason,
`many assay methodologies are carried out on plasma or
`serum which must be separated from a whole blood
`sample.
`Centrifugation is a well known method in the art by
`which plasma (before clotting) and serum (after clot-
`ting) is separated from whole blood. Stratifying whole
`blood by centrifugation, however,
`is time consuming
`and requires cumbersome laboratory equipment. The
`use of red blood cell agglutinating agents such as those
`disclosed in Van 055, et al., Vox. Sang.
`, vol. 34, pp
`351~361 (1978) can be helpful in carryingout centrifu-
`gation and other red blood cell separation techniques.
`Dojki, et al., U.S. Pat. No. 4,464,254. issued Aug. 7,
`1984, disclose a piston device capable of isolating serum
`from an already stratified blood sample. The device
`consists of a piston head connected to an open—ended
`sampling tube. The piston head is composed of a one-
`way valve under which is located a cavity containing a
`porous plastic filter body. Insertion of the piston head-
`sampling tube assembly into a test tube containing a
`stratified sample of blood allows serum to pass through
`the filter body and valve into the interior of the sam-
`pling tube. The volume and purity of the serum which
`can be separated from the whole blood is contingent
`upon the completeness of the stratification of the blood.
`Vogel, et al., US Pat. No. 4,477,575, issued Oct. 16,
`1984, disclose a device and a process using the device to
`separate serum from whole blood by causing whole
`blood to pass into and through a layer of glass fibers
`with diameters from 0.2 to 5 microns and with a density
`of 0.1 to 0.5 g/cm3. The volume of plasma or serum
`which can be separated from whole blood by this de-
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`vice is diselosed to be less than 50% of the absorption
`volume of the glass fiber layer.
`Zuk, U.S. Pat. No. 4,594,327, issued June 10, 1986,
`discloses an analytical method wherein a whole blood
`sample is combined with a red blood cell binding agent
`and the mixture is then filtered through a solid bibulous
`element to which is bound at least one specific binding
`pair member so as to remove the agglutinated red blood
`cells. The patent discloses anti-red blood cell antibodies,
`polymeric amino acids, such as polylysine, and lectins,
`such as wheat germ agglutinin, as suitable red blood cell
`binding agents for causing the aggregation of red blood
`cells in whole blood.
`.
`Hillman, et al., U.S. Pat. No. 4,753,776, issued June
`28, 1988, disclose a device and a process using the de-
`vice to separate serum from whole blood using capillary
`action to pass whole blood through a glass microfiber
`filter. The patent discloses an alternative embodiment in
`which whole blood is passed through a filter to which
`red blood cell agglutinins have been attached. Rather
`than retaining the red blood cells, however, the filter
`disclosed merely retards their flow, eventually allowing
`their escape.
`Trasch, et al., EPO Publication No. 133,895 , pub-
`lished Mar. 13, 1985, disclose a red blood cell retaining
`substrate and a process using the substrate for retaining
`red blood cells on filters thus allowing the recovery of
`plasma from whole blood. The red blood cell retaining
`substrates of the invention are stated to induce coagula-
`tion, but not hemolysis, so that the coagulated corpus—
`cular components can be removed on a filter, while the
`plasma passes through. The publication discloses alter-
`native embodiments where the retaining substrate is
`incorporated into the filter or into a pre-filter layer. The
`publication states that absorptive, porous, liquid perme-
`able carriers or filters, in the form of paper, fleece, gel
`or tissues, comprised of cellulose, wool, glass fiber,
`asbestos, synthetic fibers, polymers or mixtures of the
`same, can be used as the absorptive materials for the
`retaining zone.
`.
`Most portable technigues for the separation of serum
`or plasma are limited with respect to speed and serum
`yield efficiency. Blood separation devices utilizing glass
`fiber membranes, for example, tend to separate serum at
`a relatively slow speed and tend to retain significant
`quantities of serum or plasma in the interstices of the
`membrane. Accordingly, there exists a desire in the art
`for improved devices providing rapid and efficient
`methods for serum and plasma separatiOn.
`Another difficulty encountered in the testing of blood
`samples is that it is generally necessary to measure a
`precise test sample volume of plasma or serum for use in
`diagnostic assays. This need for precision is typically
`met by having a trained technician use a sophisticated
`pipetting apparatus or by the use of expensive auto-
`mated instruments. There are also test strip devices
`which use membrane or paper matrices to collect a
`plasma sample and transport that sample to a reaction
`zone on the test strip. Test strip devices, however, typi-
`cally provide only that sample volume capacity which
`is needed to transport sample by capillary action
`through the strip to the reaction zone, and therefore a
`low level of precision is reguired. In test strips devices,
`the plasma recipient member only collects that amount
`of sample necessary to fill the strip which in turn ends
`the migration of the sample through the strip because
`the drawing force which causes sample subject to analy-
`sis in a test strip device is limited to that amount which
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`Infopia Ex. 1018 pg. 5
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`3
`passes through a defined detection zone on the test strip
`before the strip is filled.
`SUMMARY OF THE INVENTION
`
`5,064,541
`
`The present invention relates to improved methods,
`devices and kits for separating plasma or serum from
`whole blood. Specifically, the devices of the invention
`comprise a matrix of hydrophilic sintered porous mate-
`rial in which at least one red blood cell agglutinating
`agent has been incorporated. The matrix is further char-
`acterized by a pore size selected such that individual
`blood cells will pass through the matrix but wherein
`agglutinated blood cells will be retained by the matrix.
`The devices are capable of carrying out a rapid separa-
`tion of serum or plasma from whole blood while retain-
`ing only minimal quantities of serum or plasma within
`the interstices of the matrix.
`According to one aspect of the invention, the device
`comprises a matrix of hydrophilic sintered porous mate-
`rial in which at least one red blood cell agglutinating
`agent has been incorporated. A sample of whole blood
`is applied to an inlet of the matrix and the blood cells
`within the sample come in contact with the-agglutinat-
`ing agents present in the matrix. The blood cells aggluti-
`nate and are entrapped in the interstices near the inlet of
`the matrix, while substantially blood-cell free serum or
`plasma accumulates near an outlet of the matrix. A
`receiving means, including materials such as filter paper
`or additional porous matrices, may be incorporated in
`liquid receiving relationship with the outlet of the ma-
`trix. The receiving means functions to wick the substan-
`tially blood-cell free serum or plasma from the outlet of
`the matrix, thus making the serum or plasma available
`for analysis or other purposes.
`According to an alternative aspect of the invention, a
`filter means is incorporated in liquid receiving relation-
`ship with the outlet of the matrix for improved effi-
`ciency and more rapid separation of the blood cells
`from a sample of whole blood. The filter means may
`have at
`least one red blood cell agglutinating agent
`incorporated therein in order to assist in retaining the
`blood cells. The invention also provides methods and
`devices for the analysis of selected components of blood
`plasma or serum comprising the first porous matrix of
`the invention in combination with additional matrices
`or filter means in which analytical reagents selected for
`reaction with the selected components may be incorpo-
`rated.
`
`As indicated previously, removal of red blood cells is
`of particular interest in visually red assays. Neverthe-
`less, removal of other blood cells is desirable as well,
`and is to be understood when the term "red blood cell"
`is employed herein in the context of retention in the
`matrix or removal of whole blood.
`
`invention also relates to devices and
`The present
`methods for the collection of a predetermined volume
`of plasma or serum test samples using a matrix of sin-
`tered porous material that is characterized by a repro-
`ducible fluid uptake capacity proportional to the fixed
`dimensions of said matrix, a minimal reactivity with
`plasma or serum components, and a hydrophilic internal
`surface, wherein the matrix is encased in a housing
`means whereby an entry port to the matrix is defined.
`These characteristics enable the matrix to collect and
`retain a predetermined volume of sample for analysis.
`Optionally, an exit port from the matrix is also defined
`by the encasement means.
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`The sintered porous materials used to make the col-
`lection matrix devices of the present invention include
`sintered glass, sintered steel, sintered ceramics, sintered
`plastics and equivalents thereof. A particularly pre-
`ferred material is polyethylene.
`The collection matrix can optionally be used in con-
`junction with a blood separator means which separates
`plasma or serum from a whole blood sample. Typically.
`the matrix is in liquid receiving relationship with the
`blood separator means, and the matrix thereby collects
`a predetermined volume of plasma or serum from the
`blood separator means. The collection matrix can also
`be used in conjunction with a sample receiver means to
`which the matrix transfers the predetermined volume of
`sample for analysis. Alternatively, the analysis can be
`performed upon the plasma or serum sample in the
`matrix itself.
`
`Suitable sample receiver means include reaction or
`detection vessels, such as cuvettes, test tubes, slides and
`reaction wells. The sample is eluted into the detection
`vessel by the application of an eluting buffer to the
`matrix. Other sample receiver means include absorbent
`solid phase materials having a pore size selected to
`induce the flow of sample from the matrix into the
`absorbent by capillary action. The sample receiver
`means can optionally include one or more analytical
`reagents which are reconstituted upon the transfer of
`test sample to the receiver means.
`The collection of a serum or plasma sample for analy-
`sis is performed by applying a quantity of serum or
`plasma to the collection matrix and thereby collecting a
`predetermined volume of plasma or serum.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 is a depiction of a device comprising a porous
`matrix and a filter paper receiving matrix;
`FIG; 2 is a depiction of a device comprising a first
`porous matrix, a second porous matrix, and a filter
`paper receiving matrix;
`FIG. 3 is a depiction of a device comprising a porous
`matrix, and a filter paper receiving matrix with a rea-
`gent containing zone;
`FIG. 4 is a depiction of a device comprising a first
`porous matrix, a first filter means, a second porous ma-
`trix, a second filter means, and a receiving porous ma-
`trix; and
`FIG. 5 is a depiction of a device comprising a porous
`matrix, a filter means, and a receiving porous matrix.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`invention provides improved devices
`The present
`and methods for using those devices to separate plasma
`or serum from whole blood. The devices of the inven-
`tion comprise matrices of hydrophilic sintered porous
`materials in which at least one red blood cell agglutinat~
`ing agent has been incorporated. The matrix is charac-
`terized by a pore size such that individual blood cells
`will pass through it, but wherein agglutinated cells will
`be retained by the matrix. The devices are capable of
`performing rapid separations of serum or plasma from
`whole blood while retaining only minimal
`residual
`quantities of serum or plasma within the interstices of
`the porous material.
`.
`Among the materials contemplated as being suitable
`for the matrices of the present invention are sintered
`glass, sintered steel, sintered ceramics, and sintered
`polymers of plastic, with the preferred material being
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`Infopia Ex. 1018 pg. 6
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`5
`that known as sintered polyethylene such as that de-
`scribed in British patent No. 2,186,205. Sintered poly-
`ethylene matrices commercially available from Porex,
`Inc., Fairburn, Ga. or General Polymeric Corp., West
`Reading, Pa. may be obtained which have a pore size of 5
`from about 10 microns to about 70 microns. Such a pore
`size allows individual red blood cells to pass through
`the matrix, but retains agglutinated red blood cells
`within the matrix. '
`The matrices of the present invention are hydrophilic
`so as to promote the flow of aqueous liquids through
`them. Commercially available matrices may be either
`hydrophilic or hydrophobic in nature. Hydrophobic
`matrices may be rendered hydrophilic by a variety of
`known methods. Among those methods available are
`plasma treatment or surfactant treatment of the matrix.
`Plasma treatment involves exposing the hydrOphobic
`matrix to charged gas (plasma) wherein an electronic
`charge is imparted to the solid surface rendering the
`surface wettable. Surfactant treatment involves dipping
`the hydrophobic matrix in a surfactant and letting it
`dry. This treatment assists in wetting the surface and
`interior of the matrix and results in the promotion of
`aqueous liquid flow through the matrix. It is contem-
`plated that a wide variety of commercially available
`surfactant materials would be appropriate for use with
`the present invention.
`'In the assays discussed in the
`Examples below, commercially available matrices
`which had been co-molded with surfactant were used .
`and are preferred over matrices dipped in commercially
`available surfactants.
`In general, surfactants should be selected which are
`compatible with the reactants or reagents placed within
`the matrix so as not to interfere with the preferred activ-
`ity. Additionally, it should be noted that no surfactant
`should be present in such concentrations as to cause
`hemolysis of the red blood cells. In addition, care must
`be exercised to avoid hemodilution of the plasma sam-
`ple. Hemodilution is the extraction into the plasma of
`the internal fluid of the red blood cell due to hypertonic
`conditions.
`.
`The incorporation of anti-coagulants into whole
`blood samples is particularly preferred for promoting
`the flow of plasma through the devices. Anti-coagulants
`mixed with the blood before application to the device
`prevent the blood from clotting. Separation of blood
`cells from a blood sample treated with anti coagulants
`produces plasma. Separation of red blood cells from a
`clotted blood sample produces serum. It is further con-
`templated that these anti-coagulants may be incorpo-
`rated into the matrices to prevent the blood sample
`from clotting when applied to the device. For example,
`a drop of blood from a finger stick may be directly
`applied to the device such that anti coagulants incorpo—
`rated within the device come in contact with the blood
`and prevent
`the blood from clotting. Alternatively,
`blood can be collected in a capillary tube previously
`treated with anticoagulant, and transferred to the de-
`vice in this manner. Preferred anti coagulant materials
`include heparin, EDTA and citrate.
`According to the invention, red blood cell agglutinat-
`ing agents are incorporated into the porous matrices.
`Agglutinating agents are substances which cause indi-
`vidual red blood cells to adhere to one another to form
`clumps. It is contemplated that the agglutinating agents
`may be incorporated into a matrix by means such as
`adsorption, absorption or metallic‘organic dye com-
`plexes,» although it is preferred that at least some of the
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`agglutinating agent be absorbed into the matrix such
`that it may be solubilized in the presence of a blood
`sample.
`Suitable agglutinating agents include natural and
`synthetic water soluble polymers including, but not
`limited to, those discussed in the background. Among
`the available agglutinins, preferred agglutinins include
`hexadimethrine bromide, which is available from Ald-
`rich Fine Chemicals as Polybrene ®, polylysine, and
`anti-red blood cell antibodies. It is believed that posi-
`tively charged polyelectrolytes, such as Polybrene®
`and polylysine, aggregate erythrocytes due to charge
`neutralization, changes in hydration, polymer bridging
`and osmotic interaction. IgG- or IgM-cl‘ass antibodies
`specific for red blood cell antigens cause agglutination
`by binding to similar antigenic determinants on the
`surface of two separate erythrocytes which causes the
`cells to adhere to one another. An additional enhance-
`ment of the agglutination process is achieved by incor-
`porating substances such as polyvinyl pyrrolidone
`(PVP) which apparently function as dielectrics, allow-
`ing charged cells to approach one another and be cross-
`linked by antibody and or other agglutinins.
`A high agglutinating agent concentration results in a
`longer residence time for a blood sample within the
`matrix and increases the efficiency of agglutination of
`red blood cells within the matrix. This can have the
`undesirable effect, however, of trapping a large propor-
`tion of the plasma within the matrix. Conversely, lower-
`ing the agglutinating agent concentration allows more
`plasma to be released, but may result in fewer red blood
`cells within the sample being trapped by the matrix. The
`length, volume, and porosity of the matrix, as well as
`the volume of the blood sample to be filtered by the
`matrix, in addition to the agglutinating agent concentra-
`tion affect the efficiency of entrapment of red blood
`cells within the matrix and the amount of plasma eluted
`by the matrix.
`According to a first preferred embodiment of the
`device of the present invention, the pore size of the
`matrix is selected in conjunction with the length and
`volume of the matrix, the volume of blood sample to be
`treated, and the agglutinating agent’s ability to cause the
`red blood cells to clump together, such that substan-
`tially all of the red blood cells present in a whole blood
`sample become agglutinated and are retained in the
`matrix. Removal of “substantially all" red blood cells
`present in a blood sample constitutes the removal of a
`sufficient amount of the red blood cells from the sample
`so that a clinical determination of a selected blood ana-
`lyte may be performed without interference. Prefera-
`bly, removal of “substantially all" red blood cells pres-
`ent in a blood sample constitutes the removal of at least
`about 90% of the red blood cells from the sample.
`According to one method of utilizing the first pre-
`ferred embodiment of the device of the present inven-
`tion, a sample of whole blood is applied to an inlet or
`first end of the matrix. The blood rapidly passesthrough
`the interstices of the matrix, quickly coming in contact
`with the red blood cell agglutinating agents incorpo-
`rated therein. These agents promote agglutination of
`the red blood cells which are then entrapped within the
`interstices of the matrix. This entrapment of the aggluti-
`nated red blood cells within the matrix permits the rapid
`and efficient separation of plasma or serum from the red
`blood cells. Additionally, because the matrix retains
`only a minimal amount of plasma or serum, a large
`amount of the plasma or serum may be Successfully
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`Infopia Ex. 1018 pg. 7
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`5,064,541
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`7
`harvested from the whole blood sample. Optionally, a
`filter means such as filter paper or an additional porous
`matrix may be placed in liquid receiving relationship
`with the outlet of the matrix in order to wick the serum
`or plasma from the matrix.
`FIGS. 1—2 are depictions of exemplary devices used
`to separate plasma from whole blood according to the
`first embodiment of the present invention. As illustrated
`in FIG. 1, an apparatus (10) comprises a housing (12)
`which has an entry port (13) and an exit vent (16). Lo-
`cated within the housing (12) is a device (17) comprising
`a porous polyethylene matrix (18)rwhich contains an
`agglutinating agent and is molded into a cylindrical
`shape having the dimensions of 3.5 mm in diameter and
`5 mm in height. The exact shape and dimensions are not
`critical to the invention but affect resident time and
`efficiency as described herein. Also located within the
`housing (12) is a paper matrix (20). The matrix (18) has
`an inlet (14) and an outlet (15) and is in liquid receiving
`contact with said paper matrix (20). The paper matrix
`(20) and the matrix (18) may contain the reagents neces-
`sary for the analysis of a selected blood analyte. An
`embodiment of this device is described in co~owned and
`co-pending U.S. patent application Ser. No. 335,006
`filed concurrently herewith and incorporated herein by
`reference.
`
`As illustrated in FIG. 2, an apparatus (30) comprises
`a housing (32) which has an entry port (33) and an exit
`vent (36). Located within the housing (32) is a device
`(37) comprising a first porous polyethylene matrix (38).
`Also located within the housing (32) is a second porous
`polyethylene matrix (40) in liquid receiving relationship
`with said first matrix and a paper matrix (42) in liquid
`receiving relationship with said second matrix. The first
`matrix (38) contains an agglutinating agent and has an
`inlet (34) and an outlet (35). The second matrix (40)
`contains some of the reagents necessary for the determi-
`nation of a specific blood analyte while the paper matrix
`(42) contains the other components of the reagent sys-
`tem. It is contemplated that the first matrix (38) may
`also contain reagents necessary for the analysis of a
`selected blood analyte. An exemplary dye paper rea-
`gent system is described in U.S. Ser. No. 204,443 filed
`June 9, 1988 and incorporated herein by reference.
`According to a second preferred embodiment of the
`device capable of more rapid separation of red blood
`cells, the pore size of the matrix is selected in conjunc-
`tion with the length and volume of the matrix, the vol-
`ume of blood sample to be treated, and the agglutinating
`agent’s ability to cause the red blood cells to clump
`together, such that less than all the red blood cells pres-
`ent in a whole blood sample become agglutinated and
`are retained in the matrix. In these cases where it is
`desirable to select a matrix having a relatively large
`pore size which provides a high rate of flow, but
`wherein not all the red- blood cells are retained by the
`matrix, the red blood cells remaining in the plasma or
`serum are subjected to subsequent filtration steps utiliz-
`ing secondary matrices or filters alone, or impregnated
`with red blood cell agglutinating agents, such that
`“clear" plasma or serum is produced. The removal of at
`least 97% of the red blood cells from the sample consti-
`tutes “clear" plasma or serum.
`Filter paper characterized by a pore size such that
`agglutinated red blood cells will not pass through it may
`be used to purify further the serum or plasma. Addition-
`ally, this filter paper has agglutinating agents incorpo-
`rated within it to aid in the retention of the remaining
`
`8
`red blood cells. The use of filter paper as a separate
`barrier for the retention of the red blood cells from the
`serum or plasma which flows from a matrix allows for
`a variety of filtration formats where a series of matrices
`treated with agglutinating agents are interspersed with
`pieces of filter material. Among the types of filters con-
`templated for such use are filters comprised of deriva«
`tized or underivatized cellulose, nylon, natural or syn-
`thetic membranes, or porous polyethylene matrices
`characterized by a pore size such that individual or
`agglutinated red blood cells will be retained by the
`porous matrix. Where more than one matrix is used,
`pore diameters are chosen to promote flow from one
`region to another.
`FIGS. 3—5 are depictions of exemplary devices used
`to separate plasma from whole blood according to the
`second embodiment of the present invention. As illus-
`trated in FIG. 3, an apparatus (50) comprises a housing
`(52) which has an entry port (53) and an exit vent (56).
`Located within the housing (52) is a device (57) com-
`prising a porous polyethylene matrix (58) and a paper
`matrix (66). The matrix (58) contains an agglutinating
`agent, has an inlet (54) and an outlet (55), and is in liquid
`receiving relationship with the paper matrix (66). The
`paper matrix contains a final red blood cell filtration
`region (60), an analyte reagent region (62), and a quanti-
`tative analysis region (64).
`The present invention also provides a novel means of
`collecting and retaining a predetermined amount of
`plasma or serum for analysis in a diagnostic assay. The
`novel method involves a metering matrix which enables
`the reproducible collection of discrete amounts of
`plasma or serum. This process is enabled by the use of a
`sintered porous matrix material which is selected for the
`following characteristics: a reproducible fluid uptake
`capacity that is proportional to the fixed dimensions of
`the matrix, a minimal reactivity with plasma or serum
`components, and a hydrophilic internal surface. These
`characteristics enable the matrix to collect and retain a
`predetermined volume of sample suitable for analysis in
`a diagnostic assay. Preferably, the matrix material is
`rigid for ease of handling, and optionally, the material is
`chosen as having the largest void capacity for the desig-
`nated matrix dimensions. With such a matrix, the collec-
`tion of the sample is independent of the level of the
`user‘s training, and there is no need for sophisticated
`measuring equipment.
`'
`A further advantage of the present invention is that
`the matrices can be used as components of diagnostic
`devices, such as flow through and test strip devices, to
`collect a predetermined amount of sample that is not
`dependent upon the absorptive capacity of the paper,
`fiber and nitrocellulose materials typically used in such
`devices or upon the combined absorptive capacity of
`the device components. For example,
`in a test strip
`device the length of the strip typically determined the
`volume of sample which can be absorbed, and the di-
`mensions of the test strip determine the amount of sam-
`ple which will pass through the reaction and detection
`zones on the test strip. The matrix devices of the present
`invention, however, enable the collection and retention
`of a predetermined sample volume as well as the analy-
`sis of the entire sample volume, either within the matrix
`itself or within a sample receiver means to which the
`sample is transported, after the collection by the meter-
`ing matrix of the total sample volume to be analyzed.
`There are several different materials which can pro-
`vide a volume measuring characteristic. These materials
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`Infopia Ex. 1018 pg. 8
`
`

`

`9
`include paper, derivatized cellulose, porous plastic
`membranes and sintered porous materials. However,
`not all of these materials are equally suitable for use as
`metering matrices in diagnostic devices. For example,
`while a paper matrix may have the capacity to collect a
`sample of sufficient volume, paper matrices showed
`poor reproducibility in collecting that sample volume.
`Nylon matrices also have unacceptable reproducibility.
`The poor producibility of such matrices was attributed
`to the less sturdy and less resilient nat