EXHIBIT 1020
`
`‘ U.S. PATENT NO. 5,762,871 TO NEYER
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`Infopié Ex. 1020 pg. 1
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`USOOS76287 1A
`
`United States Patent
`
`[191
`
`Neyer
`
`[11] Patent Number:
`5,762,871
`
`[45] Date of Patent:
`Jun. 9, 1998
`
`[54] MULTI-LAYER TEST DEVICE FOR
`ANALYZING THE CONCENTRATION OF
`ANALYTE IN A BLOOD SAMPLE
`
`[75]
`
`Inventor: Gebhard Neyer. Los Angeles. Calif.
`
`[73] Assignee: LXN Corp.. San Diego. Calif.
`
`[21] App]. No.: 892,697
`
`[22] Filed:
`
`Jul. 15, 1997
`
`Related US. Application Data
`
`[62] Division of Ser. No. 418,523, Apr. 7, 1995, Pat. No. 5,725,
`774.
`
`[51]
`
`Int. Cl.6 .......................... G01N 33/50; GOIN 33/52;
`C12Q 1/00; C12Q 1/54
`............................ 422/57; 210/483; 210/488;
`[52] US. Cl.
`210/489; 210/490; 210/496; 210/503; 210/504;
`210/505; 210/506; 210/508; 422/55; 422/56;
`422/60; 422/101; 436/169; 436/170
`[58] Field of Search ..................................... 210/483. 488.
`210/489. 490. 496. 503. 504. 505. 506.
`508; 422/55. 56. S7. 60. 73. 101. 102; 436/16.
`69. 169. 170. 177. 178; 530/412. 415
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`1/1971 Fetter ........................................ 23/230
`3,552,925
`1/1971 Fetter .......
`23/253
`3,552,928
`
`9/1975 Greenspan
`...... 195/1
`3,902,964
`3/1981 Kondo el al.
`.
`4,256,693
`
`..
`4,477,575 10/1984 Vogel et a1.
`9/1985 Chen ............
`4.543.338
`
`7/1987 Rapkill et 31.
`4,678,757'
`
`4,933,092
`6/1990 Aunet a].
`.
`5,064,541
`11/1991 Ieng et al.
`
`1/1992 Nitadoti e! a].
`5,084,173
`
`5,166,051
`........................... '435/7.1
`11/1992 Killeen et a].
`435/792
`5,169,757
`1271992 Yamazaki et a1.
`..
`
`436/169
`5,169,787 1211992 Knappe eta].
`
`210/767
`2/1993 Baumgardner et al
`5,186,843
`435fl.1
`
`5/1993 Maddox ..............
`5,212,060
`. 435/14
`5,306,623
`4/1994 Kiser et al.
`.
`...... 435/10
`5,366,868
`11/1994 Sakamoto
`
`..
`5,397,479
`3/1995 Kass et a1.
`210/728
`
`.....
`5.423.989
`6/1995 Alla: cl al.
`210/650
`................................ 436/87
`5,470,752
`11/1995 Burd et a1.
`
`FOREIGN PATENT DOCUMENTS
`
`2104976
`0 194 502
`O 436 897
`
`3/1994 Canada .
`.
`9/1986 European Pat. OE.
`.
`7/1991 , European Pat. Ofi.
`OTHER PUBLICATIONS
`
`Dialog Database Abstract of EPA 0 194 502 to Limbach and
`Helger. EPA published Sep. 17. 1986.
`
`Primary Examiner—John Kim
`Anomey, Agent, or Firm—Campbell & Flores LLP
`
`[57]
`
`ABSTRACT
`
`The present invention relates to a method and to a device for
`separating plasma from whole blood. The method and
`device utilize a permeable mmglass fiber matrix containing
`a polyol which is capable of clumping red blood cells. The
`matrix. in the absence of such a polyol, would otherwise be
`porous to red blood cells. The polyol-containing matrix has
`a first surface and a second surface such that a whole blood
`sample which is applied to the first surface flows direction-
`ally toward the second surface. Plasma separated from
`whole blood becomes available at the second surface of the
`matrix and can be tested for the presence of a particular
`analyte. such as glucose or fructosamine. as provided by
`multl-layer test devices of the present invention.
`
`1 3 Claims, 1 Drawing Sheet
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`.17
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`IS
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`Infopia Ex. 1020 pg. 2
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`US. Patent
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`Jun. 9, 1998
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`5,762,871
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`FIG.2
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`Infopia Ex. 1020 pg. 3
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`5.762.871
`
`1
`MULTI-LAYER TEST DEVICE FOR
`ANALYZING THE CONCENTRATION OF
`ANALYTE IN A BLOOD SAMPLE
`
`This application is a divisional of application Ser. No.
`‘ 08/418523. filed Apr. 7. 1995. U.S. Pat. No. 5.725.774.
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`
`The present invention relates generally to the chemical
`analysis of analytes present in whole blood and more
`specifically.
`to a method and to a device for separating
`plasma or serum from whole blood. thus providing a con-
`venient and accurate means for such chemical analysis.
`2. Background Information
`Presently. numerous test devices are available for the
`analysis of body fluids in order to determine the presence or
`concentration of a particular analyte. For example. tests are
`available for detecting glucose.
`fructosamine. albumin.
`calcium. urea. uric acid. bilirubin. cholesterol. and other
`soluble analytes present in whole blood or the fluid part of
`blood. namely the plasma or serum. after whole blood has
`been separated.
`Many of these test devices utilize chromogenic or other
`visual responses to indicate the presence or absence. or the
`concentration. of an analyte being detected. Cellular com-
`ponents of whole blood. and in particular the redblood cells.
`have a deep red color which substantially interferes with
`chromogenic or other visual tests. Therefore. the highly-
`colored red blood cells as well as other interfering sub-
`stances present in blood including hemoglobin and white
`blood cells are separated from the plasma or serum before a
`blood sample is assayed for a particular analyte.
`Conventionally. the plasma or serum is separated from the
`cellular material of whole blood by centrifugan'ou. The
`cellular material collects at the bottom of the centrifuge tube
`and the supernatant plasma or serum is decanted and tested
`for a particular analyte. Centrifugation. however. is time
`consuming. involves extra manipulative steps and requires
`equipment that is generally not present outside of the clinical
`laboratory. Thus. reliance on eenuifugation makes field
`testing. such as testing at the doctor’s office or at the
`patient’s home. difficult.
`‘
`Certain methods other than centrifugation have been
`developed to separate the cellular components of whole
`blood from plasma or serum. Some of the earlier methods.
`such as that described in U.S. Pat. No. 4.543.338 to Chen.
`involve the use of a carrier membrane impregnated with a
`test reagent and coated with a semipermeable membrane.
`The semipermeable membrane effectively acts as a means
`for filtering out cells or
`large molecules. such as
`hemoglobin. but allows the passage of smaller molecules
`and ions which then contact the testing reagents impregnated
`in the bibulous matrix. These methods. however. typically
`require an extra manipulative step. such as rinsing with
`water or wiping off the test device so as to remove cellular
`material retained on the semipermeable membrane. Such
`techniques can be cumbersome and laborious. Moreover. if
`the red blood cells. are not completely removed or rinsed
`from the semipermeable barrier.
`terference with the assay
`remains a problem.
`Another method for separating whole blood is described
`in U.S. Pat. No. 4.477.575 to Vogel et al. which describes
`separating plasma or serum from whole blood using a layer
`of glass fibers having a defined average diameter and
`
`2
`in U.S. Pat No.
`density. As well. Baumgardner et al.
`5.186.843 describe the use of glass fibers in a single sepa-
`ration layer. Blood separation devices utilizing glass fiber
`filters. however. tend to separate serum at a relatively slow
`speed and tend to retain significant quantities of serum or
`plasma in the interstices of the glass fiber matrix.
`Alternative approaches to blood separation involve incor-
`porating agglutinating reagents or other separation reagents
`in a matrix. For example. Aunet et al. in U.S. Pat. No.
`4.933.092. Daubney et al.
`in published Canadian Patent
`Application No. 2.104.976. and Limbach in European Patent
`Application 0 194 502 all describe the use of polymeric
`agglutinating agents. such as cationic polymers. which. for
`example. in Daubney and in Limbach are combined with
`additional agglutinating agents. such as lectins. As well.
`Barkes et al. in European Patent Application No. 0 436 897.
`describe the use of lectins and thrombin incorporated into a
`suitable carrier matrix. However. such matrices incorporated
`with these types of agglutinating agents exhibit problems
`similar to those associated with glass fiber matrices. For
`example. the separation may occur at a relatively slow speed
`and the amount of plasma or serum separated may be limited
`to 50% of the absorption volume of the matrix. often
`requiring the use of external pressure. such as in European
`Patent Application 0 194 502. in order to obtain the maxi-
`mum efiiciency and quantity of plasma or serum._
`Other separating agents. such as water-soluble salts.
`amino acids. carbohydrates and large polymers. such as
`polyethylene glycol. polyvinyl alcohol and the like. have
`been incorporated into single matrix test strips. Fetter in U.S.
`Pat. Nos. 3.552.925 and 3.552.928 describes a test device
`having a bibulous matrix impregnated with an inorganic salt
`or amino acid at a first region on the matrix and test reagents
`impregnated at an adjacent second region. While the salts or
`amino acids used in this process can separate the cellular
`components from the whole blood.
`they also introduce
`contaminating ions or molecules into the plasma or serum
`and precipitate a portion of the soluble plasma or serum
`constituents. thus rendering a quantitative assay for the
`soluble constituent unreliable. Rapkin et al. in U.S. Pat. No.
`4.678.757 describe the use of carbohydrates. such as
`mannitol. impregnated or coated onto a carrier. preferably
`coated onto an impermeable carrier. The described device.
`whether having a permeable or impermeable carrier.
`however. only.provides for capillary and longitudinal trans-
`port of the blood. Therefore. the blood separation matrices
`of Raplrin et al. are not described as being useful in test
`devices that operate primarily by gravitational force. as do
`many of the multi—layer test devices currently used by
`doctors or patients. While Kiser et al. in U.S. Pat. No.
`5.306.623 describe separation matrices which can operate by
`wicking gravity flow. Kiser et al. use large polymeric
`separating reagents. such as polyethylene glycol. polysty-
`rene sulfonic acid. hydroxypropyl cellulose. polyvinyl
`alcohol. polyvinylpyrrolidone. and polyacrylic acid. Upon
`application of a whole blood sample. such large polymers
`contained within a matrix would be solubilized. potentially
`blocking the pores of the matrix and most certainly render-
`ing the sample more viscous. thereby slowing the separation
`process and decreasing the yield of plasma.
`Based on the shortcomings of these methods. there exists
`a need for a device that provides rapid and eflicient methods
`for the separation of plasma or serum from whole blood. In
`particular. there is an increasing awareness of the impor-
`tance of. and accordingly a need for. being able to carry out
`diagnostic assays at the doctor‘s oflice or. better yet. at
`home. Therefore. there exists a need to minimize any extra
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`5,762,871
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`3
`manipulative steps. such as the rinsing or wiping of test
`devices or the application of external pressure. Moreover.
`there is a need for rapid separation which is reliable and does
`not contain interfering substances. The present invention
`satisfies these needs and provides related advantages as well.
`
`SUMMARY OF THE INVENTION
`
`The present invention relates to a method and to a device
`for separating plasma from whole blood. The method and
`device utilize a permeable non-glass fiber matrix containing
`a polyol which is capable of clumping red blood cells. The
`matrix. in the absence of such a polyol. would otherwise be
`porous to red blood cells. The polyol-containing mau'ix has
`a first surface and a second surface such that a whole blood
`sample which is applied to the first surface flows direction‘
`ally toward the second surface. Plasma separated from
`whole blood becomes available at the second surface of the
`matrix and can be tested for the presence of a particular
`analyte. such as glucose or fructosamine. as provided by
`multi-layer test devices of the present invention.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 depicts one embodiment of a multi—layer fruc«
`tosamine test device which can be used for separating
`plasma from a whole blood sample and measuring the
`concentration of fructosamine.
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`FIG. 2 exemplifies one embodiment of a glucose test
`device which is useful for separating plasma from whole
`blood and measuring the concentration of glucose.
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`DETAILED DESCRIPTION OF THE
`INVENTION
`
`There is an increasing awareness of the importance of
`being able to carry out diagnostic assays at the doctor’s
`oflice or. better yet. at home. For example. a diabetic’s blood
`glucose level fluctuates significantly throughout a given day.
`being influenced by diet. activity. and treatment. Depending
`on the nature and severity of the individual case. some
`diabetic patients measure their blood glucose levels up to
`seven times a day. Clearly. the results of these tests should
`be available to the patient immediately.
`Because of the frequent fluctuation of glucose levels in a
`given day. tests which are independent of a patient's diet.
`activity. and/or treatment and which provide longer term
`indications of blood glucose levels have been developed.
`These tests measure the concentration of glycated proteins
`or “frucosamines.” Proteins. such as those present in whole
`blood. plaSma. serum and other biological fluids react with
`glucose. under non-enzymatic conditions. to produce gly-
`cated proteins. The extent of the reaction is directly depen-
`dent upon the glucose concentration of the blood. Measure-y
`ment of serum or plasma fructosarnine levels is useful for
`monitoring diabetic control because fructosamine concen—
`trations in serum or plasma reflect an average of blood
`glucose level over approximately a half month period
`Scandinavian investigators recently showed that doctors
`and patients who were made aware of their glycated protein
`test results had better glycemic control than those who were
`unaware of such results. Moreover. it is now believed that
`glycated proteins can be the causative agents of complica-
`tions associated with diabetes. which include retinopathy.
`nephropathy. neuropathy and cardiovascular disease.
`Therefore. any delay in information transfer. such as a
`doctor's delay in reporting clinical test results to a patient.
`decreases the value of the test result. Again. this emphasizes
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`the importance in being able to perform diagnostic assays at
`the doctor‘s office or at home.
`
`For an assay to be useful in the doctor’s office or home.
`the test should be relatively free of sensitivity to small
`changes in the conditions under which the assayis carried
`out and the measurements should be accurate and reliable.
`Equally as important. if not more so. the assay must have a
`simple and convenient protocol which does not involve extra
`manipulative steps. To enhance the simplicity and conve-
`nience of such tests. the preferable body fluid for testing
`such analytes as glucose and fructosamine is whole blood
`which can simply be taken from a finger or earlobe puncture.
`Such simple and rapid determinations of an analyte in blood
`is especially desirable in the case of an emergency.
`As described above. the simplicity and accuracy of such
`tests depends to a large extent on the whole blood separation
`layer contained within the test device and the ability of the
`blood separation matrix to provide uncontaminated plasma
`or serum. The present invention provides a method and
`device. namely a blood separation-matrix. which is capable
`of such simple and accurate blood separation. Of particular
`importance. the matrix contains a polyol which causes red
`blood cells contained in whole blood to clump. The clumped
`red blood cells either can be retained in the matrix or can be
`filtered by a filter material. The method and matrix can be
`used in various test devices which analyze whole blood for
`a particular analyte. such as. the fructosamine and glucose
`test devices provided by the present invention.
`As used herein. the term “plasma” means the substantially
`colorless fluid obtained from a whole blood sample after red
`blood cells have been removed by the separation process and
`device of the present invention. Because plasma is serum
`plus the clotting protein fibrinogen. the term “plasma" is
`used broadly herein to include both plasma and serum.
`In order to obtain plasma from whole blood. the present
`invention provides a permeable non-glass fiber matrix con-
`taining a polyol which is capable of clumping red blood
`cells. The matrix is porous to red blood cells in the absence
`of such a polyol. The matrix has a first surface for sample
`application and a second surface where plasma is received or
`becomes available. If desired. the matrix additionally can
`contain a polycationic polymer. Also useful. though not
`required. is a permeable filter material or membrane sup-
`porting the separation matrix which serves as a final filter to
`red blood cells and/or provides a reagent layer for effecting
`an assay. Each of these components of the invention. as well
`as test devices which use the blood separation method and
`matrix of the present invention are disclosed in detail.
`MATRIX
`
`The separation matrix of the present invention is a per—
`meable matrix which does not contain glass fibers and;
`therefore. is termed “a permeable non-glass fiber matrix.”
`The term “permeable” means liquid-permeable. such as
`permeable to plasma. as well as permeable or porous to red
`blood cells when the matrix is provided in the absence of a
`polyol. As used herein. the phrase “matrix being porous to
`red blood cells in the absence of a polyo " means that
`without the polyol contained in or on the matrix the red
`blood cells would simply pass through the matrix. virtually
`immediately. In the absence of the polyol. red blood cells are
`not retained. by filtration or otherwise. in die matrix.
`The polyol contained within or on the matrix chemically
`reacts with the whole blood sample so as to clump the red
`blood cells. As used herein. “clump” or “clumping” means
`the collection into a mass or group. red blood cells distrib-
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`lnfopia Ex. 1020 pg. 5
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`5,762,871
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`5
`uted in a whole blood sample. While not wishing to be
`bound by any theory or mechanism the clumping can be the
`result of agglutination. coagulation. or the like. or some
`other chemical interaction between the polyol and the red
`blood cells. Thus. the present invention. is not strictly a
`filtration process. for example. based on the pore size of the
`matrix. as described. for example. in U.S. Pat No. 4.543.338
`to Chen. or as used in a glass fiber prefilter. such as that
`described in U.S. Pat. No. 4.477.575 to Vogel et al. Rather.
`it is the presence of a polyol which provides the matrix with
`its ability to separate plasma from whole blood by clumping
`the red blood cells.
`
`Surprisingly. the clumping of the red blood cells by the
`polyol does not substantially block the flow of the whole
`blood sample or plasma through the matrix. Thus. sufficient
`amounts of plasma become available at the second surface
`of the matrix. Sufficient quantities of plasma can rapidly be
`obtained for the specific applications exemplified herein.
`namely analyzing the concentration of frucotosamine or
`glucose in drops of whole blood. The present invention can
`be used with other applications and diagnostic assays as
`well. including ones which use a larger volume of blood or
`which require more plasma. With as small as a 3/15" diameter
`circle of a polyol-containing matrix of the present invention.
`as much as 10 ul of plasma can be obtained from a drop of
`blood. Therefore. a matrix of the present invention can be
`used for separating larger volumes of blood than a drop of
`blood. Accordingly. the matrix can be used in diagnostic
`applications which analyze larger quantifies of blood. such
`as for example. some known cholesterol tests.
`A useful permeable matrix can be a woven or non-woven
`material and can be an absorbent or a non-absorbent material
`which may or may not be hydrophilic. Especially suitable
`materials for the matrix include. for example. woven or
`non-woven. absorbent or non-absorbent. nylon. rayon.
`cotton. and polyester. In one embodiment of the invention.
`the matrix is a non-woven. non-absorbent polyester. The
`polyester is preferably a poly(paraphenylene terephthalate).
`such as that used in a preferred polyester sold as Sontara®
`(DuPont. Inc.. Wilmington. Del.). Another preferred matrix
`is the woven. absorbent nylon Tetex® 3-3710 (Tetko. Inc..
`Lancaster. N.Y.).
`Depending upon the porosity or other properties of the
`matrix. the clumped red blood cells either are retained in the
`matrix or are filtered out by the filter material as described
`below. Some of the above-described matrix materials. such
`as the non-woven. non-absorbent polyesters. do not have
`“pores" in the traditional sense. i.e.. that can be measured.
`for example. by pore size (microns). In the absence of a
`polyol of the present invention such materials essentially
`have no limit as the porosity and are porous to red blood
`cells. which have an average size of 5 pm. With such
`macroporous materials. if the polyol is not present the red
`blood cells pass through the matrix almost immediately. For
`those matrix materials which can be characterized based on
`pore size. the matrices used in the present invention can have
`a pore size generally of from about 2 pm to about 10 um.
`Such pores sizes can be useful for retaining the clumped red
`blood cells. Depending upon the porosity. thickness. which
`is generally 200 to 1100 um. and other properties of the
`matrix. such as absorbency. the clumped red blood cells are
`either retained in the matrix or captured in a final filter
`material as described below.
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`The polyol-containing matrix has a first surface for
`sample application and a second surface where plasma is
`received or becomes available for testing or additional
`separation. Generally.
`the first 'and second surfaces are
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`presented as opposite sides of the matrix. The whole blood
`sample flows in a direction from the first surface toward the
`second surface. under conditions which provide such direc~
`tional flow. such as. gravitation. vacuum. or external pres-
`sure. To enhance the simplicity of the method. if desired.
`separation can be performed by gravity alone. Preferably.
`the separation matrix provides for flow in a vertical
`direction. preferably by gravitation.
`Rapkin et al. in U.S. Pat. No. 4.678.757. describe the use
`of carbohydrates. such as mannitol. impregnated or coated
`onto a carrier. preferably coated onto an impenneable car-
`rier. HoweVer. the described device of Rapkin et al.. whether
`having a permeable or impermeable carrier. only provides
`for capillary and lateral transport of the blood. There can be
`divergent contact times provided by vertical flow. a rela-
`tively short period of contact. versus lateral flow. a slow
`process which can involve continuous interaction between a
`whole blood sample and a matrix. Because of these variable
`contact times. it is not predictable that what works by lateral
`flow would similarly work under vertical
`flow.
`Unexpectedly. with the present invention. even with matri-
`ces which are porous to red blood cells in the absence of a
`polyol. a matrix containing a polyol can efiectively separate
`plasma from whole blood even when the blood sample flows
`vertically through the matrix.
`
`POLYOL
`
`The separation method and devich include a permeable
`non-glass fiber matrix containing a polyol. As used herein.
`the terms “matrix containing a polyol” and “polyol-
`containing man-ix” mean that the polyol is separately added ‘
`to the matrix and is not a component originally found in the
`composition or make up of the matrix. such as cellulose filter
`paper. Further. “matrix containing a polyo ” means a polyol
`can be impregnated into the matrix or coated into or onto the
`matrix or covalently or non-covalently bound to the matrix.
`In a preferred embodiment. the polyol is impregnated into
`the man-ix.
`
`As used herein. the term "polyol" means a polyhydroxy
`alcohol which is an alkyl or aromatic containing more than
`one hydroxyl group. The term “poly” as used in “polyol"
`does not infer that the alkyl or aromatic compound is a large
`polymer made up of repeating monomeric units. but.
`instead. means that more than one hydroxyl group is present
`in the compound. As discussed more fully below. with the
`exception of polysaccharides. the polyols used in the present
`invention are simple sugars or sugar alcohols.
`oligosaccharides. or other naturally or non~naturally occur~
`ring non-polymeric alkyl or aromatic compounds.
`Therefore. the term “polyol" encompasses sugars. alcohol
`derivatives of sugars. herein termed “sugar alcohols." and
`other naturally or non-naturallyvoccurring non-polymeric
`polyols.
`‘
`As used herein. “sugar" includes monosaccharides.
`oligosaccha‘rides. and polysaccharides. A monosaccharide is
`a simple sugar which is as a linear. branched. or cyclic
`polyhydroxy alcohol containing either an aldehyde or a
`ketone group. Exemplary monosaccharides include. but are
`not limited to. mannose. glucose. talose. galactose. xylose.
`arabinose. lyxose. ribose and fructose. An oiigosaccharide is
`a linear or branched carbohydrate that consists from two to
`ten mouosaccharide units joined by means of glycosidic
`bonds. Oligosaccharides which can be used in the present
`invention include. but are not limited to disaccharides such
`as sucrose.
`trehalose. lactose and maltose. Examples of
`larger oligosaccharides which can be used in the invention
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`5 ,762,871
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`7
`include the cyclodextrins. such as alpha-cyclohexylamylose.
`beta-cycloheptaamylose. and gamma—cyclooctoamylose. as
`well as other oligosaccharides well known in the art. A
`polysaccharide is any linear or branched polymer having
`more than ten monosaccharides linked together by glyco-
`sidic bonds. Exemplary polysaccharides include. but are not
`limited to. ficoll. polysucrose. and hydroxyethyl starch.
`Encompassed within “sugar” are those sugars which are
`naturally occurring as well as those which are known but
`which have not yet been identified as occurring naturally in
`plants or animals. For example. there are five known natu-
`rally occurring aldohexoses.
`including D-glucose.
`D-mannose. D-talose. D-galactose. and L-galactose.
`However. the aldohexose structure has four chiral carbons
`and thus. sixteen possible stereoisomers. all of which are
`known. although only the five listed above have been
`identified as occurring naturally in plantsor animals. Thus.
`“sugar” encompasses enantiomers in either the D or L forms
`of a sugar as well as racemic mixtures thereof.
`A polyol of the present invention also can be a “sugar
`alcohol.” A “sugar alcohol" is an alcohol derivative of a
`mono- or an oligosaccharide which is generally formed by
`reduction of the aldehyde or ketone moiety on the mono- or
`oligosaccharide. Exemplary sugar alcohols include. but are
`not
`limited to. mannitol. sorbitol. arabitol.
`inositol.
`galactitol. erythritol. and threitol. Also included within the
`definition of “sugar alcoho ” are the alcohol derivatives of
`those mono- and oligosaocharides described above.
`Where chiral carbons are present in the sugar alcohol. the
`sugar alcohol may be in the D or L form. such as D-threitol
`or L-threitol. or in a racemic mixture of both the D and L
`forms. The sugar alcohol can. but does not have to. be
`naturally occurring. That is.
`the sugar alcohol can be a
`derivative of a knOWn. naturally occurring sugar. or.
`alternatively. it can have a D or L configuration known to
`exist but not necessarily identified as occurring in nature.
`The sugar alcohol also can be a sugar which is found
`naturally in its reduced alcohol form or it can be an alcohol
`derivative of a sugar which derivative is not known to exist
`in nature.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`8
`polyvinyl alcohol. polyvinylpyrrolidone. and polyacrylic
`acid. Upon application of a whole blood sample. such large
`polymers contained within a matrix would be solubilized.
`potentially blocking the pores of the matrix and most
`certainly rendering the sample more viscous. thereby slow-
`ing the separation process and decreasing the yield of
`plasma. Therefore. with the exception of the above-
`described' polysaccharides. the invention does not involve
`the use of large alkyl polymers as the primary separating
`agent. The matrix described in US. Pat. No. 5.306.623 is
`different from the instant invention in a number of other
`aspects as well. For instance. the Examples given in US.
`Pat. No. 5.306.623 involve the use of matrices which have
`a very small pore size. less than 1 pm. which would act as
`a filter to red blood cells in the absence of the disclosed
`polymers. As discussed above. the present invention is not
`strictly a filtration process.‘rather it involves the use of
`polyols which clump red blood cells and in the absence of
`such red blood cells the matrix would be porous to red blood
`cells. Moreover. the matrices taught by Kiser et al.. in
`addition to a polymer. also contain the test reagents. which
`reagents may substantially influence the separation and test
`results. The separation matrix of the present invention does
`not contain the test reagents. The test reagents. as disclosed
`in greater detail below. are either on the filter material or
`additional test reagent layers and the like.
`In one embodiment. to apply the polyol to the matrix. the
`polyol can simply be dissolved in an aqueous solution
`generally. at a concentration of about 20% when used alone.
`and at about 10% concentration when combined with a
`polycationic polymer. which is generally present in a con-
`centration of about 0.5% to 5% as diswssed more fully
`below. If desired. multiple layers of matrices containing
`polyol at lower concentrations. such as four layers of matrix
`containing 5% polyol. also can be used. The polyol and. if
`present. the polycationic polymer can alternatively be dis-
`solved in physiological saline (0.85% NaCl). phosphate
`buffered saline (PBS). an organic solvent. or the like.
`
`POLYCATIONIC POLYMER
`
`45
`
`In addition to sugar or sugar alcohols. the polyol can be
`a non-polymeric naturally occurring or non—naturally occur-
`ring polyol. which includes linear. branched. or cyclic alkyl
`or aromatic compounds containing more than one hydroxyl
`group. As used herein the term“non—polymeric” means the
`alkyl or aromatic compounds are not polymers. Polymers
`are defined as high molecular weight compounds consisting
`of long chains that may be open. closed. linear. branched. or
`crosslinked. which chains are composed of repeating units.
`called monomers. which may be either identical or different.
`As used herein. those polyols which are “naturally occur-
`ring" are ones which occur in nature and those which are
`“non-nattn'ally occurring" are notfound in nature. Generally.
`these naturally occurring or non-naturally occurring alkyl or 55
`aromatic compounds range in size from three to twenty
`carbons (C3 to C20). and more preferably. from three to ten
`carbons (C3 to Cm)- Examples of such naturally occurring.
`non-polymeric polyols are glycerol. a three-carbon trihy—
`droxy alcohol that occurs in many lipids. and quinic acid. a 60
`1.3.4.5-Tetrahydroxycyclohexanecarboxylic acid. which
`acid can be in the salt form. Examples of non-naturally
`occurring. non—polymeric polyols include pentaerythritol
`and dipentaerythritol.
`Kiser et al. in U.S. Pat. No. 5.306.623 describe the use of 65
`large polymeric separating reagents. such as polyethylene
`glycol. polystyrene sulfonic acid. hydroxypropyl cellulose.
`
`In addition to the polyol. a polycationic polymer can. but
`does not have to. be added to the matrix. Similar to the
`addition of a polyol to the matrix. the polycationic polymer
`can also be physically impregnated. coated into or onto. or
`covalently or non—covalently bound to the matrix. The
`polycationic polymer is also useful for clumping. as well as
`stabilizing clumped. red blood cells. the latter of which is
`described for example. in the published Canadian Patent
`Application No. 2.104.976 to Daubney et al.. which is
`incorporated herein by reference.
`The polycationic polymer component can be any polymer
`having more than one cationic site and are generally based
`on monomers which contain an amine group.
`Suitable polycationic polymers include. for example.
`hex‘adimethrine
`bromide.
`trimethylenehexamethylenediammoniumbromide.
`polylysine. polyallylamine. polyarginine. poly(N.N—
`dimethylaminoethylmethacrylate. copolymers of N.N-
`dimethylaminoethyhnethacrylate and methylmethacrylale.
`polyethyleneimine. poly(diallyldimethylammonium
`chloride). poly(1.1-dimethyl—3.S-dimethylenepiperidinium
`chloride). and mixtures thereof. The polymerized positively
`charged amino acids. such as polylysine. can have the amino
`acids in either the D or L forms. such as poly-L-lysine or
`poly-D-lysine. or a racemic mixture thereof. such as poly-
`D.L-lysine.
`.
`
`Infopia Ex. 1020 pg. 7
`
`

`

`5,762,371
`
`9
`As described above. in one