EXHIBIT 1016
`
`US. PATENT NO. 5,876,605 TOAKITAHMA ET AL.
`
`lnfopia Ex. 1016 pg. 1
`
`

`
`United States Patent
`9 Kitajima et al.
`
`[19]
`
`I|||1|||||l|||||l|||||||||l||||lllllllllll||l|l||l||llllllllllllllllllllllli
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`USOO5876605A
`
`.[11] Patent Number:
`
`5,876,605
`
`[45] Date of Patent:
`
`Mar. 2, 1999
`
`Infusionstherapie 19, 1992, pp. 187-189, Coden—1FI'RE3,
`XP000671432—Taborshi, et al.—“Application of an Auto-
`mated Plasma Filtration Device and a Blood Monitoring
`system for LDL Apheresis”.
`
`Coden,
`289-295
`pp.
`31,4,
`(1976),
`Sang
`Vos
`Vosaad—XPO00671437—Friedli, et al~“Studies on New Pro-
`cess Procedures in Plasma Fractionation on and Industrial
`
`Scale”, III Removal of Preoipitates by Filtration through a
`Horizontal Leaf Filter with Centrifugal Cleaning (Funda
`Filter) as an Alternative to Tubular—BoWl Centrifuges.
`
`Primary Exzzminer—John Kim
`Attorney, Agent, or Firm—McAulay Nissen Goldberg Kiel
`& Hand, LLP
`
`[57]
`
`ABSTRACT
`
`The method of the invention is for preparing a plasma or
`serum sample from Whole blood and includes mixing an
`aqueous solution of an inorganic salt or an amino acid or salt
`thereof with the Whole blood in an amount of 20% or less of
`the whole blood volume so that concentration of the inor-
`
`ganic salt or the amino acid or salt thereof becomes 10 to 200
`yrnol/1 ml whole blood, and then, filtering the Whole blood
`to remove blood cell components. According to the method
`of the invention, plasma separation from whole blood can be
`achieved efiiciently, and recovered volume of plasma can be
`increased. By selecting reagent, good plasma which has no
`deviation in component viewpoint, and which is possible to
`measure in the same level accuracy as a plasma obtained by
`centurifuging, can be obtained through a simple filtration
`operation.
`
`6 Claims, 2 Drawing Sheets
`
`[54] PREPARATION OF PLASMA OR SERUM
`SAMPLE FROM WHOLE BLOOD
`
`[75]
`
`Inventors: Masao Kitajima; Kenichiro Yazawa,
`both of Saitama, Japan
`
`[73] Assignee: Fuji Photo Film Co., Ltd., Kanagawa,
`Japan
`
`[211 Appl. No.: 734,430
`
`[22]
`
`Filed:
`
`Jan. 17, 1997
`
`[30]
`
`Foreign Application Priority Data
`
`Jan. 19, 1995
`
`[JP]
`
`Japan .................................... 8—007691
`
`Int. Cl.5 ................................................... .. B01D 61/00
`[51]
`[52] U.S. Cl.
`210/650; 210/645; 210/651;
`
`210/653; 210/654; 210/723; 210/767; 435/2;
`436/177; 436/178
`[58] Field of Search ................................... .. 210/645, 650,
`210/651, 653, 654, 767, 749, 723, 724,
`725, 753, 488, 489, 490, 496, 500.41, 503,
`505, 506, 507, 508; 436/175, 177, 178;
`435/2
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`1/1971 Fetter.
`1/1971 Fetter .
`210/143
`7/1987 Watanabe etal.
`210/749
`6/1,992 Sand et al.
`..... ..
`436/175
`5/1993 Genshaw .... ..
`6/1995 Allen et al.
`........................... .. 210/650
`OTHER PUBLICATIONS
`
`
`
`3,552,925
`3,552,928
`4,678,566
`5,118,428
`5,208,142
`5,423,989
`
`Database WPI—Derwent Publications AN 92—303877 & JP
`04 208 856 A—Sanwa Kagaku Kenkyusho Co.
`Database WPI—Derwent Publications AN 90~295055 & JP
`04 208 565 A—Fuji Photo Film Co. Ltd.
`
`12
`
`' '9
`
`‘"9;
`,:,oo
`5%}
`
`\
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`lnfopia Ex. 1016 pg. 2
`
`

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`U.S. Patent
`
`Mar. 2, 1999
`
`Sheet 1 of2
`
`5,876,605
`
`2Al
`
`FIG. 1
`
`Infopia Ex. 1016 pg. 3
`
`

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`U.S. Patent
`
`Mar. 2, 1999
`
`Sheet 2 of2
`
`5,876,605
`
`130
`RATIO
`
`(%)
`
`100
`
`Hot 20
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`Hot 40
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`Hot 48-
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`Hot 60
`
`
`
`Infopia Ex. 1016 pg. 4
`
`

`
`5,876,605
`
`1
`PREPARATION OF PLASMA OR SERUM
`SAMPLE FROM WHOLE BLOOD
`
`BACKGROUND OF THE INVENTION
`
`This intention relates to a method of preparing a plasma
`or serum sample from whole blood, especially to a method
`capable of obtaining a plasma or serum sample from whole
`blood having a high hematocrit value in a high separation
`rate without destroying blood cells.
`Type or concentration of blood Components, such as
`metabolites, proteins, lipids, electrolytes, enzymes, antigens,
`and antibodies, is measured, in general, using a plasma or
`serum sample obtained by centrifuging whole blood.
`However, centrifuging takes labor and time. Particularly,
`centrifuging is unsuitable for an urgent case of measuring a
`small number of samples promptly and in site inspection,
`because of requiring a centrifuge and electricity. Thereupon,
`it has been investigated to separate serum from whole blood
`by filtration.
`Several filtration methods using glass fiber filter have
`been known wherein whole blood is charged into the glass
`fiber put in a column from one side of the column, and
`plasma or serum is obtained from the other side (Japanese
`Patent KOKOKU Nos. 44-14673, 5-52463, Japanese Patent
`KOKAI Nos. 2-208565, 4-208856)H0wever, practical fil-
`tration methods capable of obtaining an amount of plasma or
`serum necessary for measuring by an automatic analyzer
`from whole blood have not been developed except a part of
`items, such as blood sugar.
`The reason is seemed that previous filtration systems do
`not satisfy the following three conditions
`1) It is diflicult to obtain a sufficient amount of plasma
`necessary for automatic analysis by filtration.
`2) Erythrocytes tend to destroy (hemolyze) through
`filtration, and accordingly, it is difficult
`to measure
`analytical items which are liable to be affected by
`hernoglobins released into plasma or serum by hemoly-
`sis or analytical items wherein quantity in blood cells is
`greater than plasma or serum, such as GOT, GPT, LDH,
`Va and K.
`_
`3) In the case of high hematocrit value (50% or more)
`samples,
`it is difficult
`to filter because of clogging
`filtering material by erythrocytes.
`For example, Japanese Patent KOKOKU No. 6-64054
`discloses plasma filtration technique by glass fiber filter
`made of a particular material, but it states that plasma
`amount capable of taking out shall be limited to 1/2 or less of
`the volume of glass fiber filter. In the Japanese patent, on the
`premise that hematocrit value is 50%, it is supposed that
`space of glass fiber filter is filled out by erythrocytes, and the
`quantity of plasma obtained by filtering up to the filled out
`state is set 50% of the volume of glass fiber filter at
`maximum However, whole blood samples having a hemo-
`tocrit value of 55 to 60% are not rare among whole blood
`samples daily dealt in clinical assay organization.
`
`SUMMARY OF THE INVENTION
`
`An object of the invention is to provide a sure means
`capable of separating plasma or serum by filtration even
`from high hematocrit value whole blood samples having a
`hematocrit Value of 60 to 70% which are a great obstacle
`upon separating plasmas from whole bloods, actually
`recorded among newborns and patients sulfering from dehy-
`dration.
`
`Another object of the invention is to provide a sure means
`capable of separating plasma or serum in an amount of 100
`pl or more, which is necessary for the measurement of plural
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`items by a usual automatic analyzer for clinical assay, from
`a sample having any hematocrit value without limiting the
`recovering amount of plasma to ‘/2 of glass fiber filter
`volume.
`_
`Still another object of the invention is to provide a means
`capable of obtaining plasma with good quality without
`destruction of blood cell components which is suitable for
`the measurement of most assay items among clinical chemi-
`cal assay and immunoserum assay, which is difiicult
`to
`realize by conventional filtration using a column.
`Problems in development of plasma-sepa/rating technique
`from whole blood are summarized in the following three
`points,
`to destroy erythrocytes (hemolyze) in plasma
`(1) Not
`separation When erythrocytes are destroyed, blood cell
`components of which quantity in blood cells is much
`greater than in plasma, such as Hb, GOT, LDH and K
`are released ‘into plasma to cause analytical error. Hb
`released into plasma interferes with optical measure-
`ment.
`
`(2) Sure plasma separation even from whole bloods
`having a high hematocrit value (50% or more) with
`difliculty in erythrocyte separation, and having ten-
`dency to hemolysis. When hematocrit value rises, sepa-
`ration by filtration becomes diflicult due to sharp
`increase of blood viscosity.
`(3) Variation of composition do not occur in plasma
`separation.
`The inventors investigated in order to solve the above
`problems and to develop a means capable of separating
`plasma in a high separation rate without hemolysis even
`from a high hematocrit value whole blood, and have
`achieved the objects by adding an agent capable of lowering
`hematocrit value to whole blood in a particular
`concentration, and then separating plasma.
`Thus, the present invention provides a method of prepar-
`ing a plasma or serum sample from whole blood which
`comprises mixing an aqueous solution of an inorganic salt or
`an amino acid or salt thereof with the whole blood in an
`amount of 20% or less of the whole blood volume so that
`concertration of said inorganic salt or said amino acid or salt
`thereof becomes 10 to 200 ,umol/1 ml whole blood, and then,
`filtering the whole blood to remove blood cell components.
`BRIEF DESCRIPTION OF DRAWINGS
`
`FIG. 1 is a sectional view of a filter unit used in Examples.
`FIG. 2 shows graphs indicating variation of various
`analytical values of plasmas obtained from whole bloods
`different in hematocrit value.
`1 Filter unit
`2 Filter holder
`
`3 Syringe
`4 Holder body
`5 Cap
`6 Filter chamber
`
`7 Sample intake
`8 Nozzle
`
`9 Suction part
`10 Glass fiber filter sheet
`11 Cellulose filter sheet
`12 Double face adhesive tape
`13 Polysulfone microporous membrane
`14 Flow area-regulating member
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`'The whole blood sample to which the method of the
`invention is applicable may contain various anticoagulant or
`
`Infopia Ex. 1016 pg. 5
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`

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`5,876,605
`
`3
`glycolysis inhibitor, such as heparin, NaF, EDTA or
`monoiodoacetate, or not. The method of the invention is
`effective especially against high hematocrit bloods, e.g.
`having a hematocrit (Hct) value of 50 to 70%, particularly
`55 to 70%.
`
`The inorganic salt and amino acid and salt thereof are an
`agent (HL agent) having an action to accelerate separation
`between blood cells and plasma to decrease hematocrit
`value.
`
`The HL agent which is the inorganic salt, amino acid or
`salt thereof is water-soluble and has a solubility of 100
`mmol/l or more, preferably 1 mol/l or more, particularly 3
`mol/l or more. Exemplary inorganic salts are combinations
`of monovalent or divalent alkals metal or alkaline earth
`metal and halogen, N03, S04, or CO3, such as NaCl, CsCl2,
`Li2SO4, CaCl2, Rb2SO4 and Cs2SO4. Exemplary amino
`acids are natural amino acids, such as Gly, Ala, Asp, Glu and
`glycinamide asparagine. The inorganic salt, amino acid and
`salt thereof are made so that pH of their aqueous solution is
`5 to 8, preferably 6 to 7.5, and therefore, the inorganic salt
`and amino acid salt may be a hydrogen salt, such as
`NaHCO3. Acidic amino acids, such as Asp and Glu, may be
`made into a salt of alkali metal or alkaline earth metal or the
`like, and basic amino acids, such as Lys and Arg, may be
`made into a salt of halogen, N03, S04, CO3 or the like.
`The HL agent be used is selected by considering subject
`matter to be measured, i.e. a component to be measured. For
`example, when sodium or chloride in blood is measured,
`NaCl is unsuitable as the HL agent. Phosphate is unsuitable
`as the HL agent for measuring inorganic phosphorus in
`blood, Moreover, HL agent which influences reactivity of
`reagents used for measurement cannot be used.
`iron,
`For example, HL agent containing magnesium,
`copper, barium, zinc, or the like cannot be used for mea-
`suring calcium, because they interfere with calcium mea-
`surement.
`
`The HL agent is used as an aqueous solution, A suitable
`concentration of the HL agent is 0.1 to 5 mol/l, preferably
`0.5 to 3 mol/l, more preferably 1 to 2.5 mol/l.
`Other components may be added to the aqueous HL agent
`solution. For example, glycerine, ethylene glycol, polyeth-
`ylene glycol or the like may be added for the purpose of
`prevention against drying. A buffer may be added for the
`purpose of pH adjustment. Furthermore, according to the
`object of analysis, various compounds may be added in
`order to facilitate separation of analyte in filtered plasma.
`Exemplary of the compounds are selective binding reagents
`for LDL choresterol, such as dextrin and tungstophosphoric
`acid, which are fractionation agent for the measurement of
`HDL choresterol. Other exemplary compounds are having
`reactivity with a particular component in plasma, such as
`various antigens and antibodies including modified ones.
`By adding the HL agent, flexibility of erythrocyte mem-
`brane decreases, and erythrocytes become resistant to defor-
`mation. It is known that erythrocyte passes through a con-
`siderably smaller space, e.g. a capillary 1-2 pm in diameter,
`than apparent size of erythrocyte during filtering whole
`blood. However, by adding the HL agent, since deformation
`of erythrocytes is restrained, filtration can be conducted very
`smoothly.
`is in
`Reduction of hematocrit value by the HL agent
`proportion to molar concentration of the HL agent in plasma
`volume of whole blood to which the HL agent is added. On
`the other hand, in a HL agent concentration of more than 200
`mmol/l, destruction of blood cells (hemolysis) tends to
`‘occur, and hemoglobin which is the main component of
`
`4
`erythrocyte elutes into plasma. Accordingly, a suitable
`amount of the HL agent added to whole blood is 10 to 200
`,umol, preferably 10 to 100 ,umol, more preferably 20 to 60
`pmol, per 1 ml whole blood. Volume of aqueous HL agent
`solution to be added is not especially limited. However, too
`large volume is undesirable, because the dilution rate of
`whole blood is great resulting in the degradation of accuracy
`in weighing. Moreover, when dilution rate is high, measur-
`ing accuracy is a problem in relation measuring sensitivity.
`Thereupon, a suitable volume of the aqueous HL agent
`solution to be added is 20% or less, preferably 10% or less,
`more preferably 5% or less of whole blood by volume ratio.
`The lower limit of the volume of the aqueous HL agent
`solution depends on the water-solubility of HL agent, etc.,
`and in general, 1% or more. In the method of the invention,
`filtering ability is improved by adding only a small amount
`of HL agent, influence on measuring sensitivity and accu-
`racy is small.
`When HL agent is added to whole blood., the HL agent is
`dissolved into plasma to elevate osmotic pressure of plasma
`to generate osmotic pressure difierence between plasma and
`the inside of blood cells. Then, force acts so as to loosen the
`diiference of osmotic pressure. That is, Water in blood cell
`is discharged into plasma to elevate osmotic pressure (i.e.
`concentration of solute) in blood cells and to lower osmotic
`pressure of plasma. As a result, volume of blood cell
`decreases and volume of plasma increases, resulting in the
`decrease of hematocrit value. By adding HL agent, Volume
`of erythrocyte decreases and flexibility of blood cell mem-
`brane decreases, and accordingly, filtration using a filtering
`material, such as porous membrane is facilitated,
`Moreover, since quantity of free plasma increases, recovery
`‘rate of plasma by filtration is improved.
`Although the lowering of hematocrit value caused by HL
`agent increases together with the increase of HL agent
`concentration, since strength of blood cell membrane is
`weak, blood cell is liable to destroy and hemolyze at high
`HL agent concentration. Since the action of HL agent is
`mainly derived from its osmotic pressure,
`the action is
`proportional to the number of solute molecules in whole
`blood (exactly in plasma). When the added amount of HL
`agent is too great, osmotic pressure diiference between the
`inside and outside of blood cell becomes too great. As a
`result, blood cell is ruptured or blood cell membrane is
`punctured to leak components in blood cell into plasma.
`In general, blood assay is based on the measurement of
`concentration of plasma component. When erythrocytes are
`destroyed or blood cell membranes are purctured, compo-
`nents in erythrocytes are leaked into plasma portion, Since
`erythrocyte contains Hb (hemoglobin)
`in a high
`concentration, when leakage occurs, plasma becomes red
`(called hemolysis) Even if the destruction of erythrocytes is
`very small, coloring of plasma caused by the leakage of Hb
`cannot be ignored, and in some analytical items, the leakage
`strongly influences analytical value. For example, in a blood
`assay of healthy persons, when 0.1% of total blood cells are
`destroyed, measured values of most enzymes, such as CPK
`and ALP, are not accurate, Some components (GOT, GPT,
`LDH, K) exist in erythrocyte in a higher concentration than
`plasma, and the measurement of those components is
`directly influenced by hemolysis. Accordingly, it is neces-
`sary to conduct separation, recovery of plasma under con-
`ditions where hemolysis occurs as small as possible.
`Whole blood of healthy person contains usually about 15
`g/dl hemoglobin
`Influence by leaked hemoglobin is
`different according to measuring method and measuring
`item.
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`Infopia Ex. 1016 pg. 6
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`

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`5,876,605
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`5
`For example, influence of hemolysis upon measurement
`of blood sugar and choresterol is small, and even when about
`150 mg/dl hemoglobin exists in plasma (corresponding to
`hemolysis of 1% of total erythrocytes), they are measured
`accurately.
`On the other hand, GOT, GPT, LDH, K and the like are
`liable to be influenced by hemolysis, and particularly, in the
`case of LDH or K, even when hemoglobin concentration is
`15 mg/dl (corresponding to hemolysis of 0.1% of total
`erythrocytes), measured values are influenced significantly
`in View of clinical diagnosis.
`Accordingly, in the case of measuring blood sugar and
`choresterol alone, plasma separation may be incomplete.
`However, in the case of measuring GOT, GPT, LDH, K, etc.,
`hemolysis must be less than 0.1%.
`Hematocrit value of healthy persons is 40 to 45%, but
`whole blood samples having a hematocrit value of 55 to 60%
`are dealt at a_frequency of several percents in daily clinical
`assay. In such high Hct samples, Hb content is 20 to 25 g/dl.
`If allowance of measurement against Hb is 15 mg/dl,
`hemolysis of high Hct samples must be restrained to less
`than 0.07%.
`When inorganic salt, amino acid or salt thereof or agglu-
`tinin such as lectin is dissolved directly in blood from a dry
`state or from impregnated into filter paper or porous
`material, followed by drying, blood contacts the dry matter.
`Blood is liable to hemolyze locally and temporarily in a
`initial stage of dissolving the dry matter in plasma.
`Accordingly, plasma obtained through the above process
`frequently contains hemoglobin which is not so much. The
`plasma has no problem for measuring blood sugar or
`choresterol, but has a problem for measuring GOT, GPT,
`LDH, K and the like. The tendency is more remarkable in
`high HCT samples.
`On the other hand, when the above compound is dissolved
`in water to make an aqueous solution with a suitable
`concentration, the degree of local concentration elevation is
`small upon mixing the aqueous solution into plasma pro-
`ceeds smoothly. As a result, hemolysis only rarely occurs.
`An amount of blood to be used is about 0.3 to 3 ml,
`usually about 0.5 to 1.5 ml, and mixing of aqueous HL agent
`solution with whole blood may be carried out only by
`several times shaking. It is supposed that the action of HL
`agent is in a moment, and the mixture reaches equilibrium
`within several seconds. Accordingly, it is not necessary to
`adjust temperature or time upon mixing.
`After mixing HL agent solution with whole blood sample,
`the whole blood sample is filtered. As the filtering material,
`various known filtering material capable of separating blood
`cells can be used. Illustrative of the filtering materials are
`glass fiber filter, microporous membranes haying blood
`cell-separating ability such as made of fluorine-containing
`polymer, of which the surface has been made hydrophilic, or
`polysulfone, laminates of glass fiber filter and microporous
`membrane, laminates of glass fiber filter and cellulose filter,
`laminates of glass fiber
`filter, cellulose filter and
`microporous membrane, laminates of fibrous porous mem-
`brane and non-fibrous porous membrane disclosed in Japa-
`nese Patent KOKAI Nos. 62-138756-8, 2-105043, 3-16651,
`etc. Preferred ones are containing glass fiber filter, and
`laminates of glass fiber filter and microporous membrane,
`laminates of glass fiber filter and cellulose filter, and lami-
`nates of glass fiber filter, cellulose filter and microporous
`membrane are particularly preferred. Filter materials con-
`taining polysulfone microporous membrane are also prefer-
`able. The most preferable ones are laminates of glass fiber
`filter, cellulose filter and microporous membrane, especially
`using polysulfone membrane as the microporous membrane.
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`Preferable glass fiber filter has a density of about 0.02 to
`0.3 g/cm3 preferably about 0.05 to 0.2 gcm3, more prefer-
`ably about 0.07 to 0.15 g/cm3, a retainable particle size of
`about 0.8 to 9 ;m, preferably 1 to 5 gm. By treating the
`surface of glass fiber with hydrophilic polymer as disclosed
`in Japanese Patent KOKAI Nos. 2-208565, 4-208856, fil-
`tration can be conducted more fast and smoothly. The
`surface of glass fiber may be treated with lectin. Two or
`more glass fiber filters may be lamirated.
`Microporous membranes having blood cell-separating
`ability of which the surface has been made hydrophilic
`separate whole blood specifically into blood cells and
`plasma without hemolysis to the degree of substantially
`influencing analytical values. A suitable pore size of the
`microporous membrane is smaller than the retainable par-
`ticle size of glass fiber filter, and is 0.2 ,um or more,
`preferably about 0.3 to 5 pm, more preferably about 0.5 to
`4.5 pm, particularly preferably about 1 to 3 gm. The void
`content of the microporous membrane is preferably higher,
`and a suitable void content is about 40 to 95%, preferably
`about 50 to 95%, more preferably about 70 to 95%. Illus-
`trative of the microporous membranes are polysulfone
`membrane, fluorine-containing‘ polymer membrane, cellu-
`lose acetate membrane, nitrocellalose membrane, etc. The
`surface of the membrane may be hydrolyzed or may be
`rendered hydrophilic by a hydrophilic polymer or an acti-
`vating agent.
`As the fluorine-containing polymer membranes, there are
`the microporous matrix membrane (microporous layer)
`composed of polytetrafluoroethylene fibrils (fines) disclosed
`in WO 87/02267, Gore-Tex (W. L. Gore and Associates),
`Zitex (Norton), Poreflon (Sumitomo Denki), etc. Other
`fluorine-containing polymer sheets usable as
`the
`microporous layer
`include polytetrafluoroethylene
`microporous membranes disclosed in U.S. Pat. No. 3,368,
`872 (Examples 3 and 4), US. Pat. No. 3,260,413 (Examples
`3 and 4), U.S. Pat. No. 4,201,548, etc., polyvinylidenefluo-
`ride microporous membranes disclosed in U.S. Pat. No.
`3,649,505 and the like, The microporous membrane of
`fluorine-containing polymer may be prepared by using a
`single fluorine-containing polymer or blending two or more
`kinds of fluorine-containing polymers of further blending
`one or more polymers not containing fluorine or fibers
`therewith. As the structure, there are unstretched one, uniaxi-
`ally stretched one, biaxially stretched one, nonlaminated
`single layer type, laminated double layer type, such as a
`membrane laminated to another membrane structure such as
`a fiber membrane In the case of nonlaminated type
`microporous membrane having fibril structure or having
`been uniaxially or biaxially stretched, microporous mem-
`brane having a great void content and a short filtering pass
`can be prepared by stretching. In microporous membranes
`having a short filtering pass, clogging rarely occurs by solid
`components (mainly red blood cells) in blood, and the
`separation time of blood cells and plasma is short. As a
`result, accuracy in quantitative analysis is improved, The
`adhesive strength of adhesive used for the partial adhesion
`to the adjacentmicroporous membrane can be strengthened
`by providing the physical activation (preferably glow dis-
`charge or corona discharge) disclosed in U.S. Pat. No.
`4,783,315 on at least one side of the microporous membrane
`of fluorine-containing polymer to render hydrophilic.
`It
`is wellknown that fluorine-containing polymer
`microporous membranes as it is have a low surface tension.
`As a result, when the membrane is used as the blood cell
`filtering layer,» aqueous liquid samples are repelled and do
`not diffuse nor permeate over the surface or into the inside.
`
`lnfopia Ex. 1016 pg. 7
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`5,876,605
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`7
`the above repelling problem has been
`In the invention,
`resolved by incorporating a sufficient amount of surfactant
`for rendering the outer surface and the inner space surface of
`the fluorine-containing polymer microporous membrane
`substantially hydrophilic thereinto. In order to impart a
`hydrophilic property sufiicient for diflusing, permeating or
`moving an aqueous liquid sample over the surface or into the
`inside of the fluorine-containing polymer microporous
`membrane without repelling to the membrane, in general, it
`is necessary that the space surface of the membrane is coated
`with a surfactant in an amount of about 0.01 to 10%,
`preferably about 0.1 to 5%, more preferably about 0.1 to 1%
`of the void volume of the membrane, For example, in the
`case of a fluorine-containing polymer microporous mem-
`brane 50 ,um in thickness, a preferred amount of surfactant
`to be impregnated is usually in the range of 0.05 to 2.5 g/m2.
`As the method of impregnating surfactant into a fluorine-
`containing microporous membrane, a common method cmo-
`prises immersing the fluorine-containing microporous mem-
`brane in the surfactant solution dissolved in a low boiling
`point (a preferable boiling point is in the range of about 50°
`C. to about 12° C.) organic solvent (e.g. alcohols, esters,
`ketones) to permeate into the inner spaces of the membrane
`substantially sufiiciently, taking the membrane out of the
`solution slowly, and then drying by blowing air (preferably
`warm air).
`As the surfactant for rendering the fluorine-containing
`polymer microporous membrane hydrophilic, the surfacet-
`ant may be nonionic, anionic, cationic or ampholytic.
`However, nonionic surfactants are advantageous for the
`multilayer analytical elements for analyzing whole blood
`samples, because nonionic surfactants have a relatively low
`hemolytic activity among the above surfactants, Suitable
`nonionic
`surfactants
`include
`alkylphenoxypolyethoxyethanol, alkylpolyether alcohol,
`polyethyleneglycol monoester, polyethyleneglycol diester,
`higher alcohol-ethylene oxide adduct (condensate), polyol
`ester-ethylene oxide adduct (condensate), higher fatty acid
`alkanol amide, etc. Examples of the nonionic surfactant are
`as follows: As the alkylphenoxypolyethoxyethanol, there are
`isooctylphenoxypol y-ethoxyethanols (Triton X-100; con-
`taining 9-10 hydroxyethylene units on average, Triton X-45;
`containing 5 hydroxyethylene units on average) and non-
`ylphenoxypolyethoxyethanols (IGEPAL CO-630; contain-
`ing 9 hydroxyethylene units on average, IGEPAL CO-710;
`containing 10-11 hydroxyethylene units on average,
`LENEX 698; containing 9 hydroxyethylene units, on
`average). As the alkylpolyether alcohol,
`there are higher
`alcohol polyoxyethylene ethers (Triton X-67; CA Registry
`No. 59030-15-8), etc.
`The fluorine-containing polymer microporous membrane
`may be rendered hydrophilic by providing one or more
`Water-insolubilized Water-soluble polymers in its porous
`spaces. The water-soluble polymers include oxygen-
`containing hydrocarbons, such as polyacrylamide,
`polyvinylpyrrolidone, polyvinylamine and
`polyethyleneimine, negative charge-containing ones such as
`polyvinyl alcohol, polyethylene oxide, polyethylene glycol,
`methyl cellulose, ethyl cellulose, hydroxyethyl cellulose and
`hydroxypropyl cellulose, nitrogen-containing ones, such as
`polyacrylic acid, polymetacrylic acid and polystyrene sul-
`fonic acid, and the like. The water-insolubilization may be
`conducted by heat
`treatment, acetal-inducing treatment,
`esterification, chemical reaction by potassium dichromate,
`crosslinking by ionizable radiation, or the like. Details are
`disclosed in Japanese Patent KOKOKU Nos. 56-2094 and
`56-16187.
`
`8
`The polysulfone microporous membrane can be prepared
`by dissolving polysulfone into dioxane,
`tetrahydrofuran,
`dimethylformamide, dimethylacetamide, N-methyl-2-
`pyrolidone or a mixed solvent thereof to obtaine a raw liquid
`for forming film, casting into film by flowing directly into a
`coagulating solution, washing, and then drying. Details are
`disclosed in Japanese Patent KOKAI No. 62-27006. In
`addition, polysulfone microporous membranes are also dis-
`closed in Japanese Patent KOKAI Nos. 56-12640,
`7 56-86941, 56-154051, etc., and they are applicable to the
`invention. The polysulfone microporous membrane can be
`rendered hydrophilic, similar to the fluorine-containing
`polymer, by incorporating surfactant or providing Water-
`insolubilized Water-soluble polymer.
`Preferable nonfibrous porous membranes, in the laminate
`of a fibrous porous membrane and a nonfibrous porous
`membrane are blushed polymer membranes composed of a
`cellulose ester, such as cellulose acetate, cellulose acetate/
`butyrate or cellulose nitrate, disclosed in U.S. Pat. No.
`3,992.158 or U.S. Pat. No. 1,421,341. The nonfibrous porous
`membrane may be a microporous membrane of polyamide,
`such as 6-nylon or 6,6-nylon, or polyethylene,
`polypropylene, or the like. Other nonfibrous porous mem-
`branes usable for the blood cell-separating filter include
`continuous microspace-containing porous membranes
`where polymer particulates, glass particulates, diatomaceous
`earth or the like are joined by a hydrophilic or non-Water-
`adsorptive polymer, such as disclosed in US. Pat. No.
`3,992,158, and U.S. 4,258,001.
`Preferable effective pore size of the nonfibrous porous
`membrane is 0.2 to 10 gm, preferably 0.3 to 5 ttm, particu-
`larly preferably 0.5 to 5 tan. The effective pore size of the
`nonfibrous porous membrane in the invention is the pore
`size measured by the bubble point method according to
`ASTM F316-70. In the case that
`the nonfibrous porous
`membrane in a membrane filter composed of blushed poly-
`mer prepared by the phase separation method, the liquid
`passages in the thickness direction are,
`in general,
`the
`narrowest at the free surface (glossy face) in the manufac-
`turing process of the membrane, and the pore size in section
`of each liquid passage stipulated a circle is the smallest near
`the free surface The minimum pore size of passages in the
`thickness direction per unit area has a distribution in facial
`direction of the membrane filter, and the maximum value
`determines filtration performance In general, it
`is deter-
`mined by the limit bubble point method.
`As mentioned above, in the membrane filter composed of
`blushed polymer prepared by the phase separation method,
`liquid passages in the thickness direction become the nar-
`rowest at the free surface (glossy face) in the manufacturing
`I process of the membrane. In the case of using the membrane
`as the nonfibrous porous membrane of the filtering material
`of the invention, it is preferable to face the glossy face of the
`membrane filter toward the side to discharge the plasma
`portion.
`The material composing the fibrous porous membrane
`may be filter paper, nonwoven fabric, Woven fabric such as
`plain weave fabric, knitted fabric such as tricot fabric, etc.
`Among them, woven fabric and knitted fabric are preferred.
`The woven fabric or the like may be treated by blow
`discharge as disclosed in Japanese Patent KOKAI No.
`57-66359.
`The void volume (per unit area) of the fibrous porous
`membrane may be equal to or different from the nonfibrous
`porous layer. The void volume correlation between them
`may be adjusted by changing either or both of void content
`and thickness.
`
`id
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Infopia Ex. 1016 pg. 8
`
`

`
`5,876,605
`
`9
`In the case that the filtering material used in the invention
`is a laminate, respective layers may be integrated by joining
`each other using partially disposed (e.g. spots) adhesive,
`according to disclosures in Japanese Patent KOKAI Nos.
`62-138756-8, 2-105043, 3-16651, etc.
`In the filtering material of the invention, it is thought that
`the filter material does not trap blood cells only by the
`surface, but catches to remove blood cells gradually by
`entangling at first large blood cell components and then
`smaller blood cell components in the space structure with
`permeating in the thickness direction in total of the filtering
`material called the volumetric filtration.
`
`In the system of the invention, density, thickness, area,
`etc. of the filtering material may be arranged acco