`
`
`
`EXHIBIT 1006
`
`US. PATENT NO. 5,135,716 TO THAKORE
`
`lnfopia Ex. 1006 pg. 1
`
`

`

`United States Patent
`
`[19]
`
`[11] Patent Number:
`[45]
`7 Date of Patent:
`Aug. 4, 1992 Thakore
`
`,
`
`llllllllllllllllllllliIllllIlllllllllllllllllllllllllllllIlllllllllllllllll
`
`USOOSl35716A
`
`5,135,716
`
`[54] DIRECT MEASUREMEN’l‘ 0F HDL
`CHOLESTEROL VIA DRY CHEMISTRY
`STRIPS
`
`[75] . Inventor: Yatin B. Thakore, East Brunswick
`Township, Middlesex County, NJ.
`
`[73] Assignee: Kingston Diagnostics, L.P., Dayton,
`NJ.
`
`[21] Appl. No: 660,429
`
`[22] Filed:
`
`Feb. 25, 1991
`
`Related US. Application Data
`
`[63]
`
`' Continuation-in-partofSer.No.379,009,1u1. 12,1989.
`
`Int. Cl.5 ............................................. G01N 21/77
`[51]
`
`[52] US. Cl.
`.............................. 422/56; 422/58;
`422/73; 435/11; 435/805; 436/17; 436/71
`[58] Field of Search .......... , ............ 422/56, 57, 58, 73;
`436/17, 71; 435/1’1, 805 .
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`‘ 3,992,158 11/1976 Przybylowicz et al.
`............. 422/56
`
`4,188,188 2/1980 Willner et ah ...........
`. 435/11 X
`4,210,557 7/ 1980 Handschuh ..
`..... 436/17
`
`4,226,713 10/1980 Goldberg .
`, 435/11 X
`
`4,366,244 12/1982 Pascal ......
`. 435/11
`
`4,786,603 11/1988 Wielinger e
`. 436/69
`.
`
`4,816,224 3/1989 Vogel et a1.
`.....
`435/170
`
`4,820,489 4/1989 Rothe et al,
`..... 422/56
`
`4,839,297
`6/1989 Frietag et a1.
`436/170
`
`4,851,335 7/1989 Kerscher et al.
`..... 435/11
`4,857,453
`8/1989 Ullman et a1.
`....... 435/7
`
`4,861,712
`8/1989 Bartlet a1.
`..... 435/13
`
`
`4,876,067 10/ 1989 Deneke ........
`. 422/56
`4,892,815
`1/1990 Kerscher et a1.
`....................... 435/7
`
`.. 422/56
`3/1990 Grenner ..................
`4,906,439
`435/7
`4,943,522 7/1990 Eisinger et al.
`
`4,987,085
`1/1991 Allen et a1.
`..
`5,034,332
`7/1991 Rapacz et a1.
`.................... 435/11 X
`
`Primary Exa’minerL—James C. Housel
`Assistant Examiner—Jeffrey R. Snay
`Attorney, Agent, or Firm—Kenneth P. Glynn
`
`answer
`[57]
`A device for determining HDL cholesterol by obtain-
`ing plasma from whole blood and determining the HDL
`cholesterOl level from the plasma. The device includes
`an inert substrate support or an active substrate support
`(eg. one or the other layers), a physical transport me-
`dium, a microporous plasma separation membrane con-
`nected to the physical transport medium, at least one
`plasma collecting test membrane, a filtering membrane,
`LDL and VLDL reactants to form LDL and VLDL
`precipitates and an optional carrier precipitation mem~
`brane. The plasma collecting test membrane has reac-
`tants which will react with HDL cholesterol and indi-
`cate the HDL cholesterol
`level quantitatively. The
`filtering membrane may be located between the micro-
`porous plasma separation membrane and the transport
`medium or between the microporous plasma membrane
`and the plasma collecting test membrane and its func-
`tion is to block the precipitated particles from reaching
`the test zone. The LDL and VLDL reactants which
`form precipitates of LDL and VLDL may be located
`anywhere upstream from the plasma collecting test
`membrane,
`i.e., within one or more of the transport
`medium, the microporous plasma separation membrane,
`the filtering membrane and the optional carriersepara-
`tiori membrane.
`
`91 Claims, 8 Drawing Sheets
`
`IO
`
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`
`
`
`lnfopia Ex. 1006 pg. 2
`
`

`

`US. Patent
`
`Aug. 4, 1992
`
`Sheet 1 of_8
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`5,135,716
`
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`US. Patent
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`Aug. 4, 1992
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`Sheet 2 of 8
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`lnfopia Ex. 1006 pg. 4
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`

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`US. Patent ‘
`
`Aug. 4, 1992
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`Sheet 3 of 8
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`5,135,716
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`

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`US. Patent
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`Aug. 4, 1992
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`lnfopia Ex. 1006 pg. 6
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`

`

`US. Patent
`
`Aug. 4, 1992
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`Aug. 4, 1992
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`lnfopia Ex. 1006 pg. 8
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`

`

`US. Patent
`
`Aug. 4, 1992
`
`Sheet 7 of 8
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`5,135,716
`
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`

`

`US. Patent
`
`Aug. 4; 1992
`
`Sheet 8 of 8
`
`5,135,716
`
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`Infopia Ex. 1006 pg. 10
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`
`
`

`

`'1
`
`5,135,716
`
`2
`cally are available but can be quite complex and expen-
`szva
`
`,
`
`DIRECT MEASUREMENT OF HDL
`CHOLESTEROL VIA DRY CHEMISTRY STRIPS
`
`REFERENCE TO A RELATED APPLICATION
`AND INCORPORATION BY REFERENCE
`
`This application is a continuation-in-part of pending
`U.S. patent application Ser. No. 07/379,009, entitled
`“Device and Method for Separation of Plasma from
`Blood and Determination of Blood Analytes” filed on
`Jul. 12, 1989 by Yatin B. Thakore and Karen L. Swan-
`son. The drawings thereof are incorporated herein by
`reference in their entirety.
`
`5
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`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention is directed to a device for de-
`termining high density cholesterol (HDL) which allows
`the user to obtain rapid, reliable results in a simple man-
`ner. More specifically, the present invention is directed
`to HDL test strips utilizing dry chemistry.
`2. Prior Art
`Measurement of high density cholesterol, particularly
`in conjunction with cholesterol measurement, has been
`proven to be an effective indicator of potential risk to
`atherosclerotic cardiovascular disease (CVD). There-
`fore,
`the determination of high density lipoprotein
`(HDL) cholesterol has become important and common
`in clinical laboratories.
`The traditional method of measuring these analytes is
`via wet chemistry, although commercial dry chemistry
`tests are available for total cholesterol. HDL choles-
`terol measurements, however, continue to be time con-
`surning.
`For a measurement of high density cholesterol, one
`needs to separate serum/plasma from whole blood by
`traditional methods of clotting or centrifugation. The
`separated plasma or serum is then added in a precise
`ratio with a precipitant system and mixed thoroughly.
`The plasma/precipitant mixture is allowed to sit for
`5-25 minutes to allow the completion of precipitate ~
`formation and agglomeration of the precipitated parti-
`cles. After this step, the mixture is centrifuged to allow
`the precipitate to form a cake at the bottom of the cen-
`trifuge tube and the supernatant containing high density
`lipoprotein (HDL) is carefully withdrawn. The choles~
`terol associated with this HDL fraction (HDL choles-
`terol) is then measured either via wet chemistry or
`could be measured by a cholesterol dry strip if designed
`to do so.
`Another common method used for HDL cholesterol
`measurement
`is ultracentrifugation wherein various
`cholesterol-containing fractions are separated in an
`ultracentrifuge. This method is even more laborious and
`time consuming and requires considerable technical
`skill. Only a few reference laboratories are equipped to
`measure HDL cholesterol in this way and this method is
`quite expensive. Electrophoresis of lipoproteins has also
`been used but this again is slow, expensive and semi-
`quantitative. It is usually used only as an adjunct to
`other quantitative methods.
`HDL cholesterol measurements therefore tend to be
`time consuming with manual methods. These steps can
`i be automated, and for a large volume of sample
`throughput as in many clinical laboratories, analyzers
`which can dispense and process the reagents automati-
`
`65
`
`Many patents have been issued which describe the .
`various physical arrangements for blood testing.*These
`include systems which involve lateral or horizontal
`movement of the blood, as well as plasma testing. For
`example, U.S. Pat. No. 4,876,067 to Deneke et a]; U.S.
`Pat. Nos. 4,861,712 to Bartl et al; 4,839,297 to Freitag et
`a1 and U.S. Pat. No. 4,786,603 to Wielinger et al, all
`assigned to Boehringer Mannheim GmbH, describe test
`carriers and methods for analytical determination of
`components of bodily fluids,
`including separating
`plasma from blood using glass fibers and the like. These
`patents, however, all teach systems which require some
`type of rotation of test pads or a portion of the test pads
`during use. U.S. Pat. No. 4,816,224 to Vogel et al also
`assigned to Boehringer Mannheim GmbH, describes a
`device for separating plasma or serum from whole
`blood and analyzing the serum using a glass fiber layer
`having specific dimensions and absorption to separate
`out the plasma from the whole blood for subsequent
`reaction.
`.
`U.S. Pat. No. 4,857,453 to Ullman et a1 describes a
`device for performing an assay using capillary action
`and a test strip containing sealed liquid reagents includ-
`ing visible indicators. U.S. Pat. No. 4,906,439 to Gren-
`ner describes a diagnostic device for efficiently and
`accurately analyzing a sample of bodily fluid using fluid
`delivery in a lateral movement via flow through chan-
`nels or grooves.
`In addition to the above patents which are representa~
`tive of the prior art showing various physical types of
`systems for blood testing and the like, recent patents
`have issued which are directed to the particular chemis- '
`try for the determination of HDL cholesterol. Thus,
`U.S. Pat. Nos. 4,851,335 to Kerscher et al and U.S. Pat.
`No. 4,892,815 also to Kerscher et al, describe specific
`types of processes and reagents for HDL cholesterol
`determination. These inventions take advantage of the
`different reactivities of HDL cholesterol versus choles-
`terol contained in other low and very low density lipo-
`proteins (LDLand VLDL). These inventionsthereby
`eliminate the traditional separation steps necessary for
`HDL cholesterol determination. The measurements are
`kinetic, meaning the rate of reaction of HDL choles-
`terol is monitored after LDL and VLDL cholesterol
`have all been reacted and requires careful control of
`time and temperature. Precisely controlled volumes of
`reagents are added at precise times in a prescribed man-
`ner. Measurement times are 3—10 minutes (U.8. Pat. No.
`4,892,815). Even though this presents a significant im-
`provement, for accurate results, it needs careful Opera-
`tor supervision if done manually or expensive instru-
`mentation if automated.
`Notwithstanding all of the above prior art, there has
`been no simple combined physical system and chemis-
`try which enables the user to simply, efficiently and
`quickly obtain HDL cholesterol readings.
`The present invention describes another approach,
`wherein the sample processing, including plasma sepa-
`ration, precipitant metering, precipitate separation as
`well as HDL cholesterol reactions are built into a strip
`such that user manipulations are minimized and HDL
`cholesterol can be measured in one to two minutes
`directly from whole blood. The method measures the
`end-point of the reaction and therefore precise time and
`temperature controls are not necessary. This method
`uses a device similar to that described in the parent
`
`.
`
`lnfopia Ex. 1006 pg. 11
`
`

`

`3
`application (of which this is a Continuation-in-Part,
`fully referenced and incorporated herein above) for
`separation of plasma and for measurement of choles-
`terol, except that specific dry chemistry for HDL deter-
`mination is used. The device employs a tangential flow
`of blood across the blood cell separation membrane.
`HDL dry chemistry precipitation reagents as well as
`. precipitate filters are built into the present invention
`device.
`
`SUMMARY OF THE INVENTION
`The present invention involves a device for determin-
`ing HDL cholesterol by obtaining plasma from whole
`blood and determining the HDL cholesterol esterol
`level from the plasma. The device includes an inert
`substrate support or an active substrate support (e.g.
`one or the other layers), a physical transport medium, a
`microporous plasma separation membrane connected to
`the physical transport medium, at least one plasma col.
`lecting test membrane, a filtering membrane, LDL and
`VLDL reactants to form LDL and VLDL precipitates
`and an optional carrier precipitation membrane. The
`plasma collecting test membrane has reactants which
`will react with HDL cholesterol and indicate the HDL
`cholesterol level quantitatively. The filtering membrane
`may be located between the microporous plasma sepa-
`ration membrane and the transport medium or between
`the microporous plasma membrane and the plasma col-
`lecting test membrane and its function is to block the
`precipitated particles from reaching the test zone. The
`LDL and VLDL reactants which form precipitates of
`LDL and VLDL may be located anywhere upstream
`from the plasma collecting test membrane, i.e., within
`one or more of the transport medium, the microporous
`plasma separation membrane, the filtering memberance
`and the optional carrier separation membrane.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The present invention described in the specification
`herein will be more fully understood when taken in
`conjuction with the drawings appended hereto, which
`show as follows.
`FIG. 1 shows a side cut view of a present invention
`device which precipitates certain lipoproteins in the
`separation membrane;
`FIG. 2 shows a side out view of a present invention
`device which precipitates certain lipoproteins in an
`extra carrier precipitant membrane.
`-
`FIG. 3 shows a side out View of a present invention
`device which includes an asymmetric membrane for the
`dual function of precipitation and filtering above and
`downstream of the microporous plasma separation
`membrane.
`FIG. 4 shows a side cut view of a present invention
`device which includes an asymmetric membrane for the
`dual function of precipitating and filtering below and
`upstream of the microporous plasma separation mem-
`brane.
`FIG. 5 shows a side cut view of a present invention
`device with an alternative arrangement using an asym-
`metric membrane for the dual purpose of filtering mem-
`brane and plasma collecting test membrane.
`FIGS. 6 through 11 show various graphs of reflec—
`tance versus HDL level based on various types of test
`pads.
`
`5,135,716
`
`4
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`5
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`
`FIG. 1 represents one embodiment of the present
`invention. The device 10 has an inert substrate 1 In this
`case, LDL and VLDL precipitating reactants are added
`to the microporous plasma separation membrane 4 and
`plasma collecting test membrane 6 contains the dry
`chemistry HDL cholesterol reagents of the type de-
`scribed above. During use, blood is added to the blood
`application area 11 of physical transport medium 3. It
`travels along the channels 2 and physical transport me-
`dium 3, in this case, a transport membrane sheet which
`is a woven mesh of monofilament polyester with 17
`micron mesh opening (Tetko, Briarcliff, N.Y.) and hav-
`ing a thickness of about 75 microns. Woven fabric,
`non-woven fabric, gauze and monofilament yarn are
`among the many choices for the transport membrane
`sheet shown as physical transport medium 3. Plasma
`separation as well as precipitation is handled by a micro-
`porous plasma separation membrane 4, in this case, 5
`micron nitrocellulose (Schleicher and Schuell, Keene,
`NH.) A filtering membrane 5 filters off the LDL and
`VLDL precipitates and prevents them from reaching
`the plasma collecting test membrane 6. Filtering mem-
`brane 5 was a 0.4 micron hydrophilic polycarbonate
`membrane (Poretics Corp, Livermore, C.A.) used
`without treatment or 0.2 micron nylon (Micron Separa»
`tions, Inc., Westboro, MA.) or 0.8 micron polysulfone
`(Gelman Sciences, Ann Arbor, M.I.). The latter two
`were saturated with 5% or 10% aqueous solution of
`polyethylene glycol (molecular weight 1000 daltons)
`and dried. Polyethylene glycol (PEG) was used as a
`wetting agent. In this embodiment, plasma collecting
`test membrane 6, as mentioned, contains, enzymes and
`chromogens for cholesterol assay so that plasma reach-
`ing it (now devoid of LDL and VLDL components)
`reacts with the reagents in plasma collecting test mem-
`brane 6, producing a colored reaction, the intensity of
`color being proportional to 'HDL cholesterol concen-
`tration. In this case, plasma collecting test membrane 6
`was a 0.45 micron nylon membrane (Micron Separa-
`tions, Inc, Westboro, M.A.). Top sheet 7 with orifice 12
`and transparent area 29 is adhered over the tops of the
`other components as shown by arrows 8 and 9. Trans-
`parent area 29 is comprised of an aperture covered with
`a transparent, oxygen permeable membrane to ensure
`oxygen transport to the oxygen-utilizing reaction taking
`place in plasma collecting test membrane 6. A drop of
`blood may be applied to the blood application area 11 of
`physical transport medium 3 through orifice 12 and the
`colorimetric reaction may be viewed through transpar~
`ent area 29. Alternatively, one or more of the layers
`may be strong enough to support the device in the ab-
`sence of an inert substrate support.
`FIG. 2 shows present invention device 20 wherein
`like parts to those. shown in FIG. 1 are like numbered.
`In the embodiment of FIG. 1, LDL and VLDL precipi-
`tation is carried out
`from the whole blood itself,
`whereas the embodiment shown in FIG. 2 alternatively
`has an' additional carrier precipitant membrane 13
`added, containing the precipitant system so that precipi-
`tation is carried out after the plasma separation step on
`the plasma alone rather than on whole blood.
`It is possible to place the precipitant elsewhere in the
`system, e.g. in the physical transport medium 3 or alter-
`natively in grooves or channels 2 of the inert substrate
`1 for precipitation from whole blood. In another alter-
`
`-
`
`Infopia Ex. 1006 pg. 12
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`5,135,716
`
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`native for precipitation from plasma, filtering mem-
`brane 5 could serve as the precipitant membrane,
`thereby eliminating carrier precipitant membrane 13,
`provided the filtering membrane pore size is appropri-
`ately chosen. This is shown in device 22 of FIG. 3 5
`wherein like parts to FIG. 2 are like numbered. A11
`asymmetric carrier separation membrane 15 placed atop
`microporous plasma separation membrane 4 serves dual
`roles of precipitation and filtration thereby replacing
`filtering membrane 5 and carrier precipitant membrane 10
`13. Also included in FIG. 3 is absorbent storage medium
`19 which absorbs excess applied blood sample to pre-
`vent excess blood from retarding the effectiveness of
`plasma collecting test membrane 6.
`Some of these variations in the basic configurations 1
`shown in FIGS. 1 and 2 will becomeclear, once the
`requirements and functions of various components are
`described in more detail in the paragraphs that follow.
`The functions of the microporous plasma separation
`membrane 4 and components 1,2 and 3 are described in
`detail in copending parent US. patent application Ser.
`No. 07/379,009 referenced above. Essentially, tangen-
`tial flow of blood on the underside of microporous
`plasma separation membrane 4 is facilitated by compo-
`nents 2 and 3. The capillary pull draws the blood on the
`underside and through the cross-section of membrane 4
`which retains the red cells while delivering clean
`plasma on the top surface which can be drawn into the
`subsequent filtering membrane 5 and/or carrier precipi-
`tant membrane 13.
`Whether precipitation is carried out on whole blood
`or plasma, the precipitant systems are adapted from
`conventional
`liquid chemistry.
`In liquid chemistry,
`which uses plasma or serum, four principal precipitation
`systems are used:
`(1) Dextran sulfate (DS) (50,000 or 500,000 dalton
`molecular weight) generally with magnesium chlo.
`ride (MgClz) as the source for divalent cations;
`(2) Heparin-manganese chloride;
`(3) Polyethylene glycol (PEG) (6000 dalton molecu-
`lar weight);
`(4) Phosphotungstic acid-sodium salt, usually in con-
`junction with Mng
`»
`In general, all the systems correlate quite well with 45
`each other as well as with the reference method of
`ultracentrifugation. All four are being used extensively
`and several commercial kits are available for wet chem-
`istry assays of HDL cholesterol using these reagents.
`Any of these system ‘could be adapted for dry chemis- 50
`try strips; however, for precipitation from blood, the
`first three are preferable since they do‘ not hemolyze the
`blood
`The choice of membrane or substrate for loading
`these precipitants is not particularly critical. In the case 55
`of polyethylene glycol of 6000 dalton molecular weight,
`however, a more open matrix is necessary (pore size of
`at least 10 microns and preferably in the 20—100 micron
`range). Otherwise PEG-6000, being waxy, clogs the
`pores and prevents HDL from reaching the plasma 50
`collecting test membrane 6 or effectively overprecipi-
`tales the HDL fraction. When using the system embod-
`ied in FIG. 1, the choice of microporous plasma separa~
`tion membrane 4 will dictate the matrix for the precipi-
`tant, with a 3-8 micron pore size membrane being a 55
`preferred choice. A 5 micron pore-size membrane of
`nitrocellulose works quite well although cellulose is
`also one of many acceptable choices.
`
`40
`
`6
`When the precipitation is carried out on plasma re-
`covered on—line from whole blood as in FIG. 2, carrier
`precipitant membrane 13 may be an open matrix or a
`microfilter and the pore size is not particularly critical
`since filtering membrane 5 is used downstream between
`this membrane and the plasma collecting test membrane
`6. The main requirements of this membrane are hydro-
`philicity, (i.e. its ability to absorb plasma readily from
`microporous plasma separation membrane 4) and the
`ability to release the plasma to the filtering membrane 5.
`From this perspective it is better to avoid membrane
`which are too fine in pore size (<0.1 micron) because
`they may hold the plasma too tightly due to very high
`capillary forces and the filtering membrane 5 down-
`stream may not be able to pull in the plasma containing
`the precipitate.
`FIG. 4 shows present invention device 23 wherein
`like parts to those shown in FIG. 3 are like numbered.
`Asymmetric carrier separation membrane 15 acts both
`as a precipitant membrane and filtering membrane and,
`in this embodinmnt, is located below and upstream from
`microporous plasma separation membrane 4 so that
`precipitation and filtering is taking place on whole
`blood. The precipitant reactants could also be in physi-
`cal transport medium 3 or anywhere upstream of micro-
`porous plasma separating membrane 4. Whether located
`upstream or downstream of carrier separation mem-
`brane 4, the smallest pore sizes of asymmetric carrier
`separation membrane 15 should be less than 1 micron
`and preferably less than 0.45 microns to assure that no
`precipitant reaches the plasma collecting test membrane
`6. It is especially desirable to use a membrane which is
`asymmetric, i.e. its pores are tapered so that its larger
`pores are below and upstream of its smaller pores. One
`could also use a composite membrane with similar char-
`acteristics. Yet another approach for filtering mem-
`brane 5 is to use a membrane with ultramicroporous
`structure with pore size in the range of 0.01 to 0.1 mi-
`crons. This can combine the functions of both filtering
`membrane 5 and carrier precipitant membrane 13 in a
`different manner. HDL particles are very small in size
`0.004-0.0l4 microns) whereas LDL particles are larger
`(0.0l8-0.03 microns) and VLDL even larger (<0.03
`microns). Such a membrane then would be able to phys-
`ically filter out larger LDL and VLDL particles with-
`out any need for precipitation or with only limited help
`from precipitants. The smaller HDL particles would
`still be allowed to reach plasma collecting test mem-
`brane 6 for subsequent assay.
`The concentrations of the precipitant can be adapted
`from those of liquid chemistries. However,in addition
`to the precipitants, it is advantageous to use hydrophilic
`non-volatile liquid or low molecular weigh additives
`such as low molecular weight polyethylene glycol (e.g.
`PEG of molecular weight 200—2000 daltons or other
`similar polyhydroxyl compounds.) Through it is not
`absolutely essential to use these compounds, it is prefer-
`able to do so, since upon loading the membrane with the
`precipitants and drying, the precipitants may bind to the
`membrane matrix and/or crystallize. The addition of
`PEG is especially useful in a two-component precipi-
`tant system consisting of polymers and co-ions (e.g.
`DS-MgCh and Heparin-MnClz). In such cases the salts
`and the polymers may adsorb differently to the mem-
`brane matrix. As a result, depending on the precipitant
`system and the membrane matrix, some trial and error
`approaches may be needed to determine the exact con-
`centratiOns and the polymerzco-ion ratio if they are
`
`lnfopia Ex. 1006 pg. 13
`
`
`
`
`
`

`

`7
`loaded in the absence of such hydrophilic components,
`since they may not be readily soluble in blood or plasma
`in a predictable manner. The non-volatile hydrophilic
`components (e.g. PEG) keep the precipitants from ad-
`sorbing and from crystallizing and permit the move-
`ment of the precipitants in a predictable, readily soluble
`form for consistent release into the plasma (or blood).
`An additional advantage of PEG is that it increases the
`"wettability" and serum uptake of a variety of mem-
`branes, particularly of cellulosic nylon and polysulfone
`types. PEG (e.g. of molecular weight of 400—2000
`daltons) can be used in concentrations of 2—20% in
`water or buffer with 5-10% concentration being in the
`optimum range. The precipitants are dissolved in the
`aqueous PEG solution at a concentration comparable to
`those used in liquid chemistry. For precipitation from
`whole blood (FIGS. 1 and 4), the precipitant concentra—
`tion would be roughly half of that used in a plasma
`precipitation method (FIGS. 2 and 3). Typically, the
`membrane is saturated with the aqueous solution of
`PEG with the dissolved precipitants and allowed to
`dry. Upon drying, the precipitant membrane is ready to
`use.
`
`The pore-size of any filtering membrane is important
`since it keeps the precipitated particles of LDL and
`VLDL from getting swept into plasma collecting test
`membrane 6 and thereby giving falsely high values for
`HDL cholesterol due to contamination of the final
`HDL sample. From experiments with wet chemistry, it
`was determined that immediately upon mixing the pre-
`cipitant solution and the serum (“serum” and “plasma”
`are used interchangeably herein) the precipitated LDL
`and VLDL particles are somewhere in the range of
`0.45—1.2 microns in size. With all precipitant systems, a
`0.45 micron pore size filter can remove all the precipi—
`tate, whereas a 1.2 micron pore size filter is adequate
`only for PEG systems. Given enough time, however,
`the precipitate agglomerates and may become quite
`large in size. Thus, a very thick filtration membrane
`may function properly with larger pore sizes than indi-
`cated above.
`.
`In general, however, a microfilter with pore size of
`less than 1 micron is adequate to serve as a filtering
`membrane with 0.2—0.8 micron pore size being an opti—
`mum range. Other requirements such as wettability and
`an ability to transfer LDL and VLDL-free serum
`plasma to reagent membrane 6 downstream are similar
`to those described for carrier precipitant membrane 13
`above. The wettability of filtering membrane 5 can also
`be enhanced if necessary by addition of wetting agents
`and/or surfactants such as low molecular weight PEG
`or derivatives of same.
`As mentioned earlier, with an appropriately sized
`microporous membrane, filtering membrane 5 can be
`eliminated and the carrier precipitant membrane may
`perform dual roles of both a precipitant carrier mem-
`brane and filtering membrane.
`,
`Conversely, one may choose to combine the func-
`tions of filtering and plasma receiving test membrane as
`shown in present invention device 25 ofrFIG. 5. In FIG.
`5, like parts are like numbered to FIG. 2. FIG. 5 embod-
`ies an alternative arrangement in which a fine pore size
`(0.02—01 micron) filtering/plasma receiving test mem-
`brane 21 serves the dual roles of filtering membrane 5
`and plasma collecting test membrane 6, thereby elimi-
`nating these two components from this embodiment.
`Again, it is preferable to; use an asymmetric membrane
`with tapered pore structure, but with the “skin” side
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`4O
`
`45
`
`50
`
`55
`
`65
`
`5,135,716
`
`8
`having finer pores facing the precipitant-containing
`carrier precipitant membrane 13 in FIG. 5. The precipi-
`tate is thereby prevented from coming in contact with
`the reagent system, which is confined to the opposite,
`more open section of the membrane.
`The pore size of plasma collecting test membrane 6 is
`not particulary critical for the embodiments shown in
`FIGS. 1—4 as long as the membrane is sufficiently hy-
`drophilic and is able to pull in HDL-containing plasma
`through its pore matrix. Additionally, the surface tex-
`ture should be relatively smooth and uniform to obtain
`smooth colors from the color-forming reactions. If a
`chromogen system is loaded from an organic liquid
`such as acetone, alcohol or toluene,
`the membrane
`should be resistant to the appropriate solvent. Good wet
`strength is also desirable. Cellulosic and nylon mem-
`branes, particularly reinforced with a fabric within the
`membrane are especially good for this purpose and the
`pore size of 0.1—1 micron usually satisfies the require-
`ments of surface smoothness and capillary pull. If the
`plasma collecting test membrane also serves as a filter—
`ing membrane as described earlier (FIG. 5), then an
`asymmetric membrane of 0.02—0.l micron is a preferred
`choice.
`
`The plasma collecting test membrane 6 (FIGS. 1
`through 4) or filtering/plasma receiving test membrane
`21 (FIG. 5) of the present invention device contains the
`enzymes cholesterol esterase, cholesterol oxidase and
`peroxidase along with buffer salts, activators, stabilizers
`and chromogen. The reagents are the same as those
`used in total cholesterol assays. The exact formulation is
`a matter of choice and also depends on the sources and
`purity of the enzymes. One typical formulation consists
`of cholesterol esterase (microbial @200 units/ml), cho-
`lesterol oxidase (Nocardia @40 units/ml), peroxidase
`(horseradish @200 units/ml) dissolved in 0.1M 2-[N-
`Morpholino] ethane sulfonic acid, potassium salt (MES)
`buffer at pH 6.7. The solution also contains 3% sodium
`cholate as activator. The reagent membrane is saturated
`with the enzyme solution, dried and then saturated in
`chromogen solution consisting of tetramethyl benzidine
`(TMB) and dioctysulfosuccinate, sodium salt (DOSS) at
`5 mg/ml and 3 mg/ml respectively in acetone (or tolu-
`ene) and allowed to dry.
`In making a device to measure HDL, one has two
`primary choices as mentioned previously:
`a) Precipitation of LDL and VLDL from whole
`blood either during or before cell separation as in
`the embodiments of FIGS. 1 and 4, respectively;
`or,
`b) precipitation from plasma as in the embodiment of
`FIGS. 2,3 and 5.
`When precipitation is from whole blood, the concen-
`tration of cells in the blood (hematocrit) may influence
`the values to some extent. In most
`liquid chemistry
`systems (with the possible exception of the PEG-6000
`system), the ratio of plasma to precipitant is critical.
`Thus, a given concentration of precipitants will give a
`good correlation for patient HDL values in a certain
`defined hematocrit range since the precipitant and
`plasma ratios will vary somewhat with the hematocrit.
`However, when the precipitation is carried out on
`plasma as in FIGS. 2, 3 and 5 this ratio is maintained
`constant in the carrier precipitant membra