EXHIBIT 1002
`
`US. PATENT APPLICATION SERIAL NO. 10/329,044 FILED BY
`
`ANAOKAR ET AL.
`
`Infopia Ex. 1002 pg. 1
`
`

`

`:E!!..31!:Z!l
`
`I253?! m"i!§§§fl:l[f3!l*'--!!-5!L!I.u 3:!!..ii“”
`
`Sunil Anaokar
`
`Gena Lynn Antonopoulos
`
`Alexandra N. Muchnik
`
`TEST STRIP AND METHOD FOR DETERMINING
`HDL CONCENTRATION FROM WHOLE BLOOD OR PLASMA
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`‘
`
`This application claims priority to US. Provisional Patent Application Serial No.
`
`10
`
`60/342,790, filed on December 21, 2001.
`
`FIELD OF THE INVENTION
`
`The present invention relates generally to testing of body fluids for concentration
`
`of cholesterol and more particularly to separating plasma or serum from Whole blood and
`
`separating LDL and VLDL cholesterolfrom HDL cholesterol in the plasma.
`
`BACKGROUND
`
`The level of cholesterol in blood is a significant indicator of risk of coronary heart
`
`disease. “Total cholesterol” includes low density lipoproteins (LDL), very low density
`
`lipoproteins (VLDL) and high density lipoproteins (HDL). It is well established from
`epidemiological and clinical studies that there is a positive correlation between levels of
`
`LDL and VLDL cholesterol (“bad” cholesterol) and coronary heart disease and a negative
`
`correlation between levels of HDL cholesterol (“good” cholesterol) and coronary heart
`
`disease. The level of total cholesterol in blood, which is a measure of the sum total of
`
`HDL, LDL, VLDL and chylomicrons, is not generally regarded as an adequate indicator
`
`of the risk of coronary heart disease because the overall level of total cholesterol does not
`
`reveal the relative proportions of HDL, LDL and VLDL. To better assess the risk of
`
`heart disease, it is desirable to determine the amount of HDL cholesterol in addition to
`total cholesterol.
`
`However, to measure HDL separately, two significant treatment steps to a whole
`
`blood sample are usually necessary. First, blood cells (especially erythrocytes) interfere
`
`15
`
`20
`
`25
`
`30
`
`Infopia Ex. 1002 pg: 2
`
`

`

`Jill- M! Elli EB! "22211 Hill ‘H-Qlli “nil-
`
`..n :11. iii?! aim“; ll'dl'i'r'i‘.‘
`
`with typical colorimetric tests and therefore must be separated from the whole blood
`sample to produce plasma or serum. Second, non-HDLs (Lg, LDL, VLDL and
`
`chylomicrons) must be removed from the plasma to be tested because reagents used to
`
`determine the level of HDL will also react with LDL and VLDL.
`
`The conventional method of removing blood cells from whole blood is
`
`centrifugation. Centrifugation is a process step requiring time and a centrifuge, and it is
`
`therefore unacceptable for blood tests that are conducted in many physicians’ offices, on-
`
`site testing by medical technicians, and testing by patients at home. Further,
`centrifugation can cause problems with separating supernatant and blood cake.
`- A significant advance to the field of diagnostic devices was ushered in with the
`
`10
`
`discovery by Vogel, et a1. (U .8. Patent No. 4,477,575) in the early 1980’s that glass fibers
`
`could be used to separate red cells from whole blood. Because of optical and chemical
`
`interference from hemoglobin in red cells, the only material that could be measured in
`
`whole blood at that time was glucose, using early test strips that required the red cells to
`be washed or wiped off afier glucose had permeated a paper-based matrix (for example,
`
`15
`
`VU.S. Patent 3,298,789 to Mast). Glass fibers separate red blood cells by physical and
`chemical adhesion of the cell surface to the glass fibers. Even today, however, the
`
`precise nature of the attraction between glass fibers and red blood cells is not clearly
`
`understood. Weak chemical bonding, van der Waals forces, hydrogen bonding or other
`
`20
`
`intermolecular forces may have a role in this attraction.
`
`The discovery that glass fibers separate blood cells, however, allowed, for the first
`time, measurement of cholesterol and other blood components in a doctor’s office instead
`
`of a reference laboratory, and the first commercial device to utilize this technology was
`
`Boehringer Mannheim’s (now Roche Diagnostics) Reflotron® instrument. This advance
`
`25
`
`was subsequently incorporated into test strips, allowing blood testing at home.
`
`Notwithstanding the significant achievement of the ‘5 75 patent, applicants have
`
`found that commercially available test strips embodying the ‘575 patent and its progeny
`
`are “lateral flow devices.” The defining feature of a lateral flow device is the presence of
`
`a sample application point that is laterally offset (along the axis of the test strip) from the
`
`30
`
`sample reading area of the test strip. For example, certain commercially available
`
`devices that appear to embody the teachings of the ‘575 patent include a blood
`
`Infopia Ex. 1002 pg. 3
`
`

`

`.Iiil. Hill 3233 iii" "3le .lleil “-ll- lLll"
`
`:1:
`
`.lefiii
`i333; Hill-ii???
`
`
`application area at one end of the elongated test strip and a test reading area at the other
`end. A whole blood sample is deposited at one end of the glass fiber blood separation
`layer, and plasma migrates to the other end at a greater rate than do red blood cells.
`
`However, it has been experimentally determined by applicants that red blood cells from
`
`the sample that is placed on the disclosed glass fiber matrix eventually migrate
`
`tangentially across the fiber matrix, albeit at a slower rate than plasma. Further, some
`
`hemolysis of the erythrocytes eventually occurs in the glass fiber layer.
`
`Furthermore, applicants have found that some commercially available total
`
`cholesterol test strips are configured such that the reaction layer is not initially in contact
`
`10
`
`with the glass fiber blood separation layer. Instead, the reaction layer is not brought into
`fluid—conveying contact with the glass fiber layer until the glass fiber layer is filled with
`
`plasma. This happens at a predetermined time afler an adequate amount of plasma, but
`
`not red blood cells, has migrated laterally to a designated location on the glass fiber layer.
`
`Timing is thus important to the successful use of such test strips. If the reaction layer is
`
`15
`
`brought into contact with the glass fiber layer too soon after depositing the blood sample
`
`on the strip, not enough plasma will have migrated to the designated area of the strip and
`
`the analyte concentration determined may be inaccurately low. On the other hand, if the
`
`reaction layer and glass fiber layer are not brought into contact soon enough, hemolyzed
`
`[and intact red blood cells will migrate to the test area and interfere with the color to be
`
`20
`
`measured from the reaction. Applicants have found these commercially available test
`
`strips to be highly accurate when used as directed. However, it would be desirable to
`
`avoid the process step of bringing the test layer into contact with the blood separation
`
`layer.
`
`Another blood separation scheme is disclosed in US. Patent No. 5,135,716
`
`25
`
`(Thakore) and the abandoned application from which it claims priority. The device
`
`described in the ‘716 patent is also a lateral flow device but operates differently than the
`
`glass fiber matrices described in the ‘575 patent. The ‘716 device purports to employ an
`
`industrial “cross-flow” or “tangential filtration” technique on a miniature scale. The
`
`blood sample is applied to one end of a physical transport medium and is moved laterally
`
`3O
`
`thereby, along the underside of a microporous plasma separation membrane. Blood is
`
`separated at the bottom surface of this microporous plasma separation membrane, and
`
`Infopia Ex. 1002 pg. 4
`
`

`

`Il
`
`in
`i! illiil 2??? iii?! "Ell! llfifll “ill-51111" .m ‘llnll iv“"‘liiL V'
`
`clean plasma is obtained on the top side of the membrane. The transport medium
`
`provides the driving force for lateral movement of blood, such that blood is swept across
`the underside of the microporous plasma separation membrane, thereby cleaning it and
`preventing it from clogging with red blood cells. However, to Applicants’ knowledge,
`there has never been a commercial test strip produced or sold under the ‘716 patent,
`likely because the blood separation technology described in the patent, among other
`
`things, is simply unworkable.
`Another alternate approach to centrifugation to separate blood cells is disclosed in
`
`10
`
`U.S. Patent No. 5,876,605 (Kitajima et al.). The method involves mixing an aqueous
`solution of an inorganic salt or an amino acid or salt thereof with whole blood in an
`amount 20% or less of the whole blood volume and then filtering the whole blood to
`
`remove blood cell components. While satisfactory results are apparently achieved with
`
`the wet chemistry method disclosed, the ‘605 patent teaches that the technique cannot be
`
`successfully adapted to dry test layers such as glass fiber matrices. ‘605 patent, column
`11 lines 1 ~30.
`
`15
`
`Test strips for precipitation and separation of non—HDL cholesterol from HDL
`cholesterols in a plasma sample are disclosed by U.S. Patent No. 5,426,030 (Rittersdorf. et
`
`al.) and its progeny. This separation technology involves a test strip with two layers in
`
`contact with one another. The first layer is made from glass fibers in the form of fleeces,
`
`20’
`
`the glass fibers having a diameter from 3 to 100 pm. The first layer is hydrophilic,
`
`having a thickness between 20—250 mm and pore sizes between 0.2—20 um, and is
`
`impregnated with a precipitating agent that precipitates non-HDLs but not HDLs. The
`second layer is preferably a mesh glass fiber layer with fibers of a diameter of 0.2 to 10.0
`um. Precipitation of non—HDL cholesterols occurs in the first layer and separation of the
`
`25
`
`non—HDL precipitants from the plasma occurs in the second layer.
`
`U.S. Patent No. 5,135,716 (Thakore), discussed above, discloses a multilayer
`
`strip, two of such layers being used for precipitating and then separating non-HDLs from
`
`plasma, respectively. The ‘716 patent also suggests that precipitation and separation of
`non—HDLs from plasma can be carried out in a single “asymmetric” carrier layer. The
`asymmetric layer essentially operates as two layers, in that the top portion of the layer
`
`30
`
`includes large pores to allow fluid movement and precipitation, whereas the bottom
`
`Infopia Ex. 1002 pg. 5
`
`

`

`will. iii}! '33; iii?! “ill lliill Niall-ii" .u 23“.. iii?! is? fill: 31131! in?
`
`portion of the layer includes smaller pores to trap the precipitants. Applicants have found
`
`that this disclosure does not rise beyond mere speculation, in that no examples or
`
`enabling disclosure of the single asymmetric layer technology to separate non-HDLs
`from plasma are found in the ‘716 patent.
`
`Yet another elaborate device to measure the concentration of HDL cholesterol
`from a whole blood sample is disclosed in U.S. Patent No. 5,213,965 (Jones) and other
`
`related and commonly assigned patents. The device includes a well in which the whole
`
`blood sample is deposited and then drawn through a capillary to a sieving pad made of
`
`fibrous material. The sieving pad achieves initial separation of blood cells from plasma
`
`10
`
`on the basis of the blood cell’s slower migration rate therethrough. The sieving pad is
`
`covered with a microporous membrane which further filters blood cells. Covering the
`
`microporous membrane is a reagent reservoir membrane containing precipitating agents
`
`for non—HDLs. On top of and extending laterally beyond the reagent reservoir is an
`
`elongate matrix which distributes the sample laterally after it leaves the reservoir.
`
`15
`
`Finally, one or more test pads are positioned above and biased apart from the elongate
`
`matrix. Plasma exits the filtering membrane and enters the reagent reservoir where non—
`
`HDLs are precipitated. The plasma and non—HDL precipitates then flow from the
`
`reservoir and migrate laterally through the elongate matrix.
`
`Undesirably, the device disclosed by the ‘965 patent relies upon not one, but two,
`separate chromatographic operations, the first being blood separation in the sieving pad,
`and the second being separation of non-HDLs across the elongate matrix. Proper timing
`
`is crucial to these chromatographic operations. Further, the device disclosed by the ‘965
`
`patent is undesirably complex. For example, it requires a well, a capillary tube, two
`
`layers to separate blood cells, and two layers to precipitate and then separate non—HDLs.
`
`Finally, the test pads must be kept spaced apart from the elongate matrix until the entire
`operation is properly timed, whereupon the test plate having the test pads thereon can be
`
`depressed against the elongate matrix. Of course, depressing the test pad creates yet
`
`another undesirable process step and introduces further potential for error.
`
`U.S. Patent No. 5,460,974 (Kozak et a1.) discloses a test device for measuring
`
`HDL cholesterol. The device relies upon a blood separation layer having incorporated
`therein about 25 to about 250 units of an agglutinin, about 50 to about 150 NlH units ofa
`
`20
`
`25
`
`30
`
`Infopia Ex. 1002 pg. 6
`
`

`

`iii. 11731 ’35:? it}? "313! Hill 41.41.11.111. m Slit; iii" iiil'ffiiii €913.13}???
`
`coagulant or a mixture thereof to agglutinize or coagulate the cellular components of the
`
`undiluted whole blood sample. The plasma is then passed into an adjacent layer by
`gravity to separate the LDL and VLDL fractions from the plasma, followed by a layer
`which filters the non-HDLS. Applicants have found that using an agglutinin or a
`
`coagulant to separate blood cells is undesirable because it affects the measured test result.
`
`It is desirable to avoid the lateral flow schemes, chromatographic operations,
`
`complex devices and the timing operations that are required for blood cell separation in
`
`the patents discussed above. It would also be desirable to achieve a blood separation
`
`mechanism that is more efficient and dependable than those listed above. It is also
`
`desirable to simplify non-HDL separation from plasma. Generally, it is desirable to
`
`provide a test strip for measuring concentration of HDL cholesterol that is more reliable,
`
`economical and easier to use than the prior art devices discussed above.
`
`SUMMARY OF THE INVENTION
`
`The present invention is a multilayer vertical flow test strip and method for using
`
`the same to measure HDL concentration from whole blood or plasma. The test strip
`
`includes a two stage blood separation mechanism, wherein a first glass fiber matrix
`separates most of the blood cells and an adjacent, second matrix, also preferably
`containing glass fibers, separates the remainder of the blood cells. The second layer also
`
`10
`
`15
`
`20
`
`precipitates and retains non-HDL cholesterol, thereby providing plasma that is
`
`substantially free of red blood cells and free of non—HDL cholesterol to a reaction layer
`
`that produces a colored response in proportion to the concentration of HDL cholesterol in
`
`the sample.
`
`In one form thereof, the present invention provides a method of determining
`
`25
`
`concentration of HDL cholesterol in awhole blood sample with a dry phase test strip.
`
`The method comprises depositing the whole blood sample at an application area of the
`
`test strip, contacting the whole blood sample with a first test layerof the test strip and
`
`separating and retaining a first portion of red blood cells from the blood sample in the
`
`first test layer. Fluid containing a remaining portion of red blood cells is then passed to a
`
`30
`
`second layer of the test strip, thesecond layer being adjacent to and in contact with the
`
`first test layer.
`
`In the second layer, the remaining portion of red blood cells is separated
`
`Infopia Ex. 1002 pg. 7
`
`

`

`.13!“ till! Iii‘EiiiLzl' “3L1!
`
`lilt'lL¥!!“-*1--!!u m till. iii}?! i
`
`
`
`and retained, and non-HDL cholesterol is also precipitated and retained, thereby
`
`producing plasma that is substantially devoid of red blood cells and non—HDL
`
`cholesterol. The plasma is passed from the second layer to a reaction layer of the test
`
`strip, the reaction layer being adjacent to and in contact with the second test layer. The
`
`reaction layer produces a colored response proportional to the concentration of HDL
`
`' cholesterol in the whole blood sample.
`
`In a preferred form, the first layer is impregnated with a salt such as sodium
`chloride (NaCl) and a sugar such as sorbitol. More preferably, the second layer is
`impregnated with phosphotungstic acid (PTA) to precipitate the non—HDLs.
`
`While the exact mechanism by which this inventive test strip works is still
`
`uncertain, the applicants have made some amazing diScoveries since filing provisional
`
`application 60/342,790, from which this applicatiOn claims priority. Surprisingly, and
`
`quite contrary to what was initially believed, the first glass fiber matrix does not provide
`
`complete separation ofblood. Instead, most of the red blood cells are retained in the first
`
`glass fiber layer, but the remainder of red blood cells is passed to and retained in the
`
`second glass fiber layer. This is quite an unexpected result because the second glass fiber
`
`layer is impregnated with phosphotungstic acid, which is known to hemolyze red blood
`
`cells. Hemolyzed red blood cells would be expected to migrate to the reaction layer and
`
`interfere with the test result. Quite surprisingly, however, the test results have been
`
`found to be quite accurate, notwithstanding that red blood cells are passed to the second
`glass fiber matrix.
`1
`One significant advantage of the blood separation mechanism of the present
`
`invention is that it is a vertical flow device, which consequently works in a dead—end or
`
`10
`
`15
`
`20
`
`vertical flow format, which is in stark contrast to the prior art lateral flow devices noted
`
`25
`
`. above. Of course, there is fluid movement, especially spreading, in all directions in
`
`applicants’ inventive test strips. Significantly, however, there is no need to allow for any
`
`net lateral movement of fluid from one side of a layer to the other, as required by prior art
`
`devices. Advantageously, applicants’ test strip can be made more compact because the
`
`large surface area of transport media needed in prior art devices for lateral movement has
`
`30
`
`been eliminated. In other words, the test layers can be vertically aligned with 'one another
`
`lnfopia Ex. 1002 pg. 8
`
`

`

`iii. 11E}! Ciili'iiiii' “22211 Kill-119113141" m SElLiEiil'
`
`
`
`and made smaller, thereby enabling a smaller and more compact test strip which requires
`
`a smaller blood sample.
`
`Another advantage of the present invention is that itavoids the time-dependent
`
`chromatographic flow schemes required by prior art test strips. As noted above, certain
`
`prior art test strips require that the test layer and blood separation layer are maintained
`
`spaced apart until a predetermined time at which plasma but not red blood cells has
`
`migrated to the contact area. With the present invention, this is unnecessary. All test
`
`layers are always positioned together. There are no moving parts in applicants’ test
`
`strips. In applicant’s test strips, separation of blood is achieved in a direction that is
`
`substantially normal (Leg orthogonal or perpendicular), not tangential, to the plane of the
`
`test layers.
`
`Surprisingly, it has been found that both precipitation and retention of non—HDLs
`
`can be conducted in a single, uniform layer. This is indeed remarkable in light of the
`
`teachings of the prior art that are replete with a two-layer or two—step technology,
`
`precipitation taking place in the first layer and separation in the second. (§;c_e, 9g” U.S. .
`
`Patent Nos-5,426,030; 5,580,743; 5,786,164; 6,171,849; 6,214,570; 5,451,370; .
`
`5,316,916; 5,213,965; and 5,213,964.) The advantages of eliminating an entire layer
`
`from a multilayer test strip are manifest. The strip is less expensive because material
`
`costs are eliminated and, of course, the strip is easier and quicker to assemble.
`
`BRIEF DESCRIPTION OF DRAWINGS
`
`The above-mentioned and other advantages of the present invention, and the
`
`manner of obtaining them, will become more apparent and the invention itself will be
`
`better understood by reference to the following description of the embodiments of the
`
`invention taken in conjunction with the accompanying drawings, wherein:
`
`Fig. 1 is an exploded perspective view of a test strip in accordance with the
`
`present invention used to determine the concentration of HDL cholesterol in a sample of
`
`whole blood;
`
`0 Fig. 2 is a sectional View illustrating the layers of the test strip of Fig. 1; and
`
`10
`
`15
`
`20
`
`25
`
`lnfopia Ex. 1002 pg. 9
`
`

`

`m.
`.
`
`, 3.11.. 'Ilifll 3553 it“ “45321! H3311 dl-=l|"3"~JI-- an Elwin iiiélitlili
`
`5 iriii‘
`
`Fig. 3 is a sectional View of a test strip in accordance with an alternate
`
`embodiment of the present invention used to determine the concentration of HDL
`
`cholesterol in a sample of serum or plasma.
`
`Fig. 3A is a perspective View of a test strip layer in accordance with the present
`invention, illustrating a plane defined by the layer;
`A
`
`Fig. 4 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 8 of this disclosure;
`Fig. 5 is a graph ofknown HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 9 of this disclosure;
`Fig. 6 is a graph of known HDL concentration versus measured reflectance for a
`
`10
`
`test strip in accordance with example 10 of this disclosure;
`
`Fig. 7 is a graph of known VHDL concentration versus measured reflectance for a
`
`test strip in accordance with example 1 1 of this disclosure;
`
`, Fig. 8 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 12 of this disclosure;
`
`Fig. 9 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 13 of this disclosure; and
`
`Fig. 10 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 14 of this disclosure.
`
`Fig. 11 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 15 of this disclosure.
`Fig. 12 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 16 of this disclosure.
`
`Fig. 13 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 17 of this disclosure.
`
`Fig. 14 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 18 of this disclosure.
`
`Fig. 15 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance With example 19 of this disclosure.
`
`Fig. 16 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 20 of this disclosure.
`
`15
`
`20
`
`25
`
`30
`
`lnfopia Ex. 1002 pg. 10
`
`

`

` €331flIEII-ill-llvilhllu
`
`1.. iliifid’liiil39:
`
`Fig. 17 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 21 of this disclosure.
`
`Fig. 18 is a graph of known HDL concentration versus measured reflectance for a
`test strip in accordance with example 22 of this disclosure.
`'
`
`Fig. 19 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 23 of this disclosure.
`
`Fig. 20 is a graph of known HDL concentration versus measured reflectance for a
`
`test strip in accordance with example 24 of this disclosure.
`
`Fig. 21 is a cross sectional view of a test strip in accordance with the present
`
`invention showing movement of blood and plasma at 1.0 seconds after the blood sample
`
`has been applied to the strip;
`
`Fig. 22 is a cross sectional view of the test strip of Fig. 21 showing movement of
`
`blood and plasma at 2.0 seconds after the blood sample has been applied to the strip;
`
`Fig. 23 is a cross sectional view of the test strip of Fig. 21 showing movement of
`
`blood and plasma at 10 seconds after the blood sample has been applied to the strip; and
`
`Fig. 24 is a cross sectional View of the test strip of Fig. 21 showing movement of
`
`blood and plasma at 60 seconds after the blood sample has been applied to the strip.
`Corresponding reference characters indicate corresponding parts throughout the
`
`several views.
`
`DETAILED DESCRIPTION
`
`10
`
`15
`
`20
`
`The embodiments of the present invention described below are not intended to be
`
`exhaustive or to limit the invention to the precise forms disclosed in the following
`
`detailed description. Rather, the embodiments are chosen and described so that others
`
`skilled in the art may appreciate and understand the principles and practices of the present
`
`25
`
`invention.
`
`Definitions
`
`“HDL” refers to high density lipoprotein.
`
`“LDL” refers to low density lipoprotein.
`
`30
`
`“VLDL” refers to very low density lipoprotein.
`
`10
`
`lnfopia Ex. 1002 pg. 11
`
`

`

`
`311.1122! 331ii32¥l§éllllill "ll-"ill" an Sill. 2333?! "iii?! '1
`illiilleiiiif
`
`
`“Non-HDL” refers to LDL, VLDL and chylomicrons, i.e., lipoproteins other than
`
`HDL that will react with a conventional cholesterol reaction membrane.
`“PTA” refers to phosphotungsticacid.
`1
`
`“Plasma” refers to the non-cellular portion of blood from which cellular
`
`components such
`
`as red blood cells are excluded.
`
`“Serum” technically differs from plasma, in that it does not include fibrinogen.
`
`However, for purposes of this application “serum” and “plasma” are sometimes used
`interchangeably.
`
`“Vertically aligned” refers to a stack of two or more test layers used in a dry
`
`phase test strip, thealayers being substantially coextensive With and aligned with one
`
`another in a stack such that no layers protrude significantly from any of the other layers.
`
`Test Device
`
`Referring now to Fig. 1, test strip 20 includes test strip holder 22 which is
`
`preferably formed by injection molding. Test strip holder includes handle 24. and end '
`
`portion 26 which is preferably hingedly attached by hinge portion 28 to second end
`
`portion 30, shown exploded away in Fig. 1. Portion 26 is foldable about hinge portion 28
`
`over portion 30 as shown. End portion 26 includes an opening 32 while end portion 30
`
`includes a complementary spaced opening 34. When end portion 26 is folded over end
`
`portion 30, openings 32 and 34 are aligned. In its folded position, opening ‘32 in holder
`
`22 defines an application window for depositing a body fluid sample while opening 34
`
`defines a test reading window in which optoelectronic measurements of chemistry test
`
`reactions are conducted.
`
`.
`
`A test strip holder essentially the same as that described with reference to Fig. 1
`
`is
`
`shown and described in US. Patent No. 5,597,532, the disclosure of which is hereby
`
`incorporated by reference. The test strip holder is not critical to the invention and other
`
`suitable embodiments of a test strip holder are contemplated by this invention. The
`
`10
`
`15
`
`20
`
`25
`
`particular test strip described herein is suitable for use with an optoelectronic instrument
`sold under the trademark Cardio Chek, commercially available from Polymer
`
`30
`
`Technology Systems, Inc., Indianapolis, IN.
`
`11
`
`lnfopia Ex. 1002 pg. 12
`
`

`

`..i
`.1
`Eligllifll Kill; 33:39 ”ill llill “nil-"Ml" m Lifll.rt“il"ill:§il3§§”
`
`Turning now to Figs. 1 and Fig. 2, there are four layers held within test strip
`
`holder 22 without requiring adhesives. Unlike the teachings of the ‘532 patent, it has
`
`been found that it is desirable to exert a compressive force upon the layers between end
`
`portion 26 and end portion 30. The preper compressive force exerted upon the layers is a
`
`design variable that can be adjusted by (l) adjusting the available space between ends 26
`
`and 30 when the strip is snapped together; (2) adjusting the size and length of rim 44,
`
`which rim depends downwardly from opening 32 and engages the top layer held between
`
`ends 26 and 30; (3) adjusting the size of protuberances 46, which also engage the layers;
`
`and (4) adjusting the depth of shelf 55. A desirable compressive force to be exerted on
`
`the test layers by the test strip reduces the height of the stack of layers by about twenty
`
`percent (20%) from the height the layers would occupy if no compressive force were
`
`exerted. The compression is obviously more extensive proximate boss or rim 44 (see
`
`Figs. 21-24). It is believed that compressing the layers removes air pockets within the
`
`test matrix and thereby improves the speed with which the physical and chemical
`
`processes take place. This, in turn, improves the precision of the test. Compression is
`
`effectuated by sandwiching the stack of layers between downwardly depending rim or
`
`boss 44 and shelf 55 (Figsl and 21). This compression causes the stack of layers to form
`
`the curved profile shown in Figs. 21-24.
`
`The top layer 36 is a disbursement or spreader mesh layer formed of, for example,
`
`woven materials such as polyester or cotton, non-woven fabric, gauze or monofilament
`
`yarn. One suitable material for spreader layer 36 is a Sefar PeCap (07—17/9) available
`
`from Sefar American, Inc., DePew, NY. Layer 36 provides rapid and even disbursement
`
`of a body fluid such as whole blood or plasma. hIt has been found that test strip 20 works
`
`without layer 36, but layer 36 is desirable because it provides a more uniform distribution
`of blood to the subj acent layer and the test results vary less when the spreader layer is
`used.
`
`Beneath and in fluid communication with disbursement or spreader layer 36 is
`
`10
`
`15
`
`2O
`
`25
`
`layer 38, whose composition and preparation are described in greater detail below. Layer
`
`38 separates blood cells (erythrocytes) from whole blood and passes a fluid containing a
`
`30
`
`remaining portion of blood cells therethrough. It has been experimentally found that
`
`about 80% - 90% of red blood cells are retained within layer 38 during the duration of the
`
`12
`
`lnfopia Ex. 1002 pg. 13
`
`

`

`ill. lift! 333; i333! “IT—Ill {[1311 L’-il~"!-elln
`
`w
`Lit iii.“ , ., l 1633;"
`
`
`
`test period. Beneath and in fluid communication with layer 38 is layer 40, whose
`
`composition and preparation are described in greater detail below. Bottom layer 42 is a
`
`reaction layer in which dry chemicals and reactants are contained for generating a visible
`
`color change in the presence of cholesterol, and layer 42 is positioned beneath and in
`
`fluid communication with layer 40 as shown. It may be desirable in some circumstances
`
`to provide additional layers between some of the aforementioned layers, for example, to
`
`improve absorption and fluid communication between layers.
`
`Blood Separation
`
`10
`
`15
`
`Layer 38 is generally a glass fiber matrix. A suitable commercial material for
`layer 38 is Ahlstrom Grade 144, thickness 0.378mm, available from Ahlstrom Filtration,
`
`Inc., Mt. Holly Springs, PA. Other glass fiber matrices could be substituted. Generally,
`layer 38 should include glass fibers with aidiameter of 0.5 to 2 microns and a density of
`
`0.1 to 0.5 g/cm’, more preferably 0.1 to 0.2 g/cm’. Layer 40 is also preferably a
`
`randomly dispersed glass fiber matrix. In the illustrated embodiment, layer 40 includes a
`
`blend of glass microfiber, cellulose fiber, and synthetic staple fiber. The glass microfiber
`
`component consists of alkali-containing or alkali—free borosilicate glass or pure quartz
`fibers, having a mean fiber diameter of 0.3 to 0.7 micrometers. The bulk density of the
`glass microfiber component is less than 0.1 g/cm3, and is typically about 0.05 g/cm3. One
`
`20
`
`suitable membrane for layer 40 is CytoSep® grade 1660 membrane, 12.9 mils thick,
`
`available from Pall Specialty Materials, Port Washington, NY. Another suitable
`
`membrane for layer 40 is paper grade 595, 0.180mm (7.1 mil) thick, available from
`
`Schleicher & Schuell, Keene, NH.
`
`Surprisingly, the inventors of the present invention have found that Separation can
`be improved by impregnating layer 38 with a salt and a sugar. Without wishing to be
`
`25
`
`tied to any specific theory, it is believed-that the ions from the salt, when acting upon the
`
`aqueous blood sample, cause water in the blood cells to be discharged into the plasma,
`thereby decreasing the volume of the red blood cells. This decrease in red blood cell
`
`volume can be crudely likened to a grape that when dehydrated contracts into a raisin.
`
`30
`
`Just as a raisin is smaller, more durable and has a skin that is less flexible than a grape,
`
`red blood cells acted on by the salt in layer 38 become smaller, more durable and their
`
`13
`
`Infopia EX. 1002 pg. 14
`
`

`

`Eilfllifll 3E}; iii?! “Pill llifll “HM-- “all--
`
`:1 .‘3'1. 'iiiiiliiilf “33513231 iii‘.’
`
`membranes less flexible. Even though the erythrocytes acted upon by the salt are
`
`smaller, they are less likely to deform and thus less likely to pass through the glass fiber
`
`matrix. Further, hemolysis (destruction of red blood cells) is reduced by the action of the
`
`salt. Also, as discussed above, it has been