EXHIBIT 1001
`
`U.S. PATENT NO. 7,087,397 TO ANAOKAR ET AL.
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`lnfopia Ex. 1001 pg. 1
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`US007087397B2
`
`(12) United States Patent
`Anaokar et al.
`
`(10) Patent No.:
`
`(45) Date of Patent:
`
`US 7,087,397 B2
`Aug. 8,2006
`
`(54) METHOD FOR DETERMINING HDL
`CONCENTRATION FROM WHOLE BLOOD
`OR PLASMA
`
`(75)
`
`Inventors: Sunil G. Anaokar, Indianapolis, IN
`(US); Gena Lynn Antonopoulos,
`Indianapolis, IN (US); Alexandra N.
`Muchnik, Indianapolis, IN (US)
`
`(73) Assignee: Polymer Technology Systems, Inc,,
`Indianapolis, IN (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 438 days.
`
`4/1995 Ziegenhorn et al.
`5,407,836 A
`5/1995 Law et :11.
`5,411,870 A
`......... .. 435/11
`6/1995 Rittersdorf et al.
`5,426,030 A *
`5,460,974 A * 10/1995 Kozak et al.
`............... .. 436/71
`5,532,172 A
`7/1996 Ziegenhorn et al.
`5,580,743 A
`12/1996 Rittersdorf et :11.
`5,597,532 A
`1/1997 Connolly
`5,786,164 A
`7/1998 Rittersdorf et al.
`5,807,696 A
`9/1998 Miyauchi et a1.
`5,814,472 A
`9/1998 Miki et 211.
`5,879,901 A
`3/1999 Futatsugi et 211.
`6,040,195 A “‘
`3/2000 Carroll et al.
`6,171,849 B1 *
`1/2001 Rittersdorf et al.
`6,214,570 B1
`4/2001 Rittersdorf et 211.
`6,844,149 B1 *
`1/2005 Goldman ..................... .. 435/4
`2003/0224471 A1* 12/2003 Jones et al. ................. .. 435/11
`
`436/514
`435/283.1
`
`
`
`(21) Appl. No.2 10/329,044
`
`(22) Filed:
`
`Dec. 23, 2002
`
`* cited by examiner
`
`Primary Examiner—Ralph Gitomer
`(74) Attorney, Agent, or Firm——Patton Boggs LLP
`
`(65)
`
`Prior Publication Data
`
`(57)
`
`ABSTRACT
`
`US 2003/0175153 A1
`
`Sep. 18, 2003
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/342,790, filed on Dec.
`21, 2001.
`
`(51)
`
`Int. Cl.
`C12Q 1/60
`435/11; 422/60
`(52) U.S. Cl.
`(58) Field of Classification Search ................ .. 435/ 11;
`422/56, 57, 60; 436/71, 170, 169, 177
`See application file for complete search history.
`
`(2006.01)
`
`(56)
`
`References Cited
`U.S. PATENT DOCUIVIENTS
`
`4,144,306 A *
`5,135,716 A *
`5,213,965 A
`5,286,626 A
`5,316,916 A
`
`3/1979 Figueras .................... .. 422/56
`8/1992 Thakore
`422/56
`5/1993 Jones
`2/1994 Law et :11.
`5/1994 Jones
`
`Amultilayer test strip and method of using the test strip for
`determining concentration of HDL cholesterol in a whole
`blood sample. The inventive test strip includes a two-stage
`blood separation mechanism, including a first glass fiber
`matrix which separates most of the blood cells and an
`adjacent, second matrix preferably also containing glass
`fibers that separates the remainder of the blood cells. The
`second layer also precipates and retains non-HDL choles-
`terol, thereby providing plasma that is substantially free of
`red blood cells and substantially free of non-HDL choles-
`terol
`to a reaction layer. Precipitation and retention on
`non~HDLs takes place by a vertical or dead—end filtration in
`a single layer. The reaction layer produces a color, the
`intensity of which is proportional to the concentration of
`HDL cholesterol in the blood sample which is applied to the
`test strip. Advantageously,
`the inventive test strip is a
`Vertical flow device, which can be made more compact and
`operates more efficiently than a lateral flow device.
`
`20 Claims, 23 Drawing Sheets
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`lnfopia Ex. 1001 pg. 2
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`U.S. Patent
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`US 7,087,397 B2
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`1
`METHOD FOR DETERMINING HDL
`CONCENTRATION FROM WHOLE BLOOD
`OR PLASMA
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims priority to US. Provisional Patent
`Application Ser. No. 60/342,790, filed on Dec. 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 sepa-
`rating LDL and VLDL cholesterol from 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.
`two significant
`However,
`to measure HDL separately,
`treatment steps to a whole blood sample are usually neces-
`sary. First, blood cells (especially erythrocytes) interfere
`with typical colorimetric tests and therefore must be sepa-
`rated from the whole blood sample to produce plasma or
`serum. Second, non-HDLs (i.e., LDL, VLDL and chylo1ni-
`crons) 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’ ofiices, on-site testing by medical teclmicians,
`a11d 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 discovery by Vogel, et al. (U.S. Pat.
`No. 4,477,575) in the early l980’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 after glucose
`had permeated a paper-based matrix (for example, US. Pat.
`No. 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
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`forces, hydrogen bonding or other intermolecular forces
`may have a role in this attraction.
`The discovery that glass fibers separate blood cells, how-
`ever, 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
`was subsequently incorporated into test strips, allowing
`blood testing at home.
`Notwithstanding the significant achievement of the ’575
`patent, applicants l1ave 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
`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 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 tan-
`gentially across the fiber matrix, albeit at a slower rate than
`plasma. Further, some hemolysis of the erythrocytes even-
`tually occurs in the glass fiber layer.
`Furthermore, applicants have found that some commer-
`cially available total cholesterol test strips are configured
`such that the reaction layer is not initially in contact with the
`glass fiber blood separation layer. Instead, the reaction layer
`is not brought into fluid-conveying contact witl1 the glass
`fiber layer until the glass fiber layer is filled with plasma.
`This happens at a predetermined time after 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 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
`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 U.S. Pat.
`No. 5,135,716 (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 differ-
`ently than the glass fiber matrices described in the ’575
`patent. The ’7l6 device ptnports to employ an industrial
`“cross-flow” or “tangential filtration” technique on a min-
`iature scale. The blood sample is applied to one end of a
`physical transport medium and is moved laterally thereby,
`along the underside of a microporous plasma separation
`membrane. Blood is separated at the bottom surface of this
`microporous plasma separation membrane,
`and clea11
`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
`
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`US 7,087,397 B2
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`3
`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 tech-
`nology described in the patent, among other things, is simply
`unworkable.
`Another altemate approach to centrifugation to separate
`blood cells is disclosed in U.S. Pat. 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 ll, lines l-30.
`Test strips for precipitation and separation of non—HDL
`cholesterol from HDL cholesterols in a plasma sample are
`disclosed by U.S. Pat. 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, the glass
`fibers having a diameter from 3 to 100 um. The first layer is
`hydrophilic, having a thickness between 20—250 um and
`pore sizes between 0.2—2O 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. Precipi-
`tation of non-HDL cholesterols occurs in the first layer and
`separation of the non-HDL precipitants from the plasma
`occurs in the second layer.
`U.S. Pat. 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 ofnon-HDLs from plasma can be carried out
`in a single “asynnnetric” carrier layer. The asymmetric layer
`essentially operates as two layers, in that the top portion of
`the layer includes large pores to allow fluid movement and
`precipitation, whereas the bottom 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 ’7l6 patent.
`Yet another elaborate device to measure the concentration
`of HDL cholesterol from a whole blood sample is disclosed
`in U.S. Pat. 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 on the basis of the blood cell’s slower migra-
`tion 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 reser-
`voir is an elongate matrix which distributes the sample
`laterally after it leaves the reservoir. 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 reser-
`voir and migrate laterally through the elongate matrix.
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`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, where-
`upon 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. Pat. No. 5,460,974 (Kozak et al.) 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 NTH units of a 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 undesir-
`able because it affects the measured test result.
`It is desirable to avoid the lateral flow schemes, chro-
`matographic 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 efiicient
`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 concentra-
`tion 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 ofthe blood cells. The second
`layer also 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 thereofl the present invention provides a
`method of determining concentration of HDL cholesterol in
`a Whole blood sample with a dry phase test strip. The method
`comprises depositing the whole blood sample at an appli-
`cation area of the test strip, contacting the whole blood
`sample with a first test layer of 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 second layer of
`the test strip, the second 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 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
`
`Infopia Ex. 1001 pg. 27
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`US 7,087,397 B2
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`5
`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 pro-
`duces 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 com-
`plete separation ofblood. Instead, most ofthe 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. Hemo-
`lyzed red blood cells would be expected to migrate to the
`reaction layer and interfere with the test result. Quite sur-
`prisingly, however, the test results have been found to be
`quite accurate, notwithstanding that red blood cells are
`passed to the second glass fiber matrix.
`One significant advantage of the blood separation mecha-
`nism of the present invention is that it is a vertical flow
`device, which consequently works in a dead-end or vertical
`flow format, which is in stark contrast to the prior art lateral
`flow devices noted above. Of course, there is fluid move-
`ment, 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 been
`eliminated. In other words, the test layers can be vertically
`aligned with one another 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 it
`avoids 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 app1icant’s test
`strips, separation of blood is achieved in a direction that is
`substantially normal (i.e., orthogonal or perpendicular), not
`tangential, to the plane of the test layers.
`Surprisingly, it has been found that both precipitation and
`retention ofnon-HDLs can be conducted in a single, tmiform
`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. (See, e.g., U.S. Pat. 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.
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`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 under-
`stood 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;
`FIG. 2 is a sectional view illustrating the layers of the test
`strip of FIG. 1; and
`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 witl1 the present invention, illustrating a plane
`defined by the layer;
`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 of known 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 test strip in accordance with
`example 10 of this disclosure;
`FIG. 7 is a graph of known HDL concentration versus
`measured reflectance for a test strip in accordance with
`example 11 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.
`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.
`
`Infopia Ex. 1001 pg. 28
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`US 7,087,397 B2
`
`7 .
`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 l.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 correspond-
`ing parts throughout the several views.
`
`DETAILED DESCRIPTION
`
`invention described
`The embodiments of the present
`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
`invention.
`
`Definitions
`
`“HDL” refers to high density lipoprotein.
`“LDL” refers to low density lipoprotein.
`“VLDL” refers to very low density lipoprotein.
`“Non-HDL” refers to LDL, VLDL and chylomicrons, i.e.,
`lipoproteins other than HDL that will react with a conven-
`tional cholesterol reaction membrane.
`
`“PTA” refers to phosphotungstic acid.
`“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 appli-
`cation “serum” and “plasma” are sometimes used inter-
`changeably.
`“Vertically aligned” refers to a stack of two or more test
`layers used in a dry phase test strip,
`the layers 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 3 0
`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
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`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 U.S. Pat.
`No. 5,597,532, the disclosure of which is hereby incorpo-
`rated 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 i