EXHIBIT 1007EXHIBIT 1007
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`U.S. PATENT NO. 5,460,974 TO KOZAK ET AL.U.S. PATENT NO. 5,460,974 TO KOZAK ET AL.
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`Infopia Ex. 1007 pg. 1Infopia Ex. 1007 pg. 1
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`USO05460974A
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`United States Patent
`Kozak et al.
`
`[19]
`
`[11] Patent Number:
`
`5,460,974
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`[45] Date of Patent:
`
`Oct. 24, 1995
`
`6/1992 McGowan ........................... 436/177 X
`8/1992 Thakore ......
`.. 436/71 X
`.
`8/1992 Hillman et al.
`436/177 X
`......
`8/1992 de Castro et al.
`436/170 X
`2/1993 Baumgardener et al.
`436/l77X
`6/1993 Patel et al.
`............
`436/170 X
`11/1993 Wilk et al.
`.......................... .. 422/73 X
`
`
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`..
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`[54] METHOD OF ASSAYING WHOLE BLOOD
`FOR HDL CHOLESTEROL
`
`[75]
`
`Inventors: Mary B. Kozak, Osceola; Andrea
`' Badke, Bristol, both of Ind.
`
`[73] Assignee: Miles Inc., Elkhart, Ind.
`
`5,118,613
`5,135,716
`5,135,719
`5,139,685
`5,186,843
`5,215,886
`5,262,067
`
`FOREIGN PATENT DOCUMENTS
`
`7/1983 European Pat. Off. ’.
`0088420
`1/1984 European Pat. Off. .
`0143574
`6/1988 European Pat. Off. .
`0295526
`6/1988 European Pat. Oif. .
`0269240
`3/1991 European Pat. Off.
`.
`0415298
`0428980 . 5/1991 European Pat. OE. .
`3117455
`2/1981 Germany .
`8805912
`8/1988 WIPO C
`
`OTHER PUBLICATIONS
`
`Clackson et al., “Precipitation of Serum Lipoproteins by
`Anionic Polyelechrolytes and Bivalent Cations”, Mono-
`graphs on Atherosclerosis, vol. II, 1982, pp. 17-39.
`
`Primary Examiner——-Donald E. Czaja
`Assistant Examiner—-Milton I. Cano
`Attorney, Agent, or Firm—Roger N. Coe
`[57]
`ABSTRACT
`
`An improved device and method of a) separating the cellular
`components of whole blood from plasma or serum, b)
`separating the low density lipoprotein (LDL) fraction and
`the very low density lipoprotein (VLDL) fraction from the
`plasma or serum, then c) assaying the plasma _or serum for
`cholesterol present in the high density lipoprotein (HDL)
`fraction are disclosed. The device includes a separation area
`that separates the cellular components of whole blood from
`the serum or plasma and separates the LDL and VLDL
`fractions from the serum or plasma, and a test area that
`assays the serum or plasma for the concentration of the HDL
`cholesterol. The method includes introducing a whole blood
`sample to a test device including a separation area compris-
`ing a first zone that separates the cellular components of the
`whole blood from the plasma or serum; and a second zone
`that separates the LDL and the VLDL fractions from the
`. plasma or serum. The essentially cell-free, LDL-free and
`VLDL-free plasma or serum then migrates to a test area.
`After the plasma or serum migrates to the test area, plasma
`or serum is assayed for HDL cholesterol and the test area is
`examined for a quantitative response to HDL cholesterol
`present in the whole blood sample.
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`13 Claims, 6 Drawing Sheets
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`lnfopia Ex. 1007 pg. 2
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`[21] Appl. No.: 959,400
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`[22] Filed:
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`Oct. 13, 1992
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`[51]
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`[52] U.S. Cl. .............
`
`Int. Cl.‘ ............................ G01N 33/92; G01N 1/18;
`G01N 21/77
`............. 436/71; 436/169; 436/170;
`436/177; 472/56; 472/57; 435/11
`[58] Field of Search .................................. 422/56, 57, 73;
`435/11; 436/13, 71, 86, 169,.170, 177
`
`[56]
`
`References Cited
`
`_
`U.S. PATENT DOCUMENTS
`6/1963 Adams et al.
`...................... 436/169 X
`3,092,465
`8/1964 Brewer
`422/101 X
`3,146,163
`1/1967 Hast
`.. 436/95
`3,298,789
`1/1971 Fetter ..
`436/169
`3,552,925
`1/1971 Fetter ..
`436/169 X
`3,552,928
`9/1971 Stone ......
`...... 422/56
`3,607,093
`..
`8/1977 Proksch et al.
`436/13
`4,045,176
`11/1977 Sodickson et al.
`436/71 X
`4,059,405
`4,126,416 11/1978 Sears ...............
`.. 436/71
`4,144,306
`3/1979 Figueras ......
`.. 422/56
`4,188,188
`2/1980 Willner et al.
`436/86 X
`4,190,628
`2/1980 Sears ...........
`...... 422/61
`.
`4,234,317 11/1980 Lucas et al.
`436/177 X
`4,256,693
`3/1981 Kondor et al.
`...... 422/56
`4,258,001
`3/1981 Pierce et al.
`422/56
`4,366,244 12/1982 Pascal
`.....
`...... 435/11
`4,409,106 10/1983 Furuta et al.
`436/177 X
`4,477,575 10/1984 Vogel et al.
`436/I77
`4,544,630 10/1985' Ziegenhorn et al.
`...... 435/11
`_ 4,678,757
`7/1987 Rapkinet al.
`......
`436/169
`4,746,605
`5/1988 Kerscher et al.
`.. 435f/
`.
`4,753,776
`6/1988 Hillman et al.
`422/101
`4,761,381
`8/1988 Blatt et al.
`..........
`436/165
`4,774,192
`9/1988 Ferminiello et al.
`422/56 X
`4,786,603
`11/1988 Vlfiellinger et al.
`.. 422/56 X
`4,801,687
`1/1989 Ngo .............
`530/387
`4,810,394
`'3/1989 Masuda
`436/177 X
`4,816,224
`3/1989 Vogel et al.
`.. 436/170 X
`....
`4,883,765
`11/1989 Tamir et al.
`...... 435/11 X
`4,892,815
`1/1990 Kerscher et al.
`...... 436/71 X
`5,064,541
`11/1991 Jeng et al.
`......
`436/177 X
`5,110,724
`5/1992 Hewett ...................................... 435/11
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`U.S. Patent
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`Oct. 24, 1995
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`Sheet 1 of 6
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`5,460,974
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`FIG.
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`FIG. 2
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`lnfopia Ex. 1007 pg. 3
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`U.S. Patent
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`Oct. 24, 1995
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`Sheet 2 of 6
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`5,460,974
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`FIG. 3
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`FIG. 4
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`Infopia Ex. 1007 pg. 4
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`U.S. Patent
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`Oct. 24,1995
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`Sheet3of6 _
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`5,460,974 ’
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`Infopia Ex. 1007 pg. 5
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`U.S. Patent
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`Oct. 24, 1995
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`Sheet 4 of 6
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`5,460,5374
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`‘FIG.6
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`Infopia Ex. 1007 pg. 6
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`L U.S.' Patent V
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`Oct. 24, 1995
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`sheeg 5 of6
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`lnfopia Ex. 1007 pg. 7
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`U.S. Patent
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`Oct. 24, 1995
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`Sheet 6 of 6
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`5,460,974 L
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`FIG‘.8
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`Infopia Ex. 1007 pg. 8
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`5,460,974
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`1 A
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`METHOD OF ASSAYING WHOLE BLOOD
`FOR HDL CHOLESTEROL
`
`FIELD OF THE INVENTION
`
`Therpresent invention relates to an improved device and
`method of a) separating the cellular components of whole
`blood from the plasma or serum, b) separating the low
`density lipoprotein (LDL) fraction and the very low density
`lipoprotein (VLDL) fraction from the plasma or serum, and
`c) then assaying the plasma or serum for a predetermined
`constituent,
`like cholesterol, present in the high density
`lipoprotein (HDL) fraction. More particularly, the present
`invention relates to an improved method of separating the
`cellular components, the LDL fraction and the VLDL frac-
`tion from a whole blood sample by utilizing a device having
`a separation area including a first zone that separates and
`retains the cellular components of the whole blood sample
`from the plasma or serum, and a second zone that separates
`the LDL and the VLDL fractions from the plasma or serum.
`The undiluted plasma or serum that exits the separation area
`then migrates to a test area that includes the reagents
`necessary to assay the plasma or serum for cholesterol. After
`the undiluted plasma or serum contacts the test area, the test
`area is examined for a response, such as a color change, to
`provide a quantitative assay for HDL cholesterol present in
`the plasma or serum. In one embodiment, the device is a dry
`phase test strip wherein a separation area, comprising one or
`more filter pads, is in contact with a test area comprising a
`test pad. A surface of the test pad is examined for a response.
`In another embodiment,
`the separation area is in fluid
`communication with the test area, such that the cell-free,
`LDL-free and VLDL-free‘ plasma or serum migrates from
`the separation area, such as through a capillary, to the test
`area to assay the HDL fiaction for cholesterol in the test
`sample.
`
`BACKGROUND OF Tl-IE INVENTION
`
`Presently, numerous test devices are available to simply
`and rapidly analyze body fluids for the presence or absence
`of a predetermined soluble constituent. For example, tests
`are available to detect glucose, uric acid or protein in urine;
`and to detect glucose, triglycerides, potassium ion or cho-
`lesterol in blood. Historically, assays of a whole blood
`sample for a predetermined soluble constituent are the most
`diflicult tests to design.
`The cellular components of whole blood, and especially
`the red blood cells, are the primary interfering substances in
`assays for a soluble constituent of whole blood. Most simple
`blood tests are chromogenic, whereby a predetermined
`soluble constituent of the whole blood interacts with a
`particular reagent either to form a uniquely-colored com-
`pound as a qualitative indication of the presence or absence
`of the constituent, or to form a colored compound of variable
`color intensity as a quantitative indication of the presence of
`the constituent. The deep red color of the whole blood
`sample interferes with these chromogenic tests, and there-
`fore the highly-colored red blood cells usually are separated
`from the plasma or serum before the blood sample is assayed
`for a predetermined soluble constituent.
`'
`The presence of red bloodlcells also can interfere with,
`various nonchromogenic blood assays, whereby the assay
`results are either inconsistent or, if consistent, are inaccurate.
`Furthermore, other cellular components, including the white
`blood cells, also can interfere in standard chromogenic
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`blood assays. Therefore, to achieve a reliable assay for a
`predetermined soluble constituent of whole blood,
`it is
`essential to separate the serum or plasma from the cellular
`components of whole blood prior to analyzing the whole
`blood sample for the predetermined soluble constituent.
`‘The assay is further complicated when the predetermined
`soluble constituent of interest is cholesterol. All cells require
`cholesterol for growth, but an excess accumulation of Clio-
`lesterol by the cells can cause various diseases, including
`atherosclerosis. Therefore, cholesterol is an important ana-
`lyte because the amount of total serum cholesterol can be
`correlated to the incidence of atherosclerosis. Accordingly,
`the assay for cholesterol in serum or plasma is one of the
`most frequently performed tests in clinical laboratories.
`Cholesterol and cholesterol esters are water-insoluble
`substances, and therefore are carried in the circulatory
`system by lipoproteins for eventual utilization by the cells.
`Lipoproteins are complex particles and contain varying
`amounts of proteins, phospholipids, cholesterol and triglyc- /
`erides. The lipoproteins in serum are classified by their
`density. These density-based classes include very low den-
`sity lipoproteins (VLDL), low density lipoproteins (LDL),
`and high density lipoproteins (HDL). Each of these lipopro-
`tein classes carry varying amounts of cholesterol, and a total
`serum cholesterol assay is a complex average of the amount
`that each lipoprotein class contributes to the total lipoprotein
`concentration of the serum.
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`_
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`It is well-known" that a specific lipoprotein class, the LDL
`fraction, is responsible for the accumulation of cholesterol in
`cells, and is more closely associated with the progression of
`heart disease, including atherosclerosis. Therefore, the early
`detection of increased levels of the LDL fraction in blood is
`of great importance. In contrast, the HDL fraction has been
`shown to be important in the removal of excess cholesterol
`from cells. Accordingly, a negative correlation exists
`between atherosclerosis and HDL cholesterol levels. Addi-
`tionally,
`the correlation between atherosclerosis and_ the
`level of LDL cholesterol in the blood is higher than a similar .
`correlation between atherosclerosis and total serum choles-
`terol levels.
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`High density lipoproteins (HllL) have been the focus of
`extensive investigation because of the inverse relationship
`between HDL cholesterol and the risk of heart infarction.
`Consequently, if the level of HDL cholesterol in the blood is
`low, an individual has an increased risk of heart infarction.
`Thus, the atherosclerosis risk of an individual.can be esti-
`mated by assaying for HDL cholesterol. The HDL choles-
`terol assay then is used to calculate the approximate amount
`of strongly atherogenic LDL cholesterol from the formula:
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`LDL cIwlerteroI=taIal chaIesterol—% total tn‘gIycerz'de.r— HDL
`clmlarlerol.
`
`In order to determine the cholesterol content of the HDL
`fraction, the other lipoprotein fractions must be removed
`from the test sample. Four general methods have been
`developed to separate the lipoprotein fractions. However,
`each method possesses disadvantages. For example, ultra-
`centrifugation is a common method, but this method is
`unsuitable for routine laboratory assays because the method
`requires special equipment, a sensitive manipulative tech-
`nique and a long separation time that can reachdays.
`Consequently, ultracentrifugation has been restricted to.
`medical research laboratories.
`'
`' In another method, LDL content is determined by a
`fractional precipitation reaction utilizing a polyanion, such
`as heparin sodium or dextran sulfate, and a divalent cation,
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`5,460,974
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`such as calcium, manganese or magnesium. In this method,
`the lipoprotein fractions are precipitated in the sequence
`VLDL, LDL, then HDL, by increasing the concentration of
`the polyanion. However, this process requires two manipu-
`lative steps because the VLDL fraction first is separated in
`, a first precipitation step, then the LDL fraction is precipi-
`tated by increasing the concentration of the polyanion.
`Therefore, the process is impractical and is not amenable to
`automation.
`In the third method, LDL cholesterol is determined from
`the Friedewald formula. Triglyceride and total cholesterol‘
`concentration, including HDL cholesterol content, of the
`sample is determined by precipitating the VLDL and LDL
`fractions from the test sample. The amount of LDL choles-
`terol
`then is calculated by the Friedewald formula. This
`method also is
`laborious and impractical. The fourth
`method, the electrophoretic separation and polyanion pre-
`cipitation method, is time-consuming and requires the use of
`an electrophoresis apparatus and a densitometer for deter-
`mining LDL cholesterol concentration. Accordingly, not one
`of- the four above-described methods is suitable for the
`routine assay of HDL cholesterol.
`In the assay of a whole blood sample for the amount of
`cholesterol in the HDL fraction, the cellular components of
`the whole blood, and the LDL and VLDL fractions of the
`plasma or serum, are separated from the whole blood
`sample. Conventionally, the plasma or serum is separated
`from the cellular material of whole blood by centrifugation.
`The cellular material collects at the bottom of the centrifuge
`tube and the supernatant plasma or serum is decanted.
`Accordingly, the interfering cellular components of whole
`blood are sufliciently removed such that a substantial back-
`ground interference is avoided. The supernatant plasma or
`serum then is subjected to one of the above-described
`methods of separating the LDL and VLDL fractions from the
`plasma or serum.
`The centrifuge method however has the major disadvan-
`tages of requiring a relatively large blood sample, usually
`from about 0.1 ml to about 5 ml, and requiring a long
`centrifuge time of approximately 5 to 10 minutes. Further-
`more, the centrifuge method requires several manipulative
`steps. Consequently, a laboratory technician either can con-
`tact a potentially-infectious blood sample or can contact
`laboratory equipment contaminated by the relatively large
`blood sample, then contract a disease.
`Overall, all the above-described separation techniques are
`best suited for large laboratories that assay a large number
`of blood samples, and for institutions, such as hospitals, that
`do not require assay results in a matter of minutes. However,
`many small laboratories and private medical ofiices do not
`have a centrifuge, ultracentrifuge or other such blood,
`plasma or serum separation devices on site. Therefore,
`simple chromogenic tests carmot be performed quickly,
`safely and easily on site, and the whole blood sample is sent
`to an outside laboratory for efiieient and safe separation and
`assay. As a result, the assay results are available in hours or
`days as opposed to minutes.
`Accordingly,
`investigators have continually sought a
`device and method of quickly, safely and easily separating
`essentially all of the interfering cellular components of
`whole blood from the plasma or serum such that the identity
`and concentration of soluble constituents in the plasma or
`serum are not altered. In addition, for cholesterol assays, a
`need also exists for a simple and inexpensive device and
`method of separating the LDL and VLDL fractions from the
`plasma or serum such that a physician can provide an
`individual a better estimation of a potential cardiovascular
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`risk than the estimation provided by present day total serum
`cholesterol assays.
`Investigators have provided several
`methods and devices for separating the interfering cellular
`components and the LDL and VLDL fractions from the
`plasma or serum. However, each method and device pos-
`sessed at least one disadvantage that made the method or
`device inaccurate, cumbersome or impractical in assaying a '
`whole blood sample for the cholesterol present in the HDL
`fraction of the serum or plasma.
`Methods other than centrifugation have been used to
`separate the cellular components of a small whole blood
`sample from the serum or plasma. One of the simpler
`methods, disclosed by Adams et al. in U.S. Pat. No. 3,092,
`465, used a bibulous, or moisture absorbing, matrix that is
`impregnated with a chromogenic testing reagent and coated
`with a semipermeable barrier. The semipermeable barrier
`screens the cellular components of the whole blood sample
`and permits passage of the smaller, soluble molecules and
`ions to contact the chromogenic testing reagent incorporated
`into the bibulous matrix. In the case of a positive test, the
`essentially colorless plasma or serum interacts with the
`chromogenic testing reagent
`to produce a color in the
`bibulous matrix. The color is observed by rinsing or wiping
`the cellular material retained on the semipermeable barrier
`from the test device. However, the rinsing or wiping tech-
`nique is cumbersome and laborious, and assay interference
`is possible if the red blood cells are not completely wiped or
`rinsed from the semipermeable barrier. In addition,
`the
`possibility of technician contact with the potentially-infec-
`tious blood sample is high. Mast, in U.S. Pat. No. 3,298,789,
`disclosed a similar device, wherein a film of ethylcellulose
`is utilized as the semipermeable barrier. Sodickson, in U.S.
`Pat. No. 4,059,405, achieved separation of the cellular
`components from the blood plasma or serum with an ultra-
`filtration membrane.
`Fetter U.S. Pat. Nos. 3,552,925 and 3,552,928 disclosed
`another method and device to assay whole blood samples for
`soluble constituents. Fetter described a test device having a
`bibulous matrix impregnated with a nonvolatile inorganic
`salt or an amino acid at a first region on the matrix and
`«impregnated with a test reagent at an adjacent second region
`of the matrix. A whole blood sample is introduced onto the
`bibulous matrix such that the whole blood first contacts the
`flrst region of the bibulous matrix including the inorganic
`salt or amino acid. The salt or amino ‘acid precipitates the
`cellular components from the blood, and the plasma or
`serum then migrates to the test reagent-impregnated second
`region of the bibulous matrix for a chromogenic interaction
`with the test reagent.
`Another prior art method of separating the cellular com-
`ponents of whole blood from the plasma or serum was
`disclosed in Vogel et al. U.S. Pat. No. 4,477,575, describing
`a process and a composition for separating plasma or serum
`from whole blood using a layer of glass fibers having a
`defined average diameter and density. However, the amount
`of plasma or serum that can be separated is limited to at most
`50%, and preferably less than 30%, of the absorption
`volume of the glass fibers. Otherwise, whole blood effec-
`tively clogs the glass fiber layer. Therefore,
`the method
`requires a high ratio of hydrophobic glass fibers to whole
`blood volume.
`In other prior art methods, the whole blood is diluted
`before assaying for a predetermined soluble plasma or serum
`constituent. The dilution of whole blood is burdensome
`because an extra manipulative step is required, and dilution
`introduces the possibility of assay error because of an
`incorrect dilution of the blood sample. The possibility of
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`technician contact with the potentially-infectious blood
`sample also is increased. For example, German Patent
`Publication DE—OS 34 41 149 disclosed a method of sepa-
`rating plasma or serum from whole blood by passing the
`whole blood through a lectin-impregnated matrix that is
`repeatedly rinsed with a diluent to dilute the plasma or serum
`before the assay is performed. The use of a lectin or a
`polymeric amino acid to separate the cellular material from
`a whole blood sample also is disclosed in European Patent
`Application No. 843076332.
`In developing a method and device for separating and
`assaying small whole blood samples, a primary consider-
`ation is the degree of sophistication of the technician per-
`forming the assay. Often it is desirable to have relatively
`untrained personnel perform routine assays and obtain accu-
`rate quantitative results. Therefore, it is important that the
`assay method minimize manipulative steps, be free of pos-
`sible interferences or contamination, minimize or eliminate
`the possibility of laboratory personnel physically contacting
`the blood sample, and provide for easy measurement. For
`example, among the several possible manipulative steps, the
`dilution of the whole blood, or the plasma or serum, prior to
`the actual assay introduces the most probable step for assay
`error or personal contact with the blood sample. Another
`common manipulative error is incomplete wiping or rinsing
`of the cellular components of whole blood from the surface
`of a device that utilizes a cell-impermeable membrane to
`separate the cellular components from the plasma or serum
`of whole blood.
`Therefore, a need exists for a method and device to
`efiiciently separate and accurately assay small volumes of
`whole blood. The method preferably avoids distinct manipu-
`lative steps to: 1) separate the cellular components from the
`plasma or serum, and then 2)
`to separate a particular
`component from the plasma or serum prior to the assay.
`Furthermore, in order to avoid dilution errors, the method
`preferably allows the assay of undiluted plasma or serum. It
`also is desirable to provide a blood separation and blood
`assay method that protects the technician from contact with
`the blood sample; thatavoids the time delays of the present
`methods; that is independent of the hematocrit value of the
`blood sample; and that yields accurate and reproducible
`results.
`.
`The ideal method includes withdrawing a whole blood
`sample in a “noninvasive” amount, such as a pin prick drop,
`and immediately depositing the undiluted whole blood
`sample on a test device that separates the cellular compo-
`nents from the undiluted plasma or serum; that separates an
`undesirable or interfering component from the plasma or
`serum; and that then assays the undiluted plasma or serum,
`absent the undesirable component, like the LDL and VLDL
`fractions, for the presence or concentration of a predeter-
`mined soluble constituent,
`like HDL cholesterol, within
`minutes. Alternatively, the test device can contact a fresh
`puncture wound and withdraw a fresh, undiluted blood
`sample from the wound for analysis. Such a separation and
`assay method and device allow medical personnel to per-
`form whole blood analyses on a more routine and more_
`confident basis.
`
`Consequently, investigators have attempted to develop
`test devices that include an element to separate, collect and
`retain the cellular components of whole blood. Examples of
`such attempts are disclosed in Vogel et al. U.S. Pat. No.
`4,477,575; Rothe et al. U.S. Pat. No. 4,604,265; Kennedy et
`al. application PCT/US86/02192; Rapkin et al. U.S. Pat. No.
`4,678,757; Terminiello et al. U.S. Pat. No. 4,774,192; Stone
`U.S. Pat. No. 3,607,093; Figueras U.S. Pat. No. 4,144,306;
`and Pierce et al. U.S. Pat. No. 4,258,001.
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`Each of the above-identified patents is directed to sepa-
`rating the cellular components of whole blood from the
`plasma or serum. However, the resulting plasma or serum
`includes the HDL, the LDL and the VLDL fractious. There-
`fore, it is still necessary to separate the HDL fraction in the
`serum or plasma from the LDL and the VLDL fractions in
`order to accurately determine the amount of HDL choles- ’
`terol
`in the whole blood sample. Some investigators
`attempted to avoid this second separation by assaying for
`HDL cholesterol in the plasma or serum indirectly, such as
`from the diiference between the total cholesterol concentra-
`tion and the sum of cholesterol in the LDL and the VLDL
`fractions, or by a similar indirect method.
`For example, Ziegenhorn et al., in U.S. Pat. No. 4,544,
`630, disclose a method of assaying for cholesterol in the
`LDL fraction in the presence of the HDL fraction by a direct
`enzymatic determination under conditions wherein the LDL
`fraction interacts with the enzymatic reagents substantially
`more quickly than the HDL fraction. Sears, in U.S. Pat. Nos.
`4,126,416 and 4,190,628, discloses methods and assay kits
`for LDL cholesterol in blood plasma by separating the LDL
`cholesterol from the other fractions by agglutinating the
`LDL fraction with a plant lectin, then detecting the amount
`‘of cholesterol in the agglutinated LDL fraction. Sears dis-
`closes that either the cholesterol in the LDL fraction canbe
`assayed or, alternatively, the cholesterol in the supernatant
`liquid, including the HDL and the VLDL fractions can be
`assayed, then the LDL cholesterol concentration can be
`determined indirectly.
`German Patent Publication DE-OS 31 17 455 discloses a
`precipitating reagent including phosphotungstic acid and
`magnesium ions to precipitate the LDL and the VLDL
`fractions. The cholesterol in the HDL fraction of the super-
`natant fluid then can be determined. If the total cholesterol
`concentration of the test sample is known, the HDL.choles-
`terol assay is used in an indirect method of assaying for LDL
`cholesterol. Not one of the above-identified references
`teaches or suggests a method or device wherein the test
`sample is the whole blood, and wherein the cellular com-
`ponents of the whole blood first are separated from the
`whole blood sample, followed by separation of the LDL and
`the VLDL fractions from the HDL fraction, in order to assay
`for HDL cholesterol without a separate precipitating, cen-
`trifuging or diluting step. As will be demonstrated more fully
`hereinafter, the present invention provides a method and a
`device to assay a small sample of whole blood without time
`consuming manipulative steps that expose a technician to a
`potentially-infected test sample and that lead to assay errors.
`McGowan, in U.S. Pat No. 5,118,613, discloses deter-
`mination of HDL lipoprotein constituents using a whole
`blood sample. First, the whole bloodvsample is anticoagu-
`lated with a compound like ethylenediarninetetraacetic acid.
`The LDL and VLDL fractions then are precipitated from the
`anticoagulated whole blood by magnesium ions and dextran
`sulfate. The LDL fraction, VLDL fraction and cellular
`material are separated from the solution by centrifuging, and
`the supernatant solution,
`including essentially the entire
`HDL fraction,
`is assayed for an HDL constituent,
`like
`cholesterol. This method includes the time-consuming and
`potentially infectious manipulative step of centrifuging.
`Other investigators have disclosed devices for assaying
`blood wherein manipulative diluting and centrifuging steps
`are eliminated. For example, Kondo et al., in U.S. Pat. No.
`4,256,693, disclose a multi-layered device to assay for
`various serum components, such as cholesterol. The Kondo
`et a]. device includes a filter layer for removing the cellular
`components of whole blood. The filter layer is positioned
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`5,460,974
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`7
`over a water-proof layer having at least one small opening.
`The plasma or serum exiting the filter layer passes through
`the small opening of the water-proof layer first to contact a
`spreading layer, then to contact a test area. The analyte of
`interest then interacts with reagents included in the test area,
`and a detectable response is observed through the bottom of
`the device. The device of Kondo et al. cannot measure for
`HDL cholesterol because all the lipoprotein fractions exit the
`filter layer to eventually saturate the test area. Accordingly,
`only total cholesterol is assayed by the device of Kondo et
`al. device. In addition, and as will be demonstrated more
`fully hereinafter, the Kondo et al. device does not include a
`capillary tube, an important feature of the present invention.
`Blatt et al., in U.S. Pat. No. 4,761,381, disclose a volume
`metering device, including a capillary channel, for metering
`a liquid sample to a reaction chamber. In the Blatt et al.
`device, the test sample is not filtered. Therefore, in an assay
`of whole blood, the cellular material interferes in a chro-
`mogenic assay for cholesterol because of uneven color
`development and hematocrit interference in high hematocrit
`regions. Furthermore, the lipoprotein fractions are not sepa-
`rated,
`thereby precluding an assay of the whole blood
`sample for HDL cholesterol.
`Hillman et al., in U.S. Pat. No. 4,753,776, disclose a blood
`separation device that includes a filter and a capillary
`channel to channel the plasma or serum exiting the filter to
`a reaction area. Hillman et al. do not teach or suggest further
`separating the VLDL and LDL fractions from the plasma or
`serum to provide a method and device to assay for HDL
`cholesterol.
`European Patent Application 168,093 discloses a binder
`for the LDL fraction. The binder is a sulfated polyvinyl
`alcohol crosslinked to a water-insoluble substrate. The EPO
`Application however does not teach separating the cellular
`components of whole blood from the plasma or serum,
`followed by separating the lipoprotein fractions in a method
`to assay for HDL cholesterol. Kerscher et al. U.S. Pat. No.
`4,746,605 teaches precipitating the HDL fraction from a test
`sample, followed by assaying the supernatant liquid for the
`LDL fraction. In contrast, the present method and device
`assay the plasma or serum for HDL cholesterol after first
`separating the cellular components and then separating the
`LDL fraction and the VLDL fraction from the plasma or
`serum.
`
`Therefore, because of the disadvantages present in the
`above—discussed methods and test devices, it is apparent that
`a simple and eifective method of separating the cellular
`components of whole blood to provide essentially cell~free,
`unaltered and undiluted plasma or serum, and of separating
`an undesirable or interfering component of the plasma or
`serum prior to assaying the plasma or serum for a predeter-
`mined analyte, like HDL cholesterol, is needed. Accord-
`ingly, the method of the present invention allows the safe,
`accurate and economical assayof a whole blood sample for
`HDL cholesterol by utilizing a test device having a separa-
`tion area including a first zone that separates and retains the
`cellular components of the whole blood sample from the
`plasma or serum, and a second zone that separates the LDL
`fraction and the VLDL fraction from the plasma or serum.
`The second zone is in intimate contact with, or is in fluid
`communication with, a test area incorporating the necessary
`reagents to assay for HDL cholesterol. The plasma or serum
`exits the separation area in an undiluted form, then migrates
`to the test area. At the test area, an interaction between the
`HDL cholesterol and the assay reagents produces a detect-
`able response, such as a color transition, that is free from
`interferences attributed to highly-colored cellular compo-
`
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`nents and to cholesterol present in the LDL and the VLDL
`fractions.
`The method and device of the present invention allow the
`assay of whole blood for HDL cholesterol without resorting
`to a lengthy and expensive extra manipulative step of
`centrifuging or diluting the test sample. The plasma or serum
`that saturates the test pad is essentially free of cellular
`material, the VLDL fraction and the LDL fraction, and is
`unaltered and undiluted, thereby allowing an accurate and
`trustworthy assay for HDL cholesterol. The method and
`device of the present invention also eliminate the disadvan-
`tages of hematocrit sensitivity; technique sensitivity due to
`wiping or rinsing the cellular components from the test
`device; and disposal of the cellular components.
`In accordance with one embodiment of the present inven-
`tion, after the whole blood sample has passed through the
`separation area to saturate the test area, a test pad, saturated
`with undilut