Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE: DIRECT ASSAY OF SKIN CHOLESTEROL IN SKIN SAMPLES
REMOVED BY TAPE STRIPPING
FIELD OF THE INVENTION
[0001] The present invention relates to a method of measuring skin
cholesterol. More particularly, the invention pertains to a method for the
direct
assay of cholesterol in skin samples removed by tape stripping, with a view to
identifying individuals at risk of having atherosclerosis as well as those at
risk
of developing atherosclerosis and similar diseases associated with and
attributable to high cholesterol levels.
BACKGROUND OF THE INVENTION
[0002] Numerous studies have shown that atherosclerosis and its
complications, such as heart attacks and strokes, are major causes of
morbidity and mortality in almost all countries of the world.
[0003] Cost effective prevention of atherosclerosis requires the
identification of individuals at risk, thereby allowing their medical
treatment
and change of life style. A desired goal is identifying those individuals
belonging to the high-risk group but there are difficulties in selecting
optimum
methods for discriminating individuals at risk.
[0004] A widely used method for identifying individuals at risk of having
atherosclerosis is based on the measurement of total cholesterol levels in
venous blood plasma (Consensus Conference on Lowering Blood Cholesterol
to Prevent Heart Disease, JAMA, 1985, 253, pg. 2080). Patients are
considered to be at high-risk if their cholesterol level is over 240 mg/dL and
there have been recent moves to lower this threshold level to lower values.
[0005] However, total cholesterol levels alone do not accurately predict
a patient's risk level. A better prediction can be made by analyzing blood
plasma lipoproteins; in particular, measurement of low density and high-
density lipoprotein (HDL) cholesterol levels is advantageous (Total and High
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Density Lipoprotein Cholesterol in the Serum and Risk of Mortality, British
Medical Journal, 1985, 290, pg. 1239-1243).
[0006] Despite their advantage, use of the above methods requires
blood sampling after a period of fasting. Additionally, the sampling is
uncomfortable, poses a risk of infection and the required analysis of plasma
lipoproteins and cholesterol is complicated and expensive. Moreover, studies
have shown that blood plasma analysis may not entirely reflect the process of
cholesterol accumulation in the arterial wall and other tissues. In many
cases,
neither plasma cholesterol levels nor even complete lipid profiles correlate
with the severity of atherosclerosis.
[0007] Significant levels of cholesterol occur in tissue as well as in
plasma and it has been shown that tissue cholesterol plays a leading role in
development of atherosclerosis. Tissues, including skin, have been identified
which accumulate cholesterol in the same way as the arterial wall and studies
have demonstrated a close correlation between cholesterol content in the
arterial wall and the skin. For example, cholesterol was extracted from
lyophilized skin samples and measured using traditional chemical and
biochemical techniques. (Nikitin Y. P., Gordienko I. A., Dolgov A. V.,
Filimonova T. A. "Cholesterol content in the skin and its correlation with
lipid
quotient in the serum in normals and in patients with ischemic cardiac
disease", Cardiology, 1987, II, No. 10, P.48-51). While useful, this method is
too complicated and painful to be employed for large scale population
screening.
[0008] U. S. Patent No. 4,458,686 describes a method of quantifying
various compounds in the blood directly under the skin or on its surface. The
method is based on measuring oxygen concentration changes
electrochemically, for instance, via polarography. In the case of non-volatile
substances that do not diffuse through the skin, it is necessary to implant
enzymes under the skin to effect oxygen changes at the skin surface. This
patent also discloses the potential of using such methods to quantify the
amount of cholesterol using cholesterol oxidase. The complex instrumentation
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and procedures needed require the services of highly skilled personnel for
making measurements, thus limiting the usefulness of the method for
screening large numbers of people.
[0009] Determination of the cholesterol content in skin gives a measure
of the extent of atherosclerosis and can be obtained through standard
laboratory analysis of skin biopsy specimens. However, there is considerable
pain involved in taking a skin sample and a risk of infection at the sampling
site. In addition, this method has other disadvantages because the thick skin
specimens incorporate several skin layers, including the outermost horny
layer (stratum corneum), epidermis and dermis. Since the dermal layer is
highly vascularized, skin biopsy samples contain blood vessels and blood
elements. They may also contain sweat and sebaceous glands and the
secretions contained therein. Additionally, subcutaneous fat is located
directly
under the derma and may also contaminate specimens. Therefore, skin
biopsy specimens are heterogeneous and their analysis may give false data
on cholesterol content in the skin.
[0010] U. S. Patent No. 5,489,510 describes a non-invasive method for
the visual identification of cholesterol on skin using a reagent having a
specific
cholesterol binding component in combination with a reagent having an
indicator component to provide a visual color change corresponding to the
presence of the component bound to cholesterol of the skin. The method
overcomes many of the objections of earlier procedures and meets many of
the desired goals required for a simple mass screening to identify individuals
at risk of having atherosclerosis. The procedure is done directly on the
paimar
skin and, while it is quick and simple, it requires all individuals to be
tested to
be present at a doctor's office or clinic where the test is conducted. This of
course limits effective large scale screening.
[0011] Molar ratios of the lipids, including cholesterol, in stratum
corneum have been determined on samples obtained by direct, solvent
extraction of skin (Norlen L., et al. J. Invest. Dermatology 72-77, 112,
1999).
High performance liquid chromatography (HPLC) and gas liquid
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chromatography in conjunction with mass spectrometry were used to separate
and analyze the lipids. The analytical methods are complex, but more
importantly, the use of corrosive and irritant organic solvent systems to
extract
human skin for routine determinations is not practical.
[0012] The lipid profile of the stratum corneum layer of skin has been
determined using a tape stripping method as described by A. Weerheim and
M. Ponec (Arch. Dermatol. Res., 191-199, 293, 2001). In this study, lipids,
including cholesterol, were solvent extracted from stratum corneum after tape
stripping of skin. The resultant lipid extract was separated by high
performance thin-layer chromatography. This method is very laborious. It
requires three consecutive solvent systems to effect the separation of the
lipids, a staining and charring method to visualize the components and a
densitometry step to determine the relative amounts of the lipids. The method
does not lend itself to the simple and rapid determination of cholesterol
levels
in large numbers of samples.
SUMMARY OF THE INVENTION
[0013] It is therefore an object of the present invention to overcome the
above drawbacks and to provide a simple and non-invasive method of
measuring skin cholesterol, which allows for effective large scale screening.
It
is also an object of the present invention to provide a tape stripping device
for
use in obtaining skin samples.
[0014] According to a first aspect of the invention, there is provided a
method of measuring skin cholesterol, which comprises the steps of:
a) providing a tape comprising a backing member coated on
at least one side thereof with a medical adhesive;
b) applying the tape onto a selected area of skin to adhere
the tape to the selected skin area;
c) stripping the tape off the selected skin area to obtain a
sample representative of an outer stratum corneum layer of the
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skin, the sample adhering to the tape so as to have exposed
skin constituents;
d) providing a source of an affinity-enzymatic compound of
formula A-C-B, wherein A is a detecting agent having affinity for
cholesterol, B is an enzymatic visualizing agent and C is a
binding agent linking the detecting agent and the visualizing
agent to one another;
e) applying a predetermined amount of the affinity-
enzymatic compound onto a predetermined surface area of the
sample and allowing the compound to remain in contact
therewith for a period of time sufficient to cause binding of the
detecting agent to cholesterol present in the exposed skin
constituents; and
f) applying a predetermined amount of a color developing
agent onto the predetermined surface area of the sample,
whereby the color developing agent reacts with the enzymatic
visualizing agent to form a colored product having a color
indicative of cholesterol level.
[0015] The detecting agent in the aforesaid method is selected from the
group consisting of steroid glycosides, triterpene glycosides, hydrophobic
proteins, polyene antibiotics and anti-cholesterol antibodies. In one aspect
of
the invention, the detecting agent is a steroid glycoside, and the steroid
glycoside is digitonin.
[0016] Further, in the aforesaid method, the enzymatic visualizing
agent is an enzyme selected from the group consisting of peroxidase, alkaline
phosphatase, urease, galactosidase, glucose oxidase and
acetylcholinesterase. In one aspect of the invention, the enzyme is
peroxidase, and the peroxidase is horseradish peroxidase.
[0017] In a further aspect of the invention, after step (e) the peroxidase
is activated with hydrogen peroxide to form an activated peroxidase, and
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wherein the color developing agent used in step (f) reacts with the activated
peroxidase to form the colored product.
[0018] In a further aspect of the invention, in step (f) a predetermined
amount of an aqueous solution containing hydrogen peroxide and the color
developing agent is applied onto the predetermined surface area of the
sample.
[0019] The color developing agent is selected from the group consisting
of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3',5,5'-
tetramethyl benzidine. In a particular aspect of the invention, the color
developing agent is 3,3'5,5'-tetramethyl benzidine.
[0020] The binding agent is a copolymer of maleic anhydride and N-
vinylpyrrolidone.
[0021] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0022] According to a second aspect of the invention, there is provided
a method of measuring skin cholesterol, which comprises the steps of:
a) providing a tape comprising a backing member coated on
at least one side thereof with a medical adhesive;
b) applying the tape onto a selected area of skin to adhere
the tape to the selected skin area;
c) stripping the tape off the selected skin area to obtain a
sample representative of an outer stratum corneum layer of the
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skin, the sample adhering to the tape so as to have exposed
skin constituents;
d) providing a source of an affinity signal-generating
compound of formula A-C-B', wherein A is a detecting agent
having affinity for cholesterol, B' is a signal-generating indicator
agent and C is binding agent linking the detecting agent and the
indicator agent to one another;
e) applying a predetermined amount of the affinity signal-
generating compound onto a predetermined surface area of the
sample and allowing the compound to remain in contact
therewith for a period of time sufficient to cause binding of the
detecting agent to cholesterol present in the exposed skin
constituents; and
f) measuring the signal generated by the indicator agent to
provide a value indicative of cholesterol level.
[0023] The detecting agent in the aforesaid method is selected from the
group consisting of steroid glycosides, triterpene glycosides, hydrophobic
proteins, polyene antibiotics and anti-cholesterol antibodies. In one aspect
of
the invention, the detecting agent is a steroid glycoside, and the steroid
glycoside is digitonin.
[0024] The indicator agent in the aforesaid method is selected from the
group consisting of dyes, fluorophores, radioisotopes, metal sol compounds
and chemiluminescent compounds. In one aspect of the invention, the
indicator agent is a dye. In another aspect of the invention, the indicator
agent
is a fluorophore. In a further aspect of the invention, the indicator agent is
a
radioisotope. In another aspect of the invention, the indicator agent is a
metal-
sol compound. In a further aspect of this invention the indicator agent is a
chemiluminescent compound.
[0025] Moreover, in one aspect of the invention, step (f) is carried out
by spectrophotometry. In another aspect of the invention, step (f) is carried
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out by colorimetry. In yet a further aspect of the invention, step (f) is
carried
out by fluorometry. A further aspect of the invention has step (f) is carried
out
by means of a radioactivity sensor. In a further aspect of this invention,
step
(f) is carried out by luminometry.
5[0026] In the aforesaid method the binding agent is a copolymer of
maleic anhydride and N-vinylpyrrolidone.
[0027] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0028] According to a third aspect of the invention, there is provided a
method of measuring skin cholesterol, which comprises the steps of:
a) providing a tape comprising a backing member coated on
at least one side thereof with a medical adhesive;
b) applying the tape onto a selected area of skin to adhere
the tape to the selected skin area;
c) stripping the tape off the selected skin area to obtain a
sample representative of an outer stratum corneum layer of the
skin, the sample adhering to the tape so as to have exposed
skin constituents;
d) providing a source of cholesterol oxidase as a detecting
agent having affinity for cholesterol;
e) applying a predetermined amount of cholesterol oxidase
onto a predetermined surface area of the sample and allowing
the cholesterol oxidase to remain in contact therewith for a
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period of time sufficient to cause oxidation of cholesterol and
formation of hydrogen peroxide; and
f) measuring the amount of hydrogen peroxide formed in
step (e), the amount of hydrogen peroxide measured being
indicative of cholesterol level.
[0029] In one aspect of the aforesaid method, step (f) is carried out by
means of an electrochemical sensor. In another aspect of the method, step (f)
is carried out amperometrically using an electrode. In a further aspect of the
method, step (f) is carried out by spectrophotometry after addition of
peroxidase and a colorimetric indicator. In one aspect, the peroxidase is
horseradish peroxidase. In a further aspect, the colorimetric indicator is
2,2'-
azino-di-(3-ethylbenzthiazoline-6-sulfonic acid). In yet a further aspect of
the
invention, the colorimetric indicator is 3,3',5,5'- tetramethyl benzidine. In
a
further aspect of the invention, the colorimetric indicator is a
multicomponent
oxidative coupling reagent of Trinder or Ngo-Lenhoff type.
[0030] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0031] The present invention also provides, in a fourth aspect thereof, a
kit for use in carrying out a method according to the first aspect. The kit
comprises:
- the aforesaid tape;
- the aforesaid source of affinity-enzymatic compound of
formula A-C-B, wherein A, B and C are as defined above; and
- a source of the aforesaid color developing agent.
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[0032] The detecting agent in the aforesaid kit is selected from the
group consisting of steroid glycosides, triterpene glycosides, hydrophobic
proteins, polyene antibiotics and anti-cholesterol antibodies. In one aspect
of
the invention, the detecting agent is a steroid glycoside, and the steroid
glycoside is digitonin.
[0033] Further, in the aforesaid kit, the enzymatic visualizing agent is
an enzyme selected from the group consisting of peroxidase, alkaline
phosphatase, urease, galactosidase, glucose oxidase and
acetylcholinesterase. In one aspect of the invention, the enzyme is
peroxidase, and the peroxidase is horseradish peroxidase.
[0034] Moreover, the aforesaid kit further includes an aqueous solution
containing hydrogen peroxide, the color developing agent being present in
said solution. The color developing agent is selected from the group
consisting of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and
3,3',5,5'-
tetramethyl benzidine. In one aspect of the invention, the color developing
agent is 3,3'5,5'-tetramethyl benzidine.
[0035] Further, in the aforesaid kit, the binding agent is a copolymer of
maleic anhydride and N-vinylpyrrolidone.
[0036] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0037] The invention further provides, in a fifth aspect thereof, a kit for
use in carrying out a method according to the second aspect. The kit
comprises:
- the aforesaid tape; and
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- the aforesaid source of affinity signal-generating
compound of formula A-C-B', wherein A, B' and C are as defined
above.
[0038] The detecting agent in the aforesaid kit is selected from the
group consisting of steroid glycosides, triterpene glycosides, hydrophobic
proteins, polyene antibiotics and anti-cholesterol antibodies. In one aspect
of
the invention, the detecting agent is a steroid glycoside, and the steroid
glycoside is digitonin.
[0039] The indicator agent in the aforesaid kit is selected from the
group consisting of dyes, fluorophores, radioisotopes, metal sol compounds
and chemiluminescent compounds. In one aspect of the inventions, the
indicator agent is a dye. In another aspect of the invention, the indicator
agent
is a fluorophore. In a further aspect of the invention, the indicator agent is
a
radioisotope. In another aspect of the invention, the indicator agent is a
metal-
sol compound. In a further aspect of this invention the indicator agent is a
chemiluminescent compound.
[0040] In the aforesaid kit the binding agent is a copolymer of maleic
anhydride and N-vinylpyrrolidone.
[0041] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0042] The invention additionally provides, in a sixth aspect thereof, a
kit for use in carrying out a method according to the third aspect: The kit
comprises:
- the aforesaid tape; and
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- the aforesaid source of cholesterol oxidase.
[0043] In the aforesaid kit the peroxidase is horseradish peroxidase. In
one aspect of the invention, colorimetric indicator is of 2,2'-azino-di-(3-
ethylbenzthiazoline-6-sulfonic acid). In another aspect of the invention, the
colorimetric indicator is 3,3'5,5'-tetramethyl benzidine.
[0044] Moreover, the backing member of the tape is formed of
polyester. The medical adhesive is a pressure-sensitive adhesive. In one
aspect of the invention, the medical adhesive is an acrylic based adhesive. In
another aspect of the invention, the medical adhesive is a synthetic rubber
elastomer adhesive. In yet a further aspect of the invention, the medical
adhesive is a silicone based adhesive. In a further aspect, the medical
adhesive comprises an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0045] Moreover, in all of the aforesaid kits, the tape is carried by a
closeable device, the closeable device having a sampling member that carries
the tape, and a closure member adapted to engage the sampling member
and retain the tape within the device. It is preferable that the tape is
sealed
within the device when the closure member engages the sampling member. In
one aspect, at least the closure member or the sampling member is provided
with a peripheral rim, and the other of the closure member or the sampling
member is provided with a peripheral groove adapted to receive the rim so
that the tape is sealed within the device. The closure member can be
connected to the sampling member by a hinge.
[0046] In one aspect of the invention, at least a portion of the sampling
member is adapted to be cut from the closeable device to form a dipstick, the
dipstick having a first end thereof devoid of tape, and a second end thereof
with the tape.
[0047] In a second aspect of the invention, at least a portion of the
sampling member is adapted to be cut from the closeable device to form a
disk, the disk having the tape provided on one face thereof.
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[0048] Further, the aforesaid kits can further comprise a cutting tool
adapted to cut the disk from the device. To show where the cutting tool is to
be applied the the closeable device can be provided with a marker on an
outside surface thereof.
[0049] Moreover, the cutting tool can be provided with a plunger to
eject the disk from the end of the cutter after the disk is cut.
[0050] The invention also provides for a tape stripping device for use in
obtaining skin samples, the device comprising:
a) a sampling member having a surface,
b) a tape provided on at least a portion of the surface of the
sampling member, the tape having a medical adhesive
presented away from the surface; and
c) a closure member adapted to engage the sampling '
member and retain the tape within the device.
[0051] It is preferable that the tape is sealed within the device when the
closure member engages the sampling member. In one aspect, at least the
closure member or the sampling member is provided with a peripheral rim,
and the other of the closure member or the sampling member is provided with
a peripheral groove adapted to receive the rim so that the tape is sealed
within the device. The closure member can be connected to the sampling
member by a hinge.
[0052] In one aspect of the invention, at least a portion of the sampling
member is adapted to be cut from the closeable device to form a dipstick, the
dipstick having a first end thereof devoid of tape, and a second end thereof
with the tape.
[0053] In a second aspect of the invention, at least a portion of the
sampling member is adapted to be cut from the closeable device to form a
disk, the disk having the tape provided on one face thereof.
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[0054] Applicant has found quite surprisingly that the measurement of
skin cholesterol can be carried out directly on the skin sample adhering to
the
aforementioned tape. The procurement of skin samples removed by tape
stripping from donor individuals allows assays to be conducted at distant and
centralized sites and also allows assays from many individuals to be run
concurrently. Thus, the method according to the invention is suitable for
large
scale screening of individuals for assessing their risk of cardiovascular
disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] For a better understanding of the present invention and to show
more clearly how it would be carried into effect, reference will now be made
by way of example, to the accompanying drawings that show a preferred
embodiment of the present invention, and in which:
[0056] Figure 1 is a top view of a sampling device as used in Example
2;
[0057] Figure 2 is a fragmentary view of the sampling device illustrated
in Figure 1, showing details of the sampling member thereof;
[0058] Figure 3 is a perspective view of a dipstick cut from the sampling
device of this invention;
[0059] Figure 4 is a perspective view of a disk cut from the sampling
device of this invention in an alternative epbodiment;
[0060] Figure 5 is a cross-sectional view of a disk from Fig. 4 in the well
of a microwell plate;
[0061] Figure 6 is a perspective view of the sampling device with
cutting tool to produce a disk of Figure 4;
[0062] Figure 7 is a cross-sectional view of the sampling device with
cutting tool to produce a disk of Figure 4; and
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[0063] Figure 8 is a cross-sectional view showing the cutting tool
placing the disk in the well of a microwell plate.
DETAILED DESCRIPTION OF THE INVENTION
[0064] Use is preferably made of a tape comprising a backing member
formed of polyester. The tape is coated on at least one side thereof with a
medical adhesive. The term "medical adhesive" as used herein refers to an
adhesive which is hypoallergic and safe for application to the skin. Such an
adhesive is preferably a pressure-sensitive adhesive, for example, an
adhesive comprising an elastomer formed of block polymers of styrene-
isoprene-styrene or styrene-butadiene-styrene.
[0065] As can be appreciated, there are many classifications and types
of adhesives. In general, any adhesive suitable for use with this invention is
a
medical adhesive as defined above to ensure there will be generally no
problems with allergic reactions when the adhesive was applied to the skin for
sampling. The inventors tested several types of adhesives for use in taking a
skin sample; the majority of these were pressure sensitive acrylic based
adhesives, but several synthetic rubber type elastomer adhesives and silicone
based adhesives were also tested.
[0066] The inventors have found that synthetic rubber adhesives based
on block copolymers of styrene and butadiene or styrene and isoprene
perform well for this invention. An example of a synthetic rubber adhesive is
a
synthetic KratonTM type adhesive (latex free) based on a block copolymer of
styrene and butadiene. Such an adhesive provided better stability for skin
samples to facilitate transportation of the samples for subsequent analysis.
[0067] A further preferred adhesive tape for use in the method of the
invention is a double-coated pressure-sensitive medical grade tape. Examples
of such a medical grade tape are those sold by 3M under Product #9877, or
by Adhesive Research, Inc. under Product #8570.
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[0068] A list of some of the other tapes that have been tested by the
inventors is shown in the accompanying table. The one requirement that is
constant is the use of a medical grade tape that is hypoallergenic. -
Adhesive Tape Product Name Supplier
MA 27Acrylic AR 8570 Adhesive Research, Inc.
MA 38 Acrylic AR 7396 Adhesive Research, Inc.
HY-3 Acrylic AR 8311 Adhesive Research, Inc.
Urethane liner
MA 65 Acrylic AR 8944 Adhesive Research, Inc.
MA 61 Ac lic AR 8890 Adhesive Research, Inc.
Acrylic AR 8968 Adhesive Research, Inc.
AS 124M Acrylic AR 8651 Adhesive Research, Inc.
Acrylic MA 38 Adhesive Research, Inc.
MA 31 Acrylic MA 31 Adhesive Research, Inc.
MA24 MA 24A Adhesive Research, Inc.
rosin tackified
pol isubut lene
Rubber solution MA 70 Adhesive Research, Inc.
Acrylic MA 46 Adhesive Research, Inc.
Acrylic #888 3M
acid free
Silicone N/A Alza Corporation
Dura esic base
Silicone/acrylic 702 Scapa Group PLC
Silicone/silicone 705 Scapa Group PLC
TABLE 1
[0069] It can be appreciated that the adhesive tapes listed in Table 1 is
not meant to be exhaustive, but merely illustrative of different adhesive
tapes
that are suitable for use with this invention at the present time, and that
other
adhesive tapes that will be apparent to those skilled in the art are
contemplated by this invention.
[0070] Double-coated pressure-sensitive tapes are generally available
with an easily removable protective liner. The liner protects the tape from
adhering until it is removed and keeps the adhesive from becoming
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contaminated. Liners may be placed on either side of the double-coated tape
or the tape may have a single liner and be wound onto itself, thereby
protecting both surfaces.
[0071] Liners with differential release properties may be used so that a
first side of adhesive may be exposed while protecting the second adhesive
surface. A double-coated tape with differential liners is particularly
advantageous for skin sampling. Removal of the first liner allows the tape to
be stuck onto the backing support of a sampling device and leaves the skin-
sampling side covered with the second liner. This second liner protects the
skin sampling adhesive area from sticking and from contamination until it is
to
be used. When required for skin sampling, the second liner is removed.
[0072] The tape can be applied onto any part of skin, but the most
suitable part is the surface of a palm because the palm does not have
sebaceous glands whose secretions contain cholesterol which may affect
results. Additionally, the skin on the palm is readily accessible for
sampling.
[0073] It is desirable to obtain uniform amounts of skin samples for
analysis. Application of the adhesive tape for sampling is typically and
routinely done using a single application of the tape to the skin. Additional
amounts of stratum corneum material can be obtained by additional
applications of the tape to the skin. Each subsequent application of the tape
to the skin results in additional skin adhering to the tape. This process
continues until the tape becomes saturated with skin material after which it
is
no longer sticky. The number of applications required to saturate a tape
depends on the type of adhesive used, but for the most commonly used
adhesive tapes, saturation is achieved with less than ten applications, for
example, but not limited to, three to seven applications. Applying tape to a
fresh area of skin for each subsequent stripping results in better and faster
saturation of the tape. Therefore, for consistent and good sampling, it is
convenient to make ten applications of a tape to the skin, using new areas of
skin for each application.
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[0074] The total amount of cholesterol present in the skin sample on
the adhesive tape is related to the size of the skin sample obtained.
Moreover,
a consistent skin sample size is required in order to compare relative levels
of
skin cholesterol between different individuals.
[0075] Obtaining consistently sized skin samples from various
individuals (or repeated samples from the same individual) is accomplished by
the following steps. First, as previously described, the skin sample is taken
by
applying the adhesive tape repeatedly to the skin such that it becomes
saturated with skin and is no longer sticky. The tape becomes saturated with
skin after about three to seven applications and ten applications are
routinely
done to ensure saturation. Next, to obtain a constant area of skin sample to
be assayed, a fixed sized area (for example, as will be hereinafter become
apparent from Examples 2 and 3) from the skin-sampling device is removed,
and immersed in standardized volumes of detector and indicator reagents, as
will also be described hereinafter.
[0076] After skin sampling, the sampling device is closed and shipped
to a central laboratory for assay of cholesterol.
[0077] When using a compound of formula A-C-B or A-C-B' for the
analysis of cholesterol in the skin samples, the detecting agent A can be for
example a steroid glycoside, a triterpene glycoside, a hydrophobic protein, a
polyene antibiotic or an anti-cholesterol antibody. Use is preferably made of
a
steroid glycoside, such as digitonin. The binding agent C, on the other hand,
is preferably a copolymer of maleic anhydride and N-vinylpyrrolidone.
[0078] In the case where use is made of a compound of formula A-C-B,
the enzymatic visualizing agent B is preferably an enzyme selected from the
group consisting of peroxidase, alkaline phosphatase, urease, galactosidase,
glucose oxidase and acetylcholinesterase. Peroxidase such as horseradish
peroxidase is preferred. In this particular case, after step (e), the
peroxidase is
activated with hydrogen peroxide to form an activated peroxidase, and the
color developing agent used in step (f) reacts with the activated peroxidase
to
form the aforesaid colored product. To this end, a predetermined amount of
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an aqueous solution containing hydrogen peroxide and the color developing
agent is applied in step (f) onto the predetermined surface area of the
sample.
Examples of suitable color developing agents which can be used in step (f)
include 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) and 3,3,5,5'-
tetramethyl benzidine. 3,3'5,5'-Tetramethyl benzidine is preferred.
[0079] In the case where use is made of a compound of formula A-C-
B', the indicator agent B' can be for example a dye, a fluorophore, a
radioisotope, a metal sol compound or a chemiluminescent compound. When
the indicator agent is a dye, step (f) can be carried out by
spectrophotometry,
such as colorimetry. When the indicator agent is a fluorophore, step (f) can
be
carried out by fluorometry. When the indicator agent is a radioisotope, step
(f)
can be carried out by means of a radioactivity sensor. When the indicator
agent is a metal-sol compound, step (f) can be carried out by colorimetry.
When the indicator agent is a chemiluminescent compound, step (f) can be
carried out by luminometry.
[0080] In the case where use is made of cholesterol oxidase as a
detecting agent having affinity for cholesterol, step (f) is preferabiy
carried out
by means of an electrochemical sensor, for instance, amperometrically using
an electrode. Step (f) can also be carried out by spectrophotometry after
addition of peroxidase and a colorimetric indicator. The peroxidase used is
preferably horseradish peroxidase. Examples of suitable colorimetric
indicators which can be used include 2,2'-azino-di-(3-ethylbenzthiazoline-6-
sulfonic acid) and 3,3',5,5- tetramethyl benzidine. A colorimetric indicator
consisting of a multicomponent oxidative coupling reagent of Trinder or Ngo-
Lenhoff type can also be used. When use is made of peroxidase and a
colorimetric indicator, the aforementioned kit for carrying out the method
according to the third aspect of the invention further comprises a source of
peroxidase and a source of the colorimetric indicator.
[0081] The method, according to the invention, achieves a simple, high-
throughput skin cholesterol assay.
EXAMPLE 1
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[0082] A double-coated pressure-sensitive medical grade tape having a
protective release liner on an upper sampling side and sold by Adhesive
Research, Inc. was used. A piece of tape 1 inch by 1 inch was cut. The piece
of tape was stuck, using the exposed, lower adhesive surface to one end of a
1 inch by 3 inch thin plastic (white polystyrene) member, leaving a 1 inch by
2
inch piece of uncovered plastic as a handle for applying the tape to the skin
and for labeling the sample.
[0083] To obtain a- skin sample, the protective liner was removed and
the exposed adhesive area applied to a clean dry section of skin. Pressure
was applied to the back of the plastic member over the adhesive area to effect
good contact of the adhesive with the skin. The plastic member with the
attached tape and stratum corneum sample was then peeled from the skin.
[0084] The sample was cut into four equal pieces each measuring p
inch by p inch. One piece was placed in a well of a 12 well tissue culture
plate, or similar container, with the skin sampling side facing up. An aliquot
of
reagent of the type A-C-B was then applied onto a predetermined surface
area of the skin sample. The A-C-B reagent used was a conjugate of digitonin
(A) linked to horseradish peroxidase (B) through a maleic anhydride-N-
vinylpyrrolidone copolymer (C). The reagent was left in contact with the skin
sample for fifteen minutes at room temperature, after which it is removed by
aspiration. Thereafter, the sample was washed with three separate aliquots of
a wash solution to remove non-specifically bound reagent. The piece was
then placed in a new, clean well of a 12 well tissue culture plate, or similar
container, with the skin sampling side facing up. An aliquot of substrate
solution was applied to the sample and left in contact with the skin sample
for
about fifteen minutes at room temperature. The substrate, solution used was
Enhanced K-Blue reagent available from Neogen Corp.(Lexington, KY,USA)
and containing hydrogen peroxide and tetramethyl benzidine as color
developing agent. An aliquot of the developed substrate solution was
removed from the well and added to an aliquot of 1 N sulfuric acid in a well
of
a 96 well microwell plate. The optical density of the resulting solution,
which is
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a measure of the amount of cholesterol in the skin sample, was read at about
450 nm on a plate reading spectrophotometer.
EXAMPLE 2
[0085] Use was made of a sampling device as shown in Figure 1. The
sampling device, vuhich is generally designated by reference numeral 10, is
formed of plastic (polypropylene) and comprises a sampling member 12
connected to a closure member 14 by an integral hinge 16. The closure
member 14 has a peripheral rim 18 and four pins 20, adapted to lock into,
respectively, a peripheral groove 22 and four holes 24 formed in the sampling
member 12. Folding the hinge 16 causes engagement of the rim 18 with the
groove 22 and of the pins 20 with the holes 24, thereby ensuring that the two
halves of the device 10 remain closed and sealed to prevent dust and
contamination of the interior surfaces. The outer surface (not shown in Figs.
1
and 2) of the closure member 14 has a flat area for receiving a label and
barcode strip, for sample identification. The sampling member 12 and closure
member 14 are respectively provided with finger-tabs 26 and 28 for opening
the device 10.
[0086] A double-coated pressure-sensitive medical grade tape 30
having a protective Kraft paper release liner 32 and sold by 3M under Product
#9877 was adhered to the central area of the sampling member 12. The
release liner 32 is wider than the adhesive tape 30, thereby defining a strip
32'
along one edge with no attached tape. This strip 32' of liner overhangs the
edge of the device to form a tab for easy removal of the liner. Immediately
before use, the liner 32 is removed using the overhanging tab 32' and this
exposes the adhesive of the tape 30 for skin sampling.
[0087] The palmar skin area for sampling was cleaned and dried. The
tape 30 with the exposed adhesive was applied onto the palm. The tape 30
was pressed against the skin by applying pressure to the back of the sampling
member 12 above the adhesive area, thereby causing adherence of the
stratum corneum layer. The device 10 was peeled away, reapplied to a new
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area of the palm and again pressed to the skin. The device is peeled away
and applied to the palmar skin in this way for a total of 10 applications.
[0088] At least two small dipsticks 40 (see Fig. 3) about four mm in
width were cut from the device 10 after application to the skin as follows.
Referring to Fig. 2, an end portion of the sampling member 12 was removed
by cutting along the portion of groove 22, which is adjacent to the tab 26.
Three cuts were then made along guide lines 36 (shown in Fig. 2) molded into
the sampling member 12, to delineate the four mm sticks, cutting from the
edge to just past the centre line. The two 4 mm wide sticks were released
from the sampling member 12 by making a third cut across the center of the
member 12, using guide line 38 molded into the member 12. Sticks 40 had a
first end portion 42 devoid of tape and a second end portion 44 with tape
having the skin sample adhered thereto.
[0089] The sticks were each placed into approximately 100 pL solution
of an A-C-B reagent in wells of a 96 well microwell plate (not illustrated).
The
reagent was a conjugate of digitonin (A) linked to horseradish peroxidase (B)
through a maleic anhydride-N-vinylpyrrolidone copolymer (C) and was used at
a concentration of approximately 1 pg/mL. The sticks were left in the solution
for about fifteen minutes at room temperature, after which they were removed
and placed into new wells of a microwell plate containing approximately 200
pL of wash solution. The microwell plate was agitated to effect washing and
after about one minute the sticks were removed to new wells containing
approximately 200 pL of fresh wash solution and again agitated for about one
minute. Washing with agitation was done a third time, after which the sticks
were removed and placed in approximately 100 pL of a substrate solution
(Enhanced K-Blue reagent). The sticks were then incubated with the substrate
solution, in the dark, for about fifteen minutes at room temperature. The
microwell plate can be shaken during this step.
[0090] After the sticks were incubated, the sticks can then be removed.
Approximately one hundred (100) pL of 1 N sulfuric acid is then added to the
wells with the substrate solution to stop further reaction, and the optical
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density of the resulting solution was read at about 450 nm on a plate reading
spectrophotometer, to provide a measure of the amount of cholesterol in the
skin sample.
EXAMPLE 3
5[0091] To allow many samples from Example 2 to be processed
together requires that the dipsticks 40 be held in a configuration that
matches
that of a standard 96 well (8 x 12) microplate. Instruments are available that
can dispense reagents into these plates and also to wash the wells, a
requirement that is necessary to prevent reagent carry-over between assay
steps. Spectrophotometers that can read the coloured solutions directly in the
wells at the final step of the assay are also readily available. However, for
such an application, the protruding part of the dipsticks from the wells, and
the
fixtures that hold them, prevent easy access to the wells for dispensing and
washing steps. This results in a dipstick assay that requires customized
equipment and/or more manual steps than conventional assays run in
microwells.
[0092] Batch processing of many samples can be achieved by
removing small disks 50 (see Fig. 4) having skin samples 70 adhered to the
adhesive tape 30 from the sampling device on one face 52 thereof (skin
sample 70 and adhesive 30 are generally illustrated in the Figures as
adhesive 30 for purposes of clarity, however, it is to be understood that the
adhesive 30 will have skin 70 thereon after application of the device to, for
example, the palm of a person), and then processing these disks in the wells
54 of a microplate 56, as shown in Fig. 5. In this manner there are no
protrusions above the well and so readily available automated liquid
dispensing and washing equipment can be used to add reagents required for
cholesterol assay. The disks are sized to fit into the wells of a microplate,
yet
remain free and not become wedged or trapped within the well. For example,
but not limited to, disks that are smaller than 6.0 mm diameter will fit into
the
wells of all commonly manufactured microplates. It can be appreciated,
however, that disks that are too small will have insufficient amount of skin
that
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will compromise assay sensitivity and reproducibility. It has been found that
disks 5 to 6 mm in diameter are best suited for assay in 96 well microplates.
However, it can be appreciated that the invention is not limited to these
dimensions, and that other disk sizes are contemplated for different wells and
microplates, as would be apparent to those skilled in the art.
[0093] In addition, when the disks are placed in the well, they should
not float with the skin-side up since this will contact the dispensing and
aspiration tubes that are inserted in the wash steps. Therefore, if the
sampling
device is constructed of materials that are less dense than water the disks
should be added to the well with the skin side down. If the sampling device is
constructed of materials that are more dense than water, then the disk is best
added with the skin side up and the height of the dispensing and aspiration
tubes adjusted so that they do not touch the skin surface.
[0094] Either a customized cutting tool or a single-hole paper punch
sized 3/16 in. (4.76 mm) can be used to remove disks from the sampling
device. The disks must be cut from the device such that any anvil-type part
used to eject the disk from a punch or cutting tool must not contact the skin.
Thus, when using a paper punch the anvil should contact the back of the
device (non-skin side) when cutting and ejecting a disk.
[0095] Referring to Figs. 6 and 7, a cutting tool 60 removes a disk from
the device 10 of Fig. I when the device 10 is in a folded over (closed)
position, as illustrated. The closed device is placed on a firm surface (not
illustrated) with the outer surface 62 of the sampling member 12 of the device
facing up. The cutting tool 60 is inserted in a circular depression 64 that
can
be provided on the outer surface 62 of the sampling member 12 of device 10
and the cutting tool 60 is then pressed down to cut through the plastic and
the
tape 30 / skin 70 sample. The cutting tool 60 is not pressed down so far,
however, so as to cut through the plastic of the closure member 14 of the
device 10.
[0096] Once a required disk 50 has been cut from the device 10, the
end 66 of the cutting tool 60 with the disk 50 is placed into a designated
well
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54 of the microwell plate 56 (see Fig. 8) and plunger 68 of the cutting tool
60
is depressed to eject the disk with the skin sample on the adhesive tape into
the well 54.
[0097] When all the designated wells of the microwell plate are
provided with a disk, the microwell plate is placed on automated plate
reader/washer and approximately 100 pL of detector reagent is added to all
the wells and the disks are incubated for approximately fifteen minutes at
generally room temperature (20-24 C). The reagent can be a solution of an
A-C-B reagent as defined in Example 2.
[0098] The detector reagent is then aspirated and approximately 250
pL of wash buffer is added to the wells. The plate can be shaken for
approximately thirty seconds after the addition of the wash buffer, removing
excess detector reagent, and then left for approximately a further ninety
seconds. The wash step can be repeated two or more times as necessary. It
is found that three wash steps are satisfactory.
[0099] After the wells are washed, approximately 100 pL of Enhanced
K-Blue substrate is added to the wells and allowed to incubate with the
washed disk for approximately fifteen minutes at generally ambient room
temperature (as previously described). The microwell plate can be shaken
during this step, and, as in Example 2, the incubation can be in the dark.
[00100] The reaction is then stopped by the addition of approximately
100 pL of 1 N sulphuric acid to the wells, and the plate is shaken to mix the
solutions. Approximately 100 pL of the stopped substrate is then removed and
transferred to the wells of a new plate and read at about 450 nm on a plate
reading spectrophotometer and analyzed as previously described to
determine the relative level of skin cholesterol for each donor.
[00101] In the above examples, it can be appreciated the invention is not
intended to be limited to the exact values specified in the Examples and that
variations from the volumes, times, temperatures, and wavelengths stated can
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be made by those skilled in the art without affecting the scope of the
invention, hence the use of the terms "approximately" and "about."
[00102] The following description is meant to be illustrative only and not
limiting. Other embodiments of this invention will be apparent to those of
ordinary skill in the art in view of this description.
[00103] While the embodiments of the invention disclosed are presently
considered to be preferred, various changes and modifications can be made
without departing from the scope of the invention. The disclosure is intended
to be illustrative and not exhaustive. This description will suggest many
variations and alternatives to one of ordinary skill in this art. All these
alternatives and variations are intended to be included within the scope of
the
claims. Those familiar with the art may recognize other equivalents to the
specific embodiments described that are also intended to be encompassed by
the claims.
