U.S. patent application number 14/712846 was filed with the patent office on 2015-11-12 for absorbent paper and use thereof for breast cancer detection.
The applicant listed for this patent is Atossa Genetics, Inc.. Invention is credited to Shu-Chih Chen, Steven C. Quay.
Application Number | 20150323537 14/712846 |
Document ID | / |
Family ID | 48168446 |
Filed Date | 2015-11-12 |
United States Patent
Application |
20150323537 |
Kind Code |
A1 |
Quay; Steven C. ; et
al. |
November 12, 2015 |
ABSORBENT PAPER AND USE THEREOF FOR BREAST CANCER DETECTION
Abstract
Biological samples of mammary fluid or components thereof are
obtained using a breast pump device coupled with an absorbent paper
or membrane, optionally facilitated by administering oxytocin to
the subject. The breast pump device stimulates expression of
mammary fluid and provides for collection of diagnostic samples on
the absorbent paper or membrane to evaluate breast disease,
including cancer. The biological sample may include fluid
containing one or more of cells or cellular components, proteins,
glycoproteins, peptides, nucleotides or other desired constituents
comprising a breast disease marker. Absorbent paper or membrane,
and methods relating to the paper or membrane, and a breast pump
device are also provided.
Inventors: |
Quay; Steven C.; (Seattle,
WA) ; Chen; Shu-Chih; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Atossa Genetics, Inc. |
Seattle |
WA |
US |
|
|
Family ID: |
48168446 |
Appl. No.: |
14/712846 |
Filed: |
May 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14352461 |
Apr 17, 2014 |
9052318 |
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14712846 |
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PCT/US2012/061733 |
Oct 24, 2012 |
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14352461 |
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61550855 |
Oct 24, 2011 |
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Current U.S.
Class: |
435/287.7 ;
422/82.09 |
Current CPC
Class: |
A61B 10/0045 20130101;
A61B 10/0041 20130101; G01N 33/6836 20130101; C12Q 1/06 20130101;
G01N 33/57415 20130101; G01N 33/683 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/68 20060101 G01N033/68 |
Claims
1.-60. (canceled)
61. A system for analyzing a nipple aspirate fluid sample from an
individual in need thereof, the system comprising: a nipple
covering comprising an absorbent paper for absorbing the nipple
aspirate fluid sample, the absorbent paper comprising
microcellulose, mixed cellulose ester paper, or nitrocellulose, and
having a diameter from about 1.0 to about 3.0 inches and a
thickness from about 0.01 to about 0.1 inches, wherein the
absorbent paper is sized to cover a nipple; and a microscope for
visualizing total protein of the nipple aspirate fluid sample,
counting the number cells of the nipple aspirate fluid sample, or a
combination thereof.
62. The system of claim 61, further comprising a colloidal metal
stain for determining the total protein of the nipple aspirate
fluid sample.
63. The system of claim 62, wherein the colloidal metal is
colloidal gold or colloidal silver.
64. The system of claim 61, further comprising: an optionally
networked computer processing device configured to perform
executable instructions; and a computer program, the computer
program comprising a software module executed by the computer
processing device to apply a model or algorithm for analyzing the
total protein content of the nipple aspirate fluid sample, the
number of cells of the nipple aspirate fluid sample, or a
combination thereof.
65. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to designate a treatment regimen for the individual.
66. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to store photomicrograms in a database of
photomicrograms.
67. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to store analysis in a database of analyses.
68. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to compare the total protein content or number of cells of
the nipple aspirate fluid sample to a standard.
69. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to transmit an analysis to a health care provider or the
individual.
70. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to transmit a diagnosis to a health care provider or the
individual.
71. The system of claim 64, wherein the computer program further
comprises a software module executed by the computer processing
device to generate a report comprising the analysis.
72. The system of claim 61, wherein the absorbent paper is a device
as shown in FIG. 1.
73. The system of claim 61, further comprising an alcohol for
cleaning a nipple and a periaureolar area of a breast.
74. The system of claim 61, wherein the individual is identified as
being at low risk for developing breast cancer where the nipple
aspirate fluid sample contains protein and is acellular.
75. The system of claim 61, wherein the individual is identified as
being at low risk for breast cancer where the nipple aspirate fluid
sample contains from about 1 pg to about 500 ng of protein and is
acellular.
76. The system of claim 61, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains at least about 300 ng of protein and two or more
cells.
77. The system of claim 61, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains between about 300 ng of protein to about 2 .mu.g of
protein and two or more cells.
78. The system of claim 61, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains between about 300 ng of protein to about 2 .mu.g of
protein, and between two and fifty cells.
79. A system for analyzing a nipple aspirate fluid nipple aspirate
fluid sample from an individual in need thereof, the system
comprising: a nipple covering comprising an absorbent paper for
absorbing the nipple aspirate fluid sample, the absorbent paper
comprising microcellulose, mixed cellulose ester paper, or
nitrocellulose, and having a diameter from about 1.0 to about 3.0
inches and a thickness from about 0.01 to about 0.1 inches, wherein
the absorbent paper is sized to cover a nipple; and a breast pump
device.
80. The system of claim 79, wherein the breast pump device is a
MASCT.TM. device.
81. The system of claim 79, wherein the absorbent paper is a device
as shown in FIG. 1.
82. The system of claim 79, further comprising: an optionally
networked computer processing device configured to perform
executable instructions; and a computer program, the computer
program comprising a software module executed by the computer
processing device to apply a model or algorithm for analyzing the
total protein content of the nipple aspirate fluid sample, the
number of cells of the nipple aspirate fluid sample, or a
combination thereof.
83. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to designate a treatment regimen for the individual.
84. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to store photomicrograms in a database of
photomicrograms.
85. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to store analysis in a database of analyses.
86. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to compare the total protein content or number of cells of
the nipple aspirate fluid sample to a standard.
87. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to transmit an analysis to a health care provider or the
individual.
88. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to transmit a diagnosis to a health care provider or the
individual.
89. The system of claim 82, wherein the computer program further
comprises a software module executed by the computer processing
device to generate a report comprising the analysis.
90. The system of claim 79, wherein the absorbent paper comprises
an L-shaped element 4 as illustrated in FIG. 1.
91. The system of claim 79, further comprising an alcohol for
cleaning a nipple and a periaureolar area of a breast.
92. The system of claim 79, wherein the individual is identified as
being at low risk for developing breast cancer where the nipple
aspirate fluid sample contains protein and is acellular.
93. The system of claim 79, wherein the individual is identified as
being at low risk for breast cancer where the nipple aspirate fluid
sample contains from about 1 pg to about 500 ng of protein and is
acellular.
94. The system of claim 79, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains at least about 300 ng of protein and two or more
cells.
95. The system of claim 79, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains between about 300 ng of protein to about 2 .mu.g of
protein and two or more cells.
96. The system of claim 79, wherein the individual is identified
for further evaluation of breast cancer or diagnosed as at medium
or high risk for breast cancer if the nipple aspirate fluid sample
contains between about 300 ng of protein to about 2 .mu.g of
protein, and between two and fifty cells.
97. A system for analyzing a nipple aspirate fluid sample from an
individual in need thereof, the system comprising: a. a nipple
covering comprising an absorbent paper for absorbing the nipple
aspirate fluid sample, the absorbent paper comprising
microcellulose, mixed cellulose ester paper, or nitrocellulose, and
having a diameter from about 1.0 to about 3.0 inches and a
thickness from about 0.01 to about 0.1 inches, wherein the
absorbent paper is sized to cover a nipple; an alcohol for cleaning
a nipple and a periaureolar area of a breast; a colloidal metal
stain for determining the total protein of the nipple aspirate
fluid sample; and a microscope for visualizing total protein of the
nipple aspirate fluid sample, counting the number cells of the
nipple aspirate fluid sample, or a combination thereof; wherein the
individual is identified as being at low risk for developing breast
cancer where the nipple aspirate fluid sample contains protein and
is acellular; the individual is identified as being at low risk for
breast cancer where the nipple aspirate fluid sample contains from
about 1 pg to about 500 ng of protein and is acellular; the
individual is identified for further evaluation of breast cancer or
diagnosed as at medium or high risk for breast cancer if the nipple
aspirate fluid sample contains at least about 300 ng of protein and
two or more cells; the individual is identified for further
evaluation of breast cancer or diagnosed as at medium or high risk
for breast cancer if the nipple aspirate fluid sample contains
between about 300 ng of protein to about 2 .mu.g of protein and two
or more cells; or the individual is identified for further
evaluation of breast cancer or diagnosed as at medium or high risk
for breast cancer if the nipple aspirate fluid sample contains
between about 300 ng of protein to about 2 .mu.g of protein, and
between two and fifty cells.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/550,855, filed Oct. 24, 2011, which application
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Breast cancer is by far the most common form of cancer in
women, and it is the second leading cause of cancer death in
humans. Despite advances in diagnosing and treating breast cancer,
the prevalence of this disease has been steadily rising at a rate
of about 1% per year since 1940. Today, the likelihood that a women
living in North America will develop breast cancer during her
lifetime is one in eight.
[0003] The current widespread use of mammography has resulted in
improved detection of breast cancer. Nonetheless, the death rate
due to breast cancer has remained unchanged at about 27 deaths per
100,000 women. All too often, breast cancer is discovered at a
stage that is too far advanced, when therapeutic options and
survival rates are severely limited.
SUMMARY OF THE INVENTION
[0004] The clinical utility of nipple aspirate fluid for helping in
breast health management has been hampered over the last 50 years
by the current clinical methodology of collecting and measuring the
presence of fluid. In fact, using current technology, up to 50% of
all women are non-secretors, that is, they are judged to not
produce nipple aspirate fluid (NAF); this, in turn, means that
these women cannot be tested for diagnosis of early stage breast
cancer using non-invasive procedures using current technology.
[0005] Additionally, paper used to collect fluid from non-secreting
and secreting women using current technology results in a
significant number of paper cuts to the nipple tissue, rendering
sample collection painful for women undergoing testing.
[0006] The present inventor identified for the first time a new
absorbent paper or membrane that significantly reduces the number
of paper cuts during sample collection. The present inventor also
identified for the first time herein, that the new absorbent paper
or membrane allows very low sample volume collection from women who
were previously considered non-secretors. Even in view of the low
sample volume, the present inventor identified for the first time
that the samples from women considered non-secretors could be
successfully assessed for protein and number of cells for early
stage breast cancer risk assessment. Prior to the present
invention, samples from women considered non-secretors were thrown
away and the women had to be tested using invasive procedures. The
embodiments presented herein, therefore, represent a significant
advance in the field of early stage breast cancer risk assessment
for a patient population which, previously, could not be assessed
using non-invasive procedures. The present methods also allow for
early stage breast cancer risk assessment of women who are
classically secretors. The present methods allow for greater
sensitivity of protein detection in a sample, thereby increasing
the ability of practitioners to detect breast cancer at an earlier
stage of disease progression. The present methods can detect
protein in nipple aspirate fluid (NAF) at a concentration of as low
as about 70 picograms (pg). This represents a significant advance
over prior techniques which detected protein in NAF samples of
secretors at a concentration of about 350 nanograms (ng).
[0007] Provided herein is a method of identifying women at low risk
of future breast cancer, comprising obtaining a sample of nipple
aspirate fluid; testing for protein in said sample; and testing for
cells in said sample, wherein the lowest risk is associated with a
sample that contains protein and is acellular.
[0008] Also provided herein is a method of identifying risk of a
patient for breast cancer or diagnosing a patient at low risk of
breast cancer, comprising: comparing, using a general purpose
computer, the concentration of protein in a nipple aspirate fluid
sample and the number of cells in said sample, to the concentration
of protein and number of cells in a population of patients known to
have breast cancer and/or to a population of patients known not to
have breast cancer; wherein the lowest risk is associated with a
sample that contains protein and is acellular or contains one
cell.
[0009] The patient population identified as being at lowest risk
for breast cancer is associated with a sample that contains protein
and is acellular or contains one cell. In one embodiment, the
sample contains no cells (i.e., is acellular). In another
embodiment, low risk is associated with a sample that contains
protein and contains one cell. The risk of breast cancer increases
with increasing number of cells.
[0010] Patients to be diagnosed with a method provided herein
include those classically designated as non-secretors and secretors
of nipple aspirate fluid (NAF).
[0011] In one embodiment, the patient is classically designated as
a non-secretor of nipple aspirate fluid.
[0012] Nipple aspirate fluid sample is collected on absorbent paper
or a membrane. Absorbent paper or a membrane that may be used in
the present methods includes paper or a membrane of the size and
dimensions as illustrated in FIG. 1. The paper or membrane may be
made of, for example, microcellulose, mixed cellulose ester paper
or nitrocellulose. This new design results in significantly fewer
cuts to nipple tissue during sample collection compared to paper
that is currently marketed for NAF collection.
[0013] The absorbent paper or membrane is washed and the effluent
is collected and assessed for number of cells. In one embodiment,
cytology of cells, if any, in said sample is analyzed using any
conventional method including, but not limited to, microscopy, flow
cytometry, immunohistochemistry, or a combination thereof. A
clinician can determine if cells, if present in the sample, are
normal contain one or more characteristics of cancer cells. In one
non-limiting example, cell samples may be stained with hemolysin
and eosin.
[0014] Protein measured by the methods described herein is total
protein content of the sample. The paper is then exposed to
colloidal gold or colloidal silver and the total protein content
(concentration) is determined.
[0015] In one aspect, if a sample contains from about 50 pg to
about 0.5 ng of protein, and does not contain cells, a patient is
identified as being at low risk for breast cancer.
[0016] Alternatively, if the sample contains at least about 300 ng
of protein and two (2) or more cells, a patient is identified for
further evaluation of breast cancer or diagnosed as at medium or
high risk for breast cancer. In one embodiment, the concentration
of protein in a sample is from about 300 ng of protein to about 2
.mu.g of protein and a patient is diagnosed at being at higher risk
for breast cancer and, in some cases, is identified for further
assessment. The cell fraction of the sample, in such a case, may
contain about 2 cells to about 50 cells. Such a cell fraction may,
in another case, may contain at least ten (10) cells.
[0017] Provided herein is an absorbent paper or membrane having the
characteristics and dimensions as illustrated in FIG. 1, where the
absorbent paper or membrane is made using microcellulose, mixed
cellulose ester paper or nitrocellulose. In one embodiment, the
absorbent paper or membrane comprises mixed cellulose ester.
[0018] A method of detecting protein in a sample of nipple aspirate
fluid (NAF) comprising collecting the NAF on an absorbent paper or
membrane and detecting the protein with a colloidal metal particle
suspension, wherein the method can detect protein at a
concentration of between 0.5 ng and 500 ng per sample. In one
embodiment, the metal particle suspension comprises colloidal
gold.
[0019] A significant advance of the present absorbent paper or
membrane is that is does not cut tissue to the extent that current
filter paper does; thus, a patient undergoing assessment is subject
to less pain and discomfort during the collection procedure. In one
embodiment, tissue exposed to the paper or membrane is nipple
tissue.
[0020] Provided herein is the use of the absorbent paper or
membrane as described herein in a method of identifying a patient
at low risk of breast cancer or diagnosing a patient at low risk of
breast cancer. In one aspect, the absorbent paper or membrane is
used in any of the methods described herein or in any other method
of nipple aspirate fluid collection for any use.
[0021] Provided herein is method of identifying risk of a patient
for breast cancer or diagnosing a patient at low risk of breast
cancer, comprising: collecting nipple aspirate fluid on absorbent
paper or membrane; washing said absorbent paper or membrane with
buffered solution to collect cells, if any; counting the number of
cells in said sample and, optionally, determining the cytology of
said cells, if any; staining said absorbent paper or membrane with
colloidal gold or colloidal silver and determining the
concentration of protein on said filter paper; and comparing, the
concentration of protein in said nipple aspirate fluid and the
number of cells in said fluid, to the concentration of protein and
number of cells in a population of patients known to have breast
cancer and/or to a population of patients known not to have breast
cancer; wherein the lowest risk is associated with a sample that
contains protein and is acellular or contains one cell.
[0022] Provided herein is a method of diagnosing risk of a patient
for breast cancer, comprising: collecting nipple aspirate fluid on
absorbent paper or membrane, wherein said NAF sample is obtained
from classical providers and non-providers; washing said absorbent
paper with buffered solution to collect cells, if any; counting the
number of cells in said sample and, optionally, determining the
cytology of said cells, if any; staining said absorbent paper or
membrane with colloidal gold or colloidal silver and determining
the concentration of protein on said filter paper; and comparing,
the concentration of protein in said nipple aspirate fluid and the
number of cells in said fluid, to the concentration of protein and
number of cells in a population of patients known to have breast
cancer and/or to a population of patients known not to have breast
cancer; wherein the lowest risk is associated with a sample that
contains protein and is acellular or contains one cell.
[0023] Also provided herein is a method of identifying risk of
developing breast cancer in an individual in need thereof,
comprising: detecting the total amount of protein and/or number of
cells in a nipple aspirate fluid sample absorbed onto an absorbent
paper as illustrated in FIG. 1, wherein said absorbent paper
comprises microcellulose, mixed cellulose ester paper or
nitrocellulose; and identifying the risk for developing breast
cancer based on the total amount of protein and/or the number of
cells of the nipple aspirate fluid sample.
[0024] In one embodiment, the sample is acellular and the patient
is identified as at the lowest risk for breast cancer. In another
embodiment, the sample contains one cell and the patient is
identified as at low risk for breast cancer. In yet another
embodiment, the sample contains more than 2 cells and the patient
is identified for further assessment.
[0025] In one embodiment, the total protein concentration of each
breast is determined and, if the total protein concentration of one
sample is greater than 300 ng, the patient is identified for
further assessment of breast cancer. Alternatively, the total
protein concentration of each breast is determined and, if the
total protein concentration of each sample is at, or below 200 ng
protein, the patient is identified as being at low risk for breast
cancer.
[0026] In one embodiment, the nipple aspirate fluid is collected on
absorbent paper or membrane following massaging of breast tissue
and suction with a mammary aspirate specimen cytology test
(MASCT.TM.) device as described in Example 1. As used herein, a
"MASCT.TM. device" refers to a device described in U.S. Pat. No.
6,287,521 by Quay et al. which is incorporated herein in its
entirety. In one non-limiting example, a sample collection device
for collecting a biological sample from a mammary organ of a
patient may comprise a breast engaging member constructed of a
non-porous material sized and dimensioned to receive at least a
nipple portion of a breast of said patient and form a suction seal
therewith; a solid phase sample collection medium in fluid
connection with said breast engaging member for receiving a sample
of expressed breast fluid; and a vacuum pump means in gaseous
connection with said breast engaging member for generating negative
pressure through the breast engaging member to facilitate breast
fluid expression, wherein said solid phase sample collection medium
is selected from the group consisting of microscopic glass slides,
capillary tubes, collection tubes, columns, micro-columns, wells,
plates, membranes, filters, resins, inorganic matrices, beads,
particulate chromatographic media, plastic microparticles, latex
particles, coated tubes, coated templates, coated beads, coated
matrices, or a combination thereof. The sample collection device
may include removable coupling means for removably coupling said
sample collection housing with said breast engaging member. In some
instances, the solid phase sample collection medium is supported by
a support member integrally or removably mounted within said sample
collection housing in fluid connection with said breast engaging
member. The support member may be disc-shaped and is interposed
between said breast engaging member and said sample collection
housing. Further, the support member may have upper and lower
retaining rings and supports a sheet of absorbent or adsorbent
material. Support member supports may be a solid phase sample
collection template including, but not limited to, capillary tubes,
coated tubes, columns, micro-columns, plates, wells and microscopic
slides, or a combination thereof. Support members define a
fluid-retaining well and include at least one air channel to allow
negative pressure to pass through the air channel to and from said
breast engaging member. The solid phase sample collection medium
may be a particulate medium contained within a cartridge removably
mounted within said sample collection housing and having a first
end of said cartridge in fluid connection with said breast engaging
member where the first end of said cartridge is covered by a porous
barrier material.
[0027] In another aspect, provided herein is a system for analyzing
nipple aspirate fluid from an individual in need thereof
comprising: an absorbent paper as illustrated in FIG. 1, the
absorbent paper comprising microcellulose, mixed cellulose ester
paper, or nitrocellulose; and a microscope for visualizing total
protein of the nipples aspirate fluid, counting the number cells
from the nipple aspirate fluid sample, or a combination
thereof.
[0028] In another aspect, provided herein is a system for analyzing
a nipple aspirate fluid (NAF) sample from an individual in need
thereof comprising: an absorbent paper as illustrated in FIG. 1 for
absorbing the nipple aspirate fluid sample, the absorbent paper
comprising microcellulose, mixed cellulose ester paper, or
nitrocellulose; and a microscope for visualizing total protein of
the nipple aspirate fluid sample, counting the number cells of the
nipple aspirate fluid sample, or a combination thereof.
[0029] In one embodiment, the total amount of protein in the nipple
aspirate fluid is detected by staining said absorbent paper with a
colloidal metal. For example, the colloidal metal may be colloidal
gold or colloidal silver.
[0030] The system may, in some embodiments, further comprise an
optionally networked computer processing device configured to
perform executable instructions; and a computer program, the
computer program comprising a software module executed by the
computer processing device to apply a model or algorithm for
analyzing the total protein content, the number of cells of the
nipple aspirate fluid, or a combination thereof.
[0031] In one embodiment, the computer program may further comprise
a software module executed by the computer processing device to
designate a treatment regimen for the individual.
[0032] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to store photomicrograms in a database of
photomicrograms.
[0033] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to store analysis in a database of analyses.
[0034] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to compare the total protein content or number of
cells of the nipple aspirate fluid to a standard.
[0035] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to transmit an analysis to a health care provider
or the individual.
[0036] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to transmit a diagnosis to a health care provider
or the individual.
[0037] In another embodiment, or in addition, the computer program
may further comprise a software module executed by the computer
processing device to generate a report comprising the analysis.
[0038] In another aspect, provided herein is a non-transitory
computer-readable storage media encoded with a computer program
including instructions executable by a computer processing device
to create an application, the application comprising a software
module configured to apply a model or algorithm for analyzing the
total protein content of a nipple aspirate fluid sample from an
individual, the number of cells of a nipple aspirate fluid sample
from an individual, or a combination thereof; and a software module
configured to designate a treatment regimen for the individual.
[0039] In yet another aspect, provided herein is a non-transitory
computer-readable storage media encoded with a computer program
including instructions executable by a computer processing device
to create an application, the application comprising: a software
module configured to apply a model or algorithm for analyzing the
total protein content and/or the number of cells of a nipple
aspirate fluid sample absorbed onto an absorbent paper as
illustrated in FIG. 1, and a software module configured to
designate a treatment regimen for the individual.
[0040] In one embodiment, the model or algorithm comprises
comparing the total protein content of the nipple aspirate fluid or
the number of cells of the nipple aspirate fluid sample to a
standard.
[0041] In yet another aspect, provided herein is a method of
identifying women at low risk of future breast cancer comprising
(a) obtaining a sample of nipple aspirate fluid on an absorbent
paper as illustrated in FIG. 1, wherein said absorbent paper
comprises microcellulose, mixed cellulose ester paper or
nitrocellulose; (b) detecting the total amount of protein in said
sample; (c) determining the number cells from said sample; and (d)
identifying a woman as low lowest risk for breast cancer wherein
the sample contains protein and is acellular.
[0042] In yet another aspect, provided herein is a method of
identifying risk of a patient for breast cancer or diagnosing a
patient at low risk of breast cancer comprising (a) collecting
nipple aspirate fluid on an absorbent paper as illustrated in FIG.
1, wherein said absorbent paper comprises microcellulose, mixed
cellulose ester paper or nitrocellulose; (b) washing said absorbent
paper with a solution to collect cells, if any; (c) counting the
number of cells in said sample and, optionally, determining the
cytology of said cells, if any; (d) staining said absorbent paper
with a metal particle suspension and determining the concentration
of protein on said absorbent paper; (e) comparing, the
concentration of protein in said nipple aspirate fluid and the
number of cells in said fluid, to the concentration of protein and
number of cells in a population of patients known to have breast
cancer, to a population of patients known not to have breast
cancer, or to a combination thereof; and (f) identifying a patient
as at risk for breast cancer or at low risk of breast cancer
wherein the sample contains protein and is acellular or contains
one cell.
[0043] In yet another aspect, provided herein is a method of
diagnosing risk of a patient for breast cancer comprising (a)
collecting nipple aspirate fluid (NAF) on an absorbent paper as
illustrated in FIG. 1, wherein said absorbent paper comprises
microcellulose, mixed cellulose ester paper or nitrocellulose,
wherein said NAF sample is obtained from classical providers and
non-providers; (b) washing said absorbent paper with buffered
solution to collect cells, if any; (c) counting the number of cells
in said sample and, optionally, determining the cytology of said
cells, if any; (d) staining said absorbent paper with colloidal
gold or colloidal silver and determining the concentration of
protein on said absorbent paper; (e) comparing, the concentration
of protein in said nipple aspirate fluid and the number of cells in
said fluid, to the concentration of protein and number of cells in
a population of patients known to have breast cancer, to a
population of patients known not to have breast cancer, or to a
combination thereof; and (f) diagnosing a patient as at risk for
breast cancer wherein the sample contains protein and is acellular
or contains one cell. In yet another aspect, provided herein is a
method of detecting protein in a sample of nipple aspirate fluid
(NAF) comprising collecting the NAF on an absorbent paper as
illustrated in FIG. 1, wherein said absorbent paper comprises
microcellulose, mixed cellulose ester paper or nitrocellulose; and
detecting the protein with a colloidal metal particle suspension,
wherein the method can detect protein at a concentration of between
0.5 ng and 500 ng per sample.
[0044] In yet another aspect, provided herein is a test material
for a risk associated with breast cancer comprising an absorbent
paper as illustrated in FIG. 1, wherein said absorbent paper
comprises microcellulose, mixed cellulose ester paper or
nitrocellulose, a sample of nipple aspirate fluid collected from a
women classically characterized as a non-yielder of nipple aspirate
fluid, and a stain the detects the presence of protein from the
nipple aspirate fluid collected on the absorbent paper.
[0045] Provided herein is an absorbent paper that is sized to cover
a nipple, comprising microcellulose, mixed cellulose ester paper or
nitrocellulose. The absorbent paper may comprise absorbent paper as
illustrated in FIG. 1. In one embodiment, the absorbent paper
comprises mixed cellulose ester paper. In another embodiment, the
absorbent paper is from about 1.0 to about 3.0 inches in diameter
and from about 0.01 to about 0.1 inches thick. In another
embodiment, the absorbent paper comprises an L-shaped element 4 as
illustrated in FIG. 1.
[0046] Provided herein is a composition comprising nipple aspirate
fluid (NAF) and an absorbent paper that is sized to cover a nipple,
comprising microcellulose, mixed cellulose ester paper or
nitrocellulose.
[0047] Provided herein is a system for analyzing a nipple aspirate
fluid (NAF) sample from an individual in need thereof comprising:
an absorbent paper for absorbing the nipple aspirate fluid sample,
the absorbent paper comprising microcellulose, mixed cellulose
ester paper, or nitrocellulose, wherein the absorbent paper is
sized to cover a nipple; and a breast pump device.
[0048] In one embodiment, the absorbent paper is from about 1.0 to
about 3.0 inches in diameter and from about 0.01 to about 0.1
inches thick.
[0049] In another embodiment, the breast pump device is a MASCT.TM.
device.
[0050] Disclosed herein, in certain embodiments, is a system for
analyzing a nipple aspirate fluid (NAF) sample from an individual
in need thereof comprising: (a) an absorbent paper for absorbing
the nipple aspirate fluid sample, the absorbent paper comprising
microcellulose, mixed cellulose ester paper, or nitrocellulose,
wherein the absorbent paper is sized to cover a nipple; and (b)
colloidal metal stain for determining the total protein of the
nipple aspirate fluid sample. In some embodiments, the absorbent
paper is from about 1.0 to about 3.0 inches in diameter and from
about 0.01 to about 0.1 inches thick. In some embodiments, the
system further comprises a microscope for visualizing total protein
of the nipple aspirate fluid sample, counting the number cells of
the nipple aspirate fluid sample, or a combination thereof. In some
embodiments, the colloidal metal is colloidal gold or colloidal
silver. In some embodiments, the system further comprises: an
optionally networked computer processing device configured to
perform executable instructions; and a computer program, the
computer program comprising a software module executed by the
computer processing device to apply a model or algorithm for
analyzing the total protein content of the nipple aspirate fluid
sample, the number of cells of the nipple aspirate fluid sample, or
a combination thereof. In some embodiments, the computer program
further comprises a software module executed by the computer
processing device to designate a treatment regimen for the
individual. In some embodiments, the computer program further
comprises a software module executed by the computer processing
device to store photomicrograms in a database of photomicrograms.
In some embodiments, the computer program further comprises a
software module executed by the computer processing device to store
analysis in a database of analyses. In some embodiments, the
computer program further comprises a software module executed by
the computer processing device to compare the total protein content
or number of cells of the nipple aspirate fluid sample to a
standard. In some embodiments, the computer program further
comprises a software module executed by the computer processing
device to transmit an analysis to a health care provider or the
individual. In some embodiments, the computer program further
comprises a software module executed by the computer processing
device to transmit a diagnosis to a health care provider or the
individual. In some embodiments, the computer program further
comprises a software module executed by the computer processing
device to generate a report comprising the analysis. In some
embodiments, said absorbent paper is a device as shown in FIG.
1.
INCORPORATION BY REFERENCE
[0051] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The features of the disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present compositions, kits and
methods will be obtained by reference to the following detailed
description that sets forth illustrative embodiments, in which the
principles of the disclosure embodiments are utilized, and the
accompanying drawings of which:
[0053] FIGS. 1A-B illustrate a 2-D image of a representative
absorbent paper or membrane described herein. Angular dimensions
are provided in inches .+-.1.degree. and in degrees. FIG. 1A
illustrates a top view of the paper or membrane. FIG. 1B
illustrates a side angle production.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Although there are currently several tests for breast
cancer, more sensitive and reliable methods are needed to detect
small, early stage, in situ carcinomas of the breast. Such methods
should significantly improve breast cancer survival, as suggested
by the successful employment of Papinicolou smears for early
detection and treatment of cervical cancer.
[0055] The methods of the embodiments provided herein may be
conducted with an appropriate breast pump device which may be used
for sample collection such as, for example a device described in
U.S. Pat. No. 5,798,266; U.S. Pat. No. 6,689,073; and U.S. Pat. No.
6,887,210, each of which is incorporated herein by reference. In
one embodiment, the device is a MASCT.TM. device.
[0056] Methods and devices for obtaining, handling, and processing
biological samples from mammary fluid are also described herein.
Preferably, these methods are non-invasive, meaning they are
non-surgical and do not involve penetration of the breast by
needles or other intrusive devices. The devices and methods
described herein are effective to collect mammary fluid alone or in
conjunction with oxytocin stimulation.
[0057] Briefly, to practice noninvasive sample collection, the
specialized breast pump devices feature a breast engaging portion
or member coupled with a vacuum pump mechanism and may be fluidly
connected with a solid phase sample collection medium.
[0058] The mammary fluid collection devices are typically provided
as a specialized breast pump which can be applied to a human or
animal breast covering the nipple and used in conjunction with a
nipple touch procedure as described in more detail below following
use of the device as described in Example 1.
[0059] The nipple touch procedure may be administered by applying a
separate absorbent paper or membrane as described herein to each
nipple.
[0060] Absorbent papers 2 (which may also be called "membranes"
herein) which may be used in the disclosed methods and may be any
material that is suitable to collect epithelial cells and
biomarkers such as, for example, proteins, carbohydrates, lipids,
nucleic acids, RNA, DNA, etc. Absorbent papers 2 include those made
of, for example, nitrocellulose, microcellulose, mixed cellulose
ester, or any other appropriate material for nipple fluid sample
collection. While FIG. 1A illustrates a circular absorbent paper,
other shapes such as, for example, ovals, squares, triangles, other
polygons, are also contemplated herein so long as the shape
accommodates sample collection.
[0061] In some embodiments, the absorbent paper 2 does not cause
papers cuts to the nipple and/or the areola. In some embodiments,
the absorbent paper 2 is shaped to avoid paper cuts to the nipple
and/or areola.
[0062] The absorbent paper 2 is formed by stamping the paper out of
large paper stock with a metal mold. The absorbent paper 2 is big
enough to cover or partially cover the nipple. In some embodiments,
the absorbent paper 2 is big enough to cover the nipple. Therefore,
an absorbent paper may be from about 1.0 inches to about 3.0 inches
in diameter or length at its average dimension A across any size of
the absorbent paper. An absorbent paper 2 may be, for example,
about 1.0, about 1.1, about 1.15, about 1.2, about 1.25, about 1.3,
about 1.35, about 1.4, about 1.45, about 1.5, about 1.55, about
1.6, about 1.65, about 1.7, about 1.75, about 1.8, about 1.85,
about 1.9, about 1.95, about 2.0, about 2.1, about 2.15, about 2.2,
about 2.25, about 2.3, about 2.35, about 2.4, about 2.45, about
2.5, about 2.55, about 2.6, about 2.65, about 2.7, about 2.75,
about 2.8, about 2.85, about 2.9, about 2.95, or about 3.0 inches
in diameter. FIG. 1A provides a non-limiting example of an
absorbent paper 2 that is 1.85 inches in diameter A. In some
embodiments, the absorbent paper 2 covers or partially covers the
areola of a breast. In some embodiments, the absorbent paper 2
covers the areola of a breast. In some embodiments, the absorbent
paper 2 partially covers the areola of a breast. In some
embodiments, the absorbent paper covers a nipple and does not
extend to the areola of a breast.
[0063] The thickness of the absorbent paper 2 may vary to allow for
optimal sample collection and includes materials that are from
about 0.01 inches to about 0.1 inches in thickness. For example,
the absorbent paper 2 may be about 0.01, about 0.02, about 0.03,
about 0.04, about 0.05, about 0.06, about 0.07, about 0.08 about
0.09, or about 0.1 inches thick. FIG. 1B provides a non-limiting
example of an absorbent paper 2 that is 0.05 inches thick. One
would understand that, while FIG. 1B illustrates the side view of
an absorbent paper 2 that is 0.05 inches thick, the thickness can
be varied as well.
[0064] The L-shaped element (which may also be identified as a
"slit" herein) is optional and is useful if the absorbent paper 2
is placed in a pressure modifying device to lower the pressure and
cause egress of fluid from the inside of the breast. In one
embodiment, the absorbent paper 2 is sized such that it fits into a
modified breast pump, and the dimensions are set accordingly.
Modified breast pumps that may be optionally used in connection
with an absorbent paper 2 are described herein.
[0065] The L-shaped element 4 in FIG. 1A is a cut out that remains
after the die cut has stamped the paper. In one non-limiting
example, the L-shaped element is 0.063 E inches across when cut out
in the stamping process. In an absorbent paper 2 that is 1.85
inches A in diameter or length at its average dimension across any
size of the device, the ends of the L-shaped element 4 are 0.25
inches B from the mid-line of the absorbent paper 2. The angle D of
the L-shaped element 4 can be any angle from 10 degrees to 170
degrees D. In one non-limiting embodiment, the angle D is 75
degrees as illustrated in FIG. 1A. The inner circle 6 illustrated
in FIG. 1A is approximately 0.75 inches C in diameter and was
designed such that the L-shaped flap 8 moves properly when used in
a breast pump device (e.g., a MASCT.TM. device described herein).
The dash symbols designating the inner circle 6 illustrate guide
lines in the figure. The slit 4 is shown by the incomplete triangle
and is shaped as illustrated to form an incomplete circle. One
would understand that the measurements described herein can be
proportionally adjusted based upon the total size of the absorbent
paper.
[0066] FIG. 1A represents the top view of one non-limiting example
of an absorbent paper 2. The dash lines are not cut lines, but
rather, are presented for ease of manufacturing to align the
L-shaped element 4 such that the center of the absorbent paper 2
fits above the nipple area and so flap 8 sufficiently covers the
nipple.
[0067] In another embodiment, the absorbent paper 2 is made of
mixed cellulose ester and is formed in the shape and dimensions as
illustrated in FIGS. 1A and 1B.
[0068] During or after administration of the breast pump device to
produce mammary fluid, a biological sample is collected from the
expressed mammary fluid using the absorbent paper, which sample may
contain one or more of whole mammary fluid, whole cells, cell
fragments, cell membranes, selected liquid, cellular or other solid
fractions of the mammary fluid, as well as proteins, glycoproteins,
peptides, nucleotides (including DNA, RNA, etc.) and other like
biochemical and molecular constituents of the mammary fluid.
[0069] In certain embodiments of the invention, the expressed
mammary fluid is contacted with a solid phase sample collection
medium fluidly connected with the breast pump, simultaneous with or
subsequent to the time of breast fluid expression. Suitable solid
phase media in this context include microscopic glass slides,
capillary tubes, coated tubes, microtiter wells or plates,
membranes, filters, affinity columns, dot blot matrices, beads,
resins, and other like media that will selectively adsorb, bind,
filter, partition or otherwise process desired components of the
mammary fluid for convenient incorporation into a desired
assay.
[0070] Provided herein is a method of identifying women at low risk
of future breast cancer, comprising obtaining a sample of nipple
aspirate fluid; testing for protein in said sample; and testing for
cells in said sample, wherein the lowest risk is associated with a
sample that contains protein and is acellular.
[0071] Also provided herein is a method of identifying risk of a
patient for breast cancer or diagnosing a patient at low risk of
breast cancer, comprising: comparing, using a general purpose
computer, the concentration of protein in a nipple aspirate fluid
sample and the number of cells in said sample, to the concentration
of protein and number of cells in a population of patients known to
have breast cancer and/or to a population of patients known not to
have breast cancer; wherein the lowest risk is associated with a
sample that contains protein and is acellular or contains one
cell.
[0072] The patient population identified as being at lowest risk
for breast cancer is associated with a sample that contains protein
and is acellular or contains one cell. In one embodiment, the
sample contains no cells (i.e., is acellular). In another
embodiment, low risk is associated with a sample that contains
protein and contains one cell. The risk of breast cancer increases
with increasing number of cells.
[0073] Patients to be diagnosed with a method provided herein
include those classically designated as non-secretors and secretors
of nipple aspirate fluid (NAF).
[0074] In one embodiment, the patient is classically designated as
a non-secretor of nipple aspirate fluid.
[0075] Nipple aspirate fluid sample is collected on absorbent paper
or membrane. Absorbent paper or membrane that may be used in the
present methods includes paper of the size and dimensions as
illustrated in FIG. 1. The absorbent paper or membrane may contain,
for example, microcellulose, mixed cellulose ester or
nitrocellulose. This new design results in significantly fewer cuts
to nipple tissue during sample collection compared to paper that is
currently marketed for NAF collection.
[0076] The absorbent paper or membrane is washed and the effluent
is collected and assessed for number of cells. In one embodiment,
cytology of cells, if any, in said sample is analyzed using any
conventional method including, but not limited to, microscopy, flow
cytometry, immunohistochemistry, or a combination thereof. A
clinician can determine if cells, if present in the sample, are
normal contain one or more characteristics of cancer cells. In one
non-limiting example, cell samples may be stained with hemolysin
and eosin.
[0077] Protein measured by the methods described herein is total
protein content of the sample. The paper is then exposed to
colloidal gold or colloidal silver and the total protein content
(concentration) is determined using conventional methods or those
described herein.
[0078] In one aspect, if a sample contains from about 50 pg to
about 0.5 ng of protein, and does not contain cells, a patient is
identified as being at low risk for breast cancer.
[0079] Alternatively, if the sample contains at least about 300 ng
of protein and two (2) or more cells, a patient is identified for
further evaluation of breast cancer or diagnosed as at medium or
high risk for breast cancer. In one embodiment, the concentration
of protein in a sample is from about 300 ng of protein to about 2
.mu.g of protein and a patient is diagnosed at being at higher risk
for breast cancer and, in some cases, is identified for further
assessment. The cell fraction of the sample, in such a case, may
contain about 2 cells to about 50 cells. Such a cell fraction may,
in another case, may contain at least ten (10) cells.
[0080] Provided herein is an absorbent paper or membrane having the
characteristics and dimensions as illustrated in FIG. 1, where the
absorbent paper or membrane is made using microcellulose, mixed
cellulose ester or nitrocellulose. In one embodiment, the absorbent
paper or membrane comprises mixed cellulose ester.
[0081] A significant advance of the present absorbent paper or
membrane is that is does not cut tissue to the extent that current
filter paper does; thus, a patient undergoing assessment is subject
to less pain and discomfort during the collection procedure. In one
embodiment, tissue exposed to the absorbent paper or membrane is
nipple tissue.
[0082] Provided herein is the use of the absorbent paper or
membrane of as described herein in a method of identifying a
patient at low risk of breast cancer or diagnosing a patient at low
risk of breast cancer. In one aspect, the absorbent paper or
membrane is used in any of the methods described herein or in any
other method of nipple aspirate fluid collection for any use.
[0083] A method of detecting protein in a sample of nipple aspirate
fluid (NAF) comprising collecting the NAF on an absorbent paper or
membrane and detecting the protein with a colloidal metal particle
suspension, wherein the method can detect protein at a
concentration of between 0.5 ng and 500 ng per sample. The metal
particle suspension may contain colloidal gold or colloidal silver.
In one embodiment, the metal particle suspension comprises
colloidal gold.
[0084] Provided herein is method of identifying risk of a patient
for breast cancer or diagnosing a patient at low risk of breast
cancer, comprising: collecting nipple aspirate fluid on absorbent
paper or membrane; washing said absorbent paper or membrane with
buffered solution to collect cells, if any; counting the number of
cells in said sample and, optionally, determining the cytology of
said cells, if any; staining said absorbent paper or membrane with
colloidal gold or colloidal silver and determining the
concentration of protein on said filter paper; and comparing, the
concentration of protein in said nipple aspirate fluid and the
number of cells in said fluid, to the concentration of protein and
number of cells in a population of patients known to have breast
cancer and/or to a population of patients known not to have breast
cancer; wherein the lowest risk is associated with a sample that
contains protein and is acellular or contains one cell.
[0085] Provided herein is a method of diagnosing risk of a patient
for breast cancer, comprising: collecting nipple aspirate fluid on
absorbent paper or membrane, wherein said NAF sample is obtained
from classical providers and non-providers; washing said absorbent
paper or membrane with buffered solution to collect cells, if any;
counting the number of cells in said sample and, optionally,
determining the cytology of said cells, if any; staining said
absorbent paper or membrane with colloidal gold or colloidal silver
and determining the concentration of protein on said filter paper;
and comparing, the concentration of protein in said nipple aspirate
fluid and the number of cells in said fluid, to the concentration
of protein and number of cells in a population of patients known to
have breast cancer and/or to a population of patients known not to
have breast cancer; wherein the lowest risk is associated with a
sample that contains protein and is acellular or contains one
cell.
[0086] In one embodiment, the sample is acellular and the patient
is identified as at the lowest risk for breast cancer. In another
embodiment, the sample contains one cell and the patient is
identified as at low risk for breast cancer. In yet another
embodiment, the sample contains more than 2 cells and the patient
is identified for further assessment.
[0087] In one embodiment, the total protein concentration of each
breast is determined and, if the total protein concentration of one
sample is greater than 300 ng, the patient is identified for
further assessment of breast cancer. Alternatively, the total
protein concentration of each breast is determined and, if the
total protein concentration of each sample is at, or below 200 ng
protein, the patient is identified as being at low risk for breast
cancer.
[0088] In one embodiment, the nipple aspirate fluid is collected on
an absorbent paper or membrane following massaging of breast tissue
and suction with a MASCT.TM. device as described in Example 1
below. As used herein, a "MASCT.TM. device" refers to a device
described in U.S. Pat. No. 6,287,521 by Quay et al. which is
incorporated herein in its entirety. In one non-limiting example, a
sample collection device for collecting a biological sample from a
mammary organ of a patient may comprise a breast engaging member
constructed of a non-porous material sized and dimensioned to
receive at least a nipple portion of a breast of said patient and
form a suction seal therewith; a solid phase sample collection
medium in fluid connection with said breast engaging member for
receiving a sample of expressed breast fluid; and a vacuum pump
means in gaseous connection with said breast engaging member for
generating negative pressure through the breast engaging member to
facilitate breast fluid expression, wherein said solid phase sample
collection medium is selected from the group consisting of
microscopic glass slides, capillary tubes, collection tubes,
columns, micro-columns, wells, plates, membranes, filters, resins,
inorganic matrices, beads, particulate chromatographic media,
plastic microparticles, latex particles, coated tubes, coated
templates, coated beads, coated matrices, or a combination thereof.
The sample collection device may include removable coupling means
for removably coupling said sample collection housing with said
breast engaging member. In some instances, the solid phase sample
collection medium is supported by a support member integrally or
removably mounted within said sample collection housing in fluid
connection with said breast engaging member. The support member may
be disc-shaped and is interposed between said breast engaging
member and said sample collection housing. Further, the support
member may have upper and lower retaining rings and supports a
sheet of absorbent or adsorbent material. Support member supports
may be a solid phase sample collection template including, but not
limited to, capillary tubes, coated tubes, columns, micro-columns,
plates, wells and microscopic slides, or a combination thereof.
Support members define a fluid-retaining well and include at least
one air channel to allow negative pressure to pass through the air
channel to and from said breast engaging member. The solid phase
sample collection medium may be a particulate medium contained
within a cartridge removably mounted within said sample collection
housing and having a first end of said cartridge in fluid
connection with said breast engaging member where the first end of
said cartridge is covered by a porous barrier material.
[0089] Prior to, or concurrent with, each assay run of the
invention, it may be useful to perform a preliminary evaluation to
verify sample origin and/or quality if sufficient sample quantity
can be obtained. The focus of such preliminary evaluations is to
verify that the sample collected from expressed mammary fluid is
indeed of mammary origin, and is not contaminated with other
potential contaminants, such as sweat from skin surrounding the
nipple. For these sample verification purposes, a variety of assays
are available which identify mammary fluid markers known to be
present in mammalian mammary fluid, and which are preferably highly
specific markers for mammary fluid (i.e. markers which are
typically always present in mammary fluid and which are absent from
all, or most of, other potentially contaminating bodily fluids and
tissues). However, an acceptable level of specificity for mammary
fluid markers within the methods of the invention is provided by
markers that are simply known to be present in mammary fluid, even
though they may be present in other bodily fluids. One such marker
is the enzyme lysozyme, which is a normal component of human serum,
urine, saliva, tears, nasal secretions, vaginal secretions, seminal
fluid, and mammary fluid. Lysozyme (muramidase) is an enzyme which
hydrolyzes beta 1,4-glycosidic linkages in the mucopolysaccharide
cell wall of a variety of microorganisms resulting in cell lysis.
Quantitative measurement of lysozyme is readily accomplished by a
well known agar plate diffusion method, described in detail in the
instructions provided with the Quantiplate.RTM. lysozyme test kit,
available from Kallestad, Sanofi Diagnostics (Chasta, Minn.),
incorporated herein by reference in its entirety.
[0090] Other mammary fluid markers for sample verification that are
more specific than lysozyme are preferred within the methods of the
invention, and can be readily incorporated within the method
embodiments based on published and generally known information.
Useful among these markers are proteins and other biological
substances that are specifically expressed or enriched in mammary
fluid. A diverse array of suitable markers in this context have
been characterized and have already been used to develop specific
antibodies, including affinity purified and monoclonal antibodies.
These antibodies can in turn be employed as immunological probes to
determine the presence or absence, and/or to quantify, selected
mammary fluid markers to verify mammary fluid sample origin and
quality. Mammary fluid markers of particular interest for use
within the invention include specific cytokeratins that are
characteristically expressed by normal and cancerous mammary
epithelial cells, against which specific panels of antibody probes
have already been developed. (See for example, Nagle, J.,
Histochem. Cytochem. 34:869-881, 1986, incorporated herein by
reference in its entirety). Also useful as mammary fluid markers
are the human mammary epithelial antigens (HME-Ags) corresponding
to glycoprotein components of the human milk fat globulin (HMFG)
protein, against which specific antibodies (e.g., anti HMFG1,
Unipath, U.K.) are also available. (See Rosner et al., Cancer
Invest. 13:573-582, 1995; Ceriani et al. Proc. Natl. Acad. Sci. USA
74:582-586, 1982; Ceriani et al., Breast Cancer Res. Treat.
15:161-174, 1990, each incorporated herein by reference in its
entirety).
[0091] The term "colloidal metal particles" used in this connection
is meant to include dispersions of particles, preferably sols,
consisting of a metal, a metal compound or nuclei coated with a
metal or metal compound. Colloidal metal particles can be prepared
following art-known procedures, such as have been described for
preparing suspensions of gold, silver, platinum or iron hydroxide
and the like. The terms "gold colloid" and "colloidal gold
composition" used herein refer to a suspension of
sub-micrometer-sized gold particles evenly dispersed in a fluid
(e.g., water or an aqueous buffer). The colloidal gold composition
utilized in the quantification assay contains highly concentrated
gold particles. In one example, the colloidal gold composition has
a gold particle concentration ranging from 3.5.times.10.sup.12 to
7.0.times.10.sup.12 particles/ml, e.g., (3.5-5.25).times.10.sup.12
particles/ml.
[0092] The present methods may be performed using conventional
techniques of immunocytochemistry such as for example immunogold
labeling. Such procedures are described, for example, in the
following texts that are incorporated by reference: (1) Colloidal
Gold-Principles, Methods and Applications, Hayat M, (1989-1990) (3
volumes), Academic Press. (Hardback); (2) Colloidal Gold-A New
Perspective For Cytochemical Marking, Beesley J (1989), Royal
Microscopical Society Handbook No 17. Oxford Science Publications.
Oxford University Press. (Paperback); (3) An Introduction To
Immunocytochemistry: Current techniques and problems, Polak J and
Van Noorden 5 (1984) Royal Microscopical Society Handbook No 11.
Oxford Science Publications. Oxford University Press. (Paperback);
(4) Immunocytochemistry-Modern Methods and Applications, Polak J
and Van Noorden 5 (1986) (2nd ed.), Butterworth Heinemann, Oxford.
(Hardback); and (5) Techniques in Immunocytochemistry, Bullock G
and Petmsz P (1982-1989) (4 volumes) Academic Press.
[0093] Procedures are also described, for example, in Marc
Moeremans, et al., "Sensitive Colloidal Metal (Gold or Silver)
Staining of Protein Blots on Nitrocellulose Membranes" in
Analytical Biochemistry 145, 315-321 (1985); Danscher et al.,
Colloidal Gold Amplification, the Journal of Histochemistry and
Cytochemistry, 31: No. 12, 1394-1398 (1983); De Mey, Colloidal Gold
Methods, Immunocytochemistry, Chapt. 6, pp. 82-112 (1983); and U.S.
Pat. No. 4,920,059 by Moermans et al.
[0094] Examples of colloidal metal particles that will bind to
proteins and nucleic acids when adjusted to the optimal pH and
concentration are the metals platinum, gold, silver and copper, and
the metal compounds for example, gold, silver, platinum, iron, or
copper compounds, such as, silver iodide, silver bromide, copper
hydrous oxide, iron oxide, iron hydroxide or hydrous oxide,
aluminum hydroxide or hydrous oxide, chromium hydroxide or hydrous
oxide, vanadium oxide, arsenic sulfide, manganese hydroxide, lead
sulfide, mercury sulfide, barium sulfate and titanium dioxide.
Colloids consisting of nuclei, coated with the above mentioned
metals or metal compounds can also be used. The particles have
similar properties as the metal or metal compound colloids, but
size, density and metal content can be optimally combined. In
general, all colloidal metal particles or metal compounds which can
be adjusted to the optimal pH for protein binding and which give a
color intensity in protein staining, sufficient to be observed by
the naked eye, can be used. Preferably, the sensitivities are equal
or superior to those obtained with the metals gold and silver.
[0095] For the staining of proteins, particularly good results are
obtained with gold, silver and iron hydroxide colloids.
[0096] The particle size of the colloidal metal or metal compound
particles may be between about 1 and about 100 nm. The appropriate
pH is the pH at which binding is maximal and the most intense color
is obtained. Maximal binding may occur when the proteins and the
colloidal metal particles have opposite net charges. In this
process, the proteins interact with colloidal metal particles at a
pH close to the pI of the protein. Adjustment of the pH can be
achieved in any of the usual ways. Addition of a stock buffer to
about a 10 mM final concentration to the colloidal metal particles
represents one method for use herein.
[0097] The appropriate concentration of the colloidal metal
particles is one that gives full color saturation within practical
incubation times (from a few minutes to one day). It can be
obtained by choosing the proper concentrations of the raw materials
with which they are prepared, or by dilution or concentration by
art-known methods.
[0098] In some instances, the absorbent paper or membrane is washed
prior to the staining procedure. This washing may be used remove
adherent interfering materials. Such washing of the absorbent paper
or membrane may be done by contacting it with a buffer solution or
water optionally supplemented with at least one substance which
promotes the staining specificity defined as specific binding of
the colloidal or metal compound particles to the proteins and the
absence of such specific binding on those parts of the immobilizing
matrix where no protein are immobilized. A detergent may be added
as needed to the colloidal metal particles, before or after
adjusting the pH. Such detergents are herein exemplified by Tween
20 (polyoxyethylene sorbitan monolaurate, Tween 80 (polyoxyethylene
sorbitan mono-oleate), Triton X-100 (octyl phenoxy polyepoxy
ethanol) and myristyltrimethyl ammonium bromide. It will be
understood that similar detergents, i.e., surface active
substances, may also be used. The detergent added to the colloidal
metal particles may also be employed to promote the specific
binding of the colloidal metal particles to the protein.
[0099] Optionally, the optical signal formed by the colloidal metal
particles may be enhanced by appropriate enhancers such as physical
developers, for example, silver- or gold-containing compounds or
modified and/or enhanced by art-known color identification methods
of metal ions after transformation of the colloidal metal into a
metal ion.
[0100] Protein can be detected and quantitated using conventional
means in the art such as, for example, reading the colored signal
produced by and characteristic for the bound colloidal metal
particles with the naked eye or using art-known spectrophotometric
techniques such as densitometry. In such procedures, the optical
density of the mixture can be measured at a wavelength ranging from
540-700 nm, and the concentration of the biomolecule is determined
based on the value of the optical density. In one embodiment, the
optical density is measured at 590 nm. Protein can be detected
using the presently disclosed methods ad a concentration of, for
example, about 0.5 pg, about 1 pg, about 100 pg, about 250 pg,
about 350 pg, about 500 pg, about 750 pg, about 1 ng, about 100 ng,
about 250 ng, about 500 ng, about 750 ng and about 1 .mu.g. In one
embodiment, protein is detected at about 0.5 ng.
[0101] Methods for washing cells from a matrix such as, for
example, absorbent paper or membrane using a buffered solution are
known in the art and contemplated herein. The effluent of such
washings may be further analyzed using methods including, but not
limited to, microscopy, immunocytochemistry and flow cytometry. For
example, after washing the absorbent paper or membrane containing
the nipple aspirate fluid sample to remove any cells, the wash
solution may be assessed using microscopy and the number of cells
in the solution is determined. The morphology of any cells present
in the solution may be determined. Additionally, cells, if present,
may be stained for one or more extracellular and/or intracellular
markers to determine if the cells have a normal profile or have one
or more markers indicative of cancer cells. For example, cells may
be analyzed for the presence or absence of BRCA1, BRCA2, p63, a
cyclin, a cytokeratin, or any other marker that may indicate that
the cells are cancer cells or normal cells based on the presence,
absence, or level of such markers. Labeled antibodies may also be
used to stain cells in such samples using conventional flow
cytometry techniques known in the art. Further methods of analyzing
cells are provided herein below in Example 2.
Digital Processing Device
[0102] In some embodiments, the methods, systems, and software
described herein include a digital processing device, or use of the
same. In further embodiments, the digital processing device
includes one or more hardware central processing units (CPU) that
carry out the device's functions. In still further embodiments, the
digital processing device further comprises an operating system
configured to perform executable instructions. In some embodiments,
the digital processing device is optionally connected a computer
network. In further embodiments, the digital processing device is
optionally connected to the Internet such that it accesses the
World Wide Web. In still further embodiments, the digital
processing device is optionally connected to a cloud computing
infrastructure. In other embodiments, the digital processing device
is optionally connected to an intranet. In other embodiments, the
digital processing device is optionally connected to a data storage
device.
[0103] In accordance with the description herein, suitable digital
processing devices include, by way of non-limiting examples, server
computers, desktop computers, laptop computers, notebook computers,
sub-notebook computers, netbook computers, netpad computers,
set-top computers, handheld computers, Internet appliances, mobile
smartphones, tablet computers, personal digital assistants, video
game consoles, and vehicles. Those of skill in the art will
recognize that many smartphones are suitable for use in the system
described herein. Those of skill in the art will also recognize
that select televisions, video players, and digital music players
with optional computer network connectivity are suitable for use in
the system described herein. Suitable tablet computers include
those with booklet, slate, and convertible configurations, known to
those of skill in the art.
[0104] In some embodiments, the digital processing device includes
an operating system configured to perform executable instructions.
The operating system is, for example, software, including programs
and data, which manages the device's hardware and provides services
for execution of applications. Those of skill in the art will
recognize that suitable server operating systems include, by way of
non-limiting examples, FreeBSD, OpenBSD, NetBSD.RTM., Linux,
Apple.RTM. Mac OS X Server.RTM., Oracle.RTM. Solaris.RTM., Windows
Server.RTM., and Novell.RTM. NetWare.RTM.. Those of skill in the
art will recognize that suitable personal computer operating
systems include, by way of non-limiting examples, Microsoft.RTM.
Windows.RTM., Apple.RTM. Mac OS X.RTM., UNIX.RTM., and UNIX-like
operating systems such as GNU/Linux.RTM.. In some embodiments, the
operating system is provided by cloud computing. Those of skill in
the art will also recognize that suitable mobile smart phone
operating systems include, by way of non-limiting examples,
Nokia.RTM. Symbian.RTM. OS, Apple.RTM. iOS.RTM., Research In
Motion.RTM. BlackBerry OS.RTM., Google.RTM. Android.RTM.,
Microsoft.RTM. Windows Phone.RTM. OS, Microsoft.RTM. Windows
Mobile.RTM. OS, Linux.RTM., and Palm.RTM. WebOS.RTM..
[0105] In some embodiments, the device includes a storage and/or
memory device. The storage and/or memory device is one or more
physical apparatuses used to store data or programs on a temporary
or permanent basis. In some embodiments, the device is volatile
memory and requires power to maintain stored information. In some
embodiments, the device is non-volatile memory and retains stored
information when the digital processing device is not powered. In
further embodiments, the non-volatile memory comprises flash
memory. In some embodiments, the non-volatile memory comprises
dynamic random-access memory (DRAM). In some embodiments, the
non-volatile memory comprises ferroelectric random access memory
(FRAM). In some embodiments, the non-volatile memory comprises
phase-change random access memory (PRAM). In other embodiments, the
device is a storage device including, by way of non-limiting
examples, CD-ROMs, DVDs, flash memory devices, magnetic disk
drives, magnetic tapes drives, optical disk drives, and cloud
computing based storage. In further embodiments, the storage and/or
memory device is a combination of devices such as those disclosed
herein.
[0106] In some embodiments, the digital processing device includes
a display to send visual information to a user. In some
embodiments, the display is a cathode ray tube (CRT). In some
embodiments, the display is a liquid crystal display (LCD). In
further embodiments, the display is a thin film transistor liquid
crystal display (TFT-LCD). In some embodiments, the display is an
organic light emitting diode (OLED) display. In various further
embodiments, on OLED display is a passive-matrix OLED (PMOLED) or
active-matrix OLED (AMOLED) display. In some embodiments, the
display is a plasma display. In other embodiments, the display is a
video projector. In still further embodiments, the display is a
combination of devices such as those disclosed herein.
[0107] In some embodiments, the digital processing device includes
an input device to receive information from a user. In some
embodiments, the input device is a keyboard. In some embodiments,
the input device is a pointing device including, by way of
non-limiting examples, a mouse, trackball, track pad, joystick,
game controller, or stylus. In some embodiments, the input device
is a touch screen or a multi-touch screen. In other embodiments,
the input device is a microphone to capture voice or other sound
input. In other embodiments, the input device is a video camera to
capture motion or visual input. In still further embodiments, the
input device is a combination of devices such as those disclosed
herein.
Non-Transitory Computer Readable Storage Medium
[0108] In some embodiments, the methods, systems, and software
disclosed herein include one or more computer readable storage
media encoded with a program including instructions executable by
the operating system of an optionally networked digital processing
device. In further embodiments, a computer readable storage medium
is a tangible component of a digital processing device. In still
further embodiments, a computer readable storage medium is
optionally removable from a digital processing device. In some
embodiments, a computer readable storage medium includes, by way of
non-limiting examples, CD-ROMs, DVDs, flash memory devices, solid
state memory, magnetic disk drives, magnetic tape drives, optical
disk drives, cloud computing systems and services, and the like. In
some cases, the program and instructions are permanently,
substantially permanently, semi-permanently, or non-transitorily
encoded on the media.
Computer Program
[0109] In some embodiments, the methods, systems, and software
disclosed herein include at least one computer program, or use of
the same. A computer program includes a sequence of instructions,
executable in the digital processing device's CPU, written to
perform a specified task. In light of the disclosure provided
herein, those of skill in the art will recognize that a computer
program may be written in various versions of various languages. In
some embodiments, a computer program comprises one sequence of
instructions. In some embodiments, a computer program comprises a
plurality of sequences of instructions. In some embodiments, a
computer program is provided from one location. In other
embodiments, a computer program is provided from a plurality of
locations. In various embodiments, a computer program includes one
or more software modules. In various embodiments, a computer
program includes, in part or in whole, one or more web
applications, one or more mobile applications, one or more
standalone applications, one or more web browser plug-ins,
extensions, add-ins, or add-ons, or combinations thereof.
Web Application
[0110] In some embodiments, a computer program includes a web
application. In light of the disclosure provided herein, those of
skill in the art will recognize that a web application, in various
embodiments, utilizes one or more software frameworks and one or
more database systems. In some embodiments, a web application is
created upon a software framework such as Microsoft.RTM. .NET or
Ruby on Rails (RoR). In some embodiments, a web application
utilizes one or more database systems including, by way of
non-limiting examples, relational, non-relational, object oriented,
associative, and XML database systems. In further embodiments,
suitable relational database systems include, by way of
non-limiting examples, Microsoft.RTM. SQL Server, mySQL.TM. and
Oracle.RTM.. Those of skill in the art will also recognize that a
web application, in various embodiments, is written in one or more
versions of one or more languages. A web application may be written
in one or more markup languages, presentation definition languages,
client-side scripting languages, server-side coding languages,
database query languages, or combinations thereof. In some
embodiments, a web application is written to some extent in a
markup language such as Hypertext Markup Language (HTML),
Extensible Hypertext Markup Language (XHTML), or eXtensible Markup
Language (XML). In some embodiments, a web application is written
to some extent in a presentation definition language such as
Cascading Style Sheets (CSS). In some embodiments, a web
application is written to some extent in a client-side scripting
language such as Asynchronous Javascript and XML (AJAX), Flash.RTM.
Actionscript, Javascript, or Silverlight.RTM.. In some embodiments,
a web application is written to some extent in a server-side coding
language such as Active Server Pages (ASP), ColdFusion.RTM., Perl,
Java.TM., JavaServer Pages (JSP), Hypertext Preprocessor (PHP),
Python.TM., Ruby, Tcl, Smalltalk, WebDNA.RTM., or Groovy. In some
embodiments, a web application is written to some extent in a
database query language such as Structured Query Language (SQL). In
some embodiments, a web application integrates enterprise server
products such as IBM.RTM. Lotus Domino.RTM.. A web application for
providing a career development network for artists that allows
artists to upload information and media files, in some embodiments,
includes a media player element. In various further embodiments, a
media player element utilizes one or more of many suitable
multimedia technologies including, by way of non-limiting examples,
Adobe.RTM. Flash.RTM., HTML 5, Apple.RTM. QuickTime.RTM.,
Microsoft.RTM. Silverlight.RTM., Java.TM. and Unity.RTM..
Mobile Application
[0111] In some embodiments, a computer program includes a mobile
application provided to a mobile digital processing device. In some
embodiments, the mobile application is provided to a mobile digital
processing device at the time it is manufactured. In other
embodiments, the mobile application is provided to a mobile digital
processing device via the computer network described herein.
[0112] In view of the disclosure provided herein, a mobile
application is created by techniques known to those of skill in the
art using hardware, languages, and development environments known
to the art. Those of skill in the art will recognize that mobile
applications are written in several languages. Suitable programming
languages include, by way of non-limiting examples, C, C++, C#,
Objective-C, Java.TM., Javascript, Pascal, Object Pascal,
Python.TM., Ruby, VB.NET, WML, and XHTML/HTML with or without CSS,
or combinations thereof.
[0113] Suitable mobile application development environments are
available from several sources. Commercially available development
environments include, by way of non-limiting examples, AirplaySDK,
alcheMo, Appcelerator.RTM., Celsius, Bedrock, Flash Lite, .NET
Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other
development environments are available without cost including, by
way of non-limiting examples, Lazarus, MobiFlex, MoSync, and
Phonegap. Also, mobile device manufacturers distribute software
developer kits including, by way of non-limiting examples, iPhone
and iPad (iOS) SDK, Android.TM. SDK, BlackBerry.RTM. SDK, BREW SDK,
Palm.RTM. OS SDK, Symbian SDK, webOS SDK, and Windows.RTM. Mobile
SDK.
[0114] Those of skill in the art will recognize that several
commercial forums are available for distribution of mobile
applications including, by way of non-limiting examples, Apple.RTM.
App Store, Android.TM. Market, BlackBerry.RTM. App World, App Store
for Palm devices, App Catalog for webOS, Windows.RTM. Marketplace
for Mobile, Ovi Store for Nokia.RTM. devices, Samsung.RTM. Apps,
and Nintendo.RTM. DSi Shop.
Standalone Application
[0115] In some embodiments, a computer program includes a
standalone application, which is a program that is run as an
independent computer process, not an add-on to an existing process,
e.g., not a plug-in. Those of skill in the art will recognize that
standalone applications are often compiled. A compiler is a
computer program(s) that transforms source code written in a
programming language into binary object code such as assembly
language or machine code. Suitable compiled programming languages
include, by way of non-limiting examples, C, C++, Objective-C,
COBOL, Delphi, Eiffel, Java.TM., Lisp, Python.TM., Visual Basic,
and VB .NET, or combinations thereof. Compilation is often
performed, at least in part, to create an executable program. In
some embodiments, a computer program includes one or more
executable complied applications.
Software Modules
[0116] The methods, systems, and software disclosed herein include,
in various embodiments, software, server, and/or database modules,
or use of the same. In view of the disclosure provided herein,
software modules are created by techniques known to those of skill
in the art using machines, software, and languages known to the
art. The software modules disclosed herein are implemented in a
multitude of ways. In various embodiments, a software module
comprises a file, a section of code, a programming object, a
programming structure, or combinations thereof. In further various
embodiments, a software module comprises a plurality of files, a
plurality of sections of code, a plurality of programming objects,
a plurality of programming structures, or combinations thereof. In
various embodiments, the one or more software modules comprise, by
way of non-limiting examples, a web application, a mobile
application, and a standalone application. In some embodiments,
software modules are in one computer program or application. In
other embodiments, software modules are in more than one computer
program or application. In some embodiments, software modules are
hosted on one machine. In other embodiments, software modules are
hosted on more than one machine. In further embodiments, software
modules are hosted on cloud computing platforms. In some
embodiments, software modules are hosted on one or more machines in
one location. In other embodiments, software modules are hosted on
one or more machines in more than one location.
Databases
[0117] In some embodiments, the methods, systems, and software
disclosed herein include one or more databases, or use of the same.
In view of the disclosure provided herein, those of skill in the
art will recognize that many databases are suitable for storage and
retrieval of metagenomic information (including metagenomic
profiles), metatranscriptome information (including
metatranscriptome profiles), and multiplex profiles. In various
embodiments, suitable databases include, by way of non-limiting
examples, relational databases, non-relational databases, object
oriented databases, object databases, entity-relationship model
databases, associative databases, and XML databases. In some
embodiments, a database is internet-based. In further embodiments,
a database is web-based. In still further embodiments, a database
is cloud computing-based. In other embodiments, a database is based
on one or more local storage devices.
[0118] In order that those skilled in the art may be better able to
practice the compositions and methods described herein, the
following example is given for illustration purposes.
EXAMPLES
Example 1
Assessment of Nipple Aspirate Fluid
[0119] This trial was a single-center study involving three (3)
healthy, non-pregnant, non-lactating female subjects. Subjects were
enrolled in the order of appearance at the clinic.
[0120] The primary trial objective was to determine the percentage
of women from age 30 to 65 that produces ductal fluid, as
determined by the presence of protein on the nitrocellulose filter
when using the MASCT.TM. device.
[0121] A secondary objective was to evaluate the nipple aspirate
fluid cytologically for the presence and type of cells (if
any).
[0122] Abbreviations
[0123] Abbreviations used herein include, for example, MAF: Mammary
Aspiration Fluid; MASCT.TM.: Mammary Aspiration Specimen Cytology
Test; NA: Not Available; ND: Not Done; NR: Not Recorded; and NAF:
Nipple Aspirate Fluid.
[0124] Methodology:
[0125] Briefly, a tared nitrocellulose filter was used to collect
ductal fluid by just touching it to each nipple (one for each
breast). Next, mammary fluid samples were aspirated using the
MASCT.TM. device with a tared sample collection unit. Both sets of
nitrocellulose filters were tested for protein using a staining
technique described below. Cells collected from washing the filters
containing nipple aspirate fluid specimens underwent cytological
examination.
[0126] Assessment:
[0127] The primary endpoint of the trial was the percentage of
women completing the trial that produce ductal fluid, as determined
by the presence of protein on the nitrocellulose filter when using
the MASCT.TM. device.
[0128] The secondary endpoint was the presence of cells in the
nipple aspirate fluid as determined by cytologic evaluation.
[0129] Results:
[0130] With regard to the protein testing done of the filters
obtained from these 3 subjects, none on the nipple wash filter
samples showed the presence of protein. All filters from the
MASCT.TM. device showed that protein was detected on the device
filter.
Overall Study Design and Plan
[0131] The MASCT.TM. device had been previously cleared for
marketing via the 510(k) regulatory pathway. This clinical study
was designed to test modifications to the MASCT.TM. device that
were made to enhance efficacy and usability and the ability to
detect protein in nipple aspirate fluid from women, including those
previously thought to be non-secreters. The clinical utility of
nipple aspirate fluid for helping in breast health management has
been hampered over the last 50 years by the current methodology of
collecting and measuring the presence of fluid. In fact, with
current technology up to 50% of all women are non-secretors, that
is, they are judged to not produce NAF.
[0132] This was a single-center study involving the enrollment of
up to 50 healthy non-pregnant, non-lactating female subjects.
Subjects were enrolled in the order of appearance at the
clinic.
[0133] Prior to entering the study, the investigator or designated
assistant explained to each subject, the nature of the study, its
purpose, procedures, expected duration, available alternatives, and
the benefits and risks involved in study participation. Each
subject was given a consent document and had the opportunity to ask
questions; and was informed of her right to withdraw from the study
at any time without prejudice. After this explanation, and before
any study-specific procedures are performed, the subject
voluntarily signed and dated an informed consent statement. Prior
to participation in the study, each subject received a copy of the
signed and dated written informed consent form and any other
written information.
[0134] Inclusion/Exclusion Criteria Review and Pregnancy
Evaluation
[0135] When necessary, each patient underwent a urine pregnancy
test prior to further participation in the study. A positive
pregnancy test would exclude the subject from participation. All
inclusion and exclusion criteria were reviewed to ensure subject
eligibility. After eligibility was established, a unique subject
identification number was assigned.
[0136] Demographics and Medical History
[0137] The following demographic and medical history was obtained
from each subject: age and ethnic origin; family medical history,
especially mother and sisters; personal medical history, including
breast cancer, benign breast conditions, and reproductive diseases
(for example, ovarian or endometrial tumors); concomitant
medications; age of menarche; age at first pregnancy; age at first
live birth; age of menopause; and height and weight.
[0138] Breast Preparation
[0139] Subjects were placed in a recumbent position. The nipple and
peri-aureolar areas of both breasts were cleansed with alcohol to
remove excess skin oils, cosmetics or epithelial debris. After the
alcohol evaporated, a warm moist compress was placed on both
breasts for 10 to 15 minutes. The compresses were removed and the
subjects were placed in a seated position. Alcohol was used to wipe
the nipple area to remove any ductal plugs that were present.
[0140] Nipple Touch Procedure
[0141] All persons handling filter materials shall wear gloves and
a protective mask to minimize the risk of filter contamination.
[0142] Device Cleaning Procedure
[0143] Prior to each subject use, the MASCT.TM. device was
thoroughly cleaned with an antimicrobial solution such as
CIDEX.RTM.. The device was not exposed to extreme temperatures or
autoclaved. The device was inspected periodically for deterioration
of the materials of the device or failure to induce negative
pressure. If either condition was observed, the unit was
replaced.
[0144] MASCT.TM. and Nipple Touch Procedures
[0145] a. Label one filter disk assembly `left` and a second filter
disk assembly `right`.
[0146] b. Weigh and record the weights of each assembly taking care
to not touch the filter with a bare hand, and using forceps as
needed.
[0147] c. Insert one assembly into the breast pump device.
[0148] d. Instruct the subject to perform manual self-breast
massage beginning with the chest wall and moving gradually to the
nipple-aureolar complex for approximately one minute.
[0149] e. The subject will then compress her breast with both hands
while the breast pump device is actuated for 60-90 seconds by the
physician or nurse practitioner.
[0150] f. Remove the filter disk assembly and weigh the MASCT.TM.
filter disks for fluid collection.
[0151] g. Using an antimicrobial solution such as CIDEX.RTM., wipe
the surfaces of the sample collection funnel and rinse out the
collection vial.
[0152] h. Repeat steps c, d, e, f and g for the second breast.
[0153] i. Store the filter disks in a refrigerator for subsequent
evaluation of protein and/or cytology evaluation. The packaging for
each filter disk assembly shall be appropriately labeled with
subject ID number and date of collection.
[0154] Variations to this collection procedure may be conducted,
but at no time will the number of massage/collection procedures
exceed that allowed in the approved labeling for the MASCT.TM.
device.
[0155] Subject Observation
[0156] Subjects will remain in the clinic and be observed for
adverse device effects for 30 minutes following aspiration. If
there are no evolving or unresolved adverse effects at that time,
the subjects will be released. At the investigator's discretion,
subjects experiencing any adverse effects at the end of this
observation period will remain at the study site until either the
effect resolves or follow-up arrangements have been made.
Conduct of the Study
[0157] All subjects in this study were studied according to the
protocol described herein.
[0158] Three (3) subjects were enrolled and the disposition of
patients is provided in the table below.
TABLE-US-00001 SUBJECT DISPOSITION BY SUBJECT Subject number
Enrolled Completed Study B01 Yes Yes B02 Yes Yes B03 Yes Yes
[0159] The following table contains individual patient data
listings for demographics. There were no deaths or serious adverse
events.
TABLE-US-00002 SUBJECT DEMOGRAPHICS Active Age Mean 50 Std. Dev.
11.79 Median 53 Range 37-60 Weight (kg) Mean 135.5 Std. Dev. 20.5
Median 135.5 Range 115-156 Height (cm) Mean 64.5 Std. Dev. 3.04
Median 66 Range 61-66.5 BMI Mean 23.03 Std. Dev. 4.1 Median 25.2
Range 18.3-25.6 N % Gender Female 3 100 Ethnicity Caucasian 3
100
Discussion and Overall Conclusions
[0160] The following results were obtained upon testing of the
individual filters used in this study.
Nipple Wash Results
[0161] None of the control nipple wash filter samples demonstrated
any presence of protein.
Breast Pump Results
TABLE-US-00003 [0162] Subject Breast Concentration of protein
Subject # 1 Left Breast 30 ng of protein Subject # 1 Right Breast
868 pg of protein Subject # 2 Left Breast 580 pg of protein Subject
# 2 Right Breast 71 pg of protein Subject # 3 Left Breast 7.1 ng of
protein Subject # 3 Right Breast 17.6 ng of protein
[0163] Based on these results, it was determined that the MASCT.TM.
device obtained nipple aspirate fluid as evidenced by the presence
of protein.
Deaths or Serious Adverse Events
[0164] There were no deaths or serious adverse events.
Subsequent Data
[0165] In a study of 31 women aged 18-65, the method of this
disclosure was used to analyze for protein and it was detected in
all 31 women (one woman had protein detected in only one breast)
giving a clinical utility of 97%. This improvement over the prior
art makes this test useful for identifying women with early,
precancerous changes when cancer prevention measures can be
instituted.
A Summary of the Efficacy Evaluation
[0166] The primary objective of the study was achieved as
determined by the presence of protein on all nitrocellulose filters
obtained when using the MASCT.TM. device. The secondary objective
of evaluating nipple aspirated fluid cytologically for the presence
and type of cells was accomplished. All samples were successfully
analyzed for cellular material.
Example 2
Cytology in Biological Samples from Mammary Fluid
[0167] This example describes the use of conventional cytological
techniques to identify and assess cells obtained from samples
described in Example 1. Following collection of the sample on the
filter paper, the filter is washed using any suitable buffered wash
solution (e.g., phosphate buffered saline) to collect cells, the
fluid may be further processed and cells may be collected by, for
example, centrifugation in a modified cytology vial. Processed
samples are then transferred to the central region of a clean glass
microscopic slide, and a cover slip is slid over the sample to
spread it along the surface of the slide. The slide is allowed to
air dry and then is fixed, for example in absolute alcohol, and
stained with standard cytological stains, such as methylene blue,
hematoxyln and eosin, or other suitable stain.
[0168] The slides are then examined by light microscopy for
evidence of atypical growth of cells and clumps of cells, using
well known methods, including those described in Diagnosis of
Non-Palpable Breast Lesions: Ultrasonographically Controlled
Fine-Needle Aspiration: Diagnostic and Prognostic Implications of
Cytology by Jacqueline Mouriquand, S. Karger Pub., July 1993;
Breast: Guides to Clinical Aspiration Biopsy by Tilde S. and Irwin
K. Kline, Igaku-Shoin Medical Pub., May 1988; Cytopathology of the
Breast (Asop Theory and Practice of Cytopathology by Shahla Masood,
American Society of Clinical Pathology, November 1995; Fine Needle
Aspiration Cytology and Its Clinical Applications: Breast and Lung
by Philip S. Feldman, American Society of Clinical Pathology,
November 1984; each incorporated herein by reference in its
entirety.
[0169] While preferred embodiments have been shown and described
herein, it will be obvious to those skilled in the art that such
embodiments are provided by way of example only. Numerous
variations, changes, and substitutions will now occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
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