U.S. patent application number 10/208364 was filed with the patent office on 2003-04-03 for method and apparatus for measuring factors in mammary fluids.
Invention is credited to Covington, Chandice.
Application Number | 20030065277 10/208364 |
Document ID | / |
Family ID | 22369400 |
Filed Date | 2003-04-03 |
United States Patent
Application |
20030065277 |
Kind Code |
A1 |
Covington, Chandice |
April 3, 2003 |
Method and apparatus for measuring factors in mammary fluids
Abstract
The present invention relates generally to breast fluid
aspirators, and more specifically to an apparatus, a system, and a
method for determining the risk of breast disease in a biological
sample obtained for the breast by means of a breast fluid
aspirator.
Inventors: |
Covington, Chandice;
(Clinton Township, MI) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
22369400 |
Appl. No.: |
10/208364 |
Filed: |
July 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10208364 |
Jul 29, 2002 |
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09488905 |
Jan 21, 2000 |
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6471660 |
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60116815 |
Jan 21, 1999 |
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Current U.S.
Class: |
600/573 |
Current CPC
Class: |
G01N 33/53 20130101;
A61K 49/0004 20130101; G01N 33/57415 20130101; A61M 1/06 20130101;
G01N 33/82 20130101; A61B 10/0045 20130101; A61M 1/064 20140204;
G01N 33/5088 20130101 |
Class at
Publication: |
600/573 |
International
Class: |
A61B 005/00 |
Goverment Interests
[0002] This invention was made with government support under NIH
Grant #NR03142 awarded by the PHS. The government has certain
rights in the invention.
Claims
What is claimed is:
1. A method for detecting a biological factor in a fluid sample
obtained from a mammary gland, comprising the steps of:
non-invasively obtaining a mammary gland fluid from a subject
comprising warming the mammary gland; massaging the mammary gland;
extracting the mammary fluid from the nipple by expression or
aspiration; and detecting the biological factor in the mammary
gland fluid.
2. The method of claim 1, further comprising the steps of:
expressing the mammary fluid by placing thumb and first fingers
behind the nipple forming a C-hold; pushing the nipple into the
chest wall; and rolling thumb and fingers forward toward the
nipple.
3. The method of claim 1, further comprising the steps of: applying
a nipple aspirator unit to the nipple, the nipple aspirator
comprising: a nipple receiving unit having a nipple receiving end
and a vacuum attachment end; a vacuum source; a vacuum line having
a first and second end wherein the first end is connected to the
nipple receiving unit at the vacuum attachment end and the second
end is connected to the vacuum source such that the vacuum source
is in vacuum communication with the nipple receiving end of the
nipple receiving unit.
4. The method of claim 1, wherein the biological factor is selected
from the group consisting of a nucleic acid, a protein, a peptide,
a glycoprotein, a lipid and a biochemical product.
5. The method of claim 4, wherein the nucleic acid is DNA or
RNA.
6. The method of claim 4, wherein the biochemical product is
.beta.-carotene or a derivative thereof.
7. The method of claim 6, wherein the biochemical product is
detected by chromatography.
8. The method of claim 1, wherein the biological factor is detected
by employing a probe that specifically interacts with the
biological factor.
9. The method of claim 8, wherein the probe is selected from the
group consisting of a monoclonal antibody, a polyclonal antibody,
and a nucleic acid.
10. The method of claim 6, wherein the biochemical product is a
carotenoid.
11. The method of claim 1, wherein the step of massaging the
mammary gland comprises massaging the mammary gland from the chest
wall towards the areola or nipple.
12. The method of claim 1, wherein the subject is a mammal.
13. The method of claim 12, wherein the mammal is a human.
14. The method of claim 1, wherein the method further comprises
warming the mammary gland prior to massaging.
15. The method of claim 1, wherein the subject is administered
oxytocin prior to massaging the mammary gland.
16. The method of claim 3, wherein the nipple receiving unit has a
tubular shape with a nipple receiving end designed to receive a
nipple.
17. The method of claim 3, wherein the vacuum source is a
syringe.
18. The method of claim 3, wherein the vacuum source is a vacuum
pump.
19. The method of claim 3, wherein the vacuum source is a wall
vacuum.
20. The method of claim 3, wherein the nipple receiving unit
comprises an inner tube and an outer tube, wherein the inner tube
and the outer tube are coaxial.
21. The method of claim 3, the nipple aspirator unit further
comprising a breast shield having the nipple receiving unit
concentrically positioned in the breast shield.
22. The method of claim 21, wherein the breast shield further
comprises a vibrating element.
23. The method of claim 21, wherein the breast shield further
comprises a massaging unit.
24. The method of claim 1, further comprising applying a nipple
aspirator unit to the nipple, the nipple aspirator comprising: a
pliable mammary gland shield configured to fit snugly over the
mammary gland of the subject, the shield having a massaging element
configured to provide physical stimuli to the mammary gland; a
nipple receiving unit centered radially in the mammary gland
shield, wherein the nipple receiving unit comprises a tubular shape
with a nipple receiving end designed to receive a nipple and a
second vacuum attachment end for attachment to a vacuum line; a
vacuum line, the vacuum line having a first end and a second end,
the first end being connected to the nipple receiving unit; and a
vacuum source for creating a vacuum connected to the second end of
the vacuum line, wherein the vacuum source is in vacuum
communication with the nipple receiving end of the nipple receiving
unit.
25. The method of claim 24, wherein the massaging element is a
vibrating element.
26. The method of claim 24, wherein the massaging element delivers
a peristaltic action, wherein the mammary gland is massaged from
the chest wall to the nipple.
27. The method of claim 24, wherein the vacuum source is a
syringe.
28. The method of claim 24, wherein the vacuum source is a vacuum
pump.
29. The method of claim 24, wherein the vacuum source is a wall
vacuum.
30. The method of claim 1, wherein the mammary gland fluid is a
breast milk.
31. The method of claim 30, wherein the breast milk is a colostrum
breast milk.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from Provisional
Application Serial No. 60/116,815, filed Jan. 21, 1999, to which
application a priority claim is made under 35 U.S.C.
.sctn.119(e).
FIELD OF THE INVENTION
[0003] The present invention relates generally to breast fluid
aspirators, and more specifically to an apparatus, a system, and a
method for determining the risk of breast disease in a biological
sample obtained from the breast by means of a breast fluid
aspirator.
BACKGROUND OF THE INVENTION
[0004] Breast cancer is one of the leading causes of disease and
death in women, with greater than 90% of breast cancer originating
in the epithelial cells of the ducts of the breast. Early detection
and treatment of breast cancer has focused on improving prognosis
and increasing the survival rates. Renewed focus on prevention and
detection of breast cancer has lead to the use of numerous
biological indicators and methods of early detection of breast
cancer risk. Such indicators include numerous oncogenic
determinants, cytokines, angiogenic factors, proteins and nucleic
acids, as well as biochemical products and lipids.
[0005] Carotenoids and retinoids are naturally occurring substances
which contain extensively conjugated polyene chains. Carotenoids
have extensively conjugated systems of carbon-carbon double bonds
which give rise to their many varied and brilliant colors. Many
carotenoids and retinoids, are biologically active. For example,
certain hydrocarbon members of the carotenoid family (most notably,
.beta.-carotene, or pro-vitamin A, one of the most abundant
carotenoids in food) are sources of retinol (one form of vitamin
A). Carotenoids protect plants from photosensitized oxidative
damage, probably by deactivating singlet oxygen. Epidemiological
evidence indicates that carotenoid intake correlates inversely with
the incidence of some types of cancer (Peto et al, Nature, 1981,
290, 201-208). Carotenoids and retinoids have been shown to retard
the development of some experimentally induced animal tumors (N. I.
Krinsky, Actions of Carotenoids in Biological Systems, Annu. Rev.
Nutr, 13, 561-587 (1993); Matthews-Roth, Curr. Top. Nutr. Dis. (New
Prot. Roles Select. Nutr.), 1989, 22, 17-38; Pure Appl. Chem.,
1985, 57, 717-722). A number of dietary intervention studies are
being carried out to try to determine the efficacy of supplemental
.beta.-carotene as a non-toxic, dietary anti-carcinogen that can
effectively decrease cancer mortality. Recently, the possibility
has begun to be examined that .beta.-carotene may be associated
with decreased incidence of coronary heart disease. Recent clinical
data using related compounds (retinoids--retinoic acid, retinol,
and retinamides) have demonstrated a role in anti-cancer therapy,
both as a therapeutic and a preventive agent (cancers of the skin,
head and neck, lung and bladder, acute promyelocytic leukemia,
leukoplakia and myelodysplastic syndromes; D. L. Hill and C. J.
Grubs, Retinoids and Cancer Prevention, Annu. Rev. Nutr. 1992, 12,
161-181). Furthermore, .beta.-carotene has antioxidant properties
at the low oxygen pressures found in tissues (Burton and Ingold,
.beta.-Carotene: an unusual type of lipid antioxidant, Science,
1984, 224, 569-573).
[0006] The protective effect of lactation and dietary carotenoids
in breast cancer development has been reported (American Cancer
Society, 1996; Holmes, Hunter, & Willett, 1995, Stoll, 1996;
and Weisburger, 1991). However, little is known about the role of
lactation in influencing transport of carotenoids into the micro
environment of the breast.
SUMMARY OF THE INVENTION
[0007] In one embodiment, the invention provides a method for
detecting a biological factor in a fluid sample obtained from a
mammary gland, comprising non-invasively obtaining a mammary gland
fluid from a subject by warming the mammary gland; massaging the
mammary gland from the chest wall towards the nipple; extracting
the mammary fluid from the nipple by expression and/or aspiration
and detecting the biological factor in the mammary gland fluid.
[0008] In another embodiment, the invention provides a method of
determining a risk of a mammary gland disease in a subject
comprising non-invasively obtaining a mammary gland fluid from the
subject, comprising warming the mammary gland; massaging the
mammary gland from the chest wall towards the areola or nipple; and
aspirating the mammary fluid; quantifying the amount of a
biological factor in the mammary fluid; comparing an amount of the
biological factor to the amount of the biological factor in a
control sample, wherein the ratio of the biological factor in the
fluid to the control sample is indicative of the risk of mammary
gland disease.
[0009] In yet another embodiment, the invention provides a method
of determining the risk of breast cancer in a subject comprising
quantifying the amount of a carotenoid in a biological sample
obtained from a mammary gland compared to an amount of a carotenoid
in a control sample, wherein if a ratio of carotenoids in the
biological sample to the carotenoids in the control sample is less
than one the ratio is indicative of a risk of breast cancer.
[0010] In another embodiment, the present invention provides a
method for increasing the amount of carotenoids in a mammary gland,
comprising warming the mammary gland; massaging the mammary gland
from the chest wall towards the areola or nipple; and aspirating a
mammary fluid from the mammary gland.
[0011] In another embodiment, the invention provides a non-invasive
method for obtaining a biological sample from a mammary organ of a
subject, comprising massaging the mammary gland tissue from the
chest wall towards the nipple; placing the thumb and first fingers
behind the nipple forming a C-hold; pushing the nipple into the
chest wall; and rolling thumb and fingers forward toward the
nipple.
[0012] In yet another embodiment, the invention provides an
apparatus for collection of a biological sample from the mammary
gland of a subject, comprising a nipple receiving unit having a
tubular shape with a nipple receiving end designed to receive a
nipple and a second vacuum attachment end for attachment to a
vacuum line; a vacuum line having a first and a second end, wherein
the second end is attached to the vacuum source and the first end
is attached to the nipple receiving unit; and a vacuum source
wherein the vacuum source is in vacuum communication with the
nipple receiving end of the nipple receiving unit.
[0013] In another embodiment, the invention provides an apparatus
for collection of a biological sample from a mammary gland of a
subject, comprising a pliable mammary gland shield configured to
fit snugly over the mammary gland of the subject, the shield having
a massaging element configured to provide physical stimuli to the
mammary gland; a nipple receiving unit centered radially in the
mammary gland shield, wherein the nipple receiving unit comprises a
tubular shape with a nipple receiving end designed to receive a
nipple and a second vacuum attachment end for attachment to a
vacuum line; a vacuum line, the vacuum line having a first end and
a second end, the first end being connected to the nipple receiving
unit; a vacuum source for creating a vacuum connected to the second
end of the vacuum line, wherein the vacuum source is in vacuum
communication with the nipple receiving end of the nipple receiving
unit.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the anatomy of the human mammary gland.
[0015] FIG. 2A shows a massaging technique for massaging the
mammary gland.
[0016] FIG. 2B shows a "C-Hold" technique used to express mammary
fluids from the human breast.
[0017] FIG. 3 is a diagram of a nipple aspirator unit.
[0018] FIG. 4 is a diagram showing the nipple receiving unit 20 in
further detail.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention discloses a method and apparatus
useful in determining and affecting the risk of breast cancer in
mammals. During the physiological process of lactation, the fluid
micro environment of the mammary gland is in a process of fluid
synthesis and drainage. Changes in the mammary gland epithelium
lining the ducts of the mammary gland during differentiation and
growth, in the preparation for milk production, is thought to alter
the susceptabilty of the cells of the mammary gland to neoplastic
changes. During lactation, the cells of the mammary gland undergo a
flushing process, whereby renewal of fluids in the mammary gland
flushes potential carcinogens in the ducts as well as brings new
fluids in contact with the cells. In the absence of such a flushing
process, waste materials, including biochemical waste products and
carcinogenic agents, accumulate and concentrate in the cellular
tissue of the mammary gland. In addition, stasis in the fluid
environment of the mammary gland, which can specifically occur
during lactation failure or upon termination of breast feeding,
causes the cellular environment of the mammary gland tissue to
become alkaline. Such alkalinity has been demonstrated to result in
increased mitotic activity and cell proliferation.
[0020] As used herein, a "biological factor" is meant to include
any number of biological active cells, proteins, chemicals (e.g.,
carotenoids), lipids, growth factors, cytokines, nucleic acid
molecules (i.e., DNA or RNA). For example, mammary gland fluid may
contain whole mammary fluid, whole cells, cell fragments, cell
membranes, various liquids, cellular or other solid fractions of
the mammary fluid, proteins, glycoproteins, peptides, nucleic
acids, lipids and other biochemical factors. For example, and not
by way of limitation, proteins including HER2 (neu), a growth
factor receptor found within tumor cells indicative of an
aggressively growing tumor, Ki67, cyclin D1 and PCNA; antigens such
as, for example, carcinoembryonic antigen (CEA) and prostate
specific antigen (PSA); lipid molecules including, for example,
cholesterol, hormones, cholesterol oxides; growth factors
including, for example, members of the TGFP superfamily, TNF, and
EGF are all capable of detection using the apparatus, kits, and
methods of the invention. Tumor growth can be evaluated using a
number of growth factor and hormone markers (e.g., estrogen, EGF,
erbB-2, and TGF-.alpha.), receptors of autocrine or exocrine growth
factors and hormone (e.g., IGF and EGF receptors) as well as
angiogenic factors such as VEGF, PDGF and others.
[0021] As used herein, a "biological sample" is meant to include
tissue, serum, plasma, mammary gland fluid, milk, nipple aspirate
and colostrum. The biological sample comprises a biological factor.
Accordingly, a control or standard sample may be a biological
sample or a synthetic sample having a known amount of a biological
factor. As used herein, a "breast disease indicator" means any
protein, peptide, glycoprotein, lipid, glycolipid, proteolipid,
nucleic acid or other biochemical or molecular factor that is
uniquely indicative of a mammary gland tissue disease, such a cell
proliferative disorder or neoplasia. Such a breast disease
indicator is measurably increased or decreased in the mammary gland
tissue, such as the epithelial cells of the ducts of the mammary
gland, compared to a normal standard sample.
[0022] A representative subset of breast disease indicators include
breast cancer markers. Such breast cancer markers that are useful
within the methods of the invention are described in Porter-Jordan
et al., Hematol. Oncol. Clin. North Amer. 8:73-100, 1994; and
Greiner, Pharmaceutial Tech., May, 1993, pp. 28-44, each of which
is incorporated herein by reference. Also included within the scope
of the invention is a non-invasive method for the detection of
mammaglobin, a mammary-specific secretory protein, and mammaglobin
nucleic acids as disclosed in U.S. Pat. No. 5,855,889, the
disclosure of which is incorporated herein by reference.
[0023] In one embodiment, the invention provides a method for the
detection of a biological factor present in the mammary gland of a
subject. In a this embodiment, the biological factor is detected in
a mammary fluid obtained from the mammary gland. In another
embodiment, the invention provides a method and apparatus for the
detection of a mammary gland disease by detecting the presence or
absence of a biological factor in a mammary gland fluid. The method
and apparatus provide for a non-invasive technique for measuring a
biological factor representing a breast disease indicator. The
presence or absence of a breast disease indicator is indicative of
the presence of a mammary gland disease.
[0024] The inventors have found that during lactation, the level of
carotenoids in mammary gland tissue is about the same level as
found in the serum of a subject. Furthermore, the concentration of
carotenoids is inversely proportional to the amount of time
post-lactation or post-fluid expression. In other words, the amount
of carotenoids in the mammary gland tissue decreases following
termination of lactation or milk-expression. As carotenoid levels
decrease, the number of oxidative scavengers also decreases,
resulting in an increase in the susceptibility of the tissue to
oxidative damage and potentially neoplastic growth. The inventors
believe that by stimulating fluid secretion from the mammary gland,
in the absence of oxytocin administration, carotenoid levels are
replenished and increased in a mammary gland fluid. In some
embodiments, oxytocin may be administered to increase fluid
secretion. The oxytocin can be exogenous oxytocin such as oxytocin
present in a pharmaceutical composition that may be administered
nasally or by buccal administration (Dawood, Ylikorkala, &
Fuchs, American Journal of Obstetrics and Gynecology, 138, 20-24.
1980). Oxytocin has a specific influence on the myoepithelium cells
of the distal ducts (Gaitan et al., Endocrinology, 1967 September;
81(3):515-20) Accordingly, such exogenous oxytocin will help fluid
collection. Alternatively, transcutaneous electrical stimulation
may be used to induce endogenous oxytocin by stimulation of the
cutaneous branches of the 4th intercostal nerve (Sarhadi et al.,
1996; Sarhadi, Shaw-Dunn, & Soutar, Br J Plast Surg 1997
December, 50(8):668-70) and the nipple using TENS (Seoud et al., J
Reprod Med, 1993 June; 38(6):438-42).
[0025] In epidemiological studies designed to investigate the
protective effect of lactation and breast cancer risk, the
independent protective effect of lactation was supported for
premenopausal, but not postmenopausal women. Mammary involution at
menopause represents a significant epithelial regression and loss
followed by replacement of ductal and lobular structures by adipose
tissue (Forbes, 1986). These anatomical changes result in a
decreased blood flow and nutrient delivery. Using the methods and
apparatus of the present invention, it is possible to increase the
amount of carotenoids in the mammary gland and/or mammary fluids of
non-lactating subjects by stimulating and/or aspirating fluid
expressed from the nipples of the subject, thus simulating the
flushing process of a lactating or breast feeding subject. As
mentioned above, decrease in carotenoids in the mammary gland
tissue deceases the number of oxidative scavengers in the mammary
gland tissue. This decrease may increase the risk of oxidative
damage to the mammary gland tissue and thus increase the chance of
cell proliferative disorders, neoplasia, and cancer. To overcome
the decrease in carotenoids levels and increase the level of such
oxidative scavengers, the invention provides a method and apparatus
for stimulating fluid "turnover" in the mammary tissue by
stimulating and/or aspirating fluid from the mammary gland of a
non-lactating subject. Without being limited to any particular
theory, it is the result of the fluid turnover in the mammary
tissue that brings carotenoids from other tissues and/or the blood
stream of the subject into the mammary tissue. The invention
provides a non-invasive method for stimulating fluid turnover in
the mammary gland of a subject in order to increase blood flow and
nutrient delivery.
[0026] In another aspect, the invention provides a method for
determining the risk of breast cancer in a subject by detecting the
amount of carotenoids in mammary gland fluid of a subject. The
subject may be any mammal, but is preferably a human. A biological
sample obtained by stimulated secretion from the mammary gland of
the subject is measured to determine the level of a biological
factor (e.g., a carotenoid level). The level of the biological
factor in the sample is measured against a standard sample. In one
embodiment, the standard sample is the level of the biological
factor (e.g., carotenoid level) in the serum of the same
subject.
[0027] In particular, during or after aspiration of the mammary
fluid, a fluid sample is collected from the nipple of the mammary
gland. The mammary fluid can be collected in any number of ways
including, but not limited to, directly aspirating the mammary
fluid into a collection device and/or rinsing the nipple with a
buffer and collecting the rinse into a suitable collection
device.
[0028] Detection and/or quantification of a biological sample of
the invention can be performed in any number of ways depending upon
the type of biological factor being measured. Generally, a
biological sample collected according to the methods of the
invention is exposed to a probe that specifically interacts with a
biological factor to be measured (e.g., a breast disease
indicator). For example, where the biological factor or breast
disease indicator is a peptide, polypeptide or protein the methods
may utilize well known ELISA immunoassay, immunoprecipitation
assays, Western blots, dot blots and affinity purification assays
to name but a few. Where the biological factor is a nucleic acid,
techniques including, for example, hybridization assays at standard
or high stringency to detect DNA or RNA using suitable non-antibody
probes can be used (e.g., Northern blots, Southern blots, dot
blots). Alternatively, PCR may be use to amplify and then detect
DNA or RNA using techniques common in the art. Where the biological
factor to be measured is a lipid or biochemical compound, the
biological sample can be extracted using extraction techniques,
such as lipid extraction techniques, and the biological factor
detected or quantified by liquid chromatography, such as
High-Performance-Liquid-Chromatography (HPLC). Such chromatography
techniques are well known in the art and are particularly suited
for the detection of carotenoids, cholesterol, cholesterol
by-products, flavorins, prostaglandins, leukotrienes and hormones.
Whole cells or cellular debris present in the biological sample
(e.g., the mammary fluid) may also be analyzed to determine the
presence or absence of a disease or disorder. Standard cell culture
techniques may be used to culture, maintain or expand a population
of cells present in the sample. Cells present in the sample may be
analyzed by histological techniques, stains, or standard microscopy
techniques that can detect, for example, morphological
characteristics of cells obtained from the fluid sample.
[0029] The invention further provides methods wherein the
biological samples, such as mammary gland fluids, are obtained
non-invasively. By non-invasive is meant that non-surgical or
non-invasive techniques are used, such that the tissue of the
mammary gland or mammary tissue is not penetrated by needles or
other devices.
[0030] To non-invasively obtain the biological sample from the
mammary gland and/or increase fluid turnover in the mammary gland,
the inventors have developed a method whereby a lactating subject
and/or a non-lactating subject can obtain mammary gland fluids from
the mammary gland. The method can be done manually or by utilizing
an apparatus of the invention, as described more fully below. In
one embodiment, the carotenoid levels in mammary gland fluid are
increased using the following procedures:
[0031] Cleansing: The mammary gland and/or nipple area are
preferably cleansed in a manner designed to remove keratin plugs
that may be blocking duct openings. For example, the mammary gland
and/or nipple may be cleansed with a detergent, or baking soda and
water.
[0032] Warming: The mammary gland is warmed sufficiently to
increase fluid flow from the gland. Warming of the mammary gland
can be done in any number of ways including, but not limited to,
warming with a heating blanket, heating bag, warmed wash cloth or
compresses, heating bottle, and other methods known in the art.
Without being limited to any particular theory, such warming may
result in the opening of pores and ducts in the mammary gland as
well as dilation of capillaries in the mammary tissue, thus
increasing fluid flow from the mammary gland.
[0033] Massaging: The mammary gland is massaged to promote or cause
fluid expression. Massaging of the mammary gland can be performed
in any number of ways so long as there is a physical stimulation to
the tissue of the breast. The stimulation maybe provided by a
device or apparatus designed to deliver a stimulatory action, or an
appendage, hand or digit of the subject. For example, the tissue
may be massaged by using a vibration device such as the apparatus
below or by other vibration devices easily identifiable by those
skilled in the art, by physical manipulation using a device or by
physical manipulation using a hand. In a one embodiment, the tissue
of the breast is massaged from the chest wall towards the nipple of
the mammary gland. The method includes gently massaging the breast
tissue around the chest wall with one hand while supporting the
breast with the other hand. Massaging is generally in the direction
from the chest wall towards the areola and nipple. This technique
uses a slight shaking or vibrating movement with the fingers, while
at the same time moving around the breast gradually until the
entire breast is massaged (FIG. 2A).
[0034] Expression/Aspiration: Fluid from the mammary gland is
extracted by expression and/or aspirations. Expression or
aspiration of mammary fluid can also be performed in any number of
ways. For example, a suction device which engages the mammary
gland, preferably the nipple, may be used to create a suction to
assist in aspirating mammary fluid from the ducts of the mammary
gland. In one embodiment, a nipple receiving unit attached to a
vacuum line (e.g., a tube or hose) is used to aspirate mammary
fluid. In a one embodiment, the nipple receiving unit is attached
to a first end of the vacuum line and a syringe is attached to a
second end of the vacuum line, wherein withdrawal of the plunger of
the syringe delivers a partial vacuum to the nipple receiving unit.
The nipple receiving unit may be of a coaxial design having an
internal cylindrical shape for receiving the nipple at a first end
and providing a vacuum at the second end. The outer cylindrical
shape providing support.
[0035] The vacuum line can be made of any material which does not
collapse under vacuum force, for example, latex tubing. The nipple
receiving unit can be made out of any material sufficiently rigid
to provide for resilience under vacuum. Preferably, the material
can withstand repeated cleanings using detergents, heat, and or
sterile gas, (for example, plexiglass or polymer materials).
[0036] To aspirate mammary fluid using the nipple aspirator unit,
the inner cylinder of the nipple receiving unit is centered over
the nipple of the subject, and held in place. A partial vacuum is
then applied to the nipple receiving unit for about 30 seconds and
released. A negative (i.e., vacuum) pressure of about -150 mm/Hg to
about -300 mm/Hg, typically about -240 mm/Hg is applied to the
nipple. In one embodiment, the plunger of a syringe attached to a
vacuum line which is in-turn attached to a nipple receiving unit is
withdrawn sufficiently to create a partial vacuum (e.g., about 10
cc of pull) for about 30 seconds.
[0037] Alternatively, expression or aspiration of mammary fluid can
be performed by manipulation of the mammary gland and the nipple by
holding the breast in a "C-hold" by positioning the thumb and first
fingers about 1 to 1.5 inches behind the nipple, with the thumb
above the nipple and the fingers below. The breast is then pushed
into the chest wall and the thumb and fingers rolled forward
towards the nipple (see FIG. 2B). If this method is performed,
squeezing the mammary gland should be avoided to prevent bruising
of the mammary tissue. Either of these techniques are continued for
a sufficient amount of time until visualization of mammary
fluid.
[0038] Once nipple aspirate or mammary fluid appears, the fluid can
be collected in any number of ways including, but not limited to,
directly aspirating the mammary fluid into a collection device
and/or rinsing the nipple with a buffer and collecting the rinse
into a suitable collection device. Suitable collection devices
include, for example, a microscope slide, a filter, matrix, or
vessel.
[0039] The present invention further provides an apparatus useful
in obtaining mammary fluids. Reference to FIG. 3, is a diagram of a
nipple aspirator unit 10 having a vacuum line 30 with a first end
31 and a second end 32, wherein first end 31 is attached to and in
vacuum communication with a nipple receiving unit 20. The vacuum
line 30 is attached at its second end 32 to a vacuum source 40.
[0040] FIG. 4 shows the nipple receiving unit 20 in further detail.
The nipple receiving unit 20 is substantially tubular and of a
solid material having at its first end a connector 25 for
connecting to a vacuum source and a second end 26 designed to fit
and receive a nipple making an air tight seal. The nipple receiving
unit may be further attached to a breast shield 27. The breast
sheild 27 can additionally have numerous features, for example and
not by way of limitation, a massaging element, a vibrating element
or any number of features designed to deliver a physical stimulus
to the mammary gland.
[0041] In addition to the methods and apparatus described above,
the invention further provides kits comprising reagents and
components for practicing the methods and assay techniques of the
invention. Such a kit may also include a carrier means being
compartmentalized to receive in close confinement one or more
containers such as vials, tubes, and the like, each of the
containers comprising one of the separate elements to be used in
the method. For example, the kits may contain components for
obtaining a biological sample, which components include a nipple
aspirator unit, a vacuum line; and a collecting device for
collecting the biological sample, which collection device may range
from a simple fluid reservoir to solid phase media for use in a
solid phase assay system.
[0042] In more detailed embodiments, the kits include reagents
and/or devices for detecting the presence of a biological factor in
a biological sample obtained non-invasively from a mammary gland.
The kits may include, for example, buffers, preservatives, and
probes. Such probes may include a monoclonal antibody, polyclonal
antibody, a nucleic acid, or enzyme. Such probes can be attached to
a solid substrate.
[0043] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only and are not intended to
limit the scope of the invention.
EXAMPLES
[0044] Women between the ages of 18 and 45 yeas of age and
non-pregnant were recruited for participation the study. Criteria
for inclusion for the breast-feeding group included delivery of
full term infant 2 months prior and that the child was as lest six
months and totally weaned from breast feeding for at least three
months. After cessation of lactation, mammary involution occurs
over a three-month period, resulting in a return to a non-secretory
alveolar lining. Women participating in this study were minimally
three-months post-wean to control of the physiologic changes that
occur during lactation and subsequent to cessation of lactation.
Criteria for inclusion for the non-breast-feeding group included
delivery of a full-term infant 24 months prior and selecting of
formula feeding the infant. Exclusion criteria or withdrawal from
the study included pregnancy.
[0045] Recruitment of subjects occurred in three-Midwest
metropolitan cities through subject referral, study recruitment
fliers at women' health clinics, WIC offices, daycare centers, and
public health clinics, and advertisements in the newspaper. Over
100 women were recruited to participate in the study, however, only
85 women met the inclusion criteria of the study. Of these, 43
women were able to express breast fluids sufficiently and had
plasma carotenoid available for analysis.
[0046] A nipple aspiration device was designed to assist in the
aspiration of breast fluid. A kit containing microtubes and the
aspiration device necessary for collection of breast fluid was
developed. A breast fluid sample collection protocol using a nipple
aspiration method to obtain breast fluids over a period of 17 days
was used. Generally, the method included washing the nipple area
with a detergent, such as baking soda and water to remove keratin
plugs. The breast is then warmed to increase fluid and blood flow
to the breast by warming the breast with a warm towel or taking a
warm shower. The breast is then massaged around the chest wall with
one hand while supporting the breast with the other hand, massaging
from the chest wall towards the areola and nipple area using a
slight shaking movement with the fingers, this is performed until
the entire breast is massaged. Fluid was expressed by positioning
the thumb and first two fingers about 1 to 1.5 inches behind the
nipple, pushing straight into the chest wall, rolling the thumb and
fingers forward and repeating the method until sufficient fluid was
expressed. A typical protocol is as follows:
[0047] Starting one week prior to the scheduled visit, the subject
will prepare breasts daily as follows: 1) Use a towel following
shower/bath to gently cleanse the nipples, removing traces of dry
skin daily; 2) Apply a solution of 1/8 teaspoon of baking soda/1/2
cup of water to the nipple for 10-20 minutes covering each with a
breast pad and securing in place with a bra. After 10-20 minutes,
the solution is washed off and any dry skin is removed. The subject
will then use of a small amount of HEB crme (hydro-emollient base
pharmaceutical creme) to the nipple. The subject then expresses
fluid from the nipple by using the C-hold technique, a nipple
aspiration device or a combination of both.
[0048] This is typically performed in the absence of oxytocin,
however, where there appears to be difficulty in expressing fluids,
exogenous oxytocin may be administered. Alternatively, endogenous
oxytocin stimulation may be achieved by electrical stimulation as
described above. Any fluid the is expressed is collect in
microtubes. All collection tubes will be labeled to coincide with
the step and breast used for aspiration. A 10 .mu.l sample from
each breast will be placed in a cytology micro-tube with
Preserv-Cyt, refrigerated for cytology studies. The remaining
fluids and serum are placed in amber micro-tubes. The serum is
centrifuged, divided, and prepared for distribution, stored at -70
C. and delivered within 48 hours for carotenoid and tocopherol
assays.
[0049] Some women were provided nipple aspiration device for
obtaining mammary fluid. These women were instructed to place the
nipple aspirator portion of the nipple aspirator centered over the
nipple and pulling the plunger of the syringe to the 10 cc mark and
holding to a count of 30. If difficulty was found in aspirating
fluid, the women were instructed to use a larger syringe and/or
pull back the plunger an additional 10 cc (i.e., 20 cc) for a count
of 30. As fluid appeared at the nipple, the fluid was collected
into a capillary tube and aliquoted into microtainers. Fluid was
stored in amber opaque microtainers to shield against light which
degrades carotenoids. Blood sampling equipment, including alcohol,
butterfly needles, and collection tubes containing EDTA and sodium
heparin were used to collect blood for carotenoid assay. Plasma was
stored in amber opaque microtainers.
[0050] Plasma and breast fluid samples were collected in amber
microtubes until assay. Blood was centrifuged immediately after
collection and the plasma withdrawn and stored in amber capillary
tubes. Carotenoid levels in both plasma and breast fluids were
determined by spectrophotometric techniques as described by Patton
et al. (1980).
[0051] Mammary fluid was collected every other day for 17 days. If
a participant was unable to collect fluid using the aspirator
within 30 minutes of the attempt, the subject was instructed to
stop and try again in two days. The first eight collections of
breast fluid were pooled into an microtube, protected from the
light, and maintained in the freezer between collections. An
initial blood sample was collected into a tube containing EDTA.
[0052] Eighty-one samples were analyzed for carotenoid level. The
mean plasma carotenoid level was 1.83 mcg/ml (SD=0.89, range
0.037-4.50 mcg/ml). The mean plasma carotenoid level reported in
this study is consistent with levels reported in epidemiologic
studies measuring plasma carotenoid (Potischman, 1990). The total
number of women with both plasma and mammary fluid carotenoid
levels numbered 43. The overall mean breast fluid carotenoid level
was 1.73 mcg/ml (SD--1.7, range 0-10.20 mcg/ml). This finding was
consistent with the mean breast fluid carotenoid level (1.94
mcg/ml) reported by Covington et al. (1998) for women post
weaned.
[0053] Age significantly influenced the relation between plasma and
breast fluid carotenoid levels, as illustrated in Table 1. A
significant negative partial regression coefficient for plasma and
age indicated a significant negative interaction. Thus the relation
between plasma and mammary fluid carotenoid levels diminished with
age. The results of the hierarchical regression did not support the
influence of parity (See Table 2). The influence of length of time
post-wean on the relation between plasma and breast fluid
carotenoids levels was supported (See Table 3). Thus the relation
between plasma and breast fluid carotenoid levels diminished with
the increasing duration of time since cessation of lactation.
[0054] Table 1
[0055] Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Age
1 Variable .beta. .DELTA.R.sup.2 p Step 1 -.37 .13* .02 Plasma
Carotenoid Step 2 .21 .04 .17 Age Step 3 -.33 .10 .03.dagger.
Plasma Carotenoid .times. Age Note *R.sup.2; .dagger.partial =
-.35
[0056]
2TABLE 2 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Parity Variable .beta. .DELTA.R.sup.2 p Step
1 -.37 .13* .02 Parity Step 3 -.11 .01 .47 Plasma Carotenoid
.times. Parity Note *R.sup.2
[0057]
3TABLE 3 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Length of Time Post-Wean Variable .beta.
.DELTA.R.sup.2 p Step 1 -.39 .16* .03 Plasma Carotenoid Step 2 .18
.03 .36 Months Post-Wean Step 3 -.48 .21 .007.dagger. Plasmid
Carotenoid .times. Months Post-Wean Note *R.sup.2: .dagger.partial
= -.51
[0058] Hierarchical regression analysis with a one-tailed test of
significance was used to examiner the influence of lactation on the
relation between plasma and breast fluid carotenoid levels. As
illustrated in Table 4, the significant beta value in Step 3 of the
hierarchical regression analysis in addition to the significant
change in the R2 when the recent lactation entered the hierarchical
regression equation with plasma carotenoid, signified the presence
of an influence of recent lactation on the relation between plasma
and breast fluid carotenoid levels. A significant positive partial
regression coefficient for plasma carotenoid.times.lactation
indicated a significant positive interaction. Thus, the influence
of lactation on the relation between plasma and mammary fluid
carotenoid levels was supported. Additionally, the longer a woman
breast fed her last child, the greater the positive relation
between plasma and breast fluid carotenoid levels. (See Table
5).
4TABLE 4 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Lactation Variable .beta. .DELTA.R.sup.2 p
Step 1 -.37 .14* .008 Plasma Carotenoid Step 2 -.16 .03 .14
Lactation Step 3 .20 .04 .09.dagger. Plasma Carotenoid .times.
Lactation Note *R.sup.2; .dagger.partial = .21
[0059]
5TABLE 5 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Cumulative Lifetime Duration of Lactation
Variable .beta. .DELTA.R.sup.2 p Step 1 -.36 .13* .01 Plasma
Carotenoid Step 2 .07 .004 .34 Plasma Carotenoid .times. Cumulative
Duration of Lactation Note *R.sup.2 .times. Parity *R.sup.2;
.dagger.p = .04, partial = -.47
[0060]
6TABLE 6 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Lactation, Age, Parity, and Length of Time
Post-Wean Serving Together as Moderators Variable .beta.
.DELTA.R.sup.2 p Step 1 -.38 .15* .04 Plasma Carotenoid Step 2 .07
.78 Age .18 Lactation -.03 Months Post-Wean .15 Parity .002 Step 3
.31 .05 Plasma Carotenoid .times. -.59.dagger. Months Post-Wean
Plasma Carotenoid .times. -.40 Age Plasma Carotenoid .times. -.16
Lactation -.16 Plasma Carotenoid .31
[0061] A significant negative partial regression coefficient for
plasma carotenoid and length of time post-wean indicated a
significant negative interaction. Thus, in the model, length of
time post-wean diminished the relation between plasma and breast
fluid carotenoid levels and altered the influence of recent
lactation and age on the relation between plasma d breast fluid
carotenoid levels.
[0062] A negative partial regression coefficient for length of time
post-wean influenced the relation between plasma and breast fluid
carotenoid levels (See Table 7). A negative partial regression
coefficient for length of time-post wean indicated a significant
negative influence. This, the influence of cumulative lifetime
duration of lactation and parity was not altered, but age did not
diminish the relation between plasma and breast fluid carotenoid
levels as reported in separate analysis, while length of time
post-wean did significantly diminish the relation between plasma
and breast fluid carotenoid levels.
7TABLE 7 Hierarchical Regression For Breast Fluid Carotenoid on
Plasma Carotenoid and Cumulative Lifetime Duration of Lactation,
Age, Parity, and Length of Time Post-Wean Serving Together as
Moderators Variable .beta. .DELTA.R.sup.2 p Step 1 -.38 .15* .04
Plasma Carotenoid Step 2 .07 .77 Age .17 Cumulative Duration -.08
of Lactation Months Post Wean .14 Parity .06 Step 3 .32 .04
.fwdarw. Plasma Carotenoid .times. -.62.dagger. Months Post-Wean
.fwdarw. Plasma Carotenoid .times. -.40 Age .fwdarw. Plasma
Carotenoid .times. -.20 Cumulative Duration of Lactation .fwdarw.
Plasma Carotenoid .times. .37 Parity Note *R.sup.2; .dagger.p =
.05, partial = -.45
[0063] The results indicate that age influenced the relation
between plasma and breast fluid carotenoid levels, the relation
between plasma and breast fluid carotenoid levels diminishing as
age increased. This was true for all women, regardless of lactation
status. The American Cancer Society (1996) reports that the
incidence of breast cancer increases with age and breast cancer
mortality rates increase for women 40-54 years of age.
[0064] Parity did not significantly influence the relation between
plasma and breast fluid carotenoid levels. Furthermore, greater
length of time post-wean significantly reduced the relation between
plasma and breast fluid carotenoid levels. The results indicate
that the relation between plasma and breast fluid carotenoid levels
diminished the longer the time post-wean. Since the longer the time
post-wean indicates a longer period of time away from the positive
influence of lactation as a potentiating process. Additionally,
post-lactation, the breast undergoes mammary involution over a
three-month period of time, in which the number of mammary alveoli
are reduced and blood flow and nutrient delivery is diminished
(Lawrence, 1994, Neville & Niefert, 1983).
[0065] Lactation had a significant positive influence on the
relation between plasma and breast fluid carotenoid levels. The
results of previous epidemiologic studies on lactation and breast
cancer risk supports a protective effect, however, the mechanism is
unknown. Lactation, a physiologic process in which blood flow and
nutrient transport to the breast increase the delivery of available
circulating plasma carotenoids into the micro-environment of the
breast in women who breast-feed, especially at shorter periods
post-wean. Lactation positively influenced the relation between
plasma and breast fluid carotenoid levels. This positive influence
was detected at 15-35 months postpartum.
[0066] The foregoing description and examples of the invention are
exemplary for purposes of illustration and explanation. It should
be understood that various modifications can be made without
departing from the spirit and scope of the invention. Accordingly,
the following claims are intended to be interpreted to embrace all
such modifications.
* * * * *