U.S. patent application number 11/116961 was filed with the patent office on 2006-02-09 for devices and methods for obtaining mammary fluid samples for evaluating breast diseases, including cancer.
Invention is credited to Steven C. Quay.
Application Number | 20060030787 11/116961 |
Document ID | / |
Family ID | 26939122 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060030787 |
Kind Code |
A1 |
Quay; Steven C. |
February 9, 2006 |
Devices and methods for obtaining mammary fluid samples for
evaluating breast diseases, including cancer
Abstract
Biological samples of mammary fluid or components thereof are
obtained using a breast pump device coupled with a solid phase
sample collection medium, alternatively facilitated by
administering oxytocin to the subject. The breast pump device
stimulates expression of mammary fluid and provides for collection
of diagnostic samples to evaluate breast disease, including cancer.
The biological sample may include whole cells or cellular
components, purified or bulk proteins, glycoproteins, peptides,
nucleotides or other desired constituents comprising a breast
disease marker. Methods, kits and adapter devices relating to the
breast pump device are also provided. Yet additional methods,
devices, accessories, and materials are provided for laboratory
handling and processing of breast fluid samples and for related
diagnostic methods.
Inventors: |
Quay; Steven C.; (Edmonds,
WA) |
Correspondence
Address: |
Jeffrey J. King;GRAYBEAL JACKSON HALEY LLP
Suite 350
155 - 108th Avenue NE
Bellevue
WA
98004-5901
US
|
Family ID: |
26939122 |
Appl. No.: |
11/116961 |
Filed: |
April 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10002540 |
Nov 13, 2001 |
6887210 |
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11116961 |
Apr 27, 2005 |
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10404866 |
Mar 31, 2003 |
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11116961 |
Apr 27, 2005 |
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09435131 |
Nov 5, 1999 |
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10404866 |
Mar 31, 2003 |
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09027362 |
Feb 20, 1998 |
6287521 |
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09435131 |
Nov 5, 1999 |
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08709207 |
Aug 27, 1996 |
5798266 |
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09027362 |
Feb 20, 1998 |
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60248134 |
Nov 13, 2000 |
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60248136 |
Nov 13, 2000 |
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Current U.S.
Class: |
600/573 ;
435/287.2; 435/6.14 |
Current CPC
Class: |
A61M 1/066 20140204;
A61M 1/06 20130101; A61B 10/0045 20130101; A61B 10/0041 20130101;
A61B 10/0096 20130101; A61M 2205/075 20130101; A61B 5/14546
20130101; C12Q 1/6886 20130101; A61M 1/064 20140204 |
Class at
Publication: |
600/573 ;
435/287.2; 435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; A61B 5/00 20060101
A61B005/00 |
Claims
1-54. (canceled)
55. A sample collection device for collecting a biological sample
from a mammary organ of a subject, comprising: a breast engaging
member for establishing a fluid connection between the engaging
member and a nipple or alveolar duct of the subject; pressure
changing means connected with said breast engaging member for
changing pressure at a surface of the nipple or within the alveolar
duct to induce or facilitate breast fluid expression from the
nipple or alveolar duct; and a solid phase sample collection medium
in fluid connection with said breast engaging member interposed
between said breast engaging member and said pressure changing
means for receiving a sample of expressed breast fluid from the
nipple or alveolar duct, wherein said sample comprises one or more
breast disease marker(s) selected from the group consisting of a
protein, a peptide, a glycoprotein, a lipid, a glycolipid, a DNA
polynucleotide, an RNA polynucleotide, or a combination
thereof.
56. The sample collection device of claim 55, wherein said
biological sample comprises one or more breast disease marker(s)
selected from the group consisting of Her 2, Ki67 Growth Factor,
Cyclin BI, Cyclin D1, Proliferating Cell Nuclear Antigen,
Transforming Growth Factor a, Tissue Plasminogen Activator, Insulin
Growth Factor Receptors, Collagenase Type IV, Laminins, Laminin
Receptor, Integrins, p53, rb, nm23, ras, c-myc, c-myb, Heat Shock
Proteins, Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14,
Alpha-Lactalbumin, Actin, CEA, HMFG, MCA, PSA, Vasopressin,
Cathepsin D, PGE2, pS2; IL-10, S-100 protein; Vimentin; Epithelial
Membrane Antigen, bcl-2, CA15-3, CA 19-9, Tn Antigen,
Alpha-lactalbumin, LASA, Gal-GalNAC, GCDFP-15, Le(y)-Related
Carbohydrate Antigen, CA 125, uPA, uPA related antigens and
complexes, uPA Receptor, PA1-1 and PA1-2, Beta-glucuronidase, CD31,
CD44 splice variants, blood group antigens including ABH, Lewis,
and MN, and genetic lesions or altered expression levels of CCND1,
EMS1, BRCA1 and BRCA2 genes, and combinations thereof.
57. The sample collection device of claim 55, wherein said solid
phase sample collection medium is in fluid contact with said nipple
or alveolar duct when the breast engaging member is applied to the
breast and positive or negative pressure is generated by said
pressure changing means.
58. The sample collection device of claim 55, wherein said pressure
changing means is provided by said solid phase sample collection
medium which operates by wicking or capillary action in contact
with the nipple or alveolar duct to facilitate expression or
evacuation of the sample of expressed breast fluid there from.
59. The sample collection device of claim 55, wherein said solid
phase sample collection medium is removably placed in fluid
connection with a breast pump.
60. The sample collection device of claim 55, 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, resins, particulate
chromatographic media, plastic microparticles, latex particles,
coated tubes, coated templates, coated beads, coated matrices, and
combinations thereof.
61. The sample collection device of claim 55, wherein said
biological sample is selected from the group consisting of whole
mammary fluid, whole cells, cell fragments, cell membranes,
proteins, glycoproteins, peptides, nucleotide components of mammary
fluid, or a combination thereof.
62. The sample collection device of claim 102, wherein said
pressure changing means functions to induce or facilitate expulsion
of breast fluid from the nipple or alveolar duct independently of,
or in conjunction with, positive pressure generated within the
alveolar duct of the breast by spontaneous or oxytocin-induced
contraction, or manual compression, of the alveolar duct.
63. A non-invasive method for obtaining a biological sample from a
mammary organ of a subject, comprising the steps of: applying a
sample collection device for collecting said biological sample from
said subject comprising a breast engaging member for establishing a
fluid connection between the engaging member and a nipple or
alveolar duct of the subject, pressure changing means connected
with said breast engaging member for changing pressure at a surface
of the nipple or within the alveolar duct to induce or facilitate
breast fluid expression from the nipple or alveolar duct, and a
solid phase sample collection medium in fluid connection with said
breast engaging member for receiving a sample of expressed breast
fluid from the nipple or alveolar duct, wherein said sample
comprises one or more breast disease marker(s) selected from the
group consisting of a protein, a peptide, a glycoprotein, a lipid,
a glycolipid, a DNA polynucleotide, an RNA polynucleotide, or a
combination thereof; and concurrent or subsequent to expression of
said breast fluid, collecting the biological sample comprising the
expressed mammary fluid or a component thereof that includes one or
more breast disease marker(s) on or within the solid phase sample
collection medium.
64. The method of claim 63, wherein said one or more breast disease
marker(s) is/are selected from the group consisting of Her 2, Ki67
Growth Factor, Cyclin B1, Cyclin D1, Proliferating Cell Nuclear
Antigen, Transforming Growth Factor a, Tissue Plasminogen
Activator, Insulin Growth Factor Receptors, Collagenase Type IV,
Laminins, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc,
c-myb, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase,
IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, CEA, HMFG, MCA, PSA,
Vasopressin, Cathepsin D, PGE2, pS2; IL-10, S-100 protein;
Vimentin; Epithelial Membrane Antigen, bcl-2, CA15-3, CA 19-9, Tn
Antigen, Alpha-lactalbumin, LASA, Gal-GalNAC, GCDFP-15,
Le(y)-Related Carbohydrate Antigen, CA 125, uPA, uPA related
antigens and complexes, uPA Receptor, PAl-I and PA1-2,
Beta-glucuronidase, CD31, CD44 splice variants, blood group
antigens including ABH, Lewis, and MN, and genetic lesions or
altered expression levels of CCND1, EMS 1, BRCA1 and BRCA2 genes,
and combinations thereof.
65. The method of claim 63, wherein said solid phase sample
collection medium is in fluid contact with said nipple or alveolar
duct when the breast engaging member is applied to the breast and
positive or negative pressure is generated by said pressure
changing means.
66. The method of claim 63, wherein said pressure changing means is
provided by said solid phase sample collection medium which
operates by wicking or capillary action in contact with the nipple
or alveolar duct to facilitate expression or evacuation of the
sample of expressed breast fluid there from.
67. The method of claim 63, wherein said solid phase sample
collection medium is removably placed in fluid connection with a
breast pump.
68. The method of claim 63, 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, resins, particulate chromatographic
media, plastic microparticles, latex particles, coated tubes,
coated templates, coated beads, coated matrices, and combinations
thereof.
69. The method of claim 63, wherein said biological sample is
selected from the group consisting of whole mammary fluid, whole
cells, cell fragments, cell membranes, proteins, glycoproteins,
peptides, nucleotide components of mammary fluid, or a combination
thereof.
70. The method of claim 63, wherein said pressure changing means
functions to induce or facilitate expulsion of breast fluid from
the nipple or alveolar duct independently of, or in conjunction
with, positive pressure generated within the alveolar duct of the
breast by spontaneous or oxytocin-induced contraction, or manual
compression, of the alveolar duct.
71. A method for determining a presence or quantity of a breast
disease marker in a biological sample of mammary fluid, comprising
the steps of: applying a sample collection device for collecting
said biological sample from said subject comprising a breast
engaging member for establishing a fluid connection between the
engaging member and a nipple or alveolar duct of the subject,
pressure changing means connected with said breast engaging member
for changing pressure at a surface of the nipple or within the
alveolar duct to induce or facilitate breast fluid expression from
the nipple or alveolar duct, and a solid phase sample collection
medium in fluid connection with said breast engaging member for
receiving a sample of expressed breast fluid from the nipple or
alveolar duct; concurrent or subsequent to expression of said
breast fluid, collecting the biological sample comprising the
expressed mammary fluid or a component thereof that includes one or
more breast disease marker(s) on or within the solid phase sample
collection medium; and detecting a presence or quantity of said
breast disease marker in said sample.
72. The method of claim 71, wherein said breast disease marker is a
breast cancer marker.
73. The method of claim 71, wherein said breast disease marker is
from the group consisting of Her 2, Ki67 Growth Factor, Cyclin B1,
Cyclin D1, Proliferating Cell Nuclear Antigen, Transforming Growth
Factor a, Tissue Plasminogen Activator, Insulin Growth Factor
Receptors, Collagenase Type IV, Laminins, Laminin Receptor,
Integrins, p53, rb, nm23, ras, c-myc, c-myb, Heat Shock Proteins,
Prolactin, Neuron-Specific Enolase, IR-14, KA 1, KA 14,
Alpha-Lactalbumin, Actin, CEA, HMFG, MCA, PSA, Vasopressin,
Cathepsin D, PGE2, pS2; IL-10, S-100 protein; Vimentin; Epithelial
Membrane Antigen, bcl-2, CA15-3, CA 19-9, Tn Antigen,
Alpha-lactalbumin, LASA, Gal-GalNAC, GCDFP-15, Le(y)-Related
Carbohydrate Antigen, CA 125, uPA, uPA related antigens and
complexes, uPA Receptor, PA1-1 and PA1-2, Beta-glucuronidase, CD31,
CD44 splice variants, blood group antigens including ABH, Lewis,
and MN, and genetic lesions or altered expression levels of CCND1,
EMS1, BRCA1 and BRCA2 genes, and combinations thereof
74. The method of claim 71, 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, resins, particulate chromatographic
media, plastic microparticles, latex particles, coated tubes,
coated templates, coated beads, coated matrices, and combinations
thereof.
Description
RELATED APPLICATIONS
[0001] This application claims the priority benefits of U.S.
Provisional Patent Application No. 60/248,134, filed by Quay on
Nov. 13, 2000 and U.S. Provisional Patent Application No.
60/248,136, filed by Quay on Nov. 13, 2000. The disclosures of each
of the foregoing priority applications are incorporated herein by
reference in their entirety.
TECHNICAL FIELD OF INVENTION
[0002] The invention relates to methods, devices, and kits for
obtaining and assaying biological samples from mammary fluid. More
specifically, the invention relates to methods, devices, and kits
for obtaining and assaying fluid and cytological samples from the
mammary glands of a mammalian subject for evaluating, diagnosing
and managing breast disease, including infections, pre-cancerous
conditions, cancer susceptibility and cancer.
BACKGROUND OF THE INVENTION
[0003] Breast cancer is by far the most common form of cancer in
women, and is the second leading cause of cancer death in humans.
Despite many recent 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.
[0004] 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. Accordingly, more sensitive
and reliable methods are needed to detect small (less than 2 cm
diameter), 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.
[0005] In addition to the problem of early detection, there remain
serious problems in distinguishing between malignant and benign
breast disease, in staging known breast cancers, and in
differentiating between different types of breast cancers (e.g.
estrogen dependent versus non-estrogen dependent tumors). Recent
efforts to develop improved methods for breast cancer detection,
staging and classification have focused on a promising array of
so-called cancer "markers." Cancer markers are typically proteins
that are uniquely expressed (e.g. as a cell surface or secreted
protein) by cancerous cells, or are expressed at measurably
increased or decreased levels by cancerous cells compared to normal
cells. Other cancer markers can include specific DNA or RNA
sequences marking deleterious genetic changes or alterations in the
patterns or levels of gene expression associated with particular
forms of cancer.
[0006] A large number and variety of breast cancer markers have
been identified to date, and many of these have been shown to have
important value for determining prognostic and/or treatment-related
variables. Prognostic variables are those variables that serve to
predict disease outcome, such as the likelihood or timing of
relapse or survival. Treatment-related variables predict the
likelihood of success or failure of a given therapeutic plan.
Certain breast cancer markers clearly serve both functions. For
example, estrogen receptor levels are predictive of relapse and
survival for breast cancer patients, independent of treatment, and
are also predictive of responsiveness to endocrine therapy.
Pertschuk et al., Cancer 66:1663-1670, 1990; Parl and Posey, Hum.
Pathol. 19:960-966, 1988; Kinsel et al., Cancer Res. 49:1052-1056,
1989; Anderson and Poulson Cancer 65:1901-1908, 1989.
[0007] The utility of specific breast cancer markers for screening
and diagnosis, staging and classification, monitoring and/or
therapy purposes depends on the nature and activity of the marker
in question. For general reviews of breast cancer markers, see
Porter-Jordan et al., Hematol. Oncol. Clin. North Amer. 8:73-100,
1994; and Greiner, Pharmaceutical Tech., May, 1993, pp. 28-44. As
reflected in these reviews, a primary focus for developing breast
cancer markers has centered on the overlapping areas of
tumorigenesis, tumor growth and cancer invasion. Tumorigenesis and
tumor growth can be assessed using a variety of cell proliferation
markers (for example Ki67, cyclin D1 and proliferating cell nuclear
antigen (PCNA)), some of which may be important oncogenes as well.
Tumor growth can also be evaluated using a variety of growth factor
and hormone markers (for example estrogen, epidermal growth factor
(EGF), erbB-2, transforming growth factor (TGF), which may be
overexpressed, underexpressed or exhibit altered activity in cancer
cells. By the same token, receptors of autocrine or exocrine growth
factors and hormones (for example insulin growth factor (IGF)
receptors, and EGF receptor) may also exhibit changes in expression
or activity associated with tumor growth. Lastly, tumor growth is
supported by angiogenesis involving the elaboration and growth of
new blood vessels and the concomitant expression of angiogenic
factors that can serve as markers for tumorigenesis and tumor
growth.
[0008] In addition to tumorigenic, proliferation and growth
markers, a number of markers have been identified that can serve as
indicators of invasiveness and/or metastatic potential in a
population of cancer cells. These markers generally reflect altered
interactions between cancer cells and their surrounding
microenvironment. For example, when cancer cells invade or
metastasize, detectable changes may occur in the expression or
activity of cell adhesion or motility factors, examples of which
include the cancer markers Cathepsin D, plasminogen activators,
collagenases and other factors. In addition, decreased expression
or overexpression of several putative tumor "suppressor" genes (for
example nm23, p53 and rb) has been directly associated with
increased metastatic potential or deregulation of growth predictive
of poor disease outcome.
[0009] Additional representative breast disease markers within
these various classes include prostaglandin E2 (PGE2);
estrogen-regulated proteins such as pS2; interleukins (e.g.,
IL-10); S-100 protein; vimentin; epithelial membrane antigen;
prostate specific antigen (PSA); bcl-2; CA15-3 (an aberrant form of
polymorphic epithelial mucin (PEM)); CA 19-9; mucin core
carbohydrates (e.g., Tn antigen and Tn-like antigens);
alpha-lactalbumin; lipid-associated sialic acid (LASA);
galactose-N-acetylgalactosamine (Gal-GaINAC); GCDFP-15;
Le(y)-related carbohydrate antigen; CA 125; urokinase-type
plasminogen activator (uPA) and uPA related antigens and complexes
(e.g., LMW-UPA, HMW-UPA, uPA aminoterminal fragment (ATF), uPA
receptor (UPAR) and complexes with inhibitors such as PA1-1 and
PA1-2); beta-glucuronidase; CD31; CD44 splice variants; blood group
antigens (e.g., ABH, Lewis, and MN); and genetic lesions or altered
expression levels of CCND1, EMS 1, BRCA1 and BRCA2 genes.
[0010] In summary, the evaluation of proliferation markers,
oncogenes, growth factors and growth factor receptors, angiogenic
factors, proteases, adhesion factors and tumor suppressor genes,
among other cancer markers, can provide important information
concerning the risk, presence, status or future behavior of cancer
in a patient. Determining the presence or level of expression or
activity of one or more of these cancer markers can aid in the
differential diagnosis of patients with uncertain clinical
abnormalities, for example by distinguishing malignant from benign
abnormalities. Furthermore, in patients presenting with established
malignancy, cancer markers can be useful to predict the risk of
future relapse, or the likelihood of response in a particular
patient to a selected therapeutic course. Even more specific
information can be obtained by analyzing highly specific cancer
markers, or combinations of markers, which may predict
responsiveness of a patient to specific drugs or treatment
options.
[0011] Methods for detecting and measuring cancer markers have been
recently revolutionized by the development of immunological assays,
particularly by assays that utilize monoclonal antibody technology.
Previously, many cancer markers could only be detected or measured
using conventional biochemical assay methods, which generally
require large test samples and are therefore unsuitable in most
clinical applications. In contrast, modem immunoassay techniques
can detect and measure cancer markers in relatively much smaller
samples, particularly when monoclonal antibodies that specifically
recognize a targeted marker protein are used. Accordingly, it is
now routine to assay for the presence or absence, level, or
activity of selected cancer markers by immunohistochemically
staining breast tissue specimens obtained via conventional biopsy
methods. Because of the highly sensitive nature of
immunohistochemical staining, these methods have also been
successfully employed to detect and measure cancer markers in
smaller, needle biopsy specimens which require less invasive sample
gathering procedures compared to conventional biopsy specimens. In
addition, other immunological methods have been developed and are
now well known in the art which allow for detection and measurement
of cancer markers in non-cellular samples such as serum and other
biological fluids from patients. The use of these alternative
sample sources substantially reduces the morbidity and costs of
assays compared to procedures employing conventional biopsy
samples, which allows for application of cancer marker assays in
early screening and low risk monitoring programs where invasive
biopsy procedures are not indicated.
[0012] For the purpose of breast cancer evaluation, the use of
conventional or needle biopsy samples for cancer marker assays is
often undesirable, because a primary goal of such assays is to
detect the cancer before it progresses to a palpable or
mammographically detectable tumor stage. Prior to this stage,
biopsies are generally contraindicated, making early screening and
low risk monitoring procedures employing such samples untenable.
Therefore, there is general need in the art to obtain samples for
breast cancer marker assays by less invasive means than biopsy, for
example by serum withdrawal.
[0013] Efforts to utilize serum samples for breast cancer marker
assays have met with limited success, largely because the targeted
markers are either not detectable in serum, or because telltale
changes in the levels or activity of the markers cannot be
monitored in serum. In addition, the presence of breast cancer
markers in serum probably occurs at the time of micro-metastasis,
making serum assays less useful for detecting pre-metastatic
disease. In contrast, fluid within the mammary glands themselves is
expected to contain much higher and more biologically relevant
levels of breast cancer markers than serum, particularly in view of
the fact that 80%-90% of all breast cancers occur within the
intraductal epithelium of these glands. Fluid within the breast
ducts is expected to contain an assemblage and concentration of
hormones, growth factors and other potential markers comparable to
those secreted by, or acting upon, the surrounding cells of the
alveolar-ductal system. Likewise, mammary fluid is expected to
contain cells and solid cellular debris or products that can be
used in cytological or immunological assays to evaluate
intracellular or cell surface markers that may not be detectable in
the liquid fraction of mammary fluid.
[0014] Previous attempts to develop non-invasive breast cancer
marker assays utilizing mammary fluid samples have included studies
of mammary fluid obtained from patients presenting with spontaneous
nipple discharge. In one of these studies, conducted by Inaji et
al., Cancer 60:3008-3013, 1987, levels of the breast cancer marker
carcinoembryonic antigen (CEA) were measured using conventional,
enzyme linked immunoassay (ELISA) and sandwich-type, monoclonal
immunoassay methods. These methods successfully and reproducibly
demonstrated that CEA levels in spontaneously discharged mammary
fluid provide a sensitive indicator of nonpalpable breast cancer.
In a subsequent study, also by Inaji et al., Jpn. J. Clin. Oncol.
19:373-379, 1989, these results were expanded using a more
sensitive, dry chemistry, dot-immunobinding assay for CEA
determination. This latter study reported that elevated CEA levels
occurred in 43% of patients tested with palpable breast tumors, and
in 73% of patients tested with nonpalpable breast tumors. CEA
levels in the discharged mammary fluid were highly correlated with
intratumoral CEA levels, indicating that the level of CEA
expression by breast cancer cells is closely reflected in the
mammary fluid CEA content. Based on these results, the authors
concluded that immunoassays for CEA in spontaneously discharged
mammary fluid are useful for screening nonpalpable breast
cancer.
[0015] Although the evaluation of mammary fluid has been shown to
be a useful method for screening nonpalpable breast cancer in women
who experience spontaneous nipple discharge, the rarity of this
condition renders the methods of Inaji et al, inapplicable to the
majority of women who are candidates for early breast cancer
screening. In addition, the first Inaji report cited above
determined that certain patients suffering spontaneous nipple
discharge secrete less than 10 .mu.l of mammary fluid, which is a
critically low level for the ELISA and sandwich immunoassays
employed in that study. It is likely that other antibodies used to
assay other cancer markers may exhibit even lower sensitivity than
the anti-CEA antibodies used by Inaji and coworkers, and may
therefore not be adaptable or sensitive enough to be employed even
in dry chemical immunoassays of small samples of spontaneously
discharged mammary fluid.
[0016] In view of the above, an important need exists in the art
for more widely applicable, non-invasive methods and materials to
obtain biological samples for use in evaluating, diagnosing and
managing breast disease including cancer, particularly for
screening early stage, nonpalpable breast tumors. A related need
exists for methods and materials that utilize such readily obtained
biological samples to evaluate, diagnose and manage breast disease,
particularly by detecting or measuring selected breast cancer
markers, or panels of breast cancer markers, to provide highly
specific, cancer prognostic and/or treatment-related information,
and to diagnose and manage pre-cancerous conditions, cancer
susceptibility, breast infections and other breast diseases.
SUMMARY OF THE INVENTION
[0017] It is therefore an object of the present invention to
provide non-invasive methods and kits for obtaining biological
samples that can be employed in assays for evaluating, diagnosing
and managing breast disease, particularly cancer.
[0018] It is a further object of the invention to achieve the above
object in assay methods and kits that are widely applicable to a
broad range of patients, and that include useful assays and kits
for screening early stage, nonpalpable mammary tumors.
[0019] It is yet another object of the invention to provide methods
and kits that utilize the aforementioned biological samples to
evaluate, diagnose and manage breast disease, preferably breast
cancer, by detecting and/or measuring selected breast disease
markers such as breast cancer markers, or panels of breast cancer
markers, to provide highly specific prognostic and/or
treatment-related information to the clinician.
[0020] The invention achieves these objects and other objects and
advantages that will become apparent from the description which
follows by providing non-invasive methods and devices for obtaining
biological samples from a mammary organ of a mammalian patient.
Through the use of novel, specialized breast pump devices of the
invention, which are fluidly connected with (i.e., by direct or
indirect coupling) a solid phase sample collection medium, the
physician can rapidly and non-invasively collect mammary fluid
samples from lactating or non-lactating female patients without
additional intervention. Alternate methods of the invention for
mammary fluid sample collection may involve administration of
oxytocin, or an oxytocin analog, in an amount effective to
stimulate or increase expression of mammary fluid induced in
conjunction with employment of the breast pump device. The oxytocin
or oxytocin analog (for example a long-acting oxytocin analog such
as carbetocin) is administered in a manner (e.g., intranasally) and
amount sufficient to reach and stimulate a target alveolar-ductal
tissue of the breast, whereby the oxytocin stimulates myoepithelial
contraction of the alveolar-ductal tissue to induce or facilitate
mammary fluid expression. Alternatively, an intramuscular or
intravascular injection of oxytocin can effect the same
myoepithelial contraction response as the intranasal administration
route. The amount, timing and/or mode of oxytocin administration
may be adjusted on an individual basis depending on such factors as
menstrual cycle stage, use of birth control or hormone replacement
therapy, pregnancy history, age of onset of menarche, ethnicity and
other factors known to affect an individual's propensity for breast
fluid expression.
[0021] Mammary fluid collection devices of the invention are
effective to induce mammary fluid expression for sample collection,
alone or in conjunction with oxytocin stimulation. These devices
are typically provided as a specialized breast pump which can be
applied to the breast covering the nipple, and which typically
directly receives the expressed mammary fluid. In preferred methods
involving use of a breast pump, negative pressure is generated on
the breast to induce expression of mammary fluid, optionally
facilitated by prior or concurrent administration of oxytocin. In
yet additional alternative methods, mammary fluid can be expressed
and collected without the aid of a breast pump, which may require
an increase of oxytocin dosage or lengthening of the post
administration time period before the mammary fluid is fully
expressed from the nipple.
[0022] During or after mammary fluid expression, a biological
sample is collected from the expressed mammary fluid, which sample
may consist 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 and RNA polynucleotides) and other like
biochemical and molecular constituents of the mammary fluid.
[0023] Sample collection can be achieved simply by receiving the
expressed mammary fluid within any suitable reservoir, such as an
ordinary sample storage container or assay vessel. In preferred
embodiments of the invention, the expressed mammary fluid is
exposed to a solid phase sample collection medium, 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,
microspheres, 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. Often it will be desirable to combine a plurality of
solid phase media for sample collection, e.g., a filter and
membrane, a membrane and a particulate medium, etc., for example to
differentially partition and adsorb selected components of the
breast fluid.
[0024] In conjunction with sample collection, the sample may be
exposed to other agents such as buffers, diluents, extraction or
chromatographic media, cross-linking agents, denaturing agents,
etc., to stabilize or otherwise prepare the sample for processing
within a desired assay.
[0025] Thus provided within the invention are methods and devices
for obtaining a biological sample from a patient and/or determining
the amount of a breast disease marker in a biological sample from
breast fluid, which employ a novel breast pump or breast pump
adapter. The breast pump functions in a similar fashion as a
conventional breast pump but also provides a solid phase sample
collection medium in fluid connection with the pump. The solid
phase sample collection medium may be integrated within the breast
pump or otherwise fluidly connected therewith, so that a sample of
expressed mammary fluid contacts the collection medium, typically
while the pump remains applied to the breast. In more detailed
aspects of the invention, methods for employing the novel breast
pump include a step of applying the breast pump to stimulate breast
fluid expression, with or without prior oxytocin or carbetocin
induction, wherein the solid phase sample collection medium is
fluidly connected with a breast engaging portion or member of the
breast pump.
[0026] According to these methods, operation of the pump results in
an expressed breast fluid sample contacting the solid phase sample
collection medium, typically while the pump remains applied to the
breast. Within the foregoing methods, additional methods are
provided which employ a novel, hand-held breast pump device,
wherein a doctor, technician or patient collecting a breast fluid
specimen can grasp and operate the hand-held pump to stimulate
expression of breast fluid and collect a specimen thereof while
keeping one hand free for additional tasks. The compact hand-held
pump design allows the device to be picked up and manipulated with
one hand, to seat the breast engaging element against the breast,
apply vacuum pressure to the breast by manual operation of the
vacuum pump to cause a suitable volume of breast fluid to be
expressed at or near the nipple, and to simultaneously collect at
least a primary sample of expressed breast fluid onto, or within,
the solid phase sample collection medium without additional manual
steps or a need to remove the device from the breast or engage two
hands in the operation.
[0027] In certain collection methods of the invention, breast fluid
expressed by use of the general purpose or hand-held breast pump is
simultaneously or subsequently diluted, filtered, washed, admixed
with fixative or other processing agents, or otherwise processed or
modified to yield a collected fluid sample partially or completely
devoid of cells, proteins and/or other selected components
originally present in the expressed fluid, to provide a processed
fluid sample for laboratory analysis. In other embodiments,
particulate components of the breast fluid, for example, cells,
cellular components and/or cellular debris, are collected after
processing and/or modification, e.g., for cytological examination.
Often, primary sample collection and/or processing in this context
is coincident with the fluid contacting one or more solid phase
collection medium(a) fluidly connected with the breast engaging
member. Depending on the type(s) of medium(a) used, preliminary
sample processing can also be achieved directly by simple operation
of the hand-held pump, without the need for additional processing
steps or removal of the breast engaging member from the subject's
breast.
[0028] In other alternative methods within the invention,
preliminary sample processing involves additional steps following
breast fluid expression. In certain embodiments, the breast
engaging member is removed from the breast after the breast fluid
is expressed and the fluid is transferred to a first solid phase
sample collection medium, typically a membrane or filter. This
initial or primary stage of sample collection may be followed by
washing or by manual transfer of selected breast fluid components
(e.g., proteins, carbohydrates, cells, or cellular debris) from the
first solid phase collection medium (e.g., a nitrocellulose
membrane) to a second solid phase medium, e.g., a fluid-containing
reservoir. Typically, preliminary sample processing in this regard
precedes final packaging of the collected sample for storage or
shipment to a lab for further processing and analysis of the
sample. In one example, whole cells or other cellular materials are
separated from expressed mammary fluid onto a nitrocellulose
membrane or a filter, which is typically secured in fluid
connection with the breast engaging member by a fixed or removable
support member mounted to the engaging member or sample collection
housing. The cells are subsequently transferred or washed in fluid
(e.g., cytology fluid) to a second solid phase sample collection
medium, for example a removable fluid reservoir connected to, or
integrated with, the breast engaging member or sample collection
housing.
[0029] In relation with these methods, various sample collection
devices and accessories for use therewith are provided within more
detailed embodiments of the invention. Typically, breast fluid
collection devices of the invention include a breast engaging
member constructed of a non-porous material that is sized and
dimensioned to receive at least a nipple portion of a human breast
and form a suction seal therewith. One or more solid phase sample
collection media are provided in fluid connection with the breast
engaging member for receiving a sample of expressed breast fluid. A
vacuum pump mechanism is provided in gaseous connection with the
breast engaging member for generating negative pressure through the
breast engaging member to facilitate breast fluid expression.
[0030] In specific embodiments of the collection device of the
invention, a sample collection housing is fluidly connected with
the breast engaging member. The solid phase sample collection
medium is often removably supported within the housing in proximity
to the nipple when the breast engaging member is applied to the
breast and negative pressure is generated by the vacuum pump
mechanism. The solid phase sample collection medium can be one or
more microscopic glass slides, capillary tubes, collection tubes,
vials, columns, micro-columns, wells, plates, membranes, filters,
resins, inorganic matrices, beads, resins, particulate
chromatographic media, plastic microparticles, latex particles,
coated tubes, coated templates, coated beads, or coated
matrices.
[0031] Optional features of the breast fluid collection device
include removable coupling means for removably coupling said sample
collection housing with said breast engaging member. In other
embodiments, the solid phase sample collection medium may be
supported by a support member integrally or removably mounted
within the sample collection housing in fluid connection with said
breast engaging member. Various types of support members, including
disposable or reuseable discs, cartridges and cassettes are
provided as an accessory for use within the invention. In yet
additional embodiments, a reciprocating mechanism for reciprocally
adjusting a position of the solid phase sample collection medium
relative to the breast engaging member is incorporated within the
device. The reciprocating mechanism may incorporate a support
member or carrier reciprocatingly mounted relative to the breast
engaging member, which support member or carrier supports the solid
phase sample collection medium. Yet another optional feature of the
device includes a breast pump adapter employing concepts of the
invention for collection of mammary fluid samples and operable in
combination with a conventional breast pump.
[0032] In other detailed embodiments of the invention, the sample
collection device is a hand-held breast pump incorporating the
breast engaging member and the vacuum pump mechanism in a compact,
structurally integrated breast fluid collection apparatus suitable
for manipulation and operation using only one hand. In certain
embodiments, hand-held breast pump comprises a modular device made
up of a plurality of components, each joined or securable in fixed
structural interconnection with one another and capable of partial
or complete disassembly from remaining components to facilitate
operation, cleaning, servicing and/or storage of the device. The
modular breast pump can include, for example, a separate breast
engaging member detachable from one or more interconnecting
components of the device for cleaning or to allow interchanging of
different engaging members to accommodate breast anatomy
differences among patients.
[0033] Within more detailed embodiments of the hand-held breast
pump, the solid phase sample collection medium can be supported by
a support member removably mounted in fluid connection with the
breast engaging member. The support member can be a removable
cassette for removable placement in fluid connection with the
breast engaging member. The support member can house any of the
above identified collection media, and may incorporate one or more
air channels that pass through a body of the support member for
passage of vacuum pressure and/or sample materials between the
breast engaging member and a sample collection housing member of
the hand-held breast pump.
[0034] Within other detailed embodiments, the hand-held device
includes a fluid-retaining recess, well or reservoir integrated or
fluidly connected with the support member or a sample collection
housing member of the device. The fluid-retaining recess, well or
reservoir may comprise an integral, defined compartment or
enclosure within the sample collection housing for receiving the
breast fluid and/or constituent samples thereof. Alternatively,
fluid-retaining recess, well or reservoir comprises a removable
fluid reservoir member of the sample collection housing, typically
provided as a rigid sample collection tube or vial removably
connected with an outer casing member of the housing. The removable
reservoir member is optionally sealably connected with the outer
casing member of the housing to form an airtight coupling
therewith. In certain embodiments, the removable reservoir member
features a circumferential ridge, fin, O-ring or other sealable
engagement means to engage and make an airtight seal against a wall
or other surface of the casing member when the vial is nested
within the casing member.
[0035] In additional detailed embodiments, the removable reservoir
member is gaseously and fluidly connected with the breast engaging
member to facilitate sample collection. For example, the vacuum
pressure from the vacuum pump means may be routed to the breast
engaging member through the removable reservoir member of the
housing, which is modified to include one or more air ports that
form a gaseous connection between a lumen of the reservoir and the
vacuum pump means. The reservoir member may function in this
context as both a conduit for vacuum pressure transmission to the
breast and a receptacle for fluid sample materials to directly
collect expressed fluid or as a secondary collection medium to
receive primarily collected sample materials washed or otherwise
transferred from a primary solid phase sample collection medium
into the reservoir. For example, a primary solid phase sample
collection medium fluidly connected with the breast engaging member
may be positioned to collect a primary sample of one or more breast
fluid components which can thereafter be washed or otherwise
transferred directly or indirectly into the removable reservoir
member, without removal or disassembly of the breast engaging
member and reservoir member.
[0036] The fluid collection reservoir may serve a dual purpose for
collection, as well as for storage, transport and/or processing of
collected breast fluid or breast fluid component samples. Relating
to this purpose, the removable reservoir member further comprises
closure means for closing the reservoir after sample collection is
completed to prevent sample contamination and spillage. The closure
means may comprises a cap adapted to sealably engage a top end of
the removable reservoir member. Where the reservoir member is
modified to include one or more air ports for transmission of
vacuum pressure between the lumen of the reservoir and the vacuum
pump means, the closure means include secondary closure means to
sealably close the air port(s) after sample collection. For
example, the secondary closure means may comprise an adhesive seal
or sticker sized and constructed to adhere to an outer wall of the
reservoir member surrounding an air port opening. Typically, the
secondary closure means comprises a combined closure and labeling
device which functions as a secondary closure mechanism to seal the
air port(s) of the removable reservoir, and as a labeling template
to provide a writing surface for sample labeling. The combined
closure and labeling tab or sticker generally includes a first,
closure-forming surface for application over the air port to form a
seal by juxtaposition or adhesive contact with an outer wall of the
removable reservoir, and a second, labeling surface opposite the
closure-forming surface made of a blank template material suitable
for receiving a stable, ink or graphite imprint. In more detailed
aspects, the secondary closure means comprises a combined closure
and labeling tab or sticker which is pre-attached to the removable
reservoir member in a first, open configuration and which can be
manually repositioned or otherwise manipulated after sample
collection to a second, closed configuration to form a seal or
closure against the air port(s).
[0037] In related aspects of the invention, a novel breast fluid
collection reservoir, e.g., a modified cytology vial, is provided
for use within a mammary fluid collection device of the invention,
which reservoir incorporates the foregoing features of the
removable reservoir member of the sample collection housing. The
novel collection reservoir thus provided is useful within the
breast fluid collection methods of the invention, as well as within
sample processing and diagnostic assay methods performed in the
laboratory subsequent to collection of a breast fluid sample.
[0038] In related aspects of the invention, methods are provided
for determining the presence or amount of a breast disease marker,
preferably a breast cancer marker, in biological samples obtained
from a mammary organ of a mammalian patient. These methods may
involve intranasal, intramuscular or intravascular administration
of oxytocin or an oxytocin analog to mammalian patients in amounts
effective to stimulate mammary fluid expression in the patient.
Once a sufficient post-administration time period has elapsed to
allow the oxytocin to reach and stimulate target alveolar-ductal
tissues, mammary fluid is collected directly from the nipple or,
alternatively, the breast is pumped, and a biological sample from
expressed mammary fluid is collected, as above. After the sample is
collected a bioassay is conducted on the sample to determine the
presence and/or amount of the breast disease marker in the sample.
Suitable bioassays in this regard include assays to detect known
markers of breast disease, such as assays employing immunological
or other suitable probes to detect specific antigens and other
markers expressed by selected pathogens, including bacterial and
viral pathogens. More preferred bioassays will detect individual
markers or panels of markers of benign breast tumors, pre-cancerous
breast disease, and/or breast cancer, such as assays employing
immunological or other suitable probes to detect specific antigens
and other markers expressed by benign, pre-cancerous and/or
cancerous alveolar-ductal cells of the breast. Preferably, the
assay will detect the presence or amount of multiple breast disease
markers in the biological sample, for example by including a panel
of immunological or molecular probe(s) that bind or react with
multiple breast cancer markers.
[0039] In yet additional aspects of the invention, clinically
useful kits are provided for determining the presence and/or amount
of a breast disease marker, preferably a breast cancer marker, in
biological samples obtained from a mammary organ of a mammalian
patient. The kits include a mammary fluid collection device in the
form of a general purpose or hand-held breast pump as described
herein. Additional kits include one or more breast pump attachments
(e.g., a detachable breast engaging member, or multiple such
attachments for use with different patients), accessories (e.g.,
replaceable fluid-retaining reservoirs), solid phase media, and/or
disposable or reusable support members, cartridges or cassettes for
holding collection media, as described herein. These and other kit
components may be provided, alone or in any combination, with or
without inclusion of the basic breast pump apparatus in the kit.
Yet additional kits include a pharmaceutical preparation of
oxytocin or an oxytocin analog in a biologically suitable carrier
for use in alternate mammary fluid collection methods of the
invention. Still other kits include on or more preparative and/or
diagnostic reagents selected from those disclosed herein, including
one or more fixatives, probes, labels and the like in separate or
common containers. In certain embodiments of the invention, kits
include compositions and/or devices for detecting the presence or
amount of one or more breast disease marker(s) in the biological
sample, often including one or more immunological or molecular
probe(s) that binds or reacts with one or more breast cancer
marker(s). The foregoing kit components are generally assembled in
a collective packaging unit, which may include written or otherwise
user-accessible instructions detailing the sample collection,
handling and/or processing methods of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 is a partial sectional view of a breast pump
employing the concepts of the invention.
[0041] FIG. 2 is a sectional view of a portion of the breast pump
as indicated in FIG. 1.
[0042] FIG. 3 is a perspective view of a support member for
supporting a solid phase sample collection medium in fluid
connection with a breast pump.
[0043] FIG. 4 is a perspective view of an alternative support
member for supporting a solid phase sample collection medium in
fluid connection with a breast pump.
[0044] FIG. 5 is a perspective view of an alternative support
member for supporting a solid phase sample collection medium in
fluid connection with a breast pump.
[0045] FIG. 6 is a perspective view of an alternative support
member for supporting a solid phase sample collection medium in
fluid connection with a breast pump.
[0046] FIG. 7 is a perspective view of an alternative support
member for supporting a solid phase sample collection medium in
fluid connection with a breast pump.
[0047] FIG. 8 is a partial sectional view of a breast pump device
employing the concepts of the invention.
[0048] FIG. 9 is a partial sectional view of a portion of a breast
pump illustrating a support member and cartridge for containing a
particulate solid phase sample collection medium.
[0049] FIG. 10 is a partial sectional view of a portion of a breast
pump illustrating a support member and an exemplary solid phase
sample collection template (coated tube).
[0050] FIG. 11 is a partial sectional view of a breast pump
employing a reciprocating mechanism to adjust positioning of a
solid phase sample collection medium within the pump.
[0051] FIG. 12 is a sectional view depicting a breast pump adapter
employing the concepts of the invention.
[0052] FIG. 13 is a sectional view depicting a breast pump adapter
employing a reciprocating mechanism to adjust positioning of a
solid phase sample collection medium within the adapter.
[0053] FIGS. 14 and 15 provide partial sectional views of a breast
pump employing a sliding reciprocating mechanism to adjust
positioning of a solid phase sample collection medium within the
pump.
[0054] FIG. 16 is a sectional view illustrating a hand-held breast
pump of the invention.
[0055] FIG. 17 is a sectional view illustrating a hand-held breast
pump of the invention.
[0056] FIG. 18 is an exploded perspective view illustrating a
hand-held breast pump of the invention employing a removable
support member and removable fluid reservoir for primary and
secondary sample collection.
[0057] FIGS. 19 and 20 provide perspective views of alternative
embodiments of a removable sample collection reservoir for use with
the hand-held breast pump of the invention.
[0058] FIGS. 21-23 provide sectional views of the removable fluid
reservoir of FIG. 19, illustrating operation of a closure/labeling
strip to seal the reservoir and provide an exterior labeling
surface for recordation of sample data.
[0059] FIGS. 24-26 provide top plan views of a modified membrane or
filter for use within the breast pump of the invention having
perforations or slits for enhancing permeability and flexibility of
the membrane or filter.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0060] As noted above, the invention provides methods and devices
10, 10' for obtaining, handling, and processing biological samples
from mammary fluid. 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. To practice the
non-invasive sample collecting method, the invention provides
specialized breast pump devices which feature a breast engaging
portion or member coupled with a vacuum pump mechanism and fluidly
connected with a solid phase sample collection medium. The mammary
fluid collection devices and methods disclosed herein are related
in certain aspects to mammary fluid collection devices and methods
disclosed in U.S. patent application Ser. No. 09/435,131, filed
Nov. 5, 1999, U.S. patent application Ser. No. 09/027,362, filed
Feb. 20, 1998, and U.S. Pat. No. 5,798,266, issued Aug. 25, 1998,
each incorporated herein by reference. The devices and methods of
the present invention are effective to induce mammary fluid
expression for sample collection, alone or in conjunction with
oxytocin stimulation.
[0061] The mammary fluid collection devices of the invention are
typically provided as a specialized breast pump 10, 10' which can
be applied to a human or animal breast covering the nipple, and
which typically receives expressed mammary fluid within a solid
phase sample collection medium, and in some cases within a
removable sample collection reservoir. During or after mammary
fluid expression, a biological sample is collected from the
expressed mammary fluid, which sample may consist 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 and RNA polynucleotides) and other like biochemical and
molecular constituents of the mammary fluid.
[0062] The breast pump devices 10, 10' of the invention function in
part by generating negative pressure applied to the nipple area of
the breast to induce mammary fluid expression. Fluid expression
induced by these breast pump devices may optionally be facilitated
by coordinate administration of the peptide hormone oxytocin, or a
functional analog thereof, in an amount effective to stimulate or
increase expression of mammary fluid induced by the breast pump
device. The oxytocin or oxytocin analog (for example a long-acting
oxytocin analog such as carbetocin) is administered in a manner
(e.g., intranasally) and amount sufficient to reach and stimulate a
target alveolar-ductal tissue of the breast, whereby the oxytocin
stimulates myoepithelial contraction of the alveolar-ductal tissue
to induce or facilitate mammary fluid expression.
[0063] During or after the mammary fluid expression step, a
biological sample is collected from the expressed mammary fluid. A
range of suitable biological samples are contemplated and will be
useful within the methods of the invention, including whole mammary
fluid, selected liquid or solid fractions of the mammary fluid,
whole cells or cellular constituents, proteins, glycoproteins,
peptides, nucleotides (including DNA and RNA polynucleotides) and
other like biochemical and molecular constituents of the mammary
fluid. Sample collection can be achieved simply by receiving the
expressed mammary fluid within a suitable reservoir, such as an
ordinary sample storage container or assay vessel.
[0064] In preferred embodiments of the invention, the expressed
mammary fluid is contacted with a solid phase sample collection
medium fluidly connected with the breast pump 10, 10', 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.
[0065] A wide range of sample collection procedures and materials
known in the art are useful within the invention. Selected methods
and materials will vary among different assays, as will be
understood and readily practiced by those skilled in the art. For
example, if the breast disease marker sought in a particular assay
is a soluble protein, it will often be desired to immobilize the
protein on a solid phase matrix or template by contacting the
target protein with a reagent having high specificity for the
protein, preferably a polyclonal or monoclonal antibody. The yields
a complex, e.g., a ligand-protein complex, an antibody-antigen
complex, or other complex in which the target protein is bound to a
specific binding partner (i.e., wherein the complex is not
dissociated upon addition of a non-specific binding partner
conventionally used as a control to determine specific binding; and
preferably wherein the binding partner binds with an affinity of kD
10-9 or greater). The binding partner that binds to the target
protein is in turn immobilized to the solid phase medium, before or
after complex formation with the target protein. Immobilization of
the binding partner, e.g., by covalent binding to a solid phase
template or matrix, can be achieved by a variety of conventional
methods known in the art.
[0066] In this manner, the target protein/binding partner complex
is adsorbed or otherwise bound directly to an insoluble matrix.
Alternatively, a variety of secondary binding partners, e.g.,
anti-isotype antibodies, may be added to bind the complex to the
insoluble matrix. The latter step depends on the nature of the
first binding partner (i.e., the binding agent that specifically
binds the target protein), for example whether the first binding
partner is a primary antibody, ligand, etc.
[0067] Particularly useful within the invention are immunoassay
which formats employ a combination of solid phase or immobilized
reagents and labeled reagents whereby the association of the label
with the solid phase is a function of the presence or absence of
reactivity with the targeted antigen. In general, such a solid
phase reagent comprises a binding substance such as an
anti-antibody (e.g., anti-IgG), or other immunobinder or other
binding agent according to the assay protocol involved, bound or
attached, covalently or noncovalently, to the solid phase matrix or
in an otherwise immobilized form.
[0068] Useful labeled reagents in solid phase immunoassays include
a binding substance such as an anti-antibody (e.g., anti-IgG), or
other immunobinder or other binding agent according to the assay
protocol involved, which is chemically coupled with a detectable
chemical moiety. Useful labels are conventional in the art and
include fluorescers, chemiluminescers, radioisotopes, and enzymes.
Enzyme labels are particularly useful and are generally selected
from alkaline phosphatase, peroxidase, and .beta.-galactosidase.
Enzyme labels are readily detectable by addition of a corresponding
chromogenic substrate and detecting the resulting color or
fluorescent response.
[0069] A variation of this protocol uses a ligand-modified form of
the targeted antigen(s) with immobilization to the solid phase
being accomplished by using a solid phase bearing an immobilized
(e.g., bound or adsorbed) binding partner to the ligand. For
example, biotin or a hapten (e.g., fluorescein) can be used as the
ligand and can be immobilized by contact with a solid phase form of
avidin or anti-hapten antibody, respectively. The addition of the
solid phase binding partner can occur at any convenient time in the
assay, such as prior to contact of sample with the
ligand-antigens(s) or thereafter.
[0070] Preferred solid phase matrices for use within the foregoing
methods include Staphylococcus aureus or Protein A or G Agarose
[e.g. Sepharose.RTM. (Pharmacia Biotech AB, Uppsala, Sweden)]
beads. Protein A and protein G are cell wall proteins isolated from
specific bacterial strains, and have specific binding sites for
certain classes of immunoglobulins. Protein A binds (to varying
degrees) most subclasses of IgG, plus IgM, IgA, and IgD. Protein G
binds nearly all subclasses of IgG, but not other classes of
immunoglobulins.
[0071] An alternative solid phase sample collection and/or assay
method utilizes a specific anti-marker primary antibody that is
covalently attached to the solid phase matrix, e.g., by covalent
linking the antibody through its free amino groups to
cyanogen-bromide-activated Sepharose particles. Insolubilized
antibody can be used to pull the corresponding marker antigen out
of solution by adsorption to its surface. In yet another
alternative format, the marker protein can be treated with a
cross-linking reagent (e.g. biotin or digoxigenin) that may be
subsequently detected by a second binding partner. In the case of
biotin, the second binding partner is avidin or streptavidin; for
digoxigenin, the second reagent is an anti-digoxigenin antibody.
Avidin and streptavidin may be coupled directly to the solid phase
medium, e.g., to agarose beads. Because the initial biotinylation
is not specific for the marker, samples are frequently
electrophoresed on, e.g., SDS PAGE, transferred to nitrocellulose
etc., and Western blotted with antibodies specific for the protein
factor.
[0072] A preferred assay method for detecting protein markers is
the well known, Enzyme Linked Immunosorbant Assay (ELISA) assay.
According to this method, a variety of coating reagents can be
adsorbed or otherwise bound directly onto a surface of a desired
solid phase sample collection medium, e.g., a microtiter plate,
well, tube, bead, test strip, plastic microparticle, latex
particle, etc., to form a coated template or matrix. These coating
reagents are typically a species-specific anti-isotype antibody
(e.g., anti-mouse-IgG) but can also include an anti-marker primary
antibody or an affinity reagent such as avidin or streptavidin. The
target protein (e.g., a soluble protein marker) is contacted with a
specific primary antibody or, alternatively, is crosslinked (e.g.,
to biotin) or otherwise modified to form a complex, and the
resulting complex is adsorbed to the coated template or matrix and
processed according to conventional assay methods.
[0073] Latex or particle agglutination methods are also to be
mentioned. Particles are coated or covalently coupled with a target
antigen, ligand, antibody or other binding partner. The particles
are then incubated with a test sample and resulting agglutination
of the particles, e.g., due to formation of ICA antibody linkages
between particles, is detected. Detection can be accomplished by
visual observation (e.g., a slide agglutination format) or
quantified by measuring turbidity changes with a spectrophotometer
or nephelometer. A well known variation of this general method
based on inhibition of particle agglutination can also be employed.
In addition, an agglutinator reagent can be prepared comprising
multiple antigens, e.g., a water soluble polymer backbone to which
are attached multiples of one or more antigens within a panel.
[0074] Alternative methods for collecting and analyzing samples
within the invention include Western immunoblot and dot-blot
methods. For application of these methods, the solid phase sample
collection medium is preferably a membrane or filter, e.g., a
nitrocellulose, polyvinylidene difluoride (PVDF), or nylon
membrane. Proteins within the breast fluid sample may be processed
(e.g., separated on SDS PAGE) or directly transferred to the
membrane, and non-specific interactions may be blocked by
incubating the membrane with, e.g., bovine serum albumin/ovalbumin
or non-fat dry milk. A primary antibody with specificity for the
protein marker is contacted with the membrane, and excess antibody
is washed, e.g., with buffered detergent. A labeled isotype
specific antibody is next contacted with the membrane, and target
protein-primary antibody-secondary antibody ternary complexes are
detected, e.g., calorimetrically.
[0075] Where the targeted protein factor includes a carbohydrate
moiety, the factor can also be adsorbed to a solid phase template
or matrix, e.g., a resin, by way of lectin-carbohydrate
interactions. Various lectins are available for this purpose that
differ in their carbohydrate binding specificity. For example,
Lectin Con A binds to mannose-containing carbohydrate structures
and with low affinity to .alpha.-glucose and
.alpha.-N-acetylglucosamine. Lectin GNA binds to terminal mannose
residues. Lectin MAA binds to .alpha.(2-3) Linked sialic acids. A
variety of other lectins collectively providing a wide range of
specificities are known in the art.
[0076] A particularly preferred solid phase sample collection
medium for use within the invention is a filter, pad or membrane
that can be directly contacted to a sample of expressed breast
fluid to adsorb, absorb, bind, partition or otherwise facilitate
sample processing or handling within a selected assay. For this
purpose, several types of transfer membranes are known, including
nitrocellulose which is the most commonly used transfer membrane.
Several commercial sources now offer nitrocellulose impregnated
with a synthetic support that improves its durability and
flexibility without altering its performance. One preferred
transfer membrane, polyvinylidene difluoride (PVDF), marketed by
Millipore (Bedford, Mass.) under the trade name Immobilon.RTM., has
slightly lower protein-binding capacity than nitrocellulose but is
mechanically stronger and compatible with many organic solvents.
This allows direct protein staining with Coomassie Blue, and direct
amino acid composition and sequence analysis of transferred
proteins, without interfering with its subsequent use for antibody
probing.
[0077] Membranes are not only useful within the invention for
protein blotting, but also for immobilization of nucleic acids.
Thus, nitrocellulose, reinforced nitrocellulose, diazotized
membranes (paper or nylon), nylon, charged nylon, or PVDF, and
DEAE-anion exchange membranes are useful for immobilizing DNA and
RNA from expressed breast fluid. In this context, the most commonly
used membranes are reinforced nitrocellulose and nylon.
Nitrocellulose has a lower background but also a lower binding
capacity than nylon and is chosen primarily when background, but
not detectability, is the main concern. Nylon, in contrast, is
ideal for lower copy number sequences, short target sequences (down
to oligomers) or for reprobing. Membranes are also available with
different pore sizes. For DNA blots, membranes with a pore size of
0.45 .mu.m are usually chosen for large fragments, but 0.22 .mu.m
for fragments of <500 bases. For RNA blots, membranes with a
pore size of 0.1 or 0.22 .mu.m are most efficient. Membranes are
available in different size specifications, including sheets,
rolls, pre-cut circles, etc.
[0078] Methods for detecting DNA on nylon without DNA purification
and processing of the samples, e.g., for detecting DNA from fluids
or whole cells, have recently been developed (Reed and Matthaei,
Nucleic Acids Res. 18:3093, 1990; and Hammermueller et al., J.
Virol. Methods 31:47, 1991; each incorporated herein by reference).
These procedures avoid enzymatic dispersion of cells, RNase and
pronase treatments to hydrolyze cellular macromolecules, etc., and
are typically based on the capacity of alkali and other reagents to
disperse and solubilize cells and hydrolyze macro-molecules
including RNA and protein, but not DNA. Positively charged modified
nylon membranes then irreversibly bind nucleic acid while remaining
suitable for hybridization.
[0079] Nucleic acid extraction and processing steps may also be
minimized by well known fast blot methods. In particular, fast blot
methods that use nylon as a solid phase take advantage of the
ability of NaOH to dissociate cells, denature DNA and immobilize
DNA. Nitrocellulose membranes have a lower binding capacity and
co-immobilization of nucleic acid and protein from neutral
solutions can be a problem. Concentrated NaI can be used to inhibit
protein immobilization, to denature DNA and to irreversibly bind
the nucleic acid to nitrocellulose without a requirement for
baking. This method can also be used for RNA.
[0080] Although it is possible to directly transfer proteins,
nucleic acids and other markers to a solid phase matrix which is in
turn directly incorporated in an assay, it may be desirable to
concentrate the target marker, e.g., by chromatography, extraction,
specific or nonspecific adsorption, etc., particularly when
sensitivity is a problem. Thus, samples can be collected and
initially processed by contacting breast fluid with a solid phase
chromatographic medium, e.g., within a cartridge comprising a
micro-column of Sepharose-coupled antibody. Up to 500-fold
increases in immunoassay sensitivity with apparent recoveries of 85
to 95% can be achieved using this approach. This and other well
known chromatographic procedures provide a powerful approach to the
quantitation of substances too dilute to be measured by routine
methods.
[0081] For sample collection and processing using chromatographic
and related methods, a particulate solid phase sample collection
medium is preferred. Various particulate media are known which
selectively adsorb, absorb, bind, or partition components of
biological samples, which media are readily adapted for collection
and processing of breast fluid samples. These particulate can be
coupled with various coating reagents known in the art, e.g.,
affinity reagents, to provided a coated medium, or may be used in
an unmodified form.
[0082] Exemplary particulate sample collection media for use within
the invention include beads, plastic microparticles, latex
microspheres, glass materials such as controlled porous glass,
granular agarose based materials, cross-linked dextran polymers,
inorganic or organic ion exchanger materials, kieselsur and other
silicate materials. Suitable materials additionally include
cellulosic materials, e.g., diethylaminoethyl (DEAE) cellulose or
diethylamino (DEA) cellulose. Also useful are natural polymeric
carbohydrates and their synthetically modified, cross-linked or
substituted derivatives, such as agar, agarose and cross-linked
dextran polymers.
[0083] Synthetic polymers which can be prepared with suitably
porous structures, such as vinyl polymers (e.g., polyethylene,
polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and
its partially hydrolysed derivatives, polyacrylates,
polyacrylamides, polymethacrylates), copolymers and terpolymers of
the above vinyl monomers among themselves and with other monomers,
polycondensates (e.g., polyesters and polyamides), and addition
polymers, such as polyurethanes or polyepoxides are also
useful.
[0084] Yet additional particulate media are prepared from inorganic
materials having a suitably porous form, such as sulfates or
carbonates of alkaline earth metals and magnesium. Examples include
barium sulfate, calcium sulfate, calcium carbonate, magnesium
carbonate, silicates of alkali and alkaline earth metals and/or
aluminum and/or magnesium, and aluminum or silicon oxides or
hydrates, such as clays, alumina, talc, kaolin, zeolite, among
others.
[0085] Also included among useful solid phase sample collection
media porous barrier materials suitable for use with breast pump
and breast pump adapter devices of the invention, for example to
enclose particulate solid phase media within a cartridge adapted
for coupling in fluid connection with a breast pump or breast pump
adapter. Such porous barrier materials are inert to and nonreactive
with markers and other analytes and reagents used in assaying for
breast disease markers, and are porous with respect to the passage
of liquids and/or particulates of a pre-selected size. Suitable
materials include various porous materials such as nylon fabric,
polyethylene and other plastic films, membranes, filters, glass
wool, sponge, styrofoam, ceramic and other porous materials.
[0086] In conjunction with sample collection, samples of expressed
breast fluid may be exposed to other agents such as buffers,
diluents, extraction or chromatographic media, cross-linking
agents, blocking agents, denaturing agents, etc., to stabilize or
otherwise prepare the sample for processing within a desired assay.
For example, the sample may be diluted (e.g., by collecting the
sample in a well or recess containing the solid phase medium wetted
or suspended in a diluent) to minimize nonspecific binding effects,
e.g., affecting a subsequent immunoassay. In the exemplary context
of sample collection for immunoassays, the avidity of the antibody
for the marker antigen is an important consideration, whereby
providing more or less diluent during sample collection and
incubation may optimize a particular antigen-antibody system being
studied.
[0087] Commonly used buffers for dilution include phosphate,
borate, or Tris-buffered saline. Usually, the choice of the buffer
is not important. Nonetheless, a careful examination of the effect
of buffer, pH, ionic strength, and divalent cations will facilitate
use of a new sample collection/assay system in order to maximize
sensitivity and resolve possible sources of interference within the
assay. Although immunoassays are usually carried out at neutrality,
doing so is not always optimal.
[0088] Nonspecific binding or adsorption, e.g., of antigens and
haptens (especially hydrophobic haptens) to glass and plastic tubes
or pipettes may markedly influence measured activity in a
particular immunoassay. With some proteins and polypeptides,
nonspecific binding in immunoassays is reduced if plastic tubes are
used. The addition of protein to the medium may also minimize
nonspecific adsorption and help avoid denaturation of highly
diluted antigens and antibodies. Therefore, assays involving
iodinated antigens are generally carried out in protein-containing
buffers. Bovine serum albumin, gelatin, lysozyme, and ovalbumin are
commonly used, usually at final concentrations of 1 to 5 mg/ml. In
some systems diluted whole serum or proteins present in the sample
itself are just as satisfactory. However, even though added
proteins are often beneficial, they should not be used
indiscriminately without making an evaluation for possible adverse
effects, for example contaminating enzymes that may degrade the
marker protein.
[0089] Other possible additives for improved sample collection and
assay methods, apart from buffer and protein, include enzyme
inhibitors and chelating agents. In assays lasting longer than 3
days, a bacteriostatic agent, such as sodium azide, 0.1 to 0.2%,
may also be incorporated into the sample collection and/or assay
medium to help avoid microbial growth.
[0090] Although a fundamental utility of the present invention lies
in the novel, non-invasive methods for obtaining biological samples
from mammary fluid, additional methods are disclosed herein that
provide useful assays for detecting and/or measuring important
breast disease markers in these samples. In this context, the
invention provides a broad range of assay methods incorporating
known procedures and reagents for determining the presence and/or
expression levels of breast disease markers, particularly breast
cancer markers, in biological samples. As incorporated within the
invention, these methods involve application of a breast pump 10,
10' to mammalian patients, optionally coupled with oxytocin
administration in amounts effective to facilitate mammary fluid
expression in the patient. After the sample is collected, a
bioassay is conducted on the sample to determine the presence
and/or amount of a selected breast disease marker, preferably a
breast cancer marker or panel of breast cancer markers, in the
sample.
[0091] As used herein, the term breast disease marker refers to any
cell, cell fragment, protein, peptide, glycoprotein, lipid,
glycolipid, proteolipid, or other molecular or biological material
that is uniquely expressed (e.g. as a cell surface or secreted
protein) by diseased breast cells, or is expressed at a
statistically significant, measurably increased or decreased level
by diseased breast cells, or in association with breast disease
(e.g. a protein expressed by an infectious agent associated with
breast disease), or is expressed at a statistically significant,
measurably increased or decreased level by diseased breast cells
compared to normal breast cells, or which is expressed by
non-diseased breast cells in association with breast disease (e.g.
in response to the presence of diseased breast cells or substances
produced therefrom). Breast disease markers can also include
specific DNA or RNA sequences marking a deleterious genetic change,
or an alteration in patterns or levels of gene expression
significantly associated with breast disease. Preferred breast
disease markers include markers of breast infections, benign
neoplasia, malignant neoplasia, pre-cancerous conditions, and
conditions associated with an increased risk of cancer.
[0092] As used herein, the term breast cancer marker refers to a
subset of breast disease markers, namely any protein, peptide,
glycoprotein, lipid, glycolipid, proteolipid, or other molecular or
biological material that is uniquely expressed (e.g. as a cell
surface or secreted protein) by cancerous cells, or is expressed at
a statistically significant, measurably increased or decreased
level by cancerous cells compared to normal cells, or which is
expressed by non-cancerous cells in association with cancer (e.g.
in response to the presence of cancerous cells or substances
produced therefrom). Breast cancer markers can also include
specific DNA or RNA sequences marking a deleterious genetic change,
or an alteration in patterns or levels of gene expression
significantly associated with cancer. In addition, breast cancer
markers can include cytological features of whole cells present in
mammary fluid, such as nuclear inclusions or cytoplasmic structures
or staining attributes uniquely expressed by, or associated with,
cancerous cells.
[0093] Among the breast cancer markers that are useful within the
methods of the invention, a subset are described in representative
review articles by Porter-Jordan et al., Hematol. Oncol. Clin.
North Amer. 8:73-100, 1994; and Greiner, Pharmaceutical Tech, May,
1993, pp. 28-44, each incorporated herein by reference in its
entirety. Other suitable markers are also widely known and can be
readily incorporated into the methods of the invention using
information and methods generally known or available in the
literature. Preferred breast cancer markers for use within the
invention include well characterized markers that have been shown
to have important value for determining prognostic and/or
treatment-related variables in human female patients. As noted
previously, prognostic variables are those variables that serve to
predict outcome of disease, such as the likelihood or timing of
relapse or survival. Treatment-related variables predict the
likelihood of success or failure of a given therapeutic program.
Determining the presence or level of expression or activity of one
or more of these markers can aid in the differential diagnosis of
patients with malignant and benign abnormalities, and can be useful
for predicting the risk of future relapse or the likelihood of
response to a selected therapeutic option.
[0094] It is important to note, however, that the invention does
not rely solely on breast disease markers that meet the stringent
requirements of sensitivity and specificity that would render the
marker immediately acceptable for clinical application to human
patients. On the contrary, a number of breast disease markers
contemplated within the invention fall short of these stringent
criteria, and nonetheless provide useful information that can be of
substantial benefit in detecting, differentially diagnosing or
managing breast cancer. Such non-clinically accepted markers are
useful for immediate application within the methods of the
invention as basic research tools, and as adjunctive tools in
clinical applications. Beyond these immediate applications, many
such markers are expected to be further developed and refined
according to the methods of the invention to the point of direct
clinical applicability, particularly in assay methods that analyze
combinations of markers to generate complementary data of greater
predictive value than data yielded by individual markers alone.
[0095] The preferred assay methods of the invention particularly
focus on breast cancer markers associated with tumorigenesis, tumor
growth, neovascularization and cancer invasion, and which by virtue
of this association provide important information concerning the
risk, presence, status or future behavior of cancer in a patient.
As noted previously, tumorigenesis and tumor growth can be assessed
using a variety of cell proliferation markers (for example Ki67,
cyclin D1 and PCNA). Tumor growth can also be evaluated using a
variety of growth factor and hormone markers (for example estrogen,
EGF, erbB-2, and TGF-a, receptors of autocrine or exocrine growth
factors and hormones (for example IGF and EGF receptors), or
angiogenic factors. In addition to tumorigenic, proliferation and
growth markers, a number of markers provide information concerning
cancer invasion or metastatic potential in cancer cells, for
example by indicating changes in the expression or activity of cell
adhesion or motility factors. Exemplary markers in this context
include Cathepsin D, plasminogen activators and collagenases. In
addition, expression levels of several putative tumor "suppressor"
genes, including nm23, p53 and rb, provide important data
concerning metastatic potential, or growth regulation of cancer
cells. A large number and variety of suitable breast cancer markers
in each of these classes have been identified, and many of these
have been shown to have important value for determining prognostic
and/or treatment-related variables relating to breast cancer.
[0096] Prior to or concurrent with each assay run of the invention,
it may be preferable to perform a preliminary evaluation to verify
sample origin and/or quality. 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.
[0097] 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 invention
based on published and generally known information. The most
preferred 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).
[0098] To conduct the breast disease marker assays provided within
the invention, a collected biological sample from mammary fluid is
generally exposed to a probe that specifically binds to a selected
breast disease or breast cancer marker, or otherwise interacts with
the marker in a detectable manner to indicate the presence or
absence, or amount, of the breast disease or breast cancer marker
in the sample. Selected probes for this purpose will generally
depend on the characteristics of the breast disease marker, i.e. on
whether the marker is a protein polynucleotide or other substance.
In preferred embodiments of the invention, the breast disease
marker is a protein, peptide or glycoprotein, all of which are
effectively targeted in breast disease marker assays using specific
immunological probes. These immunological probes can be labeled
with a covalently bound label to provide a signal for detecting the
probe, or can be indirectly labeled, for example by a labeled
secondary antibody that binds the immunological probe to provide a
detectable signal.
[0099] General methods for the production of non-human antisera or
monoclonal antibodies (e.g., murine, lagormorpha, porcine, equine)
are well known and may be accomplished by, for example, immunizing
an animal with a selected breast disease marker protein, peptides
synthesized to include part of the marker protein sequence,
degradation products including part of the marker protein sequence,
or fusion proteins including all or part of the marker protein
linked to a heterologous protein or peptide. Within various
embodiments, monoclonal antibody producing cells are obtained from
immunized animals, immortalized and screened, or screened first for
the production of an antibody that binds to the selected breast
cancer marker protein or peptide, and then immortalized. It may be
desirable to transfer the antigen binding regions (i.e., F(ab')2 or
hypervariable regions) of non-human antibodies into the framework
of a human antibody by recombinant DNA techniques to produce a
substantially human molecule. Methods for producing such
"humanized" molecules are generally well known and described in,
for example, U.S. Pat. No. 4,816,397 (incorporated herein by
reference in its entirety). Alternatively, a human monoclonal
antibody or portions thereof may be identified by first screening a
human B-cell cDNA library for DNA molecules that encode antibodies
that specifically bind to the selected breast disease marker
according to the method generally set forth by Huse et al. (Science
246:1275-1281, 1989 (incorporated herein by reference in its
entirety). The DNA molecule may then be cloned and amplified to
obtain sequences that encode the antibody (or binding domain) of
the desired specificity.
[0100] Also contemplated within the invention are bifunctional
antibodies having independent antigen binding sites on each
immunoglobulin molecule (as disclosed for example in Thromb. Res.
Suppl. X:83, 1990, and in The Second Annual IBC International
Conference on Antibody Engineering, A. George ed., Dec. 16-18,
1991; each incorporated herein by reference in its entirety), as
well as panels of individual antibodies having differing
specificities. Bifunctional antibodies and antibody panels of
particular use within the invention include antibodies and panels
of antibodies that bind to two or more selected breast disease
markers to generate complementary data of greater predictive value
than data yielded by individual markers alone.
[0101] Monoclonal antibodies are particularly useful within the
invention as labeled probes to detect, image and/or quantify the
presence or activity of selected breast disease markers. In this
context, monoclonal antibodies that specifically bind to selected
breast disease markers are provided which incorporate one or more
well known labels, such as a dye, fluorescent tag or radiolabel. By
incorporating such a label, the antibodies can be employed in
routine assays to determine expression, localization and/or
activity of one or more selected breast disease markers in a
biological sample including, or derived from, mammary fluid.
Results of these assays to determine expression, localization
and/or activity of a selected breast disease marker in a test
sample taken from a patient at risk for breast disease, or known to
have breast disease, can be compared to results from control
studies detecting and/or quantifying the same marker in biological
samples obtained from normal patients negative for breast disease.
In this manner, baseline data and cutoff values can be determined
according to routine methods to refine the assays of the invention
and adapt them for direct clinical application.
[0102] Detection and/or quantification of breast disease markers in
the biological samples of the invention can be accomplished using a
variety of methods. Preferred methods in this regard include well
known ELISA immunoassays, immunoprecipitation assays, and various
solid phase immunoassays including Western blotting, dot blotting
and affinity purification immunoassays, among other methods.
Comparable methods are disclosed herein, or are elsewhere disclosed
and known in the art, for using non-antibody probes to detect
and/or quantify the expression and/or activity of breast disease
markers. Suitable non-antibody probes for use within the invention
include, for example, labeled nucleotide probes that hybridize at
moderate or high stringency to DNA transcripts of oncogenes and
other DNA sequences associated with elevated breast disease risk,
or with mRNA transcripts encoding breast disease marker proteins.
Preferably, the nucleotide probes hybridize with a target sequence
under high stringency conditions. As used herein, "moderate
stringency" and high stringency" refers to finite ranges of
hybridization conditions that are well established in the
literature. (See, for example: Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor
Press, 1989; Hames and Higgins, eds., Nucleic Acid Hybridization: A
Practical Approach, IRL Press, Washington D.C., 1985; Berger and
Kimmel, eds, Methods in Enzymology, Vol. 52, Guide to Molecular
Cloning Techniques, Academic Press Inc., New York, N.Y., 1987; and
Bothwell, Yancopoulos and Alt, eds, Methods for Cloning and
Analysis of Eukaryotic Genes, Jones and Bartlett Publishers,
Boston, Mass. 1990; each of which is incorporated herein by
reference in its entirety. Moderate or high stringency
hybridization conditions are achieved, e.g., by adjusting the
temperature of hybridization, adjusting the percentage of
helix-destabilizing agents such as formamide in the hybridization
mix, and adjusting the temperature and salt concentration of the
wash solutions. Alternatively, stringency can be adjusted during
post-hybridization washes by varying the salt concentration and/or
the temperature. Stringency of hybridization may be reduced by
reducing the percentage of formamide in the hybridization solution
or by decreasing the temperature of the wash solution. Typical high
stringency conditions require, for example, high temperature
hybridization (e.g., 65-68.degree. C. in aqueous solution
containing 4-6.times.SSC, or 42.degree. C. in 50% formamide)
combined with a high temperature (e.g., 5-25.degree. C. below the
T.sub.m) wash and a low salt concentration (e.g., 0.1.times.SSC).
In contrast, moderate stringency conditions involve, for example,
hybridization at a temperature between 50.degree. C. and 55.degree.
C. and washes in 0.1.times.SSC, 0.1% SDS at between 50.degree. C.
and 55.degree. C., which should be sufficient to identify
polynucleotide molecules encoding I-mf from other species or to
isolate isoforms of 1-mf. In further contrast, low stringency
conditions involve, for example, low hybridization temperatures
(e.g., 35-42.degree. C. in 20-50% formamide) and intermediate
temperature (e.g., 40-60.degree. C.) washes in a higher salt
concentration (e.g., 2-6.times.SSC).
[0103] In certain preferred embodiments of the invention, cDNA and
oligonucleotide probes are employed in well known Northern,
Southern and dot-blot assays for identifying and quantifying the
level of expression of a selected breast disease marker in cell
samples collected from expressed mammary fluid.
[0104] Other suitable probes for use within the invention include
labeled ligands, binding partners and co-factors of breast disease
markers (e.g. growth factor receptor ligands, or substrates of
breast cancer associated proteases such as cathepsin D).
[0105] Measuring the level of expression of breast disease markers
according to the foregoing methods will provide important
prognostic and treatment-related information for assessing a broad
range of breast disease, including the genesis, growth and
invasiveness of cancer, in mammals, particularly humans. For
example, assays utilizing oligonucleotide probes will assist early
screening to evaluate heritable genetic lesions associated with
breast cancer, and to distinguish between pre-cancerous, early
cancerous and likely metastatic lesions in patients.
[0106] In addition to the above mentioned sample collection and
assay methods, the invention also provides kits and multicontainer
units comprising devices, components, accessories, reagents and
other related materials for practicing the sample collection and
assay methods of the invention. These kits are clinically useful
for collecting, handling and/or processing mammary fluid samples,
e.g., for determining the presence and/or amount of a breast
disease marker, preferably a breast cancer marker, in the
biological samples. The kits include a mammary fluid collection
device having a breast engaging member, vacuum pump, and sample
collection means incorporated in a general purpose or hand-held
breast pump as described herein. Additional kits include one or
more breast pump attachments (e.g., a detachable breast engaging
member, or multiple such attachments for use with different
patients), accessories (e.g., replaceable fluid-retaining
reservoirs), solid phase media, and/or disposable or reusable
support members, cartridges or cassettes for holding collection
media, as described herein. These and other kit components may be
provided, alone or in any combination, with or without inclusion of
the basic breast pump apparatus in the kit. Yet additional kits
include a pharmaceutical preparation of oxytocin or an oxytocin
analog in a biologically suitable carrier for use in alternate
mammary fluid collection methods of the invention. Preferably, the
oxytocin preparation is provided in an intranasal spray applicator
and contains approximately 40 USP units of oxytocin per ml of
liquid carrier, which carrier is a simple, inexpensive buffered
saline solution. Preferred applicators can be in any of a variety
of pressurized aerosol or hand-pump reservoir forms, with a nozzle
for directing a liquid spray of the oxytocin into a patient's
nostril. Still other kits include on or more preparative and/or
diagnostic reagents selected from those disclosed herein, including
one or more fixatives, probes, labels and the like in separate or
common containers. In certain embodiments of the invention, kits
include compositions and/or devices for detecting the presence or
amount of one or more breast disease marker(s) in the biological
sample, often including one or more immunological or molecular
probe(s) that binds or reacts with one or more breast cancer
marker(s). The foregoing kit components are generally assembled in
a collective packaging unit, which may include written or otherwise
user-accessible instructions detailing the sample collection,
handling and/or processing methods of the invention.
[0107] Kits for practicing the assay methods of the invention
include a suitable container or other device for collecting,
storing, handling and/or processing a biological sample from
expressed mammary fluid. A range of suitable collection devices is
contemplated corresponding to a wide range of suitable biological
samples that may be collected from the expressed mammary fluid. For
example, simple sterile containers or reservoirs are provided to
collect whole mammary fluid. Alternatively, a variety of solid
phase devices, including microscopic glass slides, membranes,
filters, beads and like media, are provided to receive or partition
selected liquid or solid fractions of the mammary fluid, to receive
or partition cells or cellular constituents from the mammary fluid,
or to receive or partition purified or bulk proteins,
glycoproteins, peptides, nucleotides (including DNA and RNA
polynucleotides) or other like biochemical and molecular
constituents from the mammary fluid. A wide variety of such sample
collection devices are disclosed herein, or are otherwise widely
known or described in the literature, which can be readily adapted
for use within specific embodiments of the invention. These
collection devices may be provided as a component of the breast
pump (such as a removable nitrocellulose filter placed within the
pump, optionally coupled there with by a permanent or removable
support member, to directly receive or contact the expressed
mammary fluid as it is pumped), or may be provided separately (for
example as a non-integral membrane, filter, affinity column or
blotting material to which mammary fluid or mammary fluid
components are exposed to collect a biological sample for assay
purposes). In more detailed aspects, the collection device includes
a removable, fluid-retaining reservoir as described herein
below.
[0108] In certain embodiments of the invention kits include
reagents and/or devices for detecting the presence and/or amount of
a breast disease marker in the biological sample, for example an
immunological or molecular probe that binds or reacts with a breast
cancer marker. Among these possible reagents immunological and
non-immunological probes for detecting the presence or amount of a
breast cancer marker in the biological sample. The kits may also
contain suitable buffers, preservatives such as protease
inhibitors, direct or sandwich-type labels for labeling the probes,
and/or developing reagents for detecting a signal from the label.
In one aspect, kits of the present invention contain monoclonal
antibodies useful for detecting and/or measuring a breast cancer
marker in a sample. Such antibodies may be pre-labeled, or may be
detected by binding to a secondary antibody optionally included in
the kit. The antibody reagents may be provided in a separate
container, or may be provided in combination in a series of
containers. Within yet another aspect of the invention, kits
contain sequence-specific oligonucleotide primers for detecting
polynucleotide molecules encoding breast cancer marker proteins.
Such primers may be provided in separate containers, or may be
provided in combinations of one or more primer pairs in a series of
containers. A broad selection of other kits are provided within the
invention based on general knowledge in the art and on the
description herein, including kits that contain specific
instructions for carrying out the assays of the invention.
[0109] Also provided within the invention are methods for obtaining
a biological sample from a patient and/or determining the amount of
a breast disease marker in a biological sample from breast fluid,
which methods employ a novel breast pump 10 or breast pump adapter
12, as described herein below. These methods include a step of
applying the breast pump to induce breast fluid expression, wherein
a solid phase sample collection medium is fluidly connected with
the breast pump. The solid phase sample collection medium may be
integrated within the breast pump or otherwise fluidly connected
with the pump, so that an expressed breast fluid sample contacts
the collection medium while the pump remains applied to the
breast.
[0110] To practice these aspects of the invention, the breast pump
10 (FIG. 1) and breast pump adapter 12 (FIG. 12) each have fluidly
connected therewith a solid phase sample collection medium selected
from any of the solid phase media described herein above. The
breast pump may be generally constructed according to various
conventional breast pump designs, for example according to the
general design described in U.S. Pat. No. 4,929,229 and U.S. Pat.
No. 5,007,899 to Larsson; U.S. Pat. No. 5,601,531 to Silver; U.S.
Pat. No. 3,786,801 to Sartorius; or U.S. Pat. No. 5,295,957 to Aida
et al.
[0111] As with other conventional breast pumps, the breast pump 10
of the invention includes a breast engaging portion 14 constructed
of a non-porous material. The engaging portion is sized and
dimensioned to receive at least a nipple 16 portion of a breast 17
and form a suction seal therewith. Preferably, the breast engaging
portion is sized and dimensioned to receive at least an areolar
portion of the breast, and more preferably a distal quarter to
one-half or larger portion of the breast (e.g., as shown in FIG.
1), and form a suction seal therewith. Different sizes and
dimensions of the breast engaging member may be selected, e.g., to
receive human breasts of differing sizes. Alternatively, devices
for veterinary use are provided wherein the breast engaging member
is sized and dimensioned to receive a breast of a non-human mammal.
To form a suction seal with the breast 17 as described above, the
breast engaging portion 14 of the pump 10 may be constructed in a
variety of shapes and dimensions. In one embodiment the engaging
portion is formed as a simple cylinder, tube or funnel shaped and
dimensioned to engage the nipple 16 or areolar portion of the
breast in a suction seal. Preferably, a terminal edge 18 of the
engaging portion is rounded or flared so that the edge does not
impinge uncomfortably against the skin of breast 17 when negative
pressure is applied to the breast to form the suction seal. In
preferred embodiments the engaging portion is roughly funnel shaped
to comfortably engage a distal quarter to one-half or larger
portion of the breast, as shown in FIG. 1 and form a suction seal
therewith.
[0112] The breast engaging portion 14 of the breast pump 10 can be
constructed of any suitable non-porous material which is inert to
body fluids and which has sufficient rigidity to prevent collapse
of the engaging portion when negative pressure is applied against
its inner walls 20. Preferably, the engaging portion and other
parts of the breast pump are autoclavable for sterilization
purposes. Thus, the engaging portion may be constructed of a rigid
material such as a polypropylene, polyurethane, polyvinyl plastic,
polymethyl methacrylate, and the like. Alternatively, the engaging
portion may be constructed of a semi-rigid material which prevents
collapse but allows for manual compression of at least a base 22 of
the engaging portion to massage the nipple 16 and/or areolar region
of the breast 17 to facilitate breast fluid expression. Suitable
materials in this context include rubber or synthetic elastomers,
e.g., silicon plastic (silastic) and like materials. Preferably,
the material that forms the engaging portion is transparent to
allow a physician or technician using the breast pump to visualize
the breast 17 to determine its positioning and condition during
application of the pump and to observe fluid expression from the
nipple.
[0113] The breast engaging portion 14 of the breast pump 10 is
fluidly connected to a sample collection housing 30 made of a rigid
material (preferably transparent plastic). The solid phase sample
collection medium, as described above, is supported in fluid
connection with the breast engaging portion, for example by
anchoring the solid medium to, or within, the breast engaging
portion or sample collection housing. Typically, the solid phase
sample collection medium is affixed within an interior compartment
or lumen 58 of the sample collection housing or corresponding,
fluidly connected, interior space of the breast engaging
portion-using any of a wide range of optional anchoring or
positioning means. Preferably, the solid phase medium is removably
supported in fluid connection with the breast engaging portion,
e.g., by means of a closeable retainer or replaceable cassette (see
below).
[0114] In one aspect of the invention the sample collection housing
30 or breast engaging portion 14 supports a sample collection pad,
or sheet, 38 of absorbent or adsorbent material, for example a
membrane 39 or filter 40 pad or sheet (FIGS. 2-5). Multiple pads or
sheets (of the same or different material) may be used in
combination. For example, a membrane 39 (e.g., nitrocellulose) may
be supported on a filter 40 (e.g., a paper filter) as shown in FIG.
5. In this manner, a first sheet may serve as a support member, a
wetting member, a wicking member, or a partitioning member for a
second sheet, or may introduce or remove a chemical reagent, probe,
blocking agent, buffering agent, denaturing agent, etc. therefrom.
In one aspect, the multiple sheet materials partition components of
the breast fluid (e.g., by using different materials to retain
different components of the breast fluid), thereby allowing for
collection of different samples simultaneously.
[0115] In another aspect of the invention the housing 30 or breast
engaging portion 14 supports a particulate solid phase sample
collection medium 41, for example beads, resins, microspheres,
particulate chromatographic media (e.g., agarose or silicate
media), and the like (see, e.g., FIG. 9). In yet another aspect of
the invention, the housing or breast engaging portion supports a
non-particulate solid template for sample collection, for example
one or more capillary tubes 42 (FIG. 6), coated tubes 43 (FIG. 10),
plates, wells, slides and the like formed of glass, plastic or
other suitable materials.
[0116] As shown in FIGS. 1 and 2, a preferred design of the breast
pump 10 includes a removable coupling mechanism between the
engaging portion 14 and the sample collection housing 30. A
preferred coupling mechanism includes complementary threads 44, 46,
disposed at mated connecting ends 48, 50 of the engaging portion,
and housing, respectively. Alternatively, a simple pressure fit
coupling may be provided to removably couple mated connecting ends
48, 50 of the engaging portion and housing, as shown in FIG. 9. In
yet another alternative embodiment, the connecting ends 48, 50 are
removably coupled by a hinge 52 and latch 54 that pivotally
connects the two connecting ends (FIG. 10).
[0117] The sample collection housing 30 or breast engaging portion
14 can support the solid phase sample collection medium in several
ways, as exemplified in the drawings and by a variety of equivalent
designs and configurations that will be apparent to the artisan. In
preferred embodiments of the invention, the solid phase medium is
held on or within a support member 56 adapted to support the solid
phase medium in fluid connection with the breast engaging portion,
for example a support that is fixedly interposed between the
engaging portion 14 and the sample collection housing.
[0118] Thus, in one exemplary design shown in FIGS. 1 and 2, the
support member 56 is a removable disc spanning a lumen 58 of the
housing and interposed between connecting ends 48, 50 of the
engaging portion and housing. For use in conjunction with a variety
of breast pump designs, a diameter 59 (FIG. 3) of the support
member is between about 1/4-3.0 inches, preferably about 1/2-2.0
inches, and more preferably about 3/4-1 inches. In preferred
aspects, the disc-shaped support member seats within a
circumferential groove 60 in the connecting end 48 of the housing.
A complementary circumferential groove 62 in the connecting end 46
of the engaging portion opposes the circumferential groove in the
connecting end of the housing to sandwich the disc-shaped support
member therebetween.
[0119] In this embodiment, prior to connecting the engaging portion
14 of the breast pump 30 with the housing 30, the support member is
seated therebetween (e.g. by fitting the support member within the
opposing circumferential grooves 60, 62 of the housing and engaging
portion). The force of connection (i.e. threading, pivoting or
pushing the engaging portion and housing relative to one another)
firmly sandwiches the support member in position between the
engaging portion and housing.
[0120] To facilitate this purpose, the thickness (i.e., sectional
height) 63 of the support member 56 in the present embodiment is
equal to or slightly greater than the height of a sidewall 64 of
the circumferential groove 60 of the housing 30, whereby the
support member is held in a friction fit and may be partially
compressed when the engaging portion and housing are connected.
Thus, the thickness of the support member is between about 2 mm to
5 cm, preferably about 3 mm to 2 cm, and more preferably about 4 mm
to 1 cm. Consistent with this design, the support member can be
made of a hard plastic material (e.g., a hard polyvinyl or
polyurethane), but is preferably made of a resilient, moderately
compressible material, e.g., soft plastic, rubber, or a waterproof
fiber or composite material as used in conventional plumbing and
automotive gaskets.
[0121] A disc-shaped support member 56 is well suited to support a
sheet 38 of absorbent or adsorbent material, such as a membrane or
filter. As shown in FIGS. 2 and 7, the sheet is preferably
sandwiched between an upper retainer ring 66 and a lower retainer
ring 68 of the support member to hold the sheet in place against
negative pressure that may pass through the filter when a vacuum is
applied through the engaging portion 14 and housing 30 (see below),
as well as when the nipple 16 impinges against the sheet. The upper
and lower retainer rings may be integrally joined in a disposable
refill as shown in FIG. 2, or the two retainer rings may be
separable to provide a reusable cassette for removing and inserting
replacement sheets. An example of the latter design is depicted in
FIG. 7, where the upper and lower retainer rings are releasably
interconnected, e.g., by a hinge 71 or other connecting means such
as an interlocking threading or detent fit mechanism. In this
embodiment the upper and lower rings can be opened or disconnected
to allow insertion and removal of the sheet, and juxtaposingly
closed, e.g., by a snap 72 on one ring adapted to form a detent fit
within a receptacle 74 on the opposing ring, thereby holding the
sheet in a fixed position between the two rings. To facilitate this
purpose, opposing faces 75 of the upper and lower rings may have a
rugose or otherwise decorated surface to facilitate retention of
the sheet, for example a ridge 76 or ridges to engage the sheet and
securely clamp the sheet between the two rings.
[0122] In an alternative design depicted in FIGS. 4 and 5, there is
no upper retaining ring 72 and the sheet 38 simply rests upon the
support member 56 or is removably retained against an upper surface
76 of the support member by alternative retaining means. For
example, the sheet may be fitted within a recess 78 surrounding the
upper surface of the support member that is shaped and dimensioned
to receive the sheet. The sheet may be securely fitted within the
recess, e.g., by appropriately sizing the sheet so an edge of the
sheet frictionally engages a sidewall 79 of the recess.
Alternatively, a retaining groove may be provided between the
sidewall of the recess and the upper surface of the support member
to receive the edge of the sheet and thereby retain the sheet by a
detention fit within the recess during use. In yet another
alternative design, the sheet simply rests atop the upper surface
of the support member and is removably secured thereto, e.g., by
wetting or gluing (preferably with an inert bonding agent) to
create a temporary bond between the sheet and upper support member
surface. In each of the foregoing designs, the sheet can be easily
seated within or atop the housing for sample collection and removed
thereafter for processing, e.g., by hand or using forceps or other
conventional handling tools.
[0123] In preferred embodiments of the invention, the support
member 56 includes a recess 78 which forms a fluid-retaining well,
as shown in FIG. 5. The recess can thus be filled with a desired
solution, such as a buffer, a solution containing a probe,
cross-linking agent, blocking agent, denaturing agent, etc., to
facilitate sample collection, handling, and/or processing.
[0124] Where the design of the support member 56 is such that it
spans the lumen 58 of the sample collection housing 30 or
corresponding interior compartment of the breast engaging member
14, or when the support member contains a recess 78 forming a well,
it is generally desirable to provide air channels 80 in the support
member 56 to allow negative vacuum pressure to pass from the
housing through the air channels to the engaging portion 14 of the
pump during operation, and to allow venting of the engaging portion
and housing to permit disengagement of the engaging portion from
the breast 17 after use. Preferably, one or more such air channels
are located near the periphery of the support member, as shown in
FIGS. 2, 3, 5, 6 and 9. Alternatively, one or more air channels may
be centrally located, as shown in FIG. 4. The air channels may be
positioned so that they do not communicate with the solid phase
sample collection medium, as shown in FIGS. 2, 3, 5, 6 and 9, or
they may communicate and form a gaseous connection therewith
(provided that the solid phase medium is porous and has sufficient
strength to withstand vacuum pressures transmitted through the air
channel), as shown in FIG. 4.
[0125] Alternative designs and configurations of the housing 30,
breast engaging member 14, and/or support member 56 are also
provided which vary with the type of solid phase sample collection
medium used. For example, when a particulate solid phase sample
collection medium 41 (e.g. beads, resins, or microspheres) is used,
the medium may be enclosed in a cartridge 82 removably mounted to,
or integrated within, the support member or otherwise removably
connected to the sample collection housing 30 or breast engaging
portion. As shown in FIG. 9, preferred embodiments of the invention
provide a removable engagement mechanism which allows the cartridge
or other receptacle containing the solid phase medium to be
removably engaged relative to the housing or breast engaging
member, e.g., by engaging the cartridge with a support member so
that a first end of the cartridge makes a fluid connection with the
engaging portion 14 of the pump 10. In one embodiment, the first
end of the cartridge is removably inserted through a mounting
channel 86 which passes through the support member to provide a
fluid connection between the engaging portion of the pump and the
cartridge first end. Preferably, the channel is dimensioned to
receive the first end of the cartridge in a friction fit (e.g.,
wherein a diameter of the channel is about 0.5 mm to 2 cm,
preferably about 1 mm to 1 cm, and more preferably about 3-5 mm),
whereby the cartridge can simply be pushed into the channel until
the cartridge first end is flush with, or extends slightly above,
the upper surface 76 of the support member and will remain in place
during use. For this purpose it is also preferable to form at least
the channel portion of the support member from a resilient,
moderately compressible material so that the channel yieldingly
receives and releases the cartridge in a moderate (i.e., readily
hand removed) friction fit. Alternatively, the cartridge can be
engaged relative to the housing by complementary threading or
interlocking detent fitting (e.g., a conventional key and groove
design) between the cartridge first end and the support member
channel). In yet other alternative designs the cartridge can be
permanently engaged with the support member or engaged directly to
the housing.
[0126] Design and construction of the cartridge 82 will vary
depending on the characteristics of the particulate solid phase
medium used, including the size of the particles, the function of
the particles (e.g., chromatography adsorption, affinity binding,
etc.), and whether the particles are used dry or are contained in a
solution, among other factors. Design and construction of the
cartridge will further depend on the type of breast disease
marker(s) which may be sought for detection in the sample (e.g.,
cells, proteins, lipids or nucleic acids).
[0127] In a preferred embodiment shown in FIG. 9, the cartridge is
cylindrical and contains beads or microspheres. To enclose the
beads or microspheres in the cylinder while maintaining a fluid
connection with the engaging portion 14 of the pump 10, the first
end 84 of the cylinder is covered by a semi-permeable cover 90 of a
porous barrier material (e.g., a filter or membrane) which allows
breast fluid (including or excluding selected components of the
fluid, such as cells) to pass through the cover to contact the
beads or microspheres, while preventing escape of the beads or
microspheres from the cartridge. In this manner, the cover can
partition components of the breast fluid into the cartridge, and
can also separately retain different components on the cover,
thereby allowing for collection of different samples
simultaneously. The semi-permeable cover can be affixed to the
cartridge by a variety of means, e.g., by bonding with a removable
or permanent bonding agent, or by providing a removable or integral
cover retaining ring 92 to secure the cover to the cartridge first
end 84. A second end of the cartridge features a second end cover
96 which may be integral to or removable from the cartridge, and
which may be impermeable to gas and fluids or semi-permeable as
described above for the first end cover.
[0128] In another aspect of the invention, the housing 30 supports
a non-particulate solid template for sample collection. This type
of solid phase collection medium includes, e.g., one or more
capillary tubes 42 (FIG. 6), coated tubes 43 (FIG. 10), plates,
wells, slides and the like. These templates for receiving,
adsorbing or binding a sample of breast fluid (or desired
components thereof) are preferably formed of glass, plastic or like
materials known in the art to be suitable for sample collection
(e.g., inert plastics).
[0129] To accommodate these various templates, yet additional
alternative designs and configurations of the housing 30, breast
engaging portion 14, and/or support member 56 are provided. For
example, when capillary tubes 42 are used, these may be mounted to
or integrated within the support member, or anchored by a variety
of other comparable means with respect to the housing 30. As shown
in FIG. 6, preferred embodiments of the invention utilize a support
member with one or more mounting channels 86 to removably receive a
first end of one or more capillary tubes 42, so that the end of the
tube makes a fluid connection with the engaging portion 14 of the
pump 10. Thus, the channels have a preferred diameter equal to or
slightly less than a diameter of a standard capillary tube, i.e.,
about 0.5 mm to 3 mm, preferably about 1-2 mm and more preferably
about 1.5 mm. Construction of the support member and mounting of
the tube(s) is similar to support member construction and mounting
of the cartridge as described above. When a single tube is used, it
is preferably placed centrally relative to the housing. When
multiple tubes are used they may be arrayed to collect multiple
samples simultaneously, e.g., as shown in FIG. 6.
[0130] Another alternative solid template for sample collection
provided within the invention is a coated tube 43 which is
preferably mounted relative to the housing 30 in the same manner as
described above for capillary tubes 42 (FIG. 10). The tube may be
open at both ends, or may have a semi-permeable cover at one or
both ends, as well as an impermeable second end cover, as described
above for the cartridge 82.
[0131] The coated tube has a lumenal coating 100 adapted for
adsorbing, binding, partitioning or otherwise processing the breast
fluid sample. For example, the coating may be an affinity coating
having an antibody, ligand, or other binding partner that
specifically binds a selected breast disease marker, wherein the
coating is covalently or otherwise bound to a lumenal wall of the
tube. A wide variety of useful coatings are disclosed herein or are
otherwise well known in the art. These coatings may also be used to
coat other solid phase media for use within the invention,
including templates such as wells, plates, slides, etc, including a
well formed by a recess 78 in a support member 56.
[0132] Because only small droplets of breast fluid will typically
be expressed at the surface of the nipple 16, it is generally
preferred to directly contact the expressed fluid on the nipple
with the solid phase sample collection medium. This requires
positioning of the sample collection medium close to the base 22 of
the breast engaging portion 14 of the pump as shown in the figures.
Thus, when a support member 56 is provided it is positioned so that
its upper surface 76 will directly contact the nipple during
application of negative pressure through the engaging portion to
the breast. Only approximate positioning is generally required in
this regard, because the nipple will tend to be drawn toward the
support member by the vacuum and thereby will abut the upper
support member surface.
[0133] However, because breast pump designs and breast anatomy vary
significantly, it is preferable to adjustably mount the solid phase
medium relative to the housing 30 so that it can be moved closer
to, or farther away from, the base 22 of the engaging portion 14 of
the pump 10. Thus, in preferred embodiments of the invention a
reciprocating mechanism is provided which adjustably moves the
solid phase collection medium in closer, or more distant, proximity
to the nipple when the breast pump is engaged therewith. At the
beginning of the fluid expression procedure, the collection medium
is retracted away from the nipple while negative pressure is
applied to the breast to facilitate fluid expression. Fluid
expression is visualized through a transparent engaging portion or
housing, and the collection medium is then advanced proximal to the
nipple to contact the expressed fluid.
[0134] As shown in FIG. 11, a preferred design for the
reciprocating mechanism incorporates a support member 56 to support
the solid phase collection medium, as described above. The support
member is reciprocatingly mounted relative to a rotating member 109
of the housing 30, preferably on a reciprocating carrier 110. The
support member may be removably mounted to the carrier, e.g., by
friction fitting, detention fitting or threadedly engaging the
support member to a first end 112 of the carrier, as described
above for mounting the support member to the housing and/or
engaging portion 14 of the pump 10. For example, the support member
may be mounted by friction fitting within a circumferential groove
114 at the first end of the carrier. In conjunction with this
design, the carrier is preferably in the form of an open cylinder
so that negative pressure can be effectively transmitted through
the carrier and support member to the engaging portion.
[0135] To reciprocatingly adjust the position of the carrier 110
and/or support member 56 relative to the engaging portion 14 of the
pump 10, the rotating member 109 of the housing 30 is sealably,
rotatably, and removably interconnected to the base 22 of the
engaging portion. This interconnection may be accomplished by a
variety of designs, one of which is to seat a first O-ring 116 in
opposing circumferential grooves 118, 120 in the connecting ends
48, 50 of the engaging portion, and the rotating member of the
housing, respectively. These grooves are sized and dimensioned to
receive the O-ring in an airtight seal when vacuum pressure is
applied through the housing and engaging portion of the pump,
without substantially compressing the O-ring. The O-ring is also
lubricated, e.g., with silicon grease. These features allow free
rotation of the rotating member of the housing relative to the
engaging portion of the pump, which rotation drives the
reciprocating mechanism to advance the sample collection medium
(e.g., by advancing the carrier and/or support member) to contact
the expressed breast fluid on the nipple 16.
[0136] To complete the reciprocating mechanism for the above
described embodiment of the invention, the rotating member 109 of
the housing 30 is also sealably and rotatably interconnected to a
stationary member 124 of the housing. This interconnection is
preferably achieved by seating a second O-ring 126 in opposing
circumferential grooves 128, 130 in a rear connecting end 132 of
the rotating member of the housing and a front connecting end 134
of the stationary member 124 of the housing, respectively. These
grooves are also sized and dimensioned to receive the O-ring in an
airtight seal without substantially compressing the O-ring, and the
O-ring is lubricated to facilitate free rotation of the rotating
member relative to the stationary member.
[0137] To reciprocate the carrier 110 and/or support member 56
forward and backward relative to the engaging portion 14, the
rotating member 109 of the housing 30 is provided with a lumenal,
helically oriented groove 140 dimensioned to receive a riding peg
142 extending transversely from the carrier or support member. In
addition, the rotating member of the housing is provided with a
longitudinally oriented, lumenal groove 144 dimensioned to receive
an angularly fixating keel 146 extending transversely from the
carrier or support member. In accordance with this design, rotation
of the rotating member 109 of the housing 30 drives rotation of the
carrier or support member which is angularly fixed relative to the
rotating member by the fixating keel engaged with the longitudinal
groove of the rotating member. As the rotating member of the
housing and carrier thus rotate (with the position of the engaging
portion and stationary member of the housing angularly fixed by
friction or manual or structural resistance), the riding peg rides
along the helical groove, translating the peg in the direction of
the groove and thereby causing the support member or carrier to
reciprocate forward or backward relative to the engaging
portion.
[0138] To insert and remove the solid phase medium and/or support
member 56 from the rotating member 109 of the housing 30, a
removable interconnection is provided between the rotating member
and the base 22 of the engaging portion, as described above. To
uncouple the rotating member and engaging portion, all that is
required is that these parts be pulled in opposing directions,
whereby the O-ring 116 will unseat from one of the opposing
circumferential grooves 118, 120 in the connecting ends 48, 50 of
the engaging portion and rotating member, respectively. To recouple
the rotating member and engaging portion after loading or retrieval
of the sample collection medium and/or support member, they are
simply pushed back together. To facilitate reseating of the O-ring,
it may be desired to make one of the opposing circumferential
grooves deeper than the other, so that the deeper groove retains
the O-ring when the rotating member and engaging portion are
separated, and the shallower groove more readily accepts the O-ring
when they are re-coupled.
[0139] An alternative reciprocating mechanism is provided within
the invention which uses a simple slide mechanism to reciprocate
the sample collection medium relative to the engaging portion 14 of
the pump 10, as shown in FIGS. 14 and 15. One embodiment of the
slide mechanism features a manifold 150 defining an inner lumen 152
which is not in gaseous connection with an outer lumen 154 of the
housing. This design provides for a manual slide lever 156 to
extend to the outside of the housing so that a head portion 158 of
the lever can be manually engaged by a pump operator. The slide
lever is in turn connected to the support member 56 or carrier 110
which are sized and dimensioned to allow the carrier to reciprocate
freely within the inner lumen.
[0140] In operation, the slide lever 156 is moved to a rearward
position so that the solid phase sample collection medium (e.g., a
pad or sheet 38 of absorbent material) is out of contact with the
nipple 16, as shown in FIG. 14. Negative pressure is applied
through the outer lumen 154 to the area of the breast surrounding
the nipple, the tip of which is aligned with the inner lumen.
Breast fluid expression is visualized through the transparent
engaging portion and housing, at which time the lever is manually
engaged by the head portion 158 and moved forward. Movement of the
lever causes the support member and/or carrier to move forward
until the sample collection medium contacts the expressed fluid at
the tip of the nipple. The engaging portion and housing are
removably connected, e.g., by a hinge 52 and latch 54 or other
suitable connection means, thereby allowing for easy insertion and
removal of the solid phase medium and/or support member.
[0141] In each of the foregoing breast pump designs, the engaging
portion 14 of the breast pump 10 is in gaseous connection with a
vacuum pump 160 capable of generating sustained negative pressure
in an area of the breast 17 surrounding the nipple. 16 (see FIG.
1). Any of a large variety of vacuum pumps, which are well known
for use in conjunction with breast pumps, can be used, including
manual pumps (FIG. 1), mechanically driven pumps and electrically
driven pumps. When activated, the pump generates negative pressures
of between about 50-200 mm Hg. Typically the pump will be connected
via a heavy vacuum hose 162 in connection with the engaging
portion. Generally, the hose is connected to the housing 30 which
will is in gaseous connection with the engaging portion (see, e.g.,
FIGS. 1, 8 and 11).
[0142] Pressure exerted upon the breast 17 by the pump can be
varied in accordance with well known pressure modulating mechanisms
(e.g., by providing a diaphragm or other mechanism to modulate a
diameter of an in line, pressure modulating valve). In addition,
the breast pump 10 includes a venting mechanism, e.g., a pressure
release valve 164, which the user can selectively operate to close
and vent the system before and after use, thereby selectively
applying and releasing the vacuum pressure acting on the breast. In
this regard, the system is generally vented as soon as sufficient
breast fluid expression is observed by the operator. This also
relieves pressure on seals (e.g., O-rings 116, 126), when the
reciprocating mechanism relies on a sealable and rotatable
connection between different parts of the pump (as in FIGS. 11 and
13), thereby facilitating respective rotation of the different
parts to reciprocate the support member 56 and/or carrier 110.
[0143] In yet another aspect of the invention, a breast pump
adapter 12 is provided which couples a solid phase sample
collection medium with a conventional breast pump (See FIGS. 12 and
13). As shown in FIG. 12, the adapter features a replacement breast
engaging portion 170 sized and dimensioned for removable insertion
within a breast engaging portion 14 of a conventional breast pump.
In preferred embodiments, the replacement breast engaging portion
is funnel shaped and nests within a funnel shaped breast engaging
portion of an existing breast pump. When fully nested, a terminal
edge 172 of the replacement breast engaging portion extends at
least as far as the terminal edge 18 of the breast engaging portion
of the existing breast pump.
[0144] The replacement breast engaging portion. 170 can be
removably connected to the breast engaging portion 14 of the
existing breast pump 10 by a variety of means, e.g., by friction
fitting, detention fitting or threadedly engaging the replacement
engaging portion with the breast engaging portion of the existing
pump. Preferably, the adapter 12 has a stem portion 174 that
extends into a cylindrical, connecting portion 176 of the existing
pump, and the stem portion cooperates with this part of the
existing pump to provide a removable connection mechanism. Thus, in
one preferred embodiment the stem portion features a
circumferential groove 178 dimensioned to receive an O-ring 180,
which O-ring impinges against an inner wall 182 of the connecting
portion to create a friction fit to interconnect the replacement
engaging portion with the breast engaging portion of the existing
pump.
[0145] The adapter 12 supports a solid phase sample collection
medium in fluid connection with the replacement engaging portion
170. Preferably, the solid phase medium is connected with the
replacement engaging portion by a support member 56, as described
above. The support member may be integrally or removably mounted to
the adapter, e.g., by friction fitting, detention fitting or
threadedly engaging the support member to the stem 174 of the
replacement engaging portion, in a position that will allow contact
between the nipple and solid phase medium during or after breast
fluid expression. For example, the support member may be mounted by
friction fitting within a circumferential groove 184 at a base of
the stem (FIG. 12).
[0146] In preferred embodiments of the breast pump adapter 12, a
reciprocating mechanism is provided to move the solid phase sample
collection medium relative to the replacement engaging portion 172,
in accordance with the concepts described above. As shown in FIG.
13, a preferred design for the adapter having a reciprocating
mechanism features a replacement breast engaging portion 170
sealably and rotatably nested within a rotating dial member 190,
which is in turn sized and dimensioned for removable insertion
within a breast engaging portion 14 of an existing breast pump. The
replacement breast engaging portion and rotating dial member are
preferably funnel shaped to collectively nest within a funnel
shaped breast engaging portion of a conventional breast pump. When
fully nested, a terminal edge 172 of the replacement breast
engaging portion and free edge 192 of the rotating dial member
extend at least as far as the terminal edge 18 of the breast
engaging portion of the existing breast pump.
[0147] The rotating dial member 172 is connected to a rotating
member 109 of the housing 30, preferably as a unitary insert,
whereby manual rotation of the dial member drives rotation of the
rotating member of the housing. The rotating member of the housing
is in turn rotatably coupled with an anchoring member 194 of the
housing which anchors the entire housing within the existing pump,
e.g., within a cylindrical, connecting portion 176 of the existing
pump. As shown in FIG. 13, the anchoring member of the housing is
preferably in the form of a sleeve which partially surrounds the
rotating member of the housing and is sealably, rotatably connected
therewith. The anchoring member is in turn non-rotatingly anchored
within the cylindrical, connecting portion of the existing
pump.
[0148] In one preferred embodiment the rotating member 109 of the
housing is sealably, rotatably connected with the anchoring member
194 of the housing by seating a first O-ring 196 in opposing
circumferential grooves 198, 200 at front connecting ends 202, 204
of the rotating member and the anchoring member 194 of the housing,
respectively. These grooves are sized and dimensioned to receive
the O-ring in an airtight seal between the rotating member and
anchoring member, without substantially compressing the O-ring. The
O-ring is also lubricated to facilitate free rotation of the
rotating member relative to the anchoring member. A second,
lubricated and non-compressingly seated O-ring 206 is seated in
opposing circumferential grooves 208, 210 at rear connecting ends
212, 214 of the rotating member and anchoring member of the
housing, respectively, to facilitate rotation of the rotating
member relative to the anchoring member.
[0149] To align and facilitate rotation of the rotating member 109
of the housing, the rotating dial member 190 (which drives the
rotating member of the housing) is sealably, rotatably connected
with the replacement engaging portion 170 of the adapter 12.
Preferably, the replacement engaging portion has a stem 216 which
nests within a stem-shaped base 218 of the rotating dial member.
Free rotation between these structures is achieved, e.g., by
providing a third lubricated and non-compressingly seated O-ring
220 seated in opposing circumferential grooves 222, 224 in the stem
and base of the replacement engaging portion and rotating dial
member, respectively. This rotation is also facilitated by friction
contact (by pressure and/or suction) between the replacement
engaging portion and the breast 17 of the patient, which angularly
secures the replacement engaging portion and prevents its
co-rotation with the rotating dial member.
[0150] The anchoring member 194 of the housing is in turn anchored
within the existing pump by an anchoring mechanism that angularly
secures the anchoring member within the pump, e.g., against an
inner wall 182 of the cylindrical connecting portion 176. For
example, front and rear compressible anchoring sleeves 230, 232 may
be mounted in front and rear circumferential anchoring sleeve
retainer grooves 234, 236 surrounding the anchoring member. The
anchoring sleeves are non-lubricated and are made of a
semi-compressible material such as rubber or soft plastic. This
construction creates a friction anchor between the anchoring member
and the inner wall of the connecting portion, so that the anchoring
member does not move angularly during rotation of the rotating
member 109 of the housing. Both the anchoring sleeves and retainer
grooves are preferably sharply angled at a position corresponding
to the bases of the retainer grooves (i.e., they have a rectangular
or triangular cross-section), to securely retain the anchoring
sleeves in the grooves despite strong friction against the inner
wall of the connecting portion when the anchoring member of the
housing is being inserted into the connecting portion of the
existing breast pump 10 to assemble the adapter 12 with the
pump.
[0151] Because the replacement engaging portion 170 is anchored by
friction against the breast 17, and the anchoring member 194 of the
housing 30 is anchored by friction against the inner wall 182 of
the connecting portion 176 of the existing pump 10, the rotating
member 109 of the housing rotates freely with respect to both the
replacement engaging portion and the anchoring member when an
operator manually engages the rotating dial member 190 and turns it
gently while maintaining pressure against the breast.
[0152] Relative rotation between the rotating member and anchoring
member of the housing drives the reciprocating mechanism within the
instant embodiment of the invention to advance the sample
collection medium (e.g., by advancing a carrier 110 and/or support
member 56 supporting the medium) toward the replacement engaging
portion 170 to contact the expressed breast fluid on the nipple 16.
As with previously described embodiments, the housing 30 preferably
houses a support member 56 to support the solid phase collection
medium, as described above. The support member is reciprocatingly
mounted relative to the anchoring member 194 of the housing 30,
preferably on a reciprocating carrier 110. The support member may
be removably mounted to the carrier, e.g., by friction fitting,
detention fitting or threadedly engaging the support member to a
first end 112 of the carrier, as described above. In the embodiment
shown in FIG. 13, the support member is removably engaged with the
carrier by cooperative threading 140 between the support member and
carrier. In addition, the support member may be sized and
dimensioned for receipt within the stem 216 of the replacement
engaging portion, because the replacement engaging portion and an
inner (i.e., lumenal) diameter of the stem thereof are smaller than
respective dimensions of the original engaging portion 14 and its
base 22, so that the nipple may not fully extend through the stem
to contact the collection medium within the housing. Also in
conjunction with this design, the carrier is preferably in the form
of an open cylinder and the rotating member 109 of the housing has
a vacuum port 242 so that negative pressure can be effectively
transmitted through the rotating member and carrier (and/or through
air channels 80 of the support member) to the replacement engaging
portion.
[0153] To reciprocatingly adjust the position of the carrier 110
and/or support member 56 relative to the replacement engaging
portion 170 of the adapter 12, the anchoring member 194 of the
housing is provided with a lumenal, helically oriented groove 140
dimensioned to receive a riding peg 142 extending transversely from
the carrier or support member. In addition, the rotating member of
the housing is provided with a longitudinally oriented, lumenal
groove 144 dimensioned to receive an angularly fixating keel 146
extending transversely from the carrier or support member. Lastly,
the rotating member is provided with a second, longitudinally
oriented, lumenal groove 244 to allow access of the riding peg
through the wall of the rotating member of the housing into the
helically oriented groove and to allow reciprocating passage of the
pin along the groove.
[0154] In accordance with this design, rotation of the rotating
dial member 190 drives rotation of both the rotating member 109 of
the housing 30 as well as the carrier 110 (or support member) which
is angularly fixed relative to the rotating member by the fixating
keel 146 engaged with the longitudinal groove 144 of the rotating
member. As the rotating member and carrier thus rotate (with the
position of the replacement engaging portion 170 and anchoring
member 194 angularly fixed by friction or manual or structural
resistance), the riding peg rides along the helical groove 140,
translating the peg in the direction of the groove and thereby
causing the support member or carrier to reciprocate forward or
backward relative to the replacement engaging portion.
[0155] To insert and remove the solid phase medium and/or support
member 56 from the adapter 12, removable connections can be
uncoupled between the existing pump 10 and the entire adapter unit,
between the rotating member 190 and anchoring member 194 of the
housing, or between the rotating dial member and replacement
engaging portion 170, among other access designs which will be
readily apparent to those skilled in the art.
[0156] In more detailed aspects of the invention illustrated in
FIGS. 16-26, selected features of a general purpose breast pump 10
for mammary fluid sample collection as described above are
incorporated within a hand-held breast pump device 10'--adapted for
greater fidelity and ease of sample collection. The hand-held
sample collection pump is uniquely designed and constructed to
incorporate the breast engaging member 14 and the vacuum pump
mechanism 160 in a compact, structurally integrated breast fluid
collection apparatus that can be manipulated and operated with one
hand. As with the general-purpose breast pump 10, described above,
the hand-held pump 10' incorporates the vacuum pump mechanism 160
in gaseous connection with the breast engaging element 14 to route
suction pressure from the pump mechanism, through the engaging
element, to apply negative pressure in the area of the nipple 16 of
the patient. The solid phase sample collection medium (e.g., a
membrane, filter, particulate medium, and/or a non-particulate
solid collection template such as a plastic or glass tube, well,
vial or slide) is likewise fluidly connected with the breast
engaging member--to provide for direct or indirect transmission of
the expressed breast fluid through the engaging member to contact
the solid phase collection medium.
[0157] According to these aspects of the invention, novel breast
fluid sample collection methods are provided wherein a doctor,
technician or patient collecting a breast fluid specimen can grasp
and operate the hand-held breast pump 10' to stimulate expression
of the breast fluid and collect a specimen thereof while keeping
one hand free for additional tasks, such as monitoring the patient
and recording patient information. In this regard, the compact pump
design allows the device to be picked up and manipulated with one
hand--to seat the breast engaging element against the breast of the
patient and thereafter apply vacuum pressure to the breast by
manual operation of the vacuum pump 160 to stimulate expression of
breast fluid. This action causes a suitable volume of breast fluid
to be expressed at or near the nipple 16 for sample collection. In
conjunction with these simple operation steps, the hand-held device
also allows for simultaneous collection of the expressed breast
fluid onto, or within, the solid phase sample collection medium
that is fluidly connected with the engaging member, often without
additional manual steps or a need to remove the device from the
breast or otherwise engage two hands in the operation.
[0158] The hand-held breast pump device 10' can be employed for
collection of breast fluid following oxytocin stimulation to
facilitate breast fluid expression, as described above.
Alternatively, the device can be used without oxytocin priming to
achieve breast fluid expression by vacuum pressure alone,
optionally coupled with mechanical breast stimulation, in a
substantial percentage of subjects.
[0159] In using the hand-held breast pump 10' of the invention
expressed breast fluid is typically transferred directly upon
expression to the solid phase sample collection medium, without
intervening manual steps or a requirement to remove the breast
engaging member 14 from the breast 17 before the sample is
collected. Sample collection in this manner is rapid and simple,
and promotes sanitary application of the device to minimize the
risk of patient infection and sample contamination. Thus, within
certain methods of use for the hand-held breast pump, breast fluid
is directly transferred to a solid phase collection medium, for
example a membrane, filter, reservoir or vial, integrated within
the hand-held pump. The sample may be collected as whole, undiluted
breast fluid containing constituent proteins, particulates and/or
cells. Alternatively, selected components of the expressed breast
fluid may be simultaneously or subsequently removed from the fluid
(e.g., by filtering, partitioning, or refining the breast fluid) to
yield a processed fluid sample and/or to obtain a solid phase
constituent sample. For example, various collection methods are
provided which yield separated solid components (e.g., cells or
other particulates) from the fluid. Alternate collection methods
yield soluble, suspended, or solid phase captured proteins, lipids,
carbohydrates, polynucleotides or other molecular/biochemical
components from the expressed breast fluid. In certain embodiments,
the hand-held pump device functions to separate or partition a
desired protein, lipid, carbohydrate, or polynucleotide sample
material into a solid phase collection medium, such as a membrane,
filter, or chromatographic substrate (e.g., leptin-, antibody-,
enzyme-, or ligand-coated vials, beads, etc.) In this regard,
various alternative or additional steps from known
collection/chromatographic methods can be employed during sample
collection and processing according to the invention. In this
manner, proteins, lipids, carbohydrates, polynucleotides, cells,
and non-cellular particulates may be partitioned from liquid
components of the breast fluid, or separated from one another,
simultaneous with or subsequent to expression of the fluid, by
various known methods, including membrane adsorption, filtering,
affinity chromatography, chemical processing, centrifugation, etc.
to yield a range of constituent or processed samples.
[0160] In certain collection methods of the invention, breast fluid
expressed by use of the hand-held pump 10' is simultaneously or
subsequently diluted, filtered, washed, admixed with fixative or
other processing agents, or otherwise processed or modified to
yield a collected fluid sample partially or completely devoid of
cells, proteins and/or other selected components originally present
in the expressed fluid, to provide a processed fluid sample for
laboratory analysis. In other embodiments, particulate components
of the breast fluid, for example, cells, cellular components and/or
cellular debris, are collected after processing and/or
modification, e.g., for cytological examination. These and other
alternative collection methods involving preliminary sample
processing in conjunction with use of the hand-held breast pump 10'
are optionally performed simultaneous with, or closely following
expression of the breast fluid. Often, sample collection is
coincident with the fluid contacting one or more solid phase
collection medium(a) fluidly connected with the breast engaging
member 14. Depending on the type(s) of medium(a) used, preliminary
sample processing can also be achieved directly by simple operation
of the pump, without the need for additional processing steps or
removal of the breast engaging member 14 from the subject's breast.
For example, the expressed breast fluid may be sequentially
transferred through multiple media, e.g., through a filter or
membrane into a liquid-retaining reservoir or container, thereby
separating certain components for further processing or analysis.
Alternatively, the expressed fluid may be partially processed
coincident with transfer to the solid phase medium(a) by chemical
or physical reaction (e.g., adsorption, covalent or affinity
linkage, enzymatic reaction, etc.) with the medium or a coating or
secondary processing agent admixed or linked therewith.
[0161] In yet other alternative methods within the invention,
preliminary sample processing involves additional steps following
breast fluid expression. In certain embodiments, the breast
engaging member 14 is removed from the breast after the breast
fluid is expressed and the fluid is transferred to a first solid
phase sample collection medium, typically a membrane or filter.
This initial or primary stage of sample collection may be followed
by washing or by manual transfer of selected breast fluid
components (e.g., proteins, carbohydrates, cells, or cellular
debris) from the first solid phase collection medium (e.g., a
nitrocellulose membrane 39) to a second solid phase medium, e.g., a
glass slide or fluid-containing reservoir. Typically, preliminary
sample processing in this regard precedes final packaging of the
collected sample for storage or shipment to a lab for further
processing and analysis of the sample.
[0162] In more detailed embodiments of the invention, cells or
other cellular materials useful for cytological examination are
separated or partitioned simultaneous with or sequential to breast
fluid expression onto or within a first solid phase collection
medium. In one example, whole cells are separated from the
expressed fluid onto a nitrocellulose membrane 39 or a filter 40,
which is typically secured in fluid connection with the breast
engaging member 14 by a fixed or removable support member 56
mounted to the engaging member or sample collection housing 30 or
otherwise integrated with the hand-held breast pump 10'. The cells
are subsequently transferred or washed in fluid (e.g., cytology
fluid) to a second solid phase sample collection medium (e.g., a
slide, well, tube or vial), which may also be connected to, or
integrated with, the breast engaging member or sample collection
housing as described.
[0163] To facilitate sample collection according to the foregoing
embodiments of the invention, the hand-held breast pump 10' is
typically provided as a compact, hand-held unit for ease of use and
convenience of storage. As depicted in FIG. 16, certain embodiments
of the hand-held pump comprise a modular device formed of a
plurality of components that are joined or securable in fixed
structural interconnection with one another. These components,
which include a breast engaging member 14, vacuum pump 160 and
solid phase sample collection component(s), may be partially or
completely disassembled to remove or uncouple the individual
components, or parts thereof, as desired for efficient operation,
cleaning, servicing and/or storage.
[0164] As described above for the general-purpose breast pump 10 of
the invention, the breast engaging portion 14 of the hand-held pump
10' is constructed of a rigid or semi-rigid, non-porous material
and is sized and dimensioned to receive at least the nipple 16 of
the subject's breast 17 and form a suction seal therewith (see,
e.g., FIG. 16). The breast engaging portion may be constructed in a
variety of shapes and dimensions to accommodate variations in
breast anatomy. As also described above, the terminal edge 18 of
the engaging portion is rounded or flared so that the edge impinges
comfortably and forms an effective suction seal against the skin
when negative pressure is applied to the breast. Typically, the
engaging member is constructed of a rigid plastic material which is
transparent to allow the operator to visualize the breast,
determine positioning of the device, and observe expression of
fluid from the nipple 16. Preferably, the engaging portion and
other reusable components of the pump are autoclavable for
sterilization purposes.
[0165] In more detailed embodiments of the invention shown in FIGS.
16-18, the hand-held breast pump 10' is a modular device comprised
of multiple, integrated components that are fixedly joined to one
another when the pump is assembled, but can be readily detached or
uncoupled from one another. This modular configuration of the
device allows for interchanging of parts to adapt the pump for
different patients and collection modes, and to facilitate storage,
cleaning and/or servicing of the device. Thus, in one embodiment,
the breast engaging portion 14 of the pump is provided as a
separate, funnel shaped component that is detachable from one or
more interconnecting components of the device (see, e.g., FIG. 18).
In this manner, the engaging member can be removed from the rest of
the device for cleaning and sterilization, or to allow for
interchanging of different engaging members to accommodate breast
anatomy differences among patients. Typically, the breast engaging
member is removably coupled with a surface or member of the sample
collection housing 30.
[0166] As illustrated in FIG. 18, the hand-held breast pump 10' of
the invention incorporates the solid phase sample collection medium
in fluid connection with the breast engaging member 14, typically
by use of a support member 56 affixed to, or removably connected
with, the engaging member. In selected embodiments, the support
member encloses or supports one or more pads or sheets of absorbent
or adsorbent material, for example a nitrocellulose membrane 39.
Multiple pads or sheets of the same or different material may be
used in combination (e.g., including a wetting member, a wicking
member, or a partitioning member). Alternatively, the support
member can incorporate or support a particulate solid phase sample
collection medium, for example beads, resins, microspheres,
particulate chromatographic media (e.g., agarose or silicate
media), and the like. In yet additional aspects of the invention,
the support member engages or supports a non-particulate solid
template for sample collection, for example one or more capillary
tubes, coated tubes, plates, wells, slides and the like formed of
glass, plastic or other suitable materials. In certain embodiments,
the support member may incorporate a compartment, well or reservoir
to receive or introduce sample processing agents selected from
chemical reagents, probes, blocking agents, buffering agents,
denaturing agents.
[0167] The support member 56 for use in conjunction with the
hand-held breast pump 10' is typically provided as a removable
cassette that can be inserted within the engaging member 14, often
to seat against the inner wall 20 thereof (see, e.g., FIGS. 16 and
18. In preferred aspects, the support member seats by a friction or
compression fit against the inner wall of the engaging member,
which may be facilitated by a complementary circumferential ridge
and groove design between the support member and engaging member
inner wall, as shown in FIG. 16.
[0168] In certain embodiments of the hand-held breast pump, a
disc-shaped support member 56 is provided to support one or more
sheets of absorbent or adsorbent material, such as a nitrocellulose
membrane 39, in close proximity, or in contact with, the nipple 16
when the device is in use and suction is applied to the breast. As
shown in FIGS. 16 and 18, the sheet is preferably secured by an
upper retainer ring 66 fixedly or removably seated against an
opposing surface (exemplified by a circumferential retainer groove
270) with the margin of the sheet sandwiched therebetween. This
holds the sheet in place against negative pressure that may pass
through the sheet when vacuum pressure is applied through the
engaging member and to secure the sheet in position when the nipple
impinges against it. The retainer ring may be integrally joined
with the support member which may comprise a disposable refill, or
the ring may be separable and the support member may comprise a
reusable cassette for receiving replacement sheets. In an
alternative design, there is no upper retaining ring and the
membrane 39 or filter 40 simply rests upon the support member 56 or
is removably coupled directly therewith (e.g., by wetting or gluing
to create a temporary bond between the sheet and upper support
member surface, or by appropriately sizing the sheet so that a
peripheral edge of the sheet engages a surface of the support
member in a friction or detent fit. Typically, the sheet is readily
removable from the support member for processing, e.g., by hand or
by using forceps or other conventional handling tools to disengage
the filter or membrane.
[0169] Typically, the support member 56 for use with the hand-held
breast pump device 10' includes air channels 80 that pass through
the body of the support member to allow vacuum pressure applied
from the vacuum pump 160 to reach the engaging member 14 of the
pump during operation (i.e., by passing from the pump through the
sample collection housing 30 and air channels to the engaging
member). Likewise, the air channels allow venting of the engaging
member for disengagement from the breast 17 after use. In certain
embodiments, a plurality of two-three or more air channels are
provided, which may be centrally located relative to a disc-shaped
body of the support member as depicted in FIG. 18. The air channels
can serve a dual purpose as channels for passage or transfer of
fluids and/or fluid-suspended particles, including cells and
cellular components, between the breast engaging member and the
sample collection housing 30.
[0170] In one related aspect of the invention, cellular materials
from expressed breast fluid are first collected on a primary solid
phase sample collection medium (e.g., a membrane 39 or filter 40)
mounted in fixed relation to the engaging member 14 (e.g., by a
support member 56). In a secondary sample collection or processing
step, the cellular materials are removed or flushed from the
primary sample collection medium into a secondary solid phase
sample collection medium (e.g., a solid phase sample collection
template such as a plastic or glass slide, slip, tube, well or
vial), optionally coupled with the support member and/or sample
collection housing. The cellular materials can be directly
transferred from the primary medium onto or into the secondary
medium, for example by flushing the cellular materials from the
primary medium (e.g., using physiological solutions, fixatives,
etc.) directly into the secondary medium (e.g., a recess, well,
vial or other receptacle coupled with or inserted into the support
member or housing).
[0171] In one exemplary embodiment, cellular materials are
collected from expressed breast fluid onto a primary sample
collection medium comprising a filter, particulate medium, or
nitrocellulose membrane 39. When a nitrocellulose membrane is used,
the cellular materials are washed after primary collection from the
membrane by a flushing rinse. For example, cytology fluid or
another desired rinse liquid is used to transfer the cells from the
primary collection medium into a secondary collection medium,
typically a fluid-retaining well or reservoir integrated within, or
coupled with, the support member 56 or the sample collection
housing 30.
[0172] In more detailed embodiments of the invention, a
nitrocellulose membrane 39 is employed for primary sample
collection. The membrane is typically fluidly connected with the
hand-held pump 10' by seating or mounting the filter on or within a
support member 56, as described above. Nitrocellulose membranes are
particularly well suited for collection of proteins,
polynucleotides, or other soluble or suspended constituents of
breast fluid. In preferred aspects, a nitrocellulose membrane is
selected and employed within the device for collection of
cytological specimens, particularly cells and cellular
constituents. As illustrated in FIGS. 16-18, the filter (optionally
supported by a support member) is positioned within the breast
engaging member 14 near the base 22 of the engaging member
proximate to the patient's nipple when the engaging member is
seated against the breast. Comparing FIGS. 16 and 17, activation of
suction pressure by manual operation of the device draws the nipple
16 into closer proximity to, or into actual physical contact with,
the membrane, whereby the expressed fluid is efficiently
transferred to the membrane.
[0173] Due to the fragility and fine porosity of nitrocellose
membranes 39, it may be necessary to employ additional measures to
protect the membrane against negative vacuum pressure and contact
with the nipple 16 during operation of the pump 10'. In this
regard, uniquely designed membranes and filters are provided for
use within the device which feature perforations or slits that
disrupt the planar surface of the membrane or filter to facilitate
air passage therethrough and impart structural flexibility against
mechanical perturbation. In one example, radial slits 280 emanate
from a central disc portion 282 of the membrane or filter (FIG. 24)
to allow passage of air through the membrane or filter during
vacuum pressurization and to increase structural flexibility of the
membrane or filter. Alternatively, one or more spiral perforations
284 (FIG. 25) or transverse slits 286 (FIG. 26) may be cut or
stamped in the membrane or filter to achieve similar improvements
in terms of permeability and flexibility.
[0174] Where the target constituent for collection from the
expressed fluid is whole cells or cellular components, these
materials may be partitioned onto the surface of a membrane or
filter, typically a nitrocellulose membrane 39, for further
processing and cytological examination. For this purpose,
nitrocellulose membranes are employed that have a sufficiently
small pore size to retain the cells or cellular debris on the outer
(i.e., facing the nipple) membrane surface. For example,
nitrocellulose membranes having a pore size of between about
0.5.mu. and 5.0.mu., preferably between about 1.0.mu. and 2.0.mu.,
are useful to partition whole cells on their surfaces.
[0175] As noted above, a fluid-retaining recess, well or reservoir
may be fluidly connected to either the support member 56 or the
sample collection housing 30 of the hand-held pump device 10' for
primary and/or secondary sample collection. In certain embodiments,
the fluid-retaining reservoir comprises an integral, defined
compartment or enclosure within the sample collection housing for
receipt of breast fluid and/or constituent samples thereof,
including cytology specimens that may be washed into the reservoir
after primary collection, as described above. Alternatively, the
fluid well or reservoir can be a separable component of the sample
collection housing, e.g., in the form of a flexible liner or rigid
fluid reservoir member of the housing removably connected with a
complementary housing member that partially or completely encloses
or otherwise engages the fluid reservoir member.
[0176] Thus, as depicted in FIGS. 16 and 18, certain embodiments of
the invention employ a removable fluid reservoir member 288 of the
housing 30 for secondary sample collection of breast fluid
components, including cytology specimens. In preferred embodiments,
the removable reservoir member is provided in the form of a rigid
sample collection tube or vial, exemplified by a standard cytology
vial (i.e., a container having approximately the same general shape
and dimensions as a standard cytology vial). The tube or vial is
removably connected with a complementary housing member, for
example an outer casing member 290 of the housing that partially or
completely encloses the vial. Preferably, the tube or vial is
sealably coupled with the outer casing member, for example by
partially or completely nesting the vial within the outer casing
member to form an airtight coupling therewith.
[0177] In various specific embodiments, the tube or vial engages an
inner wall 292 of the casing member 290 and forms a generally
airtight seal against it. For example, the casing member and vial
may be complementarily sized and dimensioned to provide
substantially airtight contact between the inner wall of the casing
member wall and an outer wall 294, or a top end 296 or bottom end
298, of the vial when the casing member and vial are coupled to
form the assembled housing. In certain embodiments, the outer wall
of the vial features a circumferential ridge or fin 299 that
engages and thereby makes a circumferential airtight seal against
the inner wall of the casing member when the vial is nested with
the casing member (see, e.g., FIGS. 16-20). In more detailed
embodiments, the fin or ridge is replaced by a flexible O-ring 300
that seats in a circumferential O-ring groove 302 on the outer
surface of the vial and forms a circumferential seal with the inner
casing wall.
[0178] The purpose of the sealable coupling between the outer
casing 290 and removable fluid reservoir 288 members of the housing
30 is to direct vacuum pressure from the vacuum pump 160 to the
breast engaging member 14 in a path that includes the fluid
reservoir member of the housing 30. In this manner, the removable
reservoir is both gaseously and fluidly connected with the engaging
member to facilitate secondary sample collection. To accomplish
these objectives, the removable reservoir member is modified to
include one or more air ports 310 that form a gaseous connection
between the lumen of the reservoir (which is in turn connected to
the engaging member, optionally via air channels 80 through a
support member 56) and the vacuum pump. This allows the reservoir
member to remain in place during primary and/or secondary sample
collection, to function as both a conduit for vacuum pressure
transmission to the breast and a receptacle for fluid sample
materials (e.g., to directly collect expressed fluid or as a
secondary collection medium to receive primarily collected sample
materials washed or otherwise transferred from the primary sample
collection medium).
[0179] In the latter context, the removable reservoir member 288 of
the housing 30 may communicate for both fluid and gaseous
transmission directly with the breast engaging member 14 of the
device, or indirectly by way of the air channels 80 in the support
member 56 optionally coupled with the engaging member. As noted
above, the air channels can serve a dual purpose as channels for
vacuum pressure transmission as well as transfer of fluids from the
primary sample collection medium, through the air channels, into
the removable reservoir (as exemplified by transfer of "wash" fluid
containing cells and cellular components flushed from a primary
collector, e.g., a nitrocellulose membrane, mounted in the support
member, through the air channels, and into the secondary collection
fluid reservoir). To facilitate this and related purposes, the
channels may extend through tubular basal columns 311 or other
fluid connection ports that extend from the support member toward,
or into, a lumen 314 of the reservoir member of the housing.
[0180] In the embodiment of a hand-held pump device 10' illustrated
in FIGS. 16-20, the reservoir member 288 is a cytology vial
removably, sealably coupled with the outer casing member 290 to
form the assembled housing 30. To achieve this sealable coupling,
the outer wall 294 of the vial features a circumferential fin 299,
or a flexible O-ring 300 seated in a circumferential O-ring groove
302, that forms a circumferential seal with the inner wall 292 of
the casing member (see, e.g., FIGS. 16, 19 and 20). The vial
incorporates one or more air ports 310 that communicate between the
outer wall and the inner lumen 314 of the vial to form a gaseous
connection between the lumen of the vial, the vacuum pump 160, and
the breast engaging member 14. Preferably, multiple air ports are
provided, which are located on the side of the vial positioned
below (i.e., toward the bottom end 298 of the vial) the ridge or
O-ring that forms the gaseous seal with the wall of the casing
member. Alternatively, the air ports can be located at other
positions on the vial, e.g., in a lower side wall or floor wall
318, provided the position is suitable to maintaining the desired
path of vacuum pressure flow and retaining fluids within the
reservoir. In this context, it is noted that certain embodiments of
the fluid reservoir member of the housing will dictate changes in
the position of the air port(s). For example, where a cytology
vial-shaped reservoir is selected, as shown in FIGS. 16 and 20, the
floor wall may be raised relative to the bottom end 298 of the
vial, to reduce the sample volume of the vial for handling and
processing purposes. To accommodate this and other designs, the air
ports are desirably positioned in the outer (side) wall 294 of the
reservoir between the floor wall and the sealing flange 299 or
O-ring 300.
[0181] Referring to FIGS. 19 and 20, further modifications of the
removable fluid reservoir member 288 of the housing 30 provides for
multi-purpose use of the reservoir member for sample collection in
a clinical setting, as well as sample storage, transport and/or
processing in a laboratory setting. To facilitate these latter
purposes, the removable reservoir member may be provided with
closure means for closure of the reservoir after sample collection
is completed--to prevent sample contamination and spillage. Taking
the cytology vial reservoir for example, the top end 296 of the
vial may be adapted to provide a sealable primary closure for the
main opening of the vial. For example, the vial top end can be
provided with complementary threads 320 or other closure means to
receive a conventional cap that is sized and dimensioned (e.g.,
complementarily threaded) to sealably engage the vial top end. In
conjunction with this use, and further considering the novel
adaptation of the fluid reservoir member to provide a secondary
opening (i.e., the integral air port 310 that functions as a vacuum
connection), the reservoir is also equipped with secondary closure
means to sealably close the air ports after sample collection. A
variety of air port configurations are contemplated, which can be
sealed using a commensurate variety of closure mechanisms. For
example, the air port or ports can be sealed using a flexible
(e.g., rubber) stopper shaped and dimensioned to sealably plug into
the port opening. Alternatively, the port can be closed by an
adhesive seal or sticker that adheres to the outer wall 294 of the
reservoir member surrounding the port opening.
[0182] With respect to the latter secondary closure design, the
invention provides a combined closure and labeling device 324 which
functions both as a secondary closure mechanism to seal the air
port 310 of the removable reservoir and as a labeling template to
provide a convenient writing surface for sample labeling (see,
e.g., FIGS. 21 and 22). This aspect of the invention may be
achieved, for example, by providing any of a variety of adhesive
closure/labeling tabs which may be directly applied to seal the air
port after sample collection. This simple type of closure/labeling
tab can, for example, be provided as a separate adhesive sticker
having a first, closure-forming surface 325 bearing an adhesive
coating 326 on at least a portion of said surface, for application
over the air port to form a seal, e.g., by adhesive contact with
the outer wall 294 of the removable reservoir. The adhesive coating
preferably forms a seal that is resistant to disruption by contact
with aqueous solutions and other materials present in the collected
samples, for example buffers and fixatives. The adhesive coating is
also preferably shielded before use by a protective tab or other
protective surface 327 that covers the coating and is removed
therefrom prior to use to expose the adhesive coating.
[0183] The closure/labeling device 324 has a second, labeling
surface 328 opposite the closure-forming surface 325 that is made
of a blank template material suitable for receiving a stable, ink
or graphite imprint thereon. For example, the second surface may be
made of cellulose or other fibrous material adapted for imprinting
sample data upon the surface thereof using a pen, pencil or other
writing implement. Alternatively, the second, labeling surface may
be made of plastic or other material adapted for retaining data
imprinted in permanent ink (e.g., using an alcohol-based marker).
When the adhesive tab is applied over the air port, the blank
template material of the second, labeling surface of the tab is
facing outward and at least a portion of the surface covers a
smooth portion of the outer wall of the removable reservoir 288, to
allow the doctor or technician to imprint a clear data record on
the labeling surface.
[0184] In more detailed embodiments, the closure/labeling device
324 is affixed to the removable reservoir 288 during operation of
the hand-held breast pump 10' in a first, open configuration that
leaves the air port 310 uncovered for transmission of vacuum
pressure (see, e.g., FIG. 23). The closure/labeling device which is
thus pre-attached to the reservoir can be manually repositioned, or
otherwise manipulated, after sample collection to a second, closed
configuration to form a seal or closure against the air port (see,
e.g., FIG. 21). In the exemplary embodiment shown in FIGS. 19-23,
the closure/labeling device comprises an adhesive strip that is
folded in a first, open configuration (FIG. 21) to form two layers
an inner layer 330 that is affixed to the reservoir proximate to,
but not covering, the air port, and an outer layer 332 that folds
back over the inner layer in the open configuration. In this
embodiment, the outer layer of the strip provides both the first,
closure-forming surface 325 with the adhesive coating 326 for
securing closure of the strip, and the second, labeling surface 328
formed of the blank template material opposite the closure-forming
surface. In the open configuration, the outer layer is optionally
secured in the folded-back position against the inner layer by
engagement of the labeling surface with the inner layer, for
example by a second adhesive coating 336 on the inner layer that
holds the two layers together in the folded position. Also
optionally, the adhesive coating 326 of the first, closure-forming
surface may be protected in the open configuration by folding an
end segment 338 of the outer layer 325 bearing the adhesive coating
326 for closure back on itself, so that the closure forming surface
325 provides the protective surface 327 to shield the adhesive
prior to closure (as shown in FIGS. 21 and 22). To manipulate this
closure/labeling device into the second, closed configuration, the
end segment can then be lifted and pulled outward to release the
outer layer 332 from the inner layer 330 and to unfold the end
segment to separate the adhesive coating 326 on the closure-forming
surface 325 from the protective surface 327. The outer layer 332 is
then unfolded away from the inner layer and wrapped around the
reservoir so that the closure-forming surface covers the air port
to form a closure that is water-tight or water-resistant to
effectively prevent sample spillage from the reservoir and
contamination. In this context, closure is effectuated by direct
apposition of the adhesive coating 326 to the outer wall 294 of the
reservoir surrounding the air port, or by drawing the outer layer
tight across the air port and annealing the adhesive surface back
upon the inner layer. In either case, manipulation of the
closure/labeling strip to the closed configuration positions the
labeling surface 328 to face outward for easy recordation of sample
data. In yet additional detailed embodiments, the closure/labeling
strip can be better secured against dislodgement during loading and
removal of the reservoir 288 by including one or more
circumferential guide ridges 340 (see FIGS. 19 and 20) on the
exterior of the reservoir, to shield the closure/labeling strip
from mechanical dislodgement and/or to increase the fidelity of the
closure against leakage.
[0185] In related aspects of the invention, a novel breast fluid
collection reservoir is provided for use within a mammary fluid
collection device of the invention, which incorporates the
foregoing features of the removable reservoir member of the sample
collection housing. The novel collection reservoir as described in
the foregoing passages is useful within the breast fluid collection
methods of the invention, as well as within various sample
handling, processing, and diagnostic assay methods performed in the
laboratory subsequent to collection of a breast fluid sample.
[0186] The novel breast fluid collection reservoir of the invention
is typically provided in the form of a rigid tube or vial, for
example in the form of a modified cytology vial, having a top end
which defines a primary opening for access to the sample. The
reservoir further comprises an outer reservoir wall, typically a
cylindrical sidewall of a tubular reservoir closed at a bottom end
thereof, e.g., by a floor wall, as described above. The outer
reservoir wall defines one or more air ports that communicate
between the outer wall and an inner lumen of the vial. In more
detailed embodiments, the fluid-retaining reservoir comprises a
removable fluid reservoir member of a sample collection housing of
a mammary fluid collection device. Typically, reservoir member is a
rigid sample collection tube or vial removably connected with an
outer casing member of the sample collection housing of the
collection device.
[0187] Within this aspect of the invention, the reservoir may be
adapted for removable, sealable connection with the outer casing
member of said housing, to form an airtight coupling therewith. In
certain embodiments, the fluid-retaining reservoir is a cytology
vial sealably connectable with the outer casing member to form the
airtight coupling. For example, the fluid-retaining reservoir can
be removably nested within the casing member to form a
substantially airtight contact between an inner wall of the casing,
member wall and an outer wall, or a top or bottom end, of the
reservoir member. To achieve this function, the outer wall of the
fluid-retaining reservoir may be provided with [0188] a
circumferential ridge, fin or O-ring adapted to engage and make a
circumferential airtight seal against the inner wall of the casing
member.
[0189] In more detailed aspects, the fluid-containing reservoir
member for use within the devices and methods of the invention
includes a closure device for closing the reservoir after the
sample of mammary fluid is introduced therein, to prevent sample
contamination and spillage. The closure may comprise a simple cap
adapted to sealably engage a top end of the reservoir. The cap or
similar "primary closure" may extend to cover the air port(s) of
the reservoir, which may be contiguous with the top opening of the
reservoir or separate therefrom. In alternative embodiments,
secondary closure means may be provided which are specifically
adapted for closure of the air port(s). The secondary closure means
may comprise a plug, cap or adhesive seal or sticker sized and
constructed to engage or adhere to the outer wall of the reservoir
member at or surrounding the air port opening(s) to form the
closure.
[0190] In certain embodiments, the secondary closure means
comprises a combined closure and labeling device which functions as
a secondary closure mechanism to seal the air port(s) of the
reservoir and as a labeling template to provide a writing surface
for sample labeling. Often, the secondary closure means comprise a
combined closure and labeling tab or sticker for application to the
outer wall of the reservoir to seal the air port after the sample
is collected. The tab or sticker has a first, closure-forming
surface for application over the air port to form a seal by
juxtaposition or adhesive contact with the outer wall of the
reservoir, and a second, labeling surface opposite the
closure-forming surface made of a blank template material for
imprinting written information thereon. The first, closure-forming
surface typically has a water-insoluble adhesive coating on at
least a portion of the surface. In more detailed embodiments, the
tab or sticker is pre-attached to the removable reservoir member in
a first, open configuration and can be manually repositioned or
otherwise manipulated after sample collection to a second, closed
configuration to form a seal or closure against the air port(s), as
described in detail above.
[0191] In yet additional, related aspects of the invention, methods
for breast fluid sample collection, sample handling, and/or sample
processing are provided which incorporate the novel fluid-retaining
reservoir adapted for use with a hand-held breast pump 10' of the
invention. These methods include, generally methods for collecting
breast fluid samples which involve collecting expressed mammary
fluid in a modified [0192] fluid reservoir as described above.
Additional methods involve loading and removal of a modified
fluid-retaining reservoir within a hand-held breast pump, according
to the above description. Related methods include an additional
step of securing the primary and/or secondary closure means of the
reservoir after a sample of mammary fluid, or a component thereof,
is collected therein.
[0193] Related to these methods, the invention provides additional
methods for handling or processing biological samples of mammary
fluid, or components thereof, for use in a diagnostic assay to
detect or quantify a breast disease marker in the sample. The
methods generally involve providing or obtaining the biological
sample of mammary fluid or a mammary fluid component in a
specialized fluid-retaining reservoir according to the above
description. The sample may be initially collected by oxytocin
induction or by application of a breast pump 10, 10' of the
invention without oxytocin priming. The reservoir is typically
provided as a flask, vial, or tube that has a top end defining a
primary opening for collection of, and later access to, the mammary
fluid sample, and an outer reservoir wall that defines one or more
air ports communicating between the outer wall and an inner lumen
of the vial.
[0194] In more detailed handling and processing methods, the
reservoir incorporates specialized closure means, for example a cap
that secures the top end of the reservoir and secondary closure
means to sealably close one or more air port(s) of the reservoir,
to close the reservoir after the sample is collected and thereby
prevent sample contamination and spillage. Typically, the
fluid-retaining reservoir is a modified cytology vial adapted as a
removable reservoir member integrated with a hand-held mammary
fluid collection pump 10' as described above. More detailed
handling/processing methods employing the novel reservoir include
the step of accessing said sample within the reservoir to transfer
or process the sample for detection or quantification of a breast
disease marker. Additional methods include one or more steps of
processing the sample to detect or quantify the breast disease
marker.
[0195] In additional detailed embodiments, the step of processing
the sample for marker detection comprises fixing or staining cells
or cell fragments in the sample, before or after transfer of the
sample from the reservoir, e.g., for cytological analysis. Yet
additional methods involve exposing the sample in the reservoir or
after transfer to a processing reagent, e.g., a fixative, labeling
reagent, buffer, etc., to prepare sample components, including
whole mammary fluid, whole cells, cell fragments, cell membranes,
purified proteins, bulk proteins, glycoproteins, peptides and/or
polynucleotide components, for further processing, which may
include detection or quantification as the selected breast disease
marker(s).
[0196] As noted above, certain modular designs for the hand-held
breast pump 10' of the invention feature a separate breast engaging
member 14 provided as a funnel shaped component adapted for
removable coupling to one or more interconnecting components of the
device-for cleaning and interchanging of parts. Typically, the
breast engaging member is removably coupled with the sample
collection housing 30. In one embodiment shown in FIGS. 16-18, the
housing is comprised of multiple members, exemplified by an outer
casing member 290 and a removable, fluid reservoir member 288. As
illustrated in FIG. 18, the engaging member may be directly coupled
to the fluid reservoir member, which is in turn engaged by a
sealable connection (e.g., a sealable compression fit) with the
outer casing member of the housing as described above. In this
context, one alternative coupling design is for the engaging member
to be fitted with mounting threads 340 or other coupling means to
couple with complementary threads 320 or other closure means on the
top end 296 of the removable reservoir (e.g., cytology vial). In
this manner, the closure means of the removable reservoir, adapted
to receive a conventional cap that sealably engages the reservoir
top end, serves the dual purpose of coupling the engaging member 14
with the remaining modular components of the device. Alternative
coupling means are of course contemplated as well, as exemplified
by a simple pressure fit coupling to removably couple mated ends of
the engaging member and reservoir member. To facilitate stable
coupling of the engaging member with the sample collection housing
30, the housing may be further elaborated to include flared
extensions 350 terminating in outwardly reflected feet 352
collectively shaped and dimensioned to engage an inner,
circumferential groove 354 underlying the flared or reflected
terminal edge 18 of the engaging member (see, e.g., FIG. 18).
[0197] In addition to the foregoing features, the hand-held breast
pump 10' of the invention may optionally include any of the
alternative features described above for the general-purpose breast
pump 10, including different solid phase sample collection media
and support member 56 designs. Thus, the support member for use
with the hand-held pump can also include a fluid-retaining well
which may be optionally filled with a desired solution, such as a
buffer, a solution containing a probe, cross-linking agent,
blocking agent, denaturing agent, etc., to facilitate sample
collection, handling, and/or processing. Alternative designs and
configurations of the housing 30 and/or support member 56 are also
provided which vary with the type of solid phase sample collection
medium used. For example, when a particulate solid phase sample
collection medium 41 (e.g. beads, resins, or microspheres) is used,
the medium may be enclosed in a cartridge 82 removably mounted to,
or integrated within, the support member or otherwise removably
connected to the sample collection housing 30, as described in
detail above. It is also contemplated to adjustably mount the solid
phase medium relative to the housing 30 of the hand held pump
device, so that the collection medium can be moved closer to, or
farther away from, the base 22 of the engaging portion 14 of the
pump 10. In this regard, various designs are contemplated
commensurate with the above description to provide a reciprocating
mechanism that adjustably moves the solid phase collection medium
in closer, or more distant, proximity to the nipple when the
hand-held breast pump is engaged therewith.
[0198] Within more detailed aspects of the invention, the hand-held
breast pump 10' typically incorporates a compact vacuum pump
housing 410 which structurally and functionally integrates the
vacuum pump 160 with the sample collection housing 30 and, in turn,
with the engaging member 14 (see, e.g., FIGS. 16-18). The vacuum
pump housing is in turn coupled with, or is modified to include, a
vacuum pump actuating mechanism. The actuating mechanism may be in
the form of a switch, button, lever or other actuation device
suitable for use with the selected vacuum pump. As noted above, a
variety of vacuum pumps may be incorporated within the breast pump
device, including any manual or electric, piston, hydraulic or
diaphragm pump of suitable size and dimension for incorporation in
the hand-held pump vacuum housing. In the exemplary embodiment
depicted in FIGS. 16-18, the vacuum pump is a conventional
diaphragm pump, and the pump actuation mechanism is a simple hand
lever 420 pivotally connected to the pump housing or other suitable
connection point.
[0199] Exemplifying this aspect of the invention, the pump housing
is optionally coupled with, or extended to include, an opposing
handle 422 to facilitate depression of the hand lever by gripping
and manual closure of the lever toward the handle (compare FIGS. 16
and 17). The handle is preferably molded or cast as an integral
extension of the vacuum pump housing. In more detailed embodiments,
exemplified in FIGS. 16-18, the vacuum pump housing and handle are
molded or cast together as an integral unit or modular component
with the outer casing member 290 of the sample collection housing
30. The handle defines a pivot recess 430 or detent that pivotally
receives a pivot head 432 or shaft joined or integrated with the
pump actuation lever (as shown in FIGS. 16 and 17).
[0200] In this manner the pump actuating lever 420 is pivotally
connected to the pump housing 410 or handle 422 by a pivotal
connection 430, 432 that joins the actuation lever and pump housing
in an easily assembled fashion, e.g., by snap fitting the pivot
head 432 of the lever into the pivot recess 432 of the handle. The
actuation lever easily and effectively actuates the vacuum pump 160
by depressing the lever to draw a flexible diaphragm member 440
downward, away from a primary vacuum chamber 442 connected with, or
integrated within, the sample collection housing. In the embodiment
shown in FIGS. 16-18, the primary vacuum chamber is integrally
formed as a channel within the vacuum pump housing proximate the
flexible diaphragm member and extending to a communicating port 444
opening to the inner wall 292 of the outer casing member 290 of the
housing. The remainder of the vacuum path (i.e., through the outer
casing member to the air port 310 of the removable fluid reservoir
member 288 of the housing into the lumen 314 of the reservoir,
optionally through air channels 80 of the support member 56, and to
the breast engaging member) is described above.
[0201] To engage and move the flexible diaphragm member 440 in this
fashion, the actuation lever engages a diaphragm retraction
mechanism, for example comprising a reciprocating shaft 450 or
piston sealably connected through the diaphragm to a
diaphragm-engaging head 452, to collectively translate depression
movement of the lever to downward retraction of the flexible
diaphragm member (compare, e.g., FIGS. 16 and 17). These components
of a diaphragm pump are conveniently housed within a protective
pump cover 460 which engages a complementary rim 462 or other
coupling surface of the pump housing 410 by a complementary
engagement fitting (arrows 464). This coupling may optionally serve
to sealably anchor the flexible diaphragm member, e.g., by
sandwiching a peripheral edge 466 of the diaphragm between the cap
and housing as shown in the figures. Optional pump devices,
actuation mechanisms, and pump housing designs are contemplated
within the invention, which are within the level of skill in the
art to engineer for use with the hand-held breast pump devices
disclosed herein.
[0202] As noted above, mammary fluid expression and collection
using the devices of the invention may be facilitated in certain
instances by prior or concurrent administration of the peptide
hormone oxytocin, or an analog thereof, in an amount that is
effective to stimulate myoeptithelial contraction in the alveolar
gland ducts of the breast to facilitate expression of the mammary
fluid from the nipple. Preferably the oxytocin preparation is
administered intranasally and is administered in an amount that is
intranasally effective to stimulate expression of mammary fluid
from the nipple. Alternatively, an intramuscular or intravascular
injection of oxytocin can effect the same myoepithelial contraction
response as the intranasal administration route. The amount, timing
and/or mode of oxytocin administration may be adjusted on an
individual basis depending on such factors as menstrual cycle
stage, use of birth control or hormone replacement therapy,
pregnancy history, age of onset of menarche, ethnicity and other
factors known to affect an individual's propensity for breast fluid
expression.
[0203] Oxytocin is a peptide hormone of pituitary origin that is
naturally released into the bloodstream of lactating women in
response to suckling, and stimulates contraction of myoepithelial
cells in the mammary alveoli and ducts to cause milk ejection
(Cobo, J. Perinat. Med. 21:77-85, 1993). The drug has also been
widely used for stimulating labor in pregnant women, due to its
activity of stimulating uterine contractions (Satin et al., Am. J.
Obstet. Gynecol. 166:1260-1261, 1992). For these reasons, the
pharmacology of oxytocin has been thoroughly investigated,
including detailed studies of effective dosages, half-life and
potential side effects.
[0204] For use in the present invention, an oxytocin preparation is
provided for intranasal, intramuscular, or intravenous
administration that contains oxytocin in a biologically suitable,
liquid carrier. As used herein, "oxytocin" refers to natural or
synthetic oxytocin and biologically active derivatives and analogs
thereof. Naturally occurring oxytocin from mammalian sources is of
course suitable, as are other known, naturally occurring
oxytocin-like peptide analogues and their synthetic counterparts
having similar activities for stimulating alveolar-ductal
myoepithelial contraction. Preferably, the oxytocin used within the
invention is a simple peptide hormone comprising a cyclic peptide,
the peptide having a well defined ring portion
(Cys-Tyr-Ile-Gln-Asn-Cys) and tail portion (Pro-Leu-Gly). However,
numerous derivatives and analogues are known, or readily
obtainable, in the art, e.g., derivatives or analogues having amino
acid truncations, deletions or substitutions at one or more
residues of the peptide and which exhibit substantially the same
activity as naturally occurring oxytocin (i.e., having at least
75%, and preferably 85%-95% or more, activity compared to that of
native oxytocin for stimulating alveolar-ductal myoepithelial
contraction). The most economic oxytocin preparations for use
within the invention contain a synthetic oxytocin (e.g.
Pitocin.RTM. or Syntocinon.RTM. available from various providers,
for example Sandoz (Basel, Switzerland) and United States
Pharmacopeia. Alternate benefits may be obtained with the use of a
long-acting oxytocin analog within the methods of the invention.
The utility and pharmacokinetics of such analogs, exemplified by
the peptide analog carbetocin, are described in detail in U.S.
patent application Ser. No. 09/481,058 filed Jan. 11, 2000
(incorporated herein by reference).
[0205] For use with the methods, devices and kits of the invention,
a preferred oxytocin preparation contains approximately 40 USP
units of oxytocin per ml of liquid carrier. Preferred liquid
carriers are biologically compatible solutions, such as a lactated
Ringer's solution or other physiologically balanced, sterile,
non-toxic and non-irritative solution. To administer the oxytocin
intranasally, a standard nasal squeeze bottle is used, which
delivers approximately 0.5 ml of the oxytocin preparation into the
patient's nostril when squeezed. The oxytocin is absorbed by the
nasal mucosa into the systemic circulation where it reaches and
acts specifically on the myoepithelial cells surrounding the
alveoli of the breast and making up the walls of the lactiferous
ducts, causing their smooth muscle fibers to contract and force any
fluids present into the large ducts or sinuses where it can be
expressed from the nipple spontaneously onto a sample collector or
by the further action of a breast pump. Intranasal application of
the spray preparation is therefore a practical and effective method
of administration. The half-life of oxytocin in the human
bloodstream is extremely short, estimated to be about 10-15 minutes
or less, due to its rapid removal from plasma by the kidney, liver,
and mammary gland, and the time to pharmacokinetic and clinical
steady state is readily determined depending on the mode of
administration (e.g. bolus dosage, repeat administration, or steady
infusion). (See for example, Gonser, Arch. Gynecol. Obstet.
256:63-66, 1995; and Orhue, Obstet. Gynecol. 83:229-233, 1994, each
incorporated herein by reference in its entirety). It is therefore
a routine matter to determine an appropriate concentration and dose
of the oxytocin preparation to administer an effective amount
(either intranasally effective, intravenously effective, or
intramuscularly effective) of the oxytocin to cause expression of
mammary fluid with or without the assistance of a breast pump. (See
for example, Newton, Ann. N.Y. Acad. Sci. 652:481-483; Mena,
Neuroendocrinology 61:722-730, 1995; Gonser, Arch. Gynecol. Obstet.
256:63-66, 1995; Orhue, Obstet. Gynecol. 83:229-233, 1994; Satin et
al., Am. J. Obstet. Gynecol. 166:1260-1261, 1992; and Satin et al.,
Obstet. Gynecol. 83:234-238, 1994, each incorporated herein by
reference in its entirety).
[0206] Although not all female patients are expected to be
responsive to intranasal oxytocin stimulation, an intranasally
effective amount of oxytocin for the purposes of the invention can
be readily determined. As used herein, an intranasally effective
amount of oxytocin is an amount of oxytocin sufficient to
intranasally stimulate the expression of at least 3 .mu.l of
mammary fluid in at least 50% of non-lactating female patients with
the aid of negative pressure to the nipple of between 50-200 mm Hg
applied by the breast pump (up to 45 min after a first
administration of the oxytocin spray). It may be necessary, and
indeed preferred, to administer a low, preliminary dose of oxytocin
to the patient, for example a single spray of a 40 Unit/ml oxytocin
solution in each nostril, or multiple sprays of a lower
concentration oxytocin preparation, and thereafter wait to
determine a particular patient's sensitivity. If there is no
reaction with an initial application of the breast pump after a
short post-administration period of 2-15 minutes, and preferably
2-5 minutes, a booster dose of the oxytocin spray may be
administered and the pump reapplied. In this way, the clinician can
modulate the dosage to each patient's varying sensitivity, and
thereby minimize potential adverse side effects. Alternatively, an
effective dose of intramuscular or intravenous oxytocin can be used
according to the same dosage determination and administration
principles in patients where intranasal administration fails or is
otherwise contra-indicated as a preferred mode of
administration.
[0207] As noted above, the amount, timing and/or mode of oxytocin
administration may be adjusted based on specific factors known to
render individuals more or less sensitive to induction of breast
fluid expression. These factors are generally well known in the
art, and include, for example, menstrual cycle stage, use of birth
control or hormone replacement therapy, pregnancy history, age of
onset of menarche, and ethnicity, among other factors.
[0208] Thus, in one aspect of the invention, methods for obtaining
a biological sample from a patient and/or determining the amount of
a breast disease marker in a biological sample from breast fluid
are provided which include a step of determining a menstrual stage
of the patient. Based on the determined menstrual stage, a drug
administration protocol is selected having a predetermined oxytocin
dosage, timing and/or frequency of oxytocin delivery, and/or mode
of oxytocin administration.
[0209] According to these methods, one or more variables of
oxytocin dosage, timing and/or frequency of oxytocin delivery,
and/or mode of oxytocin administration are selected depending on
whether the patient is staged within one of five approximate
menstrual phases. These phases include 1) a proliferative phase
(characterized by a tight configuration of the alveolar lumena); 2)
a follicular phase (characterized by a defined configuration of the
alveolar lumena); 3) a luteal phase (characterized by an open
configuration of the alveolar lumena, with some secretion by the
alveolar cells); 4) a secretory phase (characterized by an open
configuration of the alveolar lumena, with secretion by the
alveolar cells); and 5) a menstrual phase (characterized by a
distended configuration of the alveolar lumena, with secretion by
the alveolar cells).
[0210] It is generally not desired to conduct the methods of the
invention for patients staged in the proliferative or follicular
stage of their menstrual cycle (approximately 3-7 days and 8-14
days, respectively). However, in some circumstances sample
collection can be performed for these individuals using high and/or
repetitive doses of oxytocin and otherwise optimizing the breast
fluid expression response by selecting a particular mode of
oxytocin administration, or combination thereof (e.g., intravenous
administration followed by intranasal administration). For patients
staged in the luteal or secretory stage of their menstrual cycle
(approximately 15-20 days and 21-27 days, respectively),
intermediate dosages of oxytocin are selected and repetitive
administrations are reduced or eliminated. For patients staged in
the menstrual phase, dosages of oxytocin and repetitive
administrations are reduced even further while still providing an
effective administration protocol to yield sufficient breast fluid
expression.
[0211] Determination of effective administration protocols for
patients of different menstrual stages can also be readily achieved
within the invention. As used herein, an effective administration
protocol yields at least 3 .mu.l of expressed mammary fluid in at
least 50% of non-lactating female patients at an equivalent
menstrual stage with the aid of negative pressure to the nipple of
between 50-200 mm Hg applied by a breast pump up to 45 min after a
first administration of the oxytocin spray. Various combinations of
oxytocin dosage, timing and/or frequency of oxytocin delivery,
and/or mode of oxytocin administration are contemplated, as can be
readily determined by the skilled artisan in accordance with the
teachings herein. Likewise, it will often be preferred to
administer a low, preliminary dose of oxytocin to the patient and
thereafter wait to determine a particular patient's sensitivity,
even when an individual's menstrual stage has been determined and a
particular administration protocol selected. Thus, if there is no
reaction with an initial application of the breast pump after a
short post-administration period, a booster dose of the oxytocin
may be administered and the pump reapplied. In this way also, the
clinician can apply a first, stage specific dose of oxytocin and
thereafter modulate the dosage, period of time between booster
administrations, and/or mode of administration, to each patient's
varying sensitivity.
[0212] In other, related aspects of the invention, methods for
obtaining a biological sample from a patient and/or determining the
amount of a breast disease marker in a biological sample from
breast fluid are provided which include a step of determining a
non-menstrual stage patient sensitivity index. Examples of such
indices include 1) patient use of hormone based birth control; 2)
patient use of hormone replacement therapy; 3) patient pregnancy
history; 4) patient age of onset of menarche; and 5) patient
ethnicity. Other indices associated with sensitivity to induction
of breast fluid expression are also contemplated. These factors can
be determined by such routine steps as patient consultation,
evaluation of patient records, and clinical or laboratory-based
analysis (e.g., physical screening, measurement of sex-steroid
hormone levels, etc.) Based on a determined non-menstrual stage
sensitivity index, an effective drug administration protocol is
selected having a predetermined oxytocin dosage, timing and/or
frequency of oxytocin delivery, and/or mode of oxytocin
administration, in accordance with the methods described above. In
yet additional methods an effective drug administration protocol is
selected by first determining both a patient's menstrual stage and
at least one non-menstrual stage sensitivity index specific to the
patient, and thereafter selecting an effective oxytocin
administration protocol based on these combined indices.
[0213] In yet additional methods within the invention, it may be
preferred to conduct the foregoing sample collection methods in
conjunction with a conventional mammographic procedure. In this
manner, costs, time and patient discomfort can be minimized.
Further, by conducting the sample collection immediately following
a mammogram it is expected that breast fluid expression may be
facilitated by breast manipulation during the initial procedure.
Additional steps to facilitate breast fluid expression include
manual breast massage and application of heat packs to the
breast.
[0214] For mammary fluid collection using a breast pump 10,10' of
the invention, alone or in conjunction with oxytocin stimulation,
the breast pump is applied and negative pressure is generated on
the breast to facilitate the expression of mammary fluid. Within
the methods of the invention, negative pressures of 50-200 mm Hg
are preferred, and these pressures are maintained, preferably
intermittently, for approximately 1-15 minutes, depending on the
sensitivity of individual patients, oxytocin dosage and other
factors. The volume of expressed mammary fluid will vary depending
on a variety of factors, including the time and pressure of breast
pump administration, and other factors. For the least sensitive
breast marker assays of the invention, a volume of expressed
mammary fluid of 300-500 .mu.l is preferred to provide ample
material for conducting the assay, and these volumes will be
obtainable from a substantial proportion of women treated according
to the above methods. To express 300-500 .mu.l of mammary fluid,
some women will require repeated stimulation treatments, perhaps
requiring pooling of mammary fluid samples obtained during multiple
patient visits. However, for more sensitive assays of the
invention, e.g. solid phase immunoassays, much smaller samples of 3
.mu.l or less will be suitable to carry out the assays,
particularly in the case of breast cancer markers that are
naturally secreted into the mammary fluid and are therefore
expected to be present in very high concentrations compared to, for
example, breast epithelial cell surface antigens or intracellular
antigens that are not secreted.
[0215] The following examples are offered by way of illustration,
not by way of limitation.
EXAMPLE 1
Induction of Mammary Fluid Expression by Application of a Novel
Breast Pump/Mammary Fluid Sample Collection Device
[0216] Within the present example, a hand-held breast pump device
10' is employed to collect a primary sample of mammary fluid
components comprising whole cells and cell fragments for
cytological examination. The doctor, technician or patient collects
the breast fluid specimen by grasping and operating the hand-held
pump as described above to stimulate expression of the mammary
fluid and collect a specimen thereof. This operation is a
one-handed procedure, leaving the physician or technician free to
conduct additional activities with the other, free hand. In this
regard, the compact hand-held pump design allows the device to be
picked up and manipulated with one hand, to seat the breast
engaging element against the breast, apply sufficient vacuum
pressure to the breast by manual operation of the vacuum pump to
cause a suitable volume of breast fluid to be expressed at or near
the nipple, and to simultaneously collect at least a primary sample
of expressed breast fluid onto, or within, the solid phase sample
collection medium (e.g., a nitrocellulose filter) without
additional manual steps or the need to remove the device from the
breast or engage two hands in the operation.
[0217] In the present example, the hand-held pump device 10' is
employed to collect a primary sample of a selected mammary fluid
component comprising cells and other cellular materials, that are
retained (e.g., by retaining cells on a nitrocellulose membrane 39
or filter 40 coupled with the breast engaging member 14 or sample
collection housing 30) for further processing. The primary
collected sample of cells and other materials is then transferred
(e.g., by washing or manual transfer) to a reservoir or other solid
phase template, for further storage, processing and/or analysis. In
more specific protocols, whole cells are separated from expressed
mammary fluid onto a nitrocellulose membrane 39 or a filter 40
secured in fluid connection with the breast engaging member 14 a
removable support member 56 mounted to the engaging member or
sample collection housing 30.
[0218] The cells are subsequently transferred or washed in fluid
(e.g., cytology fluid) to a second solid phase sample collection
medium (e.g., a slide, well, tube or vial), which may also be
connected to, or integrated with, the breast engaging member 14 or
sample collection housing 30 as described above. In one protocol,
the breast engaging member is removed from the breast and a jet of
cytological fluid is directed against the filter on which the cells
are retained. This flushes the cells off of the filter into an
awaiting reservoir, typically a removable fluid-retaining
reservoir. In this regard, uniquely designed membranes and filters
are provided for use within this protocol which feature
perforations or slits that disrupt the planar surface of the
membrane or filter to facilitate air passage therethrough, to
impart structural flexibility against mechanical perturbation, and
to allow cells flushed from a first side (facing the breast) of the
filter to a second side (facing internally, e.g., toward the sample
collection housing, when the filter is mounted with the device) for
secondary sample collection. For example, radial slits 280, spiral
perforations 284 or transverse slits can be made in the filter to
allow passage of air and for cells partitioned onto the first
surface of the membrane to be flushed through the membrane into the
removable, fluid-retaining reservoir 288 seated in the sample
collection housing 30, for storage, shipment, and/or further
processing directed toward cytological examination of the secondary
collected sample.
EXAMPLE 2
Cytology in Biological Samples from Mammary Fluid
[0219] This example describes the use of conventional cytological
techniques to identify and classify breast diseases from samples
obtained as described in Example 1. Following collection of the
sample, e.g., in a fluid-retaining reservoir member, the sample may
be further processed (e.g., by centrifugation wherein the reservoir
member is in the form of 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, and other suitable stains.
[0220] 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 5 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.
EXAMPLE 3
Stimulation of Mammary Fluid Expression for Sample Collection by
Coordinate Administration of Intranasal Oxytocin in Conjunction
with Application of a Novel Breast Pump/Mammary Fluid Sample
Collection Device
[0221] The foregoing sample collection protocol in Example 1, as
well as other sample collection methods within the invention, may
be practiced solely by the use of a novel breast pump 10, 10' of
the invention. Alternatively, these sample collection procedures
may be practiced in conjunction with the use of oxytocin or
oxytocin analogs to facilitate or increase mammary fluid expression
induced by operation of the breast pump. As incorporated within the
invention, these methods involve application of the breast pump 10,
10' to the breast, optionally coupled with oxytocin administration
in amounts effective to facilitate mammary fluid expression in the
patient. After the sample is collected, a bioassay is conducted on
the sample to determine the presence and/or amount of a selected
breast disease marker, preferably a breast cancer marker or panel
of breast cancer markers, in the sample.
[0222] Oxytocin nasal solution acts specifically on the
myoepithelial elements surrounding the alveoli of the breast and
making up the walls of the lactiferous ducts, causing their smooth
muscle fibers to contract and thus force any fluids present into
the large ducts or sinuses where it can be expressed from the
nipple by the further action of a breast pump. The nasal spray is
promptly absorbed by the nasal mucosa to enter the systemic
circulation. Intranasal application of the spray preparation is a
practical and effective method of administration. Half-life of
oxytocin in the human circulation is extremely short, approximately
10-15 minutes, and oxytocin is then rapidly removed from plasma by
the kidney, liver, and mammary gland.
[0223] Because of the known effects of oxytocin to cause uterine
contractions, pregnant women should not be treated by the methods
contained herein unless the benefits of testing outweigh the risk
of inducing premature labor.
[0224] The oxytocin nasal solution contains a concentration of
natural or synthetic oxytocin, or a functional analog thereof such
as carbetocin, that is intranasally effective in a selected volume
of administered spray to stimulate expression of mammary fluid from
a nipple of the patient when a breast pump is applied to the nipple
to assist mammary fluid expression. In the present example, a
preferred oxytocin preparation containing approximately 40 USP
units of oxytocin per ml of lactated Ringer's solution is
administered into the nose with the squeeze bottle held in the
upright position while the patient is in a sitting position. One or
two sprays are administered into each nostril from a standard nasal
squeeze bottle, which delivers approximately 0.5 ml of the oxytocin
solution per spray in a fine mist when the bottle is squeezed. The
number and volume of sprays administered, as well as the
concentration of oxytocin in the solution, can be adjusted
according to well known pharmacokinetic principles (See for
example, Newton, Ann. N.Y. Acad. Sci. 652:481-483; Mena,
Neuroendocrinology 61:722-730, 1995; Gonser, Arch. Gynecol. Obstet.
256:63-66, 1995; Orhue, Obstet. Gynecol. 83:229-233, 1994; Satin et
al., Am. J. Obstet. Gynecol. 166:1260-1261, 1992; and Satin et al.,
Obstet. Gynecol. 83:234-238, 1994, each incorporated herein by
reference in its entirety) to ensure that the amount of oxytocin
administered to the patient corresponds to an intranasally
effective amount to stimulate the expression of at least 3 .mu.l of
mammary fluid in at least 50% of non-lactating female patients upon
activation of the breast pump. For example, adjustments may be
desired in the number of sprays delivered to the patient, and/or
the timing of spray delivery, so that the clinician can modulate
the dosage to each patient's varying sensitivity, and thereby
minimize potential adverse side effects. In the present example, a
preliminary dose of a single spray of the 40 Unit/ml oxytocin
solution is delivered into each nostril of the patient, and the
administering clinician waits for a short post-administration
period of 2-3 minutes. After this period the breast pump is
applied, and the clinician determines whether or not the amount of
oxytocin delivered was sufficient to facilitate or increase breast
pump-induced expression of mammary fluid. If additional fluid
expression is desired at this stage a booster dose of 1 or 2
additional sprays of the oxytocin solution can be administered in
each nostril, and the pump reapplied after a 5-10 minute
post-booster administration period.
[0225] After the intranasally effective dose of the oxytocin is
administered and the clinician has allowed a suitable
post-administration period to elapse for the oxytocin to reach and
stimulate the target alveolar-ductal tissue, the breast pump is
applied. Negative pressures of 50-200 mm Hg are applied in the area
of the nipple and are maintained, intermittently or continuously,
for approximately 1-15 minutes, depending on the sensitivity of
individual patients, oxytocin dosage and other factors.
Alternatively, oxytocin can be administered by intramuscular or
intravascular routes by well known means (Oxytocin Injection
(synthetic), USP; Wyeth-Ayerst Laboratories) to effect the same
response as intranasal administration.
EXAMPLE 4
Verification of Sample Quantity, Origin and Quality
[0226] Using either of the above methods of Example 1 or Example 3,
volumes of at least 3 .mu.l of expressed mammary fluid can be
collected in a substantial population of non-lactating female
patients. During or after the mammary fluid expression step, a
biological sample is collected from the expressed mammary fluid as
described above. For example, a nitrocellulose filter may be placed
within the breast pump in line with a path of the expressed mammary
fluid into the pump, so that the expressed fluid contacts the
filter. Upon contact of the primary sample of expressed mammary
fluid with the filter, cells, proteins and other desired components
of the mammary fluid adhere to the filter to form a filter-bound or
filter-retained biological sample for subsequent analysis. Other
suitable biological samples, including whole mammary fluid samples,
cytological samples of whole cells, membranes or other cellular
components, and samples containing proteins, glycoproteins,
peptides, nucleotides and other constituents of the primary mammary
fluid sample can be collected with appropriate modifications of the
above procedures, according to well known principles and
methods.
[0227] To ascertain that the sample of mammary fluid is of mammary
origin and is not corrupted by likely contaminants, one or more
constituents of normal mammary fluid are assayed for. In the
present example, an enzyme that is ordinarily present in mammary
fluid, lysozyme, is assayed in the mammary fluid sample to help
confirm that the sample is of mammary origin. Lysozyme (muramidase)
is an enzyme which hydrolyzes beta 1,4-glycosidic linkages in the
mucopolysaccharide cell wall of a variety of microorganisms, which
activity can be readily detected and quantified using a routine,
inexpensive assay. In the present example, Lysozyme is measured in
the primary mammary fluid sample using the Quantiplate Lysozyme
Test kit (Kallestad, Chasta, Minn.). The assay employs the reagents
and procedures provided by the manufacturer and specified in detail
in the manufacturer's instructions, with the exception that a
mammary fluid sample is substituted in place of serum, urine or
tears. Analysis of these results establishes that the sample
contains lysozyme, which is a normal component of human serum,
urine, saliva, tears, nasal secretions, vaginal secretions, seminal
fluid, and mammary fluid.
[0228] More specific assays are used in place of the lysozyme
assay, or to supplement lysozyme assay results, particularly where
clinical data for human patients are being gathered. Other mammary
fluid markers for sample verification that are more specific than
lysozyme can be readily incorporated within the invention, based on
published and generally known information. In one example, the
presence of cathepsin D is assayed using the monoclonal antibodies
and methods disclosed in Vetvicka et al., Biochem. Mol. Biol.
Int'l. 30:921-928, 1993, incorporated herein by reference in its
entirety). In another example, one or more human mammary epithelial
antigens (HME-Ags) corresponding to glycoprotein components of the
human milk fat globulin (HMFG) protein are detected in the primary
mammary fluid sample, or in the biological sample that is used in
the breast cancer marker assay, using specific antibody probes, as
described by 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). In many cases,
the sample verification assay can be incorporated within the breast
cancer marker assay in a single procedure, for example as described
below in Example 4, an assay for HME-Ags (wherein the HME-Ag
findings are indicative of sample origin/quality, and also of the
presence and/or quantity of a specific breast cancer marker in the
sample). In another example, sample verification is achieved
through a combinatorial (i.e. multiple marker) immunoassay
targeting various cytokeratins, which can be detected as a panel of
cytokeratins specifically expressed in mammary tissue sample. (See,
Nagle, J., Histochem. Cytochem. 34:869-881, 1986, incorporated
herein by reference in its entirety). One or more of these
cytokeratins (e.g. K5, K8, K18 and K19) can be simultaneously or
independently measured in the context of a breast cancer assay, and
the level of expression of the subject cytokeratin(s) can yield
information concerning the presence or status of breast cancer in a
patient. (See for example, Focus, Harvard University News Office,
Mar. 21, 1991, pp. 2-3; and Lee, Proc. Natl. Acad. Sci. USA
88:1991, each incorporated herein by reference in its
entirety).
EXAMPLE 5
Immunoassay for Human Mammary Epithelial Antigens in Biological
Samples From Mammary Fluid
[0229] Human mammary epithelial antigens (HME-Ags) are glycoprotein
components of the human milkfat globule (HMFG) and of the membrane
of the breast epithelial cell, which are released by breast tumors
and not by normal breast tissue. (Ceriani et al., Proc. Natl. Acad.
Sci. USA 74:582-586, 1977, incorporated herein by reference in its
entirety). In the present example, several HME-Ags, having
molecular weights of 150, 70, and 45 kilodaltons, are detected and
measured using specific anti-HMFG or anti-human mammary epithelial
(a-HME) probes prepared and employed as described by Ceriani et
al., Proc. Natl. Acad. Sci. USA 79:5420-5425, 1982 (incorporated
herein by reference in its entirety).
[0230] To begin the assay, biological samples from mammary fluid
collected on nitrocellulose filters coupled with a breast pump 10,
10' as generally described above are eluted electrophoretically
into phosphate buffered saline to provide a test sample, according
to standard methods. Alternatively, whole mammary fluid or other
types of biological samples obtained from mammary fluid can be
constituted in an appropriate medium or mixture to provide a test
sample for the assay. Once the test sample is thus provided, it is
then assayed according to the HME-Ags radioimmunoassay (RIA)
methods described in Ceriani et al., Breast Cancer Res. Treat.
15:161-174, 1990 (incorporated herein by reference in its
entirety).
[0231] Briefly, the RIA includes two preliminary treatments of the
biological samples to separate interfering factors: a
centrifugation step to separate out any fat present, and a second,
precipitation step to precipitate potential immunocomplexes using
polyethyleneglycol (PEG). The next steps comprise the assay proper,
where HMFG antigen bound to a solid support (microtiter plates) is
presented to stoichiometric or lesser amount of the .alpha.-HME
antibody probe, and binding of the .alpha.-HME is competed by the
biological samples from mammary fluid preliminarily treated as
above. The amount of a-HME bound to HMFG antigen on the solid phase
is determined in a final step by detection of the .alpha.-HME
antibody probe by radioiodinated, affinity-purified rabbit
anti-mouse immunoglobulin.
[0232] Solutions used in the assay are as follows: i) Phosphate
buffered saline (PBS): 176 ml 0.05M KH.sub.2PO.sub.4, 608 ml 0.05M
Na.sub.2HPO.sub.4, and 8 g NaCl brought up to 1000 ml in H.sub.2O
(pH7.4). ii) RIA buffer: 0.1% BSA, in 0.3% Triton-X-100 (Research
Prod. International Corp., Mount Prospect, IL) plus 0.05% sodium
azide in PBS. iii) Detergent buffer: 0.3% Triton-X-100 plus 0.05%
sodium azide in PBS. iv) Buffered polyethylene glycol (PEG): 6.6%
PEG (M.W. 8000) (Sigma) plus 0.05% sodium azide in PBS)
.sup.125I-labeled affinity-purified rabbit anti-mouse
immunoglobulin (R.alpha.-mouse Ig) (Antibodies, Inc., Davis,
Calif.), radioiodinated by the chloramine-T procedure as reported
(Ceriani et al., Proc. Natl. Acad. Sci. USA 79:5420-5425, 1982) and
diluted to 4.times.10.sup.6 cpm/ml, in RIA buffer. Rabbit
polyclonal anti-HMFG antibodies or rabbit anti-human mammary
epithelial antibodies (.alpha.-HME) were prepared and assayed as
described (Id.).
[0233] To prepare a standard curve for evaluating assay results,
control samples from normal human mammary fluid (exposed to
nitrocellulose filters and eluted in the same manner as the
nitrocellulose adsorbed, eluted test sample, or alternatively
provided as normal whole mammary fluid or other selected type of
sample obtained from normal mammary fluid, constituted in an
appropriate medium or mixture to provide a suitable control assay
sample) are centrifuged for 7 min at 10,240 rpm at 10.degree. C.
The upper white band formed at the top of the sample (if there is
one) is discarded. Fresh 100 .mu.g protein/ml solution of
lyophilized dilipidated HMFG (Ceriani et al., Proc. Natl. Acad.
Sci. USA 74:582-586, 1977) in detergent buffer is prepared and
sonicated at 10 second intervals for a total of 4 minutes (10 sec.
of sonication, followed by a 10 sec. silent period) using a double
step micro tip horn at 25 watts on a Sonifier Cell Disrupter 185
(Branson, Danbury, Conn.) at 4.degree. C. HMFG solutions at
concentrations of 0, 10, 33.3, 100, 333.3, and 1000 ng protein/ml
are prepared in spun female sera, and 3 aliquots of 180 .mu.l of
each HMFG level in normal female sera are pipetted into 400 .mu.l
polyethylene microcentrifuge tubes (West Coast Sci. Emeryville,
Calif.). 150 .mu.l of 6.6% PEG solution is added to each
microcentrifuge tube, and the tubes are incubated overnight on a
rotating shaker at room temperature.
[0234] Test samples are processed in a comparable manner, by
centrifuging 300-350 .mu.l of the eluted nitrocellulose filtrate in
solution (or, alternatively, of mammary fluid or other assay sample
alternative) in a 400 .mu.l microcentrifuge tube for 5-7 min. at
10,240 rpm at 10.degree. C. The microcentrifuge tubes are then cut
with a razor blade below the white band formed by the sera (if
there was one) and 180 .mu.l of remaining sera is transferred to a
new microcentrifuge tube. 150 .mu.l of a 6.6% PEG solution is then
added to each microcentrifuge tube, and the tubes are incubated
overnight on a rotating shaker at room temperature.
[0235] Day Two
[0236] (1) .alpha.-HME is diluted to its appropriate concentration
in detergent buffer. The antibody solution has stoichiometric or
lesser amounts of a-HME to 6 ng HMFG protein equivalent (prot.
eq.). Six ng of HMFG is covalently bound to microtiter plates by
the methylated BSA procedure previously described by Ceriani, in
Monoclonal Antibodies and Functional Cell Lines, pp. 398-402,
Kennet et al. (eds), Plenum Press, New York, 1984, incorporated
herein by reference in its entirety.
[0237] (2) To process test samples and control samples on the
second day, microcentrifuge tubes are centrifuged for 7 min. at
10,240 rpm at 10.degree. C. in a SHMT rotor with a Sorvall RC5C
centrifuge. In triplicate, 55 .mu.l of supernatant is pipetted into
empty microtiter plate wells (Dynatech, Alexandria, Va.), and any
precipitate pelleted is left undisturbed. 25 .mu.l of 6.6% PEG
solution is added to each well. 30 .mu.l of .alpha.-HME diluted in
detergent buffer is also added to each well, and a non-porous
Scotch.RTM. tape is placed over the wells to avoid evaporation. The
microtiter plate is then incubated overnight at room temperature on
a rotary shaker.
[0238] Day Three
[0239] The microtiter plates are centrifuged (3000 r.p.m.) for 30
minutes at room temperature to decant suspended perceptible matter.
50 .mu.l of RIA buffer is added to wells of microtiter plates
containing 6 ng HMFG and aspirated off after 5 minutes.
[0240] The total contents of microtiter plates from 1), save for
any precipitation induced by the PEG and already pelleted, are
carefully transferred to the wells of another set of microtiter
plates containing 6 ng HMFG per well (Day 2,1), above.
[0241] The microtiter plates are incubated for 3 hours with
rotating agitation at room temperature. The plates are washed 5
times with RIA buffer using Dynadrop SR-1 automatic dispenser form
Dynatech. 50 .mu.l of the radioiodinated affinity-purified rabbit
anti-mouse immunoglobulin diluted in RIA buffer is then adder per
well. The plate is covered with tape and incubated with rotating
agitation for 2 hours at room temperature. The plate is washed 5
times with RIA buffer. Wells are cut and counted in a gamma
counter.
[0242] The results of these assays will yield important information
concerning the presence and/or status of cancer in patients,
comparable in scope and value to the data provided by serum assays
conducted for the HME-Ag breast cancer marker by Ceriani et al.,
Breast Cancer Res. Treat. 15:161-174, 1990. By selecting patient
and control samples and developing and evaluating comparative data
according to the procedures followed by Ceriani and his coworkers,
the assay methods of the invention will also be readily adapted for
use in direct clinical applications to determine both prognostic
and treatment related variables in breast cancer patients. Reagents
and conditions for the assays can of course be substituted or
adjusted depending on a variety of anticipated variables, by
applying well known immunological methods and principles.
EXAMPLE 6
Competitive Radioimmunoassay for Non-Penetrating Glycoprotein in
Biological Samples From Mammary Fluid
[0243] This competitive radioimmunoassay is based on the
displacement by breast epithelial antigens contained in biological
samples from mammary fluid obtained according to the methods of the
invention of the binding of stoichiometric or lesser quantities of
the monoclonal antibody Mc5 to a solid-phase-bound antigen known as
non-penetrating glycoprotein (NPGP) contained in HMFG. HMFG is
bound to a solid support and exposed to the Mc5 antibody during an
incubation period allowing the antibody to bind the NPGP antigen in
solid phase-bound HMFG. The presence and/or level of NPGP in the
biological sample is ultimately examined by ability of the sample
to compete for Mc5 binding to the NPGP antigen in the solid
phase-bound HMFG, as detected and/or measured using a radiolabeled
goat anti-mouse antibody to bind and label the McS antibody
probe.
[0244] Buffer and other solutions and reagents in this example are
generally the same as those used for the HME-Ags polyclonal
antibody radioimmunoassay described in Example 4, above. To provide
test samples for the assay, biological samples from mammary fluid
contained on nitrocellulose filters are eluted electrophoretically
into phosphate buffered saline, according to standard methods.
Alternatively, whole mammary fluid or other types of biological
samples obtained from mammary fluid can be constituted in an
appropriate medium or mixture to provide a test sample for the
assay. Once the test sample is thus provided, it is then assayed
according to the NPGP/Mc5 radioimmunoassay (RIA) methods described
in Ceriani et al., Breast Cancer Res. Treat. 15:161-174, 1990
(incorporated herein by reference in its entirety), as follows:
[0245] 400 .mu.l of pooled normal female mammary fluid (exposed to
nitrocellulose filters and eluted in the same manner as the
nitrocellulose adsorbed, eluted test sample, or alternatively
provided as normal whole mammary fluid or other types of biological
samples obtained from normal mammary fluid constituted in an
appropriate medium or mixture to provide a test sample) to provide
a suitable control sample, which is diluted to 2.4 ml using RIA
buffer at a 1:6 concentration.
[0246] A 500 .mu.g/ml solution of lypholized HMFG is prepared in
1.times.PBS with 0.3% Triton-X-100, 0.05% sodium azide, and
sonicated using a double step micro tip horn at 25 watts on a
Sonifier Cell Disrupter 185 (Branson, Danbury, Conn.) for 4 minutes
(10 sec. sonication, 10 sec. silent period, at 4.degree. C.).
[0247] Solutions to prepare a standard curve are prepared using the
2.4 ml 1:6 normal female serum and increasing amounts of HMFG (0,
0.25, 2.5, 25, 50 .mu.g/ml HMFG, as described above in Example
4).
[0248] Each test assay sample is diluted 1:6 with RIA buffer (40
.mu.l of serum to 200 .mu.l RIA buffer) to form a diluted test
assay sample, and vortexed.
[0249] Mc5 stock solution is prepared so that it contains less than
stoichiometric amounts of antibody to 100 ng protein/well of HMFG
covalently bound to microtiter plates prepared as previously
described by Ceriani, in Monoclonal Antibodies and Functional Cell
Lines, pp. 398-402, Kennet et al. (eds), Plenum Press, New York,
1984, incorporated herein by reference in its entirety
[0250] 200 .mu.l RIA buffer are added to each well of 100 ng HMFG
and then aspirated after 5 minutes.
[0251] To prepare a standard curve, 30 .mu.l of HMFG standardizing
solutions (as in 3 above) are added in quadruplicate to a 100 ng
protein/well HMFG microtiter well.
[0252] 30 .mu.l of diluted test assay sample (or, alternatively, of
mammary fluid or other assay sample alternative) are added in
triplicate to 100 ng/well HMFG microtiter wells.
[0253] To each well 20 .mu.l of the Mc5 stock solution is
added.
[0254] Microtiter plates are covered with nonporous Scotch.RTM.
tape and incubated overnight at room temperature on a rotating
agitator.
[0255] The next day the wells are aspirated and washed 5 times with
RIA buffer.
[0256] To each well 50 .mu.l of 200,000 cpm/50 .mu.l .sup.125I-goat
anti-mouse antibody are dispensed. The wells are covered with
nonporous tape and placed on a rotating agitator for 3 hours at
room temperature.
[0257] Wells are washed 5 times with RIA buffer.
[0258] Each well is cut and the radioactivity is counted using a
gamma counter.
[0259] The results of these assays will yield important information
concerning the presence and/or status of cancer in patients,
comparable in scope and value to the data provided by serum assays
conducted for the NPGP breast cancer marker by Ceriani et al.,
Breast Cancer Res. Treat. 15:161-174, 1990. By selecting patient
and control samples and developing and evaluating data according to
the well known procedures followed by Ceriani and his coworkers,
the assay methods of the invention will be readily adapted for use
in direct clinical applications to determine both prognostic and
treatment related variables in breast cancer patients. As will be
understood by those skilled in the art, reagents and conditions for
the assays can be substituted or adjusted depending on a variety of
anticipated variables, according to well known immunological
methods and principles.
EXAMPLE 7
Solid Phase Immunoassay for Mucinous Carcinoma Associated Antigen
in Mammary Fluid
[0260] This example uses a sensitive, solid phase immunoassay to
detect the mucinous carcinoma associated antigen (MCA) in
biological samples from mammary fluid obtained according to the
methods of the invention. MCA concentrations are determined using
an antibody-bead immunoassay kit provided by Hoffman-La Roche
(Basel, Switzerland), and using the reagents and procedures
provided by the manufacturer and described in further detail in
Eskelinen et al., Anticancer Res. 9:437-440, 1989. Briefly, test
assay samples of whole mammary fluid and standards are first
incubated with MCA monoclonal antibody beads and then, after
appropriate washings, enzyme (horseradish peroxidase) labeled
secondary antibody is added. During the second incubation the
anti-MCA enzyme conjugates are attached to the antibody antigen
complex on the beads. Excess conjugates are removed by washing and,
finally, enzyme substrate is added and the color formed is
recorded.
[0261] The solid phase assay format presented in this example can
be adapted for use in a wide array of other assays to detect and/or
measure other cancer markers besides the MCA marker, with enhanced
sensitivity. In addition, the results of these assays can be
evaluated along with those of complementary assays detecting and/or
measuring different markers to yield more precise information
concerning the presence and/or status of cancer in patients, as
exemplified by the combinatorial MCA/CA 15-3 assays described by
Eskelinen et al., Anticancer Res. 9:437-440, 1989; see also
Eskelinen et al., Anticancer Res. 8:665-668, 1988, each
incorporated herein by reference in its entirety.
EXAMPLE 8
Western Analysis of Proteins From Cellular Fractions of Human
Mammary Fluid Using Polyclonal and Monoclonal Antibody Probes to
Detect Vasopressin
[0262] A variety of assays are provided by the present invention
that focus on cellular samples from human mammary fluid. In
general, these assays rely on isolation by standard separation
methods (e.g. centrifugation, sucrose gradient, etc.) of cells,
membranes or other cell components from whole mammary fluid
expressed according to the above methods. Biological samples
containing whole cells from expressed mammary fluid are
particularly useful for cytological and cytochemical examination to
detect and evaluate breast cancer in patients. Biological samples
containing purified cell membrane fractions from human mammary
fluid are particularly useful in this context, for example to
detect and/or measure breast cancer markers that are expressed by
alveolar-ductal cells as intracellular or membrane bound proteins
and are therefore not as readily detected in liquid fractions of
mammary fluid as secreted proteins.
[0263] The present example focuses on assays for detecting the
peptide hormone vasopressin in biological samples from mammary
fluid, using methods adapted from North et al., Breast Cancer Res.
Treat. 34:229-235, 1995. Specifically, this assay uses a test
sample of crude protein isolated from a pooled sample of cells
obtained from expressed mammary fluid. The cells are separated from
whole mammary fluid according to standard methods, and crude
protein is extracted from the cells by sonication in 100 volumes of
0.1 M HCl. The resulting protein suspensions are then centrifuged
at 1500.times.g for 10 min. at ambient temperature, and soluble
protein is precipitated with 40% TCA. This protein is pelleted by
centrifugation at 10,000.times.g for 2 min. TCA is then removed
from pellets by washing (.times.2) with ether. Protein is
resuspended in 0.1 M Tris HCl (pH8.7), reduced with mercaptoethanol
at 100.degree. C. for 5 min. (and in some cases S-alkylated with
N-ethyl maleimide), and subjected to SDS-PAGE electrophoresis on
15% gels at pH 9.3 using the method of Laemeli, Nature 227:680-685,
1970, incorporated herein by reference in its entirety. Separated
proteins are then electrophoretically transferred with 20 mM Tris
glycine (pH 8.0) to Immobilon PVDF membranes (Millipore, Bedford,
Mass.). These membranes are blocked with a 5% non-fat milk
solution, washed (1.times.15 min., 2.times.5 min.) with PBS
containing 0.5% Triton, and incubated with preparations of mouse
monoclonal antibody to VP-HNP, with rabbit polyclonal antibodies to
VP, with rabbit polyclonal antibodies to VAG, or with ubiquitous
mouse or rabbit IgG (negative controls) (for description of
antibodies and antibody preparation see North et al., Breast Cancer
Res. Treat. 34:229-235, 1995, incorporated herein by reference in
its entirety), for 1 h at ambient temperature. Following a second
wash in PBS-Triton (1.times.15 min., 2.times.5 min.), the membranes
are treated, respectively, with goat anti-mouse IgG-horseradish
peroxidase conjugate or goat anti-rabbit IgG-horseradish peroxidase
conjugate for 1 h, and then washed with PBS-Triton (1.times.15
min., 4.times.5 min.). Immunoreactive proteins are visualized using
an ECL Western Blotting Detection System with exposure of x-ray
film from 10 seconds to 5 min. Prestained SDS-PAGE standard
proteins are employed as molecular size markers.
[0264] Recent studies suggest that vasopressin is universally
expressed in breast carcinoma and is absent from normal breast
cells. North et al., Breast Cancer Res. Treat. 34:229-235, 1995.
These and other results indicate that vasopressin and its relatives
are important breast cancer markers that can be readily detected
using immunological assays of proteins isolated from breast tumor
cells. Accordingly, the results of the present example using cell
samples isolated from human mammary fluid are also expected to
yield important information concerning the presence and/or status
of cancer in patients.
EXAMPLE 9
Quantification of Carcinoembryonic Antigen in Biological Samples
from Mammary Fluid by Dot Immunoblotting Assay
[0265] Among the more sensitive assays of the invention, useful for
measuring low levels of breast cancer markers and for detecting
markers when only small volumes of expressed mammary fluid are
available, is the dot immunoblotting assay. In the present example,
carcinoembryonic antigen (CEA) is measured in whole mammary fluid
using an Elmotech anti-CEA monoclonal antibody kit (Mochid
Pharmaceutical Co., Tokyo, Japan) in a dot blot assay format.
Briefly, anti-CEA monoclonal antibody is diluted to appropriate
concentrations and coated on the plastic film. Aliquots (5 .mu.l)
of either standard CEA solution (0, 100, 200, and 500 ng/ml), or of
the whole mammary fluid assay sample, are smeared on the
immobilized film. Assay standards are prepared from purified
antigen preparations, in accordance with the Elmotec kit
manufacturer's instructions. If necessary, 1000 ng/ml CEA solution
is also used as a standard. After drying at room temperature, the
film is exposed to peroxidase-conjugated anti-CEA antibody for 20
min at room temperature. The film is then washed extensively with 1
M saline containing 0.5% (v/v) Tween 20. The enzyme reaction is
visualized using tetramethylbenzidine as a chromogen. The
developing solution consists of 0.05 mM tetramethylbenzidine and
0.01% hydrogen peroxide in McIlvain buffer (0.1% M
phosphate-citrate buffer), pH 5.0, containing 10% methanol. The
concentration of CEA in the mammary fluid assay sample is
determined by comparing the color intensities with a corresponding
standard.
[0266] The assay disclosed in the present example, and related
assays incorporating antibodies to other tumor markers besides CEA,
are particularly useful for measuring low levels of breast cancer
markers and for detecting markers in limited sample volumes. The
results of these assays will yield important information to
determine both prognostic and treatment related variables in breast
cancer patients. As will be understood by those skilled in the art,
reagents and conditions for the assays can be substituted or
adjusted depending on a variety of anticipated variables, according
to well known immunological methods and principles.
EXAMPLE 10
Detection of Procathepsin D and Cathepsin D Activity in Biological
Samples from Mammary Fluid
[0267] Cathepsin D is a lysosomal aspartic proteinase that has been
studied intensively as a marker for cancer processes necessary for
metastasis. In the present example, polyclonal antibodies against
procathepsin D are used to immunoprecipitate and immunochemically
detect proteins from whole mammary fluid or cell lysates from
mammary fluid, generally according to the methods disclosed in
Vetvicka et al., Biochem. Mol. Biol. Int'l. 30:921-928, 1993
(incorporated herein by reference in its entirety). Alternatively,
or as a complementary assay, the protease activity of cathepsin D
is detected, also according to the methods disclosed in Vetvicka et
al. (Id.). Briefly, pooled whole mammary fluid (preferably 3 ml if
available) is diluted with 3 ml of buffer A (50 mM Tris.HCl, 5 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 500 mM NaCl pH 7.2). The suspension is
centrifuged for 30 minutes at 10,000 g. The resulting water phase
is centrifuged again under the same conditions. The soluble part
(total of approximately 4.5 ml) is loaded on a 1 ml column of
Concanavalin A Sepharose (Pharmacia, Uppsala, Sweden) equilibrated
in buffer A, and after washing with buffer A the retained proteins
are eluted using 0.75 M methyl a-D-mannopyranoside. The fractions
(250 .mu.l) are analyzed for cathepsin D activity using the
.sup.14C hemoglobin assay as described by Lin et al., J. Biol.
Chem. 264:4482-4489, 1989 (incorporated herein by reference in its
entirety), by western blots and by silver-stained electrophoresis.
The inhibition of human milk procathepsin D is accomplished by
adding 2 .mu.l of 1 mM pepstatin A (Boehringer Manheim, Germany)
dissolved in methanol to the reaction mixture.
[0268] This assay provides but one example of many possible
embodiments of the invention that incorporate known biochemical
assays, in addition to, or supplemental to immunological assays, to
evaluate biological samples from mammary fluid to determine cancer
related variables. The fundamental methods provided herein for
obtaining samples from human mammary fluid render these assays
readily adaptable for widespread clinical application to detect
and/or measure the activity of a subject breast cancer marker
within a non-invasive screening protocol.
[0269] Those with ordinary skill in the art will appreciate that
other embodiments and variations of the invention are possible
which employ the same inventive concepts described above. Most
particularly, a wide and rapidly expanding array of useful breast
cancer markers (including proteins, DNA and RNA sequences and other
markers) and probes (including immunological, nucleotide and
biochemical probes) are readily available for adaptation and use
within the methods and kits of the invention. These markers and
probes are described or referenced to a large extent in the
literature cited and incorporated within the present disclosure, or
are elsewhere published in the literature or well known in the art.
Among these known and emerging markers and probes, useful examples
within the invention include Her 2 (also known as erbB-2 and neu).
Her 2 is a transmembrane glycoprotein growth factor receptor of the
EGF receptor family encoded by a gene located on chromosome 17q, a
region of frequent amplification in breast cancer cell lines. This
marker is highly predictive of breast cancer and can be detected in
cellular samples of the invention using known nucleotide probes to
detect genetic defects in Her 2, or to detect and/or measure mRNA
to determine overexpression of Her 2 linked to increased
proliferation of cancer cells. (See for example, Visscher et al.,
In Weinstein and Graham (eds) Advances in Pathology and Laboratory
Medicine, vol. 5, St. Louis, Mosby Yuear Book, 1992, pp. 123-161;
Barbareschi et al., Am. J. Clin. Pathol. 98:408-418, 1992; Slamon
et al., Science 235:177-182, 1987; each incorporated herein by
reference in its entirety). Protein levels of Her 2 are also
readily detected using available immunological probes. (For review
see Porter-Jordan et al., Hematol. Oncol. Clin. North Amer.
8:73-100, 1994 and articles cited on page 80 therein, each
incorporated herein by reference in its entirety). Additional
markers for use within the invention include EGF and the EGF
receptor, for which immunological and non-immunological probes and
assay methods readily adaptable within the invention are
characterized in detail at page 80-81 of Porter-Jordan et al.,
Hematol. Oncol. Clin. North Amer. 8:73-100, 1994 and in the
references cited therein, each incorporated herein by reference in
its entirety. Additional examples of proliferation markers, growth
factors and receptors, proteases, adhesion factors, angiogenic
factors, oncogenes and tumor suppressor genes that provide useful
breast disease markers and probes within the methods and kits of
the invention include Ki67 Growth Factor, Cyclin D1, Proliferating
Cell Nuclear Antigen, Transforming Growth Factor, Tissue
Plasminogen Activator, Insulin Growth Factor Receptors, Collagenase
Type IV, Laminin Receptor, Integrins, p53, rb, nm23, ras, c-myc,
c-myb, Heat Shock Proteins, Prolactin, Neuron-Specific Enolase,
IR-14, KA 1, KA 14, Alpha-Lactalbumin, Actin, IL-10, S-100 protein,
Vimentin, Epithelial Membrane Antigen, bcl-2, CA15-3, CA 19-9, Tn
Antigen, Alpha-lactalbumin, LASA, Gal-GalNAC, GCDFP-15,
Le(y)-Related Carbohydrate Antigen, CA 125, uPA, uPA related
antigens and complexes, uPA Receptor, PA1-1 and PA1-2,
Beta-glucuronidase, CD31, CD44 splice variants, blood group
antigens including ABH, Lewis, and MN, and genetic lesions or
altered expression levels of CCND1, EMS1, BRCA1 and BRCA2 genes,
and many others, for which immunological and non-immunological
binding partners, probes and assay methods are known and readily
adaptable within the invention.
[0270] Although the foregoing invention has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications are comprehended by the disclosure and may be
practiced without undue experimentation within the scope of the
appended claims, which are presented by way of illustration not
limitation.
* * * * *