U.S. patent application number 09/895031 was filed with the patent office on 2001-12-20 for methods for assessing genetic and phenotypic markers by simultaneous multicolor visualization of chromogenic dyes using brightfield microscopy and spectral imaging.
This patent application is currently assigned to The Govenment of the United States of America, Department of Health & Human Services, The Govenment of the United States of America, Department of Health & Human Services. Invention is credited to Hopman, Anton H.N., Macville, Merryn V.E., Ried, Thomas.
Application Number | 20010053958 09/895031 |
Document ID | / |
Family ID | 26734234 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053958 |
Kind Code |
A1 |
Ried, Thomas ; et
al. |
December 20, 2001 |
Methods for assessing genetic and phenotypic markers by
simultaneous multicolor visualization of chromogenic dyes using
brightfield microscopy and spectral imaging
Abstract
The present invention is directed to an improved method for
detecting a genetic marker in a biological sample comprising
contacting the biological sample with a nucleic acid probe linked
to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the genetic marker in the
biological sample. The present invention also provides an improved
method for detecting a phenotypic marker in a biological sample
comprising contacting the biological sample with a compound
comprising a detectable moiety, whereby the compound associates
with the phenotypic marker and whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
Inventors: |
Ried, Thomas; (Bethesda,
MD) ; Macville, Merryn V.E.; (Hague, NL) ;
Hopman, Anton H.N.; (Eijsden, NL) |
Correspondence
Address: |
Gwendolyn D. Spratt, Esq.
NEEDLE & ROSENBERG, P.C.
The Candler Building, Suite 1200
127 Peachtree Street, N.E.
Atlanta
GA
30303-1811
US
|
Assignee: |
The Govenment of the United States
of America, Department of Health & Human Services
Washington
DC
|
Family ID: |
26734234 |
Appl. No.: |
09/895031 |
Filed: |
June 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09895031 |
Jun 29, 2001 |
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09130078 |
Aug 7, 1998 |
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6294331 |
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60055439 |
Aug 8, 1997 |
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Current U.S.
Class: |
702/19 ;
435/6.11; 435/6.12 |
Current CPC
Class: |
C12Q 2563/107 20130101;
C12Q 2563/179 20130101; C12Q 2565/601 20130101; C12Q 1/6841
20130101; C12Q 1/6841 20130101 |
Class at
Publication: |
702/19 ;
435/6 |
International
Class: |
G01N 033/48; C12Q
001/68 |
Claims
What is claimed is:
1. An improved method for detecting a genetic marker in a
biological sample comprising: a) contacting the biological sample
with a nucleic acid probe linked to a detectable moiety, whereby
the detectable moiety can be detected by the presence of a
chromogenic dye associated with the detectable moiety; b) obtaining
a spectral image of the biological sample using brightfield
microscopy; and c) detecting the presence of the chromogenic dye,
thereby detecting the genetic marker in the biological sample.
2. The method of claim 1, wherein the biological sample is
contacted with more than one nucleic acid probe.
3. The method of claim 1, wherein the biological sample is attached
to a substrate.
4. The method of claim 1, wherein step (a) further comprises
contacting the biological sample with a cytological stain.
5. The method of claim 1, wherein step (c) further comprises
distinguishing the cytological stain from the detectable
moiety.
6. The method of claim 1, wherein the genetic marker is selected
from the group consisting of a centromere, a telomere, a general
genetic loci, a specific genetic loci, a chromosome band, a
chromosome-specific loci, a chromosome fragment, and a whole
chromosome.
7. The method of claim 1, wherein the detectable moiety comprises a
hapten.
8. An improved method for detecting a phenotypic marker in a
biological sample comprising: a) contacting the biological sample
with a compound comprising a detectable moiety, whereby the
compound associates with the phenotypic marker and whereby the
detectable moiety can be detected by the presence of a chromogenic
dye associated with the detectable moiety; b) obtaining a spectral
image of the biological sample using brightfield microscopy; and c)
detecting the presence of the chromogenic dye, thereby detecting
the phenotypic marker in the biological sample.
9. The method of claim 8, wherein the biological sample is
contacted with more than one compound.
10. The method of claim 8, wherein the biological sample is
attached to a substrate.
11. The method of claim 8, wherein step (a) further comprises
contacting the biological sample with a cytological stain.
12. The method of claim 8, wherein step (c) further comprises
distinguishing the cytological stain from the detectable
moiety.
13. The method of claim 8, wherein the phenotypic marker is
selected from the group consisting of an RNA, a protein, and an
antibody.
14. The method of claim 8, wherein the detectable moiety comprises
a hapten.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to methods of detecting genetic and
phenotypic markers in biological samples using spectral imaging and
brightfield microscopy to detect the presence of chromogenic
dyes.
[0003] 2. Background Art
[0004] In cytopathological diagnostic laboratories, cytological
specimens are routinely stained with permanent dyes such as
hematoxylin and eosin and for decades, pathologists have based
their diagnosis of disease on cyto- and histological features as
seen under a light microscope. Unlike fluorescent dyes, permanent
dyes do not fade or bleach so that second opinion diagnosis,
re-examination of archived material and even retrospective studies,
can be performed. Thus, for routine cytopathological diagnostic
purposes, fluorescence microscopy is not preferred for these
reasons as well as because of high auto-fluorescence inherent to
the tissue type or which might be induced by fixation.
[0005] Immunohistochemical and in situ hybridization methods have
become increasingly important for research and diagnosis of
disease. Also, multi-parameter cytochemical analysis is required
when rare or unique material is to be studied. Many fluorescent
markers with emission spectra ranging from blue to infra-red have
become available for multi-color detection due to advances made in
conjugation chemistry. Thus, although fluorescence microscopy could
be used for these multi-parameter applications, for the
above-mentioned reasons, it is often not possible to use methods
employing fluorescence.
[0006] The present invention overcomes previous shortcomings in the
art by providing methods for analyzing both genetic and phenotypic
markers in a single biological sample through the use of bright
field spectral imaging of chromogenic dyes. Such analyses are
valuable in a variety of clinical applications, such as, for
example, the diagnosis and characterization of cancer and the
analysis of chromosomal aberrations in pre- and post-natal
diagnostics.
[0007] An important aspect of the present invention that overcomes
a severe limitation in the art is that by using the methods
provided herein, multiple probes, both to genetic and/or phenotypic
markers, and therefore multiple chromogenic dyes can be used in the
same sample and the individual dyes can be distinguished using
spectral imaging, even where the sample has been previously stained
with a cytological- stain which otherwise would obscure the signal
from the genetic or phenotypic probes. Using these methods, a
pathologist for example, can stain a tissue sample to observe a
general morphological aspect of cells in the sample, and a
geneticist can subsequently use that stained sample to diagnose
cells in the sample for the presence of a genetic or phenotypic
marker, such as a chromosomal aberration associated with cervical
cancer, with much more clarity, accuracy, ease, and efficiency than
using previously available methods.
SUMMARY OF THE INVENTION
[0008] In accordance with the purpose(s) of this invention, as
embodied and broadly described herein, this invention, in one
aspect, relates to the present invention provides an improved
method for detecting a genetic marker in a biological sample
comprising contacting the biological sample with a nucleic acid
probe linked to a detectable moiety, whereby the detectable moiety
can be detected by the presence of a chromogenic dye associated
with the detectable moiety, obtaining a spectral image of the
biological sample using brightfield microscopy, and detecting the
presence of the chromogenic dye, thereby detecting the genetic
marker in the biological sample.
[0009] The present invention also provides an improved method for
detecting a phenotypic marker in a biological sample comprising
contacting the biological sample with a compound comprising a
detectable moiety, whereby the compound associates with the
phenotypic marker and whereby the detectable moiety can be detected
by the presence of a chromogenic dye associated with the detectable
moiety, obtaining a spectral image of the biological sample using
brightfield microscopy, and detecting the presence of the
chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
[0010] Additional advantages of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
[0011] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Triple color spectral imaging of T24 cells, using
chromosome 1, 7 and 15 centromere-specific probes. Targets were
visualized with, respectively, DAB, NF and TMB. Bottom left shows
the raw spectral image of the hybridization signals of one cell.
The colors are a result of an arbitrary RGB look-up table and do
not display the colors as seen through the microscope. Bottom
center shows the spectral image after calculation of absorption
spectra. Constituents that absorb light are displayed in the
complementary color. As a consequence, areas that don't absorb
appear black. Top shows spectra of three pixels located on the
different dyes in an absorption intensity per wavelength diagram.
The absorption peaks are wide apart and the shape of the curves
differ significantly. Bottom right shows the result of a spectrum
based classification whereby pixels with similar spectral
information are assigned the same false color. This classification
is based on the input of the spectra shown in the diagram. The DAB
spots (4) are false-colored with a brown color, TMB (2) with green
and NF (3) with red. The spectrum of the unstained nucleus differed
sufficiently from the background light to be classified in a blue
color.
[0013] FIG. 2. T24 cells hybridized for centromere of chromosome 7
and visualized with NF. The nucleus is stained with hematoxylin.
Bottom left shows the raw spectral image in arbitrary colors.
Bottom center shows the spectral image after calculation of the
absorption spectra displayed in complementary colors. Top shows the
spectra of a few pixels over the spots and the nucleus. The spectra
of the spots is the sum of the spectra for NF and hematoxylin
(compare with NF spectrum in FIG. 1 bottom left), whereas the
spectrum of the nucleus is solely hematoxylin. Based on the average
spectra of the selected pixels, a classification is performed,
shown bottom right. Spots and nuclei are false-colored in red and
blue, respectively.
[0014] FIG. 3. Double target in situ hybridization on sperm cells
of a healthy human, using a chromosome X centromere probe
visualized with DAB and a chromosome Y centromere probe visualized
with TMB, combined with histomorphologic staining with DIFF QUIK, a
commercially available eosin derivative stain. The sperm cells
contain a signal either for the X chromosome or the Y chromosome.
Top left shows the raw spectral image in arbitrary display colors.
It was difficult to determine the color by eye through the
microscope, due to weak spots and overall cytoplasmic staining and
overlapping regions as a result of the preparation (smear)
technique. Top center shows the spectral image after calculation of
the absorption spectra displayed in complementary colors. Middle
left shows the spectra of selected pixels for DAB, TMB spots and
DIFF QUIK. The spectra of the spots are mixed with the spectrum of
DIFF QUIK. Bottom left shows the classification based on the
average spectrum of these pixels, false-coloring DAB spots in
brown, TMB spots in green and DIFF QUIK staining above a certain
intensity threshold in blue. A more accurate classification was
achieved when the spectrum of the morphological staining was
eliminated from the spectral image by subtraction or division. Top
right shows the subtracted image. Bottom right shows the
classification based on the average spectrum of these pixels,
displaying just the hybridization spots in false colors brown (DAB)
and green (TMB).
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Example included therein and
to the Figures and their previous and following description.
[0016] Before the present compounds, compositions and methods are
disclosed and described, it is to be understood that this invention
is not limited to specific methods, specific nucleic acid probes,
cytological stains, detectable moities, etc., as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only
and is not intended to be limiting.
[0017] It must be noted that, as used in the specification and the
appended claims, the singular forms "a, " "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a nucleic acid probe" includes
multiple nucleic acid probe molecules.
[0018] The present invention provides methods by which the
assessment of histological or cytological preparations can be
combined with detection of genetic and phenotypic markers by using
spectral imaging.
[0019] From a technical point of view, in multi-target labeling
format, color discrimination by eye through a bright field light
microscope is difficult, if not impossible in common situations
where:1) the staining is weak; 2) signals are small (e.g. they
appear as small punctuate dots); 3) signals lay close together or
merge; or 4) signals overlap in z-direction. In addition,
cytological stains can obscure the color information from the
signals.
[0020] Color discrimination based on 3-color CCD video images can
be performed by measuring the hue value. Hue values are introduced
to specify colors numerically. Calculation is based on intensities
of red, green and blue light (RGB) as recorded by the separate
channels of the camera. The formulation used for transforming the
RGB values into hue, however, simplifies the data and does not make
reference to the true physical properties of light. In contrast,
spectral imaging analyzes light as the intensity per wavelength,
which is the only quantity by which to describe the color of light
correctly. In addition, spectral imaging provides spatial data,
because it contains spectral information for every pixel in the
image.
[0021] Thus, the present invention provides an improved method for
detecting a genetic marker in a biological sample comprising
contacting the biological sample with a nucleic acid probe linked
to a detectable moiety, whereby the detectable moiety can be
detected by the presence of a chromogenic dye associated with the
detectable moiety, obtaining a spectral image of the biological
sample using brightfield microscopy, and detecting the presence of
the chromogenic dye, thereby detecting the genetic marker in the
biological sample.
[0022] The present invention also provides an improved method for
detecting a phenotypic marker in a biological sample comprising
contacting the biological sample with a compound comprising a
detectable moiety, whereby the compound associates with the
phenotypic marker and whereby the detectable moiety can be detected
by the presence of a chromogenic dye associated with the detectable
moiety, obtaining a spectral image of the biological sample using
brightfield microscopy, and detecting the presence of the
chromogenic dye, thereby detecting the phenotypic marker in the
biological sample.
[0023] The method of detecting genetic and phenotypic markers in a
biological sample can be used for diagnosing cancer, identifying
types of cancer; determining a prognosis of a cancer, as well as
for detecting, identifying, diagnosing, characterizing and/or
determining a prognosis of a variety of other disease states or
abnormal conditions which can be detected, identified,
characterized, etc., by genetic and/or phenotypic markers in a
biological sample. The method of the present invention can also be
applied to pre- and post-natal diagnostics. For each of these
methods, the biological sample can be prepared as described herein
or by various other methods which are well known in the art for
preparing biological samples for genetic and phenotypic
analyses.
[0024] It is further contemplated that the present invention
provides methods for detecting genetic and phenotypic markers in a
biological sample for comparative cytogenetics (i.e., interspecies
studies) and karyotyping. For these methods, the biological sample
can be prepared according the methods described herein or according
to various methods well known in the art for preparing biological
samples for genetic and phenotypic analysis relating to comparative
cytogenetics and karyotyping.
[0025] Detection of phenotypic and genetic markers in a biological
sample can be by microscopy, which can be, but is not limited to,
bright-field microscopy, phase contrast microscopy, interference
contrast microscopy, Nomarski contrast microscopy, dark field
microscopy, reflection contrast microscopy, fluorescence
microscopy, infra-red microscopy, or any other type of light
microscopy. Detection can be done by visualizing the biological
sample in the microscope or by recording an image of the biological
sample photographically (e.g., by producing an image on a silver
halide emulsion film which can be developed for visualization or by
recording a digital image of the sample for visualization via an
output device, such as, for example, a computer monitor or as a
computer printout) as well as by any other means by which the
biological sample can be viewed or recorded. The spectral image of
the biological sample can be taken from the sample directly or from
a recorded image of the sample.
[0026] The biological sample of this invention can be from any
organism and can be, but is not limited to, embedded tissue
sections, frozen tissue sections, cell preparations, cytological
preparations, exfoliate samples (e.g., sputum), fine needle
aspirations, amnion cells, fresh tissue, dry tissue, and cultured
cells or tissue. It is further contemplated that the biological
sample of this invention can also be whole cells or cell organelles
(e.g., nuclei). The biological sample can be unfixed or fixed
according to standard protocols widely available in the art and can
also be embedded in a suitable medium for preparation of the
sample. For example, the biological sample can be embedded in
paraffin or other suitable medium (e.g., epoxy or acrylamide) to
facilitate preparation of the biological specimen for the detection
methods of this invention. Furthermore, the biological sample can
be embedded in any commercially available mounting medium, either
aqueous or organic, depending on the chemical properties of the
stain or any specifically developed medium, such as, for example,
as designed for TMB, based on a thin protein layer cross-linked by
formaldehyde to ensure permanent stabilization of the enzyme
reaction products (Speel et al., 1994. A novel triple-color
detection procedure for brightfield microscopy, combining in situ
hybridization with immunocytochemistry." J. Histochem. Cytochem.
42:1299-1307).
[0027] The biological sample can be on, supported by, or attached
to, a substrate which facilitates detection of phenotypic or
genetic markers. A substrate of the present invention can be, but
is not limited to, a microscope slide, a culture dish, a culture
flask, a culture plate, a culture chamber, DNA arrays, ELISA
plates, as well as any other substrate now known or developed in
the future for containing or supporting biological samples for
analysis according to the methods of the present invention. The
substrate can be of any material suitable for the purposes of this
invention, such as, for example, glass, plastic, polystyrene, mica
and the like. The substrates of the present invention can be
obtained from commercial sources or prepared according to standard
procedures well known in the art.
[0028] The detection of phenotypic and genetic markers in the
biological sample can be combined with the routine assessment of
histological and cytological specimens, generally carried out by
staining with one or more cytological stains and examining the
specimens microscopically. Thus, the present invention provides for
multi-parameter analyses of the same biological sample.
[0029] The biological sample of the present invention can be
contacted with one or more cytological stains. The cytological
stains used in the methods of this invention can be, but are not
limited to, hematoxylin, eosin, methyl green, neutral red, DIFF
QUIK (Baxter, The Netherlands), toluidine blue, alcian blue, isamin
blue, methylene blue, sudan black, periodic acid-Schiff reaction
(PAS), Masson's trichrome method, reticulin stain, Van Gieson,
Azan, Giemsa, NissI, silver and gold stains, osmium and chrom alum,
as well as any other cytological stains now known or identified in
the future. The cytological stains of this invention are available
from commercial sources or can be prepared according to standard
methods well known in the art.
[0030] The phenotypic markers identified by the methods of this
invention can be, but are not limited to, messenger RNA, gene
products, antigens, antibodies, and proteins, or fragments thereof,
which can be of, for example, tumor suppressor genes, oncogenes and
proliferation markers. Examples of gene products which can be
detected as phenotypic markers can include, but are not limited to,
gene products of p53, retinoblastoma, Ki67, PCNA, nucleolus
organizing regions and cyclins. These phenotypic markers can be
detected by methods well known in the art, including modifications
of the methods described herein to detect nucleic acid probes, such
as binding to the phenotypic marker a detectable molecule such as a
nucleic acid, a hapten, a protein, an antigen, and an antibody, or
fragments thereof.
[0031] The compound or compounds comprising a detectable marker
which are used to detect a phenotypic marker, therefore, include
any compound which can bind to, link to, hybridize to, or otherwise
associate with the phenotypic marker. For example, the compound can
be an antibody to a protein or a fragment of a protein, an antibody
to a nucleic acid, an antibody to a ligand or a fragment of a
ligand, an antibody to an antibody or fragment of an antibody
(anti-idiotype antibody), an antibody to any cellular structure or
fragment of the cellular structure, and the like. Alternatively,
the compound can comprise other molecules such as nucleic acids,
ligands, haptens, cell structures, and fragments thereof.
[0032] The genetic markers of this invention can be, but are not
limited to, centromeres, telomeres, general or specific loci,
chromosome bands, a chromosome-specific loci, chromosome fragments,
and whole chromosomes, as well as any genetic marker which detects
numerical chromosome alterations or structural chromosome
alterations such as translocations, breakpoints, microdeletions and
amplifications. For detection of these genetic markers, a nucleic
acid probe having complementarity to the nucleotide sequence of the
genetic marker is contacted with the biological sample under
conditions whereby hybridization of the nucleic acid of the genetic
marker and the nucleic acid probe can occur. These conditions can
vary, depending of the biological sample, genetic marker and
nucleic acid probe used for a given application. The hybridization
conditions for a particular application can be determined according
to protocols standard in the art. Examples of various hybridization
conditions are provided in the Examples herein.
[0033] The nucleic acid probe of this invention can be a nucleic
acid comprising the nucleotide sequence of a coding strand or its
complementary strand or the nucleotide sequence of a sense strand
or antisense strand. Thus, the probe of this invention can be
either DNA or RNA and can bind either DNA or RNA, or both, in the
biological sample. The probe can be the coding or complementary
strand of a complete gene or gene fragment. The nucleotide sequence
of the probe can be any sequence having sufficient complementarity
to a nucleic acid sequence in the biological sample to allow for
hybridization of the probe to the target nucleic acid in the
biological sample under a desired hybridization condition. Ideally,
the probe will hybridize only to the nucleic acid target of
interest in the sample and will not bind non-specifically to other
noncomplementary nucleic acids in the sample or other regions of
the target nucleic acid in the sample. The hybridization conditions
can be varied according to the degree of stringency desired in the
in situ hybridization. For example, if the hybridization conditions
are for high stringency, the probe will bind only to the nucleic
acid sequences in the sample with which it has a very high degree
of complementarity. Low stringency hybridization conditions will
allow for hybridization of the probe to nucleic acid sequences in
the sample which have some complementarity but which are not as
highly complementary to the probe sequence as would be required for
hybridization to occur at high stringency. The hybridization
conditions will vary depending on the biological sample, probe type
and target. An artisan will know how to optimize hybridization
conditions for a particular application of the present method.
Examples of hybridization conditions are described in the Examples
provided herein.
[0034] The nucleic acid probe can be commercially obtained or can
be synthesized according to standard nucleotide synthesizing
protocols well known in the art. Alternatively, the probe can be
produced by isolation and purification of a nucleic acid sequence
from biological materials according to methods standard in the art
of molecular biology (Sambrook et al. 1989. Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Pres, Cold
Spring Harbor, N.Y.). The nucleic acid probe can be amplified
according to well known procedure for amplification of nucleic acid
(e.g., polymerase chain reaction). Furthermore, the probe of this
invention can be linked to any of the detectable moieties of this
invention by protocols standard in the art.
[0035] It is further contemplated that the present invention also
includes methods for oligonucleotide hybridization wherein the
hybridized oligonucleotide is used as a primer for an enzyme
catalyzed elongation reaction such as in situ PCR and primed in
situ labeling reactions whereby haptenized nucleotides are
incorporated in situ. Additionally included are methods for in situ
hybridization, employing synthetic peptide nucleic acid (PNA)
oligonucleotide probes (Nielsen et al., 1991. "Sequence-selective
recognition of DNA by strand displacement with a
thymine-substituted polyamide." Science 254:1497-1500; Egholm et
al., 1993. "PNA hybridizes to complementary oligonucleotides
obeying the Watson-Crick hydrogen bonding rules." Nature
365:566-568).
[0036] The detectable moieties to which the nucleic acid probe of
this invention can be linked to include, but are not limited to, a
hapten, biotin, digoxigenin, fluorescein isothiocyanate (FITC),
dinitrophenyl, amino methyl coumarin acetic acid,
acetylaminofluorene and mercury-sulfhydryl-ligand complexes, as
well as any other molecule or compound which can be linked to a
nucleic acid probe and detected either directly or indirectly
according to the methods described herein.
[0037] In one method of detection, the nucleic acid or compound
moiety can be directly detected by linking the detectable moiety to
the nucleic acid probe or compound and another moiety which can
facilitate direct detection, such as an enzyme (e.g., peroxidase,
alkaline phosphatase, glucose oxidase) which produces a colored
reaction product when reacted with a suitable substrate or to
colloidal gold particles or other detectable moieties.
Alternatively, the nucleic acid or compound can be detected
indirectly by the binding of antibodies, antibody fragments or
other ligands, or the reaction of other molecules (e.g., avidin to
detect biotin) with the detectably moiety linked to the nucleic
acid or compound, including for example, enzymes such as
peroxidase, alkaline phosphatase or glucose oxidase for enzymatic
precipitation upon reaction with suitable substrates to produce a
colored reaction product, i.e., a chromogenic dye associated with
the detectable moiety. The enzyme peroxidase can also be used in
conjunction with tyramide-based detection formats.
[0038] The antibodies, antibody fragments or ligands can also be
linked to colloidal gold particles for direct detection or
subsequently enhanced with silver for indirect detection. The
detectable moieties of this invention are available from commercial
sources or can be prepared according to standard protocols well
known in the art. Methods for detecting the detectable moieties of
the present invention are common in the art. Protocols for linking
probes, detectable moieties, antibodies, ligands, etc., are also
standard in the art and are readily available to the artisan.
Additionally, the detectable moieties exemplified here can be
detected in any number of alternative detection procedures other
than those listed.
[0039] The detectable moiety of this invention can also comprise an
antibody. The antibody can be either monoclonal or polyclonal. The
antibodies of this invention can also include immunoreactive
antibody fragments. The detectable moiety can also comprise a
ligand or any other molecule that can detect the antibody or the
nucleic acid probe.
[0040] Antibodies can be made by many well-known methods (See, e.g.
Harlow and Lane, "Antibodies; A Laboratory Manual" Cold Spring
Harbor Laboratory, Cold Spring Harbor, N. Y., (1988)). Briefly,
purified antigen can be injected into an animal, with or without
adjuvants, in an amount and in intervals sufficient to elicit an
immune response. Polyclonal antibodies can be purified directly, or
spleen cells can be obtained from the animal for monoclonal
antibody production. The spleen cells can be fused with an immortal
cell line and the resulting hybridomas can be screened for antibody
secretion. A variety of immunoassay formats can be used to select
antibodies which selectively bind with a particular protein. For
example, solid-phase ELISA immunoassays are routinely used to
select antibodies selectively immunoreactive with a protein. See
Harlow and Lane (1988) for a description of immunoassay formats and
conditions that can be used to characterize antibody binding.
[0041] In some instances, it is desirable to prepare monoclonal
antibodies from various hosts, for example, for anti-species
antibodies. A description of techniques for preparing such
monoclonal antibodies may be found in Stites et al., editors,
"Basic and Clinical Immunology," (Lange Medical Publications, Los
Altos, Calif., Fourth Edition) and references cited therein, as
well as in Harlow and Lane (1988).
[0042] As described above, the antibody of the present invention
can bind an antigen which is attached to the nucleic acid probe.
The antibody itself can be linked to a detectable moiety, such as
an enzyme, and binding of antibody to an antigen attached to a
nucleic acid probe can thereby be detected directly. Alternatively,
the antibody which binds the antigen which is attached to the
nucleic acid probe can be detected indirectly, by binding a second
antibody which recognizes the first bound antibody as an antigen.
The second antibody can be linked to a detectable moiety, such as
an enzyme, thereby detecting the binding of the first antibody
indirectly.
[0043] In the present invention, the spectral image of the
biological sample on the substrate can be obtained with a device
which utilizes a common path Sagnac interferometer creating an
optical path difference based on the angle of incident light. An
interferogram is produced showing the light intensities against the
function of the optical path difference. Fourier transformation of
the interferogram recovers the spectrum. An example of this device
is a SD200 Spectracube (Applied Spectral Image, Migdal HaEmek,
Israel). Other methods to measure absorption spectra using light
microscopy can include spectrophotometry, the use of liquid crystal
tunable filters and accusto-optical tunable filters.
[0044] The spectral image of the biological sample of this
invention can be analyzed with software designed for spectral image
analysis, such as the SpCube 1.5 program (Applied Spectral
Imaging). The present invention further contemplates software
programs dedicated to the methods of this invention.
[0045] The following examples are intended to illustrate, but not
limit, the invention. While the protocols described are typical of
those that might be used, other procedures known to those skilled
in the art may be alternatively employed.
EXAMPLES
[0046] Biological specimens
[0047] T24 human bladder cancer cells (ATCC accession number ATCC
HTB 4) were grown on microscope slides under standard cell culture
conditions to 30% confluency. Cells were fixed and pretreated for
in situ hybridization as described (Speel et al., 1994). Human
spermatozoid cells were obtained from a healthy male, fixed and
smeared on a microscopic slide for pretreatment for in situ
hybridization (Martini, E., et al., 1995. Application of different
in situ hybridization detection methods for human sperm
analysis."Hum. Reprod. 10:855-861).
[0048] T24 bladder cells were hybridized with centromeric
alpha-satellite probes for chromosome 1, 7, 15 (Oncor,
Gaithersburg, Md.) or combinations thereof. The sperm cells were
hybridized with probes for X- and Y-chromosome specific loci
(Oncor, Gaithersburg, Md.). For single labeling, probes were
labeled by nick-translation with biotin (Boehringer-Mannheim,
Germany) and detected with peroxidase (PO) conjugated to avidin
(Vector, USA) or alkaline phosphatase (AP) conjugated to avidin
(Vector), depending on the enzyme substrate to be used.
Diaminobenzidine (DAB), tetra-methylbenzidine (TMB) and
amino-ethyl-carbazole (AEC) were used as substrates for PO and New
Fuchsin (NF), Fast Red (FR) and NBT/BCIP/INT (INT) were used as
substrates for AP. (Substrates were obtained commercially.) The
enzyme reaction produces a precipitate, i.e. a chromogenic dye, in
situ that is visible in a bright field light microscope.
[0049] For double labeling, one probe was labeled with biotin and
the other with digoxigenin (Boehringer-Mannheim). Digoxigenin label
was detected with polyclonal anti-digoxigenin antibody conjugated
to either PO or AP (Boehringer-Mannheim). The third label was
introduced by using fluorescein isothiocyanate (FITC) as a hapten
and detected with mouse anti-FITC antibody (Dako, Denmark) and
anti-mouse-PO or anti-mouse-AP antibody (Boehringer). In double and
triple labeling, interspecies cross-reactivity was blocked and
enzyme reactions producing the reporter signals were developed
sequentially (Speel et al., 1994).
[0050] Nuclei of T24 bladder cells were stained either lightly or
heavily with hematoxylin and sperm cells were cytologically stained
with DIFF QUIK. Simultaneous staining with cytochemical stains such
as, for example, hematoxylin (blue/purple), methyl green, eosin
(pink) or DIF Quick (red) provides histological information and
contributes to multiparameter bright-field microscopic analysis.
Specimens were covered with mounting medium (obtained commercially)
under a coverslip.
[0051] Microscopy
[0052] A Leica DM microscope was equipped with a SD200 SpectraCube
(Applied Spectral Image, Migdal HaEmek, Israel) for acquisition of
spectral images. A halogen transmission light operating at 12 V for
daylight color temperature was used in the visible range (400-700
nm) by placing a WG360 UV cut-off filter and a BG38 infrared
cut-off filter in the illumination pathway. Neutral density filters
were used to optimize the light level for spectral imaging.
Spectral images were acquired with ASI acquisition software running
on a Dell Pentium PC. Typically, a spectral image is built of 200
frames of 300 ms with an interferometer stepsize angle of 15
degrees. Spectral analysis was performed on SpCube 1.5 analysis
software (ASI).
[0053] Spectral imaging using the SD200 SpectraCube mounted on a
transmission light microscope allows for the measurement of the
absorption spectra of chromogenic dyes while retaining the spatial
information of the microscopic image. A spectral image is acquired
and for every pixel in the CCD image the absorption spectrum can be
retrieved. The so-called `optical density image` displays the
constituents of the specimen that absorb the light of certain
wavelengths. Regions that do not absorb light appear black. For
every pixel, an absorption curve can be produced, showing the
absorption intensities per wavelength. Pixel by pixel spectral data
can be utilized for subsequent mathematical operations. For
example, a spectrum-based classification would result instantly in
the pseudo-colorization of pixels with similar spectra. Defining
spectral signatures for specific regions within a specimen provides
flexibility for image analysis.
[0054] To demonstrate the improvement in clinical diagnosis
provided by the methods of the present invention as compared to
techniques available at the time the present invention was made, a
comparison was made between the bright field spectral imaging
technology of the present invention and state-of-the-art
quantitative microscopy software. For the latter procedure, a
3-chip color charged couple device (CCD) video camera and Leica
QWin software were used for image capture and quantitative hue, as
well as saturation and intensity measurements. The hue value is a
trivial but fixed number for every color, whereas the saturation
and intensity values vary dependent on the quality of the
detection. Color discrimination in a 3-color video image therefore
should be based on hue values. Hue values are displayed in a
histogram, showing the number of pixels in an image for every hue
value. Pixels with hue values which match exactly can be selected
and displayed with a single pseudo-color. Hue-classification of all
pixels in the image simultaneously is not possible in a single
operation.
[0055] For comparison of the spectral imaging method of the present
invention with quantitative microscopy, the same microscope was
equipped with a 3-chip color charged couple device camera (Sony,
Japan) controlled by QWin software (Leica Imaging, Cambridge UK),
for image acquisition and quantitative analysis, operating on a
Leica Q550 Pentium PC. Video images were acquired with a halogen
transmission light at 12 V (or 10.5V with a CB12 blue filter to
correct for daylight color temperature) and neutral density filters
for optimal video exposure times.
[0056] The in situ hybridization signals (spots) for centromere
sequences in T24 bladder cancer cells were analyzed after
single-color labeling, double-color labeling and triple-color
labeling, with and without cytological counterstaining. Single
labeling experiments without counterstain showed the spectra of the
pure dyes. The absorption spectra of the PO substrates DAB, TMB,
and AEC and of the AP substrates Fast Red, New Fuchsin and INT were
measured, showing specific spectral characteristics for each dye.
Spectral imaging of a triple-color in situ hybridization for
chromosome centromeres using TMB (green), New Fuchsin (red) and DAB
(brown) as reporter dyes resulted in good spectral separation of
the individual dyes (FIG. 1). Even the colors of small spots that
could not be easily discerned by eye were readily identified. The
absorption peaks were wide apart and the shapes of the curves
deviated clearly to allow for a spectrun-based color classification
of all spots.
[0057] In comparison, video images were acquired using a 3-chip
color CCD camera. Based on color hue values, the presence of the
three colors could be discriminated in a histogram. Hue measurement
results, however, could not be shown within the cellular context
after a single operation.
[0058] When cytological stains such as hematoxylin (blue purple)
and DIF Quick (red) are used, they are present throughout the cell
or cell compartment and are thus overlaying the hybridization
spots. The absorption spectrum of two co-localizing dyes seems to
be additive, meaning that the spectrum of the overlap is the sum of
the two pure spectra. The spectrum that is measured at the
hybridization spots is, therefore, mixed with the spectrum of the
cytological stain. The use of cytological stains, however, did not
compromise the separation of the absorption spectra of the reporter
dyes (FIG. 2). In single labeling experiments using New Fuchsin and
heavy nuclear staining with hematoxylin, the absorption spectrum of
New Fuchsin had shifted but this did not create a problem for the
classification of the hybridization signals.
[0059] In a double labeling experiment on sperm cells, using X and
Y chromosome-specific probes reported with, respectively, DAB and
TMB, and cytologically stained with DIF Quick, clear spectral
signatures of all three dyes can be defined (FIG. 3). Spectrum
based classification including all three dyes showed the
hybridization spots of X and Y in pseudo-colors which would have
been difficult from microscopic evaluation alone. The SpCube
analysis software provides for mathematical operations such as
spectrum subtraction and division. By selecting the average
spectrum of DIF Quick, a subtraction operation was executed,
eliminating the contribution of the cytological stain from the
spectral image. A similar effect can be achieved by division. The
classification image of FIG. 3 shows just the hybridization
spots.
[0060] In contrast, with the quantitative microscopy software, hue
measurements of single and double labeling experiments with
counterstaining were not consistently successful. Due to lower
color resolution, dyes of similar hue could not be discriminated in
the hue-histogram. Under influence of cytological stains, the hue
values of the hybridization spots shifted towards the hue of the
stain, which led to `drowning` of the spot in cases of intense
cytological staining or low hybridization signals. This phenomenon
could not be prevented because mathematical subtraction/division
operations can not be executed on these video images.
[0061] These data demonstrate that the bright field spectral
imaging method of the present invention provides for analysis of
absorption spectra with high precision while maintaining spatial
information. The use of cytological stains doesn't hamper spectral
analysis and thus greatly facilitates microscopic evaluation.
[0062] With quantitative microscopy, color discrimination based on
hue value using QWin software is possible by manually selecting
spots. However, cytological staining readily obscures color
discrimination. Using QWin software, the hue measurement results
can not be displayed together with spatial information in a single
operation. Thus, the data presented herein demonstrate that the
spectral imaging methods of the present invention provide higher
color resolution than 3-color video imaging, enabling the color
discrimination necessary for reliable and user-friendly
multi-parameter analysis of multi-color specimens.
[0063] Detection of phenotypic and genetic markers according to the
method of the present invention for detection, diagnosis,
characterization and prognosis of cervical cancer.
[0064] The diagnosis and staging of cancer is often not possible
without combining the results from several analyses. This
includes:1) the interpretation of histomorphology after applying
routine stains; 2) the complementation of those histomorphological
analyses with pertinent genetic markers, such as the gain of 3q in
cervical cancers as definite identifiers of tumor progression; and
3) the necessity to include phenotypic analysis by means of
immunohistochemistry with antibodies against commonly deregulated
oncogenes and tumor suppressor genes such as p53 and/or the
presence on viral genes such as human papilloma virus (HPV).
Multi-parameter analysis would benefit from the simultaneous
assessment of the above mentioned markers, which is possible, but
very difficult using fluorescence. Bright field with permanent dyes
comes with the described advantages, however, color discrimination
is a challenge. Spectral imaging overcomes these limitations by
allowing detection of multiple targets in pathological specimens
simultaneously.
[0065] As a specific example, in the progression of cervical
carcinoma in situ into invasive cervical carcinoma, an
amplification of chromosome region 3q24-28 is observed by
comparative genomic hybridization (CGH). This is the only
detectable genetic event at this stage of carcinogenesis and is
therefore suitable as a diagnostic marker for early-stage cervical
carcinoma. CGH analyses of late-stage cervical carcinomas revealed
chromosomal gains in regions of 2q and 5p. Cervical specimens can
be biopsies, smears, cytospin preparations, or sections of embedded
cells. Comprehensive diagnosis includes assessment of:1)
cytomorphology, using morphological stains such as hematoxylin and
eosin; 2) detection of HPV genome by in situ hybridization; 3)
screening for genotypic markers 3q, 2p and 5p; 4) screening for
tumor suppressor gene products p53 and Rb, both known to interact
with HPV antigens; and 5) screening for other phenotypic markers
such as Ki67 and other markers associated with the aggressiveness
of tumors.
[0066] Although the present process has been described with
reference to specific details of certain embodiments thereof, it is
not intended that such details should be regarded as limitations
upon the scope of the invention except as and to the extent that
they are included in the accompanying claims.
[0067] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
* * * * *