text:
Global Crude Oil Market Short-Term Outlook, March 2014
By Jeff Meyer
Published: 3/10/2014
This monthly short-term global crude oil market update includes a presentation and a data file. The presentation offers our latest market messages and outlooks. Each month, the analysis in this presentation will be expanded upon in a written report, either the monthly Market Briefing or quarterly World Oil Watch. The data file offers our monthly and quarterly crude oil price outlook through 2015, and annual outlook through 2035....
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MUNFORDVILLE, Ky. (AP) — Dozens of students and parents were injured Wednesday night when a tour bus carrying them on a trip to Washington, D.C., turned over in south-central Kentucky moments into the trip, officials said.
About two dozen people aboard the bus were transported for medical treatment, Hart County schools Superintendent Ricky Line said. One person suffered a head injury, Line said, and others had broken bones.
None of the injuries appeared to be life-threatening, Line said.
In addition to the driver, the bus was carrying 34 students and 20 parents. Line said the students, from the Cub Run community, were mostly sixth- to eighth-graders; some were from high school. The trip was not school-related.
"The school didn't have anything to do with it except it's our precious cargo," he said.
The bus was operated by New Image Travel of Evansville, Ind., for WorldStrides.
Rick Juelfs of New Image said company officials were on their way to the scene Wednesday night.
A phone message and email seeking comment from WorldStride were not immediately answered.
Kentucky State Police Trooper Jonathan Biven of the Bowling Green post said the bus crashed on Kentucky Route 728 in Hart County, about 70 miles south of Louisville.
The injured were being transported by ambulance or private vehicle to area hospitals to be checked out.
Hardin Memorial Hospital house manager Jerry Taylor said about seven people were brought there by ambulance with injuries that were not life-threatening.
The bus had traveled just a few miles before the wreck happened.
"It happened in the first seven minutes of a 14- to 15-hour bus trip, and it hadn't even left the county," Line said.
Line said he was relieved the injuries were not more serious and recognized the help of emergency workers, who scrambled ambulances to several different hospitals.
Dozens Injured in Kentucky Bus Crash
Students and parents hurt when tour bur overturned.
MUNFORDVILLE, Ky. (AP) — Dozens of students and parents were injured Wednesday night when a tour bus carrying them on a trip to Washington, D.C., turned over in south-central Kentucky moments into the trip, officials said.
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The invention provides a high efficiency method for combined immunocytochemistry and in situ hybridization. In one aspect, the method is used to simultaneously determining a cell phenotype and genotype by contacting a cell with an antigen-specific antibody bound to a ligand, contacting the cell with polynucleotide probe to form a complex of the probe and a nucleic acid in the cell, contacting the cell with a detectably labeled anti-ligand, and detecting the polynucleotide-probe complex and the anti-ligand-ligand complex. The presence of the anti-ligand is correlated with the presence of the antigen and the presence of the probe-nucleic acid complex is correlated with the presence of the nucleic acid in the cell.
1. A method of simultaneously determining a cell phenotype and genotype comprising:
(a) contacting the cell with an antibody bound to a ligand, wherein the antibody is specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a detectably labeled anti-ligand under conditions where the detectably labeled anti-ligand binds to the ligand;
(d) detecting the presence of the labeled anti-ligand;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the anti-ligand with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype;
wherein said step (a) precedes step (b), and step (b) precedes step (c).
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
11. The method of
12. The method of
13. A method of diagnosing a fetal abnormality comprising carrying out the process of
14. A method of simultaneously determining a cell phenotype and genotype comprising:
(a) contacting the cell with d antibody specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a binding molecule that specifically binds the antibody of step (a);
(d) detecting the presence of the binding molecule;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the binding molecule with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype;
wherein said step (a) precedes step (b), and step (b) precedes step (c).
15. The method of
16. The method of
17. The method of
18. The method of
19. The method of
20. The method of
21. The method of
22. The method of
23. The method of
24. The method of
25. The method of
26. The method of
27. A method of diagnosing a fetal abnormality comprising carrying out the process of
This application claims benefit of No. 60/140,287, filed Jun. 18, 1999.
This invention relates to new methods for characterizing the genotype and phenotype of a cell in a sample, and finds application in medicine, genetics, histology, developmental biology and cell biology.
Determination of cell phenotype by immunophenotyping or immunocytochemistry (ICC) and determination of cell genotype using hybridization-based methods (e.g., in situ hybridization assays, "ISH") are important processes in the diagnosis and study of disease. Although both ISH and ICC have been widely used for analysis, they have only infrequently been used in combination (e.g., on the same cells or different aliquots of cells from a sample). In part this is because the methods now available for simultaneously determining phenotype by antibody staining and determining genotype by ISH are cumbersome, inefficient, and of limited accuracy.
The conditions required for ISH and ICC are generally incompatible or non-optimal for the practice of the other. Carrying out ISH analysis following ICC may be inefficient due to the blocking effects of the reporting molecules used in ICC (e.g., substrate precipitate or fluorochromes). Conversely, carrying out ISH prior to ICC is unsatisfactory, e.g., because the elevated temperature and denaturation steps used for ISH are not compatible with subsequent ICC. Thus, attempts to carry out the two methods in combination have been limited. Neft et al., 1997, Acta Cytologica 41:1769-1773, carried out ISH after ICC staining for the intermediate filament protein (cytokeratin). In this study, centromeric repetitive sequences from chromosome 7 were used for the ISH DNA probes, but no data relating to hybridization efficiency and accuracy were provided. Schouten et al., 1995, Brit. J. Haematology 91:162-166, described the use of ISH with RNA probes in combination with anti-CD30 immunocytochemistry. They reported low efficiency in identifying Y chromosome containing cells and poor results when using multiple different ISH probes. Pazouki et al. 1996, Acta histochem (Jena) 98:29-37 described using ICC with an anti-hemoglobin antibody and a second-antibody detection system followed by ISH using repetitive-sequence probes (from chromosome X and Y centromeric sequences) to identify fetal cells. However, the authors provided no information on the efficiency of the method or the number of cells correctly typed.
None of these references provided a method that is specific and accurate when used for immunostaining of many types of cell antigens (e.g., cytoplasmic, membrane-associated, etc.) and many types of ISH probes (e.g., centromeric probes and unique sequence probes).
The present invention provides new and surprisingly efficient methods for analysis of cell phenotype and genotype, which may be carried out on the same cells in a sample.
In one aspect, the invention provides a method of simultaneously determining a cell phenotype and genotype by:
(a) contacting the cell with an antibody bound to a ligand, wherein the antibody is specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a detectably labeled anti-ligand under conditions where the detectably labeled anti-ligand binds to the ligand;
(d) detecting the presence of the labeled anti-ligand;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the anti-ligand with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype. According to the invention, step (a) of the method precedes step (b), and step (b) precedes step (c).
In preferred embodiments, the cell is exposed to a fixative between step (a) and step (b).
In various embodiments of the method, the cell is a human cell, such as a human fetal cell; the antibody is monoclonal; the ligand is biotin; the anti-ligand is avidin or streptavidin, which may be labeled with a reporter molecule that is fluorescent, a radioisotope, or an enzyme; the antigen is hemoglobin; and/or the polynucleotide probe is DNA, such as a probe that specifically hybridizes to sequences of human chromosomes X, Y. 13, 18 or 21. In some embodiments of the invention, signal detection is accomplished by microscope analysis.
In certain embodiments, the disclosed method is used to detect or diagnose a chromosomal abnormality in a fetal cell, a malignant cell, or a cell suspected of being malignant.
In a related aspect, the invention provides a method of simultaneously determining a cell phenotype and genotype comprising:
(a) contacting the cell with an antibody specific for an antigen suspected of being associated with the cell;
(b) contacting the cell with polynucleotide probe under conditions that allow the probe to hybridize to a target sequence in a cellular nucleic acid, thereby forming a complex of the polynucleotide probe and cellular nucleic acid;
(c) contacting the cell with a binding molecule that specifically binds the antibody of step (a);
(d) detecting the presence of the binding molecule;
(e) detecting the presence of the complex of the polynucleotide probe and cellular nucleic acid; and,
(f) correlating the presence of the binding molecule with the presence of the antigen in the cell thereby determining the cell phenotype, and correlating the presence of said complex of the polynucleotide probe and cellular nucleic acid with the presence of the target sequence in the cell, thereby determining the cell genotype. Step (a) of the invention precedes step (b), and step (b) precedes step (c). In preferred embodiments, the cell is exposed to a fixative between step (a) and step (b).
In various embodiments of the invention, the binding molecule is an anti-immunoglobulin antibody, protein A or protein G, which may be detectably labeled, for example with a reporter molecule that is fluorescent, a radioisotope, or an enzyme.
In various embodiments of the method, the cell is a human cell, such as a human fetal cell; the antibody is monoclonal; the antigen is hemoglobin; and/or the polynucleotide probe is DNA, such as a probe that specifically hybridizes to sequences of human chromosomes X, Y, or 21.
The invention further provides kits useful for practicing the combined immunocytochemistry and in situ hybridization method of the invention.
In one aspect, the invention provides a highly efficient method for determining the phenotype and genotype of a cell by carrying out both antibody staining to detect antigens in the cell or population (e.g., immunocytochemistry) and in situ hybridization to determine the genetic composition or karyotype of the cell.
Although methods for carrying out ISH and ICC are well established, the art lacks efficient and effective methods for carrying out ISH and ICC on the same cells. For optimal utility in diagnostic and research applications, a combined ICC/ISH method, should work with antigens from different cell compartments (e.g., cytoplasmic, extracellular, etc.), should be amenable to use with both repetitive-sequence and non-repetitive-sequence ISH probes, and should be highly specific and accurate.
In one aspect of the invention disclosed here, cells are analyzed by binding a cell-antigen specific antibody to a target cell, exposing the cell to a fixing agent, carrying out ISH on the cell, and subsequently detecting the antibody bound to the cell antigen. The bound antibody can be detected using a reporter molecule that binds (e.g., through an anti-ligand) to a ligand associated with the cell-antigen specific antibody. Alternatively, the bound antibody (i.e., the primary antibody) can be detected using a detectably labeled secondary antibody that specifically binds the bound cell-antigen-specific antibody. For example, a detectably labeled anti-immunoglobulin antibody can serve as the secondary antibody.
Because the present method is accurate and convenient, it is useful in a variety of clinical and research applications. In particular, the method is useful in the fields of prenatal screening of genetic traits and in cancer diagnosis. For example, in the case of prenatal diagnostics, fetal cells may be obtained by chorionic villus sampling (CVS), by amniocentesis, or from maternal blood (see, e.g., Oosterwicjk et al., 1998, Am. J. Hum. Genet. 63:1783-92), and analyzed by the method of the present invention. By way of non-limiting example, when cells are obtained from maternal blood, immunophenotyping may be used to distinguish fetal cells from maternal calls, and ISH may be used to determine chromosome copy number in the fetal cells.
In the case of cancer diagnosis, it is often desirable to distinguish a malignant cell from non-malignant cells in a sample, and to detect chromosome aberrations in the malignant cell. According to the present invention, a phenotypic marker can be used to identify a malignant cell (e.g., cytokeratin may be used as a marker for epithelial lineage cancers, such as breast, gastric, lung, prostate, esophageal, ovarian, colorectal and endometrial cancers), and ISH techniques used to identify chromosome aberrations, gene expression patterns, or the presence of viral sequences. Many other applications of the present method will be immediately apparent to those of skill upon review of this disclosure.
In Section II, infra, a number of terms are defined to provide guidance to one of skill in the practice of the invention. In Section III, the methods of the invention are described in some detail. Section IV provides examples of the practice of the invention.
a) Immunophenotyping
As used herein, the terms "immunophenotyping" and "antibody staining" are used interchangeably to refer to the use of antibodies (as defined below) that specifically bind a cell antigen to characterize the phenotype of the cell. The antibody binding may be detected directly or indirectly, including by use of enzymatic reporter molecule (e.g., a phosphatase such as alkaline phosphatase, a peroxidase such as horseradish peroxidase, and the like) that acts on a substrate to produce a detectable product to detect the antibody. Alternatively, a fluorescent or radioactive reporter molecule, or other methods may be used. As used herein, "immunocytochemistry (ICC)" refers to immunophenotyping carried out on a substrate (e.g., a slid e or the equivalent) in which a microscope is used for analysis, often in conjunction with an automated image analysis system.
b) In Situ Hybridization (ISH)
As used herein "in situ hybridization (ISH)" refers to hybridization of a polynucleotide probe to the chromosomal DNA or mRNA in a cell. The probe may be RNA, DNA, PNA (peptide nucleic acid), a chimeric nucleic acid (e.g., a DNA-RNA chimera) or the like. In preferred embodiments nonisotopic ISH is carried out. The most widely used ISH method is "fluorescence in situ hybridization" or "FISH," in which the probe is detected by fluorescein-12-dUTP are used (Wiegant et al., 1991, Nuc. Acid. Res. 372:809-812). Other examples of suitable labels include Spectrum Orange-dUTP, Spectrum Green-dUTP; Spectrum Red-dUTP and others known in the art or to be discovered in the future (see, e.g., Vysis Inc. 1999 Catalog, incorporated herein in its entirety by reference). Alternatively, biotin- or digoxigenin-labeled DNA probes are used and are visualized using reagents such as fluorochrome-conjugated avidin, streptavidin, or anti digoxigenin.
A variety of variants of the basic ISH technique are well known, and the use of these techniques in conjunction with the present invention is contemplated. ISH techniques include, but are not limited to "M-FISH" (Connor and Ferguson-Smith, 1997, Essential Medical Genetics, Blackwell Science Eds. p. 86-87); "Poly-FISH" (see, e.g., Zhen et al., 1998, "Poly-FISH: a technique of repeated hybridizations that improves cytogenetic analysis of fetal cells in maternal blood" Prenat. Diagn. 18:1181-1185); "PRINS" (see, e.g., Adinoffi and Davies, 1994, "NISH and PRINS Methods" in Nonisotopic in-situ Hybridization, R. G. Landes Company, Austin, Tex. 17-38); "Interphase FISH" (see, e.g., Oosterwijk et al., 1998, "Development of a preparation and staining method for fetal erythroblasts in maternal blood: simultaneous immunocytochemical staining and FISH analysis," Cytometry 32:170-177); "Chromosome banding" (see, e.g., Muller et al., 1997, "Toward a multicolor chromosome bar code for the en tire human karyotype by FISH" Hum. Genetics 100:271-78). Also see, Bohmeret al., 1998, "Differential development of fetal and adult heamoglobin profiles in colony culture: isolation of fetal nucleated red cells by two-color fluorescence labeling," British Journal of Haematology 103: 351-360; and Xu et al., 1998, "Improving the fixation method for preimplantation genetic diagnosis by fluorescent in situ hybridization," Journal of Assisted Reproduction and Genetics 15 (9) :570-574.
c) Antibody
As used herein, "antibody" refers to a human, nonhuman, or chimeric (e.g., humanized) immunoglobulin, or binding fragment thereof, that specifically binds to an antigen (e.g., an antigen in a cell). Suitable antibodies may be polyclonal (e.g., sera or affinity purified preparations), monoclonal, or recombinant. Useful fragments include separate heavy chains, light chains, Fab, Fab′ F(ab′)2, Fabc, and Fv fragments. Fragments can be produced by enzymatic or chemical separation of intact immunoglobulins or by recombinant DNA techniques. Often fragments are expressed in the form of phage-coat fusion proteins (see, e.g., Dower et al., WO 91/17271 McCafferty et al., WO 92/01047; and Huse, WO 92/06204). Typically, the antibodies, fragments, or similar binding agents bind a specific antigen with an affinity of at least 107, 108, 109, or 1010M−1.
Methods of preparing, manipulating, labeling, and using antibodies are well known in the art. See, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (hereinafter "Harlow") and Ausubel et al., 1999, Current Protocols In Molecular Biology Greene Publishing and Wiley-Intescience, New York, including Supplement 46 (April 1999) (hereinafter "Ausubel"), both of which are incorporated herein by reference. Many suitable antibodies are available commercially, see, e.g., Cortex Biochem, Inc. (San Leandro Calif.), Becton-Dickinson Immunocytometry Systems (San Jose, Calif.), Pharmingen (San Diego Calif.), Caltag Laboratories, Inc. (Burlingam Calif.), DAKO Corp. (Carpinteria Calif.).
As used herein, the term "primary antibody" is sometimes used to refer to an antibody that binds the cellular antigen of interest, e.g., as distinguished from a "secondary antibody" which is an anti-antibody antibody sued for indirect detection of a primary antibody.
d) Specific Binding
As used herein, "specific binding" refers to the ability of one molecule, typically an antibody or polynucleotide, to contact and associate with another specific molecule even in the presence of many other diverse molecules. For example, a single-stranded polynucleotide can specifically bind to a single-stranded polynucleotide that is complementary in sequence, and an antibody specifically binds to (or "is specifically immunoreactive with") its corresponding antigen.
e) Cell Phenotype
As used herein, "cell phenotype" has its ordinary meaning and refers to characteristics displayed by a cell, especially the expression of a particular complement of proteins, under a particular set of environmental factors, regardless of the actual genotype of the organism.
f) Cell Genotype
As used herein, "cell genotype" refers to the genetic or chromosomal complement of a cell. Information about the cell genotype includes the detection of chromosome number, the detection of any chromosome aberrations, or the detection of specific nucleotide sequence (e.g., gene sequences, genetic markers, polymorphisms, and the like) in a cell.
g) Expression Profile
As used herein, the term "expression profile" of a cell refers to the RNAs (mRNAs) expressed in the cell. Particular RNAs may be detected by ISH techniques.
h) Pathogen Profile
As used herein, the term "pathogen profile" refers to the presence of viral, bacterial, fungal or other pathogens for which pathogen-specific nucleic acids can be detected by ISH specific probes.
i)n Ligand and Anti-ligand
As used herein, the terms "ligands" and "anti-ligand" refer to pairs of molecules that specifically bind to each other. Exemplary ligand/anti-ligand pairs include any haptenic or antigenic compound in combination with an antibody (e.g., digoxigenin and anti-digoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin) as well as non-immunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, hormone-hormone receptors, IgG-protein A, and the like).
j) Detectably Labeled
As used herein, "detectably labeled" has the ordinary meaning in the art. A molecule (e.g., antibody or polynucleotide probe) can be detectably labeled by virtue of containing an atom (e.g., radionuclide), molecule (e.g., fluorescein), or complex, that due to a physical or chemical property, indicate the presence of the molecule. A molecule is also detectably labeled when it is covalently bound or otherwise associated with a "reporter" molecules (e.g., a biomolecule such as an enzyme) that act on a substrate to produce a detectable atom, molecule complex. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., Dynabeads TM), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate [FITC], Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [Amersham], SyBR Green I & II [Molecular Probes], and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agents, and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels and chemiluminescent labels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light (e.g., as in fluorescence-activated cell sorting). Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label. Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Also, simple colorimetric labels may be detected by observing the color associated with the label.
It will be appreciated that, in the practice of the invention, although not absolutely necessary, generally different labels will be used for antibody staining and ISH, so that the different probes can be easily distinguished. For example, in when both a polynucleotide ISH probe and the labeled anti-ligand probe are labeled with fluorophores, it is generally preferred that they have distinct emission patterns (wavelengths) so that they can be easily distinguished.
k) "Chromosome-specific probe" or "chromosome-specific paint" refers to a combination of detectably labeled polynucleotides that have sequences corresponding to (e.g., essentially the same as) the sequences of DNA from a particular chromosome or subchromosomal region of a particular chromosome (e.g., a chromosome arm). Typically, the chromosome-specific probe is produced by amplification (e.g., using the polymerase chain reaction) of the corresponding chromosomal DNA. A chromosome-specific probe will hybridize in an essentially uniform pattern along the chromosome or subchromosomal region from which it is derived.
l) Karyotype
As used herein, the term "karyotype" has its normal meaning and refers to the chromosome characteristics of an individual cell or cell line of a given species, as defined by both the number and morphology of the chromosomes. Typically, the karyotype is presented as a systematized array of prophase or metaphase (or otherwise condensed) chromosomes from a photomicrograph or computer-generated image. Alternatively, interphase chromosomes may be examined as histone-depleted DNA fibers released from interphase cell nuclei.
m) Chromosome Abnormality
As used herein, "chromosomal aberration" or "chromosome abnormality" refers to a deviation between the structure of the subject chromosome or karyotype and a normal (i.e., "non-aberrant") homologous chromosome or karyotype. The terms "normal" or "non-aberrant," when referring to chromosomes or karyotypes, refer to the predominate karyotype or banding pattern found in healthy individuals of a particular species and gender. Chromosome abnormalities can be numerical or structural in nature, and include aneuploidy, polyploidy, inversion, translocation, deletion, duplication, and the like. Chromosome abnormalities may be correlated with the presence of a pathological condition (e.g., trisomy 21 in Down syndrome, chromosome 5p deletion in the cri-du-chat syndrome, and a wide variety of unbalanced chromosomal rearrangements leading to dysmorphology and mental impairment) or with a predisposition to developing a pathological condition.
n) Binding Molecule
As used herein the term "binding molecule" when used to refer to the detection of an antibody (e.g., an antibody probe bound to a cell antigen) refers to a protein that specifically binds to a class of antibodies, e.g., protein A, protein G, an anti-immunoglobulin antibody (e.g., a goat anti-mouse antibody antibody), or the equivalent.
o) Efficiency and Accuracy
As used herein, the term "efficiency" refers to the ability of a given probe (antibody or polynucleotide) to enter a cell and stain the antigen or DNA (e.g., chromosome) in the cell. Efficiency is usually expressed as a percentage of the total number of target-antigen or target-chromosome containing cells in a sample, in which the probe signal can be detected.
As used herein, the term "accuracy," when referring to ISH analysis to detect chromosomes, refers to the ability to detect hybridization of the ISH probe in a cell and to detect a number of distinct signals per cell reflective of the number of target chromosomes in the cell (e.g., the number of different chromosomes containing the probe sequence). Alternatively, when distinct signals are not detected (e.g., when cells are analyzed in solution by FACS), "accuracy" refers to the ability to detect an intensity of signal reflective of the number of target molecules (e.g., the number of different chromosomes containing the probe sequence).
It will be appreciated that the results of antibody staining and ISH analysis are inherently dependent of the nature of the probe. In particular, ISH analysis of chromosome number normally produce a distribution of signal numbers. For example, in population of female cells all containing two X-chromosomes, the results of ISH using an X-chromosome probe will include cells with two signals, three signals, one signal, etc, with usually the correct number of signals (i.e., two, in this example) being detectable in the majority of cells. Thus, one way to assess the accuracy and efficiency of the method of combined immunophenotyping and genotyping disclosed herein is to compare the results (efficiency and accuracy) according to the present method with those obtained by conducting ICC and/or ISH separately using the same probes and cells. When probes are obtained commercially, the efficiency and accuracy are generally provided by the manufacturer.
III. Description
In this section, certain aspects of the invention are described. For convenience, the methods of the invention are discussed as series of steps. However, it will be understood that numerous variations may be carried out at each step without departing from the scope of the present invention.
A. Antibody Binding
In one step of the invention, a cell (or, usually, a plurality of cells) is contacted with an antibody that is specific for an antigen associated with, or suspected of being associated with, the cell or cells, so as to allow the antibody to bind the antigen.
The choice of cell and methods of preparing the cell will vary according to the particular needs of the practitioner. The method of the invention may be carried out with virtually any cell (e.g., mucleated cells) including but not limited to normal human cells, malignant or transformed cells, blood cells, fetal cells, cultured cells, biopsy samples, cryosections, touch preparations prepared from uncultured primary tumors, and the like. Cells may be obtained from aspiration biopsy or cells in bodily fluids (e.g., blood, urine, sputum and the like), amniotic fluid, maternal blood, and the like.
Typically the cell(s) are arrayed on a glass or plastic slide, coverslip, plastic dish and the like (i.e., a solid support), although this is not required. For example, the antibody may be contacted with cells in solution and the cells subsequently adhered to a support. In alternative embodiments, the entire process of ICC and ISH can be carried out in solution, and the cells deposited on a slide for microscope-based analysis, or, alternatively analyzed by flow cytometry.
Suitable methods of cell or tissue preparation and for binding antibodies to antigens include those used for conventional immunocytochemistry, and are well known in the art. See, for example, Harlow, supra, at Chapter 10. Immunophenotyping (e.g., ICC) techniques are known in the art and well within the capacity of one of ordinary skill to apply. See, e.g., Staines, 1988, J. Histochem. Cytochem. 36:145; Gillitzer et al., 1990, J. Histochem. Cytochem. 38:307; Wagner and Worman, 1988, Stain Technology 63:129; McGovern and Crocker, 1987, Am. J. Clin. Pathol. 88, 480; Graham et al., 1991, J. Clin. Pathol. 44:96; MaWhinney et al., 1990, J. Clin. Pathol. 43:591. Typically, the cells of interest are fixed (e.g., in buffered formalin) or permeablized (e.g., using by agents such as buffered detergent solution, e.g., Tween 20, Triton X, or NP 40 [<0.5% v/v] in Tris- or phosphate based buffers). Fixation or permeabilization is particularly preferred when the target antigen is not an extracellular protein (e.g., when the target is a cytoplasmic protein) to increase the accessibility of the antigen to the antibody. According to normal protocols, following antibody binding to the sample, any unbound antibody is removed in a wash step, e.g., PBS (phosphate buffered saline) or Tris-based buffer with or without non-ionic detergent.
Virtually any cell-associated antigen may be detected for example, without limitation, cytoplasmic antigens (γ-hemoglobin, ε-hemoglobin, cytokeratin), nuclear antigens (PCNA [proliferating cell nuclear antigen]), disease-associated antigens (e.g., tumor markers, HER2/neu, p53), age- or developmental stage antigens (e.g., e.g. fetal hemoglobin vs adult hemoglobin), and pathogen-associated antigens (e.g., Epstein-Barr virus, Hepatitus B virus, HIV). One advantage of the present invention is that it is effective in detecting soluble cytoplasmic proteins (e.g., hemoglobin) and membrane or extracellular proteins, as well as highly stable proteins such as cytokeratin.
According to the invention, the bound antibody is detected by detection of a label that becomes associated with the bound antibody after the ISH step is carried out. Detection of the bound antibody may be accomplished in a number of ways. In one embodiment of the invention, the antibody (e.g., the "primary antibody") is conjugated to a ligand (e.g., biotin). As is explained in greater detail below, the ligand may be bound in subsequent steps with a detectably labeled anti-ligand, so that the presence of the antigen may be detected by the associated label. A wide variety of ligands may be used, and it will be understood the choice of ligand dictates the subsequent choice of anti-ligand.
One exemplary ligand is biotin (for which, e.g., avidin and streptavidin may act as anti-ligand). Biotin is particularly useful for several reasons, including the high affinity of avidin and streptavidin for biotin, and the signal amplification possible because a large number of biotin molecules can be conjugated to a protein or nucleic acid. A number of ligands and anti-ligands can be used, including, e.g., haptenic or antigenic compound used in combination with an antibody (e.g., digoxigenin and antibody, fluorescein-isothiocyanate (FITC) and anti-FITC antibody; dinitrophenol (DNP) and anti-DNP antibody), protein A or protein G in combination with an antibody, and the like.
In another embodiment of the invention, the "primary" antibody is not conjugated to a ligand and is instead detected using a secondary antibody (i.e., an anti-antibody antibody such as a goat anti-mouse IgG antibody) which is itself labeled or otherwise detectable. In a similar embodiment, a primary antibody bound to antigen is detected by contacting the antibody with detectably labeled protein A or protein G, following the ISH step.
Numerous strategies for amplification or indirect detection of antibodies are known. See, e.g., Ausubel at Chapter 14, and the use of such methods is contemplated in the practice of the present invention. It will be apparent to those of skill that cells may be stained with multiple antibodies.
B. Fixation
In preferred embodiments of the invention, the cell(s) or tissue sample is treated with a fixative following the binding of the antibody. Such fixation has been observed to dramatically improve the ability to detect stained cells. Without intending to be bound by any particular mechanism, it is believed that the fixation step acts to immobilize the antibody in situ.
Suitable fixatives include formaldehyde-based fixatives (e.g., formaldehyde, formalin, buffered formalin, paraformaldehyde, and the like), glutaraldehyde, and others (e.g., precipitating or extractive fixatives such as acetone, methanol/acetone, methanol/acetic acid and the like. Fixation conditions used for fixation of cells prior to conventional ICC are generally appropriate in this step. It will be appreciated that fixatives and fixation conditions are selected to avoid destruction of the ability of, e.g., an antiligand (e.g., avidin) to bind the ligand (e.g., biotin) associated with the primary antibody.
C. In Situ Hybridization
In another step of the invention, the genotype of the cell is determined by in situ hybridization of a polynucleotide probe or probes capable of specifically annealing to sequence in a cellular nucleic acid and detection of the resulting hybrid, i.e., an "in situ hybridization assay." In situ hybridization assays are well known and are generally described in Angerer et al., 1987, Methods Enzymol. 152: 649-660, Ausubel et al., supra, Pinkel et al., 1988, Proc. Natl. Acad. Sci., 85:9138; EPO Pub. No. 430,402; Choo, ed., 1994, Methods in Molecular Biology Vol. 33: In Situ Hybridization Protocols, Humana Press, Totowa, N.J., and in other references listed supra, Briefly, conventional in situ hybridization assays generally comprises one or more of the following steps: (1) prehybridization treatment of the biological structure to increase accessibility of target DNA or RNA (e.g., denaturation with heat or alkali), (2) optionally (and depending on the probe) steps to reduce nonspecific binding (e.g., by blocking the hybridization capacity of repetitive sequences, e.g., using human genomnic DNA); (3) hybridization of one or more nucleic acid probes to the nucleic acid in the biological structure or tissue; (4) posthybridization washes and/or nuclease digestion to remove nucleic acid fragments not bound in the hybridization; and, (5) detection of the hybridized nucleic acid fragments. The reagents used in each of these steps and conditions for their use vary depending on the particular application.
The ISH step may be used to detect either the chromosomal DNA or mRNA of a cell. In one preferred embodiment, the detectably-labeled probe is hybridized to a chromosomal sample to detect karyotype (e.g., the presence of a Y chromosome) chromosome aberrations or chromosome number (e.g., autosomal trisomies, XXY, XYY, etc.).
ISH may be carried out using a variety of detectable or detectably labeled nucleic acid probes (e.g., 35S-labeled riboprobes, fluorescently labeled probes) capable of hybridizing to a cellular DNA (e.g., chromosomal) sequence or, alternatively, to a mRNA sequence. The probe, which may be RNA, DNA, PNA, or hybrids or chimeric molecules. As noted supra, the ISH polynucleotide probes may be labeled directly (e.g., by incorporation of fluorescently-labeled nucleotides) or indirectly (e.g., through a ligand-labeled antiligand system).
In certain embodiments, the probe is specific for a single gene (e.g., p53, HER-2/neu) or allele (e.g., p53 mutants). In other embodiments, an entire chromosome or chromosomes are stained or "painted" using probes made from highly repetitive sequences (e.g., centromeric probes) or, alternatively, using nonrepetitive sequence probes (e.g., chromosome 21 probes). When nonrepetitive probes are used it is more difficult, in conventional FISH, to achieve a high level of sensitivity and specificity. However, one limitation of centromeric probes avoided by using nonrepetitive probes is the high level of cross hybridization (e.g., between chromosome 21 centromeric sequences and chromosome 13 centromeres.
In a preferred embodiment of the present invention, the cells are treated with a formamide solution (e.g., 50% formamide in 2×SSC) before the application of the nucleic acid probe. For example, after the fixation step described in §III(B), supra, slides are washed two times 5 minutes in 70% formamide/2×SSC. Without allowing the slide to dry, probes mixed in hybridization solution (50% formamide/2×SSC) are applied onto the slide.
D. Detection of the Antibody and ISH Probes
Following the in situ hybridization step, the sample (i.e., comprising cells) is contacted with a detectably-labeled anti-ligand that binds the antibody-associated ligand described in section III(A), supra. The label associated with the anti-ligand is then be detected and the presence of the cell antigen inferred. Alternatively, the sample may be contacted with a detectably labeled or detectable secondary antibody (i.e., one that specifically binds the primary antibody bound to the cell antigen) or labeled binding molecule such as protein A, protein G, etc. to detect cells containing the target antigen (e.g., hemoglobin). The labeled or detectable secondary antibody, protein A, or protein G is then be detected and the presence of the cell antigen inferred.
The method of detection of the signals associated with the antibody probes are dependent on the specific labels or reporter groups used. In some embodiments, a microscope (e.g., a fluorescent microscope) is used to detect a precipitate, or dye (e.g., a fluorophore). When microscopic-based detection methods are used, it is often convenient to use an automated and/or computerized data collection and image analysis system. In alternative embodiments, labeled cells are detected in solution, e.g., by FACS analysis.
The annealed ISH probe is also detected following ISH, often at the same time that the antibody label is detected. The method of detecting the annealed ISH probe will also depend on the nature of the probe label. Thus, when ISH probes are labeled by incorporation of fluorescently-labeled nucleotides, detection may be accomplished by microscopic observation (manual or automatic). When detection of the ISH probe requires the application of a labeled anti-ligand (e.g. avidin-labeled rhodamine when the ISH probe is a biotinylated probe) the labeled antiligand may be added before, coincident with, or after the bound antibody is detected.
Different detectable labels (e.g., fluorescent labels with different colors or emission spectra) are selected for the antibody and the ISH probes. (It will be appreciated that if the same label is used for both antigen and chromosome sequences, it will be difficult to distinguish the signals.) Similarly, when multiple different antibody or ISH probes are used in the same sample, each probe is typically differently labeled.
It is also necessary to select the immunophenotyping reagents and an ISH labeling system that do not cross-react with each other. To illustrate this requirement, imagine a scenario in which simultaneous ICC and ISH is attempted using a biotin-labeled antibody (for ICC), a biotin-labeled polynucleotide probe (for ISH) and avidin-conjugated Texas Red for detection of the bound antibody and avidin-conjugated fluorescein for detection of the annealed polynucleotide. It will be apparent that whichever of the avidin conjugates is first added will bind both the antibody and ISH probes, rendering the results useless.
Suitable combinations of reporter molecules will be apparent to one of skill reading this disclosure. In one embodiment, fluorescent labels are used in the nucleic acid (FISH) probe and an enzymatic reporter group is used to detect the antibody staining. Illustrative embodiments are provided in Table I, infra.
As illustrated in Table I, ISH probes may be directly labeled (e.g., incorporating a fluorescently labeled nucleotide) of indirectly labeled (e.g., incorporating a biotinylated nucleotide). Directly labeled probes have the advantage of reducing the number of steps required for detection.
When cells are screened microscopically, it is sometimes desirable to counterstain the cells, e.g., to enhance detection of nuclei. Suitable counterstains (e.g., DAPI (4′-6-diamidino-2-phenylindole) are well known.
E. Kits
It is contemplated that reagents useful for practicing the present method will be provided in kit form. In one embodiment, useful kits will include, packaged together in a container, one or more of the following reagents.
(i) an antibody specific for an antigen (e.g., a human antigen), which antibody is conjugated to a ligand (e.g., biotin or digoxigenin). In one embodiment the antigen is a fetal antigen such as ε-hemoglobin;
(ii) An anti-ligand associated with (e.g., conjugated to) a detectable label (e.g., avidin, streptavidin or anti-digoxigenin conjugated to a label such as a fluorescent label.
(iii) At least one chromosome-specific polynucleotide probe suitable for ISH. In certain one embodiment the probe is specific for chromosome X, Y, 13, 18 or 21;
(iv) Other reagents useful for the practice of the method described herein.
Leukocytes were obtained from a normal blood sample of an adult male by histopaque centrifugation and deposited on poly-lysine microscope slides by manual smear. The cells were fixed on the slide for 10 minutes with a 2% formaldehyde/PBS (FB) solution. After further incubation with a 2% mouse serum (in PBS) solution the slides were reacted with anti-CD45 antibody conjugated with biotin (Becton-Dickinson Immunocytometry Systems, San Jose, Calif.) for 30 minutes at room temperature. The slides were washed twice and fixed in FB solution for three minutes.
Next the slides were washed two times for 5 minutes in 70% formamide/2×SSC. Without allowing the slides to dry, 10 μl of an XY DNA probe cocktail (Vysis Inc., Downers Grove, Ill.; CEP X SpectrumGreen and CEP Y SpectrumOrange DNA FISH probes) was applied to the slide and allowed to denature for five minutes at 85° C. followed by an overnight incubation at 37° C. The "XY" probe contained X probe that hybridized to the centromere region of chromosome X (Xp11.1-q11.1) and Y probe that hybridized to the centromere region of chromosome Y (Yp11.1 -q11.1) (Vysis Inc., Downers Grove, Ill.).
The slides were then washed three times 5 minutes at 46° C. in a pre-warmed solution of 50% formamide in 2×SSC, washed twice in PBS, and incubated at room temperature for 30 minutes with a 1:100 solution of streptavidin conjugated with alkaline phosphatase (Vector Inc., Burlingame, Calif.). After washing the slides twice in PBS, alkaline phosphatase substrate (Vector® Blue Alkaline Phosphatase Substrate Kit III, Vector Inc.) was added and incubated for 10 minutes at room temperature according to the manufacturers instructions. The slides were washed and mounted for observation. The slides were observed with Applied Imaging Corp. WinScan system (Applied Imaging Corp. Santa Clara, Calif.). The system is equipped with brightfield (for ICC) and fluorescence (for ISH).
Virtually 100% of the cells contained the Vector Blue stain and X, Y signals. This experiments demonstrates that carrying out ICC and FISH according to the method of the invention results in detection of antigen and genotype as accurately and efficiently as carrying out FISH alone or ICC alone according to conventional methods.
Umbilical cord cells were obtained from a 10-week gestation pregnancy after termination procedures. Cells were washed to remove transport media [RPMI-1640] and fixed on a slide using FB solution. After incubation with a 2% mouse serum/PBS solution, the slides were reacted with anti-epsilon hemoglobin chain antibody conjugated with biotin (Cortex Biochem, Inc. (San Leandro Calif.) for 30 minutes at room temperature. The anti-epsilon hemoglobin antibody probe specifically identifies fetal hemoglobin. The slides were washed twice and fixed in FB solution for five minutes.
The slides were then washed twice for 5 minutes with 70% formamide/2×SSC pre-warmed to 46° C. Without letting the slides dry, 10 μl of an X, Y, 21 DNA probe cocktail (Vysis Inc., Ill.) was applied to the slide, heated to 80° C. for 8 minutes to denature the target and probe DNA, and incubated at 37° C. overnight The probe cocktail contained the following chromosome-specific probes: X chromosome- CEP X SpectrumAqua, Vysis Cat. No. 32-131023; Y chromosome: CEP Y SpectrumGreen, Vysis Cat. No. 32-132024; and 21 chromosome: LSI 21 SpectrumOrange, Vysis Cat. No. 32-190002.
The following day, the slides were washed three times five minutes in 50% formamide in 2×SSC prewarmed to 46° C. The slides were washed twice with PBS and incubated at room temperature for 30 minutes with a 1:100 solution of streptavidin conjugated with alkaline phosphatase, as described in Example 1. After washing the slides twice in PBS, alkaline phosphatase substrate (Vector Blue, Vector, Inc., Burlingame) was added and incubated for 10 minutes at room temperature. The slides were then washed and mounted for microscopic analysis. Antibody staining was calibrated on a 5-point scale (neg.=no color, 4+=intense color) according to the manufacturer's instructions.
The results of this experiment are provided in Table II, below. Approximately 42% of the cells were stained with the antibody. This proportion is consistent with the percentage of cells expected to contain epsilon hemoglobin (see, e.g., Crutcher et al., 1998, "Combination of anti-embryonic and anti-fetal hemoglobin antibodies improve identification of fetal nucleated erythrocytes" 9th International Conference Prenatal Diagnosis and Therapy, Jun. 8-11, 1998). Virtually 100% of the epsilon hemoglobin positive cells also stained positive for X, Y and 21 fluorescent signals (see rows 1-4). This represents significantly better hybridization efficiency compared to conventional methods.
In control experiments in which the antibody was omitted, no antibody staining was observed (see row 5). In controls in which the fixation after antibody binding was omitted, the number of stained cells was dramatically reduced (see row 9).
These results demonstrate high efficiency and accuracy when using non-repetitive probes (in this case, chromosome 21 unique sequence probes).
HeLa cells were deposited on a slide and fixed for 2 min with a 1:1 (v/v) methanol acetone solution for two minutes followed by two wash cycles in PBS. After incubation with a solution of 2% mouse serum in PBS, the slides were reacted with anti-cytokeratin antibody conjugated with biotin (DAKO, Carpinteria, Calif.) for 30 minutes at room temperature.
The slides were washed twice and the cells fixed for 2 minutes in methanol/acetone (1:1). Next the slides were washed two times 5 minutes at 46° C. in a pre-warmed solution of 70% formamide/2×SSC. Without letting the slides dry, 10 μl of an X, 13, 18 DNA probe cocktail (Vysis Inc., Ill.) was applied to the slide, the probe was allowed to denature for eight minutes at 80° C. followed by an overnight incubation at 37° C. The probe cocktail contained the following probes: X chromosome- CEP X SpectrumAqua, Vysis Cat. No. 32-131023; chromosome 13- LSI 13 (RB1,13q14) SpectrumOrange, Vysis cat. no. 32-190001; chromosome 18- CEP 18(alpha satellite, D18Z1) SpectrumGreen, Vysis cat. no. 32-132018. The Vysis chromosome 13 probe contains unique DNA sequences spanning the RB1 gene located within band 13q14 on the long arm of chromosome 13. The chromosome 18 probe hybridizes to the centromere region (alpha satellite D18Z1) of chromosome 18 (18p11.1-q11.1).
The following day, the slides were washed three times at 46° C. for five minutes each wash in a pre-warmed solution of 50% formamide in 2×SSC. The slides were then washed twice with PBS and incubated at room temperature for 30 minutes with a 1:100 solution of streptavidin conjugated with alkaline phosphatase. After washing the sl des twice in PBS alkaline phosphatase substrate (Vector Blue, Vector, Inc., Burlingame) was added and incubated for 10 minutes at room temperature. The slides were then washed and mounted for observation
The results of this experiment are summarized in Table III, below. Virtually 100% of the cells staining positive for cytokeratin (i.e., HeLa cells) showed X, 13, and 18 fluorescent signals.
The copy number for each chromosome detected in cytokeratin-positive cells using this method was consistent with the number expected (as detailed below). Thus, these results correspond the reported ranges, with many cells having generally higher numbers of detectable chromosomes. The detection of generally higher chromosomes copy numbers using the present method than reported using prior art methods indicates that the present method is very sensitive.
Previously Reported HeLa Cell Chromosome Numbers
The copy number of chromosome 13 in HeLa cells was reported a 1 (Lin and Godstein, 1974, "Analysis of Q-banding patterns in human cell lines" J. Natn. Cancer Insti. 53:298-304); and Heneen, 1976, "HeLa cells and their possible contamination of other cell lines" Hereditas 82:217-248 or 2 (Wang and Fedoroff, 1973, "Karyology of cell in culture: trypsin technique to reveal G-band," in Kruse P. Jr. Patterson M. K. Jr. (eds): Tissue Culture Methods and Application, pp. 782-787 (Academic Press, New York); Kraemer et al., 1973, "On the nature heteroploidy" Cold Spring Harb Symp Quant Biol 38:133-144.; Nelswon-Rees et al., 1974, "HeLa-like chromosomes and type A variant G6PD isoenzyme in human cell culture producing Mason-Pfizer monkey virus-like particles" J. Natn. Cancer Inst. 53:751-757; and Mamaeba, 1983, "Cytogenetics and tissue culture," in Troshin AS (ed): Biology of the Cell in culture, pp 195-234). Chen, 1988, "Re-evaluation of HeLa, HeLa S3, and HEP-2 karyotypes" Cytogenet Cell Genet 48: 19-24 reported that 54% of HeLa cells had 2 copies of chromosome 13 and 46% had 3 copies.
The copy number of chromosome 18 in HeLa cells was reported as 1 by Nelson-Rees et al., supra, 2 by Kraemer et al., supra and Lin and Goldstein, supra (1974), 3 by Wang and Fedoroff, supra, Heneen, 1976, supra; Mamaeba, supra, and Chen, supra.
The copy number of chromosome 21 in HeLa cells was reported as 1 by Kraemer et al supra, 2 by Lin and Goldstein, supra, and Mamaeba supra, 3 by Wang and Fedoroff supra, and Heneen, supra, and 4 by Nelson-Rees et al. supra,. Chen (1988) reported that 90% of HeLa cells had 2 copies of chromosome 21 with the range of 1 to 3 copies.
The number of chromosome X of HeLa cells was identified to be 1 by Lin and Goldstein (1974), 2 by Wang and Fedoroff (1973), Kraemer et al. (1973), Nelson-Rees et al. (1974), Heneen (1976) and Mamaeba (1984). Chen (1988) reported that 76% of HeLa cells had 3 copies of chromosome X and 24% had 2 copies.
HeLa cells were mixed with XY peripheral blood lymphocytes (1:1000) and contacted with anti-cytokeratin antibody probes as described in Example 3, supra. ISH was carried out using chromosome 21 and X -specific probes as described in Example 2, supra. Virtually all of the HeLa cells (but no lymphocytes) stained positive for cytokeratin. The of the ISH staining are provided in Table IV, infra.
These results show that when HeLa cells were diluted with male lymphocytes, the HeLa cells can be detected and their genotype and phenotype can be accurately determined.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.
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May 23, 2013
April 22, 2013
The conviction of 23 Brazilian police officers for killing inmates in a prison massacre two decades ago is a "vital" step towards justice, Amnesty International has said.
More Amnesty News and Multimedia
Music and the Arts
Musicians and artists worldwide have been a vital part of Amnesty International's global efforts to bring human rights to every man, woman and child in the world.


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Added Sep 8, 2013
by Feenambulle
Paris, France
Maxi skirt in an ethnic fabric.
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Galveston House Rental Photos and Description
1 king bed, 1 queen bed, 4 twin bunk beds, sleeper sofa. 2 full bathrooms.
BeachFront Pelicans Vista
2nd row home with a beach front view!
Just a few steps to the beach.
Privately owned home.
Comfortably sleeps 10 people:
6 adults and 4 children.
Sorry, no groups over 10 people.
$249 per night plus cleaning and tax.
3 night required stay.
$500.00 security/cleaning/damage deposit.
1 small pet under 25 lbs. considered with pet fee.
Sorry, no exceptions.
Winter Holidays:
$295 per night plus cleaning and tax.
4 night required stay.
Sorry, no exceptions.
3 bedrooms:
One king bedroom
One queen bedroom
Queen sleeper sofa
4 twin bunk beds for children
2 full bathrooms
Sleeps 6 adults and 4 kids comfortably.
Pelicans Vista Details:
2nd row beachfront with great view of the Gulf of Mexico.
Sleeps 6 adults and 4 small children.
Luxury home with luxury furnishings.
Relax on the deck and watch the sunrise over the Gulf of Mexico.
Breath taking view of the Gulf of Mexico from your private patio.
The beach is just steps away.
Enjoy your kitchen complete with stove, oven, microwave, refrigerator, coffeemaker, toaster oven and blender.
Silverware, dinnerware, cups, pots and pans.
Complimentary paper products provided as well.
Washer and dryer on main floor.
Linens: sheets, blankets, pillows and towels included.
WiFi, Flat screen TV's with cable and DVD.
Call 8AM to 9PM Central time to reserve your stay.
From our past guests:
Thank you so much we had a blast and we really enjoyed the house and the kids will never forget it!!
We would love to come stay again some time and we will certainly recommend your house to all our friends. It was everything you advertised and more!
- Suitability:
- Minimum Age Limit for Renters
- children welcome
- non smoking only
- pets considered
- wheelchair inaccessible
About Taz or Jay
We are private owners and have several properties on the island listed on HomeAway.
We prefer to take care of our own properties rather than let another company do it for us.
We live nearby on the island if you need us for anything.
Taz or Jay purchased this house in 2008
Why Taz or Jay chose Galveston
Pelicans Vista has a great view of the Gulf of Mexico. It is close to Moody Gardens, the Waterpark and shopping. What else could you need?
What makes this house unique
$500.00 security deposit which is put on hold the morning of your arrival.
We release that hold when you leave our home respectfully with no damage.
If this calendar is booked, give us a call as we may have another property available!
Guestbook comments from the manager
Thanks for the best place on the island "Pelicans Vista." You explained it was a lovely home but it was much nicer and bigger than we expected. It was clean, neat, well organized and well supplied. Everything we needed to feel right at home including laundry soap for the washer and dryer (a real blessing when you have kids) and a great vacuum! The key-less lock was a brilliant idea since we didn't have to pick up a key or worry about losing a key (or locking ourselves out.) What a treat to sit on our own patio sipping coffee and watch the sunrise over the water. We will be back!


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Dog of the Day
Cat of the Day
Pet of the Day
"Life is like an elevator. On your way up, sometimes you have to stop and let some people off." -- Author Unknown
Poor guy...........lived alone and died alone.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Wolfy ~ Fuzzbutt #3My little dog ~ a heartbeat at my feet
Sparky the Fuzzbutt - PT's DOTD 8/3/2010
RIP 2/28/1999~10/9/2012Myndi the Fuzzbutt - Mom's DOTD - Everyday
Ellie - Mom to the Fuzzbuttz
To everything there is a season, and a time to every purpose under the heaven.
Ecclesiastes 3:1The clock of life is wound but once and no man has the power
To know just when the hands will stop - on what day, or what hour.
Now is the only time you have, so live it with a will -
Don't wait until tomorrow - the hands may then be still.
~~~~true author unknown~~~~
How very sad.
Rest in peace, Mr. Lancaster.
I meant," said Ipslore bitterly, "what is there in this world that truly makes living worthwhile?"
Death thought about it.
"Cats," he said eventually. "Cats are nice."
-- Terry Pratchett, Sourcery
Sad that he died alone, but his choice to live by himself seems to be what he wanted. I am sure the neighbors will remember him well, and tell his story to future generations.
I've Been Frosted
Copyright © 2001-2013 Pet of the Day.com


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© St Margaret Mary Church 2014. All rights reserved
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Margaret Mary Site
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This week's
November 30, 2014
Jesus speaks to us urgently today about being vigilant
for his coming. How will I spend this Advent time
watching and waiting for the Lord?
© St Margaret Mary Church 2014. All rights reserved
Our Vision Statement: Called into relationship by the Holy Spirit and centered in the Eucharist,
St. Margaret Mary Parish is a welcoming community, sent by Christ to serve.
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"In Manhattan, Children Still Battle 9/11-Related Illnesses"
"The day the Twin Towers crumbled, more than 25,000 kids inhaled toxic substances. Ten years later, many of them are suffering from health problems that still haven't gone away."


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Gert Garibaldi's Rants and Raves: One Butt Cheek at a Time.
Amber Kizer
Delacorte Books for Young Readers
c/o Bantam Dell Publishing Group
1540 Broadway, New York, NY 10036
"One Butt Cheek at a Time" is a book referring to the phrase "Take it one step at a time." The philosophy is that not everyone has feet, and even those that do, some cannot use them. But everyone has a butt. Therefore, saying "Take it one butt cheek at a time" would apply to everyone.
"One Butt Cheek at a Time" is about a girl who goes through life learning new things. Gert hates her name, but learns to live with it. Her best friend, Adam, is gay. They have been friends for many years. Well, Adam ends up with a boyfriend, and soon starts to neglect his friendship with Gert. Gert soon has to find a way to win her best friend back. She tries talking to Adam, but he doesn't seem to understand. Gert ends up meeting new friends when she gets assigned to a group project with her worst enemy, Jenny.
Throughout the story, Gert learns new things about her body. As a class homework assignment, Gert has to look at her private parts, using a mirror. This is so that the female can recognize if something has changed. Well, Gert decides to name hers ...
Overall, I would say that this book was very interesting--in a good way, of course. I say this because I've learned some new things by reading "Gert Garibaldi's Rants and Raves: One Butt Cheek at a Time." I really liked how the author chose to use phrases like "Holy-Mother-Of-Fake-Butter" instead of curse words. This book would be best for females fourteen and older.
Reader Opinion
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|Dec 1, 2007
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Garth Brooks Breaks His Own Single-City Ticket Sales Record in Minneapolis
Those who doubted the success of Garth Brooks' grand re-entrance into country music are kicking themselves as they learn the news that the legend has set another record.
Those who doubted the success of Garth Brooks' grand re-entrance into country music are kicking themselves as they learn the news that the legend has set another record.
Garth Brooks is returning to perform in North Carolina for the first time in more than 16 years. The country icon has announced two shows in Greensboro, N.C. in November.
At about 8:30 this morning, my cell phone rang and on the other end was another very friendly person from the Garth Brooks team. She said, "Good morning, David, I have news for you". My first reaction was to laugh because I knew exactly what was coming next.
More information on the highly anticipated forthcoming Garth Brooks album is slowly trickling in. The legend -- who is making a monstrous comeback in 2014 -- has announced the title, 'Man Against Machine,' along with the cover art.
Jason Aldean realized exactly how much he's impacting the landscape of country music when he was mentioned by a country legend. Garth Brooks recently complimented the 'Burnin' It Down' singer in a press conference, and Aldean was understandably shocked!
Friday morning at 10am, tickets will go on sale for Garth Brooks at Target Center. If recent ticket sales for this tour are any indication, they will go fast. It is recommended that you register with the ticket website ahead of time so you don't waste time during the on sale.
B105 is proud to announce the return of Garth Brooks to Minnesota! Garth is returning to Minneapolis for the fist time in over 16 years on his World Tour along with special guest Trisha Yearwood.
It was very exciting to break the news in the Northland that Garth Brooks is returning to Minnesota for the first time in 16 years, when he plays 4 shows at Target Center on November 14th and 15th. Obviously you're excited too as our Facebook post sharing the concert was viewed nearly 19,000 times in less than 24 hours.
Trisha Yearwood won't be forgetting this birthday anytime soon. While out on Garth Brooks' World Tour, her husband and a crowd of 16,000 sang her 'Happy Birthday,' in addition to a few surprises.
Just a few months into his great return to country music and Garth Brooks is already back to breaking records. The legend is proving yet again that he's the real deal, becoming the first artist ever to sell seven consecutive shows at Atlanta's Philips Arena and toppling his own ticket sales record in Chicago. Now, he's doing the same in the Sunshine State.
Garth Brooks has revealed when his new studio album will be available.
Even major country stars like Garth Brooks can use a little support onstage -- especially when that support is coming from their own family. The country legend took to the stage in Chicago over the weekend with one more familiar face added to his backing band: his daughter.


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Bo Porter on departing Robbie Grossman, Lucas Harrell
Bo Porter was effusive in his praise of Robbie Grossman, saying he fully expects the young outfielder to be back in the majors later this year. His comments were tremendously more brief about righthander Lucas Harrell, who was designated for assignment. "As far as Lucas Harrell goes, I think that some times situations run their […] [Read More]
Scott Feldman set to start on Thursday
Veteran righthander Scott Feldman, who threw seven scoreless innings Friday while mourning the death of his father, came off the bereavement list Wednesday night and is ready to start Thursday. Feldman's father Marshall succumbed to cancer on April 10 in Northern California. Feldman shuttled to Arlington for his start Friday and then headed back home […] [Read More]
Brad Peacock gets spot in rotation
Righthander Brad Peacock, who was a finalist for the fourth and fifth spot in the rotation before making the 25-man roster as a reliever, will replace Lucas Harrell in the starting rotation and start Sunday against the A's. He was 5-6 with a 5.18 ERA in 2013 with the Astros, but he finished strong with […] [Read More]
Jason Castro back at catcher
Catcher Jason Castro, who was limited to the designated hitter's role Tuesday while recovering from a right big toe bruise, was back behind the plate Wednesday night for the first time since Friday. Castro was hurt last Saturday when the Rangers' Tanner Scheppers hit him with a pitch. The All-Star catcher remained in that game […] [Read More]
Photos: George Springer's MLB debut
The highly-touted prospect is set for his MLB debut in Houston against the Kansas City Royals on Wednesday night. [Read More]
Springer's debut yields familiar result with extra-inning loss to Royals
The Astros hit the reset button Wednesday night, delivering a loud message to their players and fan base by calling up heralded outfield prospect George Springer. Owner Jim Crane's patience has run out, and general manager Jeff Luhnow emphatically declared that unproductive players will no longer stick around. Springer's promotion gave fans a reason for […] [Read More]
George Springer to start in RF, bat second
George Springer is in the Astros' lineup in right field and batting second as he makes his MLB debut. Also, Jason Castro is back at catcher after getting a "rest" at DH in Monday's game. Here's the Astros' lineup for tonight vs. the Royals: Dexter Fowler, CF George Springer, RF Jason Castro, C Jose Altuve, […] [Read More]
Why no Jon Singleton yet? Check back on April 19
Part of the reason Jon Singleton hasn't been promoted to the majors yet might be because his potential free agency has not been pushed back yet. The Astros top prospect at first base has a lot to prove after a disappointing 2013 that included a suspension for marijuana. He also had a poor spring training, […] [Read More]


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Golf Hotels – with hotels.com you can search through great selection of Golf hotels in Sooke. Book online today and enjoy last minute deals and offer for Golf Sooke hotels.
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Golf hotels within 20 kilometers of Sooke
The Westin Bear Mountain Golf Resort & Spa, Victoria
Excellent 4.4 / 5 ( 192 reviews )
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"Our stay was great. Only complaint was the spotty wifi."
A Traveller, Nov 2014, CA
Golf hotels within 35 kilometers of Sooke
Oak Bay Beach Hotel
Outstanding 4.7 / 5 ( 93 reviews )
51 people looked at this hotel in the last hour
Most recent review
"A little further from local shops and restaurants than would be preferred but the pool, hot tubs and view make up for it."
A Traveller, Aug 2014, US


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Weekly jam sessions lift spirits
Banding Together pairs professional musicians and young adults with special needs
MISSION VALLEY Playing a free concert for an audience of 16 may dishearten some musicians, but not the volunteers of Banding Together, a San Diego music therapy program for young men and women with special needs.
Every Wednesday night, San Diego singer/songwriters including Steph Johnson, Megan Combs and Steve Denyes sit in on high-spirited jam sessions with a dozen or more students ages 14 to 24 with Down syndrome, cerebral palsy, William syndrome, autism and other developmental disabilities. The students don't just sit and watch. They're jamming, too — enthusiastically singing, clapping, stomping and playing along on a number of instruments.
The musicians say the psychic pay they receive at the raucous, upbeat jams is incredible.
"I never expected to enjoy this experience as much as I have," said Ryan Ahmadi, a drummer who has volunteered at the weekly sessions for nearly two years. "Music serves as a mechanism to bond all of us together. It's such a rich and special time."
Banding Together was launched three years ago by certified neurological music therapists Julie Guy and Angela Neve, co-founders of the Musical Therapy Center of California, with offices in Mission Valley and Encinitas. Neve, 35, said they created Banding Together to offer free music therapy to families who can't afford it. Students pay no tuition for the 12-week program. Jam sessions alternate weekly between locations in Point Loma and Carlsbad.
Music is used as a medium to encourage the development of verbal, motor and social skills, such as working with others, self control, appropriate behavior in group settings and making friends, Guy said.
At last week's session in Point Loma, students began arriving at 7 p.m., many of them running into the room excitedly with wide grins on their faces and high fives for all. Sitting in a circle of chairs with several volunteers, they start the evening with a 10-minute drumming session. Guy said the hand, bongo and djembe drums are a favorite with students, who beat at varying speeds and volumes to hand commands given by different students picked each week to serve as drum leader.
For most of the hourlong jam, teen student Casey Conley will sit immobile, staring up at the ceiling. But during the drum circle, he holds an African drum up to his ear, closes his eyes and quietly thrums its surface with his fingertips. Conley's mother, Debbie, said music therapy has been a tremendous communications tool for Casey, who hasn't spoken a word for the past three years.
Casey comes to each session with his best friend, Trevor Spidell, who came alive during the drum circle, feverishly pounding on a hand drum with a joyous ear-to-ear smile. His enthusiasm is one of many small victories that occur during the fast-moving hour.
Another breakthrough comes 20 minutes later, when 13-year-old Xavier Osorio begins tapping on a keyboard with his index finger. An excited Guy, who has been accompanying the jam on her flute, explains that this is the first time in 16 weeks that Xavier has actively participated in the program.


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One Bedroom One Bath Condo Sleeps 4
Condo includes One Queen Size Bed in the Bedroom and a Queen Size Sofa Sleeper in the Living Room. Complete resort amenities of a kitchenette, one bathroom, and balcony in some units. 25' TV, Alarm Clock, Balcony in Some Units, Cable TV, Coffee Maker, Dishwasher, Garbage Disposal, Hair Dryer, Heating, Internet Access, Jacuzzi Tub, Kitchenette, Medium Refrigerator, Microwave, Oven, Place Settings, Pots & Pans, Queen Size Bed, Queen Size Sofa Sleeper, Stove Top, Toaster, VCR
Nestled among the magnificent Sierra Nevada Mountains, The Heidelberg Inn 1 Bedroom condos offer a touch of history with superb vacation amenities. The Heidelberg Inn was opened in 1928 and was used to film movies such as Road to Utopia. This beautiful resort was also where stars stayed during filming and the rooms have been graced with movie legends such as Clark Gable and Betty Grable. Since then, The Heidelberg Inn has been refurbished to reflect todays comforts.
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|Rate Period
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Payment is usually accepted in the quoted currency (USD) unless the currency and the amount is specifically agreed in advance with the owner / advertiser.
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Funeral Etiquette
What To Do Before, During and After a Funeral Service
What To Say When Someone Passes Away
What To Wear To a Funeral Service
Helping a Friend Cope With Grief
What To Say When You Don't Know What To Say
In Memory of
Harold Loyd Hanes
October 3, 1925 - June 25, 2014
Harold Loyd Hanes passed away Wednesday, June 25, 2014. He was born Saturday, October 3, 1925 in the town of Wingate Texas to Ernest J. and Elizabeth Hanes. He is survived by four Daughters and one Son. He served in the Army from 1943-1946 as an Aerial Gunner. Received an Honorable Discharge and also received a good conduct medal, Meritorious unit award , World War 1 bronze Star and Victory Medal signed by Harry Truman.
There will be a short viewing on Wednesday, July 2, 2014 at 10:00 am held at Turner & Stevens Live Oak Memorial Park and Mortuary, Monrovia, CA. followed by a Graveside.
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