Cytogenetics is a branch of genetics that is concerned with the study of the structure and function of the cell, especially the chromosomes. It includes routine analysis of G-Banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH).
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Showing posts with label FISH. Show all posts
Showing posts with label FISH. Show all posts
Sunday, February 8, 2009
Sunday, March 2, 2008
Cytogenetics, blood, bone marrow and tissue
Blood & Tissue Cytogenetics
Blood Cytogenetics: Chromosomal analysis on peripheral blood is a good way to look at the constitutional chromosomal make-up or karyotype of an individual. Peripheral blood is drawn and cultured in tissue culture media supplemented with a mitogen, PHA (phytohemagglutinin), that causes the lymphocytes to grow in culture. After 48 to 96 hours in culture, metaphase chromosomes are “harvested” and slides made for chromosome analysis. Peripheral blood can be used as a substitute for a bone marrow aspirate when one cannot be obtained and circulating blasts are present.
Bone Marrow Cytogenetics: Bone marrow cytogenetic analysis is performed on patients with leukemia, lymphoma or other hematological disorders. These patients often have acquired clonal chromosomal abnormalities that can be diagnostic and/or prognostic.
Breakage Studies: Ataxia telangiectasia (AT) is a rare, autosomal recessive chromosomal breakage syndrome characterized by progressive cerebellar ataxia, telangiectasias, immune deficiency and a predisposition to malignancy. Fanconi anemia (FA) is a rare, autosomal recessive condition characterized by growth retardation, thumb abnormalities/radial aplasia, pigmentary anomalies and hematological disorders, including eventually MDS and AML. Both disorders are associated with a DNA repair defect that leads to an increased rate of spontaneous chromosomal breakage when blood is exposed to mitomycin C and/or diepoxybutane.
Solid Tumor Cytogenetics: Cytogenetic analysis can be an important adjunct to solid tumor diagnosis and management. This is particularly true in the case of the small round blue cell tumors of childhood (Ewing’s sarcoma/PNET; rhadomyosarcomas).
Tissue Cytogenetics: Solid tissue specimens can be cultured as a source for mitotic cells for chromosomal analysis. Products of conception (POC) specimens from the first trimester spontaneous abortions are often studied. Around half of these are chromosomally abnormal, typically aneuploidies. Tissues from intrauterine fetal demises or stillborns can also be studied and may help confirm abnormal prenatal results. Tissues should be of fetal, not maternal, origin (e.g. fetal tissue, membranes, villi). Internal organs/diaphragm obtained at autopsy can be used. Skin punch biopsies may also be obtained to rule out chromosomal mosaicism or to culture for biochemical and/or molecular analysis.
From this link
Blood Cytogenetics: Chromosomal analysis on peripheral blood is a good way to look at the constitutional chromosomal make-up or karyotype of an individual. Peripheral blood is drawn and cultured in tissue culture media supplemented with a mitogen, PHA (phytohemagglutinin), that causes the lymphocytes to grow in culture. After 48 to 96 hours in culture, metaphase chromosomes are “harvested” and slides made for chromosome analysis. Peripheral blood can be used as a substitute for a bone marrow aspirate when one cannot be obtained and circulating blasts are present.
Bone Marrow Cytogenetics: Bone marrow cytogenetic analysis is performed on patients with leukemia, lymphoma or other hematological disorders. These patients often have acquired clonal chromosomal abnormalities that can be diagnostic and/or prognostic.
Breakage Studies: Ataxia telangiectasia (AT) is a rare, autosomal recessive chromosomal breakage syndrome characterized by progressive cerebellar ataxia, telangiectasias, immune deficiency and a predisposition to malignancy. Fanconi anemia (FA) is a rare, autosomal recessive condition characterized by growth retardation, thumb abnormalities/radial aplasia, pigmentary anomalies and hematological disorders, including eventually MDS and AML. Both disorders are associated with a DNA repair defect that leads to an increased rate of spontaneous chromosomal breakage when blood is exposed to mitomycin C and/or diepoxybutane.
Solid Tumor Cytogenetics: Cytogenetic analysis can be an important adjunct to solid tumor diagnosis and management. This is particularly true in the case of the small round blue cell tumors of childhood (Ewing’s sarcoma/PNET; rhadomyosarcomas).
Tissue Cytogenetics: Solid tissue specimens can be cultured as a source for mitotic cells for chromosomal analysis. Products of conception (POC) specimens from the first trimester spontaneous abortions are often studied. Around half of these are chromosomally abnormal, typically aneuploidies. Tissues from intrauterine fetal demises or stillborns can also be studied and may help confirm abnormal prenatal results. Tissues should be of fetal, not maternal, origin (e.g. fetal tissue, membranes, villi). Internal organs/diaphragm obtained at autopsy can be used. Skin punch biopsies may also be obtained to rule out chromosomal mosaicism or to culture for biochemical and/or molecular analysis.
From this link
Saturday, March 1, 2008
FISH Testing Info
Fluorescence in situ hybridization
In situ hybridization (ISH) is used to visualize defined nucleic acid sequences in cellular preparations by hybridization of complementary probe sequences. Probe sequences can be labeled with isotopes, but nonisotopic ISH is used increasingly as it is considerably faster, usually has greater signal resolution, and provides many options to simultaneously visualize different targets by combining various detection methods. The most popular protocols use fluorescence detection, as described here. These protocols have many applications, from basic gene mapping and diagnosis of chromosomal aberrations, to detailed studies of cellular structure and function, such as the painting of chromosomes in three-dimensionally preserved nuclei. This protocol describes fluorescence in situ hybridization (FISH) of biotin- or digoxigenin-labeled probes to denatured metaphase chromosomes and interphase nuclei. The hybridized probes are detected and visualized using fluorochrome-conjugated reagents.
This comes from the link below:
FISH
In situ hybridization (ISH) is used to visualize defined nucleic acid sequences in cellular preparations by hybridization of complementary probe sequences. Probe sequences can be labeled with isotopes, but nonisotopic ISH is used increasingly as it is considerably faster, usually has greater signal resolution, and provides many options to simultaneously visualize different targets by combining various detection methods. The most popular protocols use fluorescence detection, as described here. These protocols have many applications, from basic gene mapping and diagnosis of chromosomal aberrations, to detailed studies of cellular structure and function, such as the painting of chromosomes in three-dimensionally preserved nuclei. This protocol describes fluorescence in situ hybridization (FISH) of biotin- or digoxigenin-labeled probes to denatured metaphase chromosomes and interphase nuclei. The hybridized probes are detected and visualized using fluorochrome-conjugated reagents.
This comes from the link below:
FISH
FISH Definition
From NCI:
fluorescence in situ hybridization (floor-EH-sents in SY-too HY-brih-dih-ZAY-shun)
A technique used to look at chromosomes (the parts of the cell that contain genetic information in the form of DNA) or genes (specific regions of DNA in chromosomes that make RNA and proteins). Pieces of DNA containing a fluorescent dye are made in the laboratory and added to cells on a glass slide. When viewed under a microscope with a special light source, parts of chromosomes or genes that bind the pieces of DNA show up as colored. Also called FISH.
fluorescence in situ hybridization (floor-EH-sents in SY-too HY-brih-dih-ZAY-shun)
A technique used to look at chromosomes (the parts of the cell that contain genetic information in the form of DNA) or genes (specific regions of DNA in chromosomes that make RNA and proteins). Pieces of DNA containing a fluorescent dye are made in the laboratory and added to cells on a glass slide. When viewed under a microscope with a special light source, parts of chromosomes or genes that bind the pieces of DNA show up as colored. Also called FISH.
FISH Testing
FISH stands for Fluorescence In-Situ Hybridizaton. Most people with SDS have FISH testing done on their bone marrow sampls when they have their bone marrow biopsies. FISH cn also be done on peripheral blood samples.
FISH
Site above has an illustration, recorded explanation and written definition.
FISH Fact Sheet
FISH
Site above has an illustration, recorded explanation and written definition.
FISH Fact Sheet
Thursday, February 28, 2008
Routine microscopic exam: Any samples taken (blood, bone marrow, or cerebrospinal fluid) are examined under a microscope by a pathologist (doctor specializing in diagnosis of disease by laboratory tests) and are often also reviewed by the patient's hematologist/oncologist (doctor specializing in medical treatment of cancer and blood diseases).
Based on the bone marrow cells’ size, shape, and granules, doctors can classify them into specific types. A key element of this cell classification is whether the cells appear mature (resembling normal cells of circulating blood) or immature (lacking features of normal circulating blood cells). The most immature cells are called blasts.
The percentage of cells in the bone marrow that are blasts is particularly important. Having at least 20% blasts in the marrow is generally required for a diagnosis of acute myeloid leukemia. IAMLcan also be diagnosed if the blasts have a chromosome change that occurs only in a specific type of AML, even though the blast percentage doesn’t reach 20%. In order for a patient to be considered to be in remission after treatment, the blast percentage must be no higher than 5%.
Sometimes this examination does not provide a definite answer, and other lab tests are needed.
Cytochemistry: Cytochemistry studies involve placing cells from the sample on glass microscope slides, then exposing them to chemical stains (dyes) that are attracted to or react with only some types of leukemia cells. These stains cause color changes that can be seen only under a microscope. For example, one stain distinguishes AML from acute lymphocytic leukemia (ALL). The stain causes the granules of most AML cells to appear as black spots under the microscope, but it does not cause ALL cells to change colors.
Flow cytometry: This technique is often used to examine the cells from bone marrow and blood samples. It is very accurate in determining the exact type of leukemia.
Leukemia cells can be distinguished by the kinds of molecules on their surface. A sample of cells is treated with special antibodies which stick to the cells only if certain molecules are present on their surfaces. The cells are then passed in front of a laser beam. If the sample contains cells that now have antibodies attached to them, the laser will cause them to give off light, which is measured and analyzed by a computer. Groups of cells can be separated and counted by these methods.
Immunocytochemistry: During this test, as in flow cytometry, cells from the bone marrow aspiration or biopsy sample are treated with special antibodies that react only to certain molecules. But instead of using a laser and computer for analysis, the sample is treated so that certain types of cells change color. The color change can be seen only under a microscope. Like flow cytometry, it is helpful in distinguishing different types of leukemia from one another and from other diseases.
Cytogenetics: These tests involve looking at a cell’s chromosomes under a microscope. Normal human cells contain 46 chromosomes, pieces of DNA that control cell growth and metabolism.
In certain types of leukemia, 2 chromosomes may exchange some of their DNA, so that part of one chromosome becomes attached to part of a different chromosome. This change, called a translocation, can usually be seen under a microscope. Other changes in chromosomes, such as inversions, deletions, or additions, are also possible. Recognizing these changes helps to identify certain types of AML and is important in determining the outlook for the patient.
The testing usually takes about 3 weeks, because the leukemic cells must grow in laboratory dishes for a couple of weeks before their chromosomes are ready to be viewed under the microscope. The results of cytogenetic testing are written in a shorthand form that describes which chromosome changes are present.
A translocation, written as t(1;2), for example, means a part of chromosome 1 is now located on chromosome 2 and vice versa.
An inversion, written as inv(16), for example, means that part of the chromosome 16 is upside down and is now in reverse order but is still attached to the chromosome it originated from.
A deletion, written as del(7) or -7, for example, indicates part of chromosome 7 has been lost.
An addition, +8, for example, means that all or part of chromosome 8 has been duplicated, and too many copies of it are found within the cell.
Molecular genetic studies: Special tests of leukemia cell DNA can also find most translocations that are visible under a microscope in cytogenetic tests, as well as some translocations too small to be seen with usual cytogenetic testing under a microscope.
This sophisticated testing, called FISH (fluorescent in situ hybridization), is helpful in classifying leukemia because many subtypes of AML have distinctive translocations. Information about these translocations may be useful in predicting how the patient will respond to treatment.
These tests may also be used after treatment to find small numbers of leukemia cells that can be missed under a microscope.
Based on the bone marrow cells’ size, shape, and granules, doctors can classify them into specific types. A key element of this cell classification is whether the cells appear mature (resembling normal cells of circulating blood) or immature (lacking features of normal circulating blood cells). The most immature cells are called blasts.
The percentage of cells in the bone marrow that are blasts is particularly important. Having at least 20% blasts in the marrow is generally required for a diagnosis of acute myeloid leukemia. IAMLcan also be diagnosed if the blasts have a chromosome change that occurs only in a specific type of AML, even though the blast percentage doesn’t reach 20%. In order for a patient to be considered to be in remission after treatment, the blast percentage must be no higher than 5%.
Sometimes this examination does not provide a definite answer, and other lab tests are needed.
Cytochemistry: Cytochemistry studies involve placing cells from the sample on glass microscope slides, then exposing them to chemical stains (dyes) that are attracted to or react with only some types of leukemia cells. These stains cause color changes that can be seen only under a microscope. For example, one stain distinguishes AML from acute lymphocytic leukemia (ALL). The stain causes the granules of most AML cells to appear as black spots under the microscope, but it does not cause ALL cells to change colors.
Flow cytometry: This technique is often used to examine the cells from bone marrow and blood samples. It is very accurate in determining the exact type of leukemia.
Leukemia cells can be distinguished by the kinds of molecules on their surface. A sample of cells is treated with special antibodies which stick to the cells only if certain molecules are present on their surfaces. The cells are then passed in front of a laser beam. If the sample contains cells that now have antibodies attached to them, the laser will cause them to give off light, which is measured and analyzed by a computer. Groups of cells can be separated and counted by these methods.
Immunocytochemistry: During this test, as in flow cytometry, cells from the bone marrow aspiration or biopsy sample are treated with special antibodies that react only to certain molecules. But instead of using a laser and computer for analysis, the sample is treated so that certain types of cells change color. The color change can be seen only under a microscope. Like flow cytometry, it is helpful in distinguishing different types of leukemia from one another and from other diseases.
Cytogenetics: These tests involve looking at a cell’s chromosomes under a microscope. Normal human cells contain 46 chromosomes, pieces of DNA that control cell growth and metabolism.
In certain types of leukemia, 2 chromosomes may exchange some of their DNA, so that part of one chromosome becomes attached to part of a different chromosome. This change, called a translocation, can usually be seen under a microscope. Other changes in chromosomes, such as inversions, deletions, or additions, are also possible. Recognizing these changes helps to identify certain types of AML and is important in determining the outlook for the patient.
The testing usually takes about 3 weeks, because the leukemic cells must grow in laboratory dishes for a couple of weeks before their chromosomes are ready to be viewed under the microscope. The results of cytogenetic testing are written in a shorthand form that describes which chromosome changes are present.
A translocation, written as t(1;2), for example, means a part of chromosome 1 is now located on chromosome 2 and vice versa.
An inversion, written as inv(16), for example, means that part of the chromosome 16 is upside down and is now in reverse order but is still attached to the chromosome it originated from.
A deletion, written as del(7) or -7, for example, indicates part of chromosome 7 has been lost.
An addition, +8, for example, means that all or part of chromosome 8 has been duplicated, and too many copies of it are found within the cell.
Molecular genetic studies: Special tests of leukemia cell DNA can also find most translocations that are visible under a microscope in cytogenetic tests, as well as some translocations too small to be seen with usual cytogenetic testing under a microscope.
This sophisticated testing, called FISH (fluorescent in situ hybridization), is helpful in classifying leukemia because many subtypes of AML have distinctive translocations. Information about these translocations may be useful in predicting how the patient will respond to treatment.
These tests may also be used after treatment to find small numbers of leukemia cells that can be missed under a microscope.
Friday, February 22, 2008
FISH Info
Fluorescence in situ Hybridization (FISH): FISH is a molecular cytogenetic technique that can detect chromosomal abnormalities that cannot be appreciated by standard chromosomal analysis (e.g. microdeletion syndromes) or when mitotic cells are not available for chromosomal analysis (e.g. X/Y FISH for cross-sex transplants). Briefly, metaphase chromosomes or interphase nuclei are denatured on the slide, as is the fluorescently labeled DNA probe. The probe and the chromosomes/nuclei are then hybridized, slides are washed, counterstained and analyzed by fluorescent microscopy. There are a number of different types of FISH probes including unique sequence probes (e.g. microdeletion syndromes), whole chromosome painting probes, repetitive probes (e.g. centromeric alpha satellite probes, subtelomeric probes), gene fusion probes (e.g. BCR/ABL in t(9;22) in CML and ALL) and break apart probes (e.g. MLL in 11q23 rearrangements in ALL and AML).
HER2 Amplification by FISH in Breast Cancer: The HER2 oncogene is overexpressed in 25-30% of human breast cancer. In 90-95% of these cases, the overexpression is a direct result of gene amplification. This amplification correlates with a poor clinical prognosis, and therefore, amplification status has become increasingly important in therapeutic decisions for patients with breast cancer. Specifically, women with breast cancer are eligible for treatment with the monoclonal antibody trastuzumab (Herceptin, Genentech), which targets the HER2 gene product, if they show amplification and/or overexpression of HER2.
Screening for Recurrent Bladder Cancer Using FISH: Transitional cell carcinoma (TCC) constitutes 90% of human bladder cancer, 75% of which are considered “superficial”, i.e., carcinoma in situ, non-invasive papillary TCC or minimally invasion TCC. Recurrence of superficial TCC or progression to muscle invasive TCC occurs in 50-80% and 15-25% of cases, respectively. Patients with TCC are monitored with cystoscopy and/or urine cytology for recurrent disease, both of which are relatively insensitive methods of testing. Numerous studies have correlated various chromosomal aneuploidies with TCC. Fluorescence in situ hybridization (FISH) using a variety of probes has been utilized for detection of aneuploidies on cells from voided urine. The UroVysion Bladder Cancer Screening Kit (Abbott, Vysis) provides a FISH probe mixture that includes centromeric probes for chromosomes #3, #7 and #17 and a unique sequence probe for the p16 gene (located at 9p21). Specifically, concurrent extra copies of two or more chromosomes and/or loss of 9p21 is consistent with a diagnosis of recurrent TCC.
from this link: http://cytogenetics.stanford.edu/fish.html
HER2 Amplification by FISH in Breast Cancer: The HER2 oncogene is overexpressed in 25-30% of human breast cancer. In 90-95% of these cases, the overexpression is a direct result of gene amplification. This amplification correlates with a poor clinical prognosis, and therefore, amplification status has become increasingly important in therapeutic decisions for patients with breast cancer. Specifically, women with breast cancer are eligible for treatment with the monoclonal antibody trastuzumab (Herceptin, Genentech), which targets the HER2 gene product, if they show amplification and/or overexpression of HER2.
Screening for Recurrent Bladder Cancer Using FISH: Transitional cell carcinoma (TCC) constitutes 90% of human bladder cancer, 75% of which are considered “superficial”, i.e., carcinoma in situ, non-invasive papillary TCC or minimally invasion TCC. Recurrence of superficial TCC or progression to muscle invasive TCC occurs in 50-80% and 15-25% of cases, respectively. Patients with TCC are monitored with cystoscopy and/or urine cytology for recurrent disease, both of which are relatively insensitive methods of testing. Numerous studies have correlated various chromosomal aneuploidies with TCC. Fluorescence in situ hybridization (FISH) using a variety of probes has been utilized for detection of aneuploidies on cells from voided urine. The UroVysion Bladder Cancer Screening Kit (Abbott, Vysis) provides a FISH probe mixture that includes centromeric probes for chromosomes #3, #7 and #17 and a unique sequence probe for the p16 gene (located at 9p21). Specifically, concurrent extra copies of two or more chromosomes and/or loss of 9p21 is consistent with a diagnosis of recurrent TCC.
from this link: http://cytogenetics.stanford.edu/fish.html
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