Term
| What are ISH assays used for? |
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Definition
| To localize molecular information |
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Term
| What can ISH assays be performed on? |
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Definition
| Tissues, cell types, or in chromosomes |
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Term
| How is ISH interpretation performed? |
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Definition
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Term
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Definition
| protein chemistry for target detection |
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Term
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Definition
| nucleic acid chemistry for target detection |
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Term
| What are the three components of ISH? |
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Definition
| Pre-analytic, analytic, and post-analytic |
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Term
| What are the pre-analytical components? |
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Definition
| The sample (adequate amount, immediate fixation to allow sample to adhere to the slide), and test selection (only validated tests) |
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Term
| What are the analytical components? |
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Definition
| sample pre-treatment, probe, hybridization conditions, detection, sample controls, probe controls and equipment and personnel |
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Term
| What is included in the sample pretreatment? |
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Definition
| proteases remove proteins that are closely bound to DNA and RNA, this optimizes accessibility of the nucleic acids to the probe |
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Term
| What is included in the probe? |
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Definition
| the key variables are the chemical nature of the probe, label and biological specificity |
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Term
| what is included in the hybridization conditions? |
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Definition
| the stringency and specificity of the hybridization reaction is determined by conditions of hybridization and post-hybridization washes |
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Term
| What is included in detection? |
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Definition
| matched to the type of label on the probe |
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Term
| what are the post-analytical components? |
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Definition
| interpretation, report, proficiency testing and interlaboratory comparison |
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Term
| what is included in interpretation? |
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Definition
| requires evaluation of control and test samples, along with control and test probes for each run |
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Term
| what is included in the report? |
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Definition
| ISH assay can be reported separately or included as part of the integrated interpretation, probe target and specificity should be provided |
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Term
| what is included in proficiency testing and interlaboratory comparison? |
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Definition
| validity of the ISH assay must be assumed by the laboratory (compared to culture or PCR results or by participation in interlaboratory comparisons programs) |
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Term
| What is important about Automation? |
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Definition
| it plays a key role in achieving reproducible results, essential for clinical applications |
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Term
| What are the two types of automated ISH processing systems? |
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Definition
| partially automated and walk-away automated |
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Term
| What do partially automated systems perform? |
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Definition
| pretreated device, hybridization device and signal detection device |
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Term
| What do you do if there is a lack of signal in a positive sample control? |
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Definition
| could be due to omission of a step or use of incorrect reagents in a step, is signal is reduced suspect the probe and detection reagents, validation for lots to lot variation |
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Term
| what do you do if there is no signal with positive control probe on a test sample? |
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Definition
| can be due to inadequate preservation of the target, also may be due to inadequate pretreatment step |
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Term
| what do you do if there is a signal with negative control probe on a test sample |
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Definition
| prevents interpretation, can be nonspecific binding of probe or cross-contamination |
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Term
| What are research applications of ISH? |
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Definition
| mapping genes to particular chromosomes, enumerating chromosomes in chromosome spreads, detecting cells that contain infectious or altered gene sequences, and demonstrating cellular subpopulations expressing particular genes |
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Term
| what are the clinically applications in oncology? |
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Definition
| can detect gene translocation, gene amplification or gene deletion |
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Term
| What is the predominant ISH used in clinical settings? |
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Definition
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Term
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Definition
| allows multicolor probes to demonstrate the relationship of chromosomes |
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Term
| What are the commercial FISH assays available? |
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Definition
| Vysis PathVysion, Vysis UroVysion, and FISH assays for detection of lymphoma |
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Term
| What does Vysis PathVysion detect? |
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Definition
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Term
| What does Vysis UroVysion detect? |
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Definition
| aneuploidy of chromosomes 3,7,17 and 9p21 locus, and bladder cancer screening in urine cytopathology specimens |
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Term
| What do FISH assays for lymphoma detect? |
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Definition
| anaplastic lymphoma involving ALK gene, Burkitt lymphoma involving MYC gene and IGH gene, Follicular lymphoma and mantle cell lymphoma |
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Term
| What are the 5 steps of FISH? |
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Definition
1. DNA unmasking
2. probe and target denaturation
3. probe-target DNA hybridization
4. detection
5. image analysis on fluorescent microscope |
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Term
| What does FISH stand for? |
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Definition
| Fluorescence In Situ Hybridization |
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Term
| Why have different colors to fluoresce? |
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Definition
| For differentiation. Different colors for different DNA strands |
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Term
| What are the probes binding to? |
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Definition
| Green fluorescence appears on the Y chromosome (Yq12) of males and orange fluorescence occurs on the centromere region of the X chromosome (Xp11.1-q11.1) |
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Term
| What happens if you leave the slide on the illuminator or table? |
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Definition
| photobleach/no longer fluoresce |
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Term
| What are two differences between hybridization with Western Blot and hybridization with FISH? |
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Definition
1. Western blot is hybridization proteins onto a membrane that we can visualise grossly
2. with FISH we are hybridizing DNA on a glass slide that we need to visualize microscopically |
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Term
| Why is is necessary to place your samples into the "denaturation solution" in the FISH lab? |
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Definition
| The DNA in the cells need to be exposed and denatured into single strands so the probes can hybridize to the nucelotides |
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Term
| What does a male see under the fluorescence microscope for the FISH lab? |
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Definition
| one orange and one green fluoresce |
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Term
| What does a female see under the fluorescence microscope for the FISH lab? |
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Definition
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Term
|
Definition
| Cytology or molecular labs to detect protein, RNA or DNA |
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Term
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Definition
| 60-200kb fragment of DNA attached covalently to a fluorescent molecule which hybridizes to its complmentary sequence in the sample DNA |
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Term
| What is this method used for? |
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Definition
| prenatal samples, tumors, and hematological malignancies |
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Term
| What are the steps of FISH? |
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Definition
denaturation
hybridization
hybrid detection
washing
signal amplification |
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