IHC2-HTL Flashcards
Immunohistochemistry study at the HTL level
In terms of a scientific experiment, what is a control?
The benchmark, or standard, that is compared to the variable part of the experiment. When performing special stains, the control is tissue containing the structural component or organism meant to be demonstrated. If it is not observed in the control tissue, then the stain did not work properly and must be repeated.
IHC staining requires both positive and negative controls. What is a positive control?
Just as in special stains, a positive control should demonstrate the target tissue component. With IHC, that will be an antigen. It controls for false negative results.
What does it mean if the positive control slide does not have a colored precipitate?
Either the staining method did not work or the control wasn’t positive for that antigen.
Why do we have negative controls in IHC?
To check for false positive results, either nonspecific or background staining. This could be endogenous staining, cross-reactivity, or autofluorescence.
What is endogenous staining?
Tissue components, other than the target antigen, picking up the stain.
What are the different types of endogenous staining?
Endogenous enzyme activity, endogenous biotin, endogenous immunoglobulin.
What is endogenous immunoglobulin?
This occurs when the primary antibody is the same species as the tissue being studied.
What is endogenous enzyme activity?
Enzymes conjugated to antibodies for detection (horseradish peroxidase, alkaline phosphotase) can also occur naturally in tissue and react with chromogenic substrates.
What are example of endogenous enzymes?
Peroxidases are found in bloody tissue like spleen and kidney. Phophotase can be in kidney, liver, or intestines.
What is endogenous biotin?
Biotin is a vitamin used in the metabolism of macromolecules and must be blocked when using the ABC staining method.
What is autofluorescence?
Tissue components such as red blood cells, collagen, and elastin have naturally occurring fluorescence that can make diagnoses difficult when using IF staining methods.
What is cross reactivity?
Unintended epitope binding, especially with secondary antibodies.
There are 2 types of negative controls in IHC. What are they?
1-Controls that do not contain the target antibody, and therefore should not show a chromogen reaction. 2-Controls that get a buffer wash step instead of a primary antibody application, and therefore should not show a chromogen reaction.
Why do we want control slides to come from the same laboratory that performs the IHC staining?
Because the control blocks will have the same fixation and processing as the patient tissue.
Immunofluorescence is usually done on frozen sections. IHC was originally done on frozen sections, why?
Because epitope retrieval methods for formalin fixed tissue had not been perfected.
True or False: Using frozen sections for immunos means they are unfixed.
Not necessarily true. Frozen sections may remain unfixed or be fixed with Acetone, Alcohol, Cold NBF, formalin vapors, etc.
What fixatives should not be used on paraffin embedded tissue, if IHC will be performed.
Glutaraldehyde, Chromates, Bouins.
What is a tissue microarray?
A control block containing multiple cores from different tissues. It will have a mixture of positive and negative tissues, as well different antigen expression levels. Often called a sausage block. It is a good idea to use a microarray for antibody and equipment validation.
True or False: Antibodies can be diluted in buffer solution.
True. Rarely are antibodies used at full strength. They can be bought in concentrated form and diluted in the lab or bought in a prediluted form.
What are antibodies diluted in?
PBS (Phosphate Buffer Saline) is the most common diluent. Sometimes TBS (Tris-Buffered Saline), normal saline or blocking buffer. It should be something that will reduce background staining. That is the purpose of diluting antibodies.
How do you determine how much to dilute an antibody?
There is often a suggested dilution in the concentrated antibody package insert (data sheet). Otherwise, start at 1:1000 and working down to 1:500 or up to 1:5000.
What is a diagnostic panel in IHC?
A panel is an algorithm that uses
a collection of antibodies to answer specific disgnostic questions. (Identify tissue abnormalities, differentiate between malignant and benign tumors, determine tumor type and prognosis.) This will be based on gross and microscopic morphologies and patient clinical history.
What is a RTU antibody?
Ready to use as opposed to concentrated. Concentrated antibodies need to be diluted. Pictured are automatic and manual staining kits.
True or False: Most cancers are carcinomas.
True. Almost 90% of all malignancies are carcinomas. (Neoplasms of epithelial origins.)
What is the difference between keratin and cytokeratin?
They are the same. The protein keratin forms hair and nails and the stratum corneum layer of the skin. Cytokeratin makes up the intermediate filaments of the cytoskeletons of epithelial cells. Therefore, cytokeratins are cytoplasmic staining antibodies.
What are the main two cytokeratins used to identify primary tumor sites?
CK7/CK20.
True or False: You can check for overfixation with formalin by doing a Vimentin.
True. Vimentin is sensitive to overfixation with formalin.
True or False: LCA and CD45 are the same marker.
True-Known as Leukocyte Common Antigen, it is a set of high molecular weight glycoproteins on the surface of most WBCs. Tonsil is a good control. As it is lymphoid tissue, B and T cells will stain strongly.
If CD45 stains both B lymphocytes and T lymphocytes, what antibodies would you use to differentiate between the them?
CD20 for B cells, CD3 for T cells. In the image the follicles containing B cells are staining with CD20 and not with CD3. It is the T cells that are staining with CD3.
Cytokeratins are cytoplasmic staining. What markers are membranous staining?
CD markers. CD stands for Cluster of Differentiation Each numbered cluster of CD antibodies recognize a different cell surface molecule.
What type of lymphoma do positive CD15 and CD30 markers indicate?
Classical Hodgkin’s Lymphoma. These markers identify the Reed-Sternberg cells.
True or False: ER/PR and Her2 are diagnostic markers.
True. These are markers that determine treatment.
True or False: Estrogen Receptor and Progesterone Receptor are nuclear staining.
True. Breast cancers that are positive for either or both of these hormone receptors tell us that the patient may respond to hormone therapy.
True or False: Her2 is nuclear staining.
False. Her2 is membranous staining and it stands for human epidermal (growth factor) receptor 2. Positive Her2 staining means the protein Her2 is being overexpressed. Her2 positive breat cancers grow faster and spread more than Her2 negative cancers. Patients that are Her2 positive may qualify for herceptin treatment
If FISH stands for Fluorescent In Situ Hybridization, what does SISH and CISH stand for?
Silver In Situ Hybridization and Chromogenic In Situ Hybridization. (CISH is the one that’s like IHC).
What is ISH? CISH Her2 is shown.
In situ hybridization. A technique that uses a complementary nucleic acid probe to find a specific nucleic acid sequence in tissue. CISH (which is just the basic chromogenic in situ hybridization) uses chromogens (like DAB) the same way IHC does.
Can ISH have the same targets as IHC?
Yes. IHC uses antibodies to locate proteins in cells. ISH uses probes to locate the genes coding for the expression of those protein.
Can you stain 2 antibodies on one section of tissue?
Yes, it is called dual staining IHC. Here CD15 is brown and CD30 is red.
Can you stain more than 2 antibodies on one section of tissue using traditional IHC methods?
Yes, it is called multiplexing. So, even if you just use 2 colors, you can make a cocktail of some of them. This PIN4 is staining HMWCK and p53 together brown and AMACR red.
Can you use 2 probes on one section of tissue for in situ hybridization?
Yes, and of course that is called DISH. What else?
What about FISH multiplexing?
What about it?
