Exam 2 Flashcards
what is the purpose of periodic acid-Schiff (PAS) stain?
detection of polysaccharides, mucins, and basement membranes (carbohydrates)
what are some principles of periodic acid-Schiff (PAS) stain?
- Reaction is based on oxidation of certain tissue elements to aldehydes by periodic acid
- Schiff reagent is prepared by treating basic fuchsin with sulfurous acid
- Metabisulfite rinses are used to remove excess Schiff reagent and prevent false colorization of the tissue elements because of oxidation of any adsorbed reagent
what is the quality control for periodic acid-Schiff (PAS) stain?
- section of kidney is most sensitive control
- If procedure is used to demonstrate glycogen, use a section of liver containing glycogen or a section of cervix
what is the purpose of Mayer mucicarmine stain?
staining of epithelial mucin in tissue
what are some principles of Mayer mucicarmine stain?
- Staining pattern was comparable to that of Alcian blue
- Stains carboxylated and sulfonated mucins, but not neutral mucins
- This technique is specific for epithelial mucins, it has frequently been used in the identification of adenocarcinomas (cancers in glands that make mucus or fluid: lung, breast, prostate, colon)
what is the quality control for Mayer mucicarmine?
Section of un-autolyzed colon, small intestine, or appendix
what is the purpose of Alcian blue, pH 2.5?
staining of acid mucopolysaccharides
what are some principles for Alcian blue, pH 2.5?
- Alcian blue is a copper phthalocyanin basic dye that is water soluble and colored blue because of its copper content
- Believed to form salt linkages with the acid groups of acid mucopolysaccharides
what is the quality control for Alcian blue, pH 2.5?
A section of un-autolyzed small intestine, appendix, or colon should be used as a positive control
what is the purpose of Alcian blue, pH 1.0?
demonstration of sulfated mucosubstances
what are some principles of Alcian blue, pH 1.0?
- When used in a 0.1N hydrochloric acid solution (pH 1.0) Alcian blue stains only sulfated acid mucopolysaccharides and sulfated sialomucins (glycoproteins)
- Acid mucopolysaccharides and sialomucins that are carboxylated only will not be stained, because they are not capable of ionization at pH 1.0
what is the quality control for Alcian blue, pH 1.0?
Section of un-autolyzed small intestine, appendix, or colon should be used as a positive control
what is the purpose of Alcian blue with hyaluronidase?
to differentiate epithelial from connective tissue mucins
what are some principles of Alcian blue with hyaluronidase?
- Staining will disappear or be dramatically reduced when tissue sections containing hyaluronic acid, chondroitin sulfate A, or chondroitin sulfate C (“connective tissue” mucin) are digested with testicular hyaluronidase
- Glycoproteins (“epithelial” mucins) will not be affected
what is the quality control for Alcian blue with hyaluronidase?
- 2 sections of umbilical cord should be used as a control (“with” and “without”)
- Section of small bowel, appendix, or colon may be used as a second control to demonstrate epithelial mucins
what is the purpose of Alkaline Congo red stain?
demonstration of amyloid (starch-like) structures
what are some principles of Alkaline Congo red stain?
- Benzidine derivative that can react with cellulose
- Pretreatment with alkali aids in the release of native internal hydrogen bonds between adjacent protein chains
- Amyloid is a linear molecule & this configuration allows azo and amine groups of the dye to form hydrogen bonds with similarly spaced hydroxyl radicals of the amyloid
- Whether the binding of the dye occurs with the polysaccharide or the protein component of amyloid is indefinite at this time
what is the quality control for Alkaline Congo red stain?
- sections of amyloid must be used
- Better not to keep too many control sections cut because staining intensity has been reported to decrease with age of sections
- Massive, presumably long-standing deposits give less intense biochemical reactions than small, newly formed deposits
what is the purpose of Crystal violet stain?
Good rapid screening method for amyloid but is not as specific as Congo red method
what are some principles of Crystal violet stain?
- Exact mechanism of amyloid staining with crystal violet has not been defined
- It is assumed that the “metachromatic” staining of amyloid is because of the mucopolysaccharide content - however amyloid will induce only weak metachromasia with thionine and toluidine blue
- Addition of acid to solution prevents overstaining
- Low sensitivity & is rarely used
what is the quality control for Crystal violet stain?
section containing amyloid must be used
what is the purpose of Masson’s Trichrome stain?
- differentiate collagen from smooth muscle in tumors
- identify increases in collagenous tissue in diseases such as cirrhosis of the liver
what are some principles of Masson’s Trichrome stain?
- The mechanism of the stain is not totally understood and may be related in part to the size of different dye molecules
- Sections are first stained with acid dye such as Biebrich scarlet (all acidophilic tissue elements such as cytoplasm, muscle, & collagen will bind to the acid dyes
- The pH of the phosphotungstic/phosphomolybdic acid solution probably increases selective collagen staining & aids in diffusion or removal of Biebrich scarlet
what is the quality control for Masson’s Trichrome stain?
not needed, but if desired, can use uterus, small intestine, fallopian tube, or appendix
what is the purpose of van Gieson picric acid-acid fuchsin stain?
Serves as excellent counterstain for other methods such as the Verhoeff elastic technique
what are some principles of van Gieson picric acid-acid fuchsin stain?
- Picric acid provides the acidic pH necessary and also acts as a stain for muscle & cytoplasm
- Saturated picric acid solutions are important in the preparation of the stain & again for selective staining of collagen
- If picric acid is not saturated, collagen may stain pale pink to pale orange, and collagen, cytoplasm, and muscle may all stain the same color
what is the quality control of van Gieson picric acid-acid fuchsin stain?
not needed, but if desired, can use uterus, small intestine, appendix, or fallopian tube
what is the purpose of Verhoeff elastic stain?
- Used to demonstrate pathologic changes in elastic fibers, which includes atrophy of elastic tissue, thinning or loss that may result from arteriosclerotic changes: reduplication, breaks, or splitting that may result from other vascular diseases
- May also be used to demonstrate normal elastic tissue, as in identification of veins and arteries
- To determine whether or not the blood vessels have been invaded by tumor
what are some principles of Verhoeff elastic stain?
- Tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine
- Both ferric chloride and iodine serve as mordants, but also have an oxidizing function that assists in converting hematoxylin to hematein
- It is called a regressive method due to the requirement of sections being overstained & then differentiated
- Elastic tissue has strongest affinity for iron-hematoxylin complex & will retain dye longer than other tissue elements, which allows other elements to be decolorized & elastic fibers to remain stained
- Counterstain is van Gieson solution typically, but others can be used
what is the quality control for Verhoeff elastic stain?
Most laboratories use a section of aorta embedded on edge, but can use cross-section of muscular artery
what is the purpose of Cressyl violet stain?
Identification of neurons in tissue sections or the demonstration of loss of Nissl substance (chromatolysis)
what are some principles of Cressyl violet stain?
- used at an acidic pH
- Staining is restricted to nuclei and to DNA and RNA containing structures
- Contrast of Nissl substance & nuclei with the unstained background is enhanced
what is the quality control for Cressyl violet stain?
spinal cord section
what is the purpose of acid-fast stain?
detection of acid-fast bacteria in tissue sections
what are some principles of acid-fast stain?
- Lipoid capsule of acid fast organism takes up carbol-fuchsin and resists decolorization with dilute mineral acid
- Carbol-fuchsin is more soluble in the lipids of the cell wall than in acid alcohol but is readily removed from bacteria that lack the waxy capsule
- Carbol-fuchsin methods provide a specific way of identifying mycobacteria
what is the quality control for acid-fast stain?
- Tissue containing acid-fast organisms
- Millipore-filtered water should be used in floatation bath & negative control from the same day’s workload must be run (cut on same microtome and using the same water bath as is used for diagnostic case)
what is the purpose of PAS for fungi?
detection of fungi in tissue
what are some principles of PAS for fungi?
- Polysaccharides present in fungal cell walls are oxidized by the periodic acid to aldehydes
- Aldehydes react with Schiff reagent to yield rose-colored fungi
what is the quality control for PAS for fungi?
section containing fungi
what is the purpose of Prussian blue stain?
Detection of ferric (Fe3+) iron in tissues
what are some principles of Prussian blue stain?
- Detects ferric ion in loosely bound protein complex
- Iron that is strongly bound will not react
- Sections are treated with an acidic solution of potassium ferrocyanide, and any ferric iron present reacts to form an insoluble bright blue pigment called Prussian blue
what is the quality control for Prussian blue stain?
- Section containing ferric ion must be used
- Excessive amounts of iron are not desirable in the control because the reaction product is slightly soluble & may contaminate the incubating solution, giving a background stain on all sections
what is immunohistochemistry?
- a study that determines the origin of a tumor, prognosis, and treatment
- important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease
what is an antibody?
a host protein (immunoglobulin) produced in response to the presence of foreign molecules, organisms, or other agents in the body
what is an antigen?
a molecule made up of proteins, carbohydrates, or other polymers, and is capable of producing an immune response in animals or cell cultures for the production of antibodies
what is an epitope?
a region of an antigen where the antibody binds.
An antibody may target more than one antigen, but is specific for only one epitope
what are polyclonal antibodies?
produced by multiple immune cells in the host animal after exposure to a single antigen
what are monoclonal antibodies?
produced by a single clone of plasma cells, resulting in one type of antibody that will react with only a single specific epitope on the antigen against which it was raised
what should be seen in a good immunohistochemical stain?
- specific staining pattern
- strong, crisp staining of only target cells & no diffuse staining of tissue support matrix
- no cross-reactivity, or staining of, non-target cells
- no diffusion of reaction product onto surrounding support tissue
- no nonspecific staining resulting from endogenous biotin or enzymes such as peroxidase
- counterstain should contrast target cells, but not mask delicate staining
what are 2 common antigen retrieval methods?
- ) heat induced epitope retrieval
2. ) enzyme-induced epitope retrieval
what are some advantages to epitope retrieval?
- ability to further dilute antibodies
- exposure to epitope sites not previously detectable
- more intense reactions with decreased incubation times
- more uniform staining
- decreased background staining
- day to day consistency to stains
- improved standardization
what is immunofluorescence?
a technique that makes it possible to visualize antigens in tissue sections or in live cell suspensions
what is a fluorochrome?
a dye that absorbs light and then emits its own light at a longer wavelength
what is fluorescence?
a phenomenon of absorption and emission of light
what are the most common fluorochromes used in immunofluorescence techniques?
- fluorescein isothiocyanate (FITC)
- rhodamine
what kind of specimens are used in immunofluorescence?
frozen kidney and skin
how can tumor differentiation be performed?
through enzyme immunohistochemical techniques
how does enzyme histoimmunochemistry work?
The enzyme, in the presence of a substrate and a chromogen, provides the indicator system to visualize the location of the antibody
what is the direct method of immunohistochemical staining?
A labeled antibody of known specificity is used to identify antigens in the patient’s tissue (chromogen binds to primary antibody)
- NO secondary antibody or blocking agent for secondary anti-body (would need blocking peroxidase if using horse radish peroxidase for enzymatic reactions)
what is the indirect method of immunohistochemical staining?
- The patient’s serum is added to tissue sections containing known antigens to test the patient for the presence of antibodies to those antigen
- The patient’s serum may also be added to a known bacterium to detect the presence of bacterial antibodies in the patient
- A labeled antibody (ex: anti-human Ig) must be used to detect the bound antibodies from the patient’s serum
- utilizes blocking agent and secondary antibody
what is a positive control and what is it for?
- The optimum control is one prepared in the lab under the exact same conditions as the diagnostic tissue
- Because fixation varies and affects the reactions so dramatically, commercially prepared control slides are not ideal to determine if a negative reaction on the diagnostic slide is a true-negative reaction or if it is the result of overfixation
what is a negative control used for?
they are run by substituting the primary antibody for either non immune serum from the same species as the primary antibody or the diluent used for the primary antibody
which procedure is tissue stained for a predetermined period of time?
progressive
which type of stain is Gill’s hematoxylin?
progressive
in which procedure is acetic acid the acidifier?
regressive
which procedure does tissue uptake stain at a slower rate?
progressive
is eosin negatively charged?
yes
does eosin bind positively charged proteins?
yes
which special stain would you use to stain carbohydrates?
PAS (periodic acid-Schiff) stain
which special stain would you use to stain nerves?
Cresyl violet
which of the following is the correct blocking agent for IF with the following primary/secondary combo?:
- primary: mouse anti-human MHP
- secondary: goat anti-mouse IgG
a. normal mouse serum
b. normal human serum
c. normal goat serum
d. peroxidase
c. normal goat serum
does practically any tissue have an internal control for Crystal violet?
no
an additional blocking step must be performed for what kind of reactions?
enzymatic reactions that involve horse radish peroxidase (HRP) for colorimetric reaction
what is required to prevent endogenous peroxidases?
peroxidase block (usually 3% hydrogen peroxide soln)
what would happen if you did not block the endogenous peroxidases?
background, nonspecific color throughout the tissue instead of only at the sites with primary antibody bound
how can antigen retrieval be done?
- enzymatically (proteolytic cleavage)
- heat and/or pressure (pressure cooker)
