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Histo 2 > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

what is the purpose of periodic acid-Schiff (PAS) stain?

A

detection of polysaccharides, mucins, and basement membranes (carbohydrates)

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2
Q

what are some principles of periodic acid-Schiff (PAS) stain?

A
  • 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
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3
Q

what is the quality control for periodic acid-Schiff (PAS) stain?

A
  • 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

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4
Q

what is the purpose of Mayer mucicarmine stain?

A

staining of epithelial mucin in tissue

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5
Q

what are some principles of Mayer mucicarmine stain?

A
  • 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)
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6
Q

what is the quality control for Mayer mucicarmine?

A

Section of un-autolyzed colon, small intestine, or appendix

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7
Q

what is the purpose of Alcian blue, pH 2.5?

A

staining of acid mucopolysaccharides

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8
Q

what are some principles for Alcian blue, pH 2.5?

A
  • 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
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9
Q

what is the quality control for Alcian blue, pH 2.5?

A

A section of un-autolyzed small intestine, appendix, or colon should be used as a positive control

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10
Q

what is the purpose of Alcian blue, pH 1.0?

A

demonstration of sulfated mucosubstances

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11
Q

what are some principles of Alcian blue, pH 1.0?

A
  • 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
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12
Q

what is the quality control for Alcian blue, pH 1.0?

A

Section of un-autolyzed small intestine, appendix, or colon should be used as a positive control

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13
Q

what is the purpose of Alcian blue with hyaluronidase?

A

to differentiate epithelial from connective tissue mucins

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14
Q

what are some principles of Alcian blue with hyaluronidase?

A
  • 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
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15
Q

what is the quality control for Alcian blue with hyaluronidase?

A
  • 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
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16
Q

what is the purpose of Alkaline Congo red stain?

A

demonstration of amyloid (starch-like) structures

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17
Q

what are some principles of Alkaline Congo red stain?

A
  • 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
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18
Q

what is the quality control for Alkaline Congo red stain?

A
  • 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
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19
Q

what is the purpose of Crystal violet stain?

A

Good rapid screening method for amyloid but is not as specific as Congo red method

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20
Q

what are some principles of Crystal violet stain?

A
  • 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
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21
Q

what is the quality control for Crystal violet stain?

A

section containing amyloid must be used

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22
Q

what is the purpose of Masson’s Trichrome stain?

A
  • differentiate collagen from smooth muscle in tumors

- identify increases in collagenous tissue in diseases such as cirrhosis of the liver

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23
Q

what are some principles of Masson’s Trichrome stain?

A
  • 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
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24
Q

what is the quality control for Masson’s Trichrome stain?

A

not needed, but if desired, can use uterus, small intestine, fallopian tube, or appendix

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25
Q

what is the purpose of van Gieson picric acid-acid fuchsin stain?

A

Serves as excellent counterstain for other methods such as the Verhoeff elastic technique

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26
Q

what are some principles of van Gieson picric acid-acid fuchsin stain?

A
  • 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
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27
Q

what is the quality control of van Gieson picric acid-acid fuchsin stain?

A

not needed, but if desired, can use uterus, small intestine, appendix, or fallopian tube

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28
Q

what is the purpose of Verhoeff elastic stain?

A
  • 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
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29
Q

what are some principles of Verhoeff elastic stain?

A
  • 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
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30
Q

what is the quality control for Verhoeff elastic stain?

A

Most laboratories use a section of aorta embedded on edge, but can use cross-section of muscular artery

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31
Q

what is the purpose of Cressyl violet stain?

A

Identification of neurons in tissue sections or the demonstration of loss of Nissl substance (chromatolysis)

32
Q

what are some principles of Cressyl violet stain?

A
  • 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
33
Q

what is the quality control for Cressyl violet stain?

A

spinal cord section

34
Q

what is the purpose of acid-fast stain?

A

detection of acid-fast bacteria in tissue sections

35
Q

what are some principles of acid-fast stain?

A
  • 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
36
Q

what is the quality control for acid-fast stain?

A
  • 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)
37
Q

what is the purpose of PAS for fungi?

A

detection of fungi in tissue

38
Q

what are some principles of PAS for fungi?

A
  • Polysaccharides present in fungal cell walls are oxidized by the periodic acid to aldehydes
  • Aldehydes react with Schiff reagent to yield rose-colored fungi
39
Q

what is the quality control for PAS for fungi?

A

section containing fungi

40
Q

what is the purpose of Prussian blue stain?

A

Detection of ferric (Fe3+) iron in tissues

41
Q

what are some principles of Prussian blue stain?

A
  • 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
42
Q

what is the quality control for Prussian blue stain?

A
  • 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
43
Q

what is immunohistochemistry?

A
  • 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
44
Q

what is an antibody?

A

a host protein (immunoglobulin) produced in response to the presence of foreign molecules, organisms, or other agents in the body

45
Q

what is an antigen?

A

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

46
Q

what is an epitope?

A

a region of an antigen where the antibody binds.

An antibody may target more than one antigen, but is specific for only one epitope

47
Q

what are polyclonal antibodies?

A

produced by multiple immune cells in the host animal after exposure to a single antigen

48
Q

what are monoclonal antibodies?

A

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

49
Q

what should be seen in a good immunohistochemical stain?

A
  • 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
50
Q

what are 2 common antigen retrieval methods?

A
  1. ) heat induced epitope retrieval

2. ) enzyme-induced epitope retrieval

51
Q

what are some advantages to epitope retrieval?

A
  • 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
52
Q

what is immunofluorescence?

A

a technique that makes it possible to visualize antigens in tissue sections or in live cell suspensions

53
Q

what is a fluorochrome?

A

a dye that absorbs light and then emits its own light at a longer wavelength

54
Q

what is fluorescence?

A

a phenomenon of absorption and emission of light

55
Q

what are the most common fluorochromes used in immunofluorescence techniques?

A
  • fluorescein isothiocyanate (FITC)

- rhodamine

56
Q

what kind of specimens are used in immunofluorescence?

A

frozen kidney and skin

57
Q

how can tumor differentiation be performed?

A

through enzyme immunohistochemical techniques

58
Q

how does enzyme histoimmunochemistry work?

A

The enzyme, in the presence of a substrate and a chromogen, provides the indicator system to visualize the location of the antibody

59
Q

what is the direct method of immunohistochemical staining?

A

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)

60
Q

what is the indirect method of immunohistochemical staining?

A
  • 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
61
Q

what is a positive control and what is it for?

A
  • 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
62
Q

what is a negative control used for?

A

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

63
Q

which procedure is tissue stained for a predetermined period of time?

A

progressive

64
Q

which type of stain is Gill’s hematoxylin?

A

progressive

65
Q

in which procedure is acetic acid the acidifier?

A

regressive

66
Q

which procedure does tissue uptake stain at a slower rate?

A

progressive

67
Q

is eosin negatively charged?

A

yes

68
Q

does eosin bind positively charged proteins?

A

yes

69
Q

which special stain would you use to stain carbohydrates?

A

PAS (periodic acid-Schiff) stain

70
Q

which special stain would you use to stain nerves?

A

Cresyl violet

71
Q

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

A

c. normal goat serum

72
Q

does practically any tissue have an internal control for Crystal violet?

A

no

73
Q

an additional blocking step must be performed for what kind of reactions?

A

enzymatic reactions that involve horse radish peroxidase (HRP) for colorimetric reaction

74
Q

what is required to prevent endogenous peroxidases?

A

peroxidase block (usually 3% hydrogen peroxide soln)

75
Q

what would happen if you did not block the endogenous peroxidases?

A

background, nonspecific color throughout the tissue instead of only at the sites with primary antibody bound

76
Q

how can antigen retrieval be done?

A
  • enzymatically (proteolytic cleavage)

- heat and/or pressure (pressure cooker)

Histo 2 (2 decks)

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