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QIHC maryjw79 @ gmail email 4 access > STAINING > Flashcards

STAINING Flashcards

1
Q

What is the Direct staining method?

A

A labeled Ab of known specificity is used to identify Ag in patient tissue.

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2
Q

What is the Indirect staining method?

A

The patient’s serum is added to tissue sections containing known Ag to test the patient for the presence of Ab to those Ag. The patient’s serum can also be added to bacteria.

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3
Q

What is the Unlabeled/ Soluble Enzyme Immune Complex method?

A

A 3 step method involving a primary Ab, secondary Ab and a soluble enzyme-antienzyme complex.

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4
Q

What is the Avidin-biotin complex (ABC) method?

A

The primary Ab is followed by a secondary biotinylated Ab which bond irreversibly to Avidin and forms a complex.

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5
Q

What are three main benefits of the ABC method?

A
  1. There is low background staining
  2. Sensitivity can be up to 40X other immunoperoxidase methods
  3. Antibody may be used at higher dilutions than in other methods
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6
Q

What is polymeric detection?

A

The “gold standard” of IHC staining in which either a single strand (monomeric) or multiple strand (polymeric) molecules are fused with the secondary Ab which eliminates the need for ABC or patient serum. We did this in Vancouver.

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7
Q

Which detection system works better: monomeric or polymeric?

A

Monomeric, because there is increased sensitivity and Ab binding site penetration with single strand molecules. Turnaround time is also decreased.

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8
Q

What is the general procedure for polymeric detection?

A

Antibody-polymer-chromogen.

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9
Q

What neoplasm does BCL2 test for?

A

Follicular lymphoma

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10
Q

What neoplasm does CD117 test for?

A

Gastrointestinal stromal tumor

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11
Q

What neoplasm does CD15 test for?

A

Hodgkin’s lymphoma

Reed-Sternberg cells

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12
Q

What neoplasm does CD20 test for?

A

B cell lymphoma

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13
Q

What neoplasm does CD3 test for?

A

T cell lymphoma

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14
Q

What neoplasm does CK20 test for?

A

Colon carinoma

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15
Q

What neoplasm does Cytokeratin (Pankeratin) test for?

A

Carcinoma in general

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16
Q

What neoplasm does Desmin test for?

A

Leiomyoma (smooth muscle tumor, common in uterus)

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17
Q

What neoplasm does ER test for?

A

Breast carcinoma

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18
Q

What neoplasm does Glial Fibrillary Acidic Protein (GFAP) test for?

A

Glioblastoma multiforme

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19
Q

What neoplasm does Glial Fibrillary Acidic Protein (GFAP) test for?

A

Glioblastoma multiforme

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20
Q

What neoplasm does Gross Cystic Disease Fluid Protein (GCDFP-15) test for?

A

Breast ductal carcinoma

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21
Q

What neoplasm does HER2 test for?

A

Breast carcinoma

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22
Q

What neoplasm does HMB-45 test for?

A

Melanoma

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23
Q

What neoplasm does CD45 test for?

A

Lymphoma

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24
Q

What is another term for CD45?

A

Leukocyte common antigen

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25
Q

What neoplasm does Melan-A test for?

A

Melanoma

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26
Q

What neoplasm does PR test for?

A

Breast ductal carcinoma

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27
Q

What neoplasm does S100 test for?

A

Schwannoma

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28
Q

What neoplasm does Thyroid Transcription Factor (TTF1) test for?

A

Lung adenocarcinoma

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29
Q

What neoplasm does Thyroid Transcription Factor (TTF1) test for?

A

Lung adenocarcinoma

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30
Q

What neoplasm does Vimentin test for?

A

Seminoma

Over fixation

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31
Q

Define Quenching

A

Blocking of naturally occurring excess endogenous enzymes found within a cell.

ex: RBCs causing excessive background staining

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32
Q

Define Quenching

A

Blocking of naturally occurring excess endogenous enzymes found within a cell.

ex: RBCs causing excessive background staining

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33
Q

When is “Quenching” performed?

A

Beginning of procedure, after depar and antigen retrieval

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34
Q

What is the most notable exception to the “usual” Quenching protocol?

A

CD34 because it will be dissolved by the hydrogen peroxide.

It should be applied after the primary antibody and before the second antibody

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35
Q

What is the purpose of a primary antibody?

A

It’s an antibody that binds to antigens in the patient’s tissue

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36
Q

What is the purpose of a secondary antibody?

A

It binds to the primary antibody and becomes conjugated into a complex

ex: ABC method

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37
Q

What are the two different detection systems?

A

Biotin-advin (strep)

Polymer

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38
Q

Define flurophore?

A

Molecules that glow upon excitation with UV light

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39
Q

Define enzyme

A

Proteins that catalyze reactions

They convert soluble compounds into an insoluble deposited chromogen

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40
Q

Define substrate

A

Molecules that react with enzymes in order to activate them

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41
Q

Define chromogen

A

Get trapped by the reaction of an enzyme with its substrate

It is deposited at the antigen site

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42
Q

What are commonly employed reagents for Quenching peroxidase activity?

A
  1. Hydrogen peroxide in water/TBS/PBS
  2. Hydrogen peroxide in methanol
  3. Commerically prepared solutions (ex: UltraBlock, DEEB, BloxAll)
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43
Q

What are commonly employed reagents for Quenching Alkaline phosphatase activity?

A
  1. Levamisole in water
  2. Commercially prepared solutions
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44
Q

How are slides that are to be stained with reagents that employ horseradish peroxidase as the ‘label’ pre treated?

A

Incubated with weak solution of Hydrogen Peroxide (0.5-2%)

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45
Q

What is the scientific basis for “protein blocking”?

A

Inhibit non specific binding of charged elements to antibody reagents

This causes background staining

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46
Q

What are some commonly used protein blockers?

A
  1. Powdered milk/albumin
  2. BSA
  3. Casein (milk protein)
  4. Denatured mouse/rabbit/sheep/goat serum
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47
Q

What kind of tissues can immunofluorescence be performed on?

A

Paraffin-fixed tissues

Cell monolayers (such as ‘Cytospin’ and ‘ThinPrep’ preparations)

Frozen sections

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48
Q

What are the benefits of immunofluorescence?

A
  1. All of the fluorescent channels can easily be viewed separately and then merged to form a pseudocoloured image
  2. Very weak staining from the primary antibody can be observed in isolation
    without any interference from other signals
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49
Q

What are the disadvantages of immunofluorescence?

A
  1. Tissue sections display a degree of autofluorescence due to various tissue components being naturally fluorescent,
    such as collagen.
  2. Formaldehyde fixation also increases the degree of autofluorescence

Autofluorescence can mask the signal from fluorescent reporter labels, making the interpretation of fluorescence results difficult

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50
Q

What is the disadvantage of using immunofluorescent?

A

Fluorescence from the chromophore fades with time, especially if it is exposed to excitation light for extended periods

The slides cannot be used to provide a permanent record of the staining
results and so photographic documentation/digitization of the tissue sections is necessary.

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51
Q

What is the advantage of using FITC?

A

FITC is an apple-green fluorescence and is
rarely seen as autofluorescence in mammalian tissues

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52
Q

What is the ideal fluorochrome to antibody ratio?

A

The ideal fluorochrome to antibody
ratio is between 2 and 4:1

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53
Q

How can cross-reactivity be checked in immunofluorescene?

A

Incubating the conjugated antibody directly on a tissue section containing human immunoglobulin and
examining the slide for fluorescence

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54
Q

What is the light source in fluorescent microscopes?

A

Mercury vapor

Xenon

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55
Q

Define Immunofluorescene

A

Utilizing the specificity of antibodies with fluorescent dyes to recognize an antigen

Allows visualization of the distribution of the antigen through fluorescent dyes with a fluorescence microscope

56
Q

What are two classes of immunofluorescence techniques?

A

Direct (Primary)

Indirect (Secondary)

57
Q

What are two classes of immunofluorescence techniques?

A

Direct (Primary)

Indirect (Secondary)

58
Q

Describe the steps in the direct labeling detection system

A
  1. Apply conjugated primary antibody - attaches to the epitope
  2. Apply substrate chromogen - attaches to the primary antibody
59
Q

Describe the steps in the indirect labeling detection system

A
60
Q

Describe the steps in the peroxidase anti-peroxidase (PAP) detection system

A
61
Q

Describe the steps in the alkaline phosphatase anti-alkaline phosphate (APAAP) detection system

A
62
Q

Describe the steps in the avidin-biotin (ABC) complex detection system

A
63
Q

Describe the steps in the labeled streptavidin-biotin (LSAB) detection system

A
64
Q

Describe the steps in the labeled polymer detection system

A
65
Q

What is the rationale for use of the direct labeling method?

A
66
Q

What are the source of errors in the direct labeling method?

A
67
Q

What controls can be used in the direct labeling method?

A
68
Q

What is the rationale for use of the indirect labeling method?

A
69
Q

What are the source of errors in the indirect labeling method?

A
70
Q

What controls can be used in the indirect labeling method?

A
71
Q

What is the rationale for use of the PAP method?

A
72
Q

What are the source of errors in the PAP method?

A
73
Q

What controls can be used in the indirect labeling method?

A
74
Q

What is the rationale for use of the APAAP method?

A
75
Q

What are the source of errors in the APAAP method?

A
76
Q

What controls can be used in the APAAP method?

A
77
Q

What is the rationale for use of the ABC method?

A
78
Q

What are the source of errors in the ABC method?

A
79
Q

What controls can be used in the ABC method?

A
80
Q

What is the rationale for use of the LSAB method?

A
81
Q

What are the source of errors in the LSAB method?

A
82
Q

What controls can be used in the LSAB method?

A
83
Q

What is the rationale for use of the labeled polymer method?

A
84
Q

What are the source of errors in the labeled polymer method?

A
85
Q

What controls can be used in the labeled polymer method?

A
86
Q

Which IHC methods are most sensitive?

A
87
Q

Which IHC methods are the least sensitive?

A
88
Q

What technique is used to amplify staining (applied prior to the chromogen)?

A
89
Q

What two reagents are commonly used to amplify staining?

A
90
Q

How does pH influence the IHC reaction?

A
91
Q

How does temperature influence the IHC reaction?

A
92
Q

How does time influence the IHC reaction?

A
93
Q

How does kinetics influence the IHC reaction?

A
94
Q

Define background as it relates to immunoenzyme procedures.

A
95
Q

Define endogenous substance

A
96
Q

What is the effect of antigen diffusion on immunoenzyme background?

A
97
Q

What is the effect of contaminating antibodies on immunoenzyme background?

A
98
Q

What is the effect of cross reactivity on immunoenzyme background?

A
99
Q

What is the effect of endogenous biotin on immunoenzyme background?

A
100
Q

What is the effect of endogenous enzyme activity on immunoenzyme background?

A
101
Q

What is the effect of Fc receptors on immunoenzyme background?

A
102
Q

What is the effect of hydrophobic interaction on immunoenzyme background?

A
103
Q

What is the effect of ionic interaction on immunoenzyme background?

A
104
Q

What are antibody diluents

A
105
Q

What is the importance of the pH for diluents?

A
106
Q

What are wash buffers?

A
107
Q

What two primary types of phosphate are used to buffer saline in immunoenzyme staining?

A
108
Q

What two surfactants are utilized in wash buffers and antibody diluents?

A
109
Q

Why is hematoxylin used in IHC?

A
110
Q

What is the disadvantage of regressive staining pertaining to IHC?

A
111
Q

What is the effect of alcohol on AEC?

A
112
Q

What is the effect of alcohol on DAB?

A
113
Q

What are the ingredients in Harris Hematoxylin?

A
114
Q

What are the advantages of using Harris hematoxylin with AEC?

A
115
Q

What are the disadvantages of using Harris hematoxylin with DAB?

A
116
Q

What are the disadvantages of using Harris hematoxylin in IHC?

A
117
Q

What are the ingredients in Gill Hematoxylin?

A
118
Q

What are the advantages of using Gill hematoxylin with AEC?

A
119
Q

What are the disadvantages of using Gill hematoxylin with DAB?

A
120
Q

What are the disadvantages of using Gill hematoxylin in IHC?

A
121
Q

What are the ingredients in Mayer Hematoxylin?

A
122
Q

What are the advantages of using Mayer hematoxylin with AEC?

A
123
Q

What are the disadvantages of using Mayer hematoxylin with DAB?

A
124
Q

What are the disadvantages of using Mayer hematoxylin in IHC?

A
125
Q

What are the three main staining patterns in tissues?

A
  1. Nuclear
  2. Cytoplasmic
  3. Nuclear and cytoplasmic
126
Q

List antibody that has nuclear staining

A
127
Q

List an antibody that has cytoplasmic staining

A
128
Q

List an antibody that has nuclear and cytoplasmic staining

A
129
Q

Identify the staining location of the most sensitive antibodies?

A
130
Q
A
131
Q

Define procedure contol

A
132
Q

Lectin histochemistry is useful for the demonstration of what type of cells?

A

Cells that are not easily distinguishable by ordinary dye-based staining:

-microglial cells in nervous tissue
-nerve fibers in the peripheral nervous system
-muscle fibers types in paraffin sections
-capillary blood vessels in various organs
-parts of renal tubules

133
Q

What are the four steps of IHC staining?

A
  1. Fixation and processing
  2. Blocking non-specific/background staining
  3. Detection systems
  4. Antigen retrieva
134
Q

What is the purpose of fixation in IHC?

A

Optimize preservation because this affects morphologic and immunohistologic results.

135
Q

What are the properties of direct conjugate-labeled antibody method?

A
  1. Uses one labelled antibody, which binds directly to antigen
  2. Purity or specificity - can’t have both
  3. Rapid and easy
  4. Monoclonal
  5. Sensitivity is low due to low signal amplification.
136
Q

What are the properties of indirect or sandwich detection system

A
  1. Versatility is increased
  2. The primary antibody used at a higher working dilution to achieve successful staining
  3. The secondary antibody is prepared with high order of specificity and affinity
  4. More sensitive due to signal amplification through several secondary antibody reactions with different antigen sites in the primary antibody
137
Q

Describe the streptavidin-peroxidase complex as detection system

A

Peroxidase enzyme oxidizes DAB turning it into a brown pigment

This pigment precipitates out of solution as a brown solid and is located at the site of the antigen.

QIHC maryjw79 @ gmail email 4 access (16 decks)

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