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QIHC maryjw79 @ gmail email 4 access > DETECTION SYSTEMS > Flashcards

DETECTION SYSTEMS Flashcards

1
Q

What is photobleaching?

A

A fluorophore loses its ability to fluorescence due to the buildup of reactive oxygen particles due to its natural activity.

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2
Q

How can photobleaching be reduced?

A

1) limiting the amount of free oxygen radicals in the environment

2) decreasing the excitation light in intensity and duration

3) Using a low concentration of fluorochrome with high-quantum efficiency

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3
Q

What are some common applications of IF in pathology?

A
  1. FISH to detect gene aberrations in cells.
  2. The detection of single monolayer organisms such as bacteria and parasites.
  3. The visualization of cell structures by super resolution microscopy.
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4
Q

Define ISH

A

In-situ hybridization

Technique used to identify gene amplifications, deletions and translocations as well as chromosomal copy number changes.

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5
Q

What are the three different ISH techniques?

A

FISH (fluorescent)
CISH (chromogens)
SISH (silver)

Named for the system that is used to identify the probe

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6
Q

Why is CISH used more commonly than FISH?

A
  1. The type of microscope (brightfield) used for CISH is more readily available than the microscope used for FISH (fluorescence).
  2. Brightfield CISH allows for better visualization of tissue structures.
  3. CISH signals do not fade overtime like FISH.
  4. Documentation by visual acquisition is simpler with brightfield than with fluorescent scopes decreasing the evaluation time for CISH vs FISH.
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7
Q

What are the two major categories of fixation?

A

Chemical

Drying

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8
Q

What are the two most commonly used fluorochromes in IF?

A

FITC

Rhodamine

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9
Q

What are the two most common enzymes used for antibody visualization?

A

Horseradish phosphatase (HRP)

Alkaline phosphatase (AP)

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10
Q

How are DAB and AEC commonly used?

A

Visualize the sites of antibody binding by forming a colored compound

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11
Q

In Avidin-biotin methods, what follows the primary antibody?

A

The biotinylated secondary antibody

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12
Q

How does alkaline phosphatase function in some IHC methods?

A

As the enzyme

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13
Q

Define fluorochrome

A

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

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14
Q

How is horseradish peroxidase used in some Avidin-biotin methods?

A

As the enzyme

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15
Q

What is the most common substrate used for alkaline phosphatase labeled antibody?

A

naphthol-AS-phosphate

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16
Q

What chromogen is commonly used to demonstrate alkaline phosphate?

A

Fast red violet LB

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17
Q

What chromogen is INSOLUBLE in alcohol?

A

DAB

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18
Q

If 3-amino-9-ethylcarbazole (AEC) is used as the chromogen, what kind of hematoxylin should be used as the counterstain?

A

Mayer

Hematoxylin must not contain alcohol

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19
Q

How must tissue for immunofluorescence be handled?

A

Fresh, Frozen

DO NOT fix

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20
Q

Define neoplasm

A

New, uncontrolled and abnormal growth of tissue

Three types:
1. Benign
2. Premalignant
3. Malignant

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21
Q

What does a completely negative Vimentin result indicate?

A

Tissue has been over fixed in formalin

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22
Q

Besides the heat employed, what is the other most important factor of HIER?

A

Chemical composition

pH

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23
Q

How is ficin used in IHC?

A

A proteolytic enzyme of vegetable origin widely used for red cell antibody detection

Can be used as a trypsin substitute in immunoperoxidase staining

EIER

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24
Q

What is the main benefit of polymeric detection methods?

A

Serum and avidin-biotin blocking steps not needed

Quicker

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25
Q

Why must antibodies be detected with a labeled method?

A

Antibodies cannot be seen with a microscope unless they are labeled or flagged by some method that permits their visualization

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26
Q

Define the Direct-Conjugate-Labeled Antibody Method

A

Attaching a label to an antibody by chemical means and then directly applying this labeled conjugate to
tissue sections

The label is attached directly to the antibody with specificity for the antigen

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27
Q

What are the benefits of the direct conjugate labeled method?

A

Rapid
Ease of performance

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28
Q

What are the disadvantages of the direct conjugate labeled method?

A

Need to conjugate each of the appropriate primary antibodies separately to detect different antigens

The primary antibody be used at a relatively high concentration due to a lack of sensitivity

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29
Q

Define the Indirect Antibody Method

A

The primary antibody is unlabeled

The method uses a labeled secondary antibody with specificity against the primary antibody

a.k.a. “sandwich”

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30
Q

Fluorescent staining advantages

A

Rapid

Works well in multiplex

Non-masking

Fluorescent labels have a small molecular footprint making them easy to conjugate with an antibody

Allows analysis of proteins in fresh, frozen or fixed tissues

Can detect bacteria/parasites

Wider dynamic range

No enzyme amplification needed

Allows visualization of cell structures by super resolution microscopy

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31
Q

Fluorescent staining disadvantages

A

Fades over time

Hard to view cell morphology

Requires ability to view with fluorescent microscope

Need a specific filter to visualize each fluorophore

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32
Q

How can you prevent autofluorescence?

A

Do not use aldehydes, (formalin, glutaraldehyde) as this can result in high autofluorescence

Can be minimized by wash in sodium borhydride in PBS prior to antibody incubation

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33
Q

Describe autofluorescence

A

The natural emission of light by biological structures

Caused by flavin coenzymes and reduced pyridine nucleotides

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34
Q

When evaluating fluorescent-stained frozen sections, morphologic detail of tissue can be enhanced by examining slides using?

A

Combining fluorescent microscopy with phase contrast microscopy

A phase contrast microscope alters the phase relationships of light passing through and around an object, and as a result makes individual components (e.g. nuclei and cytoplasm) visible

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35
Q

What is the classic preparation of tissue for immunofluorescence?

A

Frozen sections of unfixed tissue

Antigenic reactivity is minimally impaired and fluorescent antibody staining is strongest

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36
Q

How can you combat autofluorescene?

A

Use probes that maximize the fluorescence signal relative to the autofluorescence

Use optical filters that maximize the fluorescence signal relative to the autofluorescence

Have a well calibrated and maintained microscope

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37
Q

What is fluorescence compensation?

A

Emission signals of the fluorphore overlap

The overlapping color must be removed or it will give a false level

The overlapping color is electronically removed using single positive controls

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38
Q

How can fluorescence compensation be prevented?

A

By using fluorophores that do not have overlapping emission spectra

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39
Q

What are the benefits of HRP enzymes in IHC?

A

Generate sharp and dense precipitates
Detects intracellular targets
Detects highly delineated locations

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40
Q

What are the benefits of AP enzymes in IHC?

A

Allows visualization of underlying tissue structure

Yield a more diffuse and translucent precipitate

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41
Q

What is the substrate when using HRP enzyme based detection in IHC?

A

Hydrogen peroxide

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42
Q

What is the substrate when using AP enzyme based detection in IHC?

A

naphthol-AS-phosphate

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43
Q

What two factors create staining inconsistencies when using AP enzyme based reactions?

A

Sensitive to heat

Sensitive to light

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44
Q

What chromogens are used in the HRP based detection in IHC?

A

DAB

AEC

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45
Q

What chromogens are used in the AP based detection in IHC?

A

Fast-red-violet LB

Fast-red TR

Fast-blue BBN

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46
Q

Define blocking

A

To block tissue endogenous peroxidase activity (needed when the tissue contains many RBCs)

To block nonspecific background staining occurring as a result of antibody attachment to highly charged collagen and connective tissue elements

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47
Q

How do you block for endogenous peroxide activity?

A

Use 3% Hydrogen Peroxide prepared in absolute methanol

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48
Q

How do you block for nonspecific staining?

A

Add nonimmune serum from the same species in which the secondary antibody is produced to the tissue before applying primary antibody

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49
Q

What are some examples of nonimmune proteins used for blocking?

A

Casein

Nondry fat milk

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50
Q

What wash buffers can be used with an HRP based detection system?

A

PBS

TBS

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51
Q

What wash buffer should only be used with AP based detection systems?

A

Use TBS only

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52
Q

What are the benefits of using wetting agents?

A

The addition of a wetting agent will help with staining

Improve rinsing steps

Slow down bacterial growth

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53
Q

What are some examples of wetting agents?

A

Tween-20

Triton-X

Brij-35

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54
Q

What temperature should the wash buffer be used at?

A

Room temperature

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55
Q

What are the two commonly used wash buffers?

A

PBS

TBS (tween)

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56
Q

What must be checked and maintained in the wash buffer?

A

pH

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57
Q

When must the pH of a wash buffer be checked and documented?

A

Each time a new lot is used

Each time a new batch is prepared

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58
Q

What decides the use of a certain chromogen?

A

The choice of chromogen is directed by the enzyme used

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59
Q

What are some commonly used chromogens?

A

DAB brown color
AEC red color
Silver black used in ISH
Fast-red fuchsia
Fuchsin fuchsia
BCIP blue
NBT blue
INT blue

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60
Q

What can be used to block endogenous alkaline phosphate?

A

Levamisole

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61
Q

What chromagen can be used if a deeper intense color is preferred?

A

Nitro BlueTetrazolium (NBT)

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62
Q

When using an IHC alkaline phosphatase staining method, what is the mechanism that produces the color?

A

An enzyme that hydrolyzes the substrate to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes.

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63
Q

A blue-black end product color is produced when nickel is added to which of the following substrates?

A

DAB

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64
Q

What are the two primary probe for in situ?

A

DNA
RNA

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65
Q

Explain pretreatment options in ISH

A

The purpose of pretreatment is to prepare the tissue section for hybridization

i.e. antigen retrieval using heat or enzymes

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66
Q

Explain hybridization options in ISH

A

The purpose of hybridization is to drive probe binding to the target - nucleic acids seek complement binding

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67
Q

Explain stringency wash options in ISH

A

The purpose of stringency wash is to remove free and nonspecifically bound probe

Destabilizes poor matches

Goal is to keep probe hybridized but remove all nonspecific binding

Encourages nucleic acids to form single strand

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68
Q

Explain high stringency wash in ISH

A

High hybridization temperatures
Low concentration of salt in the buffers

Enables the hybridization of highly homologous nucleic acid sequences

If too high, probes will not bind to the target

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69
Q

Explain low stringency wash in ISH

A
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70
Q

What variables are usually modified to alter the stringency in ISH?

A

Washing steps can be optimized by adjusting temperature, salt, and detergent concentration in the wash buffer to minimize non-specific interactions

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71
Q

List the steps in an ISH procedure

A
  1. Drying
  2. Depar
    3.Pretreatment
  3. Denaturation
  4. Hybridization
  5. Stringency wash
  6. Detection
  7. Counterstain
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72
Q

Identify which steps of an ISH procedure differ from an IHC procedure

A

IHCs do not have
1. Denaturation
2. Hybridization
3. Detection

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73
Q

Define enzyme

A
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74
Q

Define substrate

A
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75
Q

List the two most frequently used enzymes in IHC

76
Q

Explain the principle of enzyme IHC methods

77
Q

Explain the purpose of enzyme IHC methods

78
Q

List the advantages of enzyme IHC methods

79
Q

List the disadvantages of enzyme IHC methods

80
Q

What is the substrate for horseradish peroxidase?

A

Hydrogen peroxide

81
Q

What are the advantages of using horseradish peroxidase?

82
Q

What are the disadvantages of using horseradish peroxidase?

83
Q

What is the substrate for glucose oxidase?

84
Q

What are the advantages of using glucose oxidase?

85
Q

What are the disadvantages of using glucose oxidase?

86
Q

What is the substrate for Beta-galactosidase?

87
Q

What are the advantages of using Beta-galactosidase?

88
Q

What are the disadvantages of using Beta-galactosidase?

89
Q

What is the substrate for alkaline phosphatase?

90
Q

What are the advantages of using alkaline phosphatase?

91
Q

What are the disadvantages of using alkaline phosphatase?

92
Q

Define endogenous blocking

93
Q

Explain endogenous enzyme blocking

94
Q

What is the microscopic result if an enzyme block is not used?

95
Q

What is the endogenous block used for horseradish peroxidase-based detection system?

96
Q

Where in tissues is endogenous peroxidase found?

97
Q

What is the endogenous block used for alkaline phosphatase-based detected system?

98
Q

Where in tissues is alkaline phosphatase found?

99
Q

What is the blocking process for endogenous biotin that has been increased by using retrieval methods?

100
Q

Which tissues contain high levels of endogenous biotin?

A

liver, kidney, pancreas, mammary gland, skeletal muscle, salivary gland and adipose tissue

101
Q

Explain protein blocking

102
Q

List examples of protein blocking

103
Q

What color is the end product of 3,3’-diaminobenzidene (DAB)?

104
Q

What are the advantages of using 3,3’-diaminobenzidene (DAB)?

105
Q

What are the disadvantages of using 3,3’-diaminobenzidene (DAB)?

106
Q

What are the advantages of using 3-amino-9-ethylcarbazole (AEC)?

107
Q

What are the disadvantages of using 3-amino-9-ethylcarbazole (AEC)?

108
Q

What color is the end product of 3-amino-9-ethylcarbazole (AEC)?

109
Q

What color is the end product of 4-chloro-1-naphtol (CN)?

110
Q

What are the disadvantages of using 4-chloro-1-naphtol (CN)?

111
Q

What are the advantages of using 4-chloro-1-naphtol (CN)?

112
Q

What color is the end product of p-phenylenediamine dihydrochloride (Hanker Yates)?

113
Q

What are the disadvantages of p-phenylenediamine dihydrochloride (Hanker Yates)?

114
Q

What are the advantages of p-phenylenediamine dihydrochloride (Hanker Yates)?

115
Q

What color is the end product of Naphthol AS MX phosphate?

116
Q

What are the advantages of Naphthol AS MX phosphate?

117
Q

What are the disadvantages of Naphthol AS MX phosphate?

118
Q

What color is the end product of Fast red?

119
Q

What are the advantages of Fast red?

120
Q

What are the disadvantages of Fast red?

121
Q

What color is the end product of Fast blue?

122
Q

What are the advantages of Fast blue?

123
Q

What are the disadvantages of Fast blue?

124
Q

What color is the end product of New fuchsin?

125
Q

What are the advantages of New fuchsin?

126
Q

What are the disadvantages of New fuchsin?

127
Q

What color is the end product of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)?

128
Q

What are the advantages of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)

129
Q

What are the disadvantages of 2-(p-isophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT)

130
Q

What color is the end product of Nitro blue tetrazolium (NBT)?

131
Q

What are the advantages of Nitro blue tetrazolium (NBT)?

132
Q

What are the disadvantages of Nitro blue tetrazolium (NBT)?

133
Q

What color is the end product of Tetranitro blue tetrazolium (TNBT)?

134
Q

What are the advantages of Tetranitro blue tetrazolium (TNBT)?

135
Q

What are the disadvantages of Tetranitro blue tetrazolium (TNBT)?

136
Q

List four chromagens compatible with horseradish peroxidase

137
Q

List three chromagens compatible with alkaline phosphatase

138
Q

List three chromagens compatible with glucose oxidase

139
Q

Identify which chromagens require special disposal

140
Q

What methods can be used to test the reactivity of a chromagen?

141
Q

Describe the types of specimens which may be used for immunofluorescene staining

142
Q

What are the advantages of IF techniques?

143
Q

What are the disadvantages of IF techniques?

144
Q

Explain the methods used for FFPE tissue used for IF staining

145
Q

Explain the methods used for frozen tissue sections for IF staining

146
Q

What are the advantages of using frozen sections versus FFPE tissues for IF staining?

147
Q

What are the disadvantages of using frozen sections versus FFPE tissues for IF staining?

148
Q

What are the fixative options for IF staining?

149
Q

What are the principles of fluorescent antibody techniques?

150
Q

Explain the principles of the double layer technique

151
Q

List the steps in the double layer technique

152
Q

Explain the principles of the sandwich technique

153
Q

List the steps in the sandwich technique

154
Q

Explain the principles of the complement incubation technique

155
Q

Lists the steps of the complement incubation technique

156
Q

What is co-localization?

157
Q

What is a fluorophore?

158
Q

What is a fluorochrome?

A

Fluorescent dyes

159
Q

What are the 3 fluorochomes commonly used in IF to label the anitbody?

A
  1. Fluorescein isothiocyanate (FITC)
  2. Tetramethylrhodamine isothiocyanate (TRITC)
    3.
160
Q

What color is Fluorescein isothiocyanate (FITC)?

161
Q

What color is Tetramethylrhodamine isothiocyanate (TRITC)

162
Q

What color is

163
Q

What is autofluorescence?

164
Q

List three sources of naturally occurring autofluorescence in tissue components

A
  1. Collagen
    2.
    3.
165
Q

What reduction method can be used for autofluorescence in collagen?

166
Q

What reduction method can be used for autofluorescence in ?

167
Q

What reduction method can be used for autofluorescence in ?

168
Q

List the most common source of chemically induced autofluorescence

169
Q

List the reduction methods for autofluorescence

170
Q

Define pre-bleaching

171
Q

Define photobleaching

172
Q

Define CISH

173
Q

Define FISH

174
Q

What are the two primary main probes in ISH?

175
Q

How do the probes differ in ISH?

176
Q

Lists the steps in an ISH procedure

177
Q

Which steps in the ISH procedure differ from an IHC procedure?

178
Q

Explain pretreatment options in ISH

179
Q

Explain denaturation options in ISH

180
Q

Explain hybridization options in ISH

181
Q

Explain stringency wash options in ISH

182
Q

Define high stringency wash in ISH

183
Q

Define low stringency wash in ISH

184
Q

What variables are usually modified to alter the stringency in ISH?

185
Q

What are the two aspects to blocking of background staining of tissues?

A
  1. Nonspecific antibody binding
  2. Presence of endogenous enzymes
186
Q

What are the methods of blocking nonspecific background staining?

A
  1. Quenching endogenous peroxidase by an H202 methanol solution
  2. Blocking endogenous biotin by using the avidin-biotin blocking agent, or skim milk
  3. Normal serum will act as a secondary Ab to block nonspecific binding sites
187
Q

Examples of chromagens

A

Peroxidase
AEC
DAB (diaminobenzidine)
Vector S-G
Vector - VIP
Vector Nova Red
Alk Phos
New Fuchsin
Fast Red
BCIP/NBT
Vector red
Vector black

QIHC maryjw79 @ gmail email 4 access (16 decks)

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