Special Histopath Technique Flashcards
1
Q
Histopath Technique for
Routine
Special
A
Routine
-Manual/Automated Processing
Special
Frozen Section
Ag-Ab based
-Immunohistochemistry
-Immunofluorescence
Molecular Technique
-FISH
2
Q
A type of tissue process in which tissue is rapidly freeze and sectioned in a cryostat machine and then stains for microscopic examination. The process takes 10-15 minutes.
A
Frozen section
3
Q
For rapid diagnosis during intraoperative surgery. Provides a real-time information.
A
Frozen section
4
Q
Preparation for immunohistochemistry (IHC) and immunofluorescence.
A
Frozen section
5
Q
Quick-freeze makes the tissue hard enough to section with microtome.
A
Frozen section
6
Q
Frozen Section process takes?
A
10-15 mins
7
Q
a chamber which houses a microtome. Inside temperature ranges from -18 to -70˚C.
A
Cryostat
8
Q
Temperature of Cryostat
A
-18 to -70C
9
Q
Methods of freezing
A
Liquid Nitrogen
Isopentane
Carbon Dioxide gas
Aerosol sprays
10
Q
Most common method of freezing
A
Liquid Nitrogen
11
Q
Liquid Nitrogen Temp
A
-190C
12
Q
Isopentane Temp
A
-150C
13
Q
Carbon Dioxide Gas Temp
A
-70C
14
Q
Aerosol spray temp
A
-50C
15
Q
Method of freezing that is used in cold knife procedure
A
Carbon Dioxide Gas
16
Q
Method of freezing for quick freezing spray
A
Aerosol Sprays
17
Q
acts as a embedding medium. in cryostat
A
Water
18
Q
Cold knife procedure, commonly used as freezing agent
A
CO2 gas
19
Q
Cold knife procedure temperature of
Knife
Tissue
Environment
A
-40 to -60C
-5 to -10C
-10C
20
Q
Different temperature is employed between tissues and the knife.
A
Cold knife procedure
21
Q
Refrigerated apparatus used fresh tissue microtomy.
A
Cryostat procedure
22
Q
Cryostat cold chamber is maintained at temperature of
A
-15 to -30 C.
23
Q
Optimum working temperature for cryostat procedure
A
-18 to -20
24
Q
Size of the tissue section in frozen section
A
5-10 micra
25
Stain Used in Frozen Section
Hematoxylin and Eosin Stain
26
It is also requested if lipids, fats, or some nervous system elements are to be demonstrated.
Frozen section
27
provides initial diagnosis thereby should not be a replacement for paraffin-embedded section as it has also disadvantages.
Frozen section
28
Special way of preserving tissues by rapid freezing of fresh tissue
Quenching -160C to -180C
29
Ice water molecules are the removed through desiccation or transferring the frozen tissue block in vacuum a higher temperature for 24-48 hours
Sublimation -30 to -40C
30
Special way of preserving tissues by rapid freezing of fresh tissue (Quenching -160C to -180C).
Ice water molecules are the removed through desiccation or transferring the frozen tissue block in vacuum a higher temperature for 24-48 hours (Sublimation -30 to -40C).
Freeze Drying
31
Quenching Temp
-160 to -180
32
Sublimation Temp
-30 to -40
33
Machine used in Freeze Drying
Laboratory Bench-Top Vacuum Freeze Dryer
34
Similar with freeze drying but with addition of dehydration step.
Freeze substitution
35
Frozen tissue is dehydrated in expensive vacuum drying apparatus fixed in _____ or in _______ dehydrated in ______
Rossman’s formula
1% acetone
absolute alcohol.
36
Machine used in Freeze Substitution
Leica Automated Freeze Substitution System
37
It is a microscopic-based technique used to demonstrate specific cellular components particularly the protein ( epitope or antigen) in tissue samples.
Immunohistochemistry
38
Immunohistochemistry technique is based on
antigen and antibody principle for detection
39
An enzyme labeled antibody (IgG) binds to antigenic biomarkers on the surface of target tissue then a change in color is observed under light microscope.
Immunohistochemistry
40
Specimen for Immunohistochemistry
Formalin fixed paraffin embeddedd
Frozen section
41
Components of Immunohistochemistry
Primary antibody
Secondary antibody
Chromogen
42
Antibody-IgG type, binds specifically to the target antigen.
Primary antibody
43
binds to only one epitope of the antigen (specific).
Monoclonal
44
binds to multiple epitope of an antigen (sensitive).
Polyclonal
45
binds to the primary antibody, conjugated with enzymes or fluorescence.
Secondary antibody
46
a substrate when added produces insoluble colored precipitate.
Chromogen
47
exposing the epitope of Ag to allow the binding of antibody. Done in FFPE tissues prior to Ab labelling.
Antigen Retrieval
48
breaks down formalin cross-linking to allow better exposure of antigenic sites. Commonly used enzyme are Trypsin and Protease.
Proteolytic Enzyme Digestion-
49
tissue section is subjected to microwave oven heating to retrieve antigen lost during tissue processing. Boiling usually for 20 minutes.
Microwave Antigen Retrieval-
50
the same with microwave technique with less time required and better retrieval of antigen. Autoclave and water bath can also be used.
Pressure cooker Antigen Retrieval-
51
Commonly used enzyme for Proteolytic Enzyme Digestion
Trypsin
Protease
52
Immunohistochemistry work flow (Simple)
Ag retrieval
Addition of Primary Ab
Application of Secondary Ab
Addition of Chromogen
53
Immunohistochemistry work flow (Complex)
FFPE tissue
Deparaffinization
Rehydration
Antigen Retrieval
Blocking
Primary Ab addition
Secondary Ab addition
Counterstain
Dehydration
Coverslip
54
Immunohistochemistry techniques
Direct technique
Indirect technique
Peroxidase anti peroxidase (PAP) technique
Avidin-Biotin complex (ABC) technique
Labeled Streptavidin Avidin Biotin (LSAB) technique
55
One step method
Uses primary labelled antibody directly attach to the target antigen on the surface of the tissue.
Direct technique (traditional)
56
Label used for Direct Technique (traditional)
Fluorochrome or Horseradish Peroxidase (HRP)
57
More sensitive than direct technique
It uses
1. primary unlabeled antibody
2. secondary labeled antibody directed to primary antibody.
Indirect Technique
58
commonly used enzymes in Indirect Technique
Horseradish peroxidase
59
Peroxidase Anti Peroxidase (PAP) technique components
Antigen molecule
Primary Ab
Secondary Ab
Peroxidase anti peroxidase complex
60
A type of indirect method which uses
1.1. Peroxidase anti-peroxidase complex (Ab against HRP)
2. Secondary antibody
3. Primary unlabeled antibody (from rabbit)
Peroxidase anti peroxidase (PAP) technique
61
bridge in PAP complex by secondary antibody.
primary unlabeled antibody
62
This reaction visualized by the use of hydrogen peroxide as an enzyme substrate, and a chromogen Diaminobenzidine (DAB) resulting to insoluble dark brown reaction.
Peroxidase anti peroxidase (PAP) technique
63
100-1000 X sensitive than indirect method since it uses more enzyme in the which give a higher degree of reaction.
Peroxidase anti peroxidase (PAP) technique
64
Uses avidin (from egg white) that has affinity to biotin a low molecular weight vitamin that easily conjugate to Ab and enzymes.
Avidin-Biotin complex (ABC) technique
65
Uses
1. Primary antibody
2. Biotinylated secondary antibody
3. Avidin-biotin enzyme complex or labeled streptavidin
Avidin-Biotin complex (ABC) technique
66
When the chromogenic enzyme substrate is applied, it yields a colored precipitate at the site of the reaction.
Avidin-Biotin complex (ABC) technique
67
Avidin-Biotin complex (ABC) technique composition
Antigen
Primary antibody
Biotinylated secondary antibody
Biotin
Avidin
Streptavidin
68
Uses
1.Primary antibody
2. Boitinylated secondary antibody
3.Streptavidin-enzyme conjugate
Labeled Streptavidin Avidin Biotin (LSAB) technique
69
gives a better amplification and detection than ABC technique.
Labeled Streptavidin Avidin Biotin (LSAB) technique
70
Immunohistochemistry immunostains
High molecular weight Cytokeratin (HMWCK)-
CD30
CD31
CD56
CK20
71
Immunohistochemistry immunostains that detects epithelial neoplasm.
High molecular weight Cytokeratin (HMWCK)-
72
Immunohistochemistry immunostains that is used to diagnose Hodgkin lymphoma.
CD30
73
Immunohistochemistry immunostains that is use to identify endothelial tumor.
CD31
74
Immunohistochemistry immunostains for the diagnosis of leukemia and small cell carcinoma.
CD56
75
Immunohistochemistry immunostains for merkel carcinoma and gastrointestinal carcinoma.
CK20
76
Based also on antigen-antibody principle for detection.
Immunofluorescence
77
Fluorochrome is used instead for enzyme.
Immunofluorescence
78
Immunofluorescence Color for
1. Fluorescein
2. Rhodamine
Apple-Green
Rhodamine
79
Reaction is observed as a fluorescence under fluorescence microscope.
Immunofluorescence
80
Provides better image quality
Frozen section or culture cells can be used as a specimen.
Immunofluorescence
81
A fluorochrome used to demonstrate mucin
Stain last only for 2 hours
Fluorescent Acridine Orange Technique
82
Fluorescent Acridine Orange Technique stain result for
1. Acid mucopolysaccharides
2. Fungi
3. Background
Black
Greenish Red
Reddish Orange
83
Used for demonstration of certain type of carcinoma.
Cytokeratin (CK) 8
84
Flourescent staining for DNA and RNA color result for
1. Rhodamine
2. Acridine Orange (DNA);(RNA)
Orange Red Emmision
DNA-Yellow-green
RNA- Orange Red
85
Uses fluorescent-labelled probe (short single stranded nucleic acid) which is complimentary to the target DNA or mRNA in a tissue section
Fluorescence In-situ hybridization (FISH)
86
Fluorescence In-situ hybridization (FISH) uses?
fluorescent-labelled probe
87
It is based on the principle of hybridization- complementary binding between a probe and target nucleic acids.
Fluorescence In-situ hybridization (FISH)
88
It targets the nucleic acids instead to the antigen express on the surface of the cell.
Fluorescence In-situ hybridization (FISH)
89
Frozen section, paraffin section and smears can be used.
Detection is through radioactivity or fluorescence microscope.
Fluorescence In-situ hybridization (FISH)
90
Fluorescence In-situ hybridization (FISH) uses what type of section?
Frozen section
Paraffin Section
Smears
