FINALS: HISTOPATHOLOGY Flashcards
(44 cards)
IMMUNOHISTOCHEMISTRY
- X ___, special procedure in ___
- involves ___
- routine
- disease diagnosis
- antigen-antibody reactions
PURPOSE OF IHC:
- Most common form of Ab used in IHC
- For identification of tissue / cellular antigens / phenotypic markers
- Detection of organisms (microbes: cause diseases) in cytologic procedures (sputum, body fluids, FNA)
ANTIGENS (found in ___)
- cells
- to be detected
ANTIBODIES (class of ___, ___)
Most common Ig used?
- serum proteins
- immunoglobulins
- IgG
TYPES OF ANTIBODIES USED IN IHC
A. ___
- produced by ___
- react w/ ___
- from ___
Main Source:
Other Source:
- POLYCLONAL
- different cells
- various isotopes
- laboratory animal sources
- RABBITS - immunized to produce Abs
- GOATS, PIGS, SHEEP
TYPES OF ANTIBODIES USED IN IHC
B. ___
- more ___
- produced from ___
- reacts only w/ ___
Main Animal Source:
B. MONOCLONAL
- specific
- individual clone of plasma cell
- 1 specific type of isotope
- MICE
LABELLING OF ANTIBODIES
1. USE OF ___
Most widely used?
Alternative?
- ENZYMES
- HRP (HORSE RADISH PEROXIDASE)
- AP (ALKALINE PHOSPHATASE)
Required in use of enzymes to see ___?
Required by chromogens; ___?
- CHROMOGENS -> color developers
- positive result
- SUBSTRATE
- consumed by enzyme
Ab + ___ to react w/ antigen
E + C + S
Enzyme: HRP (HORSE RADISH PEROXIDASE)
Substrate: ___
Chromogen:
1. ___
Color:
Suitable Counterstains:
- ___
Color:
Suitable Counterstains:
Enzyme: HRP (HORSE RADISH PEROXIDASE)
Substrate: HYDROGEN PEROXIDASE (H2O2)
Chromogen:
1. DAB (3,3’ DAB diaminobenzidine)
Color: Brown
Suitable Counterstains: Hematoxylin, Methyl green, Methyl blue
- AEC (3-amino-9-ethylcarbazole)
Color: Brick red
Suitable Counterstains: Hematoxylin, Methyl blue
Enzyme: AP (ALKALINE PHOSPHATASE)
Substrate: ___
Chromogen:
1. ___
Color:
Suitable Counterstains:
- ___
Color:
Suitable Counterstains:
Enzyme: AP (ALKALINE PHOSPHATASE)
Substrate: Phosphate
Chromogen:
1. BCIP (5-Bromo-4-Chloro-3-Indolyl Phosphate) / NBT (Nitro Blue Tetrazolium)
Color: Bluish violet / Black violet
Suitable Counterstains: Nuclear fast red, Brilliant green
- FAST TR RED SALT
Color: Rose color
Suitable Counterstains: NONE
Used to observe end products?
Fluorochrome dye used?
- LIGHT MICROSCOPE
- FLUORESCENCE MICROSCOPE
HEMATOXYLIN AS COUNTERSTAIN
- ___; ___
MAYER’S OR HARRIS HEMATOXYLIN - ___
MAYER’S - ___
HARRIS - ___; ___
- traditional counterstain; nuclear stain
- Immunohistochemistry
- Reddish violet
- Red; commonly used bc tissue morphology are well-defined
INCUBATION - ___
Incubation time & temperature to link Ab w/ ___
- required to link Ag to Ab
- 30-60 MINS AT ROOM TEMPERATURE
- peroxidase
LABELLING OF ANTIBODIES:
2. USE OF ___
- requires ___
Most common:
- FLUROCHROME LABEL
- FLUORESCENCE MICROSCOPE
- FITC (FLUORESCENCE ISOTHIOCYANATE)
LABELLING OF ANTIBODIES:
3. USE OF ___
GOLD - ___ end-product (under ____
- COLLOIDAL METALS
- pink
- light microscope
LABELLING OF ANTIBODIES:
4. ___
To detect ___ (traditionally use ___)
- Can bind w/ ___; can also be ___
Source:
- RADIO LABEL
- LECTINS
- antigen
- antibody
- tissue carbohydrates
- labeled
- PLANT / ANIMALS
SPECIMENS FOR IHC:
1. ___
- for ___
- done ___ (while ___)
- tissue prepared as ___ & must be fixed for a few sections using ___ / ___
Preparation: Cut tissue using ___ / ___
- FROZEN SECTION
- rapid diagnosis
- intraoperatively
- patient is in OR
- cryostat section
- METHANOL / ACETONE
- freezer microtome / CRYOSTAT
PURPOSE OF FIXATION:
- To preserve the position of antigens being detected
- To prevent destruction of labile antigenic sites
SPECIMENS FOR IHC:
2. ___ LIKE ___ & ___
Preferred fixative for IHC?
Required for processed specimens?
Purpose:
- PROCESSED SPECIMENS
- Formalin fixed, Paraffin embedded
- FORMALDEHYDE
- ANTIGEN RETRIEVAL
- Increase accessibility of Ag to Ab
2 GENERAL METHODS OF ANTIGEN RETRIEVAL:
1. ___
- ___ / ___
MOST COMMON ENZYMES USED:
Both can break ___
Forms crosslinks?
- HIER (HEAT INDUCED EPITOPE RETRIEVAL)
- PIER (PROTEASED INDUCED EPITOPE RETRIEVAL) / PROTEOLYTIC ENZYME RETRIEVAL
- TRYPSIN, PROTEASE
- formalin crosslinks
- 10% FORMALIN AS FIXATIVE
PRE-TREATMENT
BLOCKING - only for ___, X ___
1. ___
- ___
- uses ___
- ___
- all ___ & reduces ___
- uses ___
- processed specimen
- frozen section
- PEROXIDASE BLOCK
- endogenous peroxidases
- 3% H2O2 (HYDROGEN PEROXIDE)
- PROTEIN BLOCK
- non-specific sites
- background
- 10% NORMAL SERUM
PURPOSE OF USE OF CONTROL AS REQUIREMENT IN IHC:
- To test for specificity of Abs involved
- To prevent misinterpretation due to false + & false - results
POSITIVE CONTROL - tissue section having the ___
NEGATIVE CONTROL - omit ___ from ___ / replace ___ by an __ directed against an ___
INTERNAL TISSUE CONTROL / BUILT IN CONTROL -contains ___ not only in the ___ but also in ___
- Ag being detected
- primary Ab
- staining sched
- primary Ab
- Ig
- unrelated Ig
- target Ag
- tissue elements
- adjacent normal tissue elements
