Histotechnique Flashcards
Preparation of tissue slices or Sections that can be examined visually With transmitted light
Histotechnology
Ideally, the tissue on the slide should have the same structural features as it had in the body
Histotechnology
Accessioning
Suitable container (2)
*Covered or closed
*Large enough to hold the specimen without distribution
Accessioning
Fixative
*Adequate amount
*1:20 ratio
Accessioning
Properly labeled (4)
- Patient’s name, age, sex
- Specimen details
- Type of source of specimen
- Date and time of collection
Accessioning
Histopath requisition form (6)
- Patient’s details
- Specimen details
- Clinical or differential diagnosis
- Brief clinical history
- Surgical Operation done
- Other patient info
Accessioning
Gross Examination (3)
1.Done by pathologist
2.Description of the specimen
3.Cutting small sections and placing it in plastic cassettes
Tissue processing
9 steps, 1 optional step
- Fixation
*Decalcification (Optional) - Dehydration
- Clearing
- Infiltration
- Embedding
- Sectioning
- Deparaffinization
- Staining
- Mounting
What Tissue processing step
o Tissue specimen is placed in solutions of chemicals that cross- link proteins and inactivate. degenerative enzymes
o Preserve tissue structure
o Prevent degeneration and autolysis
o Duration : (12-72 hours), 2-4 hours : very small specimens
FIXATION
In Fixation step
Duration : ________________, in very small specimens:_____________
12-72 hours, 2-4 hours
Fixatives of Fixation
Aldehydes (2)
l. buffered isotonic 37% formaldehyde (most used) (Formalin)
2. Glutaraldehyde : electron microscopy
Fixatives of Fixation
Alcohols (2)
methanol
ethanol
o Optional procedure after fixation
o Removal of calcium ions from a bone or calcified tissue
o Makes bone soft and easy to cut
o Duration: days to weeks
DECALCIFICATION
Decalcifying agents (3 types)
Strong mineral acids
Weak organic acids
Chelating agents
Decalcifying agents
Strong mineral acids (2)
- Nitric acid
- Hydrochloric acid
Decalcifying agents
Weak organic acids (2)
- Formic acid
- Thiochloroacetic acid
Decalcifying agents
Chelating agents (1)
EDTA
What Tissue processing step
o Water is extracted through gradually by transfers through a series of increasing ethanol
o Done in increasing concentrations to avoid Tissue distortion
o 70% > 90% > 100%
DEHYDRATION
What Tissue processing step
o Ethanol is replaced by an organic solvent miscible with both alcohol and the embedding medium
o Gives the tissue a translucent appearance
CLEARING
Clearing agents (2)
- Xylene
- Toluene
What Tissue processing step
o AKA Impregnation
o Tissue is placed in a melted paraffin in an oven
o Evaporates the clearing solvent
o Promotes infiltration of paraffin into the tissue cavities/spaces
INFILTRATION
Automated tissue Processing Machines (2 types)
- Tissue transfer machine/ rotary type
- Fluid transfer machine/vacuum type
What Tissue processing step
o Casting or blocking
o Specimen is placed in a mold then filled with melted paraffin wax
o Ensure proper orientation of the specimen
o Mold is cooled to promote solidification
EMBEDDING
What Tissue processing step
o Block is placed in microtone for cutting
o 3-10 um thick for light microscopy
o <1 um thick for electron microscopy
SECTIONING
What Tissue processing step
o Removal of paraffin from the slide
o Allows the tissue to successfully interact with the stain
o Incomplete removal of paraffin may lead to poor staining
DEPARAFFINIZATION
What Tissue processing step
o Used to highlight important features of the tissue as to enhance the tissue contrast
o Anionic( negative charge): basophilic
o Cationic (positive charge): acidophilic
o Hematoxylin and Eosin (H&E): most commonly used tissue stain
o Periodic acid-Schiff (PAS) reaction
o Sudan black
o Metal Impregnation
STAINING
In STAINING, the most commonly used tissue stain is
Hematoxylin and Eosin (H&E)
In Staining
Anionic is ____________ charged: _________________
negative, basophilic
In Staining
Cationic is ____________ charged: _________________
positive, acidophilic
What Tissue processing step
o Placing of a protective glass coverslip on the slide with clear adhesive
MOUNTING
