HISTOPATH TISSUE PREPARATION Flashcards
is a process whereby a
selected tissue specimen is immersed in a watch
glass containing isotonic salt solution, carefully
dissected or separated, and examined under the
microscope, either unstained by phase contrast or
bright field microscopy, or stained with differential dyes
teasing or dissociation
Small pieces of tissue not more than one mm. in
diameter are placed in a microscopic slide and
forcibly compressed with another slide or with a
cover glass
squash preparation (crushing)
Process of examining sections or sediments, whereby cellular materials are spread lightly over a slide by means of a wireloop or applicator or by making an apposition smear with another slide.
smear preparation
w/ an applicator stick or a platinum loop,
the material is rapidly and gently applied in a direct or zigzag line throughout the slide
streaking
kind of smear that should be avoided
too thin and too thick smear
a selected portion of the material is
transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous strands apart with an applicator stick.
spreading
For fresh sputum ____
bronchial aspirates and thick
mucoid secretions
done by placing a drop of secretions
or sediment upon one slide and facing it to another clean slide
pull-apart
pull-apart is used for
Serous fluid, conc. Sputum, enzymatic lavage
samples from GIT and blood smears
the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination by phase contrast microscopy or stained for light microscopic study
touch preparation (impression smear)
utilized when a rapid diagnosis of
the tissue is required
frozen section
frozen section is used for
For lipid, carbohydrates and nervous tissue elements
For rapid pathologic diagnosis during surgery
For diagnostic and research enzyme histochemistry
For silver stains (neuropathology) immunofluorescent and immunohistochemical staining
Commonly Used Methods of
Freezing
- Liquid nitrogen
- Isopentane cooled by liquid nitrogen
- Carbon dioxide gas
- Aerosol sprays
- adequate for freezing small
pieces of tissue except muscle
aerosol sprays
used for histochemistry and during operative procedures
Liquid nitrogen
disadvantage of liquid nitrogen
soft tissue is liable to crack due to the
rapid expansion of the ice w/in the tissue, producing ice crystals or freeze artifacts.
Overcools urgent biopsy blocks, causing damage to both..
block and blade if sectioning is done at -70 C or
below
Non-fatty unfixed tissues are sectioned well at
temperatures between
-10 C and -25 C
TISSUE PROCESSING
- Numbering
- Fixation
- Dehydration
- Clearing
- Infiltration
- Embedding
- Trimming
- Section-Cutting
- Staining
- Mounting
- Labeling
Identify properly all the specimens received without the need of writing the patient’s name to the accompanying specimen tag
Numbering
where is the details written in numbering
Entering the details of the specimen in alog book
how to do numbering
In numbering, the specimen number is preceded by either S (surgical), A (autopsy) or C (cytology). The year is also indicated. Example: S98-7677
After numbering the pathologist will describe the
_____ of the specimen. The MT will
write down the ____ at the back of the
request
gross description and description
Specimen size for processing:
3x2x0.5cm (length) and 3-5mmthick
the function of fixation
Preserve the tissue- stop all cellular activities
Prevent breakdown of cellular elements (prevents postmortem decomposition (autolysis) and prevents putrefaction)
Coagulate or precipitate protoplasmic substances
for conventional bright field light microscopy, we follow the following steps:
0.5 x 0.5 cm tissue is added to a small jar containing the fixative
adding tissue to a small jar containing fixative is to:
prevent autolysis
terminate cell metabolism
kill bacteria
harden tissue
fixative used is usually
Formalin 10% solution
tissues are fixed:
to preserve cells and tissues constituents
to prevent their degredation
with fixative’s volume 20x greater
Bouin’s Fixative / Formalin for 24 hrs
TWO basic mechanisms in fixation
additive fixation
Non- additive fixation
chemical constituent of the fixative is
taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein
additive fixation
example of additive fixatives
Formalin, mercury and osmium tetroxide
fixation-fixing agent is not incorporated
into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H bonds of certain groups w/in the CHON molecule.
non-additive fixation
non-additive fixatives example
alcohol fixatives
FACTORS INVOLVED IN FIXATION
pH
Temperature
Thickness of section
Osmolality
Concentration
Duration of Fixation
the pH in fixation
6-8
temperature in fixation
Surgical spe - room temp.
tissue processors- 40 deg celsius
electron microscopy & histochem - 0-4 deg celsius
Formalin at 60 deg celsius is for
rapid fixation of urgent biopsy
Formalin 100 deg celsius is for
TB
Thickness of section for EM
1-2 mm^2
Thickness of section for LM
2cm^2 or not more than 0.4 cm
Thickness of section for Brain
10% buffered formalin for 2-3 weeks
osmolality for slightly hypertonic solution
around 400-450mOsm
osmolality of isotonic solutions
340 mOsm
concentration in fixation
Formaldehyde- 10%
Glutaraldehyde- 3%
Glutaraldehyde - 0.25% immunoelectron microscopy
duration of fixation for buffered formalin
2-6 hours
duration of fixation for EM
fixation 3 hrs and then placed in a buffer
PRACTICAL CONSIDERATION OF FIXATIVES
SPEED
PENETRATION
VOLUME
DURATION
formalin penetration
1mm/hr
volume of fixatives
10-25 times that of the tissue
the maximum effectiveness for volume of fixatives
20x the tissue volume
duration for fixatives
uterus & intestinal tract take longer time than small or loosely textured such as biopsies and scrapings
Characteristics of good fixatives
Cheap
Stable
Safe to handle
Kill cell quickly with minimal distortion
Inhibit bacterial decomposition and autolysis
Minimum shrinkage
Penetrate rapidly and permit application of stains
Isotonic, insoluble to hypotonic
FIXATIVE TYPES
Simple
Compound
what are the simple fixatives
Aldehydes
Metallic Fixatives
Lead Fixatives
what are the Aldehyde Fixatives
Formaldehyde, Glutaraldehyde
what are the Metallic Fixatives
Mercuric Chloride, Chromate (potassium dichromate, chromic acid)
what are the Lead Fixatives
Picric, Acetic, Acetone, Alcohol, Osmic Acid
2 or more fixatives
Compound Fixative
Fixative according to action
Microanatomical Fixatives and Cytologic Fixatives
permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.
Microanatomical Fixatives
types of Microanatomical Fixatives
- 10% formal saline
- 10% neutral buffered formalin
- Heidenhain ‘s Susa
- Formal sublimate (formal corrosive)
- Zenker ‘s solution
- Zenker-formal (Kelly ‘s solution)
- Bouin’s solution
- Brasil’s solution
Are those (fixatives) that preserve specific parts and particular microscopic elements of the cell itself
Cytologic Fixatives
the types of cytologic fixatives
- Nuclear
- Cytoplasmic
- Histochemical
Preserves nuclear structures
Nuclear Fixatives
Fluids in Nuclear Fixatives
Flemming’s Fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenheins Susa
are those that preserve cytoplasmic structures in particular
Cytoplasmic Fixatives
Fluids in Cytoplasmic Fixatives
Flemming’s fluid without acetic acid
Kelly’s fluid
Formalin with “post-chroming”
Regaud ‘s fluid (Muller ‘s fluid)
Orth ‘s fluid
are those that preserve the chemical constituents of cells and tissues.
Histochemical Fixatives
fluids in Histochemical Fixatives
Formal Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid
Bouin’s Fluid composition
Picric Acid - 75 mL
Formalin - 25mL
Glacial Acetic Acid - 5mL
- it has rapid and even penetration
- fixed tissue gives brilliant staining with trichome methods
- used to demonstrate glycogen
- good for GIT biopsies
Bouin’s Fluid
Involves thermal coagulation of tissue proteins for rapid diagnosis
Heat Fixation
Heat Fixation is used for..
For frozen tissue sections
For bacteriological smears
For nuclear and cytoplasmic detail
Heat Fixation destroys
RBC
Heat Fixation dissolves
starch and glycogen
Is the process of placing an already fixed tissue in a second fixative
secondary fixation
secondary fixation Is the process of placing an already fixed tissue in a second fixative in order to:
Facilitate & improve demonstration of particular subs.
Make special staining techniques possible
Ensure further and complete hardening and preservation of tissues
Done before dehydration and on deparaffinized sections before staining (10% formalin/10% formol saline as primary fixative)
Is a Form of secondary fixation whereby a primarily fixed is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and in cytologic preservation of tissues
POST-CHROMATIZATION
Removing excess fixative from the tissue after fixation
Washing Out
remove excess chromates, formalin, osmic acid
Tap Water
wash excess amount of picric acid
50 to 70% Alcohol
remove excess mercuric fixatives
Alcoholic Iodine
Factors Affecting Fixation
Size and thickness
Presence of mucus
Presence of fat
Presence of blood-
Cold Temp
larger tissue requires more fixatives and longer fixation time
Size and thickness
maybe washed with saline solution
presence of mucus
cut in thin section and fixed longer
presence of fat
flushed out with saline
presence of blood
inactivates enzymes
cold temp
FIXATION IS ENHANCED BY
Size and thickness of tissues
agitation
Moderate heat (37-56 C) accelerates fixation but hastens autolytic changes and enzyme destruction
Principles and precautions in handling and fixation of Specimen
Fix Autopsy ASAP
Fix surgical specimen ASAP
Proper labeling and Identification
Tissues size not more than 5mm thick except lung edema (1-2cm thick)
Volume 20x except for osmium tetroxide 5-10x
For prolong fixation vol not less than 50-100x
Hollow organs (stomach) shld be completely opened before fixing
Lungs may be covered with several layers of gauze
Human brain (2 weeks) may be suspended by acord tied under circle of willis to prevent flattening
Water should not be used for glycogen-containing tissue
Works as a physical agent similar in mechanism to vacuum, oven and agitation to increase the movement of molecules and accelerate fixation.
MICROWAVE TECHNIQUE
MICROWAVE TECHNIQUE used to..
Used to accelerate staining, decalcification, immunohistochemistry and electron microscopy
the tissue in microwave technique..
Tissue is heated right thru the block in a very short time.
For antibodies demonstration
Formalin-fixed and paraffin embedded sections may be used
Prepared as Cryostat section and fixation limited to a few seconds in absolute methanol or acetone.
IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES
preserve the maximum enzyme activity at its localization
ENZYME HISTOCHEMISTRY
Fixed in 4% formaldehyde or formol saline
ENZYME HISTOCHEMISTRY
Fresh frozen cryostat sections may be fixed in..
acetone or formaldehyde and washed in distilled water prior to enzyme staining
primary fiaxatives in EM
Osmium tetroxide, glutaraldehyde and paraformaldehyde
Osmium tetroxide, glutaraldehyde and paraformaldehyde is performed at
4C
For electron histochemistry and electron immunocytochemistry ____ is useful
karnovsky’s paraformaldehyde-glutaraldehyde is useful
