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The Gross Room/

Surgical Cut-up
• Tissues from the body taken for diagnosis of disease
processes must be processed in the histopathology
laboratory to produce microscopic slides that are
viewed under the microscope by pathologists

• The persons who do the tissue processing and make


the glass microscopic slides are histotechnologists
Specimen Reception
• To receive samples securely and safely
• Specimen labeled properly attached to body
of container
• Request form
• Correlation of specimen against the clinical
request form is mandatory
Specimen Accessioning
• Tissue specimens received in the surgical
pathology laboratory have a request form that
lists the patient information and history along
with a description of the site of origin
• Usual method of specimen identification is
simply the year with a sequential numbering
system.
• In cases of multiple specimens from a single
patient, a single laboratory number is suffice.
Gross Examination
• Good lighting
• Good ventilation
• Non-absorbent wipe
clean surface
• Appropriate protective
clothing
• Comfortable
environment for
pathologist and support
staff
Gross Examination
• Tissues are examined by a pathologist,
pathology assistant, or pathology resident

• Gross examination consists of :


– describing the specimen – size, shape, number,
color , consistency
– placing all or parts of it into a small plastic cassette
which holds the tissue while it is being processed to
a paraffin block

– Initially, the cassettes are placed into a fixative


Gross Examination
• Labeling of tissues with unique identification
number of code assigned to the tissue sample
accessioned in the lab

• Tissue inside the cassette should be dissected to


3-4mm in thickness

• Care must be taken not to overfill the cassette


impeding the flow of reagents around the tissue
Gross ONLY examination
• Non tissue – bullets, implants, foreign body
• Medico legal chain of evidence

• Tissues unlikely to require a detailed diagnosis


and may be examined grossly
– Tonsils, traumatic injuries, teeth
– Incidental resections such as ribs, fat and vessels
INKING the Margins
• When a malignancy is
suspected, then the
specimen is covered with
ink --- to mark the margins
of the specimen

• Different colored inks to


identify different areas if
needed

• When sections are made


and processed, the ink will
mark the actual margin on
the slide
Tissue Processing
• After the removal of a tissue sample from the
patient, a series of process must take place to
ensure the final microscopic slides are of
diagnostic quality

• Producing quality slides for diagnosis is NOT


an accident; it requires skills that are
developed through continued practice and
experience
Tissue Processing
• Permanently preserved
• Stained for demonstration of specific
structures
• Mounted on glass slides with coverslips for
permanent keeping
Tissue Processing
• is designed to remove all extractable water
from the tissue, replacing it with a support
medium that provides sufficient rigidity to
enable sectioning of the tissue without
damage or distortion
Tissue Processing
1. Fixation
2. Dehydration
3. Clearing
4. Infiltration (Impregnation)
5. Embedding
6. Trimming
7. Section-cutting
8. Staining
9. Mounting
10. Labeling
• FIXATION is the most important step in the
processing of tissue sample

• The size & type of specimen n the tissue


cassette determines the need for complete
fixation and processing
– 3-4mm thickness
– Do not overfill the cassette
Factors Influencing the Rate of
Processing
• Agitation
– The rate of exchange is dependent upon the
exposed surface of the tissue that is in contact
with the processing reagent
– Automated processors – vertical or rotary
oscillation or pressurized removal and
replacement of fluids at timed intervals
– Efficient agitation can reduce the overall
proccessing time by 30%
Factors Influencing the Rate of
Processing
• Heat
– Increases the rate of penetration & fluid exchange
– It must be used sparingly to reduce the possibility
of shrinkage, hardening and brittleness of the
tissue
– Temperatures limited to 45˚C can be used
effectively ; higher temp may be deleterious to
subsequent immunohistochemical staining
Factors Influencing the Rate of
Processing
• Viscosity
– Is the property of resistance to the flow of a fluid
– The smaller the size of the molecules in the
solution, the faster the rate of fluid penetration
(low viscosity)
– If the molecular size is larger, the rate of exchange
is slower (high viscosity)
Factors Influencing the Rate of
Processing
• Vacuum
– Using reduced pressure to increase the rate of
infiltration
– If used on the automated processor should not
exceed 15 inches of Hg (mercury) to prevent
damage and deterioration to the tissue
Fixation
• To preserve the tissue
• To prevent breakdown of cellular elements
• To coagulate or precipitate protoplasmic
substances that may leak out during subsequent
steps of tissue processing

• carried out as soon as possible after removal of


the tissues (in the case of surgical pathology) or
soon after death (with autopsy) to prevent
autolysis
Two Basic Mechanisms Involved in
Fixation
• Additive fixation
– The 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 (Ex. Formalin, mercury and osmium
tetroxide)
Two Basic Mechanisms Involved in
Fixation
• Non-additive fixation
– The 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 within the
protein molecule (Ex. alcohol fixatives)
Main Factors Involved in Fixation
• Hydrogen ion concentration – pH 6 to 8

• Temperature – traditionally room temp ;


tissue processors 40˚C ; Electron microscopy &
histochemistry 0-4˚C

• Thickness of section - 1 to 2mm2 for EM and 2


cm2 for light microscopy
– Except in lung edema – 1 to 2 cm thick
Main Factors Involved in Fixation
• Osmolality – best results usually in hypertonic
solutions 400 to 450mOsm

• Concentration
– 10 % formaldehyde
– 3% glutaraldehyde

• Duration of fixation – 2 to 6 hours ; if


prolonged causes shrinkage & hardening
• Specimen should be placed in fixative as soon
as it is removed from the body
• Rate of penetration approximately 1mm per
hour & slows down as it goes deeper
• Volume is 10-25x the volume of tissue to be
fixed
– EXCEPT osmium tetroxide – only 5-10x
• Duration depends on the structure of tissue
Effects of Fixatives
• Harden soft & friable tissues and make
handling and cutting sections easier
• Make cells resistant to damage & distortion
• Inhibit bacterial decomposition
• Increase optical differentiation
• Acts as mordants or accentuators to hasten
staining or inhibit certain dyes
• Reduce risk of infection
A good fixative is …
• Cheap, stable, safe to handle
• Minimum distortion & shrinkage
• Inhibit bacterial decomposition & autolysis
• Minimum shrinkage of tissue
• Rapid and even penetration of tissues
• Harden tissues for cutting of sections
• Isotonic
• Permit staining
Types of Fixative
• According to COMPOSITION
– Simple
• aldehyde (formaldehyde, glutaraldehyde)
• metallic fixative (mercuric chloride, chromate
fixatives, potassium dichromate, chromic acid, lead
fixatives, picric acid, acetic acid, acetone, alcohol,
osmium tetroxide, heat
– Compound
Types of Fixative
• According to ACTION
– Microanatomical
• 10% Formol saline, 10% Neutral buffered formalin,
Heidenhan’s Susa, Formol sublimate, Zenker’s
solution, Kelly’s solution, Bouin’s solution, Brasil’s
solution
Types of Fixative
• According to ACTION
– Cytological
• Nuclear: Flemming’s fluid, Carnoy’s fluid, Bouin’s
fluid, Newcomer’s fluid, Heidenhain’s Susa
• Cytoplasmic: Flemming’s fluid without acetic acid,
Kelly’s fluid, Formalin with “post-chroming”,
Regaud’s fluid (Muller’s fluid), Orth’s fluid
• Histochemical: 10%Formol saline, acetone,
absolute ethyl alcohol, Newcomer’s fluid
• LIPID fixation
– cryostat or frozen section followed by
general lipid stains.
– Mercuric chloride & potassium dichromate
• Carbohydrate fixation – alcohol fixative
• Protein fixation – neutral buffered formol
saline or formaldehyde
• Glycogen fixation – alcohol based, such as
Rossman’s fluid or cold absolute alcohol
Types of Fixative
• According to METHOD
– PHYSICAL
• Heat fixation
• Microwave fixation
• Freeze drying and freeze substitution
– Chemical
ALDEHYDE FIXATIVES
• 10% Formaldehyde
– usual fixation time 24 hrs
– one of the most widely used – compatible with
many stains, penetrates tissues well
– Disadvantages :
• irritating fumes and upon contact to skin
• Forms pigment granules - precipitates may be removed
by filtration or addition of 10% methanol
• Unsuitable for electron microscopy
ALDEHYDE FIXATIVES
• 10% Formol Saline
– saturated formaldehyde diluted to 10% with NaCl
– Recommended for fixation of CNS & general post
mortem tissues for histochemical examination
– Slow fixative - > 24 hrs or more
ALDEHYDE FIXATIVES
• 10% neutral buffered formalin or phosphate
buffered formalin
– Recommended for preservation and storage of
surgical, post-mortem & research specimen
– Prevents precipitation of pigments
– Best fixative for tissues containing iron pigments
and elastic fibers
ALDEHYDE FIXATIVES
• Formol – Corrosive (Formol-Sublimate)
– recommended for routine post mortem tissues
– Excellent for many staining procedures including
silver reticulum methods
ALDEHYDE FIXATIVES
• Alcoholic Formalin (Gendre’s fixative)
– Can be used for rapid diagnosis, fixes &
dehydrates at the same time
– Good for preservation of glycogen & for micro-
incineration technique
– Fix sputum
ALDEHYDE FIXATIVES
• Glutaraldehyde
– Buffered glutaraldehyde , followed by secondary
fixation in osmium tetroxide is satisfactory for
Electron microscopy
– 2-5% - small tissue fragments ; 4 % - larger tissues
– Recommended for enzyme histochemistry and
electron microscopy
METALLIC FIXATIVES
• Mercuric chloride
– most common metallic fixative (5-7% aqueous
solution)
– Mercury deposits are removed from
deparaffinized sections before staining, by treating
with 0.5% iodine solution in 70% ethanol for 5-10
minutes.
– Routine fixative of choice for preservation of cell
detail in tissue photography
Mercuric chloride
• Zenker’s fluid
– With glacial acetic acid to prevent turbidity &
formation of dark precipitate
– Recommended for fixing small pieces of liver,
spleen, connective tissue fibers & nuclei
– Recommended for trichrome staining
– De-zenkerization – alcoholic iodine
Mercuric chloride
• Zenker – formol (Helly’s solution)
– Microanatomic fixative for pituitary gland, bone
marrow & blood containing organs
– Pigments removal - saturated alcoholic picric acid
or NaOH
• B-5 fixative – bone marrow biopsies
• Heidenhain’s Susa solution
– Recommended mainly for tumor bx sp. skin
– Excellent cytologic fxative
Chromate Fixatives
• 1-2 % Chromic Acid
• 3% Potassium dichromate
• Regard’s (Muller’s ) fluid
– Recommended for demonstration of chromatin,
mitochondria, mitotic figures, Golgi bodies, RBC and
colloid-containing tissues
• Orth’s fluid
– Recommended for study of early degenerative
processes & tissue necrosis
– Demonstrates ricketsiae & other bacteria
– Preserves myelin better than buffered formalin
Lead Fixatives
(4% aqueous solution)
• Recommended for acid mucopolysaccharides
• Fixes connective tissue mucin
• Takes up CO2 to form insoluble lead carbonate
– Removed by filtration or adding acetic acid
PICRIC ACID FIXATIVES
• Excellent fixative for glycogen demonstration
• Yellow color – removed by treatment with
another acid dye or lithium carbonate
– Brasil’s Alcoholic Picroformol Fixative
– Bouin’s solution
• recommended for embryos & pituitary biopsies
• Excellent fixative for preserving soft & delicate tissues
• Preferred fixative for tissues to be stained by Masson’s
trichrome for collagen, elastic or connective tissue
GLACIAL ACETIC ACID
• Normally used in conjunction with other
fixatives
• Solidifies at 170 C
• Very useful in the study of nuclear
components of the cell
• Causes tissue to swell which is used to
counteract the shrinkage produced by other
components (mercury)
• Contraindicated for cytoplasmic fixation
ALCOHOL FIXATIVES (70-100%)
• May be both as a fixative & dehydrating agent
• Excellent for glycogen demonstration
• Contraindicated when lipids are to be studied
– 100% Methyl alcohol – excellent for fixing dry &
wet smears, blood smears & bone marrow tissues
– 95% Isopropyl alcohol
– 70% Ethyl alcohol
ALCOHOL FIXATIVES (70-100%)
• Carnoy’s fixative
– Recommended for fixing chromosomes, lymph
glands & urgent biopsies
– Considered to be the most rapid fixative
– Also used to fix brain tissue – dx rabies
• Newcomer’s fluid
– Recommended for fixing mucopolysaccharides
and nuclear proteins
– Acts both as nuclear & histochemical fixative
OSMIUM TETROXIDE (OSMIC ACID)
• Fixes conjugated fats & lipids permanently
• Used extensively for neurological tissues
• Adequately fixes electron microscopy
• Poor penetration – only for 2-3mm thick
• Addition of sat. aq mercuric chloride will
prevent its reduction with formation of black
deposits
OSMIUM TETROXIDE (OSMIC ACID)
• Flemming’s solution
– Most common chrome-osmium acetic acid fixative
– Excellent for nuclear structures
– Permanently fixes fat
• Flemming’s solution w/o acetic acid
– Recommended for cytoplasmic structures
particularly mitochondria
Trichloroacetic acid

• Precipitates proteins & poor penetrating


agent
• Weak decalcifying agent – softening effect on
dense fibrous tissues
• Marked swelling effect on tissues serves to
counteract shrinkage produced by other
fixatives – sometimes incorporated into
compound fixatives
Acetone

• Used it at ice cold temp from –5 0C to 40 0C


• Study of water diffusible enzymes especially
phosphatases & lipases
• Fixing brain tissues – dx of rabies
• Dissolves fat
• Preserves glycogen poorly
Secondary FixatIon
• Placing an already fixed tissue into a second
fixative
• Before dehydration & on deparaffinized
section before staining
– Facilitate & improve demonstration of particular
substances
– Special staining techniques
– Ensure further & complete hardening and
preservation of tissues
Post – chromatization
• Form of secondary fixation
• a primarily fixed tissue is placed in 2.5 to 3%
potassium dichromate for 24 hrs
– Act as mordant for better staining effects
– To aid in cytologic preservation of tissues
Heat fixation
• Thermal coagulation of proteins
• Suitable for frozen tissue sections
• Preparation of bacteriologic smears
• Produces considerable tissue shrinkage &
distortion
• Destroys RBC
• Dissolves starch and glycogen
Microwave technique

• Used to accelerate staining, decalcification,


immunohistochem & EM
• Blocks are placed in microwave, 450 watts,
550C for 1.5 to 4 minutes
• Useful in preserving neurochemical
substances, such as acetylcholine
• Rapid fixation
• Reduces time taken for IHC and ISH
Washing-out
• Removing excess fixative to improve staining
and remove artifacts .
• Several solutions may be used :
– Tap water
– 50-70 % alcohol
– Alcoholic iodine
Improper fixation

• Autolysis - Delay fixation or insufficient


fixative
• Removal of substances; loss or inactivation
of enzymes - Wrong choice of fixative
• Artifact pigments - Incomplete washing
• soft & feather like - Incomplete fixation
• Brittle and hard - Over fixation
Fixation artifacts

• Eliminated or reduced by fixation in phenol


formalin
• Use of neutral buffered formalin and phenol
formalin almost completely strops the formation
of formalin pigment

• “crush artifact” – partial coagulation of partially


fixed protein by ethanol or incomplete wax
impregnation
"technicon" tissue processor
• Tissue blocks
contained in
baskets through a
series of reagents

• Vertical oscillation
or the mechanical
raising and lowering
of the tissue
provides the
agitation needed
Dehydration
• Removal of unbound water and aqueous fixatives
from the tissue components
• Should be accomplished slowly
• Dehydrating agents : ethanol, ethanol acetone,
methanol, isopropyl, glycol & denatured alcohols
• Specimens are processed through a graded series
of reagents of increasing concentration
– usually done with a series of alcohols – ethanol 70%
to 95% to 100%.
Clearing
• consists of removal of the dehydrant with a
substance that will be miscible with the
embedding medium (paraffin)
• the appearance of tissue is translucent

• Most common - xylene


Infiltration (impregnation)

• the tissue is infiltrated with the embedding agent


• almost always paraffin
• Paraffins can be purchased that differ in melting
point, for various hardnesses, depending upon the
way the histotechnologist likes them and upon the
climate (warm vs. cold).
Embedding

• Tissues that come off the tissue processor


are still in the cassettes and must be
manually put into the blocks by a technician
who must pick the tissues out of the
cassette and pour molten paraffin over
them.

• The tissues must be aligned, or oriented,


properly in the block of paraffin.
Tissue embedding – molten paraffin

Specimen orientation is important for


demonstration of proper morphology.
Embedding
Sectioning
• Once the tissues have been embedded, they must be
cut into sections that can be placed on a slide.
• This is done with a microtome.

• The microtome is nothing more than a knife with a


mechanism for advancing a paraffin block standard
distances across it.

• Microtomes have a mechanism for advancing the


block across the knife. Usually this distance can be set,
for most paraffin embedded tissues at 6 to 8 microns.
Sectioning with microtome knife
floated on a warm water bath that helps
remove wrinkles… Then, picked up on a glass
microscopic slide
Unstained section on a slide
Tray of unstained slides in
drying oven
H & E staining
• Hematoxylin is the oxidized product of the
logwood tree known as hematein.

• In order to use it as a stain it must be


"ripened" or oxidized.
– done naturally by putting the hematein solution on the
shelf and waiting several months
– by buying commercially ripened hematoxylin or by putting
ripening agents in the hematein solution.
H & E staining

Eosin is an acidic dye with an affinity for


cytoplasmic components of the cell.

Hematoxylin (nuclei)
Eosin (cytoplasm)
Slides being stained –
automated stainer
Automated stainer
Coverslipping
• The stained section on the slide must be covered
with a thin piece plastic or glass :

– protect the tissue from being scratched


– provide better optical quality for viewing under
the microscope
– preserve the tissue section for years to come.
Mounting (coverslip) and Labeling
Cryostat
• When the tissue is frozen, the
water in the tissue turns to ice
and in this state the tissue is
firm, the ICE acting as the
embedding medium
• Refrigerated cabinet in which
a specialty microtome is
housed.
• Cold chamber kept at an
atmospheric temp. of
-10 to -20C

Method of choice : isopentane


and liquid nitrogen
• The consistency of the
FROZEN SECTION frozen block can be
altered by varying the
temperature of the tissue.

• Majority of non fatty


unfixed tissues section
well at -20 to -25 C.

• Sectioning of fixed tissue


requires a block temp of
-10C or warmer
Staining for frozen section
"progressive" stain in which the section is left in contact with
the stain until the desired tint is achieved.
Pathologists at work