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HISTOPATHOLOGICAL TECHNIQUES (Lecture) Considerations in tissue examination

Examination of fresh tissue  Structural and chemical components of cells to be
HISTOLOGY - the microscopic study of the normal studied
tissues of the body  Nature and amount of tissue to be evaluated
HISTOPATHOLOGY -the microscopic study of tissues  Urgency of result
affected by disease  Advantage of fresh tissue examination
- demonstration of minute structural alterations in  Observe protoplasmic activities
tissues as the result of disease  Disadvantage of fresh tissue examination
 Not permanent, therefore liable to changes
Sources of tissue study
1. Cadavers Different cassettes having different color
2. Autopsy - post-mortem examination COLOR PARTS
3. Animal Yellow Liver and renal
4. Biopsy - remove a piece of tissue or a sample cells Green Routine
White Bones
Scope Grey Skin
 Useful in establishing the pathogenesis and Pink Lymph nodes
pathology of any disease caused by bacteria, virus,
chlamydia, rickettsia, mycoplasma, parasite, toxin, Histology Procedure
poisons etc.
 There are certain diseases in which Specimen
histopathological examination of tissues is the Accessioning
only alternative to diagnose the disease. e.g.
Bovine spongiform encephalopathy.
The agent of this disease takes a very long Gross
incubation period and very difficult to isolate and Examination
there is no immune response and inflammation in
animal.
(Therefore, histopathology remains the only alternative
Tissue
for confirmatory diagnosis.)
Fixation
 In some cases, tissues from dead animals are
only available material for laboratory diagnosis.

Sample collection and preservation Tissue Processing

Collection of samples: A. Dehydration
 Small piece of tissue (as early as possible) B. Clearing
 Piece is removed with sharp knife C. Impregnation
 At the time of tissue collection, it should be kept in
mind that the representative tissue piece should
include the part of lesion and a part of normal tissue, Tissue
 Tissues should be collected directly in the fixative Embedding
and not in any other pot or water.
 The tissue pieces from hollow organs like intestines,
oviduct etc., should be cut transversely. Tissue
Sectioning
Labelling
Tissue is accompanied by a tag or label, bearing the lab
number given to specimen at start, through all stages.
Slide
Items required for proper tissue and storage Staining
1. Ice
2. Cryotubes
3. Cryomold Specimen Accessioning
4. Biopsy cassette  Tissue specimens received in the surgical pathology
5. Liquid nitrogen laboratory -lists the patient information and history
6. NBF Container along with a description of the site of origin.
7. Needle  The specimens are accessioned by giving them a
8. Scalpel number that will identify each specimen for each
9. Petri culture dish patient.
10. 1.5mL tube
11. 50 mL tube Gross Examination
 Tissues removed from the body for diagnosis arrive
in the Pathology Department and are examined by a
pathologist.
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Gross examination consists of describing the specimen *A special
and placing all or parts of it into a small plastic cassette method of smear
which holds the tissue while it is being processed to a preparation
paraffin block. whereby the
 Initially, the cassettes are placed into a fixative. surface of freshly
cut piece of tissue
Methods is brought into
1. Teasing or Dissociation contact and pressed in
 A process whereby a selected tissue specimen is the surface of the slide
immersed in a watch glass containing isotonic salt -May be stained for light microscopy or
solution, carefully dissected or selected and unstained for Phase Contrast Microscopy
examined under the microscope -Cells are examined in their actual intercellular
 Can be examined unstained via Phase Contrast relationship
Microscopy or stained via Bright Field Microscopy
4. Frozen section
2. Squash Preparation (Crushing)  Purpose:
 A process whereby small  For rapid diagnosis of tissue
pieces (less than 1 mm) of  Recommended when lipids and nervous tissue
tissue are placed on a glass elements are to be demonstrated
slide and forcibly  Method:
compressed with another  Makes use of very thin slices 10-15u cut from
slide fresh tissue frozeb on a microtome with CO2 or
 A vital stain may be placed on a Cryostat
at the junction of the slide  Cryostat- cold chamber (-10 to -20 deg Celsius)
and the cover slip  Applications
 Rapid pathologic diagnosis during surgery
 Diagnostic and research enzyme
3. Smear Preparation histochemistry
 A process of examining sections or sediments,  Diagnostic and research demonstration of
whereby cellular materials are spread lightly over a soluble substances such as lipids and
slide carbohydrates
 Methods of Smear Preparation  Immunofluorescent and immunohistochemical
 Streaking staining
-With an applicator stick  Some specialized silver stains in
or platinum loop the neuropathology
material is rapidly spread  Rapid freeze: slow freezing causes distortion due to
and gently applied in a ice crystal artifacts
direct or zigzag line  Liquid nitrogen (most rapid and commonly
throughout the slide available)
 Isopentane cooled by liquid nitrogen
 Spreading  CO2 gas (freezing microtome)
-A selected portion if the  Aerosol sprays (not for muscle tissue)
material is transferred to a  Liquid nitrogen
clean slide and gently spread  Overcools: cracks tissue, biopsy blocks
into a moderately thick (-70deg Celsiums)
film by teasing the  Sectioning requires equilibration at cryostat
mucus strands with an temp (-10 to 125 deg C)
applicator stick  Vapor phase forms around the tissue causing
uneven cooling making diagnostic
interpretation difficult
 Isopentane: liquid at room temperature
 Pull Apart  Prevents vapor phase due to its high thermal
- Done by placing a drop of conductivity
secretion or sediment upon
one slide and facing it to Processing of tissues
another clean slide Fresh vs Preserved tissue examination
Two slides are then pulled apart  Most fresh tissue are very delicate
with a single uninterrupted motion  Easily distorted
 Easily damaged
 Preserved tissues provides the efficiency of:
 Of staining for better demonstration
 Touch Preparation  Permanent keeping when mount on slides with
- Also called impression smear coverslips

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 Fixation  Isotonic (340mOsm) and hypotonic fixatives:
 Dehydration cell swelling and poor fixation
 Clearing  Best: slightly hypertonic solutions
 Infiltration (impregnation) (400-450mOsm)
 Embedding
 Trimming Practical considerations
 Section Cutting  SPEED: Tissue are placed in a fixative as soon as
 Staining they are removed
 Mounting  Prevent autolysis and putrefaction
 Labelling  Pentration:
 Formalin: 1 mm per hour, slows down as it
Fixation goes deeper
- a process by which the constituents of cells and the  Volume: 1
tissues are fixed in a physical and partly chemical state  0-25x the volume of the tissue
so that they can withstand subsequent treatment with  Maximum effectiveness at 20x
various reagents with minimal loss of architecture.  Duration of fixation:
- Prevent autolysis and degradation of tissue-such that  Fibrous organs take longer
they can be observed both anatomically and  Fixation time can be cut down by heat, vacuum,
microscopically following sectioning. agitation or microwave
**Fixation should be carried out as soon as to prevent  Refrigeration
autolysis and putrefaction.  To slow down decomposition if the tissue
cannot be fixed immediatedly
Aims of fixation  Brain cells deteriorate very quickly, BM mitosis
 Rapid and even penetration. up 30 mins after death
 To preserve cells and tissues in a life like manner as
possible. Types of fixation
 Stabilize labile elements. PHYSICAL
 Must be rigid to allow sectioning. 1. Heat fixation
 Must allow staining. - simplest method, often used in microbiology to fix
 Optical contrast must be induced for morphological bacteria prior to Grams staining
examination. - in histopathology, heat is primarily used to accelerate
other forms of fixation as well as the steps of tissue
Effects of fixation processing.
 They harden soft and friable tissues and make the
handling and cutting of sections easier. (accelerated 2. Microwave fixation
by the action of alcohol) - Microwave heating speeds fixation and can reduce
 They make cells resistant to damage and distortion times for fixation of some gross specimens and
caused by hypotonic and hypertonic solutions. histological sections from more than 12 hours to less
 They inhibit bacterial decomposition. than 20 minutes
 They increase optical differentiation of cells and - harmful if used with formalin-fixed tissues due to the
tissue components. production of large amount of harmful vapors.
 They act as mordants or accentuators to promote
and hasten staining. Remedy:
 They reduce the risk of infections. a. must be used with hoods or microwave
processing system.
Factors affecting the quality of fixation b. Use commercial glyoxal-based fixatives which do
1. Buffers and pH (pH6-8) not form vapors when heated at 55°C
2. Duration of fixation (2-6 hours, buffered formalin)
 Prolonged: shrinkage and hardening of tissue 3. Freeze-drying and Freeze-substitution
3. Size of specimen - rarely used in the laboratory
 1-2sqmm EM a. Freeze-drying is a useful technique for studying
 2sqcm Light Microscopy soluble materials and small molecules
 no more than 4mm thick - tissues are cut into thin sections, immersed in liquid
4. Temperature of Fixation nitrogen, and the water is removed in a vacuum chamber
 0-4degC: EM, histochemistry at −40°C. The tissue can be post-fixed with
*Mast cells: room temp formaldehyde vapour.
 Chemical reactions are more rapid at higher b. Freeze- substitution: specimens are immersed in
temperatures fixatives at −40°C, such as acetone or alcohol,
*Heat fixation in bacteriology which slowly remove water through dissolution of
5. Concentration of fixative ice crystals, and the proteins are not denatured;
 10% Formaldehyde bringing the temperature gradually to 4°C will
 3% Glutaraldehyde complete the fixation process
6. Osmolality
 Hypertonic solutions: cell shrinkage

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CHEMICAL
1. Coagulant fixatives: fixes the histomorphology of
cells by coagulating the proteins in the cell like collagen
and lipoproteins
 Disadvantage: not used in ultrastructural analysis
because fixation results in cytoplasmic flocculation
as well as poor preservation of mitochondria and
secretory granules
a. Dehydrant coagulant fixatives:
Principle:
- most commonly used are alcohols and acetone
b. Acid coagulants:
Principle: rapidly denatures and precipitates proteins by
destroying hydrogen and other bonds.
- acetic acid, TCA
Picric acid fixatives
 Bouin‟s solution- recommended for fixation of
embryos
 Brasil‟s Alcoholic Picroformol
2. Non- coagulant cross-linking fixatives
- Principle: fixes the tissue by forming cross-links within
and between proteins and nucleic acids as well as
between nucleic acids and proteins
Types According to COMPOSITION
A. Simple fixatives- only one component
1. ALDEHYDE FIXATIVES
a. Formaldehyde
-Pure formaldehyde: a vapor that, when
completely dissolved in water, forms a solution
containing 37–40% formaldehyde; this
aqueous solution is known as „formalin.
b. - 10% neutral buffered formalin (NBF) is the most
common fixative used in diagnostic pathology.
c. 10% formol-saline- 40% Formaldehyde + 10%
sodium chloride-preservation and storage of
surgical, post mortem and research specimen
d. Formol-corrosive- for routine post mortem tissues
e. Glutaraldehyde
-made up of two formaldehyde residues, linked
by three carbon chains
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3. Osmium tetroxide – pale yellow powder that dissolves
in water (up to 6% at 20oC) to form a strong oxidizing
agent
a. Flemming‟s solution- most common
Chrome-Osmium acetic acid fixative
b. Flemming‟s solution without acetic acid – made
up only of chromic and osmic acid, recommended
for Mitochondria
B. Compound fixatives- are made up of two or more
fixatives which was added together to obtain optimum
fixation effect.
Types According to ACTION
a. micoanatomic fixatives
b. cytological fixatives
c. cytoplasmic fixatives
d. histochemical fixatives
Fixation for selected individual tissues:
1. eyes
- Eyes may be fixed in NBF, usually for about 48
hours; to speed fixation one or two small windows
can be cut into the globe (avoid the retina and iris)
after 24 hours. After gross description, the anterior
(iris) and posterior (e.g. optic nerve) are removed
with a new, sharp razor blade and the components
of the globe are fixed for an additional 48 hours, or
more, in buffered formaldehyde, before being
processed. Embedding may be incelloidin or
paraffin.
2. brain
- Conventionally this fixation takes at least 2 weeks.
- Fixatives may also be enhanced by the use of
microwave technology
- Alcoholic formalin should not be used for fixation if
immunohistochemistry is to be performed
3. breast
-Clinical samples should be fixed in 10% NBF for
between a minimum of 6–8 hours and a maximum
of 72 hours, and should be sliced at 5mm intervals
after appropriate gross inspection and margins
designation.
4. lymphoid tissue
- Special care should be taken with all lymphoid
tissue, as many organisms (e.g. Mycobacterium
tuberculosis and viruses) may sequester
themselves in the lymphoid reticular system. The
lymphoid tissue is usually sliced and a
representative sample of fresh tissue taken for
special studies (e.g. flow cytometry or molecular
analysis). The rest of the lymph node is fixed in NBF,
though some laboratories fix part of
the tissue in B5 or zinc
5. lungs
-Lung biopsies are typically fixed in NBF. The lungs
from autopsies may be inflated by and fixed in NBF
via the trachea or major bronchi, and in our
experience these lungs can be cut within 2 hours.
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6. testis
-Biopsies of the testes are fixed routinely in NBF

7. muscle biopsies
-The tissue for routine histological assessment is
fixed in NBF and embedded so the fibers of the
specimens are viewed in cross-section and
longitudinally. After processing this is stained with
H&E, a trichrome stain, and Congo red if amyloid is
suspected

8. renal biopsies
-Renal core biopsies should be subdivided into
three and each piece should contain adequate
numbers of glomeruli. Each portion is then
preserved, depending upon the method to be used
for analysis:
• NBF for routine histology
•Buffered glutaraldehyde (pH 7.3) for ultrastructural
analysis
•Snap frozen in isopentane and liquid nitrogen for
immunofluorescence examination.

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