Histopathology

1. Gross Examination, Selection, Collection
and Fixation of Specimen
Dr. Sohaib Aslam
PhD Pathology
Ass. Professor
Dr Ghulam Abbas
PhD Poultry Nutrition
Ass. Professor
Riphah college of Veterinary Sciences, Lahore

2. 2
1 Specimen Identification
Grossing
Collection
Labeling2
3
4
Fixation5
Outline

3. The laboratory diagnosis of an infectious disease
begins collection of a clinical specimen for
examination or processing in the laboratory
Rule: right one, collected at the right time,
transported in the right way to the right laboratory.
Proper collection
first step accurate laboratory Dxs.
 Collection and transportation of specimens
two important aspects.
b/4 antimicrobial agents.
Prevention of contamination of the specimen

4. Identification and Labelling
Specimen Identification
• Animal/patient information
• History
• Description of site of origin.
Labeling
• Give each specimen a number

5. Gross Examination
In order to establish a diagnosis and to select relevant portions for
microscopic examination
Diagnoses on the basis of gross examination 90 % of specimens
In the remaining 10 % the skilled pathologist can be close to the
diagnosis
Two mandatory prerequisites before examining
• Knowledge of the clinical history
• Thorough knowledge of the anatomy of the organ

6. Principles
of macroscopic assessment or gross examination of a specimen or lesion
• Location of the lesion
• Size, weight, length
• Shape and architecture
• Color and consistency
• Describe cut surfaces and identify pathological changes

7. Selection and Collection of Specimen
• A basic rule is that the fewer sections are taken, the fewer slides will need to be
reviewed.
• Without delay after death/sampling & fix
• Selected tissue should contain portion of lesion
• Take youngest lesion from periphery
• Take specimen from more than one areas of affected organs
• Cut must be made quickly, sharply and accurately
• Optimum thickness 5-6mm
• No pitching, squeezing, bending or crushing
• Wash blood with normal saline

8. Gross Specimen Photography
• Acquiring Gross Pathology Skills
1. See as many gross specimens as possible
2. Specifically study the specimen for the features already discussed
3. Correlate the microscopic findings in each section with the gross findings of the
specific area from which the section(s) have come.

9. Grossing of Specimen
• Cutting up specimens is known as grossing
• Grossing is an art
• Sectioning the tissue to be processed for diagnosis
• Cut the organ at interval of 1 cm thickness
• Histopathological sections ~5-6mm
• Put in metal cassettes/capsules and then in fixative
• Small specimen can be wrapped in tissue paper and then in cassette & fixed

10. Instruments Used in Grossing

11. Handling of Specimen
Proper specimen handling from the animal/patient to the time a
completed slide
Handling in such a way that specimens deemed acceptable
• Identification of the patient sample (labeling)
• Specimen container to be used (Wide-mouthed screw-capped,
leaked proof)
• Type and volume of fixation
• Transport packing, temperature and method
• Write “Biological Material”

12. Wide mouth screw-capped bottle

13. Fixation of Tissues
The major objective to maintain clear and consistent
morphological features as living stat
In order to visualize the microanatomy of tissues,
• Stained sections of tissue must maintain the microscopic
relationships among cells, cellular components (e.g. the
cytoplasm and nuclei)
• Extracellular material with little disruption of the organization
of the tissue
• Maintaining the tissue's local chemical composition.
• Prevent drying of tissue
Each fixative has some disadvantages, like molecular loss,
swelling, shrinkage, hardening of tissues and color verification

14. Conti..
The fixative acts to 'fix at a point in time' by minimizing the loss or
enzymatic destruction of cellular and extracellular molecules, maintaining
macromolecular structures and protecting tissues from destruction by
• Microorganisms
• Long-term storage
• Infectious agents
• Prevent the micro-architecture by activity of catabolic enzymes
The most important characteristic of a fixative is to support high quality and
consistent staining with hematoxylin and eosin (H&E)

15. Ideal Fixatives
Prevent autolysis & bacterial
decomposition
Preserve tissue in their natural state &
fix all components
Make cellular components insoluble in
liquids encountered in tissue processing
 Preserve tissue volume
 Avoid excessive hardness of fixed
tissue
 Enhance staining of tissues
Non-toxic & non-allergic

16. Types of Fixatives
• Fixatives can be classified in different ways depending on their mechanism of
action.
• In the most general terms, there are physical and chemical methods of fixation.
1. Physical fixation processes includes augmenting a fixative with heat or
microwaving, Cryopreservation is another physical method
2. The chemical fixatives can be classified a few ways
I. Additives that form cross- links e.g. Formaldehyde, zinc sulfate,
glutaraldehyde, picric acid and mercuric chloride.
II. Non-additive those that denature, most commonly accomplished by
dehydration e.g. methyl and ethyl alcohol

17. Classification of Fixatives
• A. Tissue fixatives
• a. Buffered formalin b. Buffered gluteraldehyde
• c. Zenker’s formal saline d. Bowen’s fluid
• B. Cytological fixatives
• a. Ethanol b. Methanol c. Ether
• C. Histochemical fixatives
• a. Formal saline b. Cold acetone c. Absolute alcohol

18. Factors Influencing Fixation
Factors influencing fixation Recommendations Possible Negative effects
Specimen dimensions 3 mm tissue sections are ideal
4 mm maximal thickness
Thicker sections result in incomplete
fixation
Time of fixation Varies by fixative:
Formalin—requires 6–8 h
Bouin’s—not more than 18 h
B-5 2–4 h then transfer to formalin
Under-fixation can result in tissue
distortion and poor staining;
over-fixation with some fixatives
(alcohols, B-5) can make
tissue brittle or result in loss of
antigenicity
Penetration rate Depends on the diffusion
characteristics
of each fixative:
Formalin penetrates at about 1 mm/h
Fascia and capsules are naturally
occurring physical barriers to
fixatives and can dramatically
decrease penetration. They
must be incised prior to fixation
Temperature Room temperature is ideal for the
majority of tissue fixation and up to
45 °C during processing
Heat increases the rate of fixation but
also speeds up autolysis, which can
result in poor morphology and
staining

19. Factors influencing fixation Recommendations Possible Negative effects
Volume ratio Generally accepted as 15–20:1 fixative to
tissue ratio
The concentration of active reagent in
fixative diminishes as the chemical
reaction of fixation occurs. If depleted,
fixation will cease no matter how long
tissue remains in the fixative
pH and buffers Breakdown of formaldehyde results in
formic acid which decreases pH. Buffers
help avoid this by maintaining pH
between 6.8 and 7.2
Formic acid reacts with hemoglobin to
produce formalin pigment that deposits in
tissue and can be misinterpreted as
microorganisms or other pigments
(melanin, iron); this can be removed from
tissues in the staining process by short
immersion of slides in Lugol iodine
Osmolality Do not place tissue in water or leave in
saline for excessive periods of time Cell
lysis can occur if placed in hypotonic
solution If immediate fixation is not
possible, refrigerate,
place on saline soaked gauze, or immerse
in isotonic saline for a short period
Cell lysis can occur if placed in hypotonic
solution

20. Characteristics of an Ideal Fixative
• Useful for a wide variety of tissues.
• Preserve small and large specimens
• Penetrate and fix tissues rapidly
• Have a shelf life of at least one-year
• Compatible with automated tissue processors
• Readily disposable or recyclable
• Support long-term tissue storage
• Giving excellent microtomy of paraffin blocks
• Cost effective, non-toxic and non-allergic
• The most important is to support high quality and consistent staining
with hematoxylin and eosin (H&E)

21. Common Fixatives: Uses and Limitations
Fixative Primary/recommended use Limitations
Alcohol Routine cytology Cases where gout
is suspected
Fixation for frozen sections, smears,
and touch preps
Methanol and ethanol cause cell
shrinkage and will make tissue
brittle specimens if over fixed
Alcoholic formalin Completion fixation with
incompletely fixed tissue; primary
fixative for fatty specimens (allows
for easier detection of lymph)
Acidic pH can allow for formation
of formalin pigment precipitates
B-5 Hematopoietic and lymphoid tissue Sections require removal of mercury
pigment prior to staining; tissue
cannot be stored in this; low
molecular weight or no extractable
nucleic acid
Bouin’s Gastrointestinal and
genitourinary tissue
Slowly removes small calcium and
iron deposits; lysis of erythrocytes;
low molecular weight or no
extractable nucleic acid

22. Fixative Primary/recommended use Limitations
Decalcifying solution
(acid based)
Large bone sections where
future molecular testing is not
required
Poor staining with low molecular
weight or no extractable nucleic
acid. Prolonged immersion can
completely dissolve specimen
Formalin Routine processing Dissolves uric acid crystals; can
dissolve breast microcalcifications if
fixed >24 h prior to processing;
reduced high molecular weight
nucleic acids with time.
Unbuffered formalin can allow for
formation of formalin pigment
precipitates
Michel transport medium Renal biopsy transport Requires
tissue to be washed with PBS
Cases requiring immunofluorescence
prior to processing
Zenker’s Bone marrow biopsies Poor antigen preservation for IHC;
slow penetration; contains mercury;
lyses red blood cells
Common Fixatives: Uses and Limitations

23. Neutral Buffered Formalin
• 10% Neutral buffered formalin (NBF) mostly used
• 40% formaldehyde gas in 100 w/v of distilled water= 100% formalin
• 10 mL of 100% formalin + 90 mL distilled water = 10% formalin
• 10 mL of 100% formalin + 90 mL distilled water + excessive Mg or Ca
carbonates = 10% NBF formalin
Mechanism of Action
• It forms cross links between amino acids of proteins nearby making them
insoluble
• Make 4mm thick tissue fix in 8 h
Advantages
• Rapid penetration, easy availability and cheap
• Does not over-harden the tissue, easily fixes lipids and carbohydrates
• Support the acid-base dyes

24. Disadvantages:
• Irritant to the nose, eyes and mucous membranes
• Formation of precipitate of paraformaldehyde (which can be
prevented by adding 11- 16% methanol)
• Formation of black formalin pigment (acid formaldehyde hematin