Histopathology and Cytology

HISTOPATHOLOGY AND CYTOLOGY
Histopathologic technique
HISTOPATHOLOGY
• Histopathologic examination of properly
processed tissues can confirm or dispute clinical
impression and other diagnostic test, and guides
the clinician on the treatment of disease.
• Histopathologic examination ensures that
resection margins are adequate (especially with
malignant tumors), determines the extent of
lymphatic involvement and direct spread, and
assists the staging of disease.
• Autopsies determine the cause of death and
provide objective data foe medical practice audit
on adequacy of treatment or complications of
disease.
PROCESSING OF TISSUES
A better and more effective means of
studying tissues, whether normal or
abnormal, is by examination of their
section and smears which have been:
1. permanently preserved
2. stained for demonstration of
specific structures,
3. mounted on a glass slides with
coverslip for permanent keeping.
Processing of solid tissues in histopathology
section
Procedure in order
1. Fixation
2. Dehydration
3. Clearing (dealcoholisation)
4. Impregnation and embedding
5. Trimming and section cutting
6. Staining
7. Mounting
Fixation
• The preservation of fresh tissues for
examination.
• Inadequate or poor fixation will in poorly
processed tissue.
• Primary aim of fixation is to preserve the
morphologic and chemical integrity of the
cell in as life-like manner as possible.
• Secondary purpose is to harden and protect
the tissue from the trauma of further
handling, so that it is easier to cut during
gross examination.
Examples of Fixative:
1. Formaldehyde
2. Glutaraldehyde
3. Mercuricchloride
4. 10 % Formal saline
Consideration in fixation
1. The amount of fixative must be adequate, approximately 20X the
volume of the tissue specimen.
2. Autopsy material must be fixed as soon after death.
3. Surgical specimen should be fixed as soon as possible after removal.
4. All tissue specimen must be properly labeled and identified.
5. Tissues must not be more than 5 mm thick
6. Drying should be avoided to prevent shrinkage and distortion of
tissue
7. Hollow organs must be packed with cotton soaked in fixative before
being immersed in fixative solution.
Dehydration
• Process of removing intracellular and extracellular water from the
tissue following fixation and prior to wax impregnation.
• Dehydration time should be as brief as possible to minimize the risk
of cellular constituents.
• Amount of dehydrating agent should not be less than 10X the volume
of the tissue to ensure complete penetration of the tissue.
Examples of Dehydrating agent:
1. Alcohol (ethyl alcohol)- in
increasing strengths usually
70%, 95%, and 100%
2. Acetone
3. Tertahydrofuran
Clearing( dealcoholization)
Process whereby alcohol or dehydrating
agent is removed from the tissue and
replaced with a substance that will
dissolve the wax with which the tissue is
to impregnated.
Examples of Clearing agent:
1. Xylene ( most common)
2. Tuolene
3. Benzene
4. Chloroform
Impregnation and Embedding
IMPREGNATION (infiltration)
• process whereby the clearing agent is
completely removed from the tissues and
replaced medium that will completely all the
tissue cavities,
• thereby giving the firm consistency to the
specimen and allowing easier handling and
cutting of suitably thin sections without any
damage to the tissue.
EMBEDDING ( Casting or blocking)- process by

which the tissue is placed in a precisely arranged
position an a mold containing a medium which is
then allowed to solidify.
Common impregnation and embedding
media
1. Paraffin wax
2. Celloidin
3. Gelatin
4. Plastic
Paraffin-embedded tissues (tissue blocks)
Time schedule of manual tissue processing:
Steps Time
Fixation (10 % Buffered formalin) 24 hours
Dehydration
70 % alcohol 6 hours
100 % alcohol 1 hour
Clearing
Xylene I 1 hour
Xylene II 1 hour
Impregnation (4 changes of paraffin wax) (15 minutes each changes)
Embedding (Paraffin wax) 1 hour
Trimming and section cutting
After embedding, the paraffin embedded tissue is trimmed and cut into
uniformly thin slices or “sections” to facilitate studies under the microscope.
• MICROTOME- machine used in cutting tissues at a predetermined

thickness.
• TRIMMING- excess wax is cut off from the tissue block to expose the tissue
surface in preparation for actual cutting.
• SECTIONING- process whereby tissues are cut into uniformly thin slices or
“sections” usually at 4-6 microns in routine histologic procedures.
Trimming and section cutting
After cutting, sections are floated on a water bath to let the sections
flattened, a slide is put with adhesive usually Mayer’s Egg Albumin,
labeled and immersed in a water bath and section is fished out and
drained, then dry the slides in an incubator at 37 C or on a hot plate at
45 C.
Staining
• Process of applying dyes on the section to see and study the
architectural pattern of the tissue and the physical characteristics of
the cells.
• H an E ( Hematoxylin & Eosin) Staining technique:
• Most common used for micro anatomical studies of tissues
Staining procedure
1. Xylene 3 mins.
2. Xylene 3 mins.
3. Absolute ethyl alcohol 2 mins.
4. 95% ethyl alcohol 2 mins.
5. Rinse in running water 1 min
6. Harris Hematoxylin 5 mins.
7. Wash in tap water to remove excess stain
8. 1 % acid alcohol 10-30 secs.
9. Rinse in tap water
10. Ammonia water 5 mins.
11. Wash in running water 5 mins.
12. 5 % aqueous Eosin 5 mins
13. Wash in tap water
14. Dry
MOUNTING
• A mounting media is usually a syrup fluid applied
between the section and the cover slip after staining,
setting the section firmly, preventing the movement of
the cover slip.
• It protects the stained section from getting scratched,
and from bleaching or deterioration due to oxidation,
thereby preserving the slides for permanent keeping
Examples of Mounting media
1. Canada Balsam
2. Glycerin jelly
3. Brun’s fluid
4. Farrant’s medium
DIAGNOSTIC CYTOLOGY
Diagnostic Cytology
• Microscopic examination of cells from different body sites for
diagnostic procedures.
• EXFOLIATIVE CYTOLOGY- deals with the microscopic study of cells

that has been desquamated from epithelial surfaces. Exfoliated cells
may be found in the smears that have spontaneously been shed or
physically removed from the epithelial and mucous membrane.
Diagnostic Cytology
Purpose:
1. Detection of malignant cells in the body fluids, used for staging
cancer.
2. Detection of precancerous lesions in women. ( Pap smear)
3. Assessment of female hormonal status in case of sterility and
endocrine disorders.
4. For detection of infectious agents.
Diagnostic Cytology
Specimen:
1. Cervico-vaginal smear (Pap smear)
2. Nipple discharge
3. Gastric or bronchial secretion
4. Pleural and Peritoneal fluid
5. Sputum
6. Urine sediment
7. CSF
Diagnostic Cytology
Smear preparation:
1. Smears should be made from fresh material.
2. The material should be smeared evenly on clean slide. Label with
patient’s name, age and date and type of specimen, and identifying
number.
3. Prepared smears must be immediately fixed by quick immersion in
a fixative solution or sprayed with fixative.
Common staining Procedure: Papanicolau Stain (Pap’s stain).

Histopathology vs cytology
Collection is invasive. Collection is less invasive.

Preparation is complex. Preparation is more simple.
Deals with architectural pattern of tissues. Deals with morphology and chemistry of cells.

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HISTOPATHOLOGY AND CYTOLOGY

EMBEDDING ( Casting or blocking)- process by

• MICROTOME- machine used in cutting tissues at a predetermined

• EXFOLIATIVE CYTOLOGY- deals with the microscopic study of cells

Common staining Procedure: Papanicolau Stain (Pap’s stain).

Collection is invasive. Collection is less invasive.

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