Introduction to Histology

Introduction to Histology

Histology is the study of cellular organization of

body tissues and organs

Histology specimens are usually obtained from:

Small tissue biopsies
Larger specimens removed at surgery
Tissues from autopsy
Cytological Specimen
FNACs
Tissue/Cellular scrapings

Gross Examination

Cassette Bottom

Cassette Top

Gross examination is done to describe

the specimen.

The specimen is then cut into

representative sections and is put in
small plastic cassette to hold the tissue

Done by a pathologist, pathology

assistant or resident to:
Describe the specimens size,
shape, color and any apparent
abnormalities.

Accession Number

Description of margins and

their orientation
or accurate diagnosis later.

Fixation

Fixation preserves the cell morphology and detail.

A fixative brings about the cross linking of proteins

to convert them into a state where the tissue detail
and information is fixed.

Fixation is the most important factor for quality IHC.

Most common fixatives are NBF, Zinc-based formalin

fixatives and Bouins fluid etc

Fixation is a trade-off between good morphology

and good antigenicity

Based on mechanism of action, fixatives are

classified into:

Small intestine well preserved

Aldehyde
Mercury fixatives
Alcohols
Autolyzed Small intestine
Notice how is missing the epithelium
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Oxidizing agents

Aldehyde Fixatives
Formaldehyde (Formalin) and Glutaraldehyde

Formalin:
Standard solution is 10% neutral buffered
formalin. Buffering prevents any acidity that
would promote autolysis and cause
precipitation of formol-heme pigment in the
tissues

Aldehyde
fixative

Penetrates the tissue well, but is relatively slow

Fixes the tissue by forming cross-linkages
between amino acid residues (particularly
lysine)
Cross-linking does not greatly harm the
structure of proteins and most of the
antigenicity is not lost; therefore,
formaldehyde is good for most of the
immuno-histochemistry techniques

Alcohol Fixatives
Methanol and Ethanol

Preserve tissue by denaturing and

precipitating proteins

Not used routinely for tissues (cause too

much of brittleness and hardness)

Good for Cytologic smears because they act

quickly and give good nuclear detail

Spray cans of alcohol fixatives are marketed

to physicians doing PAP smears

Mercury Fixatives

Contain Mercuric chloride

Penetrate is poor and might cause tissue hardness

Give excellent nuclear detail

Generally used for hematopoitic and reticulo endothelial tissues

Oxidizing Agents

Include permanganate fixatives (potassium permanganate),

dichromate fixatives (potassium dichromate), and osmium tetroxide

Cross-link proteins, but cause extensive denaturation

Used very infrequently

Processing (1/2)

Tissue is then processed into a form to

provide enough physical support and enable
cutting into 2- 6 micron sections.

Routinely, this is done by infiltrating and

surrounding the tissue with paraffin wax.

Tissues and the fixatives preserving them

contain water; because water and paraffin
are not miscible, a series of steps must be
taken to remove the water. This technique is
called processing.

Processing (2/2)
Removal water by ethanol

Dehydration:
Removes water from the tissue through
the passing of tissue in a series of alcohol
from 70% to 100%.

Fixed
Tissue

Fixation

70%

95%

10
0%

Dehydration

Xylene is used to clear the dehydrant

alcohol that is miscible with both alcohol
and paraffin. The tissues are usually put
through 3 changes of the clearing agent.

SP06
12345 1A

Paraffin
tissue block
prepared for
microtomy.

Embedment

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Clearing:

Paraffin
wax

Impregnation

Xylene

Clearing

Impregnation:
Tissues are then put through changes of
melted paraffin wax.

Automated Tissue Processor

Tissue Chamber:
Holds the tissue

Containers with fluids:

Holds the various fluids

Tissue Chamber

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Containers With Fluids

Tissue from processors are manually placed

in embedding molds and aligned properly

Tissues are processed by Alcohol, xylene

and wax sequentially.

Embedding

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Tissues from the processor are in molten

paraffin and are still in the cassettes; they
must be manually put into the embedding
molds

The mold and cassette are then filled with

more molten

The paraffin is then allowed to solidify by

putting the embedding molds on a
refrigerated surface

Once the paraffin is solid, the entire block,

with the cassette is popped out of the mold
and is ready for thin sectioning using
microtome

Sectioning

Picking up of sections from water bath

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Microtome : These are mechanical devices

for cutting uniform sections of tissue of
appropriate thickness.

The cut sections are floated on a warm

water bath to remove the wrinkles.

Then they are picked up on a glass

microscopic slide.

Tissue section on glass slide

Frozen Sections

Frozen sections are used for rapid diagnosis of a

pathological process (benign vs. malignant
Detect on the next course of action
Random analysis of tissue sample

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Tissue is snap frozen in a fluid called OCT and

sectioned in a cryostat (refrigerated microtome);
sections are picked up with a glass slide and
stained.

Baking

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The slides with the cut tissues are placed in

an oven.

Baking removes the water between the

section and the slide

Enables proper adherence of the tissue on

the slide

H&E Staining

Hematoxylin (H): A basic dye stains acidic groups (Heterochromatin,

glycosaminoglycans) blue.

Eosin (E): Acidic dye and stains proteins red.

Coverslip /Mounting

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Protects and preserves the stained tissue section.

Provides better optical quality for viewing under microscope.

Diagnosis

Subjective evaluation

Comparison of tissue obtained in the patient specimen to its normal counterpart

Involves years of training in pattern recognition

Criteria:
Nuclear size and shape
Chromatin pattern
Cytoplasmic size and shape
Cytoplasmic organization
Cellular and Tissue organization

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Definitions

Histology: The microscopic study of tissue, their formation, structure and function.

Histopathology: The study of structural and functional abnormalities that are

expressed as diseases of organs and systems.

Anatomic Pathology: Visual analysis of the microscopic anatomy of the patients

cells or tissues in order to identify disease processes in these cells or tissues.

Clinical Pathology: Analysis of patients specimens (usually fluids) for elements in

order to identify disease processes in the body e.g. Biochemistry, Hematology,
Microbiology and Virology.

Cytology: The study of cells.

Cytopathology: The study of disease in cells i.e. observing the cellular level changes
at microscopic level when compared to normal cells.

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