Specimen Processing in Histopathology

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 Outline
 Specimen identification and labeling

 Grossing & Fixation

 Dehydration

 Clearing

 Impregnation

 Embedding

 Microtomy

 Staining and Mounting

 The specimen is accessioned by giving them a number that will identify

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 Information must be provided
 Patient Identification

 Identification of individual requesting examination

 Procedure date

 Adequate clinical history and physical examination

 Organs resected or biopsied

 Orientation if required.

 Prior surgery or pathologic diagnosis

 Prior treatment

 Mention if rapid diagnosis is required.

 Complex series of chemical events to preserve the basic structure of the tissue.

 The purpose of fixation is to preserve tissues permanently.

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 Ideal Fixatives
 Prevent autolysis & bacterial decomposition

 Preserve tissue in their natural state & fix all components

 Preserve tissue volume

 Avoid excessive hardness of fixed tissue

 Enhance staining of tissues

 Non-toxic & non-allergic

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 Formalin
Routinely, 10% formalin is used which is prepared by mixing 10ml of
100% formalin in 90ml of distilled water.
Mechanism of action:
 It fixes 4 mm thick tissue in 8 hours.
 The fixative should be 10 times more in volume then the specimen.
Advantages:
 Rapid penetration

 Easy availability & cheap

 Does not over harden the tissue

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Grossing
Grossing of specimen is important stage in surgical pathology.
It involves:
 Accurate naked eye description of intact specimen
 Correct method of sectioning
 Gross examination of cut surface
 Selection of proper tissue blocks for microscopy
 Instructions for embedding & block making.

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 Grossing is an art
A knowledge of what needs to be taken for microscopic study is crucial for final
diagnosis.

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 Grossing room
 Large room, well illuminated and properly ventilated.
 Cutting board placed inside a metal box designed in such a fashion
that all the fluids flow directly into the sink.
 Shelves for specimen container.
 Ready access to a sink with hot and cold water.
 Ready access to formalin
 Box of instruments, Box with cassettes, labels.
 Large formalin container, photographic facilities.
 Large table with sink for large specimens

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 Instruments used in grossing

Dissecting
scissors
Large scissors

scalpel
Saw

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 Instruments used in grossing

BLADE

GLOVES

Ruler Cutting
board
Forceps

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 Inking
 Resection
margins

 Embedding instructions

 Orientation

 Distinguish between samples

 Identify the cut surface

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 General principles of grossing
 Specimen identification
 Identify all the anatomical structures present
 Orientation markers should be identified
 Measurements: Length, height
 Examine the external surface.
 Cut all the organs at intervals of 1cm thickness
 Describe cut surface, identify pathologic process
 If suspected lesion is present, measure, describe with color.

 Surgical margins
 Histological sections

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 Small biopsies
 Number of fragments, aggregate dimension
 Greatest dimension of largest fragment
 Shape of the fragment
 Color and consistency
 Should not be cut or inked
 All small biopsies must be supported within the cassette to prevent
tissue loss during processing.
 All fragments are submitted.
 Small fragments may be dipped in eosin to make them more visible.
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 Lymph node
1. If the lymph node is received in the fresh state, cut 2–3mm slices
 Take a small portion for culture
 Make imprints smears from the cut surface
 Rest of tissue fix in formalin, and submit for histology.
2. If the specimen is received in formalin, submit representative sections.
Description
 State whether node received fresh or fixed
 Size, number of node and condition of capsule
 Appearance of cut surface: color, nodularity, hemorrhage.
Sections for histology
 One to three sections including capsule, depending on size of node
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 Tissue processing
Stage of tissue processing:
• Fixation
• Dehydration
• Clearing
• Infiltration
• Embedding

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 Fixation
 Most commonly used reagent for the fixation is 10% neutral
buffered formalin

 The tissue bits should of the size of 2-3 micrometer in thickness.

 Tiny biopsies or small specimen can be wrapped in a filter paper

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 Dehydration
Removal of free water and aqueous fixatives from tissue components.

Dehydration should be accomplished slowly

The various dehydrating agents used are;

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 Clearing
Clearing agents act as an intermediary between dehydration & infiltration
solution
Choice of a clearing agent depends upon the following:
• Rapid penetration of tissue
• Speedy removal of dehydrating agent
• Ease of removal by melted paraffin wax
• Minimal tissue damage
• Cost and convenience

Clearing agents: xylene

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 Infiltration & embedding
Paraffin wax

Most popular infiltration and embedding medium

It permeates the tissue in liquid form and solidify rapidly when cooled

The paraffin wax increases harden the tissue & helps in easy sectioning
of the tissue.

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Overnight Tissue Processing

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Station Reagent Time Temp
1 10% formalin 1 hour 38 degree C
2 10% formalin 1 hour 38 degree C
3 50% alcohol 1 hour 38 degree C
4 70% alcohol 1 hour 38 degree C
5 90% alcohol 1 hour 38 degree C
6 90% alcohol 45 min 38 degree C
7 100 % alcohol 1 hour 38 degree C
8 100 % alcohol 45 min 38 degree C
9 Xylene 1 hour 60 degree C
10 Xylene 30 min 60 degree C
11 Paraffin 1 hour 60 degree C
Embedding involves the enclosing of properly processed, correctly oriented specimen in a
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 Embedding

upport medium that provide external support during microtomy.

It must fill the matrix within the tissue, supporting cellular components.

It should provide elasticity and resisting distortion during sectioning

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Embedding Procedure

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 Microtomy
A technique by which tissue is sectioned and attached on the surface for
microscopic examination with Microtome.

TYPES OF MICROTOMES:
• Sliding
• Rotary
• Rocking
• Freezing
• ultramicrotome

Microtome knives:
• Disposable blades
• Glass and diamond knives
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Rotary Microtome
 Most commonly used.
 Block holder moves up and down
While the knife remains fixed.
 Suitable for cutting of small
Tissues
Parts of a Microtome (Rotary):
 Block holder
 Knife clamp screws
 Knife clamps
 Block adjustment
 Thickness gauge
 Angle of tilt adjustment
 Operating handle.

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Staining (H & E)

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 Mounting media
 DPX ( Distrene Dibutyl phthalate Xylene)
 Canada Balsam

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