Tissue processing in
Histopathology
Maryum Shafiq
Sample collection & preservation:
1) Collection of sample:
Surgery/Biopsy/Autopsy
The tissue is removed from the animal body or plant part
Placed in a fixative which stabilizes the tissues to prevent decay.
The most common fixative is formalin (10% formaldehyde in water)
Small piece of tissue (as early as possible)
Piece is removed with sharp knife
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,
Tissues should be collected directly in the fixative and not in any other pot or water.
The tissue pieces from hollow organs like intestines, oviduct etc., should be cut
transversely.
Labelling:
Tissue is accompanied by a tag or label, bearing the lab number given to
specimen at start, through all stages.
Preservation:
Tissues are saved in different cases having different colors:
Yellow (liver, renal)
Green (routine)
White (bones)
Grey (skin)
Pink (lymph nodes)
Specimen accessioning:
Tissue specimens received in the
surgical pathology laboratory -lists
the patient information and history
along with a description
of the site of origin.
The specimens are
accessioned by giving them
a number that will identify
each specimen for each
patient
Gross examination:
Tissues removed from the body for diagnosis arrive in the Pathology Department and
are examined by a pathologist.
Gross examination consists of describing the specimen and placing all or parts of it
into a small plastic cassette which holds the tissue while it is being processed to a
paraffin block.
Initially, the cassettes are placed into a fixative
Proper identification and orientation of the specimen.
• Un-labelled specimen should never be processed.
• A properly completed histopathology requisition form containing patient’s name,
age, sex, relevant clinical data, surgical findings, nature of operation and name of
tissue submitted.
• Careful search and examination of all the tissue submitted in order.
Fixation:
To preserve tissues permanently in as life-like a state as possible.
• Fixation should be carried out as soon as possible after removal of the tissues or
soon after death to prevent autolysis.
• There is no perfect fixative.
• The fixatives depend on the type of tissue present and features to be demonstrated.
• Five major groups of fixatives
– Aldehydes
– Alcohols
– Oxidizing agents
– Picrates
Tissue processing:
It is impractical to place the entire organ under a routine light microscope for
study- Too large, but also opaque-impossible to examine its micro-components.
A small portion -specific tissue or organ –excised from a given organ and
processed for microscopic analysis.
Distortions, and loss of components due to the preparation process are almost
always present.
Stages:
Dehydartion
Clearing
Impregnation
Automatic tissue processor:
A tissue processor is a device that prepares tissue samples for sectioning and
microscopic examination in the diagnostic laboratory.
Microscopic analysis of cells and tissues requires the preparation of very thin, high
quality sections (slices) mounted on glass slides and appropriately stained to
demonstrate normal and abnormal structures.
The ATP machine plays a big role in the preparation of the tissue by passing them
through various chemicals; a major process called TISSUE PROCESSING
The first automatic tissue processors were introduced during the first half of the 20th
century. In the USA, they were produced under the name of Auto-Technicon and in
the UK under the name of Histokine, and later by other companies.
These devices have slowly evolved to be safer to use, handle larger specimen
numbers, process more quickly and to produce better quality outcomes.
Overview:
The ATPM works by following through an already established processing steps.
Tissues to be processed are cut into small pieces to ensure the tissue fits into the
tissue cassettes
Smaller tissues (2-4 um) will be processed faster than the whole tissue or organ.
These tissue cassettes are packed into the oscillating tissue basket to tissue prior to
fixation.
PARTS OF THE ATPM – using TP 1050 Leica model
(i) Oscillating tissue basket
(i) 10 beakers or jars
The transparent beakers is for processing fluids
(ii) 2 thermostatically controlled beakers
The coated beaker is for parrafin wax
(iii) An electric rotor at the base
(iv) Lifting mechanism
(v) Time disc and alarm system
(vii) Control unit - with display screen and control buttons
Working principle:
Most ATPMs are easy-to-program interface. The Leica processor model has ten 1.8L
(60.9oz.) reagent beakers and two 1.8L wax baths.
The tissue basket oscillates up and down in each station at three-second intervals to
ensure thorough and even mixing of the reagents and optimum tissue infiltration.
Infiltration time is separately programmable for each station. Up to nine programs
may be run with immediate or delayed starting times.
When it’s time for tissue to be transferred to the next beaker or jar, the cover of the
machine is raised up, and the lifting mechanism carefully removes the tissue basket
and gently transfers it to the next beaker.
When the infiltration time for any particular station is exceeded, a warning message
will display, indicating the station number and excess time. Controls are arranged by
functionality with an LCD to indicate operational parameters. Reagent container lids
have seals to minimize operator exposure to hazardous fumes.
Tissue basket immediately immerses in a station in the event of power loss to protect
samples from drying out. When power is restored, program will resume. In the event
of long-term power failure, wax is liquified. Capacity of tissue basket is 80 cassettes.
Vacuum configurations hasten infiltration, allowing pressure to be applied to any
station in either manual or automatic operation. Fume control configurations extract
fumes with a fan and pass them through an internal carbon filter.
For added efficiency, these models feature a two-part containment shield surrounding
the reagent container platform.
Processing time schedule:
Processing schedule varies and it depends oh the following:
(i) Nature and size of tissue
(ii) Urgency
Beaker I – fixative (formalin) 1-2 hours
Beaker 2 – fixative 1 hour
Beaker 3 – fixative. 30- 45 minutes
Beaker 4 – 70% alcohol. 30 minutes
Beaker 5 – 90% alcohol. 30 minutes
Beaker 6 – Absolute alcohol. 1 hour
Beaker 7 – Absolute alcohol. 1 hour
Beaker 8 – Methanol 30 minutes
Beaker 9 – Xylene. 1-2 hours
Beaker 10 – Xylene 45 minutes – 1 hour
Wax bath I - (done at 45°c) 2 hours
Wax bath II - 2 hours
Remember that the nature of tissue, size and urgency determines the processing
time schedule.
All the processes are performed by the ATPM from start to finish except embedding
which is best done manually. This wasn’t possible in the 18th and 19th century.
Advantages:
(i) It’s very efficient
(ii) Saves time and energy to operate
(iii) Cost effective and user friendly
(iv) Can process different tissues same time
(v) The machine does the transfer of tissue from one bath to another. Before now, this
wasn’t possible
Thank You
