An Introduction to Automated Tissue
Processing
A Lecture Transcription
Chapter: An Introduction to Automated Tissue Pro-
cessing
This chapter provides a comprehensive overview of the principles and procedures in-
volved in automated tissue processing, a critical step in preparing biological specimens
for microscopic examination in a histology laboratory.
1 Preparing the Specimen for Processing
Before automated processing can begin, the tissue specimen, which has been previously
selected during a process called "grossing," must be properly contained.
1.1 The Tissue Cassette
The small, properly trimmed tissue sample is placed into a labeled plastic container called
a tissue cassette. This cassette protects the tissue and allows for its identification
throughout the entire process.
1.2 The Tissue Basket
Multiple tissue cassettes are then loaded into a tissue basket. This basket is designed to
hold the cassettes securely and allow for the free circulation of various chemical solutions
(reagents) that the tissues will be subjected to.
2 The Automated Tissue Processor
Once loaded, the tissue basket is placed into an automated machine that handles the
subsequent steps.
2.1 Overview of the Histokinetic Machine
The machine demonstrated is an automated histokinetic tissue processing machine
(Model TP1020). This equipment automates the lengthy process of preparing tissue for
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embedding by systematically moving the specimens through a series of different chemical
solutions according to a pre-set program.
2.2 Open vs. Closed Processing Systems
There are two primary types of automated tissue processors:
Open System (Carousel Type): The machine shown is an open system. In this de-
sign, the containers of chemical solutions (reagents) are stationary, arranged in a
circular "carousel" formation. The tissue basket is moved automatically from one
container to the next at programmed intervals.
Closed System (Fluid-Transfer Type): In a closed system, the tissue basket remains
stationary in a single chamber. The various reagents are pumped into and then
drained out of this chamber in the correct sequence. Closed systems are becoming
more common as they reduce technician exposure to chemical fumes.
3 The Four Stages of Tissue Processing
The core function of the automated processor is to take the tissue through four essential
stages. This machine utilizes twelve stations to accomplish these four processes.
3.1 Stage 1: Fixation
• Purpose: To preserve the tissue and prevent its decay or decomposition. While
the tissue has likely undergone initial fixation, this step ensures it is thoroughly
fixed.
• Reagent: 10% Neutral Buffered Formalin.
• Procedure: The machine immerses the tissue basket in two separate containers of
10% formalin, typically for one hour each. This ensures complete fixation.
3.2 Stage 2: Dehydration
• Purpose: To remove all the water from the tissue. This is essential because the
next step involves a reagent (xylene) that is not miscible with water, and the final
embedding medium (paraffin wax) is hydrophobic.
• Reagent: A graded series of alcohol, from lower to higher concentrations.
• Procedure: The tissue is moved through a series of alcohol baths with increasing
concentrations (e.g., 70%, 80%, 90%, and two changes of 100% absolute alcohol).
This gradual increase in alcohol concentration is crucial to prevent the violent diffu-
sion of fluids that would cause tissue distortion and damage (cell shrinkage). Each
step typically lasts for one hour.
3.3 Stage 3: Clearing
• Purpose: To remove the dehydrating agent (alcohol) and replace it with a sub-
stance that is miscible with both alcohol and the final embedding medium (paraffin
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wax). This process is called "clearing" because it makes the tissue translucent by
increasing its refractive index.
• Reagent: Xylene is the most commonly used clearing agent.
• Procedure: The tissue basket is immersed in two separate containers of xylene,
typically for one hour each.
3.4 Stage 4: Infiltration (Impregnation)
• Purpose: To completely fill all the spaces within the tissue with a supporting
medium, which provides a firm consistency for sectioning (cutting very thin slices).
• Reagent: Molten paraffin wax.
• Procedure: The tissue basket is immersed in two separate containers of molten
paraffin wax. The wax infiltrates all the spaces previously occupied by water and
then xylene, providing internal support to the tissue structure.
4 Operating the Automated Processor
Proper operation ensures consistent and high-quality results.
4.1 Loading and Programming
The tissue basket is placed onto the machine’s spindle. The machine features a keypad
that allows a technician to select or create specific programs. A key feature of automation
is the ability to program a delayed start, allowing the machine to run overnight and have
the tissues ready in the morning.
4.2 The Role of Agitation
The machine continuously moves the tissue basket up and down within each solution. This
movement, known as agitation, ensures that the reagents constantly circulate around
the tissues, which facilitates faster and more thorough penetration.
4.3 Temperature Regulation
The final two stations containing paraffin wax are heated. The machine is equipped with
thermostats to maintain the wax at its precise melting point (approximately 60-62°C).
This ensures the wax remains in a liquid state for proper infiltration. If the temperature
is too high, it can damage the tissue.
5 Laboratory Best Practices
5.1 Specimen Handling and Labeling
Every cassette must be meticulously labeled with a unique identification number. Mixing
up specimens can lead to a catastrophic misdiagnosis for a patient.
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5.2 Reagent Management
The volume of reagents in each container must be checked regularly to ensure it is suf-
ficient to completely submerge the tissue basket. The quality of the reagents must also
be monitored and changed on a regular schedule to prevent contamination and ensure
effective processing.
