Del Rosario, Ma. Veronica M. Prof. Cecilio L.

Ichon RMT, MaSPED

BSMT-3 September 28, 2021

Activity 7
Tissue Processing
(Steps for Paraffin Sections)
OBJECTIVES

1. To identify tissue processing and its importance

2. To enumerate the different reagent use in paraffin section
3. To analyze the steps in tissue processing for paraffin sections
4. To illustrate the routine fo r tissue processing

DRAWING

Routine for Tissue Processing

 QUESTIONS AND ANSWERS

1. Define tissue processing

“Tissue processing” describes the steps required to take an animal or human tissue from
fixation to the state where it is completely infiltrated with a suitable histological wax and can
be embedded ready for section cutting on the microtome. Tissue processing can be
performed manually (hand processing), but where multiple specimens must be dealt with, it
is more convenient and much more efficient to use an automated tissue processing machine
(a “tissue processor”). These devices have been available since the 1940’s1 and have
slowly evolved to be safer in use, handle larger specimen numbers, process more quickly,
and to produce better quality outcomes.

There are two main types of processors: the tissue-transfer (or “dip and dunk”) machines
where specimens are transferred from container to container to be processed, and the fluid-
transfer (or “enclosed”) types where specimens are held in a single process chamber or
retort and fluids are pumped in and out as required. Most modern fluid-transfer processors
employ raised temperatures, effective fluid circulation and incorporate vacuum/pressure
cycles to enhance processing and reduce processing times.

If tissue is completely fixed, processing problems are less likely to occur. Once the tissue
has been fixed, it must be processed into a form in which it can be made in to thin
microscopic sections. The usual way this is done is with paraffin. Tissues embedded in
paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10
microns, usually 6-8 routinely. The technique of getting fixed tissue into paraffin is called
tissue processing. The main steps in this process are dehydration and clearing. First, the
water from the tissues must be removed by dehydration.

This is usually done with a series of alcohols; say 70% to 95% to 100%. Sometimes the
first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major
disadvantages. The next step is called "clearing" and consists of removal of the de hydrant
with a substance that will be miscible with the embedding medium (paraffin). The
commonest clearing agent is xylene. Toluene works well, and is more tolerant of small
amounts of water left in the tissues,.

Chloroform used to be used, but is a health hazard, and is slow finally, the tissue is
infiltrated with the embedding agent, almost always paraffin. Paraffin s can be purchased
that differ in melting point, for various hardness , depending upon the way the
histotechnologist likes them and upon the climate (warm vs. cold). A vacuum can be applied
inside the tissue processor to assist penetration of the embedding agent.

Processing of tissue is an important step because poorly processed tissue badly affects
the section cutting and staining. The basic aim of processing is to remove water from the
tissue section and to impregnate the tissue with another medium that can give support to the
tissue. The various factors influence the tissue processing such as size of the tissue,
temperature, viscosity, and negative pressure. Tissues are processed mainly by automated
tissue processors which are of two types: tissue transfer processor and fluid transfer
processor. Manual tissue processor is now obscured. It is used only in small laboratory and
in emergency. Microwave processing accelerates the rate of processing. The present
chapter covers all these aspects of tissue processing in histopathology.
2. Give the importance of tissue processing

Most laboratory supervisors would emphasise to their staff the importance of

tissue processing. It is worthwhile to stress that use of an inappropriate processing
schedule or the making of a fundamental mistake (perhaps in replenishing or
sequencing of processing reagents) can result in the production of tissue specimens
that cannot be sectioned and therefore will not provide any useful microscopic
information. This can be disastrous if you are dealing with diagnostic human tissue
where the whole of the specimen has been processed (“all in”). There is no spare
tissue. There is no diagnosis. There is however a patient to whom an explanation has
to be provided.
Although mechanical or electrical faults occasionally occur in tissue processors,
processing mishaps where tissues are actually compromised, mainly occur because
of human error. It is important to emphasise the value of proper education and
training for those carrying out tissue processing and the need to apply particular care
when setting up a processor for any processing run.

3. Give the steps in tissue processing for Paraffin Section

1) Obtaining a fresh specimen

 Fresh tissue specimens will come from various sources. It should be noted that
they can very easily be damaged during removal from the patient. It is important
that they are handled carefully and appropriately fixed as soon as possible after
dissection.

2) Fixation

 The specimen is placed in a liquid fixing agent (fixative) such as formaldehyde

solution (formalin). This will slowly penetrate the tissue causing chemical and
physical changes that will harden and preserve the tissue and protect it against
subsequent processing steps.

3) Dehydration

 All the fixating solution and tissue fluid is removed.They are replaced
with organic solvents such as xylol or toluol. Organic solvents are used
because they are miscible

4) Clearing
 The purpose of clearing is to remove dehydrating agents from tissues and to
prepare the tissues for impregnation with the embedding agent.
 5) Wax Filtration

 Is the process in which the tissues or the specimens are enclosed in a mass of
the embedding medium using a mould. Since the tissue blocks are very thin in
thickness they need a supporting medium in which the tissue blocks are
embedded.

6) Embedding or blocking out

 Is the process in which the tissues or the specimens are enclosed in a mass of the
embedding medium using a mould. Since the tissue blocks are very thin in thickness they
need a supporting medium in which the tissue blocks are embedded. 

4. Give an idea on proper handling of specimens

 Use an Appropriate Schedule

 Maintain Reagent Quality
 Use High-Quality Wax
 Avoid Hazardous Reagent
 Orientate Specimens Carefully
 Choose an Appropriate Mold
 Handle Specimens Gently
 Avoid Excessive Heat
 Check Temperatures Regularly
 Do Not Over-fill Molds

REFERENCES

1) Clayden EC. Practical section cutting and staining. Edinburgh: Churchill Livingstone,
1971.
2) Hopwood D. Fixation and fixatives. In Bancroft J and Stevens A eds. Theory and
practice of histological techniques. New York: Churchill Livingstone, 1996.
3) Carson FL. Histotechnology. 2nd ed. Chicago: ASCP Press, 2007.
4) Winsor L. Tissue processing. In Woods A and Ellis R eds. Laboratory histopathology.
New York: Churchill Livingstone, 1994;4.2-1 - 4.2-39.

SIGNATURE:

Ma. Veronica M. Del Rosario