TISSUE PROCESSING

CECILIA LEKPOR

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 1

 LEARNING OBJECTIVES
• At the end of the lecture, students should be able to

❖Explain the term tissue processing

❖The major steps in tissue processing?
❖Explain manual and automatic processing
❖Factors affecting Tissue Processing
❖Chemical or alterations that can be done to improve tissue processing

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 2

 OVERVIEW
❖Tissues from the body taken for diagnosis of disease processes must be processed
in the histology laboratory to produce sections of slides that are viewed under the
microscope by pathologists
❖Gross examination of the tissue is done by 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

❖Without processing tissue, it is not permanent, it lacks colour and it is

difficult to cut thin sections for microscopy
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 3
 OVERVIEW-CONT’
❖The term tissue processing refers to the treatment of tissue necessary to
impregnate into a medium, so that the tissue is rendered sufficiently firm
yet elastic for tissue sections of desirable thickness to be cut on a microtome

❖The techniques for processing tissues, whether

• Biopsies,
• Larger specimens removed at surgery or
• Tissues from autopsy follow these steps:
➢Specimen Accessioning
➢ Gross examination
➢ Fixation
➢Tissue processing (Dehydration, Clearing, Infiltration and embedding)
➢Sectioning
11/10/2023 and Staining Tissue processing MLP 309- Cecilia Lekpor 4
 HISTO TECHNIQUES
❖ Histological techniques deals with the preparation of tissue for microscopic
examination

❖The aim of a good histological technique is to preserve microscopic anatomy of

tissue and make them firm, so that sections can be made

❖ After staining, the section should represent the anatomy of the tissue as close as
possible to their structure in life

❖The person/s who process the tissue and makes the microscopic slides is the
HISTOTECHNOLOGISTS

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 5

HISTOPATHOLOGICAL TECHNIQUE-CONT
❖There are 3 main techniques used in preparing sections from
tissues for microscopy:

➢The paraffin technique (It is the most common method)

➢The celloidin technique (It is the most perfect method)

➢The freezing technique (It is the most rapid method)

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 6

 INITIAL PROCESSES
➢Selection of tissue block and accessioning

➢Special tissue treatment after the use of certain fixatives

➢Treatment of hard and bony tissues

➢completion of fixation (re-fixation)

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 7

 SELECTION OF TISSUE
❖The pathologist usually does the gross examination of the tissue by describing the
specimen (organ type, size, shape, consistency and colour) 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

❖The histotechnologist/BMS records the description behind the request card that
accompanied the specimen

❖The size of the tissue selected should be 3cm in maximum dimension but should
not be more than 4mm thick

❖Indication of whether part or whole tissue sample taken should be made

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 8
 SPECIAL TREATMENT OF TISSUES
❖Picric-acid containing fixatives:
❖ It does almost as well as mercurial fixatives with nuclear detail but does not
cause as much hardness

➢Picric acid in tissues may hinder wax infiltration ribbons

➢Tissues left in fluid for longer than 12 to 24 hours become hard and brittle and
difficult to section

➢Tissues fixed in alcoholic picric acid-based fixatives should be washed in 100%

alcohol
➢Those fixed in non-alcoholic picric acid fluids should be washed repeatedly in
50-70% alcohol 11/10/2023
Tissue processing MLP 309- Cecilia Lekpor 9
POTASSIUM DICHROMATE- K2Cr207
➢Potassium dichromate may be used to identify adrenal medullary tumours, both
macroscopically and microscopically

➢It reacts with adrenal medullary catecholamines, producing a black or brown

precipitate which is water-insoluble and insoluble green-brown precipitate with
reaction with alcohol

➢Thorough washing of the tissue fixed in dichromate is required to avoid forming

an oxide in alcohol which cannot be removed later (over night washing preferred)

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 10

Osium tetroxide (OsO3)
❖Osium tetroxide (OsO3)- reduced to black Os in ethanol and so tissues fixed in
OsO3-based fixatives should be washed for 5 hours in running water and
dehydration started in 30% ethanol

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 11

 TREATMENT OF BONY TISSUE
❖Bone or tissues containing calcified material require decalcification prior to
processing to enable standard Microtomy

❖ Various decalcifying fluids can be used, including acids, e.g., nitric acid,
chelating agents, e.g., ethylene diamino-tetraacetic acid (EDTA)
❖Tissues such as uterus, leiomyomas, keratinized and calcified tissues
are softened by fixing in:

➢4% phenol in 70% alcohol

➢Mixtures of glycerol, alcohol and aniline

➢ Commercially
11/10/2023
eg “Mollifex”
Tissue processing MLP 309- Cecilia Lekpor
prepared solutions 12
 PRINCIPLES OF TISSUE PROCESSING
❖The importance of tissue processing is to embed the tissue in a solid medium firm
enough to support the tissue and give it sufficient rigidity to enable thin section to
be cut and yet soft enough not to damage the knife or the tissue

❖The aim of tissue processing is to substitute the aqueous fixative within the
specimen by non-water-miscible paraffin wax

❖To ensure good tissue processing:

➢ It is essential that tissue slices are no more than 2–3 mm thick for rapid/urgent
processing schedules

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 13

PRINCIPLES OF TISSUE PROCESSING-CONT’
➢Tissue of thickness 3–5 mm should be for routine, overnight and weekend
schedules

➢Tissue crammed into the processing cassette prevent the reagents from circulating
adequately

➢Tiny biopsies may be placed into commercially available biopsy bags or mesh
biopsy cassettes which, in turn, are placed inside the processing cassette

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 14

 PROCESSING PROCEDURE
❖The main steps involved in tissue processing

➢Dehydration
➢Clearing
➢Infiltration AND Embedding

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 15

 DEHYDRATION
➢Most tissue fixatives are made in aqueous solution and the function of the
dehydrating agent is to remove free and bound water molecules from the tissue
specimen. The first step in processing is dehydration

➢Water is not miscible with paraffin wax, so dehydration is necessary in order not
to interfere with the next steps-clearing and infilteration

➢The most employed dehydrating agent, for routine paraffin processing, is 99.85%
ethanol, due to its cost, is purchased by laboratories in the form of the less
expensive 99% industrial methylated spirit (IMS) containing 2% methanol

➢Tissues are dehydrated by using increasing strength of alcohol; e.g., 60%,70%,

80%, 90% and 100%

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 16

 DEHYDRATION-CONT
➢The duration for which tissues are kept in each strength of alcohol depends
upon the size of the tissue, fixative used and the type of tissue

➢Delicate tissues may need to be processed slowly from 50% ethyl alcohol,
whereas tissues that have been fixed in an alcoholic reagent such as
Carnoy’s fixative may be immersed directly into several changes of 99%
alcohol

➢ Most routine histological processing schedules employ 70% alcohol as the

first step in tissue dehydration

➢Generally, tissue blocks of 1 mm thickness require 30 minutes in each

graded alcohol and tissue blocks of 5 mm thickness require up to 90
minutes in each graded alcohol
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 17
 DEHYDRATING AGENTS
❑Alcohols are clear, colorless, flammable, hydrophilic liquids, miscible with water
and, when anhydrous, with most organic solvents
❑In addition to their role as dehydrants, alcohols also act as secondary coagulant
fixatives during tissue processing
❖EHTANOL
➢Supplied as 99.85% ethanol and 99.85% ethanol denatured with 2% methanol
➢ Ethanol is a rapid, efficient and widely applicable dehydrant

➢Processing times in absolute ethanol should be minimal

➢ Progressive removal of bound water from carbohydrates and proteins during

prolonged immersion in absolute ethanol causes tissues to harden excessively and
become brittle making section difficult to cut
11/10/2023
Tissue processing MLP 309- Cecilia Lekpor
18
 DEHYDRATING AGENTS-CONT’
❖METHANOL
➢Good ethanol substitute but rarely used for routine processing because of its
volatility, flammability and cost
➢ It is a poor lipid solvent and will not dissolve nitrocellulose unless mixed with
acetone. In microwave processing it tends to harden tissues more than ethanol
❖ISOPROPANOL
➢Available as 99.8% (absolute) isopropanol
➢Slightly slower in action and not as hygroscopic as ethanol, but a far superior lipid
solvent
➢Completely miscible with water and most organic solvents, melt paraffin wax and
readily expelled from tissues and wax baths
➢ Shrinks and hardens tissues less than ethanol
➢Used to dehydrate hard, dense tissues, which can remain in the solvent for
extended
11/10/2023 periods without harm 19
Tissue processing MLP 309- Cecilia Lekpor
 DEHYDRATING AGENTS-CONT’
❖ACETONE
➢Colorless flammable liquid with low toxicity and is freely miscible
with water and organic solvents

➢It is a fast, effective dehydrant though it may cause tissue shrinkage; it

may also act as a coagulant secondary fixative

➢ Acetone is the best dehydrant for processing fatty specimens

➢Tissues are dehydrated through four changes of acetone, the last of

which should always be fresh
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 20
 CLEARING
❖The clearing agent is a reagent that is miscible with paraffin wax and acts as a
link between the dehydrating agent (non-miscible) and the paraffin wax itself

❖Xylene, is the most used clearing agent. Usually, 2-3 changes of xylene removes
the dehydrating agent and renders tissue translucent because they have similar
refractive index to that of the tissues making them visible under the microscope

➢Other clearing agents used for routine tissue processing are hydrocarbons such as
toluene, Benzene chloroform and petroleum solvents
➢Toluene is a possible carcinogen
➢chloroform has a narcotic effect

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 21

 CLEARING –CONT’
❖Benzene-This is an excellent clearing agent. It is rapid, causes minimal
shrinkage, does not render tissues excessively hard or brittle
➢It is highly flammable and not recommended for use as a clearing agent because
of its toxicity – causes aplastic anaemia – and has carcinogenic properties
➢Both Benzene and Toluene are carcinogenic and toxic in nature

❖Cedar wood oil- is the best but very costly and difficult to eliminate from tissues
during wax infiltration
➢ It is particularly useful for processing dense tissues such as uterus, certain
carcinomas and has a role in forensic histopathology in processing the hardened
skin margins of electrical burns and bullet wounds

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 22

 CLEARING-CONT’
➢Xylene is a potential carcinogen, may cause headaches as a side-effect to
inhalation of the vapour where adequate extraction facilities are not provided
(usually in cover slipping and manual staining areas)
➢Xylene is probably the most widely used clearing agent

➢ It is compatible with all the major enclosed tissue processors and is relatively
cheap
➢Prolonged exposure to xylene may cause excessive tissue hardening and tissue
processing schedules should be formulated to minimize this effect

➢Tissues of 1–2 mm thickness require two or three changes of xylene over 0.5–1
hour
➢ Specimen blocks of 3–5 mm thickness require three changes over a 2–4 hours
period for normal processing
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 23
 INFILTRATION
➢Paraffin wax is the infiltration and embedding medium of choice for routine
histopathology
➢Is the saturation of tissue cavities and cells by a supporting substance which is
generally, but not always, the medium in which they are finally embedded

➢Paraffin wax is categorized by its melting point

➢Melting points, although not wholly accurate, are in the range 40–70℃ and, as a
rule, the higher the melting point the harder the wax

➢In histopathology, paraffin wax of melting point 56–58℃ is normally employed

➢Softer tissues benefit from infiltration by lower melting point waxes and harder
tissues from infiltration by those
11/10/2023 with
Tissue processingaMLP
higher melting point
309- Cecilia Lekpor
24
 INFILTERATION-CONT’
➢Infiltration times are greatly reduced by the application of a vacuum.
This is essential for specimens such as lung, where the vacuum forces
out trapped air molecules from the tissue

➢Additives have been included in some paraffin waxes to improve their

crystalline consistency by reducing their hardness and brittleness,
hence, improve their microtomy characteristics

➢Additives such as plasticizers, thermoplastic resins, plastic polymers

and dimethyl sulphoxide (DMSO), aids infiltration and allows thin
sectioning of tissues
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 25
 PROCESSING METHODS
❖There are two methods of processing tissues

➢Manual processing

➢Automatic Processor- using machines

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 26

 MANUAL PROCESSING
❖Manual tissue processing is usually undertaken for the following reasons:
➢Power failure or breakdown of a tissue processor
➢When there is a need for a non-standard processing schedule especially for:

▪ Rapid processing of an urgent specimen, delicate material, very large or thick

tissue blocks
▪ Hard, dense tissues (nitrocellulose methods)
▪ Special diagnostic, teaching or research applications
▪ Quick processing for very small tissues which require special attention
▪ Usually done using wide –mouth containers with reagents and the tissue in a
cassette is moved from on reagent to the other
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 27
 AUTOMATIC TISSUE PROCESSOR
❖There are two broad types of automatic tissue processors
➢Carousel-type ‘open’ tissue processors

➢ Enclosed processors-vacuum impregnation processors (VIP) ,

Microwave processors and Ultrasound processors

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 28

 CAROUSEL TYPE PROCESSOR
❖There is transfer of tissues contained within a basket, through a series of
stationary reagents (12) arranged in-line or in a circular carousel plan

❖Electronic devices that can allow the machine to be programmed so that at a

given time, the central arm moves the sample basket to the next solution

❖Agitation is produced by a rotational or dunking motion and heat is available on

the final wax baths

❖Open processors release reagent vapour into the atmosphere as the tissue basket
moves between stations and, therefore, should be contained within a well-
ventilated area/extraction facility
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 29
CAROUSEL TYPE
PROCESSOR

11/10/2023
Tissue processing MLP 309- Cecilia Lekpor 30
 THE ENCLOSED PROCESSOR
➢Enclosed processors are stand-alone/modular units which fully contain
reagent fumes during tissue processing by means of water/vapour traps
and charcoal adsorption filters

➢ Reagents are pumped into and out of a processing chamber

➢ Agitation is provided in the form of tidal flow of reagents in and out

of the chamber or by a side-to-side rotational motion

➢A vacuum and heat created in the tissue container aids in processing

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 31
 ENCLOSED (VIP) TISSUE PROCESSOR
Control Panel

Sample
Chamber

Reagent baths

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 32

ENCLOSED (VIP) TISSUE PROCESSOR

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 33

PROCESSING PROTOCOLS

❖Protocol schedules are done either indicating the position of the

reagent, name, time and condition of processing

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 34

 OVERNIGHT PROCESSING SCHEDULE
POSITION REAGENT TIME (Minutes) OPERATING CONDITION
1 10% Formalin 60 Heat/Agitation/Vacuum
2 70% Alcohol 45 Heat/Agitation/Vacuum
3 80% Alcohol 60 Heat/Agitation/Vacuum

4 90% Alcohol 60 Heat/Agitation/Vacuum

5 95% Alcohol 90 Heat/Agitation/Vacuum
6 100% Alcohol 90 Heat/Agitation/Vacuum
7 100% Alcohol 90 Heat/Agitation/vacuum
8 100% Alcohol 120 Heat/Agitation/Vacuum
9 Xylene 90 Heat/Agitation/vacuum
10 Xylene 120 Heat/Agitation/Vacuum
11 Wax 90 Heat/Agitation/Vacuum
Tissue processing MLP 309- Cecilia Lekpor 35
12 11/10/2023 Wax 120 Heat/Agitation/Vacuum
 SHORT SCHEDULE PROCESSING
Postion Reagent Time Operating Condition
(Minutes)
1 Carnoy’s fluid 45 Heat/Agitation/Vacuum

2 100% 15 Heat/Agitation/Vacuum

3 100% 15 Heat /Agitation/Vacuum

4 100% 15 Heat/Agitation/Vacuum

5 100% 15 Heat/Agitation/Vacuum

6 100% 15 Heat/Agitation/Vacuum

7 100% 15 Heat/Agitation /Vacuum

8 Xylene 10 Heat/Agitation/vacuum

9 Xylene 15 Heat/Agitation/Vacuum

10 Xylene 5 Heat/Agitation/Vacuum

11 Wax 30 Heat/Agitation/Vacuum
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 36
 RAPID TISSUE PROCESSING
❖Acetone+ Isopropyl alcohol(60-70%)

❖Acetone+ Isopropyl alcohol+ Mineral oil

❖Paraffin + Mineral Oil

❖Paraffin

➢Time spent in each tank is 15min

➢The over all processing time is 1 hour

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 37
FACTORS AFFECTING TISSUE PROCESSING
❖Certain factors affect tissue processing
❖During impregnation, the following factors may affect tissue
impregnation:

➢Nature of processing reagents

➢ Tissue characteristics
➢Processing conditions

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 38

 NATURE OF PROCESSING REAGENTS
❖Viscosity: Viscosity affects the rate at which fluids can penetrate tissues.
The larger the molecule the higher is the viscosity and the slower the rate of
penetration

➢A reason for the common use of paraffin wax as an impregnating medium

is its low viscosity when heated and hence it’s comparative quickness in
impregnation

❖Concentration: High concentration of fluid outside the tissue causes rapid

reagent diffusion with related strong diffusion currents. These may shrink
and disrupt tissues

➢To prevent this, tissues are processed through graded series of solutions
strength
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 39
 TISSUE CHARACTERISTICS
❖Tissue thickness:
➢Block thickness affects the rate of reagent diffusion and processing
time.
❖Tissue Type:
➢Fatty tissues usually require extended processing time as lipids inhibit
the diffusion of processing reagents

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 40

 PROCESSING CONDITION
❖Temperature, pressure and agitation reduce the duration of tissue processing and
improve infiltration
❖TEMPERATURE
➢At low temperatures processing reagents are more viscous and hence their tissue
diffusion rates are slower

➢ An elevated processing temperature increases the kinetic energy of reagent

molecules, decreases reagent viscosity and increases the rate of diffusion of
reagent into the tissue

➢ Gentle heating of the dehydration and clearing reagents, in the range 37–45℃,
will substantially reduce processing times but may increase tissue shrinkage
because of heat on collagen
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 41
 PROCESSING CONDITION-CONT’
➢High temperatures at the infiltration stage may cause undue tissue shrinkage and
hardening
➢ This can be resolved by employing temperatures of 2–3℃ above the melting
point of the infiltration media to minimize these effects
❖VACUUM AND PRESSURE
➢Modern enclosed tissue processing machines incorporate a switchable vacuum
and pressure cycle
➢Application of a vacuum enhances dehydration, clearing and infiltration

➢Vacuum applied during infiltration stages helps to remove air from porous tissue

➢ Tissues, particularly lung, are de-aerated, and the solvent boiling point is reduced,
thus 11/10/2023
facilitating evaporation of the reagent
Tissue processing MLP 309-from the molten infiltration medium
Cecilia Lekpor 42
 PROCESSING CONDITION-CONT’
❖AGITATION
➢The maximum surface area of tissue should be available for fluid/reagent
exchange and circulation during processing
➢This is not achieved in situations where tissue cassettes lie at the bottom of a
container, static in the reagent or are packed tightly into the processing basket
➢Maximum surface area for fluid exchange is impaired and circulation of reagent
around the tissue is impeded
➢The reagent surrounding the tissue remains at a lower concentration and a much
longer time is required for satisfactory processing

➢To achieve consistent processing results, the tissue cassettes should be loosely
packed, suspended and agitated within the medium to facilitate the exchange of
dilute reagent from the tissues with the more concentrated reagent replacing it
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 43
 PROBLEMS IN TISSUE PROCESSING
❖“Floaters" are small pieces of tissue that appear on a slide that do not belong
there--they have floated in during processing

➢ Floaters may arise from sloppy procedure on the cutting bench-- dirty towels,
instruments, or gloves can have tissue that is carried over to the next case
➢ Therefore, it is essential that you do only one specimen at a time and clean
thoroughly before opening the container of the next case

❖What to do:
➢Guard against unrecognized floaters by always separating like specimens in the
numbering sequence

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 44

PROBLEMS IN TISSUE PROCESSING
➢For example, if you have three cases with prostate chips, separate them in
accessioning with totally different specimens such as uterus or stomach

➢That way, if numbers are transposed or labels written wrong or tissue carried over,
then you will have an obvious mismatch

➢During embedding, If reusable moulds are employed, you must be aware that
tissue may potentially be carried over and appear as "floaters“, thoroughly clean
moulds before use

➢Always be sure that you properly identify the tissue! This means that you make
sure that the accession number on the request card matches that on the tissue
casettes
11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 45
 REFERENCES
• HistoPATHMedical Laboratory Technology 4th Edition

• Manual of Histological Techniques-John D Bancroft

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 46

•

THANK YOU

11/10/2023 Tissue processing MLP 309- Cecilia Lekpor 47