MODULE 2.

1: TISSUE PROCESSING
3. SMEAR PREPARATION
• process of examining sections or sediments whereby
COURSE CONTENT
cellular materials are spread lightly over the slide by
I Introduction to tissue processing
A Overview the means of wire loop or applicator stick or by making
B Methods of tissue examination an opposition smear with another slide.
i Fresh tissue • useful in cytological exam particularly for cancer
ii Preserved tissue diagnosis.
C Receiving, custody and identification of tissues TYPES OF SMEAR PREPARATION
D Grossing and section cutting
INTRODUCTION TO TISSUE PROCESSING
HISTOLOGY VS HISTOPATHOLOGY
• HISTOLOGY is the microscopic study of the normal
tissues of the body while
• HISTOPATHOLOGY is the microscopic study of
tissues affected by disease.
• The procedures adopted for the preparation of material
such studies are known as histologic or
histopathologic technique.
TISSUE PROCESSING
• Tissues from the body taken for diagnosis of disease
processes must be processed in the histology
laboratory to produce microscopic slides that are
viewed under the microscope by pathologists. •
HISTOTECHNIQUES
• The techniques for processing the tissues, whether
biopsies, larger specimens removed at surgery, or
tissues from autopsy
• Histotechnologists.
PRINCIPLE :
- Remove extractable water from the cell/tissue and
replaced it with support medium that provides sufficient
rigidity
- Enables sectioning of tissue without parenchymal
damage
- AIM: Preserving the tissue with minimal distortion. streaking
- In order to cut thin sections of the tissues: • The material is gently & rapidly applied in direct or
o have suitable hardness and consistency zigzag line same as in microbiological smears.
when presented to the knife edge spreading
o can be imparted by infiltrating and • portion of material transferred to a clean slide and
surrounding the tissue with paraffin wax, gently spread into a moderately thick film by teasing
colliding or low viscosity nitrocellulose, mucous strand using applicator stick.
various types of resins or by freezing. Pull-apart
METHODS OF FRESH TISSUE EXAMINATION • Two slides one on top of the other the specimen spread
evenly in between two slide.
1. TEASING OR DISSOCIATION
4. TOUCH PREPARATION/ IMPRESSION SMEAR
• process whereby selected tissue specimens are
immersed in solution of isotonic salt carefully dissected • surface of a freshly cut tissue is brought into contact &
and separated and then examined under the pressed on the surface of slide examined by phase
microscope either unstained by Phase contrast or contrast.
Bright field microscopy or stained with differential • used for breast secretions and bone marrow.
stained. FROZEN SECTION
2. SQUASH PREPARATION • It is utilized to get a rapid/ immediate diagnosis of a
pathologic process.
• a process whereby small pieces of tissue not more than • it is especially recommended when lipids and nervous
1 mm in diameter are placed in a slide and forcibly tissue elements are to be demonstrated.
compressed with another slide or with a cover slip. • frozen sections are usually done on muscle and nerve
biopsies as well as on surgically removed tumors.

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TWO METHODS OF PREPARING FROZEN DEHYDRATION

SECTIONS:
1. COLD KNIFE PROCEDURE
• uses conventional freezing microtome gas supply of
carbon dioxide gas from a CO2 cylinder, or by using an
equipment known as cryostat.
• selected block of tissue, aprrox. 3-5mm thick.
• Dewline – point at which sections may then be cut at
10 um thickness.

Temperatures of cold knife in cold environment:

• Knife = -40deg to -60deg
• Tissue = -5deg to -10deg
• Environment = 0deg to -10deg
2. CRYOSTAT PROCEDURE
• this method makes use of Cryostat, an apparatus used • Removal of water and aqueous fixative from the tissue
in fresh tissue microtomy. • Should be accomplished slowly →gradual increase in
• cryostat consists of an insulated microtome housed in concentration; gradual increase in time
an electrically driven refrigerated chamber with a temp. Excessive dehydration – tissue becomes hard, brittle and
near -20deg where microtome, knife, specimen and shrunken
atmosphere are kept at the same temperature. Incomplete dehydration – impair penetration of clearing agent
• optimum working temp. of cryostat is -18° to -20°C. making the tissue unreception to infiltration
• Typical dehydration sequence (not more than 4mm
SPECIAL PROCESSING TECHNIQUES
thick) would be:
1. FREEZE-DRYING 70% ethanol 15 min
• is a special way preserving tissues by rapid freezing 90 % ethanol 15 min
(quenching) of fresh tissue at -160°C and subsequently 100% ethanol 15 min
removing ice water molecules (dessication) by 100% ethanol 15 min
transferring frozen tissue into vacuum chamber at high 100% ethanol 30 min
temp. e.g. -40°C (sublimation) 100% ethanol 40 min
• tissue thickness around 2mm
Advantages:
CLEARING
✓ It produces minimum tissue shrinkage, and allows
tissues to be processed in a fresh sate.
✓ It causes minimal chemical change on the cells, most
especially on protein components, and less
displacement of tissue and cellular constituents.
✓ Important in enzyme studies.
2. FREEZE-SUBSTITUTION
• is a process of dehydration, performed at temp. low enough
to avoid formation of ice crystals and to circumvent the
damaging effects observed after ambient-temp
dehydration.
• it is fixed in Rossman’s formula or in 1%Acetone and
dehydrated in abs. alcohol. • Intermediary between dehydration and infiltration.
Advantages: • Clearing agent must be miscible to both the
• Relatively more economical and less time-consuming dehydrating reagent used and the infiltrating medium.
than freeze drying. • Translucent appearance of tissue
• For best morphological and histochemical o when the dehydrating agent has been entirely
preservation. replaced by clearing agent.
PRESERVED TISSUES • Typical clearing sequence (not more than 4mm thick)
• A better and more effective means of studying tissues would be:
whether normal or abnormal is by examination of their Xylene 20 min
sections and smears which have been permanently Xylene 20 min
preserved, stained for demonstration of specific xylene 45 min
structures and mounted on glass slides with cover slips INFILTRATION IN PARAFFIN
for permanent keeping. • The melted wax will penetrate inbetween the cells of
• The aim of a good histopathological technique is to the tissues.
produce microscopic preparation of tissue, usually • This process of wax infiltration is a necessary step to
stained, that represent as closely as possible their harden the tissues before their embedding.
structure in life. • Infiltrating/ embedding medium almost always a
HISTOPATHOLOGIC TECNIQUES Paraffin.
FIXATION o Typical paraffin infiltration sequence (not
• Stabilizes cellular and tissue protein. more than 4mm thick) would be:
• Resistant to autolysis by inactivating lysosomal enzymes. Paraffin wax 30 min
• Specimen must be dissected to 2-4mm thick. Paraffin wax 30 min
• Must not overflow the cassette. Paraffin wax 45 min
• Different processing time for large and small tissue cuts.

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EMBEDDING • The specimens are accessioned by giving them a

• Impregnated tissues are placed in a mold with their number that will identify each specimen for each
labels and then fresh melted wax is poured in it and patient.
allowed to settle and solidify. GROSS EXAMINATION & DISSECTION
• Once the block has cooled sufficiently to form a surface • Gross examination consists of describing the specimen
skin it should be immersed in cold water to cool it and placing all or parts of it into a small plastic cassette
rapidly. which holds the tissue while it is being processed to a
• After the block has completely cooled it is cut into paraffin block.
individual blocks and each is trimmed. • Initially, the cassettes are placed into a fixative.
• Correct orientation of tissue in a mold is the most • When a malignancy is suspected, then the specimen
important step in embedding. is often covered with ink in order to mark the margins
SECTION – CUTTING of the specimen.
• This is done with a microtome. • Different colored inks can be used to identify different
• Good microtomy techniques will minimize artifacts that areas if needed.
can lead to difficult diagnostic interpretation of special • sections are made and processed, the ink will mark the
stains. actual margin on the slide.
• One of the most directly correlated factors is thickness
in which specimen is cut.
STAINING
• They must be stained to create contrast.
• Dried in a 60°C then passed through another series of
chemical reagents.
• Xylene remove the paraffin and absolute alcohol
removes xylene.
• Different staining procedures are done depending on
the tissue component that is being studied
MOUNTING OF TISSUE SECTIONS
• Temperature of the warm bath should be kept at 5 –
10°C below the melting point of the embedding wax.
• Ribbon must not be left in the bath for more than 1 – 2
mins
• Dangerous mistake that can take place in mounting
sections from floatation baths is “shedding” of friable
small tissue fragments.
FACTORS AFFECTING THE RATE OF TISSUE
PROCESSING:
• Tissue Density and Thickness – variable tissue
density affects infiltration and subsequent sectioning of
tissues.
• Agitation – increase exposure of tissue
• Heat/Temperature – Increase the rate of penetration
and fluid exchange SPECIMEN DISSECTION PLANS
• Viscosity – resistance to the flow of fluid; reagent used
in tissue processing must have the same viscosity • Small samples
• Vacuum and Pressure – remove reagent from the • Rarely need dissection
tissue thereby facilitate rapid infiltration of embedding • Load all
reagent • Must be placed in nylon bag or filter paper
• Tissue markers (eosin) to highlight from background
RECEIVING, CUSTODY AND IDENTIFICATION OF
SURGICAL PROCEDURES TO OBTAIN SPECIFIC
TISSUES
TYPES OF TISSUES:
SPECIMEN RECEPTION:
FINE NEEDLE ASPIRATION
• Separate room → act as interface between the
pathology lab and the hospital/staff &/or the client
• Key point – receive samples safely and securely
• The specimen’s identify must be secured prior to entry
to the pathology laboratory
• Correlation of the specimen against the
• clinical request form is MANDATORY
• Check for corroborative data, validate with multiple
sources of cross references
• “Two-person” rule
• Once fully validated and properly identified can the
specimen passed to the dissection room.
• Specimens are received, sorted, entered into the
Laboratory Information System (LIS), labeled with • Is the simplest, least invasive test and uses smallest
barcoded labels and processed. needle to simple remove cells from the area of
abnormality.
•

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CORE NEEDLE BIOPSY

• Removes not only cells, but also a small amount of the

surrounding tissue.
INCISIONAL & EXCISIONAL BIOPSY

• Where tissue is scooped or spooned to remove tissue

or growths from body cavity such as endometrium or
cervical canal.

REFERENCES

Notes from the discussion by

CPU-College of Medical Laboratory Science PowerPoint

presentation.
• Incisional biopsy takes out even more surrounding
abnormality tissue but not all while excisional biopsy
generally removes the entire area in question.
PUNCH BIOPSY

• Is considered the primary technique for obtaining

diagnostic full-thickness skin specimens. 3- to 4- mm
cylindrical core of tissues sample.
SHAVE BIOPSY

• Where small fragments of tissue are “shaved” from a

surface (usually skin)
CURRETINGS

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