Tissue Handling

Krisna Murti

Department of Anatomic Pathology, Faculty of Medicine

University of Sriwijaya
Cellular Pathology
 Levels of Study

 Organisms
 Organs
 Tissues
 Cellular
 Ultrastructure
 Molecular
 Histopathology
• Microscopes as tools

• Staining for H&E, histochemistry or

immunohistochemistry to show specific chemical
contains in cells or tissues

• Observe structural alterations in cells / tissues due to

particular diseases
 Tissue Processing Steps

• Obtaining a fresh specimen

• Fixation

• Dehydration

• Clearing

• Wax infiltration

• Embedding or blocking out

 Pre-analytic

 Collection of samples
 Management and labelling of samples
 Adequate packaging
 Transport
 Pre-analytic

 Collection of samples
 Management and labelling of samples
 Adequate packaging
 Transport
 Histopathological Materials
(Samples/Specimens)
• Specimens are taken under anesthesia in living
human/animal or postmortem Types of Biopsies
 During surgery
• Biopsy: excise, endoscopy, cystoscopy  Puncture (needle biopsy)
 Aspiration
• Operation : appendices, ovary, breasts  Excisional
 Incisional
 Scrape
• Extirpation : Lymph nodes

• Curettage : specific for abnormalities in endometrium,

cervix and utery
Eg. abortus, mola hidatidosa, hormonal
abnormality, malignancies
 Histopathological Materials
(Samples/Specimens)

• Numerous techniques can be used to prepare tissue for

microscopical examination depend on:
o Structures examined
o Nature of examined tissues
o Urgency of investigation
o Fresh or preserved specimens

• Fresh tissue can be examined as:

• Smear: e.g. screening of cancer cervix
• Frozen section using cryostat as urgent conditions e.g. during
surgery
Pathobiology: Mechanism of Disease

• Epidemiology
• Etiology - Causes
• Pathogenesis - Evolution
• Morphology - Structural Changes
• Clinical Significance – Functional Changes
• Management
• Complications
• Prevention
Fixation is very important!!!

Why???
 Autolysis Begins Immediately

Excision
T
Nucleases Autolysis is
i
Proteases accelerated by
m Autolysis increased
e Lipases
temperatures
Saccharidases
Fixation
Dehydration
Embedding
Examination and
Diagnosis

Ancillary Studies
 Cold Ischemic Time
Excision of tissue

• The duration is calculated from when the tissue is

removed from the body to when the tissue is placed
into fixative

• Should be as short as possible (one hour or less)

• Produce deleterious effects such as deterioration of

an epitope

• Ischemic epitopes cannot be recovered using

antigen retrieval techniques
 Fixation Procedures

• Formalin 10% buffer pH 7.0 - 7.4

• The whole tissues rinse in fixation

• Volume of formalin is 10-20 X tissues volume

• If the tissues are too large … lamellation every

0.5-1 cm to ensure fixation enters the tissues

• Duration of fixation 24 – 72 hours

• Penetration of fixation: 1mm/hour fixation

• The chemical reaction rates are slower than

the penetration rates
Proteases
• Destroy protein antigens
• Not all protein antigens are destroyed at the same rate

Protease digestion of tissue

• Has been virtually completely replaced by antigen retrieval approaches for

immunohistochemistry

• Still a major component of many in-situ hybridization protocols

Nucleases
• Destroy nucleic acids
• RNA destroyed much more quickly than DNA
• Found in different levels in different tissues
Pancreas and Eosinophils have extremely high levels
of ribonucleases

Stability
• In general DNA>Protein>RNA
• Some proteins are essentially as stable as DNA
 Fixation

• A chemical process by which biological tissues are preserved

from decay (autolysis or putrefaction)

• Fixation terminates any ongoing biochemical reactions

• Increases the mechanical strength or stability of the treated

tissues
 Purpose of Fixation

• In general is to preserve a sample of biological material (tissue or

cells) as close to its natural state as possible in the process of
preparing tissue for examination

• To achieve this several conditions must be met

The specific aims of fixation are:

• Prevent postmortem degeneration
• Prevent autolysis
• It is effective against hydrolytic enzymes
• Stop the bacterial effect
• Harden the tissues, as fixation causes coagulation of proteins
• Fixation has a mordanting effect, facilitating subsequent staining of tissues
 Features of Fixatives

• Acts to disable intrinsic biomolecules – particularly proteolytic enzymes – which would

otherwise digest or damage the sample.

• Protect a sample from extrinsic damage as fixatives are toxic to most common
microorganisms (bacteria in particular) which might exist in a tissue sample or which might
otherwise colonise the fixed tissue

• Alter the cells or tissues on a molecular level to increase their mechanical strength or
stability.

• The increased strength and rigidity can help preserve the morphology (shape and structure) of
the sample as it is processed for further analysis such as Nuclear Morphometry System
 Features of Fixatives

Note:
• Even the most careful fixation does alter the sample and
introduce artifacts that can interfere with interpretation of
cellular ultrastructure

• A prominent example is the bacterial "mesosome", which was

thought to be an organelle in gram-positive bacteria in the
1970s, but was later shown by new techniques developed for
electron microscopy to be simply an artifact of chemical
fixation
Characteristics of A Good Fixative Agents

1. Kill the cell quickly without shrinkage or swelling

2. Penetrate the tissue rapidly
3. Inhibit bacterial decay and autolysis
4. Harden the tissue
5. Render it insensitive to subsequent treatment as staining
6. Allow tissue to be stored for long time
7. Simple to prepare
8. Economic
 Fixation Process

• Fixation is usually the first stage in a multistep process to prepare a sample of

biological material for microscopy or other analysis

• The choice of fixative and fixation protocol may depend on the additional
processing steps and final analyses that are planned

• For example
 Immunohistochemistry utilizes antibodies which bind to a specific
protein target
 Prolonged fixation can chemically mask these targets and prevent antibody
binding
 In these cases, a 'quick fix' method using cold formalin for around 24 hours is
typically used
 Types of Fixation
• Heat fixation:
After a smear has been allowed to dry at room temperature, the slide is gripped by tongs or a clothespin
and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the
slide.
Heat-fixation method can be successfully used for preparing Gram-negative and Gram-positive
bacteria samples for studies

• Perfusion:
• Fixation via blood flow
• The fixative is injected into the heart with the injection volume matching cardiac output
• The fixative spreads through the entire body, and the tissue doesn't die until it is fixed
• Advantage of preserving perfect morphology
• Disadvantages that the subject dies and the cost is high (because of the large volume of fixative needed for larger
organisms)

• Immersion:
• The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of the tissue
to be fixed
• The fixative must diffuse through the tissue in order to fix, so tissue size and density, as well as the type of fixative
must be taken into account
• Using a larger sample means it will take longer for the fixative to reach the deeper tissue.
 Types of Fixative Agents

• Crosslinking fixatives
• Aldehydes
• Oxidising agents
• Precipitating fixatives
• Ethanol
• Methanol
• Acetone
• Other fixatives
• Picric acid
• Mercuric chloride
 Crosslinking fixatives “Aldehyde”

• Creating covalent chemical bonds between proteins in tissue

• Proteins solubilize to the cytoskeleton, and provides additional rigidity
to the tissue
• The most commonly used fixative in histology is the crosslinking fixative
formaldehyde (also named formalin)
• Usually used as a 10% Neutral Buffered Formalin (NBF), that is approx.
3.7%-4.0% formaldehyde in phosphate buffered saline
• Formaldehyde is a gas at room temperature, formalin-formaldehyde gas
dissolved in water (~37% w/v)-is used when making the former fixative
 Crosslinking Fixatives “Aldehyde”

• Paraformaldehyde is a polymerized form of formaldehyde, usually

obtained as a fine white powder, which depolymerises back to formalin
when heated
• Fixes tissue by cross-linking the proteins, primarily the residues of the
basic amino acid lysine
• Its effects are reversible by excess water and it avoids formalin
pigmentation
• Other benefits
 Long term storage
 Good tissue penetration
 It is particularly good for immunohistochemistry techniques
 Can be used as a fixative for cell smears
 Crosslinking Fixatives “Aldehyde”

• Another popular aldehyde for fixation is glutaraldehyde

• Similar mechanism to formaldehyde
• Larger molecule, may not penetrate thicker tissue specimens as
effectively as formaldehyde
• May offer a more rigid or tightly linked fixed

• Some fixation protocols call for a combination of formaldehyde and

glutaraldehyde, so that their respective strengths complement one another

• These crosslinking fixatives – especially formaldehyde – tend to preserve

the secondary structure of proteins and may protect significant amounts of
tertiary structure as well
 Crosslinking Fixatives “Oxidizing agents”
• The oxidising fixatives can react with various side chains of proteins and
other biomolecules, allowing the formation of crosslinks which stabilise tissue
structure

• Osmium tetroxide is often used as a secondary fixative when samples are

prepared for electron microscopy

• It is not used for light microscopy as it penetrates thick sections of tissue very
poorly

• Potassium dichromate, chromic acid, and potassium permanganate all

find use in certain specific histological preparations
Precipitating Fixatives “Denaturing fixatives”

• The precipitation and aggregation of proteins is a very different process from

the crosslinking

• by reducing the solubility of protein molecules and by disrupting the

hydrophobic interactions which give many proteins their tertiary structure

• The most common precipitating fixatives are ethanol, methanol and Acetone

• Acetic acid is a denaturant that is sometimes used in combination with the other
precipitating fixatives

• The alcohols, by themselves, are known to cause shrinkage of tissue during

fixation while acetic acid alone is associated with tissue swelling, so combining
the two may result in better preservation of tissue morphology
Oxidizing Agents
• React with various side chains of proteins and other biomolecules, allowing formation of crosslinks that
stabilize tissue structure
• Cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary
fixatives

Osmium tetroxide
Often used as a secondary fixative when samples are prepared for electron microscopy
Not used for light microscopy as it penetrates thick sections of tissue very poorly

Potassium dichromate, chromic acid, and potassium permanganate

all find use in certain specific histological preparations

Mercurials
• Mercurials such as B-5 and Zenker's fixative have an unknown mechanism that increases staining
brightness and give excellent nuclear detail
• Fast but penetrate poorly
• Produce tissue shrinkage
• Their best application is for fixation of hematopoietic and reticuloendothelial tissues
• Note that since they contain mercury care must be taken with disposal
Oxidizing Agents

Picrates
• Penetrate tissue well to react with histones and basic proteins to form crystalline
picrates with amino acids and precipitate all proteins
• It is a good fixative for connective tissue, preserves glycogen well, and extracts
lipids to give superior results to formaldehyde in immunostaining of biogenic and
polypeptide hormones
• Causes a loss of basophilia unless the specimen is thoroughly washed following
fixation

HOPE fixative
• Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives
formalin-like morphology, Excellent preservation of protein antigens for
immunohistochemistry and enzyme histochemistry
• Good RNA and DNA yields
• Absence of crosslinking proteins
Procedures in Sending The Specimens

1. Fill in forms completely

2. Send the specimens in adequate fixation
(formalin 10% buffer, amount is 10-20 X tissue
volume)
 Analytic

Tissue Processing
The steps required to take 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

• Manually (hand processing)

• Automated tissue processing machine
(“tissue processor”)
 Principles of Tissue Processing

• Preservative action

• Provides stability

• Protects from infection

• Prevents autolysis

• Permits sectioning and staining

Identification of Surgical Specimen

• Clinical Data-Form
• Adequate specimen
• Proper Fixative-
Lamellation for big size
• 10% buffered Formalin
 Gross/Macroscopic Examination

• Direct examination by eyes or by touching

• Observation and technique of morphology

• Gross appearance:
• Sizes and shape: enlargement / smaller
• Consistency: soft, hard, solid, fragile
• Color: pale, yellow, brownish,
• Weight
• Surface
• Edge of section
Gross/Macroscopic Examination

Description:
• Specimen weight and
measurement
(approximately)
• Consistency
• Photo *
• Cut section
Inking the margins

• To mark surgical margin

• Spread of lesion
• Malignancy
• Adequacy of removal
• Different colors to identify
margins
Preparation for Processing

• Edge of lesions
• Wall of cysts
• Include normal areas
• Avoid necrotic area
• Whole specimen if small
• Direction, mark
Preparation for Processing

• Specimen placed in porous plastic

cassettes
• Size is (2 x 2 x 0.5) cm
• Thickness is no more than 5mm
• In 10% formalin
 Processing
• Dehydration; removal of water, in graded
series of alcohol 50%, 70%, 95%, 100%
• Clearing; removal of alcohol with “xylene”
that will be miscible with the embedding
medium (paraffin)
• Impregnating with paraffin
• Embedding
 Dehydration
 Removal of water
 Replacing formalin with alcohol in
gradual sequence (70, 95, 100%)
 Paraffin is able to enter tissue

A typical dehydration sequence for specimens not more

than 4mm thick would be:

• 70% ethanol 15 min

• 90-95% ethanol 15 min
• 100% ethanol 15 min
• 100% ethanol 15 min
• 100% ethanol 30 min • Automatic tissue processor
• 100% ethanol 45 min • Overnight process
 Clearing and Impregnating

• Removal of alcohol with “xylene”

that will be mixable with the
embedding medium (paraffin)

• Impregnating with paraffin

 Precausion of Pre-embedding in Wax/Paraffin

The wax is clear of clearing agent

No dust particles must be present

Immediately after tissue embedding, the wax must be rapidly cooled to reduce
the wax crystal size
 Embedding
Process by which tissues are surrounded by a medium such as agar, gelatin, or wax which when solidified
will provide sufficient external support during sectioning

Procedures:
• Form paraffin block then taken for sectioning
• Tissue cassette is placed in melted paraffin
• Fill mold with hot paraffin
• Tissue is placed in mold
• Coolling down

paraffin block
 Preparation of Sectioning

• Microtome
• Ribbon
• Warm water
• Glass slides
• Cover slides
Sectioning

• Microtome
• Thickness: 3-10 microns
• Ribbon of sections
• Taken on hot water bath
 Microscope slide preparation

• Picking up floating sections • Taking the section onto slide

onto slides • Flat, no air bubbles, no stretch
• Common “float” artefact or breaks

Water bath
 Staining Procedure

• Machine

• Deparaffinize and hydrate to water • Manual

• Mayer's hematoxylin for 15 minutes
• Wash in running tap water for 20 minutes
• Counterstain with eosin from 15 seconds to 2 minutes depending
on the age of the eosin, and the depth of the counterstain desired
• For even staining results dip slides several times before allowing
them to set in the eosin for the desired time
• Dehydrate in 95% and absolute alcohols, two changes of 2
minutes each or until excess eosin is removed. Check under
microscope
• Clear in xylene, two changes of 2 minutes each
• Mount in Permount or Histoclad
Automated staining

• Routine stain H&E

• Hematoxylin (basic)
• Eosin (acidic)
• Nucleus is acidic and
cytoplasm is relatively
basic
• Special stains
 Cover Slipping

• Clearing agent– xylene

• Thin glass coverslips to protect
the section
• Using mounting media (Eg. DPX,
Resins, Canada balsam etc.)

*
 Staining with Haematoxylin-Eosin (Routine)

Results
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying different tissue components
Microscopic Examination

Interpretation?
Post-analytic
 Reporting
Staining results
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying
different tissue components
• Additional sections
• Deeper / Thinner sections
• Special Stains/techniques
• Reference
• Discussions with Clinicians
• Diagnosis
• Report Typing
Reporting and Data Recording
Take Home Messages
Thank you