HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Specimen Collection and Handling

Image-guided biopsy
Important Terminologies
• Combines an imaging procedure - such as X-ray,
1. Pathologist computerized tomography (CT), magnetic resonance
• Someone who studies the causes, nature and imaging (MRI) or ultrasound—with a needle biopsy
effects of diseases
Skin biopsy
2. Biopsy
• A sample of tissue taken from the body in order to • A skin (cutaneous) biopsy removes cells from the
examine it closely surface of your body
• Is used most often to diagnose skin conditions, including
3. Lesion melanoma and other cancers
• In physiology, a structural or biochemical change in
an organ or tissue produced by disease processes Skin biopsy procedures include:
or a wound
1. Shave biopsy
4. Tumor • During shave biopsy, the doctor uses a tool similar
• Refers to a mass to a razor to scrape the surface of your skin

• Medical Technologist – receives the specimen then 2. Punch biopsy

informs the pathologist • During a punch biopsy, the doctor uses a circular
• Surgeon – collects the sample tool to remove a small section of your skin’s deeper
layer
REASONS WHY BIOPSIES ARE PERFORMED • Recommended; Obtain 3 layers of skin (Epidermis,
dermis and subcutaneous tissue)
1. To look for cancer • Punch biopsy: 3-4 mm cylindrical core
2. Help check for cancer spread
3. Check for rejection of a transplanted organ 3. Incisional biopsy
4. To diagnose a health problem • the doctor uses a scalpel to remove a small area of
5. To help determine the best therapy option skin
TYPES OF BIOPSIES 4. Excisional biopsy.
• the doctor removes an entire lump or an entire area
Bone marrow biopsy of abnormal skin
• A needle suctioning out liquid bone marrow from Surgical biopsy
hipbone • If the cells in question can’t be access with other biopsy
• Recommended if an abnormality is detected in the blood procedures or if other biopsy results have been
or if the doctor suspects cancer has originated in or inconclusive, your doctor may recommend a surgical
traveled to the bone marrow biopsy
Endoscopic biopsy 1. Excisional
a. Removes entire mass/lesion
• The doctor uses a thin, flexible tube (endoscope) with a
light on the end to see structures inside the body 2. Incisional
• Special tools are passed through the tube to take a small a. Removes a portion of the mass/lesion; for rapid
sample of tissue to be analyzed diagnosis
• For GI conditions
Importance Of Correct Specimen Collection And
NEEDLE BIOPSY Transport For Histological Examinations
Needle biopsy procedures include:
• Incorrect patient or specimen identification and
Fine-needle aspiration labeling errors may lead to issuing of erroneous reports
• Tissue architecture and especially cellular detail can be
• During fine-needle aspiration, a long thin needle is obscured by improper fixation, making proper tissue
inserted into the suspicious area diagnosis virtually impossible
• Disadvantage: Cells are only seen
• Incorrect orientation or lack of orientation of a
Core needle biopsy specimen, lack of proper identification of orientation
• A larger needle with a cutting tip is used during core sutures in the request form, or lack of clearly excised
needle biopsy to draw a column of tissue out of a margins (e.g.mesorectum) makes it difficult for the
suspicious area. pathologist to comment on surgical excision margins
• Includes cells and surrounding tissue
• Results of ancillary investigations maybe necessary in
Vacuum-assisted biopsy order to make an accurate histopathological diagnosis
• A suction device increases the amount of fluid and cells
that is extracted through the needle • If the report is urgently required, it should be indicated
in the request form.

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• Some types of specimens may require a special 14. Avoid specimen trauma
fixative, or need to be sent unfixed (fresh) for certain
investigations 15. Avoid cross-contamination (always clean equipments)

• It maybe necessary to inform the laboratory prior to 16. Take care with biopsy pads
sending specimens for special or urgent investigations
(eg. intraoperative frozen section) 17. Choose appropriate tissue cassette

Guidelines For Handling And Transport Of 18. Avoid overloading of tissues

Histopathological Specimens
19. Clearly label tissue cassettes
Small biopsy samples
ROUTINE PROCESSING OF HISTOLOGICAL
• The specimen should be collected into a widemouthed SPECIMENS
container with a well-fitting lid containing an adequate
amount of 10% formalin saline (formalin) to completely Biopsy specimen
submerge the specimen
• After its removal, biopsy specimen is put in a container
• The container should be accurately labelled including with a mixture of water and formaldehyde (formalin) or
patient name, age, sex, and ward some other fluid to preserve it

Large biopsy samples • The container is labeled with the patient's name and
other identifying information (e.g. hospital number
• The specimen should be placed in a widemouthed and birthdate) and the site of biopsy (exactly where on
container with a well-fitting lid, containing an adequate the body it was taken from)
amount of 10% formol saline (formalin) to completely
submerge the specimen • It is then sent to the pathology lab

• The container should be larger than the specimen, • Next, the pathologist looks at the specimen without a
preferably a bucket (Do not squeeze the specimen into microscope (gross examination)
the container)
AUTOPSY SPECIMEN
• The container should be accurately labelled including
patient name, age sex Fixed tissues
• A complete autopsy should be performed on all fatal
Frozen sections cases associated with a respiratory disease outbreak.

• Used mainly lot intraoperative diagnosis which will • Lung tissue should also be received from any non-fatal
influence the course of the operation case where a biopsy is performed.

• Tissue sent for frozen section needs immediate and • On all fatal cases, tissues should be collected from all
quick processing and reporting, for which the laboratory major organs and fixed in formalin or embedded in
needs to be ready paraffin

Dos and Don’ts of Specimen Transport Collection and

Transport The following tissues are particularly important:

1. Avoid mechanical trauma (do not shake the specimen) 1. Central (hilar) lung with segmented bronchi
2. Right and left proximal and distal bronchi, upper airways
2. Prevent specimen drying (prior to processing tissue (e.g. epiglottis, larynx, trachea)
must be fixed within less than an hour) 3. Representative pulmonary parenchyma from right and
left lung
3. Avoid heat damage
• Paraffin blocks are usually shipped at room
4. Avoid chemical damage (careful with choice of temperature; they should not be frozen. However, if
formalin/fixative solutions) the weather is
• extremely hot, shipping with a cold pack might prevent
5. Label the specimen properly incidental melting of the paraffin.

6. Ensure prompt fixation • DO NOT FREEZE FIXED TISSUES. For fatal cases, a
preliminary autopsy report should be provided with the
7. Use sufficient fixative and a suitable container tissues.

8. Check for fixative pH (if too acidic may form References:

artifacts/pigments)
Retrieved from:
9. Expedite large specimen fixation Unit 3: Specimen Collection and Handling
PPT of Maria Benneth Caberoy-Palec, RMT, MSMT
10. Avoid unnecessary delays Lecture Discussion of Prof. Joana Rose Saltin, RMT
September 7, 2022
11. Handle specimen gently

12. Check for fixation status

13. Prepare thin slices during cutting

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 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Fixation
Penetration
Steps in Tissue Processing
1. FIXATION ➢ Formalin diffuses into the tissue at the rate of
2. DECALCIFICATION approximately 1mm/hr and slows down as it goes
3. WASHING OUT deeper into the tissues
4. DEHYDRATION
5. CLEARING Volume
➢ 10-25 times the volume of tissues to be fixed
Methods ➢ 10x the basic volume

Chemical Types of Fixatives

Heat Based on action

1. Microwave oven (450watts) method ✓ Preserves the anatomy of the section
2. Flaming of bacterial smears
Microanatomical (all fixative with few exceptions)
Cold method
1. Quenching (rapid freezing. 30sec-1 min) Cytological
2. Fresh frozen
3. Freeze drying Nuclear
4. Freeze substitution preserves nuclear structures (e.g. chromosomes )

FACTORS INVOLVED IN FIXATION 1. Osmium tetroxide (Flemming's fluid)

2. Ethanol (Carnoy's fluid)
1. Hydrogen ion concentration 3. Picric acid (Bouin's fluid)
4. Newcomer's fluid
➢ pH: 6.0-8.0 5. Mercuric chloride (Heidenhain's fluid)
2. Temperature
Cytoplasmic
preserves cytoplasmic structures
➢ 20-22°C - routine, mast cells
➢ 0-4°C- electron microscopy and some histochemistry
1. Osmium tetroxide (Flemming's without Hac)
➢ 40°C- used in tissue processors
2. Mercuric (Helly’s/ Zenker-formol)
➢ 45°C - formalin-fixation of RNA
3. Formalin with “post-chromation”
➢ 55°C- formalin-fixation of DNA
4. Chromate (Regaud's/Muller's/ Orth's fluid)
➢ 60°C- formalin fixation of very urgent
➢ 100°C - formalin fixation of tissues with tuberculosis
Histochemical
✓ preserves the chemical constituents of the cells and
3. Thickness of section
tissues
➢ 1-2 mm2 - electron microscope
1. Acetone (lipase & phosphatase)
➢ 2 cm2- light microscopy
2. Absolute ethyl alcohol (carbohydrate;glycogen)
➢ 0.4 cm- light microscopy (thinness)
3. Alcoholic formaldehyde (carbohydrate in human skin)
4. Picric acid (glycogen)
4. Osmolality
5. Rossman's fluid (glycogen)
6. 10% formol-saline (protein)
➢ slightly hypertonic solutions (400-450 mOsm) produce
7. Newcomer's fluid (carbohydrate;glycogen)
best results
8. Baker's formol-calcium (phospholipids)
9. Digitonin (cholesterol for ultrastructural demonstration)
Duration of Fixation
10. Imidazole osmium tetroxide (post-fixation of lipids)
➢ 2-6 hours – Buffered formalin
Based on active components
➢ 3 hours – glutaraldehyde in electron microscopy
Aldehyde group
Refrigeration
✓ satisfactory for routine paraffin sections, electron
microscopy and for histochemical and enzyme studies
➢ Slows down decomposition

Concentration Formalin solution (37%-40% formaldehyde (formalin)

gas in water)
➢ 10% Formaldehyde ➢ 5-10% solution-working solution
➢ 3% glutaraldehyde ➢ most common
➢ 0.25% glutaraldehyde ➢ Preserves fat, mucin, glycogen hemosiderin & elastic
fibers
Speed ➢ Preserves lipids
➢ For metallic impregnation
➢ Specimen should be placed in a fixative as soon as it is ➢ "Soft fixative"
removed from the body to prevent autolysis and ➢ "Tolerant fixative"
putrefaction ➢ Relatively slow (24 hrs. or more)

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Acrolein ➢ Extensively used for CNS
➢ Electron microscopy ➢ Destroyed by sunlight
➢ Toxic
➢ Lacrimatory 1. Flemmings fluid with HAC
2. Flemming's fluid without HAC
Hydroxyadipaldehyde 3. Zetterqvist fluid
➢ Electron microscopy 4. Palade's fluid
➢ Toxic
➢ Lacrimatory Alcoholic fixatives

Mercuric salts ➢ Causes denaturation & precipitation of protein by

removal of hydrogen bond leading to dehydration
➢ Rapid action (slower beyond 3-4mm depth) ➢ 70-100% concentration
➢ Excellent trichome and brilliant metachromatic staining ➢ Fixative & dehydrant
➢ Best for tissue photography
➢ Always leaves a brown precipitate (HgCh) in all tissue 1. 100% methyl alcohol
➢ Makes tissue unduly hard & brittle 2. 70-100% ethanol
➢ Marked shrinkage 3. Camoy's fluid
4. Newcomer's fluid
Formalin preparations 5. 95% isopropyl alcohol

1. 10% aqueous formalin Picric acid

2. 10% formol saline ➢ Excellent cytoplasmic fixative for demonstration of
3. 10% NBF glycogen
4. Formol-corrosive (formol-sublimate) ➢ Best for trichome staining
5. Alcoholic formalin (Gendre's fluid) ➢ Leaves a yellow stain to the tissue Highly explosive in
6. Alcoholic-acetic-acid-formalin (AAF) the dry form

Glutaraldehyde 1. Bouin's fluid

2. Bouin-Hollande
1. 2.5% (small tissues & needle biopsies;fixed in 2-4 hrs at 3. Brasil's alcoholic picroformol
room tempt.) 4. Alcoholic picric acid
2. 4% (larger tissues less than 4 mm;fixed in 6-8 hrs up to 5. Rossman's fluid
24 hrs) 6. Gendre's fluid

Mercuric chloride Trichloroacetic acid

most common metallic fixative ➢ Poor penetration
➢ Combined with other fixatives
Mercuric chloride preparations ➢ Marked swelling of tissue

1. 5-7% saturated HgCh Acetone

2. Acidic sublimate ➢ Histochemical
3. Buffered sublimate (B-4) ➢ Rapid fixation for brain tissues
4. B-5 ➢ 0-5°C
5. Saturated alcoholic HgCl ➢ Used in freeze-drying & freeze-substitution techniques
6. Schaudinn's-HgCh-alcohol (sublimate alcohol)
7. Huber's SECONDARY FIXATION
8. Olmacher's fluid and Fluid of Carnoy & Lebrun
9. Formaldehyde sublimate ✓ The process of placing an already fixed tissue in a
10. Acetic-HgCh-formalin second fixative
11. Sublimate preparations ✓ Done before dehydration and on deparaffinized sections
a. Zenker's fluid- before staining usually with 10% formalin or 10% formol
b. Helly's fluid saline as a primary fixative

Chromates Purposes:
➢ Good cytoplasmic fixative
➢ Leaves a "stubborn" brown precipitate ➢ To facilitate and improve the demonstration of particular
substances
1. 1-2% chromic acid ➢ To make special staining techniques possible
2. 3% potassium dichromate ➢ The secondary fixative acts as a mordant
3. Orth's fluid ➢ To ensure further and complete hardening and
4. Variant's of Orth's preservation of tissues
5. Regaud's (Muller's)
6. Ciaccio's fluid Post-chromatization
7. San Felice's fluid
8. Kolmer's fluid ✓ A form of secondary fixation whereby a primarily fixed
9. Held's fluid tissue is placed in aqueous solution of 2.5-3% potassium
dichromate for 24 hours
Lead
➢ 4% aqueous solution of basic lead nitrate Purposes:
➢ Lillie's alcoholic lead nitrate formalin ➢ To act as a mordant for better staining effects
➢ To aid in cytologic preservation of tissues
Osmium tetroxide
➢ Best for electron microscopy WASHING-OUT
➢ Good cytoplasmic (golgi bodies & mitochondria) result The process of removing excess fixative from the tissue after
but poor nuclear staining fixation

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 Purposes: References:
➢ To improve staining
➢ To remove artifacts from the tissues Retrieved from:

Required in: Module 2.3: Steps in Tissue Processing

➢ Prolonged fixation in unbuffered formalin PPT by Prof. Maria Benneth Caberoy-Palec, RMT, MSMT
➢ Fixation of blood-containing tissue (eg.spleen) in Discussion by Prof. Joana Rose Saltin
unbuffered formalin September 14, 2022
➢ Fixation in HgCl2 fixative

Reagents used:
1. Tap water
2. 50-70% alcohol
3. Alcoholic iodine

ARTIFACT/FIXATION PIGMENT

Hematin/"formalin" pigment
➢ Fine, diffused, dark brown granules; yellow needle-like in
cluster

Washing-out agent:
1. 10 vol. HaD, Alcoholle picric acid
2. 0.2% NaOH/KOH in 70% alcohol
3. Tap water

Hemozain
➢ Similar to hematin
➢ Located In the liver, spleen. & brain of patients with
malaria

HgCl2
➢ Irregular brown crystals
➢ Extracellular
➢ Monorefringent-fresh
➢ "Maltese cross" birefringence- seen in tissues secondarily
fixed in HgCl2, on prolonged storage

Washing-out agent
1. Iodine
2. Bleached

Chromic oxide
➢ Fine,yellow-brown precipitate
➢ Monorefringent
➢ Extracellular
➢ Caused by failure to wash specimen in water after
chromate/potassium dichromate fixation
➢ Used 1% acid alcohol to remove chromic oxide

"Crush" artefact
➢ Found in surgical specimens (eg. Liver biopsy)
➢ Shown as intense eosinophilic staining at the center of
the tissue
➢ Due to partial coagulation of partially fixed protein by
ethanol or by incomplete wax infiltration

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 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Fixation
Advantages
Practical Considerations of Fixation • It is cheap, readily available, easy to prepare and
relatively stable, especially if stored in buffered solutions
a. Speed • It is compatible with many stains
b. Penetration • It does not overharden tissues
c. Volume • Penetrates tissues well.
a. Consider volume to fully penetrate tissue
d. Duration of Fixation Formaldehyde/ Formalin Derivatives

Mechanism of Action of Chemical fixatives 1. 10% neutral Buffered Formalin

a. To avoid formalin pigments (?)/ artifacts due to
1. Crosslinking acidification
a. (e.g., Aldehydes) that act by creating covalent
chemical bonds between proteins in tissue. b. It prevents precipitation of acid formalin pigments
on postmortem
b. This anchors soluble proteins to the tissue.
cytoskeleton, and lends additional rigidity to the
tissue.
2. 10% Formol-saline
c. Hardening due to chemical bonds of proteins in a. This is a simple microanatomical fixative made up
tissue and fixative sol. of saturated formaldehyde (40%, by weight
volume) diluted to 10% with sodium chloride
2. Precipitating (or denaturing)
a. (e.g., alcoholic fixatives) that act by reducing the b. It preserves microanatomic and cytologic details
solubility of protein molecules and (often) by with minimum shrinkage and distortion
disrupting the hydrophobic interactions that
give many proteins their tertiary structure. c. Large specimens may be fixed for a long time
provided that the solution is changed every three
b. The precipitation and aggregation of proteins is months.
a very different process from the crosslinking
that occurs with the aldehyde fixatives. d. It preserves enzymes and nucleoproteins.
e. It demonstrates fats and mucin.
According to COMPOSITION f. It does not over-harden tissues, thereby facilitating
dissection of the specimen.
A. Simple Fixatives
are made up of only one component substance. 3. Zine formalin

1. Aldehydes 4. Formol-corrosive
a. Formaldehyde a. Formol-Sublimate
b. Glutaraldehyde
2. Metallic Fixatives b. Cytological structures and blood cells are well
a. Mercuric chloride preserved. There is no need for "washing-out".
b. Chromate fixatives Tissues can be transferred directly from fixative to
3. Picric acid alcohol.
4. Acetic acid
5. Acetone 5. Paraformaldehyde
6. Alcohol a. a polymerized form of formaldehyde, usually
7. Osmium Tetroxide obtained as a fine white powder, which
depolymerizes back to formalin when heated.
B. Compound Fixatives
b.Glutaraldehyde
are those that are made up of two or more fixatives which • made up of two formaldehyde residues, linked by a
have been added together to obtain the optimal combined three- carbon chain.
effect of their individual actions upon the cells and tissue
constituents. • Glutaraldehyde is a larger molecule than formaldehyde,
and so its rate of diffusion across membranes is slower
According to Composition than formaldehyde.

1. Aldehyde Advantages
• It has a more stable effect on tissues
a. Formaldehyde
• It preserves plasma protein better
• most widely used fixatives is 10% formalin, made from
• It preserves cellular structures better
formaldehyde
• It does not cause dermatitis
• Principle: Crosslinking between lysine residues
• Cross-linkages in the proteins, particularly between
lysine residues. Disadvantages:
• This cross-linkage does not harm the structure of • It is more expensive
proteins greatly, so that antigenicity is not lost. • It is less stable

Formalin – derived from formaldehyde solution PRECIPITATING/ ALCOHOLIC FIXATIVES

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 • It may produce considerable shrinkage of tissues
Alcohol • it is a soft fixative and does not harden some
cytoplasmic structures
• Rapidly denatures and precipitates proteins by
destroying hydrogen and other bonds
Other Metallic Fixative Derivatives
• It must be used in concentrations ranging from 70-100%
because concentrated solutions will produce lysis of Zenker's solution
cells
Zenker-Formol (Helly's) Solution
• not used routinely for tissues because they cause too
much brittleness and hardness
Lillie's B5 Fixative
• Spray cans of alcohol fixatives are marketed to
physicians doing PAP smears Heindenhain's Sua Solution

3. Oxidizing Agents
• can be used to fix and preserve glycogen, pigments,
blood, tissue films and smears
Osmium Tetroxide
100% Methyl alcohol • This is a pale-yellow powder which dissolves in water to
o excellent for fixing dry and wet smears, blood form a strong oxidizing solution.
smears and bone marrow tissues.
• Causes the complete denaturation of protein.
• ex. Flemming's solution, Fleming solution without acetic
95% isopropyl acid

Ethyl alcohol Chromate Fixatives

Carnoy's fixative Chromic Acid

o good nuclear staining and differentiation. • Used in 1-2 aqueous solution, usually as a constituent of
o It preserves Nissl granules and cytoplasmic granules a compound fixative.
well. • It is a strong oxidizing agent.
o preserves nucleoproteins and nucleic acids. • Precipitates all proteins and adequately preserves
carbohydrates.
Clarke's solution
o used on frozen sections and smears Potassium Dichromate
o preserves nucleic acids but extracts lipids • is used in a 3% aqueous solution.
• preserves lipids and mitochondria
Formol-acetic alcohol • Ex. Regaud's (Muller's) Fluid, Orth's fluid
o Similar to alcoholic formalin
Picric Acid
Alcoholic formalin Used in strong saturated aqueous solution.
o Alcohol + formalin It also dyes the tissues.
o Alcohol – ppt; Formalin – cross-linkages
o for fixation or post-fixation of large fatty specimens
Advantages:
(particularly breast), because it will allow lymph
It is an excellent fixative for glycogen demonstration.
nodes to be more easily detected as it clears and
extracts lipids
Ex. Bouin's solution, Hollande's solution, Genre's solution,
Brasil's alcoholic picroforol fixative
Gendre's fixative
4. Lead fixatives
• newcomer's fluid
• recommended for mucopolysaccharides and fixes
connective tissue mucin.
2. Metallic Fixatives
5. Glacial Acetic acid fixatives
Mercuric Chloride
• fixes and precipitates nucleoproteins.
• Most common metallic fixative, frequently used in
6. Trichloroacetic acid
saturated aqueous solutions of 5-7%
• precipitates proteins.
• Penetrates poorly and produces shrinkage of tissues, so
7. Acetone
is usually combined with other fixative agents
• Used at iced cold temperature ranging from -5'C to 4'C.
Advantages
Advantages:
• It penetrates and hardens issues rapidly and well
• It is recommended for the study of water diffusible
• Nuclear components are shown in detail
enzymes especially phosphatases and lipases.
• It precipitates all proteins
• It is used in fixing brain tissues for diagnosis of rabies.
• It is recommended for renal issues. fibrin, connective
tissues and muscles
Disadvantages:
• It preserves glycogen
• It produces inevitable shrinkage and distortion.
• It preserves but does not precipitate proteins
• It dissolves fat
• It evaporates rapidly.
Disadvantage:
• Fumes are irritating to the nose and eyes and may
8. Heat fixation
cause sinusitis.

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• involves thermal coagulation of tissue proteins for rapid References:
diagnosis, usually employed for frozen tissue sections
and preparation of bacteriologic smears. Retrieved from:

B. According to Action: Module 2.2: Fixation

PPT and Discussion by Prof. Saltin
Microanatomical Fixatives September 17, 2022
• are those that permit the general microscopic study of
tissue structures without altering the structural pattern
and normal intercellular relationship or the tissue

Cytological Fixatives
• Are those that preserve specific parts and particular
microscopic elements of the cell itself.

Nuclear fixatives
• those that preserve the nuclear structures in particular
and they usually contain glacial acetic acid as their
primary component.

Cytoplasmic fixatives
• are those that preserve cytoplasmic structure in
particular and they must never contain glacial acetic
acid.

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 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Decalcification and Post-decalcification

METHODS USED TO CHECK FOR COMPLETENESS OF
Decalcification and Post-decalcification DECALCIFICATION

Principle Physical (mechanical)

➢ Hydrolysis of calcium salt releasing calcium ion (Ca++) ➢ Touching, bending, squeezing, pressing, pricking
➢ Removal of calcium ion by different methods.
Radiologic (x-ray)
Methods ➢ Most accurate
1. Use of dilute acid ➢ Not recommended for Hg ++ -fixed tissue
2. Chelation
3. Ion-exchange resin (1-14 days) Chemical (calcium-oxalate test)
4. Electrophoresis ➢ Ca++ in the decalcifying fluid is tested
➢ The decalcifying fluid is to be prepared with distilled
Use of dilute acid water
➢ Involves presumptive and confirmatory tests.
Calcium salt + dilute acid -> Ca++ + dilute acid ->
insoluble salt Presumptive test

HNO3
1. 5-10% HNO3 (12-24)
2. Formol - HNO3 (2-7 days)
3. Perenyi’s fluid (2-7 days)
4. Phloroglucin - HNO3 (12-24 hrs)

HCl
1. 5% HCl
2. Von Ebner’s fluid
3. Formic acid Confirmatory test
4. HSO4
5. Chromic acid
6. Citric acid- acetate buffer (pH 4.5)

Chelation

Calcium salt + EDTA -> Ca++ + EDTA -> Ca++ - EDTA

chelate

➢ EDTA combines with Ca++ forming an insoluble non- POST-DECALCIFICATION

ionized complex. ➢ The process of neutralizing chemically or removing the
➢ Recommended for immunohistochemistry, enzyme study acid from the tissues.
and EM
➢ Inactivates alkaline phosphatase activity. 1. Tissue is washed in running water
2. Neutralize with saturated lithium carbonate or 5-10%
Ion-exchange resin (1-14 days) aqueous sodium bicarbonate fro several hours.
3. Do not place EDTA-decalcified tissue directly in 70%
Calcium salt + formic acid -> Ca++ + resin -> Ca++ binds alcohol
to resin 4. Tissue softeners may sometimes be required
5. Perenyi’s fluid act as tissue softener and decalcifying
➢ Ammonium form of polysterence resis attracts Ca++ agent.
from formic acid- containing decalcifying fluid.
➢ Resin may be re-used by eluting the bound resin with
the use of N/10 HCl
➢ Minimal cell & tissue distortion

Electrophoresis (electrical ionization)

Calcium salt + HCl -> Ca++ + cathode -> Ca++ migrates

to cathode.

FACTORS AFFECTING DECALCIFICATION

1. Concentration and volume of decalcifying agent used.
2. Temperature
3. Agitation References:
4. Tissue size & thickness
5. Ideal time Retrieved from:

Module 2.4: Decalcification and Post Decalcification

PPT and Discussion by Prof. Joana Rose Saltin
September 21, 2022

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 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Dehydration, Clearing & Embedding

CLEARING (DE-ALCOHOLIZATION)
Dehydration
➢ Is the process whereby alcohol or a dehydrating agen is
➢ The process of removing intercellular and extracellular removed from the tissues.
water from the tissue. ➢ In frozen sections, glycerin and gum syrup are used
when the tissue is to be cleared directly from water.
Characteristics of an ideal dehydrating agent:
➢ It should dehydrate rapidly Characteristics of a good clearing agent:
➢ It should not evaporate very fast
➢ It should be able to dehydrate even fatty tissues. ➢ It should be miscible with alcohol
➢ It should not harden tissues excessively ➢ It should be miscible with, and easily removed by melted
➢ It should not remove stains paraffin wax and/or by mounting medium
➢ It should not be toxic to the body ➢ It should not produce excessive shrinkage, hardening or
➢ It should not be a fire hazard damage of tissue
➢ It should not dissolve out aniline dyes
DEHYDRATING AGENTS: ➢ It should not evaporate quickly in a water bath
➢ It should make tissues transparent
Alcohol
➢ A 37 C temperature will hasten dehydration time and is CLEARING AGENTS:
especially used for tissue sections that require urgent
examinations such as fragmentary biopsies. Organic solvents
➢ Rapid
Ethanol (ethyl alcohol) ➢ Produces hardening, brittleness, and shrinkage of
➢ Ascending concentration (from 70-100%) tissues
➢ May start with 30% for very delicate specimen.
Xylol (30 mins. - 1 hour)
Methanol (methyl alcohol) ➢ Most popular
➢ For blood films ➢ Colorless clearing agent
➢ Fast
Butanol (butyl alcohol) ➢ Hardens tissues on prolonged exposure (limited to 3
➢ Plant & animal microtechnique hours)
➢ Becomes “milky white” when tissue is inadequately
Acetone dehydrated.
➢ 1.5- 2 hours
➢ Substitute for alcohol Toluene (1-2 hours)
➢ It is not carcinogenic
Cellosolve (ethylene glycol monoethyl ether) ➢ It is expensive
➢ Dehydrates rapidly
➢ Ethylene glycol ethers are combustible at 110-120 F Benzene
➢ Rapid (15 mins. - 1 hour)
Triethyl phosphate ➢ Produces minimum shrinkage
➢ Fast ➢ It is miscible with absolute alcohol
➢ Does not harden tissue ➢ It is highly flammable
➢ Produces very little distortion ➢ It is carcinogenic
➢ Minimal shrinkage
Chloroform
Dioxane (diethylene dioxide) ➢ Slower than xylol
➢ Acts both as a dehydrant and clearant ➢ It has no hardening effect
➢ Produces less shrinkage than alcohol ➢ It produces minimum shrinkage
➢ Prolonged exposure will not affect consistency or ➢ It is suitable for large specimens
staining properties ➢ It is relatively toxic to the liver on prolonged inhalation
➢ It is miscible with absolute alcohol
Tetrahydrofuran (THF)
➢ Non-toxic Amyl acetate
➢ Vapor causes nausea, dizziness, anesthesia ➢ Very good clearing agent
➢ It produces skin irritation and conjunctival irritation ➢ It is not toxic
➢ Softeners (4% phenol, glycerol/ alcohol mixture
(“Mollinex”) may be added to the dehydrating agent to Carbon tetrachloride
soften tendon, nail, or dense fibrous tissue ➢ Produces considerable hardening
➢ Is miscible in both water and paraffin, lower alcohols, ➢ It is toxic on prolonged inhalation
ether, chloroform, acetone, benzene, and xylene.
Carbol- xylene
➢ It is very rapid
➢ It is used prior to permanent mounting

Methyl benzoate & Methyl salicylate

➢ It is slow
➢ Are used in double-embedding technique

Oils

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➢ It allows indefinite stay of tissues Impregnation and Embedding
➢ Used for large and hard tissues
THREE METHODS USED TO PROCESS PARAFFIN WAX
Cedar wood oil (2-3 days) IMPREGNATION AND EMBEDDING
➢ It produces no shrinkage
➢ Used for CNS tissues, smooth muscle, skin, and By manual processing
eyeballs
By automatic processing
Aniline oil ➢ Makes use of an automatic tissue processor
➢ It is recommended for embryos, insects and very ➢ It fixes, dehydrates, clears, and infiltrates tissues
delicate specimens
By vacuum embedding
➢ It involves the wax impregnation under negative
Clove oil
atmospheric pressure inside an embedding oven
➢ It produces brittlenes but with minimum shrinkage of
➢ Promotes a more rapid wax penetration od tissue
tissues
➢ It has slow infiltration with paraffin wax
➢ It removes aniline dyes and dissolves celloidin Recommended for:
➢ Urgent biopsies
➢ Delicate tissues such as lung, brain, connective tissues,
Oil of Bergamot
decalcified bones, eyes, spleen, and central nervous
system
Oil of Oreganum (Spanish hop oil/ thyme oil)
SUBSTITUES FOR PARAFFIN WAX
Terpineol (Artificial oil of Lilac)
Paraplast
➢ Mixture of paraffin and plastic
➢ Its melting point: 56-57 degrees celsius

Synthetic waxes

Embeddol
Its melting point: 56-57 degrees celsius

Bioloid
Recommended for eyes

Tissue mat
Paraffin plus rubber

Ester wax
Can be used for impregnation without prior checking of the
tissue

Water soluble waxes

Carbowax
➢ A polyethylene glycol (PEG)
➢ Water miscible
➢ For study of fats and enzymes
➢ Solid at room temperature

Celloidin

Used for:
➢ Hollow tissues (e.g. lung)
➢ Hard and dense tissues (e.g. bones and teeth)
➢ Large tissue sections of whole embryo and whole organs
(e.g. eyeball)

Two forms:
1. Amber chips (parlodion)
2. Lint- moistened/ Low viscosity nitocellulose (LVN)

Two methods:

Wet celloidin method

Recommended for bones, teeth, large brain sections, and
whole organs

Dry celloidin method

For eyeballs

LAA. ADE. MLAD. JABB. AMAL. CARC. SCMT. JAJA. MDS.KA 2

 Plastic/ epoxy resin EMBEDDING (CASTING/BLOCKING)
➢ Uses polyester plastic, acrylic plastic (methyl ➢ It is the process by which the impregnated tissue is
methacrylate & glycol methacrylate) or epoxy placed into a precisely arranged position in a mold
plastic/resin containing a medium which is then allowed to solidify.
➢ For electron microscopy studies
➢ Orientation
Gelatin/ Agar the process by which a tissue is arranged in precise
positions in the mold during embedding, on the
➢ Seldom used except when: microtome before cutting, and on the slide before
o Dehydration is to be avoided staining.
o When tissues are to be subjected to histochemical
and enzyme studies ➢ Double embedding method
the process in which tissues are first infiltrated with
➢ It is water soluble celloidin and subsequently embedded in paraffin wax.

Uses molds:

Leuckhart L-pieces
➢ Recommended for routine use
➢ Disadvantage: it is too slow and cumbersome

Compound embedding unit

➢ Advantage: embeds more specimens at a time

Plastic embedding ring and base mold

Tissue-Tek
➢ Equipped with a warm plate to manage the impregnated
specimens and a cold plate for rapid solidification of the
block.

Disposable molds
1. Peel-away
2. Plastic ice trays
3. Paper boats

References:

Retrieved from:

Module 2.5: Dehydration, Clearing, Embedding

PPT and Discussion by Prof. Joana Rose Saltin
September 24, 2022

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 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES (LEC)

Microtomy
Cryostat
Microtomy ➢ a refrigerated apparatus used in fresh tissue microtomy.
➢ It consists of a rotary microtome , kept inside a cold
The process by which processed tissue is trimmed and cut chamber which has been maintained at a temperature
into uniformly thin slices betwen negative 5 to negative 30 degrees celsius
(average is negative 20 degrees celsius), cutting
Instrument used for the process: sections of 4 microns

Microtome ➢ Used for:

o Fluorescent antibody staining techniques
Three parts: o Histochemical enzyme studies
1. Block holder (chuck) o Rapid preparation or urgent tissue biopsies for
2. Knife carrier and knife intraoperative diagnosis
3. Pawl, ratchet feed wheel and adjustment screws
Ultrathin microtome
Principle: ➢ Used for cutting tissue sections of 0.5 micra for electron
A spring-balanced teeth or pawl is brought into contact with, microscopy.
and turns a ratchet feed wheel connected to a micrometer
screw, which is in turn rotated, moving the tissue block at a Most common application of microtomes:
pre-determined distance towards the knife for cutting
sections at uniform thickness Traditional Histology Technique
Tissues are hardened by replacing water with paraffin,
Kinds:
Cryosecitoning Technique
Rocking microtome Water-rich tissues are hardened by freezing and cut in the
frozen state with a freezing microtome or microtome-cryostat
➢ For cutting serial sections paraffin embedded tissue
Electron Microscopy Technique
Rotary Microtome
After embedding tissues in epoxy resin, a microtome with a
glass or gem grade diamond knife is used to cut very thin
➢ Invented by Minot in 1885-86
sections
➢ Used to cut paraffin embedded tissues
➢ Different from rocking microtome in that:
o The knife and the block holder are brought together Botanical Microtomy Technique
by upward and vertical motions Hard materials like wood, bone, and leather require a sledge
o It is heavier and more stable than the rocking microtome.
microtome
o It is more complex in design and construction Microtome Knives
o It is more expensive
Three basic types or shapes:
Sliding Microtome
1. Plane-concave knife (25 mm in length)
➢ Invented by Adams in 1789 a. One side of the knife is flat while the other is
➢ For cutting of celloidin-embedded tissues concave

2. Biconcave knife (120 mm in length)

Two types:
a. Both sides are concave
1. Base sledge microtome
2. Standard sliding microtome
3. Plane-wedge knife (100 mm in length)
➢ Different from base sledge in that the blocks remains
a. Both sides are straight
stationary while the knife is moved backward and
forward during the process of sectioning
Bevel angle
➢ It is more dangerous becaus of the movable knife.
➢ The angle formed between the cutting edges
➢ 27 degrees - 32 degrees
Freezing microtome
Clearance angle
➢ Invented by Queckett in 1848
➢ The angle between the block face and lower facet of
knife
➢ Used for cutting unembedded or gelatin-embedded
➢ 10 degrees - 15 degrees
blocks or frozen in liquefied carbon dioxide, liquefied
nitrogen, Freon, and other refrigerants

➢ Used to cut undehydrated tissues in a frozen state:

o When rapid diagnosis is required.
o When histological demonstration of fat is needed.
o When certain neurological structures are to be
studied.
o When sensitive tissue constituents to be studied
are damaged or destroyed by heat.

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 HONING AND STROPPING
Care and handling of the strop
➢ Badly nicked knives with blunted ends have to undergo ➢ Leather strops are usually dry and require oiling before
sharpening in order to: they are used
o Ensure optimum sectioning of tissue blocks
o Prevent gross irregularities on the tissue sections ➢ Strops are usually treated with vegetable oil (castor oil)
o Prevent tears or striae in tissue sections applied into the back of the strop (not the surface)

Sharpening of the knife involves two stages: ✓ Sharpening of knives are no longer practices in most
laboratories because of the availability of disposable
Honing (hard sharpening) knives
➢ Removal of gross nicks on the knife edge (coarse o Cheaper to use
honing) o Have a sharp cutting edge
o To remove blemishes.
Magnetic knives
➢ Grinding the cutting edge of the knife on a stone (Honing ➢ Suitable for use in the cryostat
proper) o Glass knives: for electron microscopy
o To acquire an even edge o Diamond knives: for electron microscopy
➢ Makes use of a hone (“oil stones”)
o A natural sharpening stone or hard grinding
surface (carborundum) which serves to remove
nicks and irregularities on the knife edges

Types of Hone:

1. Belgium yellow
a. For manual sharpening
b. Gives the best result

2. Arkansas
a. Gives more polishing effect

3. Fine carborundum
a. Much coarser than the 1st two

Care handling of a hone

➢ The surface of a hone is wiped clean with a soft cloth
moistened with xylene
➢ It is then covered with a thin film of mineral and clove oil,
xylene, liquid paraffin or soapy water.
➢ After its use, the hone must be washed with warm soapy
water, dried and kept in a box

Direction
➢ Edge first, with a “heel to toe” direction

No. of strokes
➢ 10-20 strokes (plane-wedge knife)
➢ 20-40 strokes (biconcave knife)

Lubricant
➢ Facilitates honing and removes metal cutting

1. Light oil
2. Dilute paraffin solution
3. Xylene

Stopping
➢ Process whereby the “burr” formed during honing is
removed and the cutting edge of the knife is polished.

➢ Makes use of a strop

o Is a leather strip used to remove the “burrs”
created by honing

Purpose:
To polish and sharpen the cutting edge of the knife
References:
Direction:
Edge, last, with a “toe to heel” direction Retrieved from:

No. of strokes Module 2.6: Microtomy

➢ 40-120 double strokes PPT and Discussion by Prof. Joana Rose Saltin
➢ 80-120 strokes on each side (biconcave knife) September 24, 2022

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