RECEIVING SPECIMEN AND

GROSS EXAMINATION
RECEIVING SPECIMEN
• What to check?
• Appropriate label: name, type of specimen, time and date
• Eligibility of the writing
• Completeness of request form
• Radiologic findings esp. for orthopedic specimens
• Volume and time of the fixation
• Requesting physician
• Test requested
• Number of specimens received
• Type of specimen received: tissue, slides, blocks, fluid
• Name and signature of the person submitting the specimen
• If not sure with anything, call the help of your pathologist
RECEIVING SPECIMEN
Specimen Identification/Request form
• Patient information
• History
• Clinical impression
• Surgical operation performed
• Description of site of origin

Labeling
• Give each specimen a control/identification number
• Easier to find whenever needed
Sample Case 1
• 21 y/o female
• Breast mass
• History: 3 months prior to consult, the patient noted a mass on her breast. The
physician decided to do a biopsy to know if it is malignant or benign hence
admission.

Specimen Identification/Request form

• Patient information
• History
• Clinical impression
• Surgical operation performed
• Description of site of origin
Sample Case 2
• 65 y/o female
• Breast mass to consider malignancy s/p incision biopsy
• History: patient has 3 months history of nipple discharge, skin dimpling and
peanut-sized breast mass which is non-movable, non-painful

Specimen Identification/Request form

• Patient information
• History
• Clinical impression
• Surgical operation performed
• Description of site of origin
TYPES OF SURGICAL SPECIMENS
• Excision biopsy specimen- the whole affected area is removed

• Incisional biopsy specimen- tissue is removed for diagnosis

from the affected area

• Punch biopsy- punches are used to remove a small piece of

suspicious tissue for examination
PUNCH BIOPSY
TYPES OF SURGICAL SPECIMENS
• Shave biopsy- small fragment of tissue is “shaved” from a
surface using a blade (usually skin)

• Curettings- tissues are removed in small pieces from the lining

of the organ (usually from uterus or cervix)

• Core biopsy- small tissue samples are removed using a special

needle, sometimes through the skin (percutaneously)
Shave biopsy
Curettings
Core needle biopsy
GROSS EXAMINATION
• Cutting up specimens is known as grossing
• Grossing is an art
• Sectioning the tissue to be processed for diagnosis
• Cut the organ at interval of 1 cm thickness
GROSS EXAMINATION
Mandatory prerequisites before examining
• Knowledge of the clinical history
• Thorough knowledge of the anatomy of the organ

Just by inspection, an experienced pathologist has diagnosis already in

her/his mind and that how sectioning at a particular tissue is obtained.
GROSS EXAMINATION
• The fewer sections are taken, the fewer slides will need to be reviewed
• Gross without delay after fix
• Selected tissue should contain portion of lesion
• Take specimen from more than one areas of affected organs
• Cut must be made quickly, sharply and accurately
• Optimum thickness of sections taken: 5 mm
• Wash blood with normal saline
GROSS EXAMINATION
Macroscopic description includes:
• Location of the lesion
• Size, weight, length
• Shape and architecture
• Color and consistency

• Describe cut surfaces and identify pathological changes

GROSS EXAMINATION
• Histopathological sections- 5 mm (average)
• Put in cassettes/capsules and then in fixative
• Use pencil to label the cassettes
• Small specimen can be wrapped in tissue paper and then in cassette
& fixed
FIXATION
FIXATION
• When tissues are removed from the body, blood supply is cut off which
signals the start of autolysis and putrefaction, hence fixation

• process by which the tissue are fixed in a physical and chemical state so
that they will withstand subsequent treatment with various reagents with
minimum loss of architecture

• Factors to consider:
• Tissue
• Penetration of fixative
• Correct choice of fixative
EFFECTS OF FIXATION
• Inhibition of autolysis
• Hardening of tissue
• Solidification of colloid material
• Optical differentiation
• Effects on staining
• Loss of material during fixation
• Tissue shrinkage
Properties of ideal fixative
• Prevent autolysis and bacterial decomposition
• Preserve tissues in their natural state
• Make cellular component insoluble to reagents
• Preserve tissue volume
• Enhance staining of tissue
• Non-toxic
• Not expensive
• Carried out at room temperature
Properties of ideal fixative
• Does not deteriorate, stable
• Easily prepared
• Penetrate tissues quickly
• Raises refractive indices
• Fixes all constituents of tissues

• IN SHORT, NO IDEAL FIXATIVE

Speed of Fixation
• Speed of fixation of most fixative is almost 1mm/hr
• Several hours are needed for fixation alone
• Best thickness- 4 to 5 mm
Amount of fixative fluid
• Should approximately 20 times the volume of the specimen
• Should surround the specimen
Factors affecting fixation
• Size and thickness of tissue
• pH- ideal is 6-8
• Mucinous containing tissues
• Tissue with large amount of blood
• Fatty tissues
• Accelerated by agitation and warmer temperature
• Concentration of fixative
• Formalin is best at 10%;
• Glutaraldehyde is generally made up at 0.25% to 4%.
Tissue fixatives
• ALDEHYDES: Formalin, Glutaraldehyde
• MERCURIALS: Zenker’s fluid, Helly’s fluid, Heidenhain’s susa
• CHROMATE FIXATIVES: Regaud’s/Moller’s fluid, Orth’s Chromic acid ,
Potassium dichromate
• PICRATES: Bouin’s fluid
• ALCOHOLS: Methanol, Ethanol, Isopropranolol, Carnoy’s- most rapid,
for chromosomes, Newcomer’s- for polysaccharides
• OSMIUM: Flemming’s
 Neutral Buffered Formalin
• 10% Neutral buffered formalin (NBF) mostly used
• 40% formaldehyde gas in 100 w/v of distilled water= 100% formalin
• 10 mL of 100% formalin + 90 mL distilled water = 10% formalin
• 10 mL of 100% formalin + 90 mL distilled water + Mg or Ca carbonate=
10% neutralized formalin (avoid formation of calcium artifacts in tissues)
• 10 mL of 100% formalin + 90 mL distilled water + Sodium phosphate
monobasic and sodium phosphate dibasic= 10% NBF
Neutral Buffered Formalin
Mechanism of Action
• It forms cross links between amino acids of proteins nearby making them
insoluble

Advantages
• Rapid penetration, easy availability and cheap
• Does not over-harden the tissue, easily fixes lipids and carbohydrates
• Support the acid-base dyes
• Ideal for immunohistochemistry staining
Formalin
• Formation of black formalin
pigment (acid formaldehyde
hematin)
• Brown, granular material,
extracellular, birefringent
• Progressive in deposition
• Often seen after several days
• Action of acid formalin on blood
• Avoided by using buffered
formalin
Glutaraldehyde
● Used for Electron Microscopy

● Advantage:
● Most efficient cross linking agent for collagen
● More rapid fixation than formalin.

● Disadvantage:
● Poorer penetration
● False positivity with PAS
● More costly
MERCURIC CHLORIDE
● Zenker’s fluid: cytologic and nuclear
chromatin stain, trichrome staining
● Helly’s fluid: bone marrow and spleen,
cytoplasmic stain, aka Zenker’s formol
● White crystalline substance
● Advantages: rapidly penetrates & hardens
tissue
● Disadvantages: Extremely poisonous &
corrosive to metals, pollutant to the
environment
MERCURIC PIGMENT
• Powerful protein precipitant
• Mercury pigment – brown to
black granular deposit
CHROMATE FIXATIVES
• Regaud’s: chromatin, mitochondria, mitotic figures
• Orth’s fluid: rickettsia, chromaffin tissues like adrenal medulla,
mitochondria, golgi apparatus
• Chromic acid: preserve proteins
• Potassium dichromate: mitochondria
• Chromate pigments: fine, yellow brown
PICRIC ACID
• Bouin’s solution
• Bright yellow crystalline substance
• explosive properties
• Enhances cytoplasmic staining
• Acts as mordant
• Much shrinkage but little hardening
• Recommended for fixation of testis
and connective tissues
• Has a mild decalcifying property
NUCLEAR AND CYTOPLASMIC FIXATIVES
• NUCLEAR “BFNCH” • CYTOPLASMIC “HORFF”
• Bouin’s • Helly’s
• Flemming’s with acetic acid • Orth’s
• Newcomer’s • Regaud’s
• Carnoy’s • Flemming without acetic acid
• Heidenhain’s susa • Formalin with post chroming
DECALCIFICATION
DECALCIFICATION
Process of removing inorganic calcium content of the bone /tissue
before processing the specimen after fixation for easier tissue
processing and microtomy
 DECALCIFICATION

• Examination of bone marrow, bone and other tissues with

calcifications
• Ratio of specimen to the decalcifying agent- 1:20
• Criteria for decalcifying agent
• Complete removal of calcium salts
• No damage to tissues
• Lack harmful effects on staining reactions
• Easily removes calcium salt (time-saving)
FIXATION BEFORE DECALCIFICATION
• To protect the cellular and fibrous elements of specimen from damage
• Extend fixation times for bone specimens before decalcification
•Buffered formalin is a satisfactory fixative for bone
•For the preservation of bone marrow Zinc formalin mixtures or Bouin’s fluid
 TYPES OF DECALCIFYING AGENTS
• CHEMICAL METHOD
• STRONG ACIDS- recommended concentration: 5-10%
• NITRIC ACID- Perenyi’s fluid, phloroglucin
• HYDROCHRLORIC ACID- Von ebner’s
• WEAK ACID
• FORMIC ACID
• ACETIC ACID
• PICRIC ACID
• CHELATING AGENTS: EDTA (ethylenediaminetetraacetic acid)
• ION-EXCHANGE RESIN METHOD: quick and more efficient
• ELECTROLYTIC METHOD: heat may cause charring of specimen
• ELECTROPHORESIS METHOD: calcium are attracted to negative electrode
 DECALCIFYING AGENT: STRONG ACIDS
NITRIC ACID
• 5-10% in distilled water
• Change nitric acid solutions daily until bubbles ceased
• May have yellow discoloration- formation of nitrous acid
• Rapid but may damage tissues if left for a long time

HYDROCHLORIC ACID
• 5-10% in distilled water
• Formalin should be washed from specimen to avoid formation of bis-
chloromethyl ether (a carcinogen)
 DECALCIFYING AGENT: WEAK ACIDS
FORMIC ACID
• Widely used
• Routine: 10% of formic acid in distilled water is recommended
• Large volume is used and renewed every 48 hours
• Slower but much gentler

PICRIC ACID AND ACETIC ACID

• Cause tissue swelling if used alone
• Incorporated in Carnoy’s and Bouin’s fixative
CHELATING AGENTS
• Ethylenediaminetetraacetic acid (EDTA)- work by capturing the
calcium ions from tissue
• Process is very slow & gentle
• Very high quality morphology is required
• Preferred for decalcifying bone for electron microscopy
• 14% concentration
• pH 7.0
DECALCIFICATION ARE AFFECTED BY THE
FOLLOWING FACTORS:
1. Size of the specimen to be decalcified
2. Density of calcium present in the specimen
3. Concentration of decalcifying agent
4. Temperature
5. Agitation
6. Suspension- fresher solutions are more effective hence the renewal
of decalcifying agent after sometime
 DECALCIFICATION
Method to determine completion of progress of decalcification

1. X-ray method- most reliable method but may not be available for all

2. Ammonia method- drop by drop in the decalcifying solution where

cloudiness indicates the presence of calcium
TISSUE PROCESSING
TISSUE PROCESSING
• Fixed tissues undergo infiltration of different reagents producing a
specimen with suitable hardness and consistency for microtomy
• Requires 12-24 hours done in many stages
• Dehydration
• Clearing
• Impregnation
• Embedding
• Can be done through:
• Manual tissue processing
• Automated tissue processing
CONVENTIONAL TISSUE PROCESSOR
Sequence of tissue processing
1. Dehydration
• Uses increasing strength of alcohol- 70%, 80%, 90%, 100%
• Water in the tissues are replaced by alcohol
• Delicate tissues gets high degree of shrinkage
• Ratio of tissue to alcohol: 1:10

2. Clearing
• Paraffin wax is not alcohol soluble hence, clearing agent is used to replace
alcohol from the tissues
• Clearing agents: xylene, chloroform, benzene, carbon tetrachloride and
toluene
Sequence of tissue processing
3. Impregnation
• Occur at melting point temperature of paraffin wax
• Volume: 25-30 times the volume of tissues
• Clearing agent is replaced by paraffin wax

4. Embedding
• Impregnated tissues are placed in molds with their labels with melted paraffin
wax----allowed to solidify---let it cool
• Immersed in cold water---remove from mold---trimmed
2.
MICROWAVE TISSUE PROCESSOR
MICROWAVE TISSUE PROCESSING
• Three main goals
• reducing cost of reagents
• reducing time taken
• eliminating noxious materials from the process

• Principle
• electromagnetic field causes excitation of atoms which achieves its
revolution. This produces energy as intensity from inside the materials. This
intensity improves the pace of dispersion of liquids all through the tissue
blocks or segments significantly more actually rather than regular warming
MICROWAVE TISSUE PROCESSING
• Microwave works as a physical agent
• Similar in mechanism to vacuum/oven
• Heat agitation to expedite the movement of molecules
• Accelerate fixation, decalcification, tissue processing and staining
• Works well with immunohistochemistry and electron microscopy
MICROTOMY
 MICROTOME
• Derived from the
Greek. mikros,
meaning “small”, and
temnein, meaning
“to cut”)
• It is a mechanical
device for cutting
thin uniform slices of
tissue sections
FULLY AUTOMATED ROTARY MICROTOME
Two motors that drive both the fine and the coarse advance hand-wheel
TYPES OF MICROTOMES
⚫ Rotary
⚫ Rocking
⚫ Rotary Rocking
⚫ Sliding
⚫ Vibrating
⚫ Freezing
⚫ Saw
⚫ Cryostat
⚫ Ultra
⚫ Laser
ROCKING MICROTOME
 ROCKING MICROTOME
• Name derived from the rocking
action of the cross arm
• Oldest in design, cheap, simple to
use, extremely reliable, minimum
maintenance
• Knife is fixed, the block of the
tissue moves through an arc to
strike the knife.
• Steady backward and forward
movement of the handle gives
ribbons of good sections.
 SLIDING MICROTOME

• The knife or blade is stationary, the

specimen slides under it during
sectioning
VIBRATING MICROTOME
⚫ Designed to cut fresh unfixed tissue
⚫ high speed vibration produced in a safety razor blade
provides the cutting power
⚫ Greatest application in enzyme histochemistry & ultra
structure histochemistry.
⚫ Tissues are cut at very slow speed to avoid
disintegration.
ULTRA MICROTOME
 ULTRA MICROTOME

⚫ These are used exclusively for electron microscopy .

⚫ Prepare ultrathin sections .
⚫ It has been reported that sections can be cut as thin as 10 nm
⚫ Knives are usually made from glass, diamond or sapphire.
⚫ The block is brought to the knife edge under microscopical control
and as each section is cut it is floated on to a water bath adjacent to
the knife edge
TYPES OF KNIVES BASED ON COMPOSITION
⚫ STEEL KNIVES
⚫ GLASS KNIVES
⚫ DIAMOND KNIVES
⚫ SAPPHIRE KNIVES
STEEL KNIVES
GLASS KNIVES
 DIAMOND KNIVES
• Manufactured from gem quality diamonds.
• Very expensive the knives are extremely durable,
because of the hardness factor of the diamond, and are
used primarily for cutting ultrathin, resin sections.
 SAPPHIRE KNIVES

⚫ Manufactured from one piece of solid sapphire

artificially produced from an alumina monocrystal
under computer controlled thermal conditions.
TYPES OF KNIVES BASED ON CHARACTERISTICS
⚫Non-corrosive knives
⚫Tungsten carbide
⚫Disposable blades
 NON- CORROSIVE KNIVES
These are manufactured from hardened, heat
treated stainless steel free from all impurities and
containing 12 to 15% chromium.
 TUNGSTEN CARBIDE KNIVES

⚫ 100 times harder than hardened tool

steel
⚫Excellent resistance but are brittle
because of their extreme hardness
and should be handled carefully
 DISPOSABLE BLADES
⚫ manufactured from high quality stainless steel
⚫ the edge of disposable blades can be coated with
platinum6 or chromium7 to enhance strength and
prolong cutting life
 DISPOSABLE BLADES
⚫ Disposable blades need to be held rigid in a special holder to
prevent vibration during the cutting stroke.
⚫ These knives consistently produce high quality sections virtually
free from compression.
 MICROTOMY
⚫ Apply ice to the block surface to make the wax hard
which would have become soft by frictional heat.
⚫ There should be a smooth continuous plastic flow of the
sections in the form of a ribbon
 MICROTOMY
⚫ When the ribbon comes off it is held gently with a fine moistened
brush or forceps and then transferred to waterbath.
⚫ Section is then floated on water bath (temp 5-6° below
melting pt. of wax) to remove creases
 MICROTOMY
• Clean or albuminised slide is half submerged in water and
section is picked up using a dissecting needle.
• The slide is then set in an upright position to drain
• Slides are kept in incubator (37°overnight for plain slides and 60°
for 2 hours for albuminised ones)
 SLIDE ADHESIVES
• Most commonly used adhesive is Albumin.
• Others are Starch paste and Chrome gelatin.
• Albumin solution is prepared by mixing equal parts of glycerin,
distilled water and white of eggs, then filtered through coarse
filter paper and a crystal of Thymol is added
 WHY NEED ADHESIVE?
There are occasions when sections may tend to float from the slide and these are:

1. When sections are submitted to strong alkali solutions during staining.

2. Cryostat sections for immunofluorescence, immunocytochemistry and
urgent diagnosis.
3. Tissues from the CNS.
4. When sections are submitted to high temperatures.
5. Tissues containing blood clot.
6. Tissues which have been decalcified.
 HOT PLATE
• For delicate tissues a lower temperature is desired for drying so as to avoid
splitting and cracking of the section due to excess heat: 370C for 24 hours
or longer is recommended
• On Hot Stage which temperature is maintained at 45-50 degree 30
minute is sufficient.
SHORT EXERCISE: FIXATIVE
1. Neutral buffered formalin A. Contains picric acdi
2. Glutaraldehyde B. Contains mercuric chloride
3. Helley’s fluid C. Used for electron
4. Zenker’s fluid microscopy
5. Bouin’s fluid D. Used as standard fixative
SHORT EXERCISE: MICROTOMY (IDENTIFY)

1. 2. 3.
SHORT EXERCISE: TISSUE PROCESSING

ENUMERATE STAGES OF
CONVENTIONAL TISSUE
PROCESSING (3)