PRECAUTIONS IN

HANDLING,
ACCEPTANCE &
FIXATION OF SPECIMENS
Acceptance of Specimen
 Important Responsibilities of a Med-Tech

 Record pertinent patient data

 Evaluate and inspect request form
 Evaluate and inspect specimen
Acceptance of Specimen
 Request Form:
 Name of the patient, age, sex, room number, address,
civil status
 Type of specimen
 Examination desired
 Clinical diagnosis
 Requesting physician or surgeon
Acceptance of Specimen
 NUMBERING is the first and an important step in
all histopathologic techniques,

 Purpose: to identify properly all the specimens

without writing the name of the patient to the
accompanying tag of the specimen to be processed
Acceptance of Specimen
 Evaluate and inspect the request form and
specimen
 Correct request form
 Written legibly
 Proper container – tight fitting & transparent
 Proper fixative – volume
 Properly labeled
Fixation
 First and most crucial step in histotechnology
 AIM:
 To preserve the morphologic and chemical integrity of
the cell in as life-like manner as possible
 Stop all activities of the cell, such autolysis
 To harden and protect the tissue from the trauma of
further handling, so it may be easier to cut during gross
examination
 Stabilize protein = mechanical strength
Fixation: Main Factors
 Main factors
 Hydrogen Ion Concentration
 Temperature
 Thickness of section
 Osmolality
 Concentration
 Duration of fixation
Fixation: Effect in General
 Effects of Fixatives in General
 Harden soft and friable tissues and make the handling and
cutting of sections easier.
 Make the cells resistant to damage and distortion during the
process
 Inhibit bacterial decomposition.
 Increase the optical differentiation of cells and tissue
components
 Act as mordents or accentuators
 Reduce the risk of infections during handling and actual
processing of tissues.
 Fixation: Ideal Characteristics
 Cheap.
 Stable.
 Safe to handle.
 Kill the cell quickly thereby
producing minimum
distortion of cell
constituents.
 Inhibit bacterial
decomposition and autolysis.
 Produce minimum shrinkage
of tissue.
NOTE: No single fixative posses all the qualities.
 Permit rapid and even
penetration of tissues.
 Harden tissues
 Isotonic
 Make cellular components
insoluble to hypotonic
solutions and render them
insensitive
to subsequent processing.
 Permit the subsequent
application of many staining
procedures
Fixation: Types – COMPOSITION
 Simple Fixative  Compound
Fixative
Fixation: Types – COMPOSITION
 Simple Fixative  Metallic Fixatives
 Aldehydes
 Mercuric chloride
 Chromate fixatives
 Formaldehyde  Potassium dichromate

 Glutaraldehyde  Chromic acid

 Lead fixatives
 Metallic Fixatives  Picric acid
 Mercuric chloride  Acetic acid

 Chromate fixatives  Alcohol

 Osmium tetroxide (Osmic acid)

 Potassium dichromate
 Heat
 Chromic acid
Fixation: Types – ACTION
 Microanatomical Fixatives
 Permit the general microscopic study of tissue structures without
altering the structural pattern and normal intercellular relationship of
the tissues in question
10% Formol saline
10% Neutral buffered formalin
Heidenhain’s Susa
Formol sublimate (Formol corrosive)
Zenker’s solution
Zenker-formol (Kelly’s solution)
Bouin’s solution
Brasil’s solution
Fixation: Types – ACTION
 Cytological Fixatives
 Preserve specific parts and particular microscopic elements of
the cell itself.
 Nucler Fixatives
 preserve
the nuclear structures (e.g., chromosomes)
 Contain glacial acetic acid as their primary component (pH <=

Flemming’s fluid
Carnoy’s fluid
Bouin’s fluid
Newcomer’s fluid
Heidenhain’s Susa
Fixation: Types – ACTION
 Cytological Fixatives
 Preserve specific parts and particular microscopic elements of the cell
itself.
 Cytoplasmic Fixatives
 Preserve cytoplasmic structures
 No glacial acetic acid (pH >= 4.6)
 destroy mitochondria and Golgi bodies
Flemming’s fluid without acetic acid
Kelly’s fluid
Formalin with “post-chroming”
Regaud’s fluid (Muller’s fluid)
Orth’s fluid
Fixation: Types – ACTION
 Histochemical Fixatives
 Preserves the chemical constituents of cells and tissues
10% Formal Saline
Absolute Ethyl Alcohol
Acetone
Newcomer’s fluid
Fixation: Formaldehyde
 A gas produced by the oxidation of methyl alcohol
 Souluble in water (extent of 40% by weight)

 Routine: 10% (1:10 dilution)

 Duration: 12-24 Hours

 Turbid after prolonged storage (Formation of

paraformaldehyde)
 Removal of Formlin Pigments:
 Karadeswitsch’s Method; Lillies’s Method; Picric Acid
Method
Fixation: Formaldehyde
Advantage
 Cheap
 Readily available & stable
 Penetrates tissue well
 Does not over hardens
tissue
 Preserves fat & mucin
 Best for nervous tissue
Disadvantage
 Irritating
 Skin (allergic dermatitis)
 Eyes
 Produce considereable
shrinkage of tissue
 Reduce both basophilic &
eosinophilic staining cells
 Forms abundant brown
artifacts, pigments & granules
Fixation: Simple
Aldehyde
Fixation Best used Fixation Best used
Glutaraldehyde Small fragment; Paraformaldehyde Excellent for
Small needle routine paraffin
biopsies sections

Metallic Fixatives
Fixation Best used Fixation Best used
Mercuric chloride Nuclei; CT
Reco: Renal, fibrin
& CT
Fixation: Simple
Fixation Best used Fixation Best used
Chromic fixatives Carbohydrates Potassium Certain lipids
dichromate
Lead fixatives Mucopolysaccharid Picric Acid Glycogen
es
Acetic Acid Nucleoprotein Acetone fixatives Enzyme studies
fixatives Phosphates
Lipases
Brain tissue (rabies)
Alcohol fixatives Small tissue Osmium tetroxide Fats & lipids
fragemts fixatives Nuclear stainig
Glycogen
Fixation: Compound - Microanatomical

Fixation Best used Fixation Best used

10% Formol saline Nervous system 10% Buffered Post mortem
General post formalin surgical research
mortem materials specimen
Heidenhein’s Susa Skin biopsies Formol sublimate Routine post
solution mortem materials
Formol Saline Post mortem Zenker’s solution Post mortem
sublimate materials materials
Zenker’s Formol Pituitary tissue and Bouin’s solution Embryos
(Helly’s Fluid) bone
Fixation: Compound - Cytological
Fixation Best used Fixation Best used
Flemming’s Nuclear structure Carnoy’s fluid Chromosomes
solution study
Lympnodes
Urgent studies –
glycogen
Boiun’s fluid Embryo Newcomer’s fluid Mucopoplysacchari
Glycogen des
Nuclear protein
Chromosomes
Fixation: Compound - Cytoplasmic
Fixation Best used Fixation Best used
Flemming’s fluid Cytoplasmic Champy’s fluid Mitochondria
w/o acetic acid structures Golgi elements
Fats
Regaud’s fluid Mitochondria Orth’s fluid Early degenerative
(Moller) process
Tissue necrosis
FAULTS OCCURRING
DURING
TRIMMING/CUTTING OF
PARAFFIN BLOCKS
Mary Christelle G. Aquitania
UST Medical Technology Intern
Trimming
 Process wherein the paraffin block is exposed for
actual cutting after when the wax is solidified and
removed from the mold
 Sides, top and bottom of the tissue block are
trimmed until leveled perfectly and all sides are
parallel to each other
 Routine histologic procedures: Cut between 4-6μ.
Faults before Section-Cutting
FAULTS
Brittle or hard tissue
Clearing agent turns milky as
soon as tissue is placed in it
REASON REMEDY
Prolonged fixation
Prolonged dehydration
Prolonged clearing Tissue may be softened by
soaking in a small dish
Prolonged paraffin infiltration
containing water with
Overheated paraffin oven detergent, phenol or Molliflex
Drying out of tissue before
actual fixation
Water not completely Repeat dehydration with
removed (incomplete absolute alcohol, then repeat
dehydration) clearing

Blocked is trimmed down

Clearing agent is not
Upon trimming, tissue smells
completely removed due to
of clearing agent
insufficient impregnation
nearest to the tissue. The
remaining wax id melted on
embedding oven and paraffin
impregnation is repeated,
changing the paraffin at least
once before blocking.
Faults before Section-Cutting
FAULTS REASON REMEDY
Repeat clearing; id object has
Tissue is opaque, section cutting is already been embedded, prolong
difficult due to the presence of Insufficient clearing oven and paraffin impregnations
alcohol repeated, changing the paraffin at
least once before blocking
Insufficient dehydration, therefore
Tissue shrinks away from wax when
incomplete clearing and Repeat the whole procedure
trimmed
impregnation

Tissue is soft when block is trimmed Incomplete impregnation Repeat whole procedure

Air holes on tissue during trimming Incomplete impregnation Repeat impregnation

On trimming, wax appears

Contaminated wax Re-embed in freshly filtrated wax
crystalline

Block not cooled rapidly enough

Paraffin block, after cooling, is Repeat paraffin impregnation, then

Insufficient paraffin impregnation
moist and crumbles re-embed
Faults during Section-Cutting
FAULTS REASON REMEDY
Surfaces and edges of the
Re-trim the block
block are not parallel
Horizontal surface of the
Re-adjust and re-orient the
block is not parallel to the
block
knife
Coat horizontal edges of the
Sections fail to form ribbons
Paraffin wax is too hard block with wax of lower
melting point
Knife is tilted to much Reduce the tilt
Readjust the thickness of the
Sections are too tick sections
Hone and strop
Sections roll up on cutting Knife is blunt Sharpen the knife
so that they adhere and get Tilt of knife is too great Reduce the tile
broken against the knife
Knife edge is dirty Clean the knife edge
edge
Faults during Section-Cutting
FAULTS REASON REMEDY
Adjust the knife so that the
Blunt of dull spot on the knife,
knife will present a uniformly
producing an irregular knife
sharp edge to the block, or
edge
sharpen
Ribbon is curved, crooked or
Edges of the block are not
uneven instead of straight Re-trim the block
parallel but round wedge shape
Knife is not parallel to the block Readjust knife and block
Repeat impregnation using pure
Paraffin is impure
wax
Knife is blunt or dull Re-sharpen the knife
Cool the block on ice water until
Paraffin block Is warm and soft
firm
Sections are compressed, Knife edge is coated with
Clean the knife edge
wrinkled or jammed paraffin
Sections are too thin Readjust thickness of section
Microtome set screw is loose Tighten the screw
Tilt of knife is too vertical Reduce the tilt
Faults during Section-Cutting
FAULTS REASON REMEDY
Sections are squashed (width of
Bevel of knife is lost due to Re-sharpen, using a knife back
each section is less than that of
incorrect sharpening or automatic knife sharpener
block)
Bubble or dirt formed in the Re-embed in freshly filtered
embedding medium wax if necessary
Once embedded in paraffin wax,
A hole is formed in the section
Hard spot in tissue due to decalcification is impractical;
calcium use a base-sledge microtome
with a wedge knife
Tilt of knife is too great or bevel
is not cleared, hence object is
Reduce the tilt
compressed against the knife
edge clamp set screw on knife
Section of unequal thickness are Or blockholder is loose
Tighten the screw
produced Blocks are too large
Cut blocks into smaller
Block are too hard
fragments
Soften the blocks in detergent or
phenol
Faults during Section-Cutting
FAULTS
Sections adhere to the knife or
other parts of the microtome
Nicks or damage on the knife
Ribbon is split or lengthwise
vertical scratches are seen on
sections
Sections are lifted from the
knife on upstrokes
REASON
Static electricity due to low
static electricity, or boil water in
atmospheric humidity
Knife edge is dirty
Knife edge is dull
Knife tilt is too great
edge
Dirty embedding
Knife edge is dirty
Tilt of knife is too great
Knife tilt is too great
Knife is dull
Paraffin is too soft or room
temperature is warm
REMEDY
Breather out or blow gently on
the block and knife to breakup
the room to increase the
humidity
Clean the knife edge
Sharpen the knife
Reduce the tilt
Sharpen the knife
Re-embed in filtered wax
Clean knife edge with xylene
Reduce the tilt
Reduce the tilt
Sharpen the knife
Cool paraffin wax in ice water
Faults during Section-Cutting
FAULTS REASON REMEDY
Tilt of knife is too small,
paraffin block is therefore
Resistance is felt on the lower
compressed against the base of Increase the tilt
part of the section during cutting
the knife towards the end of
stroke
Horizontal or parallel lines or Knife edge vibrate due to Treat with phenol during
furrows across the section hardness of tissue processing or collodionize
(“Chatters”) are seen, forming
Tilt of knife is too great Reduce the tilt
thin and thick zones
Knife is blunt Sharpen knife
Knife is not clamped properly Adjust the knife
Tilt of knife is too great Reduce the tilt
Section cut is sometimes thin, Tighten adjusting and locking
Knife or block holder is loose
sometimes thick screws
Knife tilt is too small that block
is compressed by bevel and Increase the tilt
section is not cut
Faults during Section-Cutting
FAULTS REASON REMEDY
Tilt of knife is too slanted or too Readjust the angulation of the
Knife makes a hard metallic big knife
scrapping or ringing sound on Take fresh block treated with
Tissue is too hard
backstroke, when section is cut phenol during processing
Knife blade is too thin Change the knife
Frozen tissue crumbles and
comes off the block holder Freezing is not adequate Refreeze the tissue block
when cut
Frozen tissue chips into
Tissue is frozen too hard Warm the tissue with fingers
fragments when cut