HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES STAINING METHODS

FRESH TISSUE EXAMINATION, SPECIAL TISSUE PROCESSING, FIXATION.

1. Polychrome methylene blue
PRELIM: WEEK 3 LEC (02/10/2023) 2. Alcoholic pinacyanol
METHODS OF FRESH TISSUE EXAMINATION 3. Thionine
1. TEASING/DISSOCIATION 4. Hematoxylin and Eosin: progressive (no decolorizer)

TISSUE PROCESSING
1. Fixation 6. Trimming
2. Dehydration 7. Section/ Microtomy
2. CRUSHING / SQUASH PREPARATION 3. Clearing/ Dealcoholization 8. Staining
4. Infiltration/ Impregnation 9. Mounting
5. Embedding 10. Labelling

NUMBERING/LOGGING/RECEIVING
3. SMEAR PREPARATION o S (surgical), A (autopsy), C (cytology)
- Streaking o Use PENCIL in writing the description
- Spreading o Specimen size for processing 3X2 CM AND 3-5 MM THICK
- Pull apart
- Touch preparation FIXATION/PRESERVATION
o FIRST AND MOST CRITICAL STEP
4. FROZEN SECTION o Process of preserving cells and tissue constituents
- normally used when a rapid diagnosis of a tissue is required. o The tissue is preserved by preventing degeneration,
- Freezing of UNFIXED TISSUE is best for frozen section. putrefaction, decomposition and distortion
- Freezing of FIXED TISSUE is used to localize hydrolytic o PRIMARY AIM: to preserve the morphologic and chemical
enzymes and other antigens. integrity of the cell in as life-like manner as possible.
o Formal (formol) calcium o SECONDARY AIM: to harden and protect the tissue from the
▪ Derivative of formaldehyde trauma of further handling.
▪ Fixed at 4°C for 18 hours o STABILIZATION OF PROTEINS: Most important reaction for
APPLICATIONS: maintaining tissue morphology
1. Rapid pathologic diagnosis during surgery 2.
2. Enzyme histochemistry EFFECTS OF FIXATIVES IN GENERAL
3. Demonstration of soluble substances such as lipids and o Harden soft and friable tissue
carbohydrates o Make tissue resistant to damage and distortion
4. Immunofluorescent and immunohistochemical staining o Inhibit bacterial decomposition
5. Some specialized silver stains, particularly in neuropathology o Increase optical differentiation of cells
FREEZE DRYING: without use of FREEZE SUBSTITUTION: similar o Act as mordants or accentuators
any chemical fixative to freeze drying but: o Reduced the risk of infection
▪ QUENCHING: rapid freezing
(- 160°C) ▪ Frozen tissue is submerged to CHARACTERISTICS OF A GOOD FIXATIVE
▪ SUBLIMATION: removal of ROSSMAN’S FORMULA (1% ▪ Cheap ▪ Produce minimum shrinkage
▪ Stable ▪ Rapid penetration of tissue
water in the form of ice acetone) and dehydrated using
▪ Safe to handle ▪ Harden tissue
(-40°C vacuum) absolute alcohol. ▪ Prevent distortion ▪ Isotonic w/ minimal effect on tissue
▪ Inhibit bacterial decomposition ▪ Permit staining
TWO METHODS OF PREPARING FROZEN SECTION
1. COLD KNIFE PROCEDURE MECHANISM OF ACTION OF FIXATIVE
o almost any microtome can be used 1. ADDITIVE FIXATION - becomes part of the tissue by formation of
o uses carbon dioxide cross links or complexes. Ex. Formalin, Hg, osmium tetroxide
o Optimum condition for sectioning: 2. NON-ADDITIVE FIXATION - NOT incorporated in the tissue,
- KNIFE = -40° to -60° C stabilizes tissue by removing of the bound water. Ex. Alcoholic
- TISSUE = 5° to -10° C fixatives
- ENVIRONMENT = 0° to -10° C
- If tissues are frozen too hard = chip into fragments when FACTORS INVOLVED IN FIXATION
cut. Remedy: Surface of the block maybe be softened by pH ▪ 6-8
warming slightly with the finger. TEMPERATURE ▪ Autotech (40°C)
- If tissues have not been sufficiently frozen = thick and ▪ Traditionally (RT)
crumble, block may come away from the stage. ▪ EM and histochem (0-4°C)
Remedy: More bursts of carbon dioxide gas should then be ▪ Rapid examination (60°C)
▪for tissue w/ TB: (100°C)
given to refreeze the block.
THICKNESS: ▪ EM 1-2mm2
▪ LM 2cm2 (no more than 0.4 cm/4mm thick)
2. CRYOSTAT PROCEDURE (COLD MICROTOME) ▪ brain tissue - suspended whole 2-3 weeks (circle of
o OPTIMUM WORKING TEMPERATURE = willis)
-18° to -20°C ▪ Large solid tissue- open or sliced thinly
o CRYOSTAT – a refrigerated cabinet in OSMOLALITY ▪ slightly hypertonic solution around 400- 450mOsm
which a modified microtome is housed. CONCENTRATION: ▪ 10% formalin
o All the controls to the microtome are ▪ 3% Glutaraldehyde
operated from outside the cabinet. ▪ 0.25% Glutaraldehyde (IEM)
o Presently, the ROTARY MICROTOME is the type of choice. TIME DURATION ▪ Primary fixation (2-6 hours)
▪EM: Fixation of 3 hours
COMMONLY USED METHODS OF FREEZING
o OPTIMAL CUTTING TEMPERATURE (OCT): best mounting media for cryostat PRACTICAL CONSIDERATIONS
o LIQUID NITROGEN: most rapid 1. Speed
o ISOPENTANE cooled by liquid nitrogen 2. Rate of penetration: Formalin 1mm/hour
o CARBON DIOXIDE GAS 3. Volume: 10-25 times that of the tissue
o AEROSOL SPRAYS = adequate for freezing small pieces of tissue EXCEPT muscle. ▪ 20:1= maximum effectiveness
o Disadvantage: ▪ 5-10:1 = Osmium tetroxide
- Soft tissue is liable to crack ▪ 50-100:1 = prolonged fixation (museum preparation)
- Overcools urgent biopsy blocks that can damage the block 4. Duration
and the blade if sectioning is done at -70 deg C and below

[SARANILLO, KA.]
 TYPES OF FIXATIVE ACCORDING TO COMPOSITION PICRIC ACID FIXATIVE
1. SIMPLE FIXATIVE – ex. Formaldehyde, glutaraldehyde, chromate o HIGHLY EXPLOSIVE when dry
fixatives, lead fixatives, picric acid, acetic acid o Excessive YELLOW STAINING of tissues
2. COMPOUND FIXATIVE o NEVER WASH IN WATER before dehydration
o For glycogen (excellent)
TYPES OF FIXATIVE ACCORDING TO ACTION a. Bouin’s - recommended for fixation of
1. MICROANATOMICAL FIXATIVE – 10% Formol saline, 10% embryos and pituitary biopsies
Neutral buffered formalin (NBF), Heidenhain’s SuSa, Formol b. Brasil’s alcoholic picroformol fixative –
sublimate, Zenker’s solution, Zenker’s formol, Bouin’s solution, Excellent fixative for glycogen and less
Brasil’s solution messy then Bouin’s solution (excellent)
2. CYTOLOGICAL FIXATIVE
a. NUCLEAR FIXATIVE – contains glacial acetic acid ex. Bouin’s GLACIAL ACETIC ACID
fluid, Flemming’s fluid, Newcomers fluid, Carnoy’s fluid, o It is normally used in conjunction with other fixatives to form a
Heidenhain’s SuSa (BFNCH) compound solution
b. CYTOPLASMIC FIXATIVE – ex. Helly’s fluid, Orth’s fluid, o Solidifies at 17°C
Regaud’s fluid, Flemming fluid without acetic acid, Formalin o Fixes & precipitates nucleoproteins, chromosomes, & chromatin
with post chroming (HORFF) material
3. HISTOCHEMICAL FIXATIVE – ex. 10% Formol saline, Absolute
Ethyl Alcohol, Newcomers fluid, Acetone (FANA) ALCOHOL FIXATIVES
a. Lipid Fixation – fixatives containing mercuric chloride and 1. Methyl alcohol
potassium dichromate in cryostat section - for fixing dry and wet smears (PBS and BM tissues)
b. Carbohydrate Fixation – Alcoholic fixative for glycogen 2. Ethanol
(Rossman’s fluid or cold absolute alcohol) - simple fixative incorporated with compound fixatives for
c. Protein Fixation – neutral buffered forma better results, preserves but does not fix glycogen
(Disadvantage: polarization)
ALDEHYDE FIXATIVES 3. Isopropyl alcohol
1. Formaldehyde (Formalin) – gas produced by the oxidation of - for fixing touch preparation
methyl alcohol. Concentrated solutions should not be 4. Carnoy’s fluid
neutralized (explosion) Stock solution: 37-40% Working solution: - for fixing chromosomes, lymph glands and urgent biopsies
10% (no buffer: unstable) Formalin pigments: (MOST RAPID;1-3 hours)
a. Paraformaldehyde 5. Alcoholic Formalin (Gendre’s fixative)
o White crystalline precipitates - to preserve sputum
o Due to prolonged standing 6. Newcomer’s
o Removed by: 10% METOH/filtration - for fixing mucopolysaccharides and nuclear proteins. Give
b. Acid formaldehyde hematin better reaction in Fuelgen stain than Carnoys
o Brown/black granular deposits that may obscure
microscopic details OSMIUM TETROXIDE
2. 10% Formol Saline – diluted in 10% NaCl CNS o Pale yellow powder which dissolves in water (up to 6% at 20°C)
3. 10% Neutral Buffered Formalin (NBF) or PO4 buffered formalin to form a strong oxidizing solution
- Best general tissue fixative o Inhibits hematoxylin
- Best fixative for tissue containing iron granules w/ double o Produce black precipitate crystals (osmium oxide)
phosphate buffer 1 mm/hr = rate of tissue penetration o For lipids
4. Formol corrosive (formol sublimate) a. Flemmings solution – most common chrome-osmium
- for routine post mortem tissues w/ HgCl2 acetic acid fixative used (FIXATIVE & DECALCIFYING AGENT),
5. Glutaraldehyde - EM permanently fixes fat, for nuclear structures (excellent)
6. Karnovsky’s paraformaldehyde – glutaraldehyde b. Flemming’s solution without acetic acid (improve
- EM: electron histochemistry & electron immunocytochemistry cytoplasmic details) – recommended for mitochondria
7. Acrolein – Mixture w/ formaldehyde/formaldehyde
8. Formol-calcium – Lipids (frozen section) TRICHLOROACETIC ACID
o Precipitates proteins
METALLIC FIXATIVES o Swelling effect counteract shrinkage by other fixatives
1. MERCURIC CHLORIDE (BOSCHZZ) o Weak decalcifying agent (softening effect
a. B5 fixative – For BM biopsies. FT: 1 ½ -2 hours
b. Ohlmacher’s ACETONE
c. Schaudinn’s o Used at ice cold temperature from -5°C to 4°C
d. Carnoy-Lebrun o For diffusible enzymes such as phosphatases and lipases
e. Heidenhain Susa – For tumor biopsises esp of o For fixing BRAIN TISSUE (Rabies Diagnosis)
the skin. FT 3-12 hrs
f. Zenker (fixing small pieces of liver,spleen, CT HEAT FIXATION
fibers and nuclei) FT: 12-24 hrs g. Zenker’s o Thermal coagulation of tissue proteins
formol (helly’s solution) – Fixative for pituitary o For bacteriologic smears
gland, BM and blood containing organs o Bacteriologic smears
o Microwave: 45-55°C
2. CHROMATE FIXATIVES o Underheating: poor sectioning
a. Regaud’s (mollers) (molliflex) – to demonstrate chromatin, o Overheating (>65°C): vacuolation, overstained cytoplasm
mitochondria, mitotic figures, golgi bodies, RBC a. Fixatives for enzyme histochemistry
b. Orth’s fluid – to study early degenerative processes and - 4% formalin or formol saline, acetone or formalin for
tissue necrosis, preserves myelin better cryostat section
c. Chromic acid - preserves CHO b. Fixatives for EM
d. Potassium dichromate (K2CrO4 ) - preserves lipids and - Glutaraldehyde, PtCl3, PtCl3 – formalin (Zamboni’s),
mitochondria (pH 4.5-5.2) AuCl, Osmium tetroxide, 10% NBF = acceptable but not
recommended
3. LEAD FIXATIVES c. Fixative for electron histochemistry and electron
- Fixes CT mucin and is recommended for acid immunocytochemistry
mucopolysaccharides - KARNOVSKY’S PARAFORMALDEHYDE
GLUTARALDEHYDE

[SARANILLO, KA.]
 FIXATION TERMINOLOGIES
1. SECONDARY FIXATION- process of placing an already fixed tissue
in a second fixative
2. POST-CHROMATIZATION - fixation whereby a primarily fixed
tissue is placed in Aqueos solution of 2.5-3% potassium
dichromate
3. WASHING OUT - Removing excess fixative
a. Tap water (chromates) - Helly’s, Zenker’s, Flemming’s
b. 50-70% alcohol- picric acid (Bouin’s solution)
c. Alcoholic iodine – mercuric fixatives

FACTORS AFFECT FIXATION OF TISSUES

RETARDED BY:
1. Size and thickness of the tissue specimen
2. Presence of mucus
3. Presence of fat 1. Size and thickness of tissue
4. Presence of blood 2. Agitation
5. Cold temperature 3. Moderate heat (37-56°)

PRINCIPLES AND PRECAUTIONS IN HANDLING FIXATIVES

1. Autopsy/surgical materials should be fixed as soon as after death
2. All tissue must be properly labelled
3. If tissue are refrigerated, avoid 0°C
4. Tissue should not be more than 5mm thick, except in lung edema (1-
2 cm thick)
5. Purulent, exudates or transudates should be kept for bacteriologic
culture
6. Amount of fixative must be adequate 20:1
7. CONTAMINATION of tissue should be avoided
8. WASH the tissue before staining
9. SOLID ORGANS must be injected with fixatives
10. HOLLOW ORGANS should be packed with cotton soaked in fixative/
or opened
11. AIR- FILLED LUNGS may float on fixative, to avoid this cover the lungs
in several layers of gauze soaked with fixative
12. HUMAN BRAIN should undergo intravascular perfusion (avoid
washing out of blood with Ringers lactate). Suspended in a cord tied
in the circle of Willis (Fixation time: 2 weeks)
13. EYES should not be dissected before they are fixed.
14. Frozen section may lead to formation of ICE CRYSTALS
15. When handling MUSCLE TISSUES, it should be stretched for 30 min.
to avoid rigor
16. WATER SHOULD NOT BE USED FOR GLYCOGEN containing tissue
17. HARD TISSUES may be washed out overnight with running water, and
immersed in 4% aqueous phenol for 1-3 days (LENDRUMS METHOD)

DIFFICULTIES ENCOUNTERED BECAUSE OF IMPROPER FIXATION

CAUSE
Failure to arrest early autolysis of cells failure to fix immediately
Removal of substances soluble in fixing agent wrong choice of fixative
Presence of artifact pigments incomplete fixation
Tissue are soft and feather like incomplete fixation
Loss or inactivation of enzymes wrong choice of fixative
Shrinkage and swelling of cells Overfixation
blocks are brittle and hard prolonged fixation

PIGMENT COLOR REMOVED BY:

Acid Brown/black a. Saturated picric acid
formaldehyde granules b. Alcoholic KOH
hematin c. Kardasewitsch method
d. Lillie’s method
Mercuric chloride Black granules Alcoholic iodine
pigment
Chromate Fine, yellow brown Acid-alcohol
pigment
Osmium tetroxide Black precipitate Cold H2O
pigment crystals
Crush artifact Intense eosinophilic staining at the center of the
tissue (H & E)
Due to partial coagulation of partially fixed protein

[SARANILLO, KA.]