HISTOPATHOLOGY

HISTOTECHNOLOGY
- Performed by the histotechnologist to provide the pathologist a good quality tissue section on
which to base patient’s diagnosis.
- Done in a slide for microscopic examination by the pathologist

TYPES OF SPECIMEN
1. AUTOPSY- from a DEAD sample
2. BIOPSY/SURGICAL- from a LIVING sample

*NOTE: MOST tissue specimen require MANDATORY SUBMISSION to the laboratory for examination

EXCLUDED SPECIMEN FOR MANDATORY SUBMISSION (p 4-5, LO)

 FATS from liposuction
 FORESKIN removed from circumcision
 NORMAL TOENAILS or FINGERNAILS incidentally removed

PRE-ANALYTIC FACTORS:
1. Warm Ischemia – Initial insult a tissue suffers when blood supply is interrupted (sudden cut off
of blood supply)
2. Cold Ischemia – state of lack of oxygen once tissue is removed from the body.

PURPOSE OF TISSUE PROCESSING

1. Through microscopic examination, the pathologist can determine the disease process involved.
The result is often used to diagnose disease, provided with a good quality tissue section.
2. To ensure that the pathologist receives good quality tissue sections, MORPHOLOGY and
CHEMICAL INTEGRITY of cells/tissues are necessary to maintain from the time it is removed
from the body until the time of processing.

PROCESSES INVOLVED:
1. GROSS EXAM carried out by the pathologist
2. DISSECTION
3. TISSUE PROCESSING- done by the medical technologist/histotechnologist
*For gross exam only:
 Accessory digits
 Varicose veins
 Prosthetic breast implants

FIXATION – first and most critical step

- Process of preserving morphology and chemical integrity of cells and tissues as close to original
as possible
- Killing, penetration and hardening of tissues
- Immersion of tissue specimen in a fixing agent
- PRIMARY GOAL: To preserve the tissue to prevent autolysis (postmortem decomposition) due
to bacterial attack, putrefaction and action of enzymes.
- SECONDARY GOAL: To harden tissues to facilitate cutting and to protect tissues from trauma
of further handling.

MECHANISMS: (*all fixatives are additive fixatives except acetone and alcohol containing fixatives)
1. ADDITIVE FIXATION- chemical components of fixatives are taken in by the tissues and
become part of the tissue to prevent autolysis and stabilize the protein. (e.g Formalin,
mercury, osmium tetroxide)
2. NON-ADDITIVE FIXATION – fixing agent doesn’t combine with tissue. Tissues are preserved
when fixatives alters tissue structure. (e.g Acetone, Alcohol)
 HISTOPATHOLOGY

FACTORS INVOLVED

1. Hydrogen Ion (H+) Concentration

 Satisfactory pH = 6-8
 Unbuffered formalin – little effect to tissue
 Low pH – dark pigment production, thus obscuring cell details. Remedy is to buffer at pH= 7.0
using acetate, phosphate, TRIS buffer.

2. Osmolality
 Slightly hypertonic (but in practice, isotonic)
 Isotonic – holding solution for frozen or kidney biopsies during transport
 Hypertonic = Shrinkage
 Hypotonic = Swelling, leading to poor fix’n

3. Temperature
 Surgical specimens = ROOM TEMPERATURE (conventional)
 AUTOTECHNICON – automatic tissue processor used in hospitals that can do fixation,
dehydration, clearing and infiltration; calibrated to 40°C
 Ideal temp. for Electron Microscopy and Histochemistry, 0-4°C
 Formalin heated to 60°C = rapid fix’n of VERY URGENT BIOPSIES
 Formalin heated to 100°C = fix tissues with TB

4. VOLUME
 10-25x the volume of spx
 Max. effectiveness = 20x the vol. of spx
 Formalin-Spx ratio, 15-20 : 1
 Museum prep’n = should NOT be less than 50-100x the vol. of spx
 When osmium tetroxide is used, fixative agent should only be 5-10x the vol. of spx

5. THICKNESS
 Fixative agent penetrate tissues @ a rate of 1mm/hr or 3.6mm/hr or 7.2mm every 4 hours
 Tissue block should NOT be more than 5mm or must be 4-5 mm
 When placed in a tissue cassette, spx MUST be 3mm
 When using a lung spx, 1-2cm
 Specimen for EM = 1-2 mm2
 Specimen for LM = 2 cm2

6. CONCENTRATION
 Formaldehyde, 10% solution
 Glutaraldehyde, 3% solution
 For immunoEM, 0.25% glutaraldehyde

7. Duration of fixation
 Spx MUST be fixed within 20-30 minutes from the time blood supply is interrupted/cut off
 Fixation time (6-48 hrs) – time period the tissue is exposed to fixing agent
 For EM = 3 hours

FACTORS THAT PROLONG/RETARD FIX’N

1. Size and thickness of tissue – the larger the tissue, the more fixative to be used and the
longer the fixation time
2. Cold temperature – Inactivation of enzymes
3. Presence of mucus Slow penetration, Slow and poor fixation
4. Presence of blood Flushed with NSS before fixation
*Brain Fix’n – undergo INTRAVASVULAR PERFUSION (washing out of blood using Ringer’s
Lactate)
5. Presence of fat – Must be cut thinly and fixed longer
 HISTOPATHOLOGY

FACTORS THAT ACCELERATE/ENHANCE FIX’N

1. Size and thickness of tissue - the smaller the tissue, the lesser fixative to be used and the
shorter the fixation time
2. Agitation – use of autotechnicon for continuous mixing
3. Heat - @37°C

FACTORS TO CONSIDER IN CHOOSING THE RIGHT FIXATIVE

1. Need for immediate exam – FAST ACTING
2. Type of tissue to be processed
 Liver – Zenker’s fluid
 BM – B5
3. Tissue structure to be studied
 Chromosome – glacial acetic acid
 Glycogen – Brasil’s
4. Staining technique to be applied – fluid vs. stain, must be compatible
5. Type of section to be made – serial or individual

GENERAL EFFECTS OF FIXATIVES

1. Make the tissue resistant to damage
2. Harden the tissue
3. Inhibit bacterial decomposition
4. Reduce risk of infection during handling
5. Act as mordant that facilitates & hastens staining procedure
6. Promote optical differentiation of cells/tissues

CHARACTERISTICS OF A GOOD FIXATIVE (Refer to book by Gregorios)

TYPES OF FIXATION

A. Physical Methods
1. Heat Fixation
 carried out for frozen sections and fixing bacterial smears
 involves thermal coagulation of CHONs
2. Microwave
 accelerate staining and decalcification
 may be used for neurochemical substances (Acetylcholine)
3. Freeze-drying – Special way of preserving tissues by rapid freezing (QUENCHING)
 Must be 2mm
 Immersed in freezing agents for 2-3s (e.g. Liquid nitrogen, Isopentane,
Propaneisopentane)
 Dehydration – tissue is placed in a vacuum drying chamber @ -40°C
4. Freeze substitution
 No vacuum drying chamber
 Tissue is immersed in Rossman’s solution or 1% Acetone and dehydrated using
absolute alcohol
B. Chemical Methods – immersion of tissue in fixing solution, most common
1. Coagulant fixatives
 Capable of forming NETWORKS to allow rapid penetration
 Maintains cellular architecture and tissue morphology at light microscopic level
 Mitochondria and secretory granules poor preservation
 Alcohol (methanol, ethanol), acetone, TCA, picric acid
2. Cross-linking fixatives / non-coagulant
 capable of forming GEL, making process SLOWER
 Formaldehyde-based, combined with other agent
 Useful for specific tissues (e.g. Alcoholic formalin for fatty tissues like breast)
 HISTOPATHOLOGY

TYPES OF FIXATIVES

I. According to composition
a. Simple fixatives – one component substance only
1. Aldehydes
a. Formaldehyde b. Glutaraldehyde
2. Metallic Fixatives
a. Mercuric Chloride c. Lead fixatives
b. Chromate Fixatives
o Potassium Dichromate
o Chromic Acid
b. Compound fixatives - Fixative + Fixative

II. According to action

A. Microanatomical fixatives
 permit general microscopic study of tissue structure without structural and
intercellular alteration
 most fixatives are under this category
B. Cytological fixatives - preserve specific parts of cell
1. Nuclear – with HAc, pH= less than 4.6
2. Cytoplasmic – without HAc, pH= more than 4.6
C. Histochemical fixatives – preserve chemical (enzyme) constituents

ACCORDING TO ACTIVE COMPONENT SUBSTANCE

A. Trichloroacetic acid – fixative and decalcifying agent (compound fixative)

Advantages:
1. Precipitates protein
2. Weak decalcifying agent
Disavantage:
1. Poor penetration (for small pieces of tissues or bones only)

B. Glacial acetic acid – compound fixative that solidifies @ 17°C

Advantages:
1. Fixes and precipitates nucleoproteins.
2. Precipitates chromosomes and chromatin materials.
3. Collagen-containing tissues tend to swell.
Disadvantage:
1. Contraindicated for cytoplasmic fixation (destroys mitochondria and Golgi apparatus)

C. Acetone – used at ice cold temp. (-5-4°C), fixative and dehydrating agent
Advantages:
1. For the study of phosphatases and lipases (water diffusible enzymes)
2. Fixation of brain tissues for rabies diagnosis
3. Solvent for metallic salts
Disadvantages:
1. Dissolves fat
2. Poor glycogen preservation
3. Volatile / evaporates rapidly
4. For small tissues only
 HISTOPATHOLOGY

D. Picric acid fixatives

 Used in strong saturated aqueous solution (1%)
 Produce a YELLOW COLOR, removed by an (1) acid dye, (2) LITHIUM CARBONATE
(3) immersion in 70% alc. and 5% sodium thiosulfate and wash with water.
GENERAL ADVANTAGES
1. For glycogen demonstration
2. Allows brilliant staining with the trichrome method
3. Suitable for ANILINE STAINS (Malloy’s / Heidenhain’s / Masson’s)
4. Precipitates all proteins
GENERAL DISADVANTAGES
1. Causes RBC hemolysis
2. Not suitable for frozen sections
3. Must never be washes in water before dehydration
4. Highly explosive when dry
5. Alters and dissolves lipids
6. Interferes with AZURE EOSIN method of staining

 Bouin’s –fixation of EMBRYO & PITUITARY BIOPSY

Advantages
1. Minimal distortion of micro-anatomical structures
2. For ENDOMETRIAL CURETTINGS
Disadvantages
1. Not suitable for fixing kidney structures, lipid, mucus
2. Destroys cytoplasmic structures (mitochondria)
3. Reduces FEULGEN rxn

 Brasil’s
Advantages
1. Better and less messy than Bouin’s
2. Excellent for glycogen fixation

 Hollande’s solution
1. For GIT biopsies and endocrine tissues

E. Alcohol fixatives
 denatures and precipitates CHONs by destroying H-bonds and other bonds
 Concentration : 70-100%
 Absolute Alc.- preserve glycogen, pigments, blood, tissue films and smears
 80% Alc. – for photographic work and demonstration of the original color of spx.

GENERAL ADVANTAGES
1. For small tissue fragments
2. Fixative and dehydrating agent
3. For glycogen preservation
4. Preserves nuclear stains
GENERAL DISADVANTAGES
1. Low concentration causes RBC hemolysis
2. Dissolves fats and lipids
3. Causes GLYCOGEN POLARIZATION (accumulation of granules at both end of the cell)
 HISTOPATHOLOGY

 100% Methyl Alc.

Advantages:
1. Both fixative and dehydrating agent.
2. Fixes dry and wet smears, blood smear and bone marrow tissues.
Disadvantages:
1. Slow penetration
2. Can over harden and make tissues difficult to cut due to prolong fixation (>48hrs)

 Ethyl alcohol
 Simple fixative, with concentrations of 70-100% but frequently incorporated into
compound fixatives for better results.
 Low conc. = RBC hemolysis, Inadequate WBC preservation
Advantages:
1. Preserves but does not fix glycogen
2. For HISTOCHEMISTRY esp. enzyme studies (nucleoprotein & nucleic acid)
3. Fixes blood, tissue films and smears.
Disadvantages:
1. Causes GLYCOGEN POLARIZATION
2. STRONG REDUCING AGENT, and should not be mixed with strong oxidizing
agent (Potassium Dichromate, chromic acid and osmium tetroxide)
3. Less hemosiderin preservation

 Isopropyl alcohol – for fixing TOUCH PREPARATIONS

 Carnoy’s fluid – fixes chromosomes, lymph glands, URGENT BIOPSIES and brain
tissues for rabies diagnosis.

Advantages:
1. MOST RAPID FIXATIVE
2. Both FIXATIVE and DEHYDRATING agent
3. Preserves NISSL BODIES, CYTOPLASMIC GRANULES,
NUCLEOPROTEINS and NUCLEIC BODIES.
4. Excellent for GLYCOGEN FIXATION.
Disadvantages:
1. Produces RBC hemolysis
2. Dissolves FAT, LIPIDS and MYELIN
3. Leads to POLARIZATION, unless cold temp. is used (-70°C)
4. Dissolves ACID-SOLUBLE GRANULES & PIGMENTS

 Newcomer’s fluid
Advantages:
1. Both NUCLEAR and HISTOCHEMICAL agent
2. Produces better reaction with FEULGEN’S stain that Carnoy’s
3. Fixes MUCOPLOYSACCHARIDES and NUCLEAR PROTEINS

 Clarke’s solution – better results with H&E, preserves NUCLEIC ACID

 Methacarn – causes less hardening that Carnoy’s

 Rossman’s solution – good for CT MUCINS and UMBLICAL CORD

 HISTOPATHOLOGY

F. Metallic fixatives
1. Lead fixatives – used in 4% aqueous solution for basic lead acetate
Advantages:
1. For ACID MUCOPOLYSACCHARIDES
2. Fixes CONNECTIVE TISSUE MUCIN
Disadvantage
1. Takes up CO2 forming insoluble lead

2. Chromate fixatives
 Chromic acid
 used in 1-2% aqueous solution, precipitates all proteins
 adequately preserves CHO
 STRONG OXIDIZING AGENT

 Potassium dichromate – used in 3% aqueous solution

Advantages:
1. Preserves MITOCHONDRIA (pH= 4.5-5.2) and LIPIDS
(*acidic solution causes mitochondria destruction but cytoplasm, chromatin
bodies and chromosome fixation)
2. Fixes but does not precipitate cytoplasmic structures

 Regaud’s / Moller’s fluid

Advantages:
1. For the demonstration of chromatin, mitochondria, mitotic figures, Golgi
bodies, RBC and colloid-containing tissues.
Disadvantages:
1. Always be FRESHLY PREPARED
2. Tissue should not be thicker than 2-3mm
3. Produces SUB-OXIDE precipitates
4. Blackens tissue fragments due to prolong fixation
5. Poor glycogen penetration (contraindicated for CHO)
6. Reduced intensity PAS reaction

 Orth’s fluid
Advantages:
1. Demonstration of RICKETTSIAE
2. Recommended for the study of EARLY DEGENERATIVE PROCESSES and
TISSUE NECROSIS
3. Better MYELIN preservation
Disadvantages:
1. SAME AS REGAUD’S / MOLLER’S

3. Mercuric chloride
 MOST COMMON metallic fixative, used frequently in sat. aqueous solution of
5-7%
 Combined with other fixatives and used widely as SECONDARY FIXATIVE
 Produces BLACK PRECIPITATES of mercury

REMOVAL OF MERCURY DEPOSITS

1. Treat section with 0.5% iodine solution in 70% ethanol for 5-10 minutes
2. Treat with water
3. Decolorize or 5mins. in sodium thiosulfate
4. Wash in running water
 HISTOPATHOLOGY

MERCURIC CHLORIDE POST-FIXATION

1. Poor ultrastructural preservation
2. Good trichrome staining
3. Satisfactory immunoperoxidase techniques

General advantages:
1. Fine nuclear components details
2. Precipitates ALL proteins
3. For CYTOPLASMIC STAINING (in lieu of formaldehyde)
4. Excellent Trichrome staining
5. Used in TISSUE PHOTOGRAPHY
6. Brilliant metachromatic staining of cells
7. Recommended for renal tissues, fibrin, connective tissues and muscles
General disadvantages
1. Use off thin sections only of not more than 5mm in thickness
2. Causes RBC hemolysis and removal of much iron from hemosiderin
3. INERT to fats and lipids
4. Forms BLACK GRANULAR DEPOSITS
5. Corrosive to metals
6. Difficult cutting of frozen section due to inadequate freezing of fatty tissues
*Precautions:
1. Black deposits can be removed by adding SAT. IODINE SOLUTION IN 96%
ALC.
2. Use of METALS and JEWELRIES should be AVOIDED.

 Zenker’s fluid – HgCl2 + glacial HAc* (*prevent turbidity and formation

of dark precipitate)
 recommended for liver, spleen, connective tissue fibers and nuclei

Advantages:
1. Recommended for TRICHROME STAINING
2. Permits BRILLIANT STAINING of nuclear and connective tissue
fibers
3. Act as a MORDANT
Disadvantages:
1. Not stable after addition of glacial HAc
2. Poor penetration
3. Causes RBC hemolysis and removal of iron from hemosiderin
4. Tend to form MERCURIC PIGMENT deposits or precipitates
*Precautions and practical considerations
1. Mercury deposits can be removed by immersing tissues in alc. Iodine
solution (DE-ZENKIRIZATION)
2. Chemically = TREATMENT WITH SODIUM THIOSULFATE

 Zenker formol (Helly’s solution)

Advantages:
1. Excellent microanatomic fixative for pituitary gland, bone marrow,
spleen, liver
2. Better than Zenker in terms of fixation and staining
3. Preserves cytoplasmic granules
Disadvantages:
 HISTOPATHOLOGY

1. Same with Zenker’s but the color of the pigment produced is BROWN
due to RBC hemolysis from prolonged fixation of >24 hrs

 Heidenhain’s Susa – for TUMOR BIOPSIES (skin), excellent

CYTOLOGIC FIXATIVE
Advantages:
1. Rapid and even penetration and fixation
2. Produces shrinkage and hardening of tissues due to COUNTER-
BALANCE
3. Permits SILVER IMPREGNATION
4. Permits easier sectioning of large blocks of fibrous connective tissues
Disadvantages:
1. Tissue should not be more than 1cm thick
2. Weigert’s method is NOT possible
3. Deposits tend to form on tissues
*Precaution
1. After using fixative, TRANSFER DIRECTLY to a high-grade alc. e.g.
96% or absolute alc.

 B-5 fixative – for BONE MARROW BIOPSIES

Advantages:
1. Good fixative for cytology of bone marrow biopsies
2. Rapid fixation, 1.5-2 hrs.
*Precaution
1. Forms precipitate but with no consequence
2. Overfixation hardens the tissue and makes difficult cutting.

G. Zinc sulfate
 Replacement for mercuric chloride due to concerns of the mercuric chloride
 Preserves tissue antigenicity
Advantages
 Moderate health risks
 Preserves antigenicity
Disadvantages
 Precipitate zinc, carbonates, phosphates
 May cause clog to the tissue processor line
 Respiratory, skin, eye irritant
 Ingestion of 10g of zinc may cause death

H. Osmium tetroxide
 Pale yellow powder
Advantages
 Fixes conjugated fats and lipids
 Fixes myelin and peripheral nerves
 Adequately fixes materials for ultrathin sectioning in EM
 Precipitates and gels CHONs
Disadvantages
 Inhibits hematoxylin
 Forms black precipitate after contact with organic matter and sunlight exposure
 Can cause irritation, conjunctivitis and blindness* (*due to black osmic oxide)
 Very expensive
 Extremely volatile
 HISTOPATHOLOGY

 Flemmings with HAc

 Most common chrome osmium acetate
 For nuclear preparation
Advantages
 Fixes nuclear structures (chromosomes)
 Permanently fixes fats
Disadvantages
 Chromic-osmic acid depresses staining power of hematoxylin
 Tend to form artifact pigments
 Very expensive

 Flemming’s without HAc

 For cytoplasmic structures (mitochondria)

I. Aldehyde fixatives
 Glutaraldehyde
 Made up of two formaldehyde residues
 2.5 % sol’n – for small or needle biopsies, 2-4 hrs. @ RT
 4% sol’n – for large tissues, 6-24 hrs
Advantages
 Firmer texture of central nervous tissues
 Preserves plasma proteins
 For enzyme histochemistry and EM
 Does not cause dermatitis
Disadvantages
 More expensive
 Less stable
 Reduces PAS positivity of reactive mucin

 37-45% Formaldehyde (100% formalin)

 10% formalin – most commonly used
 pH = 7, with phosphate buffer
Advantages
 cheap, readily available, fast acting
 compatible with many stains
 preserves fats, mucin, glycogen
 for colored tissue photography and mailing spx
 Tolerant fixative
Disadvantages
 Can cause allergic dermatitis
 If unbuffered ;
a. Reduces basophilic/eosinophilic staining
b. Forms abundant brown pigment
 Prolonged fixation ;
a. Can cause bleaching, dispersal of fats and loss of glycogen

 10% Formol saline –with NaCl

 Fixes CNS tissues, general post-mortem tissues for histochemical
examination
Advantages
 Preserves microanatomic and cytologic details
 Preserves enzymes and nucleoproteins
 Demonstrates fats and mucin
 For silver impregnation
 HISTOPATHOLOGY

Disadvantages
 Slow fixative
 Metachromatic reaction of amyloid is reduced

 10% Neutral buffered formalin (NBF)

 For the preservation and storage of surgical, post-mortem, research spx
Advantages
 Prevents ppt’n of acid formalin pigments
 Best fixative for tissues containing iron pigments and elastic fibers
Disadvantages
 Time consuming
 Inert towards lipids (neutral fats, phospholipids)
 Reactivity of myelin to Weigert’s iron hematoxylin is reduced

 Formol-corrosive (Formol-sublimate) – formaldehyde + mercuric chloride

 For post-mortem tissues
Advantages
 Excellent for silver reticulum methods
 Brightens cytoplasmic and metachromatic stains
 No washing out
Disadvantages
 Forms HgCl2 deposits

 Alcoholic formalin (Gendre’s fixative) – formaldehyde + ethanol + glacial HAc

 For immunoperoxidase studies and for EM
Advantages
 Fix’n time if halved
 For rapid diagnosis (fixes and dehydrates)
 For glycogen preservation and micro-incineration
 Fixes sputum (coagulates mucus)
Disadvantages
 Gross-hardening of tissues
 Partial lysis of RBC
 Formaldeehyde = little cross linking
 Glutaraldehyde = effective at cross linking

 Glyoxal (40% aqueous sol’n)

 Smallest aldehyde
Advantage
 Rapidly penetrating ;
a. Surgical – 4-6 hours
b. Small biopsy – 45 minutes
Disadvantage
 Carcinogenic