Steps in

Histopathology
Maricelle D. Manlutac, RMT, MPH, MLS(ASCPi)
Faculty
Examination of Fixed Tissues:
Histopathologic Techniques/Steps

1.Numbering 7. Blocking

2. Fixation 8. Trimming

**Decalcification 9. Sectioning

3. Dehydration 10. Staining

4. Clearing 11. Mounting

5. Impregnation 12. Labeling

6. Embedding
Numbering
Enter all data in the logbook/record book
• Patient information (Name, Age, Address, Sex, CS,
Birthdate, Hospital number/Bar code)
• Type of specimen
• Desired Examination
• Clinical diagnosis
• Requesting Physician

Get the corresponding number: Accession number

Ex. AUF-HP-2016-S-001
Specimen Worksheet

 Also known as Gross Worksheet / Histopathology Form

 Provides information: orientation in embedding and

special instructions (Special stains, etc.)

Contains (but not limited to):

a. Accession number

b. Number of slides (ex. FNAB)

c. Blocks per case

Fixation
 Most critical step: preserving fresh tissue for examination
“Quality of the section on the slide is only as good as the quality
of the fixed tissue specimen”

 Life-like manner preservation as possible

 Harden and protect the tissue from trauma

 Prevents degeneration, decomposition, putrefaction, and

distortion of tissues - protein stabilization

 Risk of infection is reduced

1º Aim: Preserve the morphologic and chemical integrity of the cell in a

life-like manner
2º Aim: Harden and protect the tissue from the trauma of further
handling
Fixation
Purpose:
1. To preserve the tissue
 Stop all cellular activities

2. To prevent breakdown of cellular elements

 Inactivation of lysosomal hydrolytic enzymes

3. To coagulate or precipitate protoplasmic substances

 Render tissue components insoluble that may leak out during
histologic handling
GENERAL EFFECTS
 Confers chemical stability on the tissue

 Hardens the tissue (helps further handling)

 Halts enzyme autolysis

 Halts bacterial putrefaction

 May enhance later staining techniques

Mechanisms involved in Fixation:
A. Additive fixation
 Chemical constituent of fixative is taken in & becomes part of
the tissue
 Stable proteins: form cross-links or molecular complexes

Example: formalin, mercury, osmium tetroxide

B. Non-additive
 Fixing agent is not incorporated into the tissue
 Stable protein: removing the bound water attached to H-bonds

Example: Alcoholic fixatives

Mechanisms involved in Fixation:
 Aldehydes: cross-linkages formed in the proteins
 Mercurials: give excellent nuclear detail. Their best
application is for fixation of hematopoietic and
reticuloendothelial tissues.
 Alcohols: protein denaturants
 Oxidizing agents: cross-link proteins, but cause
extensive denaturation
 Picrates: nuclear detail
Characteristics of a Good Fixative
 Readily available

 Cheap

 Stable

 Safe to handle

 Kills quickly

 Minimum tissue shrinkage

 Rapid & even penetration

 Hardens tissues for easier cutting

Factors involved in Fixation:

1. Hydrogen Ion Concentration

 Ideal pH is between 6 and 8

2. Temperature
 Surgical specimen: Room temperature
 EM and histochemistry: 0-4 Celcius

3. Thickness of section
 Tissue blocks: small, thin, within prescribed tissue
processor
Factors involved in Fixation:
4. Osmolality
 Best: Slightly hypertonic solutions
 More or less isotonic

5. Concentration
 Formaldehyde: 10% solution
 Glutaraldehyde: 3% solution

6. Duration of Fixation
 Primary fixation in buffered formalin: 2-6 hours
 Prolonged fixation- shrinkage and hardening
 EM: 3 hours fixation holding buffer
 Recommended size of tissue: 2cm2 or no more than 4 mm
Consideration for Fixation:
 Speed
 Specimen should be placed in fixative as soon as removal
from the body

 Penetration
 Rate: 1 mm per hour

 Volume
 Traditional volume: 10-25 x the volume of the tissue
 Maximum effectiveness: 20 x the volume of the tissue

 Duration of Fixation
 Fibrous organs uterus and intestine longer
 Cut down duration: heat, vacuum, agitation, or microwave
Types of Fixative
 According to COMPOSITION
 Simple : Aldehydes, Metallic fixatives
 Compound

 According to ACTION
 Microanatomical
 Cytological : Nuclear & Cytoplasmic
 Histochemical

 According to effects on tissue protein: Precipitant (P)

or non-precipitant (NP)
According to effects on tissue
protein
Precipitant Non-precipitant
 Chromium trioxide  Acetic acid

 Ethanol  Formaldehyde

 Mercuric chloride  Osmium tetroxide

 Methanol  Potassium dichromate

 Picric acid
Simple Fixatives
 Aldehydes  Picric Acid
 Formaldehyde
 Acetic Acid
 Glutaraldehyde
 Acetone
 Metallic Fixatives
 Mercuric Chloride  Alcohol
 Chromate Fixatives  Osmium Tetroxide /
 Lead Fixatives Osmic Acid

 Heat
Microanatomical Fixatives
 10 % Formol Saline  Zenker’s

 10 % Neutral Buffered  Zenker – Formol (Helly’s)

Formalin
 Bouin’s
 Heidenhain’s Susa
 Brasil’s
 Formol Sublimate (Formol
Corrosive)
Cytological Fixatives
 Nuclear:  Cytoplasmic
 Flemming’s  Flemming’s w/o acetic
 Carnoy’s acid
 Bouin’s  Helly’s
 Newcomer’s  Formalin w/ post
chroming
 Heidenhain’s
 Regaud’s (Moller’s)
 Orth’s
Histochemical Fixatives
 Formol Saline 10%

 Absolute Ethyl Alcohol

 Acetone

 Newcomer’s Fluid
I. Aldehyde Fixatives
I.a Formaldehyde (HCHO)
 Oxidation of methyl alcohol

 Fixation time: 12-24 hours

 Cheap, readily available, easy to prepare, stable,

compatible w/ stains, penetrates tissues well, preserves fat,
mucin, glycogen, for tissue photography

Polarization of HCHO Turbidity Paraformaldehyde

Precaution of HCHO:
 Irritating fumes, prolonged fixation may bleach tissues
 Well ventilated room

 Not neutralized if concentrated – explosion

 Bleaching
 prevented by changing formalin

 Form abundant brown artifacts, pigments, and granules

 Removed by: A. Kardasewitsch’s Method – ethyl alcohol
B. Lillie’s Method – Hydrogen peroxide
C. Picric Acid Method- Saturated Picric Acid Solution
I. b Glutaraldehyde
 Aldehyde with MW 100 / Formaldehyde residues linked by
3 straight carbon chains

 For LM, EM, cellular and plasma protein

 Small fragments and needle biopsies: 2.5% solution

 Large tx ≤ 4 mm thickness: 4% solution

Advantage vs. HCHO: more stable effect, less tissue

shrinkage, less irritating

Disadvantage: more expensive, slow penetration

II. Metallic Fixatives
II. a Mercuric Chloride
 Most common metallic fixative; 5-7 %

 For tissue photography, recommended for renal tissues,

fibrin, CT, muscles

Disadvantage: hardens outer layers only, black granular

deposits formed, corrosive to metals
Removal of Mercuric Chloride Granules

Bring sections to water

Alcoholic Iodine for 1-2 mins

(0.5mL of iodine in 100mL of 70% alcohol)

Rinse with water

5% Sodium Thiosulfate- Hypo solution for 1-2 mins

2-5 minutes wash with running

water
II. b Chromate Fixatives
 Strong oxidizing agent- do not combine with alcohol and
formalin

 Chromic Acid – preserves CHO and strong protein precipitant

Potassium Dichromate (K2Cr2O7)

 Used in 3% aqueous solution
 preserves lipids, mitochondria
II. c Lead Fixatives
 4% aqueous solution

 Mainly for MPS

 Precipitates protein

Forms insoluble lead carbonate

 Filtering
 Adding HAc
III. Picric Acid Fixatives
 Solubility: 1.7 grams dissolved in 100mL dH2O at 15 ° C

 Chemically: 2,4,6-trinitrophenol

 Best for Glycogen fixation

Removal of Picric Acid Yellow Color

• Lithium carbonate in 70% alcohol then wash

• 70% ethyl alcohol followed by 5% Sodium Thiosulfate then

wash
IV. Acetic Acid Fixative
 Used at – 40 ° C

 Solidifies at 17 degrees C  glacial

 For nucleoproteins, chromosomes

 Contraindicated in cytoplasmic fixatives 

destroys mitochondria & golgi
V. Acetone
 Used at – 5 to 4° C

 Fixing brain tissues for rabies

 For Enzyme studies

 Phosphatases
 Lipases

 Dissolves fat, evaporates rapidly, preserves

glycogen poorly
VI. Alcohol Fixatives
 Denatures/precipitates CHONs (destroys H bonds)

 Concentration: 70-100%

 For small tissue fragments and glycogen

Disadvantage:

Polarization – causes glycogen granules to move towards

the poles / ends of cells
VII. Osmium Tetroxide Fixative
 Osmic acid

 Pale yellow

 Dissolves in water: strong oxidizing agent

 Acid vapor  conjunctivitis, osmic oxide in cornea 
blindness

 Fixes fats, for EM, and excellent in nuclear staining

 Expensive, poor penetration, reduced w/ sunlight 

black deposit; dark bottle
VIII. Heat Fixation
 Thermal coagulation of tissue proteins

 Heat fixation generally preserves overall morphology but

not internal structures

 Heat denatures the proteolytic enzyme and prevents

autolysis

Ex. bacteriologic smears

Compound Fixative

I. Microanatomical
A. 10% Formol-Saline
 Nervous system and general post mortem

B. 10% Buffered Formalin

 Surgical post mortem research

C. Heidenhain’s Susa Solution

 Skin Biopsy

D. Formol Sublimate
 Routine post mortem materials
Compound Fixative
I. Microanatomical
E. Formol Saline Sublimate
 Post mortem

F. Zenker’s Solution
 Nuclei; Trichrome technique

G. Zenker’s Formol (Helly’s Fluid)

 Pituitary tissues and bone

H. Bouin’s Solution
 Embryo
Compound Fixative
II. Cytological
1. Nuclear Fixative

A. Flemming’s Fluid
 Nuclear structures and fats

B. Carnoy’s Fluid
 Most rapid of all fixative
 Fixation time: ½ - 3 hours
 Chromosomal studies, lymph nodes, and urgent glycogen studies

C. Bouin’s Fluid
 Embryos and glycogen
 Kidney is badly distorted

D. Newcormer’s Fluid
 MPS, nuclear protein, and chromosomes
Compound Fixative
II. Cytological
2. Cytoplasmic Fixative

A. Flemming’s Fluid (Without Acetic Acid)

 Removal of acetic acid preserves cytoplasmic structures

B. Champy’s Fluid
 Mitochondria, golgi elements, and fats
 Require small amount of fixative: 5-10x

C. Regaud’s Fluid (Moller)

 Mitochondria and yolk

D. Orth’s Fluid
 Study of early degenerative process and tissue necrosis
 Rickettsia and other bacteria
Secondary Fixation
 Fixed tissue in a second fixative
 improvement the demonstration of particular substances
 make special staining technique possible
 ensure further & complete hardening & preservation of
tissues

Post-Chromatization
 Type of secondary fixation placed in 2.5-5% K2Cr2O7 for 24
hours
 Mordant for better staining effects
Removal of Fixation
“Washing-Out”
 improve staining and removal of artefacts
1. Tap water
: used to remove excess chromate, formalin or
osmic acid
2. 50-70% Alcohol
: picric acid (Bouin’s)
3. Alcoholic Iodine
: used to remove excessive mercuric fixatives
Problems Encountered:

1. Failure to arrest autolysis

2. Removal of substance soluble in fixative
3. Presence of artefacts
4. Soft and leathery tissue
5. Shrinkage and swelling of tissue
6. Brittle and hard tissue