Histopathologic Techniques

Frederick R. Llanera, MD, FPSP, ASCPi, AMT, RMT

Pathologist, Philippine Heart Center
Faculty, University of Santo Tomas
Guest Lecturer, University of Minnesota

Examination of Fresh Tissues

Teasing or Dissociation
Squash Preparation (Crushing)
Smear Preparation (Streaking, Spreading,
Pull Apart, Touch or Impression Smear
Frozen Section

FS indications
- rapid diagnosis (guide for intra-

operative patient management)

- to optimally process tissues for special
studies for diagnosis, treatment, or
research
- to confirm that lesional tissue is present for
diagnosis on permanent sections (sample
adequacy)

FS limitations

Limited section sampling

Ice crystal or freezing artifact
Inferior quality compared to paraffin
sections
Lack of special studies (time constraint)

Special stains, immunohistochemistry, culture

Lack of consultation for difficult cases

Consider these during RFS:

Relevant clinical information / history

Type of tissue or location of biopsy
To determine beforehand what information
the surgeon requires from the FS and how
the information will be used.
Optimal turn-around time is </= 15 mins

Consider these during RFS:

Coordination between lab and OR

(personnel involved)
Check cryostat (-17C)
No fixative used
Protection of laboratory personnel
Selecting part of the tissue for FS

Examination of Fixed Tissues Histopathologic Techniques /

Steps:

Numbering
Fixation
Dehydration
Clearing
Impregnation
Embedding

Blocking
Trimming
Sectioning
Staining
Mounting
Labelling

Fixation

Kills, hardens, preserves tissues for the next

histopath steps
life like appearance prevention of
degeneration, putrefaction, decomposition,
distortion protein stabilization (cross links
formed between fixative and proteins)
Reduce risk of infection
Promotes staining
Inhibit bacterial decomposition

Fixation

To preserve the tissue

Stop all cellular activities

To prevent breakdown of cellular elements

Inactivation of lysosomal hydrolytic enzymes

post mortem decomposition (autolysis); or
by chemically altering, stabilizing, and making
tissue components insoluble
Prevention of putrefaction after death
(bacterial / fungal colonization & overgrowth)

Fixation

To coagulate or precipitate protoplasmic

substances

Additive fixation chemical constituent of

fixative is taken in & becomes part of the
tissue by cross links or molecular
complexes stable protein (formalin,
mercury, osmium tetroxide)

Fixation

To coagulate or precipitate protoplasmic

substances

Non additive fixation removes bound

water by attaching to H bonds of certain
groups within the protein molecule new
cross links are established (alcoholic
fixatives)

Microwave Technique

Physical agent like vacuum, oven (heat)

and agitation to increase movement of
molecules and accelerate fixation
Accelerates staining, decalcification,
immunohistochemistry and electron
microscopy
Oscillation frequency 2450 mHz

Microwave advantages:

Tissue is heated right through the block in

a very short time (main advantage)
Non chemical technique (less
interference)
Rapid
Lesser time for immunohistochemistry and
in-situ hybridization

Microwave disadvantages:

Penetrates 10-15 mm only

No significant cross linking of protein
molecules; subsequent chemical fixation
may be needed
Viable spores/pathogens (alcohol based
fixatives or microwaving alone)

Special tissue processing

Tissues that must be submitted unfixed

Tissues for frozen section evaluation

Gout: uric acid dissolves in formalin may use 100%
ethanol instead
Tissues submitted for infectious disease and cytogenetic
studies
Lymph nodes for lymphoma work-up
Muscle and nerve biopsy
Kidney biopsies
Tissue submitted for analysis of lipids

Processing bone marrow

biopsies

The fixative used is very important.

Submit entire needle biopsy after
fixation in Bouins fluid overnight, which
is mildly acidic and removes calcium.
Serially number eight slides and cut
sections at 4 microns.
Stain slides 1 & 5 with H&E; slides 2 &
6 with reticulin stain, and slides 3 & 7
with iron.

Store slides 4 and 8

Fixative

Cheap
Stable
Safe to handle
Kills quickly
Minimum tissue
shrinkage
Rapid & even
penetration

Hardens tissues for

easier cutting
Isotonic

Types of Fixative
According to composition
- Simple Aldehydes, metallic fixatives
- Compound
According to action
- Microanatomical
- Cytological Nuclear & Cytoplasmic
- Histochemical

Simple Fixatives

Aldehydes

Formaldehyde
Glutaraldehyde

Metallic Fixatives

Mercuric Chloride
Chromate Fixatives
Lead Fixatives

Picric Acid
Acetic Acid
Acetone
Alcohol
Osmium Tetroxide /
Osmic Acid
Heat

Microanatomical Fixatives

10 % Formol Saline
10 % Neutral Buffered
Formalin
Heidenhains Susa
Formol Sublimate
(Formol Corrosive)

Zenkers
Zenker Formol
(Hellys)
Bouins
Brasils

Cytological Fixatives

Nuclear:

Flemmings
Carnoys
Bouins
Newcomers
Heidenhains

Cytoplasmic

Flemmings w/o acetic

acid
Hellys
Formalin w/ post
chroming
Regauds (Mollers)
Orths

Histochemical Fixatives

Formol Saline 10%

Absolute Ethyl Alcohol
Acetone
Newcomers Fluid

Formaldehyde

Methanol oxidized
Cheap, readily available, easy to prepare,
stable, compatible w/ stains, penetrates
tissues well, preserves fat, mucin,
glycogen, for tissue photography
Irritating fumes, prolonged fixation may
bleach tissues

Formaldehyde precautions:

Paraformaldehyde formation
Well ventilated room
Not neutralized if concentrated explosion
Buffered or neutralized by adding
magnesium carbonate/CaCO3 wide
mouth bottle
Bleaching prevented by changing formalin

10 % Formol Saline

Penetrates and fixes tissues well,

minimum shrinkage & distortion, does not
overharden tissues
Slow (>24 h)

10% Neutral Buffered Formalin

Na dihydrogen PO4, Disodium H PO4

For preservation and storage of surgical,
post mortem and research specimens
Best fixative for Fe pigments, elastic fibers
Longer to prepare time consuming, inert
towards lipids

Formol corrosive/formol sublimate

Formol mercuric chloride

Minimum shrinkage and hardening
No need for wash out from fixative to ROH
Slow
Forms mercuric chloride deposits

Glutaraldehyde

For LM, EM
Adv vs. HCHO: more stable effect, less
tissue shrinkage, less irritating
Disadv: more expensive, slow penetration

Mercuric Chloride

Most common metallic fixative; 5-7 %

For tissue photography, recommended for
renal tissues, fibrin, CT, muscles
Disadv: hardens outer layers only, black
granular deposits formed (removed by
adding iodine), corrosive to metals

Mercuric Chloride

Zenkers (HgCl2 + Glacial HAc) liver,

spleen, CT fibers, nuclei; poor penetration,
wash thoroughly in running H20
Zenker-Formol (Hellys)HgCl2 , K2Cr2O7
for pituitary, BM, spleen, liver; brown
pigment producedremove by picric/NaOH
Heidenhains Susa HgCl2, NaCl, TCA for
skin biopsies; place in high grade ROH

Mercuric chloride

(new) B-5 fixative for bone marrow

biopsies
- HgCl2, anhydrous Na acetate

Dezenkerization

HgCl2 deposits are removed by alcoholic

iodine solution prior to staining
Oxidation w/ Na to mercuric iodide,
removed by treatment with Na thiosulfate:

Bring slides to water. Immerse in Lugols

iodine (5mins), running water (5mins), 5% Na
thiosulfate (5mins), running water (5mins),
proceed with required water soluble stain

Chromate Fixatives

Chromic Acid preserves CHO

K2Cr2O7 preserves lipids, mitochondria
Regauds (Mollers) 3% K2Cr2O7 for
chromatin, mitochondri, Golgi, RBC,
colloid, mitotic figures; slow, not for fats
Orths 2.5% K2Cr2O7 for Rickettsia,
bacteria, myelin

Lead Fixatives

For acid MPS

Fixes connective tissue mucin
Forms insoluble lead carbonate remove
by filtering or adding HAc

Picric Acid fixatives (yellow)

Bouins (picric, HCHO, glacial) for embyros,

glycogen, does not need washing out; poor
penetration, not good for kidneys, mitochondria,
hemolyzes RBC
Brasils alcoholic picroformol (w/TCA) good for
glycogen; better & less messy than Bouins
Remove yellow color by 70% ethanol followed
by 5% sodium thiosulfate & running water
Highly explosive when dry

Glacial Acetic Acid

Solidifies at 17 degrees C glacial

For nucleoproteins, chromosomes
Contraindicated in cytoplasmic fixatives
destroys mitochondria & golgi

Alcohol Fixatives (fixative/dehyd)

- Denatures/ppt CHONs (destroys H bonds)
Methanol BM / bld smears, slow
Ethanol strong reducing agent
Carnoys-absolute ROH, CHCl3, glacial
HAc (most rapid); RBC hemolysis
Alcoholic Formalin (Gendres) - sputum
Newcommers isopropyl ROH, propionic
acid, petroleum ether, acetone, dioxane
for MPS

Alcohol Fixatives (fixative/dehyd)

Disadavantage:
Polarization causes glycogen granules
to move towards the poles / ends of cells

Osmium Tetroxide (Osmic Acid)

Fixes fats, for EM

Expensive, poor penetration, reduced w/
sunlight black deposit; dark bottle
Acid vapor conjunctivitis, osmic oxide in
cornea blindness
Inhibits hematoxylin
Extremely volatile
Flemmings (w/ and w/o acetic acid)

TCA

Weak decalcifying agent

Poor penetration

Acetone

Use at ice cold temp (-5C to 4C)

Fixes brain for rabies
Dissolves fat, evaporates rapidly,
preserves glycogen poorly

Heat Fixation

Thermal coagulation of tissue proteins

For frozen sections / bacteriologic smears

Post Chromatization

Secondary Fixation

To demonstrate some substances better

May act as mordant for special staining
To ensure further and complete hardening
and preservation of tissues

Washing out

Tap water
50 70 % alcohol
Alcoholic iodine

Fixation

Retarded by:

Large size
Mucus
Fat
Blood
Cold

Enhanced by:

Small / thin tissue

Agitation
Moderate heat (37 to
56 degrees C)

Decalcification

Bones, teeth, calcified tissues

tuberculous lungs, arteriosclerotic vessels
Poor cutting of hard tissues / knife
damage
Know patients case - if too large use
saw
Change decalcifying agent regularly

Decalcification*

grating sensation during cutting = place

block in 10 % HCl for 1 hour
Rapid decalcification produces effect on
nuclear staining (failure of nuclear
chromatin to take up hematoxylin)

Decalcification

Acids
Chelating Agents
Ion Exchange Resins (Ammonium form of
polystrene resin)
Electrical Ionization (Electrophoresis)

Decalcification

Acids HNO3, HCl, formic, TCA,

sulfurous, chromic, citric
Chelating Agents EDTA - slow
Ion Exchange Resins (Ammonium form of
polystrene resin) 1 14 days spread
on bottom of container
Electrical Ionization (Electrophoresis)
attraction of Ca to negative electrode

Acids

Most common
Stable
Easily available
Cheap
Nitric, hydrochloric, formic, TCA,
sulfurous, chromic, citric acid

Nitric Acid (5-10%)

Most common
Fastest
Disadvantage: inhibits nuclear stain
combine with formaldehyde or alcohol
Aqueous nitric acid 10%, formol nitric acid,
Perenyis, Phloroglucin nitric acid

Nitric Acid

Aqueous nitric acid 10% = 12-24 hours

Concentrated nitric acid w/ distilled water

Rapid, with minimal tissue distortion (if
prolonged)
Yellow color imparted

Nitric Acid

Formol Nitric Acid = 1 3 days

Rapid acting
Good nuclear staining
Less tissue destruction than 10% aqeuous
nitric acid
Use fume hood
Lessen yellow tissue discoloration by 5%
sodium sulfate or 0.1 % urea

Nitric Acid

Perenyis = 2-7 days

10% nitric acid, 0.5% chromic acid, absolute

ethyl alcohol
Decalcifies and softens
Good nuclear and cytoplasmic staining
Maceration avoided by chromic/ethyl
Disadv: slow, difficult to assess complete
decalcification by chemical means

Nitric Acid

Phloroglucin Nitric Acid = 12 24 hours

Conc nitric + phloroglucin = dense white

fumes, then add 10% nitric acid
Most rapid
Disadv: poor nuclear staining
* when decalcification is complete, acid must
be removed by 3 changes of 70 to 90%
ethanol

HCl

Slower action, greater tissue distortion

Good nuclear staining
* rapid proprietary solutions- w/ HCl
* slow proprietary solutions - w/ buffered
formalin/formic acid
Von Ebners fluid NaCl, HCl, H20

Good cytologic staining

Formic Acid

Better nuclear staining with less tissue

distortion & * safer to handle than nitric
and HCl
2-7 days - slow
Fixative & decalcifying agent
Excellent nuclear & cytoplasmic staining

Formic acid sodium citrate solution (better

nuclear staining than nitric acid)