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
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.
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 LabellingFixation
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
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
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
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
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
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
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
Processing bone marrow
biopsies
The fixative used is very important. Submit entire needle biopsy after
fixation in Bouin’s 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.
Fixative
Cheap Stable Safe to handle Kills quickly Minimum tissue shrinkage Rapid & even
penetration
Hardens tissues for
easier cutting
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 HeatMicroanatomical Fixatives
10 % Formol Saline 10 % Neutral Buffered Formalin Heidenhain’s Susa Formol Sublimate (Formol Corrosive) Zenker’s Zenker – Formol (Helly’s) Bouin’s Brasil’sCytological Fixatives
Nuclear: Flemming’s Carnoy’s Bouin’s Newcomer’s Heidenhain’s Cytoplasmic Flemming’s w/o acetic acid Helly’s Formalin w/ post chroming Regaud’s (Moller’s) Orth’s
Histochemical Fixatives
Formol Saline 10%
Absolute Ethyl Alcohol Acetone
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
Formaldehyde – precautions:
Paraformaldehyde formation Well ventilated room
Not neutralized if concentrated – explosion Buffered or neutralized by adding
magnesium carbonate/CaCO3 – wide mouth bottle
10 % Formol Saline
Penetrates and fixes tissues well,
minimum shrinkage & distortion, does not overharden tissues
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
Formol corrosive/formol sublimate
Formol mercuric chloride
Minimum shrinkage and hardening
No need for wash out from fixative to ROH Slow
Glutaraldehyde
For LM, EM
Adv vs. HCHO: more stable effect, less
tissue shrinkage, less irritating
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
Zenker’s (HgCl2 + Glacial HAc) – liver,
spleen, CT fibers, nuclei; poor penetration, wash thoroughly in running H20
Zenker-Formol (Helly’s)–HgCl2 , K2Cr2O7
for pituitary, BM, spleen, liver; brown
pigment produced–remove by picric/NaOH
Heidenhain’s Susa – HgCl2, NaCl, TCA
Mercuric chloride
(new) B-5 fixative for bone marrow
biopsies
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 Lugol’s
iodine (5mins), running water (5mins), 5% Na thiosulfate (5mins), running water (5mins),
Chromate Fixatives
Chromic Acid – preserves CHO
K2Cr2O7 – preserves lipids, mitochondria Regaud’s (Moller’s) – 3% K2Cr2O7 – for
chromatin, mitochondri, Golgi, RBC,
colloid, mitotic figures; slow, not for fats
Orth’s – 2.5% K2Cr2O7 – for Rickettsia,
Lead Fixatives
For acid MPS
Fixes connective tissue mucin
Forms insoluble lead carbonate – remove
Picric Acid fixatives (yellow)
Bouin’s (picric, HCHO, glacial) – for embyros,
glycogen, does not need washing out; poor
penetration, not good for kidneys, mitochondria, hemolyzes RBC
Brasil’s alcoholic picroformol (w/TCA) – good for
glycogen; better & less messy than Bouin’s
Remove yellow color by 70% ethanol followed
by 5% sodium thiosulfate & running water
Glacial Acetic Acid
Solidifies at 17 degrees C glacial For nucleoproteins, chromosomes
Contraindicated in cytoplasmic fixatives
Alcohol Fixatives (fixative/dehyd)
- Denatures/ppt CHONs (destroys H bonds)
Methanol – BM / bld smears, slow Ethanol – strong reducing agent
Carnoy’s-absolute ROH, CHCl3, glacial
HAc (most rapid); RBC hemolysis
Alcoholic Formalin (Gendre’s) - sputum
Newcommer’s – 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
TCA
Weak decalcifying agent Poor penetration
Acetone
Use at ice cold temp (-5C to 4C) Fixes brain – for rabies
Dissolves fat, evaporates rapidly,
Heat Fixation
Thermal coagulation of tissue proteins
Secondary Fixation
To demonstrate some substances better May act as mordant – for special staining To ensure further and complete hardening
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 patient’s case - if too large – use
saw
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)
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) –
Acids
Most common Stable
Easily available Cheap
Nitric, hydrochloric, formic, TCA,
Nitric Acid (5-10%)
Most common Fastest
Disadvantage: inhibits nuclear stain –
combine with formaldehyde or alcohol
Aqueous nitric acid 10%, formol nitric acid,
Nitric Acid
Aqueous nitric acid 10% = 12-24 hours Concentrated nitric acid w/ distilled water Rapid, with minimal tissue distortion (if
prolonged)
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%
Nitric Acid
Perenyi’s = 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
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 Ebner’s 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
