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 processingSpecial 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 Processing bone marrow biopsiesbiopsies

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. 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 Heidenhain’s Susa Formol Sublimate

(Formol Corrosive)

Zenker’s Zenker – Formol

(Helly’s) Bouin’s Brasil’s

Cytological 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 Newcomer’s 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

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 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 Lugol’s

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 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, 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)

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

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 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 Flemming’s (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 patient’s 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,

Perenyi’s, 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

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

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 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 nuclear staining than nitric acid)