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

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)

To prevent breakdown of cellular elements

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 Mercuric Chloride Chromate Fixatives Lead Fixatives

Metallic 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:

Cytoplasmic

Flemmings Carnoys Bouins Newcomers Heidenhains

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:

Enhanced by:

Large size Mucus Fat Blood Cold

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)