Tissue Processing 10.

Labelling
• The longest phase in routine histopathology ⁃ Tissue slides are labeled on the frosted areas with
• Tissue must be adequately prepared and set in a assigned tissue numbers or codes
paraf n block after xation and before staining ⁃ Tissue slides should be properly labeled

1. Fixation
⁃ Small pieces of tissues are immersed in a solution Labelling of Tissues
called xative(10% neutral buffered formalin / 37% • A unique identi cation number or code is assigned
formaldehyde) after removal from the body. to the tissue sample accessioned in the laboratory
⁃ To avoid tissue digestion by enzymes present within
the cells or bacteria Completion of Fixation before Processing
⁃ To preserve cell and tissue structure in a life like • The tissue should be dissected to 3-4 mm in
manner thickness
⁃ 1-6hrs • A rule of thumb for most specimens is the size of
2. Dehydration small coin.
⁃ Tissue is transferred through a series of
increasingly concentrated alcohol solutions called Post- xation Treatment
as dehydrating agents, ending in 100%, which • Special xation techniques may require additional
removes all water. steps before processing is initiated
⁃ Commonly used : alcohol • Picric acid xatives should be placed directly into
⁃ Best: ethanol 70% alcohol for processing
⁃ 2 hours- auto • Carnoy’s uid should be place directly into 100%
⁃ 5-6 manual alcohol
3. Clearing Post- xation Treatment of Common
⁃ Alcohol is removed in the tissue by immersing in a Fixatives used in Histopathology
clearing agent. Fixative Post-Fixation Comment
⁃ Xylene
Treatment
⁃ 2 hours - auto
⁃ 5-6- manual Zenker’s, Thorough To remove
4. In ltration Helly’s, and rinsing in water chromic acid
⁃ Tissue is then placed in melted paraf n until P reagents that may form
becomes completely in ltrated with the substance. containing insoluble
5. Embedding chromate precipitate
⁃ The paraf n- in ltrated tissue is placed in a mold
with melted paraf n and allowed to harden To remove
⁃ Note: The medium used in in ltration of tissue is the mercury
same medium used for embedding. pigments
6. Trimming
⁃ The resulting paraf n block is trimmed to expose Bouin’s, Washing in To remove
the tissue for sectioning (slicing) on a microtome. Gendre’s, and ethanol until picric acid
7. Section - Cutting reagents supernatant is which may
⁃ Tissue block is sliced into thin lms using a containing faintly yellow or prevent wax
microtome which are placed on glass slides and picrate clear in ltration and
allowed to adhere, de-paraf nized, and stained. to get good
8. Staining ribbons on
⁃ Methods of staining have been devised that not sectioning
only make the various components conspicuous but
also permit distinctions to be made between them. Hollande’s and Brief washing in To remove
⁃ Cell components with a net negative charge reagents water phosphate salts
(anionic) stain more readily with basic dyes and are containing which may
termed basophilic phosphate precipitate in
⁃ Cationic components have af nity for acidic dyes tissues and
and are termed as acidophilic cause
9. Mounting sectioning
⁃ Stained tissue slides are mounted with a cover slip problems
using a mounting media.
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 Principles of Tissue Processing THICK SPECIMEN
• Stages of tissue processing:
• Dehydration Process Time
• Clearing
• In ltrating Fixation 10% Bu ered 24 hours
• Embedding Formalin

Factors In uencing the Rate of Dehydration 70% alcohol 6 hours

Processing
95% alcohol 12 hours
• Tissue density
• Agitation 100% alcohol 2 hours
• Heat
100% alcohol 1 hour
• Viscosity
• Vacuum 100% alcohol 1 hour
• Fixation
• Solvent e ects Clearing xylene or 1 hour
• Tissue Modi ers toluene
• Tissue porosity
xylene or 1 hour
Properties of the Processing toluene
Reagents
• Concentration of the reagents must be Impregnation Para n Wax 15 mins
increased in graded increments
Para n Wax 15 mins
• In ltrating and embedding media must ll all
spaces within the tissue to support section
Para n Wax 15 mins
cutting
• Processing reagents having miscibility with Para n Wax 15 mins
water, solvents and embedding media should
still be used in graded series Embedding Para n Wax 3 hours
• Polarity of the reagents must be changed
gradually,from highly polar aqueous xative and
dehydrant to the non-polar embedding medium SMALL BIOPSY
to lessen undue tissue e ect
• High viscosity of the clearing and in ltrating Process Time
agents results in slow di usion through tissues
10% Formalin 10 mins
Methods of Tissue Processing
Manual Tissue Processing 95% Alcohol 10 mins
• Rarely used, circumstances requiring manual
tissue processing: 100% Alcohol 10 mins
• Power failure or equipment malfunction
• Large tissue sample requiring more time than Xylene 10 mins
can be allocated on an automated processor
• Small biopsies needing rapid diagnosis Para n 10 mins
• Can be accelerated using laboratory-grade
microwave ovens or ultrasonics
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 Automatic Tissue Vacuum Embedding
Processing • Involves wax impregnation under negative
atmospheric pressure inside embedding oven
Tissue-transfer (dip and dunk) • Hasten removal of air bubbles and clearing
• Uses carousel-type processors which xes, agent from tissue block
dehydrates, clears and in ltrate tissues, • Promoting more rapid penetration of wax in the
decreasing time and labor needed tissue
• Length of time the specimens were submerged • Recommended for urgent biopsies, for delicate
in each reagent was electronically programmed tissues (lungs, brain, CT, decalci ed bones,
eyes, spleen and CNS)
Fluid-transfer (enclosed) • Gives the fastest result
• Enclosed, self-contained vacuum tissue
• processor Processor Maintenance Tips
• Reagents and melted para n are • Any spillage or over ow should be wiped away
• moved sequentially into and out of the immediately
• retort chamber using vacuum pressure • Accumulation of wax on any surface should be
Advantages: removed
• vacuum and heat can be applied at any stage • Temperature of the para n bath should be set
• Customized schedules at 3C above the melting point of the para n
• Fluid spillage contained • Timing should be checked when placing tissue
• Fumes eliminated cassettes in the processor, especially when
delayed schedules are selected
Microwave ovens
• Shortens processing time Advantages of Newer Technology in
• Stimulate di usion of the solutions into the Processing
tissue • Custom programs speci c to tissue being
• Processing is dependent on the thickness and processed
density of the specimen • Rapid schedules
• Reagents: ethanol, isopropanol and para n • Fluid and fume containment
• Graded concentrations not require • Environmentally friendly reagents
• Clearing agents not necessary • Delay schedules
• NO formalin and xylene
Advantages: Automated Processing Schedules
• Provides uncompromised morphology and • Overnight processing
antigenicity of specimens • Schedule for processing eyes
• Increased e ciency • Rapid processing schedule for small biopsies
• Environmentally-friendly reagents
Disadvantages: Sample schedule for Overnight Processing
• Process is labour intensive Reagents Time Pressure Temperature
• Lab-grade microwave are costly
• Requires calibration and monitoring 10 % 1 hour On 38 C
formalin
Rapid Tissue Processor
10 % 1 hour On 38 C
• Uses microwave technology, vacuum formalin
in ltration, and proprietary reagents
• Reagents: acetone, isopropanol, polyethylene 50% 1 hour On 38 C
glycol, mineral oil, para n alcohol/
formalin
Advantages:
• Toxic vapors eliminated 70% alchol 30 min On 38 C
• environmentally safe reagents
Disadvantages: 95% alcohol 30 min On 38 C
• Costly processor
• Grossing of specimen requires standardized 95% alcohol 40 min On 38 C
dissection
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 100% 40 min On 38 C • Permit the subsequent application of many
alcohol staining procedures to facilitate easier and more
pro table examination.
100% 40 min On 38 C
alcohol
TWO BASIC MECHANISMS
Xylene 40 min On 38 C Additive Fixation
• Chemical constituent of the xative is taken in
Xylene 40 min On 38 C and becomes a part of the tissue
• Forms crosslinks and gives stability to proteins
Para n 30 min On 60 C • Ex Formalin, mercury, osmium tetroxide
Non additive xation
Para n 20 min On 60 C
• the xing agent is NOT taken in, but changes
the tissue composition and stabilizes the tissue
Para n 40 min On 60 C
by removing the bound water attached to
hydrogen bonds of certain groups within the
Para n 40 min On 60 C
protein molecule
• Ex: alcoholic xatives
FIXATION
• First and most critical step in tissue processing. Factors A ecting the Quality of Fixation
• Bu ers and Hydrogen ion concentration (pH)
Fixatives • pH <5.7 brown-black insoluble crystalline
• Property of forming cross-links between birefringent pigment forms
proteins. • pH 3-5 increase pigment formation
• Stabilization of proteins is the most important • pH 7- optimal pH for bu ering formalin
reaction in maintaining tissue morphology. • Temperature
• 1° aim: to preserve the morphologic and • Surgical sp: Room temp
chemical integrity of the cell in as life-like • EM and some histochemistry: 0 - 4° C
manner as possible. • Thickness of section
• Stopping all cellular activities • Formalin penetrates 3-4mm /hr
• 2° aim: to harden and protect tissue from the • NBF penetrates 1mm/hr
trauma of further handling so that it is easier to • EM: 1-2 sq. mm
cut during gross examination. • LM: 2 sq. cm or not more than 0.4cm
• To prevent breakdown of cellular elements • Osmolality and ionic composition
(autolysis and putrefaction) by inactivating • Hypertonic or hypotonic solutions lead to
lysosomal enzymes or by chemically shrinkage and swelling respectively
components insoluble. • Best result are obtained at slightly hypertonic
• To coagulate or precipitate protoplasmic (400-450 mOsm)
substances • 10% NBF - 1500 m
• Osmonic composition of uids should as
Characteristics of a Good Fixative isotonic as possible
• Cheap • Concentration of xative
• Stable • Formaldehyde: 10%
• Safe to handle • Glutaraldehyde: 3%
• Kill the cell quickly • Duration of xation
• Inhibit bacterial decomposition and autolysis • Ideal time to perform xation after interruption
• Produce minimum shrinkage of tissue of blood supply: 20 to 30 minutes
• Permit rapid and even penetration of tissues • Usual xation time: 24 hours
• Harden tissue • Additives
• Isotonic • Addition of electrolytes and nonelectrolytes to
• Make cellular components insoluble to xatives improves morphology of the xed
hypotonic solutions and render them insensitive tissue
to subsequent processing.
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 • Ex. Calcium chloride, potassium thiocyanate, • Recommendation for incomplete xation
ammonium sulfate, and potassium dihydrogen • Increase time in xative solution
phosphate • Change to another xative (zinc formalin,
• •Mechanism of action: gloyoxal)
• •react directly with proteins causing • Place formalin-alcohol in the rst three stages of
denaturation the processing cycle
• Or react independently with xative and cellular • Ensure that grossed sections are < 3mm for
constituents good reagent penetration
• Nonelectorlyte substances: ex. Sucrose, • Amount of xative is 15-20 times that of the
dextran, and detergent tissue
• Change the formalin solution frequently to
Practical Considerations of Fixation prevent depletion
• Speed • Do not pack tissue section tightly in the
• Penetration cassettes
• Volume • Use agitation of cassettes in formalin holding
• Correct Fixative to Tissue Ratio15-20:1 solutions during or after grossing
• Duration of Fixation
Reactions of Cellular Components to
Consequences of Delayed, Incomplete, or Fixation
Poor Fixation Nucleus
• Loss or total disappearance of nuclear • Formalin does not react well with either DNA or
chromatin RNA unless xation temperature is elevated at
• Disappearance of some cells 65C for DNA and 45C for RNA
• Cell shrinkage with artifactual space around • Formalin xation of the nucleus results in the
cells formation of nuclear bubbling
Proteins
Factors that Affect Fixation of Tissues • Fixation disrupts tertiary con guration of protein
• Retarded by: by changes that occur with protein bonds
• Size and thickness of tissue specimen Lipids
• Presence of mucus • Generally lost during the procedure
• Presence of fat • Fixative of choice: osmium tetroxide and
• Presence of blood chromic acid
• Cold temperature Carbohydrates
• Enhanced by: • Some carbohydrates are lost during xation
• Size and thickness of tissue specimen • Glycogen is preserved due to entrapment in
• Agitation xed proteins
• Moderate heat (37-56 C)
TYPES OF FIXATIVE
Factors to Consider in Selecting Fixatives • According to Composition
• Need for immediate examination • Simple Fixatives
• Tissue structure or component to be studied • One component only
• Type of tissue to be processed • Aldehydes and Metallic Fixatives
• Staining technique to be applied • Glacial Acetic Acid
• Type of section to be made • Solidi es at 17°C
• Fixes nucleoprotein, destroys mitochondria and
Dif culties Encountered Because of Golgi bodies
Improper Fixation • Causes tissue to swell
• Failure to arrest early autolysis • Compound Fixatives
• Removal of substances soluble in xing agent • 2 or more simple xatives
• Presence of artefact pigments on tissue sections • Zenker’s uid
• Tissues are soft and feather-like in consistency - Composed of glacial acetic acid and mercuric
• Loss/inactivation of enzymes needed forstudy chloride
• Shrinkage & swelling of cells & tissue structure
• Tissue blocks are brittle and hard
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 - Glacial acetic acid 10% NEUTRAL BUFFERED FORMALIN
• Causes tissue to swell • Most commonly used and BEST general
- Mercuric chloride tissue xative
• Causes tissue to shrink • For surgical, post mortem, research specimens
• Usually buffered with phosphate buffer
According to Action (pH=7.2-7.4)
Microanatomical • Contains iron pigments
• For general microscopic study of tissue • 4– 24 hours
structures Formol-Corrosive (Formal-Sublimate)
• 10% Formol saline • For routine postmortem tissues
• 10% NBF • Fixes fats and lipids
• Heidenhain’s Susa • Fixation time: 3 to 24 hours
• Bouin’s Solution Alcoholic Formalin (Gendre’s Fixative)
Cytological • Also called alcoholicBouin’s
• Speci c parts and particular microscopic • Cause better retention of carbohydrate
elements of the cell itself (glycogen) in tissues
• NuclearFixatives • Fixes sputum
• Preserve nuclear structures • Fixation time: between 4 hours and overnight
• Contain Glacial acetic acid followed by washing in 70% ethanol, followed by
• pH ≤4.6 95% ethanol
• Flemming’s Glutaraldehyde
• Carnoy’s • For enzyme histochemistry and Electron
• Bouin’s Microscope studies
• Cytoplasmic xatives • Should be stored at 4C and pH 5
• Does NOT contain Glacial HAc • 2 concentrations
• pH > 4.6 • 2.5% for small tissue fragments and needle
• Kelly’s uid biopsies
• Orth’s • RT for 2– 4 hours
• Histochemical xatives • 4% for large specimens (<4mm thick)
• Preserve chemical constituents of cells • RT for 6– 8 hours up to 24 hours
andtissues FORMOL CALCIUM
• Acetone • For preservation of Fats and Lipids
• Newcomer’s uid (nuclear and histochem) Karnovsky’s paraformaldehyde-
glutaraldehyde solution and Acrolein
ALDEHYDE FIXATIVES • For electron cytochemistry
FORMALDEHYDE
• Formed from oxidation of methanol METALLIC FIXATIVES
• High concentration (37-40%) Mercuric Chloride
• Tend to over harden the outer layer of the tissue • Most commonly used METALLIC xative
and affect staining adversely • Advantages
• Should be diluted 1:10 or 1:20 to make 10% or • Permits brilliant metachromatic staining of cells
5% solution or combined with another • Routine xative of choice for tissue photography
• Fixation time: 24 hrs • Disadvantages
10% PERCENT FORMOL SALINE • Produce black granular deposit (except for
• Made from saturated formaldehyde diluted to Heidenhain Susa w/c is removed by De-
10% with NaCl Zenkerization)
• For CNS tissues and general post mortem • Corrode metal (except nickel alloy)
tissues for histochemical exam • Decrease the amount of demonstrable glycogen
• 24 hours at 35°C or 48 hours at 20-25°C • Causes considerable lysis of cell
• Causes tissue shrinkage
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 • Examples of Metallic Fixatives GLACIAL ACETIC ACID
• Zenker’s Fluid (with Glacial HAc) • Used in conjunction with other xatives
• Good xative for blood (congested) sepcimens • Solidi es at 17°C
• For trichrome staining
• For xing small pieces of liver, spleen, CT ALCOHOL FIXATIVES
bers and nuclei •Rapidly denatures and precipitates proteins
• Helly’s solution •Must be used in concentrations (70 to 100% )
• For pituitary gland, BM extramedullary •Less conc. Produce cell lysis
hematopoiesis, blood containing organs •Both xative and dehydrating agent
and intercalated discs •POLARIZATION
• Heidenhain’s Susa Solution •General disadvantage movement of glycogen
• for tumor biopsies (skin) granules towards the ends/poles of cells
• B5 Fixative METHANOL 100%
• For bone marrow, lymph nodes, spleen and • For blood smears and BM
hematopoietic tissues ETHANOL
CHROMATE • Most commonly used smear xative for
• Appear as yellow brown deposits exfoliative cytology
• Chromic Acid • Fixation time: 18-24 hours
• For carbohydrates ISOPROPANOL 95%
• Strong oxidizing agent hence strong reducing • For touch preparations
agent must be added (formaldehyde) CARNOY’S FLUID
• 3% Potassium Dichromate • Most RAPID xative (1– 3 hours)
• Fixes mitochondria at pH 4.5– 5.2 • For chromosomes, lymph glands and urgent
• Regaud’s (Muller’s) Fluid biopsies
• For chromatin, mitochondria, mitotic gures, • Also for brain tissue and diagnosis of rabies
Golgi bodies, RBC and colloid-containing tissue Newcomer’s Fluid
demonstration • For MPS and nuclear proteins
• Orth’s Fluid • Both nuclear and histochem xative
• For Rickettsia and other bacteria • Fixation time: 12-18 hours at 3C
• For study of early degenarative process OSMIUM TETROXIDE FIXATIVES
LEAD FIXATIVES •Should be kept in DARK-COLORED,
• Generally for Acid MPS chemically clean bottle to prevent
• For CT mucin evaporation and reduction by sunlight and organic
PICRATE FIXATIVES matter
•For glycogen demonstration •Inhibits hematoxylin and makes counterstaining
•Dyes tissues, but the yellow color can be dif cult
removed by treatment with •Produces BLACK PRECIPITATE (Osmium
another acid or lithium carbonate oxide)
•Highly explosive when dry •Prevention: Addition of saturated aq. Mercuric
•Must NEVER be washed in water before chloride
dehydration •Remedy: black osmic oxide crystals may be
Bouin’s Solution dissolved in cold water
• Excellent general xative for connective tissue •PRECAUTION : may cause conjunctivitis or
stains blindness
• disadvantage: destroys membranes, intact FLEMMING’S SOLUTION
nuclei cannot be recovered • Most common chrome-osmium acetic acid
• for embryos and pituitary biopsies xative
• For fatty tissue: Bouin’s solution (75 ml) plus • For nuclear preparation of section
95% ethanol (25ml) Flemming’s Solution without Acetic
• Require 48 hours for good sections of lipomas acid
or liposarcomas • Made up only of chromic and osmic acid
Brasil’s Alcoholic Picroformol Fixative • For cytoplasmic structures (mitochondria)
• For glycogen • The removal of acetic acid improves the
cytoplasmic detail of the cell
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TRICHLOROACETIC ACID
•Incorporated into compound xatives
•Poor penetrating agent, suitable for small pieces
of tissues or bones only
ACETONE
•Used at ice cold temperature ( -5°C to 4 °C)
•For the study of water diffusible enzymes
(phospatases and lipases)
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