Histotechniques

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Histotechniques

• Dr Mulazim Hussain Bukhari
• MBBS, DCP, MPhil, FCPS, PhD
• Associate Prof Pathology
• King Edward Medical University, Lahore

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Tissue Processing

• Specimen Accessioning
• Gross Examination
• Fixation
• Tissue Processing
• Sectioning
• Frozen Sections
• Staining
• H and E staining
• Cover slipping
• Decalcification
• Artifacts in Histologic Sections
• Problems in Tissue Processing

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Safety in the Lab

• The lab should be well illuminated and
  well-ventilated.
• Rules and Regulations governing
• formalin and
• hydrocarbonds
• such as xylene
• and toluene.
• Limits set by the Occupational Safety and Health
  Administration (OSHA) that should not be
  exceeded.
• These limits should be revised and revived to
  reduced any mishap

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Cont.

• Every chemical compound used in the laboratory
  should have a materials safety data sheet on file
  
• that specifies the nature,
• toxicity,
• and safety precautions to be taken when handling
  the compound.
• The laboratory must have a method for disposal of
  hazardous wastes.
• Health care facilities processing tissues often
  contract this to a waste management company.
• Tissues that are collected should be stored in
  formalin
• and may be disposed by incineration
• or by putting them through a "tissue grinder"
  attached to a large sink (similar to a large
  garbage disposal unit).

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Cont.

• Check the sharpness of scalpel, scissors and
  quality of other ones like ruler, probes weighing
  machines
• Every instrument used in the laboratory should
  meet electrical safety specifications and have
  written instructions regarding its use.
• Flammable materials may only be stored in
  approved rooms and only in storage cabinets that
  are designed for this purpose.

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Cont.

• Fire safety procedures are to be posted.
• Safety equipment including fire extinguishers,
• fire blankets,
• and fire alarms should be within easy access.
• A shower and eyewash should be readily
  available.
• No smoking, eating or movements in the labs
• Use disposable gloves

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Cont.

• Laboratory accidents must be documented and
  investigated with incident reports and industrial
  accident reports.
• Specific hazards that you should know about
  include
• Bouin's solution is made with picric acid. This
  acid is only sold in the aqueous state. When it
  dries out, it becomes explosive.

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Sodium azide

• Many reagent kits have sodium azide as a
  preservative.
• You are supposed to flush solutions containing
  sodium azide down the drain
• with lots of water, or
• there is a tendency for the azide to form metal
  azides in the plumbing.
• These are also explosive.

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Drainage

• Benzidine, benzene, anthracene, and napthol
  containing compounds are carcinogens and should
  not be used.
• Mercury-containing solutions (Zenker's or B-5)
  should always be discarded into proper
  containers.
• Mercury, if poured down a drain, will form
  amalgams with the metal that build up and cannot
  be removed.
• Hazards of usually used formalin

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Objective

• Tissues from the body taken for diagnosis of
  disease processes must be processed in the
  histology laboratory to produce microscopic
  slides that are viewed under the microscope by
  pathologists.
• The techniques for processing the tissues,
  whether biopsies, larger specimens removed at
  surgery, or tissues from autopsy
• The persons who do the tissue processing and make
  the glass microscopic slides are
  histotechnologists

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Specimen Accessioning

• Tissue specimens received in the surgical
  pathology laboratory have a request form that
  lists the patient information and history along
  with a description of the site of origin.
• The specimens are accessioned by giving them a
  number that will identify each specimen for each
  patient

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Grossing

• Describing the specimen
• Placing all or parts of it into a small plastic
  cassette
• When a malignancy is suspected
• Inking a gross specimen for margins

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Fixation

• Types of fixatives (AMAPO)
• Aldehydes
• Mercurials
• Alcohols
• Picrates
• Oxidizing agents

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Fixation - factors affecting fixation

• There are a number of factors that will affect
  the fixation process
• Buffering
• Penetration
• Volume
• Temperature
• Concentration
• Time interval
• Position of tissue

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Buffering

• Fixation is best carried out close to neutral pH,
  in the range of 6-8.
• Hypoxia of tissues lowers the pH, so there must
  be buffering capacity in the fixative to prevent
  excessive acidity.
• Acidity favors formation of formalin-heme pigment
  that appears as black, polarizable deposits in
  tissue.
• Common buffers include phosphate, bicarbonate,
  cacodylate, and veronal.
• Commercial formalin is buffered with phosphate at
  a pH of 7.

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Penetration

• Penetration of tissues depends upon the
  diffusability of each individual fixative, which
  is a constant.
• Formalin and alcohol penetrate the best, and
  glutaraldehyde the worst.
• Mercurials and others are somewhere in between.
• One way to get around this problem is sectioning
  the tissues thinly (2 to 3 mm).
• Penetration into a thin section will occur more
  rapidly than for a thick section

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Volume

• The volume of fixative is important.
• There should be a 101 ratio of fixative to
  tissue.
• Obviously, we often get away with less than this,
  but may not get ideal fixation.
• One way to partially solve the problem is to
  change the fixative at intervals to avoid
  exhaustion of the fixative.
• Agitation of the specimen in the fixative will
  also enhance fixation.

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Temperature

• Increasing the temperature, as with all chemical
  reactions, will increase the speed of fixation,
  as long as you don't cook the tissue.
• Hot formalin will fix tissues faster, and this is
  often the first step on an automated tissue
  processor.

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Concentration of fixative

• Concentration of fixative should be adjusted down
  to the lowest level possible, because you will
  expend less money for the fixative.
• Formalin is best at 10
• glutaraldehyde is generally made up at 0.25 to
  4.
• Too high a concentration may adversely affect the
  tissues and produce artefact similar to excessive
  heat.

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Time interval

• Also very important is time interval from of
  removal of tissues to fixation.
• The faster you can get the tissue and fix it, the
  better.
• Artefact will be introduced by drying, so if
  tissue is left out, please keep it moist with
  saline.
• The longer you wait, the more cellular organelles
  will be lost and the more nuclear shrinkage and
  artefactual clumping will occur

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Kaiserling formula for preservation for surgical
specimens for museum

• Formalin pure 5 liter
• Distilled water 22.5 liter
• Potassium acetate(CH3COOK ) 250gm
• Chloral hydrate 50 gm
• 
  27 liter
• Potassium acetate is used in mixtures applied for
  tissue preservation, fixation, and mummification.
  Most museums today use the formaldehyde-based
  method recommended by Kaiserling in 1897 and
  containing potassium acetate.
• For example, Lenin's mummy was soaked in a bath
  containing potassium acetate

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Gough Sections

• Whole organs may be sectioned on paper by the
  methods of Gough and Wentworth.
• These sections provide valuable information on
  whole organ structure and serve as links between
  mounted museum specimens and histologic sections.

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Colour restoration

• Small amount of sodium hydrosulphite to preserve
  the colour.
• If the container is properly sealed, the colour
  restoration is then permanent.
• For photography, the procedure is to first wash
  and clean the specimen.
• It is then soaked in an excess of 60 ethanol
  until the colour has been restored satisfactorily

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Characteristics of Fixatives

• Chemical Fixatives
• Freeze Substitution
• Microwave Fixation

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Ideal Fixative

• Penetrate cells or tissue rapidly
• Preserve cellular structure before cell can react
  to produce structural artifacts
• Not cause autofluorescence, and act as an
  antifade reagent

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Chemical Fixation

• Coagulating Fixatives
• Crosslinking Fixatives

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Coagulating Fixatives

• Ethanol
• Methanol
• Acetone

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Coagulating Fixatives
Advantages

• Fix specimens by rapidly changing hydration state
  of cellular components
• Proteins are either coagulated or extracted
• Preserve antigen recognition often

Disadvantages

• Cause significant shrinkage of specimens
• Difficult to do accurate 3D confocal images
• Can shrink cells to 50 size (height)
• Commercial preparations of formaldehyde contain
  methanol as a stabilizing agent

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Crosslinking Fixatives

• Glutaraldehyde
• Formaldehyde
• Ethelene glycol-bis-succinimidyl succinate (EGS)

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Cross-linking Fixatives

• Form covalent crosslinks that are determined by
  the active groups of each compound

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Principles of Fixation

• Once tissues are removed from the body, they
  undergo a process of self-destruction or
  autolysis
• which is initiated soon after cell death by the
  action of intracellular enzymes causing the
  breakdown of protein and eventual liquefaction of
  the cell.

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Principles of Fixation

• Autolysis is independent of any bacterial action,
  
• retarded by cold,
• greatly accelerated at temperatures of about 30C
  and
• almost inhibited by heating to 50C

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Cont.

• Autolysis is more severe in tissues which are
  rich in enzymes,
• such as the liver,
• brain and kidney,
• and is less rapid in tissues such as elastic
  fibre and collagen.

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Cont.

• By light microscopy, autolysed tissue presents a
  washed-out' appearance with swelling of
  cytoplasm,
• eventually converting to a granular, homogeneous
  mass which fails to take up stains.

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How Autolysed tissue looks like

• The nuclei of autolytic cells may show some of
  the changes of necrosis
• including condensation (pyknosis),
• fragmentation (karyorrhexis) and
• lysis (karyolysis)
• D/D these are not accompanied by an inflammatory
  or cellular response.

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How Autolysed tissue looks like

• There may be diffusion of intracellular
  substances of diagnostic significance, such as
  glycogen which is lost from the cells in the
  absence of prompt and suitable fixation.
• Autolysis also causes desquamation of epithelium
  which separates from its basement membranes.

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Bacterial Action on dead tissue

• Bacterial decomposition can also produce changes
  in tissues that mimic those of autolysis and is
  brought about by bacterial proliferation in the
  dead tissue.

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Bacterial Action on dead tissue

• Such bacteria may normally be present in the body
  during life such as the non-pathogenetic
  organisms present in the bowel, or may be present
  in diseased tissues at the time of death such as
  in septicaemia.

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The objective of fixation

• is to preserve cells and tissue constituents in
  as close a life-like state as possible and to
  allow them to undergo further preparative
  procedures without change.
• Fixation arrests autolysis and bacterial
  decomposition and stabilizes the cellular and
  tissue constituents so that they withstand the
  subsequent stages of tissue processing.
• Aside from these requirements for the production
  of tissue sections, increasing interest in cell
  constituents and the extensive use of
  immunohistochemistry to augment histological
  diagnosis has imposed additional requirements.

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Cont.

• Fixation should also provide for the preservation
  of tissue substances and proteins.
• Fixation is, therefore, the first step and the
  foundation in a sequence of events that
  culminates in the final examination of a tissue
  section.

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Common pitfalls of fixation

• It is relevant to point out that fixation in
  itself constitutes a major artifact.
• The living cell is fluid or in a semi-fluid
  state, Whereas fixation produces coagulation of
  tissue proteins and constituents, a necessary
  event to prevent their loss or diffusion during
  tissue processing the passage through hypertonic
  and hypotonic solutions during tissue processing
  would otherwise disrupt the cells.
• For example, if fresh unfixed tissues were washed
  for prolonged periods in running water, severe
  and irreparable damage and cell lysis would
  result.
• In contrast, if the tissues were first fixed in
  formalin, subsequent immersion in water is
  generally harmless.

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Summary of objective

• Fixation
• Confers chemical stability on the tissue
• Hardens the tissue (helps further handling)
• Halts enzyme autolysis
• Halts bacterial putrefaction
• May enhance later staining techniques
• Introduces a 'consistent artifact'

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Aldehydes

• include formaldehyde (formalin) and
  glutaraldehyde.
• Tissue is fixed by cross-linkages formed in the
  proteins, particularly between lysine residues.
• This cross-linkage does not harm the structure of
  proteins greatly, so that antigenicity is not
  lost.

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Cont.

• Therefore, formaldehyde is good for
  immunoperoxidase techniques. Formalin penetrates
  tissue well, but is relatively slow.
• The standard solution is 10 neutral buffered
  formalin.
• A buffer prevents acidity that would promote
  autolysis and cause precipitation of formol-heme
  pigment in the tissues.

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Formaldehyde

• Formaldehyde, as 4 buffered formaldehyde (10
  buffered formalin), is the most widely employed
  universal fixative particularly for routine
  paraffin embedded sections.
• It is a gas with a very pungent odor, soluble in
  water to a maximum extent of 40 by weight and is
  sold as such under the name of formaldehyde (40)
  or formalin (a colorless liquid).

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Formaldehyde

• Formaldehyde is also obtainable in a stable solid
  form composed of high molecular weight polymers
  known as paraformaldehyde.

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Cont.

• Heated paraformaldehyde generates pure gaseous
  formaldehyde which, when dissolved in water,
  reverts mostly to the monomeric form.
• Aqueous formaldehyde exists principally in the
  form of its monohydrate, methylene glycol,
  CH2(OH)2, and as low molecular weight polymeric
  hydrates or polyoxymethylene glycols.
• It has been suggested that the hydrated form,
  methylene glycol, is the reactive component of
  formaldehyde but this has been disputed.

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Preparation of 10

• Four per cent formaldehyde or 10 buffered
  formalin is commonly prepared by adding 100 ml of
  40 formaldehyde to 900 ml distilled water with 4
  g sodium phosphatase, monobasic and 6.5 g sodium
  phosphate, dibasic (anhydrous).

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Formaldehyde solutions

• 10 neutral buffer formalin (4
  formaldehyde)REAGENTS REQUIRED1 40
  formaldehyde 100 ml2 Distilled water 900 ml3
  Sodium dihydrogen orthophosphate 4 g4 Disodium
  hydrogen orthophosphate (anhydrous) 6.5 g
  (sodium hydrosulphite)
• METHODPrepare, using quantities indicated.
  Fixation time 24-72 hours

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Buffered formaldehyde-glutaraldehyde 200 mOsm38

• REAGENTS REQUIRED1 -Sodium dihydrogen
  orthophosphate 1.6 g2-Sodium hydroxide 0.27
  g3-Distilled water 88 ml4-40 formaldehyde 10
  ml5-50 glutaraldehyde 2 mlMETHODPrepare,
  using quantities indicated. Fixation time 16-24
  hours.

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Formol saline

• REAGENTS REQUIRED1- 40 formaldehyde 100 ml2
  -Sodium chloride 9 g3 -Tap water 900 ml
• METHODPrepare, using quantities indicated

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Some other forms of Fixatives

• Baker's formol-calcium (modified)REAGENTS
  REQUIRED1 40 formaldehyde 100 ml2 Distilled
  water 900 ml3 10 calcium chloride 100 ml4 7 g
  of cadmium chloride is sometimes added to the
  mixture
• METHODPrepare, using quantities indicated.
  Fixation time 16-24 hours.
• Formol salineREAGENTS REQUIRED1 40
  formaldehyde 100 ml2 Sodium chloride 9 g3 Tap
  water 900 ml
• METHODPrepare, using quantities indicated.
• Alcoholic formaldehydeREAGENTS REQUIRED1 40
  formaldehyde 100 ml2 95 alcohol 900 ml3 0.5 g
  calcium acetate may be added to this mixture to
  ensure neutrality
• METHODPrepare, using quantities indicated.
  Fixation time 16-24 hours.

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Fixatives - general usage

• Formalin is used for all routine surgical
  pathology and autopsy tissues when an H and E
  slide is to be produced.
• Formalin is the most forgiving of all fixatives
  when conditions are not ideal, and there is no
  tissue that it will harm significantly.

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Zenker's fixatives

• Zenker's fixatives are recommended for
  reticuloendothelial tissues including
• lymph nodes,
• spleen,
• thymus, and
• bone marrow.
• Zenker's fixes nuclei very well and gives good
  detail.
• However, the mercury deposits must be removed
  (dezenkerized) before staining or black deposits
  will result in the sections

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Bouin's solution

• Bouin's solution is sometimes recommended for
  fixation of
• testis,
• GI tract, and
• endocrine tissue.
• It does not do a bad job on hematopoietic tissues
  either, and doesn't require dezenkerizing before
  staining

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Glutaraldehyde

• Glutaraldehyde is recommended for fixation of
  tissues for electron microscopy.
• The glutaraldehyde must be cold and buffered and
  not more than 3 months old.
• The tissue must be as fresh as possible
• Preferably sectioned within the glutaraldehyde at
  a thickness no more than 1 mm to enhance fixation

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Alcohols

• Alcohols, specifically ethanol, are used
  primarily for cytologic smears.
• Ethanol (95) is fast and cheap.
• Since smears are only a cell or so thick, there
  is no great problem from shrinkage, and since
  smears are not sectioned, there is no problem
  from induced brittleness.
• Note For fixing frozen sections, you can use
  just about anything--though methanol and ethanol
  are the best

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Glutaraldehyde

• Glutaraldehyde causes deformation of alpha-helix
  structure in proteins so is not good for
  immunoperoxidase staining.
• However, it fixes very quickly so is good for
  electron microscopy.
• It penetrates very poorly, but gives best overall
  cytoplasmic and nuclear detail.
• The standard solution is a 2 buffered
  glutaraldehyde

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Glutaraldehyde

• First used in 1962 by Sabatini et al
• Shown to preserve properties of subcellular
  structures by EM
• Renders tissue autofluorescent so less useful for
  fluorescence microscopy, but fluorescence can be
  attenuated by NaBH4.
• Forms a Schiffs base with amino groups on
  proteins and polymerizes via Schiffs base
  catalyzed reactions

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• Forms extensive crosslinks - reacts with the
  ?-amino group of lysine, ?-amino group of amino
  acids - reacts with tyrosine, tryptophan,
  histidine, phenylalanine and cysteine
• Fixes proteins rapidly, but has slow penetration
  rate
• Can cause cells to form membrane blebs

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Glutaraldehyde

• Supplied commercially as either 25 or 8
  solution
• Must be careful of the impurities - can change
  fixation properties - best product from
  Polysciences (Worthington, PA)
• As solution ages, it polymerizes and turns
  yellow.
• Store at -20 C and thaw for daily use. Discard.

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Summary of Formaldehyde

• Crosslinks proteins by forming methelene bridges
  between reactive groups
• The ratelimiting step is the de-protonation of
  amino groups, thus the pH dependence of the
  crosslinking
• Functional groups that are reactive are amido,
  guanidino, thiol, phenol, imidazole and indolyl
  groups
• Can crosslink nucleic acids

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Cont.

• Therefore the preferred fixative for in situ
  hybridization
• Does not crosslink lipids but can produce
  extensive vesiculation of the plasma membrane
  which can be averted by addition of CaCl2
• Not good preservative for microtubules at
  physiologic pH

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• Protein crosslinking is slower than for
  glutaraldehyde, but formaldehyde penetrates 10
  times faster.
• It is possible to mix the two and there may be
  some advantage for preservation of the 3D nature
  of some structures.

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Mercurials

• fix tissue by an unknown mechanism.
• They contain mercuric chloride and include such
  well-known fixatives as B-5 and Zenker's.
• These fixatives penetrate relatively poorly and
  cause some tissue hardness, but are fast and give
  excellent nuclear detail.

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• Their best application is for fixation of
  hematopoietic and reticuloendothelial tissues.
• Since they contain mercury, they must be disposed
  of carefully

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Alcohols

• including methyl alcohol (methanol) and ethyl
  alcohol (ethanol), are protein denaturants and
  are not used routinely for tissues because they
  cause too much brittleness and hardness.
• However, they are very good for cytologic smears
  because they act quickly and give good nuclear
  detail.
• Spray cans of alcohol fixatives are marketed to
  physicians doing PAP smears, but cheap hairsprays
  do just as well

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Oxidizing agents

• include permanganate fixatives
• Potassium permanganate,
• dichromate fixatives
• Potassium dichromate,
• Osmium tetroxide.
• They cross-link proteins, but cause extensive
  denaturation.
• Some of them have specialized applications, but
  are used very infrequently.

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Bouins Solution (Picrates)

• include fixatives with picric acid.
• Foremost among these is Bouin's solution.
• It has an unknown mechanism of action.
• It does almost as well as mercurials with nuclear
  detail but does not cause as much hardness.
• Picric acid is an explosion hazard in dry form.
• As a solution, it stains everything it touches
  yellow, including skin.

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On the left with HE staining black mercuric
chloride precipitate is seen in this lymphoma
fixed in B-5 and not properly dezenkerized. This
precipitate is seen on the right under polarized
light microscopy.
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Removal of Pig

• Formalin pigment
• 1. Dewax the sections, rinse in 100 alcohol,
  rinse in 70 alcohol, rinse in distilled water.
• 2. Treat in saturated alcoholic picric acid for
  30 minutes to 2 hours.
• 3. Wash well in running tap water.
• 4. If yellow staining of the section persists
  rinse in dilute lithium carbonate.
• 5. Rinse in tap water.
• 6. Continue with method.

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Mercury pigment

• 1. Dewax the sections, rinse in 100 alcohol,
  rinse in 70 alcohol, rinse in distilled water.
• 2. Treat in Lugol's iodine for 2 minutes.
• 3. Decolourise in 5 sodium thiosulphate for 5
  minutes.
• 4. Wash well in running tap water.
• 5. Continue with method.

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Dichromate pigment

• 1. Dewax the sections, rinse in 100 alcohol,
  rinse in 70 alcohol, rinse in distilled water.
• 2. Treat in 2 HCl in 70 alcohol 16-24 hours.
• 3. Rinse in tap water.
• 4. Continue with method.

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Tissue Processing

• Once the tissue has been fixed, it must be
  processed into a form in which it can be made
  into thin microscopic sections.
• The usual way this is done is with paraffin.
• Tissues embedded in paraffin, which is similar in
  density to tissue, can be sectioned at anywhere
  from 3 to 10 microns, usually 6-8 routinely.
• The technique of getting fixed tissue into
  paraffin is called tissue processing
• Dehydration
• Clearing

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Dehydration

• Wet fixed tissues (in aqueous solutions) cannot
  be directly infiltrated with paraffin.
• First, the water from the tissues must be removed
  by dehydration.
• This is usually done with a series of alcohols,
  say 70 to 95 to 100.
• Sometimes the first step is a mixture of formalin
  and alcohol.
• Other dehydrants can be used, but have major
  disadvantages.
• Acetone is very fast, but a fire hazard, so is
  safe only for small, hand-processed sets of
  tissues.
• Dioxane can be used without clearing, but has
  toxic fumes

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Clearing

• Removal of the dehydrant with a substance that
  will be miscible with the embedding medium
  (paraffin).
• The commonest clearing agent is xylene.
• Toluene works well, and is more tolerant of small
  amounts of water left in the tissues, but is 3
  times more expensive than xylene.
• Chloroform used to be used, but is a health
  hazard, and is slow.
• Methyl salicylate is rarely used because it is
  expensive, but it smells nice (it is oil of
  wintergreen).

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Embedding

• Finally, the tissue is infiltrated with the
  embedding agent, almost always paraffin.
• Paraffins can be purchased that differ in melting
  point, for various hardnesses, depending upon the
  way the histotechnologist likes them and upon the
  climate (warm vs. cold).
• Wax hardness (viscosity) depends upon the
  molecular weight of the components and the
  ambient temperature.
• High molecular weight mixtures melt at higher
  temperatures than waxes comprised of lower
  molecular weight fractions.
• Paraffin wax is traditionally marketed by its
  melting points which range from 39C to 68C.

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Other agents

• A product called paraplast contains added
  plasticizers that make the paraffin blocks easier
  for some technicians to cut.
• A vacuum can be applied inside the tissue
  processor to assist penetration of the embedding
  agent.
• methyl methacrylate,
• glycol methacrylate,
• araldite, and epon.
• Methyl methacrylate

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General Embedding Procedure

• METHOD1 Open the tissue cassette, check against
  worksheet entry to ensure the correct number of
  tissue pieces are present.
• 2 Select the mould, there should be sufficient
  room for the tissue with allowance for at least a
  2 mm surrounding margin of wax.
• 3 Fill the mould with paraffin wax.
• 4 Using warm forceps select the tissue, taking
  care that it does not cool in the air at the
  same time.

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• 5 Chill the mould on the cold plate, orienting
  the tissue and firming it into the wax with
  warmed forceps. This ensures that the correct
  orientation is maintained and the tissue surface
  to be sectioned is kept flat.
• 6 Insert the identifying label or place the
  labelled embedding ring or cassette base onto the
  mould.
• 7 Cool the block on the cold plate, or carefully
  submerge it under water when a thin skin has
  formed over the wax surface.
• 8 Remove the block from the mould.
• 9 Cross check block, label and worksheet

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Sectioning tissues

• Turn on the water bath and check that the temp is
  35-37ºC.  
• Use fresh deionized water (DEPC treated water
  must be used if in situ hybridization will be
  performed on the sections). 
• Blocks to be sectioned are placed face down on an
  ice block or heat sink for 10 minutes.
• Place a fresh blade on the microtome.
• Insert the block into the microtome chuck so the
  wax block faces the blade and is aligned in the
  vertical plane. Set the dial to cut 4-10 µM
  sections. 
• The blade should angled 4-6º.  

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• Face the block by cutting it down to the desired
  tissue plane and discard the paraffin ribbon.
• If the block is ribboning well then cut another
  four sections and pick them up with forceps or a
  fine paint brush and float them on the surface of
  the 37ºC water bath.
• Float the sections onto the surface of clean
  glass slides.
• If the block is not ribboning well then place it
  back on the ice block to cool off firm up the
  wax. 
• If the specimens fragment when placed on the
  water bath then it may be too hot.
• Place the slides with paraffin sections in a 65C
  oven for 20 minutes (so the wax just starts to
  melt) to bond the tissue to the glass.
• Slides can be stored overnight at room
  temperature

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H and E staining

• Hematoxylin is the oxidized product of the
  logwood tree known as hematein.
• Since this tree is very rare nowadays, most
  hematein is of the synthetic variety.
• In order to use it as a stain it must be
  "ripened" or oxidized.
• This can be done naturally by putting the
  hematein solution on the shelf and waiting
  several months, or by buying commercially ripened
  hematoxylin or by putting ripening agents in the
  hematein solution.

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Cont.

• Hematoxylin will not directly stain tissues, but
  needs a "mordant" or link to the tissues. This is
  provided by a metal cat ion such as iron,
  aluminum, or tungsten.
• The variety of hematoxylins available for use is
  based partially on choice of metal ion used.
• They vary in intensity or hue. Hematoxylin, being
  a basic dye, has an affinity for the nucleic
  acids of the cell nucleus.

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Cont.

• Hematoxylin stains are either "regressive" or
  "progressive".
• With a regressive stain, the slides are left in
  the solution for a set period of time and then
  taken back through a solution such as
  acid-alcohol that removes part of the stain.
• This method works best for large batches of
  slides to be stained and is more predictable on a
  day to day basis.
• With a progressive stain the slide is dipped in
  the hematoxylin until the desired intensity of
  staining is achieved, such as with a frozen
  section.
• This is simple for a single slide, but lends
  itself poorly to batch processing.

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Eosin

• Eosin is an acidic dye with an affinity for
  cytoplasmic components of the cell.
• There are a variety of eosins that can be
  synthesized for use, varying in their hue, but
  they all work about the same.
• Eosin is much more forgiving than hematoxylin and
  is less of a problem in the lab.
• About the only problem you will see is
  overstaining, especially with decalcified tissues

99
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100
Decalcification

• Some tissues contain calcium deposits which are
  extremely firm and which will not section
  properly with paraffin embedding owing to the
  difference in densities between calcium and
  parffin.
• Bone specimens are the most likely type here, but
  other tissues may contain calcified areas as
  well.
• This calcium must be removed prior to embedding
  to allow sectioning.
• A variety of agents or techniques have been used
  to decalcify tissue and none of them work
  perfectly.
• Mineral acids,
• organic acids,
• EDTA, and
• electrolysis have all been used.

101
Strong mineral acids

• nitric and
• hydrochloric acids
• rapid
• damage cellular morphology,
• so are not recommended for delicate tissues such
  as bone marrow.

102
Organic acids

• acetic and
• formic acid are better suited to bone marrow,
  since they are not as harsh.
• However, they act more slowly on dense cortical
  bone.
• Formic acid in a 10 concentration is the best
  all-around decalcifier.
• Some commercial solutions are available that
  combine formic acid with formalin to fix and
  decalcify tissues at the same time.

103
EDTA and Electrolysis

• EDTA can remove calcium and is not harsh (it is
  not an acid)
• but it penetrates tissue poorly and
• works slowly and is
• expensive in large amounts.
• Electrolysis has been tried in experimental
  situations where calcium had to be removed with
  the least tissue damage.
• It is slow and not suited for routine daily use.

104
Procedure

• Specimens should be decalcified in hydrochloric
  acid/formic acid working solution 20 times their
  volume.
• Change to fresh solution each day until
  decalcification is complete.
• It may take 24 hours up to days or months
  depending on size of the specimens.
• See below for the testing procedures
• Once the decalcification is complete, rinse
  specimens in water briefly and transfer to
  ammonia solution to neutralize acids left in
  specimens for 30 minutes.
• Wash specimens in running tap water thoroughly up
  to 24 hours.
• Routine paraffin embedding.

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End-Point of Decalcification

•      X-ray (the most accurate way)
•      Chemical testing (accurate)
•      Physical testing (less accurate and
  potentially damage of specimen)

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Chemical Test 

•      5 Ammonium Hydroxide Stock
•      Ammonium hydroxide, 28 --------------------
  5 ml
•      Distilled water -----------------------------
  ----- 95 ml
•      Mix well
•  5 Ammonium Oxalate Stock
•      Ammonium oxalate ----------------------------
  5 ml
•      Distilled water -----------------------------
  ---- 95 ml
•      Mix well
•  Ammonium Hydroxide/Ammonium Oxalate Working
  Solution
•      Use equal parts of the 5 ammonium hydroxide
  solution and the 5 ammonium oxalate solution. 

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Procedure

•  Insert a pipette into the decalcifying solution
  containing the specimen.
• Withdraw approximately 5 ml of the hydrochloric
  acid/formic acid decalcification solution from
  under the specimen and place it in a test tube.
• Add approximately 10 ml of the ammonium
  hydroxice/ammonium oxalate working solution, mix
  well and let stand overnight.
• Decalcification is complete when no precipitate
  is observed on two consecutive days of testing.
  Repeat this test every two or three days. 

108
Physical Tests

• The physical tests include bending the specimen
  or inserting a pin, razor, or scalpel directly
  into the tissue.
• The disadvantage of inserting a pin, razor, or
  scalpel is the introduction of tears and pinhole
  artifacts.
• Slightly bending the specimen is safer and less
  disruptive but will not conclusively determine if
  all calcium salts have been removed.
• After checking for rigidity, wash thoroughly
  prior to processing.
•