9: Decalcification & Tissue Processing

Decalcification

Removal of calcium salts

Decalcification: technique for removing _ from bone or other
_ tissue so that good-quality paraffin _ can be prepared
without compromising tissue _

mineral, calcified, sections, integrity

Decalcification is carried out…

after the specimen has been fixed, prior to routine processing

Principle of decalcification

Criteria for decalcifying agents

Complete removal of calcium salts, cause no damage to tissue, no adverse effect on subsequent process, minimal time for removal of calcium salts

Decalcification can be achieved by…

Acid decalcification, chelating agents, ion-exchange resins, electrophoretic removal using current

Decalcification agents: Strong acid decalcifiers

Nitric acid, HCl (usually at 5-10%)

Decalcification agents: Weak acid decalcifiers

Formic acid, acetic acid, picric acid (not used alone, found as components in some fixatives)

Chelating agents

Bind to calcium ion present in bone, carry out decalcification

Chelating agent examples

EDTA (ethylene diamine tetra-acetic acid) (disodium salt), binds ionised calcium

Using chelating agents takes___, they’re used as a _ solution and as a _ decalcifier for _ tissue with calcium present

days to months, simple, surface, paraffin

Ion exchange resins result in _ decalcifyin

Advantages of electrophoretic decalcification

Shortened time for compelte decalcification, better preservation of soft tissue

Disadvantages of electrophoretic decalcification

Limited number of specimens processed at a time, impracticality

Factors affecting decalcification

Concentration, temperature, agitation, fluid access

Factor: concentration

Affects rate of calcium removal, will be depleted as it combines with calcium (use larger volume, renew several times during process)

Factor: temperature

Proportional relationship

Factor: agitation

Increases rate of decalcification

Factor: fluid access

Determination of decalcification end point

Poking, prodding, bending

Chemical test

X-ray (ideal method)

Decalcification end point chemical test: Ammonium _ solution added to sample of decalcified solution (neutralised with __), precipitation indicates _ of calcium

oxalate, ammonium hydroxide, presence

Tissue processing takes place..

between fixation and embedding in paraffin block

True or false: fixed tissue is the same as a paraffin block

False

Tissue processing: All tissues must be _ before they can be ___. For permanent tissues peparation, they are taken through a series of _ and ___ in a stable medium to provide the necessary support for _.

supported, sectioned for microscopy, reagents, infiltrated and embedded, microtomy

Most widely used tissue processing method for histological studies

Paraffin wax method

Prior to tissue processing, tissue sample must be…

well-fixed, suitable size for processing, not ‘squeezed’ into processing cassette

Tissue processing pre-requisite: Oversized samples will become…

distorted, likely suffer from poor processing

Tissue processing pre-requisite: Fragile, friable, and minute fragments must be…

secured by wrapping in foam pads

Factors affecting tissue processing

Type of tissue, reagents used, agitation/fluid access, temperatureheat, vaccum

Type of tissue

Size, thickness, density, lipid content

Tissue processing: type of tissue (density and lipid content)

Bone: decalcification prior to processing

Fatty tissue: de-fatting prior to processing

Tissue processing: Reagents used

Dependent on type of investigation, to fix or not (formalin or not)

Tissue processing: agitation/fluid access

Enabling faster and more effective processing, automatic processors, provides fluid access from all directions

Tissue processing: temperature

Use of heat to increase or decrease fluid penetration and thus processing times

Tissue processing: vacuum

Use of pressure during wax impregnation: removes air bubbles, decreases boiling point of wax and thus viscosity, lowers boiling point of clearing agents

Stages of processing

Fixation, dehydration, clearing, wax infiltration or impregnation

Tissue processing: dehydration

Remove water (either bound or free in tissue), usually with a series/gradient of alcohols (70% 80% 90% 100%) to remove all water and prevent damage

Dehydration: various components of the call are also removed by this process, such as…

Lower ethanol concentration…

High ethanol concentration…

lipids, water soluble proteins removed, other lipids may be dissolved

Dehydrating agents

Ethanol or alcohol, methanol, iso-propyl alcohol, acetone

Dehydration: Ethanol is…. but….

hydrophilic, dissolves lipids

Dehydration: Methanol is… but is…

hydrophilic, flammable and high toxic

Dehydration: Iso-propyl alcohol is… but is…

hydrophilic, can be used with microwave methods for processing, used for xylene free processing

flammable

Dehydration: Acetone is… but is…

rapid in action

flammable, has poor penetration, causes brittleness with long exposure

Tissue processing: clearing

Removal of dehydrant (de-alcoholisation) from tissue in preparation for wax infiltration

Clearant: reagent must be…

totally miscible with both de-hydrating reagent and infiltrating and embedding medium

Choice of clearing agent depends on

Processor ___ and processing ___

Safety factors

Availablility

_ of removing __

_ of removal of __

Minimal ___

system to be used, conditions, speed, dehydrating agent, ease, molten wax, damage to tissue

Most commonly used clearing agent

Xylene

Other clearing agents

Iso-propyl alcohol

Chloroform, cedar oil and citrus fruit oils, benzene, toluene

Xylene is… but…

economical and doesn’t interfere with staining, causes irritation and tissue damage (long-term effects)

Iso-propyl alcohol is good for… but is…

xylene-free processing, flammable

Chloroform is… but…

slow acting, causes irritation, health hazard, causes secitoning difficulty

Cedar oil is… but…

less hazardous, can interfere with some staining procedures

Citrus fruit oil is… but…

less hazardous and preserved fine tissue structures, can interfere wth some staining procedures

Benzene and toluene give… but are…

good tissue structure preservation, not ideal for routine use (more toxic and less economical than xylene)

Tissue processing: wax infiltration or impregnation

Clearing agent completely replaced with embedding medium (wax) which creates stability and rigidity to enable microtomy

Considerations for wax

Stable at RT, no tissue damage, safe to handle, readily available, used for all tissue types, no interference with staining techniques, relatively low melting point

Duration of infiltration depends on…

Size and type of tissue, clearing agent used, use of vacuum

Infiltration agents

Paraffin wax, resin, celloidin (nitrocellulose compound), plastic/polyester wax (gelatin, agar)

Manual processing

Routine, obsolete, for tissue restoration, resin processing

Automatic processing

Used routinely, has various mechanisms i.e. vacuum, agitation, pressure

A

Screen

B

Molten wax

C

Carbon filter (black box)

D

Condensate bottle (blue bottle)

Tissue processing: Dehydration

Tissue samples are dehydrated through a series of graduated alcohol to remove water

Tissue processing: Clearing step

Alcohol is replaced by clearing agent (e.g. Xylol), makes tissues translucent

Tissue processing: Wax infiltration

Xylol replaced with hot parrafin wax, penetrates tissues and occupies all spaces

Processing schedules are _ to suit tissue __

customised, types and sizes

Urgent (small specimens) cycle duration

2–4 hours

Overnight cycle duration

8–12 hours

Large specimen (whole organs) cycle duration

24 hours