Tissue Processing: Fixation-Decalcification-Dehydration

1. fixation

2. dehydration

3. clearing

4. impregnation / infiltration

5. embedding

6. trimming

7. section-cutting

8. staining

9. mounting

10. labelling

steps in tissue processing

fixation

first & most critical step in tissue processing

produce microscopic preparations of tissue that represent as closely as possible their structure in life

aim of a good histopathological technique

preserve morphologic & chemical integrity of the cell as it is in the living state

primary aim of fixation

harden & protect tissue from trauma → easier to cut during gross exam

secondary aim of fixation

• speed

• penetration

• volume

• duration of fixation

practical considerations of fixation

1 mm per hour

formalin diffuses into the tissue at what rate

10-25x volume of the tissue

amount of fixative used has been ___ the volume of the tissue to be fixed

• heat

• vacuum

• agitation

• microwave

fixation time can be cut down by

• additive fixation

• non-additive fixation

mechanisms of fixation

additive fixation

chemical constituent of the fixative is taken in and becomes part of the tissue by—forming cross-links or molecular complexes and giving stability to protein

pH 6-8

pH (hydrogen ion concentration) where satisfactory fixation occurs

room temperature

fixation is traditionally carried out at what temperature

40ºC

many laboratories use tissue processors at what temperature

0-4ºC

electron microscopy ideal temperature

60ºC

formalin heated to ___ for rapid fixation of very urgent biopsy, but distortion may happen

100ºC

formalin heated to ___ to fix tissues w/ tuberculosis

10% buffered formalin for 2-3 weeks

brain is usually suspended whole in

hypertonic solutions (400-450 mOsm)

optimum osmolality for fixation

• 10% formaldehyde

• 3% glutaraldehyde

concentration normally used for fixation

low concentrations of glutaraldehyde

an ideal concentration for immune-electron microscopy

2-6 hours

duration of fixation using buffered formalin

3 hours

duration of fixation for electron microscopy before placing in a holding buffer

• shrinkage

• hardening of tissue

• inhibit enzyme activity and immunological reactions

effects of prolonged fixation

microwave technique

physical agent like vacuum, oven (heat), and agitation increases movement of molecules and accelerated function

microwave technique

used to accelerate staining, decalcification, immunohistochemistry, and electron microscopy

• simple

• compound

fixative according to composition

• microanatomical

• cytological

  • nuclear

  • cytoplasmic

• histochemical

fixative according to action

• size & thickness

• presence of mucus, fats, blood

• cold temperature

fixation is retarded by

secondary fixation

placing previously fixed tissue in a second fixative

improve demonstration of a particular substance

purpose of secondary fixation

post chromatization

fixed tissue is placed in an aqueous solution of 2.5-3% potassium dichromate for 24 hrs

2.5-3% potassium dichromate

aqueous solution used in post chromatization

that acts as mordant for better staining effect & preservation of tissues

washing out

done to remove excess formalin or fixative from the tissue after fixation for better staining effect & to remove artifacts from the tissue

tap water

used in washing out chromates

tap water

used in washing out formalin

tap water

used in washing out osmic acid

50-70% alcohol

used in washing out picric acid

alcoholic iodine

used in washing out mercuric fixatives

decalcification

removing calcium to make tissue flexible & easier to cut

1. selection of tissue

2. fixation

3. decalcification

4. acid neutralization

5. thorough washing

stages of decalcification

after fixation

decalcification is done

surface decalcification

needed when partially decalcified bone / unsuspected mineral deposits in soft tissue are found during paraffin sectioning

face side down in 5% hydrochloric acid (HCl) for 30-60 mins

after finding a calcified section, the exposed surface in a paraffin is placed

• acids

• chelating agents

• ion exchange resins

• electrical ionization (electrophoresis)

calcium may be removed b

acid decalcifying agents

most widely used agents for routine decalcification of large amounts of bone tissues

• nitric acid

• hydrochloric acid

• formic acid

• citric acid

• trichloroacetic acid

• sulfurous acid

• chromic acid

mineral acids

chelating agents

substances which combine w/ calcium ions & other salts → form weakly associated complexes & facilitate removal of calcium salt

EDTA (Versene)

most common chelating agent → but a very slow decalcifying agent

7-7.4

ideal pH for EDTA

detailed microscopic studies

EDTA is used as a chelating agent only in

EDTA

used as an anticoagulant & water softener

1-3 weeks

EDTA decalcification time for small specimens

6-8 weeks

EDTA decalcification time to totally decalcify dense cortical bone

EDTA disodium salt + distilled water + formaldehyde

EDTA formula

EDTA

excellent bone decalcifier for immunohistochemical or enzyme staining, & electron microscopy

magnesium chloride

EDTA inactivates alkaline phosphatase activity → restored by adding

ion exchange resin

ammonium form of polystyrene resin

ion exchange resin

hastens decalcification by removing calcium ions from formic acid containing decalcifying solutions

ion exchange resin

not recommended for fluids containing nitric acid or hydrochloric acid

electrophoresis

process whereby positively charged calcium ions are attracted to a negatively electrode → removed from the decalcifying solution

electrophoresis

decalcification time is shortened due to heat & electrolytic reaction

electrophoresis

satisfactory for small bone fragments

88% formic acid + concentrated hydrochloric acid + distilled water

solution for electrophoresis

• concentration of decalcifying agent

• volume of decalcifying agent

• too rapid removal of calcium salts → complete digestion of tissue specimen → marked swelling & hydrolysis of bony matrix → poor staining

• temperature

factors influencing decalcification

more concentrated

concentration of acid solutions → decalcify bone more rapidly

20:1

decalcifying agent to tissue ratio

greater amount

volume of decalcifying agent → increase speed of decalcification

heat

hasten decalcification, but also increases damaging effects of acids on tissue

37 deg C

decalcification temperature → impair nuclear staining of Van Gienson’s stain for collagen fibers

55 deg C

decalcification temperature → undergo complete digestion in 24-48 hours

room temperature, 18-30 deg C

optimum temperature for decalcification

• X-ray or radiological testing

• chemical testing

• physical or mechanical testing

how to determine end point of decalcification

x-ray or radiological testing

most ideal & most reliable in determining end point of decalcification

chemical testing

recommended for routine purposes in determining end point of decalcification

precipitation w/ ammonium oxalate

chemical testing in determining end point of decalcification principle

physical or mechanical testing

determining end point of decalcification done by touching or bending the tissue w/ the fingers to determine consistency of tissues

physical or mechanical testing

determining end point of decalcification by pricking tissue w/ a fine needle or a probe to produce needle tract artifacts & destroy important cellular debris

• saturated lithium carbonate solution

• 5-10% aqueous sodium bicarbonate solution

after decalcification → remove acid by immersing the decalcified bone in

30 minutes

in post decalcification, adequate water rinsing for small samples can be accomplished in

1-4 hours

in post decalcification, adequate water rinsing for larger specimens can be accomplished in

• thoroughly washed in water

• stored in formol saline w/ 15% sucrose

• stored in phosphate-buffered saline w/ 15-20% sucrose @ 4 deg C before freezing

acid decalcified tissues for frozen sections must be

70% alcohol

tissues decalcified in EDTA solutions SHOULD NOT be placed directly into ___, formation of EDTA precipitate in it & within the tissue

formol saline or phosphate buffered saline

rinsing the decalcified tissue w/ water or storing overnight in ___ will prevent formation of crystalline precipitate

tissue softeners

used to soften hard tissues—ease sectioning & avoid microtome damage

1. Perenyi’s fluid

2. molliflex

3. 2% HCl

4. 1% HCl in 70% alcohol

tissue softeners

dehydration

process of removing intercellular & extracellular water from the tissue

increasing strengths

many dehydrating agents are ALCOHOL that are generally used in ___ to remove aqueous tissue fluids w/ little disruption to the tissues

70%-95%-100%

dehydration strength

30% ethanol

recommended dehydrating agent for delicate tissues

1. alcohol

2. acetone

3. dioxane

4. cellosolve

5. triethyl phosphate

6. tetrahydrofuran

commonly used dehydrating agents

alcohol

most common dehydrating agents

unequal impregnation of tissue

concentrated alcohol (95% or absolute) tend to harden only the surface of tissue → deeper parts are not completely penetrated →

≤70% gradually increased to 95% alcohol is used

remedy for unequal impregnation of tissue due to use of concentrated alcohol