DECALCIFICATION PROCEDURE:
BONE 1. Specimens should be decalcified in hydrochloric
acid/formic acid working solution 20 times their volume.
o special procedure under fixation
2. Change to fresh solution each day until decalcification is
o Process of removing calcium salts from the tissue and
complete.
making them suitable for sectioning.
• It may take 24 hours up to days or months depending on
o Calcium is responsible for the hardness of the tissue.
size of the specimens.
3. Wash specimens in running tap water thoroughly.
EXAMPLE OF CALCIUM SALTS:
4. Routine paraffin embedding.
1. Calcium phosphate (CaPO4)
2. Calcium carbonate (CaCO3)
METHODS OF DETERMINING THE END POINT OF
3. Calcium fluoride (CaFl2) DECALCIFICATION
• Calcium salts must be removed prior to embedding to 1. X-ray/Radiological (the most accurate way)
allow better sectioning.
2. Chemical method (convenient)(clear-complete)
3. Physical method (less accurate and potentially damage
TISSUES SUBJECTED TO DECALCIFICATION: the specimen)
1. Bones
2. Teeth X-RAY/RADIOLOGICAL METHOD
3. Atheromatous aorta o most accurate and best method
4. Calcified eyeball due to tumor o expensive
5. Calcified tuberculous foci o good but not always convenient
6. Urinary stones/calculi o Cannot be used when radio opaque metallic salts such as
mercuric chloride have been used in fixation.
7. Tumor of the meninges
INTERPRETATION:
8. Other calcified tissues
o black dots - presence of calcium
o transparent - no calcium
PRINCIPLE OF DECALCIFICATION
o Insoluble calcium salts are converted into soluble
calcium salts by the action of the decalcifying agent so that WHAT TO DO WHEN RECEIVING A HARD SPECIMEN?
the tissue will become soft.
1) Clean the bone, remove muscles interference.
o Chelating agent binds to calcium ions present in the
2) Fix the bone in 10% formalin for 1 day.
bone and decalcification process is carried out.
3) Decalcify with 5% HNO3 for 1 week.
PURPOSE OF DECALCIFICATION
4) Test the end point of decalcification.
o Done to assure that the specimen is soft enough.
5) Embed in a solid medium.
o To allow cutting with the microtome knife.
o Failure to decalcification results in torn, ragged sections
and damage to the cutting edge of the microtome knife. DEHYDRATION
• the process of removing intracellular and extracellular
water from the tissue following fixation and prior to wax
CRITERIA OF A GOOD DECALCIFYING AGENT:
infiltration.
1. Complete removal of calcium.
• this is usually done with a series of alcohol, 70% , 95% to
2. Less damage to tissue, cells and fibers. 100%.
3. Subsequent staining should not be altered. • sometimes the first step is a mixture of formalin and
alcohol.
4. Short time required for decalcification.
• amount of dehydrating agent: at least 10 times the
volume or more
Characteristics of a good dehydrating agent: Reminder when using Alcohol
1. Must dehydrate rapidly without producing considerable 1. The strength of initial alcohol required in each
shrinkage or distortion of tissues. concentration will depend upon the size, nature of the
tissue and fixative used.
2. Should not evaporate very fast.
- Small and delicate tissue: Lower concentration and
3. Should be able to dehydrate even fatty tissues.
shorter intervals
4. Should not harden tissues excessively.
- Concentrated alcohols produce shrinkage, make tissues
5. Should not remove stains. hard & brittle
6. Should not be toxic to the body. 2. Length of storage: The tissue may be stored in 70% -
80% alcohol (not for longer periods of time)
7. Should not be a fire hazard.
- Less than 70% concentration may macerate the tissue
- Longer storage using 70%-80% may interfere the staining
Commonly Used Dehydrating Agents properties of the tissue
1. Alcohol 3. Temperature: hastens at 37 degrees Celsius
2. Acetone ➢ Duration of dehydration should be kept to the
3. Dioxane minimum consistent with the tissues being processed.
4. Cellosolve ➢ Tissue blocks 1 mm thick should receive up to 30
minutes in each alcohol, blocks 5 mm thick require up to
5. Triethyl phosphate 90 minutes or longer in each change.
6. Tetrahydrofuran ➢ Tissues may be held and stored indefinitely in 70%
ethanol without harm.
Dehydrating agents:
Alcohols CLEARING
1. Ethanol • Removal of the dehydrating agent and replacement of a
fluid (clearing agent) that is miscible with both the
2. Methanol dehydrating agent and embedding medium (paraffin wax).
3. Isopropanol • Most common clearing agent is xylene
Glycol-ethers • Makes tissue transparent
1. Ethoxyethanol • Increase refractive index
2. Polyethylene glycols • Low boiling points
3. Cellosolve • Most are flammable liquids
4. Dioxane • Over clearing may cause tissue brittleness
Other dehydrants: ➢ Chloroform is also used, but is a health hazard, and is
1. acetone slow.
2. triethyl phosphate ➢ Methyl salicylate is rarely used because it is expensive,
but it smells nice (it is oil of wintergreen).
3. tetrahydrofuran
Characteristics of a good clearing agent:
Ethyl alcohol - the best dehydrating agent
1. Should be miscible with alcohol.
* not expensive
2. Should be miscible with paraffin wax and/or mounting
* not poisonous and not toxic medium.
* no untoward effects 3. Should not produce excessive tissue shrinkage and
hardening.
* fast acting
4. Should not dissolve aniline dyes.
* penetrates tissue easily
5. Should not evaporate quickly.
6. Should make tissue transparent.
Choice of a clearing agent depends upon the following:
1. The type of tissues to be processed, and the type of
processing to be undertaken.
2. The processor system to be used
3. Intended processing conditions such as temperature,
vacuum and pressure. (T,V,P)
4. Safety factors
5. Cost and convenience
6. Speedy removal of dehydrating agent
7. Ease of removal by molten paraffin wax
8. Minimal tissue damage
Other clearing agents:
1. Esters
2. n-Butyl Acetate
3. Terpenes
4. Limonene
5. Terpineol
➢ Clearing agents also remove a proportion of tissue fat,
which presents a barrier to wax impregnation.
➢ Prolonged clearing time causes the tissue to become
brittle.
➢ Incomplete clearing results in uneven hematoxylin-
eosin staining and poor nuclear chromatin patterns.
