Decalcification Flashcards
rinse the decalcified spxs with running tap water.
common practice of post-decalcification
Removal of excess acid is done by immersing the decalcified tissue in: • Saturated Lithium carbonate • 5- 10% Aqeous Sodium bicarbonate
Post-decalcification
uses litmus paper, NH4H2O & NH4 Oxalate
Chemical Method/Calcium Oxalate Test
KODAK X-OMAT or Faxitron – x – ray paper
X-Ray/ Radiologic Method
simple, reliable, recommended for routine purposes
Chemical Method/Calcium Oxalate Test
very expensive; most reliable; most ideal
X-Ray/ Radiologic Method
– inaccurate a.k.a. FLEXIBILITY METHOD
Physical/ Mechanical
Physical/Mechanical ; X-Ray; Chemical
Ways of measuring the extent of decalcification
Increase in size and consistency will require longer periods for complete decalcification.
Size and Consistency
more concentrated acid solutions decalcify bone more rapidly, but are more harmful to the tissue.
Concentration
Concentration, Fluid Access, Size and Consistency, Agitation, Temperature
Factors Influencing the Rate of Decalcification
o Action is too slow for routine purposes
Disadvantages Citric Acid Citrate
o Permits excellent nuclear and cytoplasmic staining o Does not produce cell or tissue distortion
Advantages Citric Acid Citrate
o 7% Aqueous solution Citric acid (monohydrate): 5 mL o 7.4% Aqueous solution Ammonium Citrate (anhydrous): 95 mL o 1% Aqueous solution Zinc Sulfate: 0.2 mL Chloroform (as preservative) – a few drops
Citric Acid-Citrate Buffer Solution (6 days)
o Both fixative and decalcifying agent o Used for decalcifying minute bone spicules
Advantage of Chromic Acid
o Chromic acid: 15 mL o Osmium Tetroxide: 4 mL o 2% Glacial Acetic Acid: 1 mL
Chromic Acid (Flemming’s Fluid)
suitable only for minute pieces of bone
Sulfurous Acid
o Weak decalcifying agent, not used for dense tissues o Very slow-acting, not recommended for urgent examinations
Disadvantages Trichloroacetic Acid
o Permits good nuclear and cytoplasmic staining o Does not require washing out
Advantages Trichloroacetic Acid
o Trichloroacetic acid: 5 gm o 10% Formal saline: 95 mL
TRICHLOROACETIC ACID
o Relatively slow, not suitable for urgent specimens o Requires neutralization with 5% sodium sulfate
Disadvantages of Formic Acid
o Both a fixative and decalcifying agent o Permits excellent nuclear and cytoplasmic staining o Recommended for small pieces of bones and teeth o Suitable for most routine surgical specimens
Advantages of Formic Acid
o Formic acid: 10 mL o 10% Normal saline: 90 mL
Formic Acid (2-7 days)
Best Decalcifying Agent (Less Tissue Distortion). Only recommended for routine tissue biopsies (not suitable for urgent biopsies
Formic Acid (2-7 days)
pH 4.5; No Tissue Distortion; excellent staining
Citric Acid-Citrate Buffer Solution (6 days)
BOTH FIXATIVE and DECALCIFYING AGENT; Environmental Toxin
Chromic Acid (Flemming’s Fluid)
Very Weak Decalcifying Solution
Sulfurous Acid
Good Nuclear Staining; Weak Decalcifying Agent
Trichloroacetic Acid (TCA) (4-8 days)
BOTH FIXATIVE and DECALCIFYING AGENT; BEST GENERAL DECALCIFYING AGENT; Moderate-Acting
Formic Acid (2-7 days)
o Extent of decalcification cannot be measured by chemical test
Disadvantage of Von Ebner's Fluid
o Permits good cytologic staining o Moderately rapid decalcifying agent o Does not require washing o Recommended for teeth and small pieces of bone
Advantages of Von Ebner's fluid
o Saturated aqueous solution of NaCl: 50 mL o 36% Concentrated HCl: 8 mL o Distilled Water: 50 mL
Von Ebner's Fluid
o Nuclear staining is poor o Prolonged decalcification produces extreme tissue distortion o Yellow color must be neutralized with 5% sodium sulphate and thoroughly washed with running tap water (24 hours) o Complete decalcification cannot be determined by chemical means
Disadvantages of Phloroglucin-Nitric Acid
o Most rapid decalcifying agent o Recommended for urgent cases
Advantages of Phloroglucin Nitric Acid
o Concentrated Nitric Acid: 10 mL o Phloroglucin: 1 gm o 10% Nitric Acid: 100 mL
PHLOROGLUCIN-NITRIC ACID
o Slow decalcifying agent for dense bones o Complete decalcification cannot be determined by chemical test
Disadvantages of Perenyi's Fluid
o Recommended for routine purposes o Decalcifies and softens tissues at the same time o Nuclear and Cytoplasmic staining is good o Maceration is avoided due to the presence of chromic acid and alcohol
Advantages of Perenyi's Fluid
Prolonged decalcification, damage tissue stainability, imparts a yellor color with nitrous acid, strong acids tend to be more damaging to tissue antigens (histochemical staining)
Disadvantages of Nitric Acid
Rapid in Action, Produces minimum shrinkage, produce good nuclear staining, recommended for urgent biopsy, needle, and small biopsy
Advantages of Nitric Acid
A method of dealing with small unexpected deposits of calcium that may be encountered with Paraffin blocks. Placed face down the tissue block on a pad of cotton or gauze saturated with 10% HCl for one hour.
Surface Decalcification
SURFACE DECALCIFICATION, Recommended for small pieces of bones and teeth
Hydrochloric Acid
Most Rapid Decalcifying Agent, prevents excessive softening of bones
Phloroglucin-Nitric Acid (12-24 hours)
BOTH TISSUE SOFTENER and DECALCIFYING AGENT Staining is enhanced Good for tissues with small calcium deposits
Perenyi’s Fluid (2-7 days)
Most Common, Fastest decalcifying agent
Nitric Acid (12-24 hours)
faster than the routine procedure.
Microwave oven decalcification
Removal of calcium by using a diluted mineral and along with ... to keep the decalcifying fluid free of calcium.
ION EXCHANGE RESIN
Chelates calcium to form a soluble salt
Function of chelating agent
EDTA (pH: 7)
Most common chelating agent
14 Days
Dense bone tissue usually require up to DAYS or longer to complete the process.
24-48 HOURS
The ideal time required for decalcifying tissue
55°C
Tissue will undergo complete digestion within 24-48 hours
37°C
Impaired nuclear staining of Van Gieson’s stain for collagen fibers
18-30 degrees celsius
Optimum room temperature
1:20
Rate of Tissue and Decalcifying agent
Heat will serve to HASTEN decalcification but it also INCREASES the damaging effects on tissues.
true
If not done, it can result to poor cutting of tissues and damages to the knife that is used in sectioning.
DECALCIFICATION
Must be done to Bones, Teeth, Calcified tissues such as atherosclerotic plaque in blood vessels and tuberculous lungs.
DECALCIFICATION
REMOVAL OF CALCIUM ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut.
DECALCIFICATION
CAN YOU MAKE THIN SECTIONS FROM BONE AND TEETH?
NO, because this organs are calcified. Remove first the calcium to make the organs soft for easy sectioning