MIDTERM LEC: DECALCIFICATION Flashcards
Follows fixation
DECALCIFICATION
Removal of calcium or lime salts from calcified tissues
DECALCIFICATION
Inadequate decalcification poor cutting of hard tissues an damage to the knife edge during sectioning
DECALCIFICATION
Hastened by heat and agitation
DECALCIFICATION
With a common 2-day duration
DECALCIFICATION
Calcifications cause a grating sensation during sectioning
Remedy:
Remove block from the chuck, and place face down on cotton or gauze with 10% HCl
Hematoxylin-stained microcalcifications =
dark purple granular
masses with light purple halos
DECALCIFICATION IS DONE:
Bone
Teeth
Teratoma (means monster)
Calcified tissues: tuberculous organs, arteriosclerotic vessels
DECALCIFICATION GROSSING:
Done before fixation
Double gloves, eye protection glasses, face mask
Specialized table for bone processing
Bone specimens are grossed in fresh state
Bone dust particles can be loaded with blood or infectious malts (osteomyelitis, gangrene)
Fine fret-saw saw hand razor
Factors Affecting Decalcification:
- Concentration
- Tissue-to-volume ratio
- Temperature
- Mechanical agitation
- Size & consistency of tissue sample
HIGH=faster, but may be more harmful to tissue
Concentration
Tissue-to-volume ratio
optimum: 1:20
HIGH=faster, but may be more harmful to tissue
Temperature
TEMPERATURE OPTIMUM:
18 to 30 degrees Celsius
Hastens decalcification
Mechanical agitation
Larger specimens will slow the rate of decalcification
Size & consistency of tissue sample
Methods of Decalcification:
- Acids
- Chelating Agents
- Ion Exchange Resin
- Electrical ionization (electrophoresis)
Most widely used, stable, easily available, relatively inexpensive:
- Nitric acid
- Hydrochloric acid
- Formic acid
- Trichloroacetic acid
- Sulfurous acid
- Chromic acid
- Citric acid
Most common and fastest agents
Nitric acid
Removed by 70% ROH during dehydration
Nitric acid
Imparts yellow coloration d/t nitrous acid formation
[Remedy: add Urea or Sodium thiosulfate/sulfate]
Nitric acid
NITRIC ACID TYPES:
- 10% Aqueous Nitric Acid
- Formol-Nitric Acid
- Perenyi’s fluid
- Phloroglucin Nitric acid
Urgent, needle and small biopsies
10% Aqueous Nitric Acid
Has formalin (allows less destruction)
Formol-Nitric Acid
Urgent biopsies
Formol-Nitric Acid
Has chromic acid and ROH (thus, no tissue breakup)
Perenyi’s fluid
Also a tissue softener
Perenyi’s fluid
Most rapid decalcifying agent
Phloroglucin Nitric acid
Complete decalcification cannot be determined through chemical means
Phloroglucin Nitric acid
Removed with 3 changes of 70-90% ETOH
Phloroglucin Nitric acid
Slower and causes more distortion compared to HNO3
Hydrochloric acid
Provides good nuclear staining
Hydrochloric acid
Recommended for surface decalcification if used in 1%
with 70% ROH
Hydrochloric acid
HCl + 36% NaCl
Von Ebner’s
1 TYPE OF Hydrochloric acid:
Von Ebner’s
For teeth and small bones
Von Ebner’s
Only weak acid used as a primary decalcifying agent
Formic Acid
Complete decalcification cannot be determined through chemical means
Von Ebner’s
For routine decalcification of post-mortem research tissues, small pieces of bone and teeth, immunohistochemical staining
Formic Acid
```
Better nuclear staining, less tissue distortion than HNO3
Formic Acid
Addition of citrate faster decalcifying rate
Formic Acid
P: autopsy, BM, cartilage, research tissues
Formic acid-Sodium citrate solution
Small bone spicules
Trichloroacetic acid
Good nuclear staining, does not require washing out
Trichloroacetic acid
Weak decalcifying agent and very slow
Trichloroacetic acid
Very weak agent thus for minute pieces of bone pieces only
Sulfurous acid
Both a fixative and decalcifying agent
Chromic acid (Flemming’s Fluid)
Minute bone spicules
Chromic acid (Flemming’s Fluid)
DADV: highly corrosive to skin, carcinogenic, and environmental toxin
Chromic acid (Flemming’s Fluid)
Excellent nuclear and cytoplasmic staining but too slow
Citric acid-citrate buffer solution
pH 4.5
Citric acid-citrate buffer solution
For immunohistochemistry, enzyme staining, and electron microscopy
CHELATING AGENTS
Principle: Use of other salts to form weakly dissociated complexes with calcium salts for ease of removal
CHELATING AGENTS
CHELATING AGENTS Duration:
1-3 weeks for small specimens; 6-8 weeks for longer & dense bones
CHELATING AGENTS Optimum pH:
7-7.4
CHELATING AGENTS Examples:
Cal-Ex & Versene (EDTA)
Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue
ION EXCHANGE RESINS
Ion exchange resin (ammonium form of polystyrene resin) is spread over the bottom of container put tissue on top add
decalcifying agent (20 to 30 times the volume of the tissue)
ION EXCHANGE RESINS
Not recommended for fluids with mineral acids such as HNO3
and HCl
ION EXCHANGE RESINS
ION EXCHANGE RESINS Duration:
1 to 14 days
Calcium ions (positively charged) moves to cathode (negative electrode)
ELECTROPHORESIS
Uses 88% formic acid
ELECTROPHORESIS
For small bone fragments
ELECTROPHORESIS
Shorter time for calcium removal because of heat and electrolytic reaction involved
ELECTROPHORESIS
Decalcification must be frequently monitored to avoid maceration of tissue. The methods of measuring extent of decalcification are as
follows:
- Physical/Mechanical Test
- Radiologic MethodChemical or X-Ray
- Chemical Method
MEASURING EXTENT OF DECALCIFICATION
Touching and bending tissue using fingers; and poking using fine needle or a probe
Physical/Mechanical Test
MEASURING EXTENT OF DECALCIFICATION
Rubbery consistency, and soft = Tissue is decalcified
Physical/Mechanical Test
MEASURING EXTENT OF DECALCIFICATION
DADV: vague and inaccurate artifact production, destruction of cellular details, small calcified foci may not detected
Physical/Mechanical Test
MEASURING EXTENT OF DECALCIFICATION
ADV: most ideal, most sensitive, and most reliable method; can detect smallest focus of calcium
Radiologic Method or X-Ray
MEASURING EXTENT OF DECALCIFICATION
Rinse decal agent from tissue Put tissue on waterproof polyethylene sheet on top of X-ray film Expose to X-ray for 1 minute at 30kV Leave until film is developed
Radiologic Method or X-Ray
MEASURING EXTENT OF DECALCIFICATION
Opaque result = Incomplete decalcification
Radiologic Method or X-Ray
MEASURING EXTENT OF DECALCIFICATION
DADV: very expensive, never used with HgCl2-fixed tissues (radio-opacity, = xrays do not pass through)
Radiologic Method or X-Ray
MEASURING EXTENT OF DECALCIFICATION
Simple, reliable, and convenient
Chemical Method
MEASURING EXTENT OF DECALCIFICATION
PCPL: precipitation of calcium hydroxide or calcium oxalate
Chemical Method
MEASURING EXTENT OF DECALCIFICATION
Addition of strong ammonia to the discarded decalcifying fluid (until solution becomes ALK).
Cloudiness = presence of Ca in the discarded fluid
Chemical Method
MEASURING EXTENT OF DECALCIFICATION
If no cloudiness, add saturated aqueous solution of ammonium oxalate, and let it stand for 30 minutes
Cloudiness = incomplete decalcification
Chemical Method
POST-DECALCIFICATION
Removal of decalcifying agent through:
- Immersion in saturated lithium carbonate or 5-10% aqueous sodium bicarbonate solution for several hours;
- Rinsing in tap water (small spx=30 mins; large spx=1-4 hours);
- For frozen section, acid decalcified tissue is stored in formol saline with 15% sucrose or PO4-buffered saline (PBS) with 15- 20% sucrose at 4 OC before freezing
TISSUE SOFTENERS
Performed prior to dehydration or sectioning; for unduly hard tissues that may damage microtome knives. The agents are as follows
- Perenyi’s
- 4% Aqueous Phenol
- Molliflex (Effect: tissues appear swollen/soapy [not a negative effect])
- 2% HCl
- 1% NaCl in 70% ROH