HISTOPATHOLOGY

DECALCIFICATION (24-48 hrs)

 Process of removing calcium or lime salts from the tissue following fixation and before
impregnation to:
 Ensure and facilitate normal cutting of sections
 Prevent obscuring the micro-anatomic detail of sections by dust and other debris
 Carried out at room temperature, 25°C
 Ideal tissue thickness is 1-3 mm
 TYPES OF SPECIMENS:
 Amputated limbs
o Sent to the lab immediately after surgery secondary to tumor, inflammation,
gangrene
o Delivered without fixative
o Relevant portions are fixed and if not processed asap, refrigerate @ 4-6°C
 Resected specimens
o LESS URGENT with established diagnosis
o Bones with benign or low-grade malignant tumor and arthritic femoral heads
 PURPOSE OF BONE RADIOGRAPHY (option prior to decalcification)
 To examine nature of a lesion
 To determine extent of a lesion
 To provide a map of a lesion prior to selection for processing
 To check the progress of decalcification endpoint test
 To confirm the presence of prosthetic devices, metal or glass fragment implanted by
trauma
 First step in preparing bone for decalcification
 Immersion to 10% Neutral Buffered Formalin
 Carnoy’s should not be used prior to decalcification due to its toxicity
 Inadequate decalcification
 Torn and ragged sections and damages to the cutting edge of microtome
 Carried out by using chemical agents:
 Acids – formation of soluble calcium salts
 Chelating agents – bind to calcium ions
 Specimens causing interference in the evaluation and examination of sections:
 Bones, teeth, tuberculous organs, teratomas
 Bones and calcified tissues (tuberculous organs and arteriosclerotic vessels):
 Cut into small pieces using fine fret saw and trimmed with hand razor
 Paraffin-embedded and frozen tissues
 Undergo sectioning of small foci of microcalcification which appears dark blue granule
masses with lighter purple halos
 Small foci calcification in paraffin embedded block after trimming:
 Causes resistance and grating sensation when sectioned with microtome knife
 REMEDY: Remove block from the chuck and place face down on a pad of cotton or
gauze saturated with 10% HCl for 1 hour
 Effects of rapid decalcifying agents on H&E staining
 Failure of the cell nuclei (chromatin) to take up Hematoxylin
 Eosin – produce deep, brick red stain
 Reduced by post-decalcification and removal, adjustment of the staining procedure
 HISTOPATHOLOGY

METHODS OF DECALCIFICATTION

A. Use Of acids
1. STRONG INORGANIC ACIDS
Indications (Used for):
1. Needle biopsy and small biopsy spx
2. Urgent diagnoses (<24hrs)
3. Large and mineralized cortical bone spx monitored by decalcification endpoint test

Disadvantages:
1. Loss of enzymes
2. Damage of tissue antigens for immunohistochemical staining
3. Conc. agents destroy tissue causing swelling
4. Use of old stocks = DISTORTION

A. NITRIC ACID
 Fastest and most common
 Used as simple aqueous solution (5-10%) or combined with other agents
 For routine purposes
 ADVANTAGE: very rapid producing minimal distortions
 DISADVANTAGE: inhibits nuclear stains and destroys tissues
 Imparts YELLOW COLOR
 REMEDY: addition of 5% sodium thiosulfate or 0.1% urea prior to
decalcification

 10% AQUEOUS NITRIC ACID – 12-24 hrs – CONC. NITRIC ACID + DISTILLED H2O
Advantages:
 For urgent biopsy, needle and small biopsy
 Produces good nuclear staining
 Removed by 70% alcohol
Disadvantages:
 Imparts YELLOW COLOR with nitrous acid
 Damage tissue stainability and tissue antigens for immunohistochemical staining

 FORMOL NITRIC – 1-3 days - CONC. NITRIC ACID + FORMALDEHYDE

Advantages:
 Rapid acting, for URGENT BIOPSIES
 Relatively good nuclear staining
Disadvantages
 Used inside a fume hood
 Staining reaction is interfered by nitrous acid formation
*Removed by adding 5% sodium sulfate or 0.1% urea

 PERENYI’S FLUID – 2-7 days - NITRIC ACID + CHROMIC ACID + ETHYL ALC.
Advantages:
 Both decalcifying and tissue softener
 For routine purposes
 Nuclear and cytoplasmic staining is good
Disadvantages
 Relatively slow, not recommended for urgent work
 Formation of precipitate when added with ammonia
*Removed by adding glacial acetic acid + 0.5mL sat. aqueous ammonium oxalate
 HISTOPATHOLOGY

 PHLOROGLUCIN-NITRIC ACID – 12-24 hrs - CONC. NITRIC ACID + PHOTOGLUCIN

Advantage
 MOST RAPID, for urgent works
Disadvantages
 Poor nuclear staining, causes distortion
 Removal of acid requires three changes of 70-90% ethanol

B. HYDROCHLORIC ACID – 1- 3 days

Advantage
 Produce good nuclear staining and recommended for surface decalcification if used in
1% solution with 70% alcohol
Disadvantage
 Slower action than the nitric acid

 8% AQUEOUS HCl – 1-3 days – HCl + DISTILLED H2O

 Surface decalcification of blocks
 Prolonged decalcification (24hrs) causes impaired staining especially in high temp.

 VON EBNER’S FLUID – NaCl + HCl

Advantages
 Relatively good cytologic staining
 Moderately rapid agent
 Recommended for teeth and small pieces of bones

2. WEAK ORGANIC ACIDS

A. FORMIC ACID – 2-7 days

 Moderate acting agent, better nuclear staining, safer than nitric and HCl
 For POSTMORTEM RESEARCH TISSUES
 Added with citrate to accelerate dehydration
 Carnoy’s and Bouin’s – incidental, albeit, weak decalcifiers that are used in urgent
cases
 Picric acid and acetic acid – not used alone as decalcifying agents, causes tissue
swelling
Advantages
 Both fixative and decalcifying agent
 For immunohistochemical staining of most routine surgical spx
Disadvantages
 Relatively slow agent
*Hastened by increasing the solution to 25mL but causes opaque solution and interferes
with staining results
 Requires neutralization with 5% sodium sulfate and washing out
 FORMIC ACID-SODIUM CITRATE SOLUTION – 3-14 days
Advantage
 For autopsy materials, bone marrow, cartilage and tissues studied for research
purposes

B. TRICHLOROACETIC ACID (TCA) and PICRIC ACID – 4-8 days

Advantages:
 Recommended for small spicules of bone
 HISTOPATHOLOGY

 Good nuclear staining

 Does not require washing out
 Excess acid can be removed by 90% alc. improving tissue dehydration
Disadvantages
 VERY SLOW-ACTING agent, weak decalcifying agent and not used for dense tissues

C. SULFUROUS ACID
 Very weak decalcifying solution
 Suitable only for minute pieces of bones

D. CHROMIC ACID (FLEMMING’S FLUID)

Advantages:
 Both fixative and decalcifying agent
 For minute bone spicules
Disadvantages:
 Hematoxylin nuclear staining is inhibited
 Tends to undergo reduction, form precipitate, require frequent changes
 Environmental toxin
 Highly corrosive to skin and mucous membranes
 Carcinogenic

E. CITRIC ACID-CITRATE BUFFER – 6 days

Advantages:
 Contains chloroform as preservative
 Permits excellent nuclear and cytoplasmic staining
 Does not produce cell or tissue distortion
Disadvantage
 Slow acting agent/ weak, for small pcs. of tissues

B. Use of Chelating Agents

1. ETHYLENE DIAMINE TETRAACETIC ACID (EDTA)
 Commercially known as VERSENE
 Recommended for detailed microscopic studies
 Combines with CALCIUM and MAGNESIUM
 EDTA + Ca2+ = anticoagulant and water softener
 Small spx: 1-3 weeks
 Dense cortical bone: 6-8 weeks
 Changed every 3 days
 Recommended pH: 7-7.4
 pH = 8, OPTIMAL BINDING
Advantages:
 EXCELLENT BONE DECALCIFIER
 Recommended for IMMUNOHISTOCHEMICAL, ENZYME STAINING, EM
 Forms minimal histological artifacts, caused by production of CO 2 bubbles
 Extent of decalcification can be measured by routine chemical test
Disadvantages:
 Very slow acting agent, not recommended for urgent and routine purposes
 Inactivates alkaline phosphatase activity, restored by adding Magnesium chloride
 Causes slight tissue hardening, expensive and not routinely used
 HISTOPATHOLOGY

C. Ion Exchange Resin

 Ammonium form of polystrene – Most common
 Decalcify tissues from formic acid containing decalcifying solutions
 Not recommended for fluid containing mineral acids like nitric acid and HCl
PROCESS
 RESIN: ½ inch thick at the bottom of the container
*Hastens Ca2+ removal and increases tissue solubility
 Decalcifying agent: 20-30 times the volume of the spx
 Immerse spx for 1-14 days
 Measure degree of decalcification by physical or X-ray method

Advantages
 Produces minimal cell and tissue distortion
 Spx can be kept in the solution for up to 20 days
 Well-preserved cellular detail
 Forms minimal histological artifacts due to CO 2 bubble production
Disadvantages
 Degree of decalcification cannot be measured by chemical means
 VERY SLOW, not recommended for routine and urgent purposes
 Causes SLIGHT TISSUE HARDENING

D. Electrolytic Method / Electrical Ionization

 Process whereby positively charged Ca 2+ ions are attracted to a negative electrode and
subsequently removed
 MOST RAPID, decalcify up to 8HRS
 Dependent upon the supply of ELECTRIC CURRENT
PROCESS
 Suspend spx using platinum anode in a jar
 Change decalcifying solution after 8HRS
 Rinse apx in ALK. Water
 Immerse in Lithium Carbonate before staining

Advantage
 For SMALL BONE FRAGMENTS
Disadvantage
 Prolonged decalcification: MACERATION AND DESTRUCTION OF TISSUE
 Good cytologic and histologic details are not always preserved in tissue

FACTORS AFFECTING DECALCIFICATION RATE

1. Concentration and Volume of decalcifying agent

 More concentrated = Rapid decalcification, but harmful to tissue and may cause complete
digestion of spx
 Recommended fluid to tissue volume ratio: 20:1

2. Temperature
 Heat @ 37°C = hastens decalcification, but impaired nuclear staining of Van Gieson’s stain
for collagen fibers
 Recommended temperature = 18 - 30°C
 At 55°C = complete digestion of tissue within 12-48hrs
 HISTOPATHOLOGY

3. Agitation
 Mechanical agitation: influences fluid exchange, accelerate and speeds up process
 Gentle fluid agitation: low-speed rotation, rocking, stirring, bubbling air into solution
 Sonication vigorously: disruption of tissue

4. Suspension
 Wrap spx with gauze bag and use with thread that is dipped in melted paraffin wax

5. Other factors
 Age, type of bone, size of the spx

FACTORS TO CONSIDER IN CHOOSING THE RIGHT DECALCIFIER

 Urgency of case – need for immediate examination

 Degree of mineralization
 Staining technique to be applied / stain to be used

TESTS TO MEASURE THE COMPLETENESS OF DECALCIFICATION

(DECALCIFICATION-ENDPOINT TESTS)

1. Physical / Mechanical
 Touching or bending of tissue with the fingers to determine consistency
 Pricking the spx with fine needle pr a probe
 Both methods can disrupt soft tumor = false positive microfractures = misdiagnosis

2. Chemical Method / Calcium Oxalate Method

 Simple, reliable, convenient method recommended for routine purposes but INACCURATE
 Detection of Ca2+ by precipitation of insoluble Ca(OH)2 or CaOx

3. X-ray / Radiological Method

 MOST IDEAL, SENSITIVE AND RELIABLE method but VERY EXPENSIVE
 With Ca2+ = OPAQUE
 Without Ca2+= CLEAR
 Use of FAXITRON and KODAK X-OMAT X-RAY FILM
 Not suitable for mercuric chloride fixed tissues
PROCESS
 Rinse acid decalcifying agent from the sample
 Place on waterproof polyethylene sheet on top of the x-ray film
 Expose to x-ray for 1 minute, 30Kv
 Left in place until film is developed

4. Bubble Test
 Addition of CaCO3 (Ca2+ + CaCO3 = CO2, bubble formation in the surface of spx)
 Inaccurate, unreliable
 HISTOPATHOLOGY

POST DECALCIFICATION

 Chemical Neutralization = immersion of decalcified bone in sat. lithium carbonate solution or

5-10 aqueous sodium bicarbonate solution
 Rinsing with running tap water
 Small samples : 30minutes
 Large samples : 1-4hours
 Acid decalcified tissues for frozen sections
 Stored in saline containing 15% sucrose or phosphate-buffered saline with 15-20%
sucrose @ 4°C before freezing
 Tissues decalcified in EDTA
 Store overnight in formol saline or PBS

TISSUE SOFTENERS
 4% aqueous phenol for 1-3 days (Lendrum’s method – immersion in 4% phenol)
 Molliflex – swollen and soapy
 1% or 2% HCl in 70% alcohol
 Perenyi’s fluid