TISSUE DECALCIFICATION
INTRODUCTION (1)
• Bone consists of cells (osteocytes) surrounded by a
calcified matrix containing Type 1 collagen fibres.
• Calcium and phosphate combined with hydroxyl ions to
form hydroxyapatite crystals
• Hydroxyapatite crystals [Ca10(PO4)6(OH)2] which are
deposited between the fibrous elements.
• These crystals are dissolved out during the process of
decalcification which, if performed correctly, leaves
cohesive tissue with the physical characteristics of
dense fibrous connective tissue.
INTRODUCTION (2)
• Preservation of hard tissues close to the living state is essential for
understanding of cellular and subcellular structures and functions.
• The cutting of thin sections by ordinary methods is impossible in the case of
tissues such as teeth, bone, teratomas containing bony tissue, lesions that have
become partly calcified, odontomes and bony lesions.
• Such tissues must be treated to remove calcium phosphate by a process known
as “decalcification”, thereby making the tissue soft enough to be cut by the
microtome
Indications for bone biopsy
• Confirm the diagnosis of a bone disorder
• Determine if a bone tumour is malignant or benign
• Find the cause of ongoing bone pain
• determine the cause of an unexplained infection or inflammation
Specimen of Decalcification
• 'J' Needle Biopsy
• Bone Marrow trephine biopsy
• Sections from bone tumours
• Sections from underlying bone in tumours of jaw or chest
What precedes decalcification
• Fixation
• Complete fixation helps protect cellular and fibrous elements of the
bone from damage caused by acids in decalcifying fluids.
• For adequate fixation: reduce bone size, remove excess skin or soft
tissue arround lesion.
• Cut, fix and decalcify
• Suitable fixative:10% NBF
DECALCIFICATION: What it means
• Removal of calcium ions from bone through histological process
thereby making the bone flexible and easy for pathological
investigation
• It is to ensure hard bony tissue specimens are soft enough to allow
cutting with a microtome knife
• Inadequate decalcification can result in torn, ragged sections and
damage to cutting edge of the microtome knife
METHODS OF DECALCIFICATION
• Acid decalcification
• Chelating agents
• Ion exchange resin method
• Electrolytic method
• Microwave decalcification
• Surface decalcification
ACID DECALCIFIERS
• Commonest method used in dissolving calcium in an acid solution.
• Strong acids: 5-10% (recommended) aqueous Nitric acid and HCL. Used with
dense cortical bone.
o X’tics: rapid, cause tissue swelling and can cause tissue damage if used
longer than 24–48 hours, not suitable for
enzyme/immunohistochemistry and delicate tissue like the bone marrow
• Weak acids: Formic, acetic acid and picric acid
• X’tics: acetic and picric used as additives bcos they can cause tissue
swelling when used alone.
• Formic acid is slower and gentler than nitric and HCL. Suitable for routine
specimen and IHC
Chelating method
• EDTA commonly used, binds calcium ions and gradually depletes the crystal size
of the outer layer of the hydroxyapatite.
• Advantages:
• Deposits of iron and other metals may also be removed,
• tissues are not hardened after decalcification
• Good moprhology and improve staining for EM/IHC
• Disadvantages:
• Time consuming
• Costly
Ion exchange resin method
• Consists of a polymeric matrix and a functional group with a mobile ion
which can be exchanged with other ions. The most common synthetic
structures are
• Cross-linked polysterene
• Cross-linked polymethacrylate
• Phenol-formaldehyde
• IER in decalcifying fluids are used to remove calcium ion from the fluid.
Thereby ensuring a rapid rate of solubility of calcium in this fluid.
• X’tics: faster, preserve tissue better, cellular details are preserved
Other approaches
• Electrolytic method:
• This is based on the principle of attracting calcium ions to a negative
electrode in decalcifying solution.
• Microwave decalcification:
• Decalcification time is reduced with no apparent adverse effects on structural
and preservation or antigenicity.
End point Testing
• In order to achieve optimal results when processing calcified tissues, it is important to
determine the point at which decalcification is complete.
• While incomplete decalcification can lead to tissue distortions (and possibly a damaged
microtome), over-decalcification causes problems with staining, in particular nuclear
staining
• Physical test; Unreliable , cause damage and distortion of tissue. “Bubble test”
• X-ray : best method but costly, tissue fixed in mercuric chloride containing fixatives
cannot be tested as they will be radio opaque
• Chemical method
• It is done to detect calcium in decalcifying fluid when decalcification is considered
complete. Eg : Calcium Oxalate test
Treatment following decalcification and prior to
processing
• Various method for neutralising residual acid decalcifier before processing.
• Generally a short effective wash in tap water should be sufficient as any
remaining acid will be removed during processing.
• Treatment over night in 5% Lithium or sodium sulphate can be used.
• It is important to remove the bulk of the decalcifier to avoid contaminating the
processing reagents and the processor with acid
Criteria of selecting good decalcifying agents
• Complete removal of calcium
• Less of damage to tissue, cells and fibers
• Subsequent staining should not be altered
• Speed of removal of Ca salts
Factors influencing the rate of decalcification
• Type of tissue and thickness
• Concentration
• Temperature
• Agitation
• Suspension
SAFETY
• Acids used to make decalcifying solution should be
handled carefully, concentrated acid is very hazardous
• Wear apron, gloves and goggles for handling however
small the quantity.
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