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Decalcification Process-LectureNotes3
Medical Technology (Our Lady of Fatima University)
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DECALCIFICATION PROCESS
Two types of adult bones:
Compact Bone
• Known as the strong bone
• Supports the shaft of long bones like femur, tibia, and external surface of the flat bones
Trabecular or Spongy Bone
-Is composed of mesh of bone strands which forms the weight-bearing structure of the
diaphysis, epiphysis, or vertebrae
• Note: the main bulk of bone is approximately 70% mineral and 30% organic components by
weight with sparse bone cells.
3 Major Components of the Bone:
Mineral - main target of decalcification
-During the initial mineralization phase, the mineral in the bone is approximately 38% calcium
deposited as amorphous calcium phosphate, later on, it is transformed into hydroxyapetite
with the addition of hydroxyl ion.
-With the addition of calcium, it forms a crystal lattice of needle-like crystals in which
carbonate, citrate, and floride ions as well as magnesium, potassium, And strontium can be
substituted or included.
Cells
The three types of cells are the osteoblasts, osteocytes, and osteoclasts
• Osteoblast-they are appear as plump cells with eccentric nuclei distal to the bone surface and
supported by a basophilic cytoplasm. They formed the osteoid.
• Osteocytes-reside in the lacunae of the bone which are connected to each other and to the
vascular spaces by canaliculi.
Osteoclast- they are multinucleated giant cells supported by a cytoplasm rich in mitochondria
and alkaline phosphatase. -bone resorption for calcium to be back in circulation
Organic Extracellular Matrix
-It is composed of collagen fibers and ground substance
-The bone collagen differ from the collagen in the body because it becomes mineralized and is
laid down in bands or lamellae roughly parallel to one another.
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Decalcification
-Is the removal of calcium ions or lime salts from the organic extracellular matrix, calcified
collagen, and surrounding tissues of the bone.
Examples: bones, teeth, tuberculous organs, arteriosclerotic vessels, eyeball
• Functions:
-ensure & facilitate normal cutting of sections
-prevents obscuring micro anatomic detail by bone dust & cellular debris
-more concentrated acid solutions decalcify bone more rapidly
-high concentrations and greater amount of fluid will increase the speed of the process
-prevent damage of microtome when cutting
-calcium hides cancer cells
• CONSIDERATIONS
-cut tissues into small pieces with fine fret-saw & trimmed with hand razor
• PRECAUTIONS
Hide important details
• Inadequate decalcification results
• Heat: may increase rate of decalcification
• Ideal decalcification time: 24-48 hours 1-2 days
• Dense bone tissues: 7 days or longer
• Optimum temperature: 18-30 °C/Room temperature 25
• @ 37 C= impaired nuclear staining of Van Gieson's stain
• @ 55 C= tissues will undergo complete digestion within 24-48 hours
• Ratio of fluid to tissue volume: 5-10:1
Factors Affecting the Rate of Decalcification
• Concentration and Volume vary according to decalcifying agent
• Temperature
• Agitation
• Suspension - sample must be immersed in decalcifying
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• Other factors:
- Patient's age - older the harder
- Type of bone - spongy/compact
- Standard Size of the specimen - larger the longer
Types of Specimen
Amputated Limbs
• These are often sent to the laboratory immediately after surgery secondary to tumor,
inflammation, and gangrene.
• They are often delivered without fixative, and must be taken care of as soon as possible
• If it is not possible to treat the specimen for several hours after its arrival, it should be
refrigerated at 4*C-6*C.
Resected Specimens
-These include bones with benign or low grade malignant tumors and arthritic femoral heads.
- They are usually with established diagnosis and are considered less urgent.
Selection of Specimens
• In urgent cases of suspected tumor or infection, a sample with the length mineralization
should be selected.
• For metabolic bone, the iliac crest trephine biopsies are used.
• If an X-ray of the specimen is available, a representative sample can be selected through a
"map" diagram of the lesion.
The main purpose of bone radiography are:
1. To examine the nature of a lesion.
2. To determine the extent of a lesion.
3. To provide a map of a lesion prior to selection for processing.
4. To check the progress of decalcification endpoint test.
5. To confirm the presence of prosthetic devices, metal or glass fragments implanted by trauma
Sawing of Specimens
• A good band saw is an essential equipment in a histopathology laboratory • An inexpensive,
lightweight hobby shop or handyman's bench hand saw is also useful
• Sawing should bent a slow even rate to match the speed of the blade cutting into the bone.
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• Pushing the bone produces uneven cuts, and may jam or break a blade.
• After sawing, clean the bone for any bone dust or debris adhering to slab surfaces using a
slow stream of water and soft brush.
• Care must be taken so as not to push debris into marrow spaces.
TYPES OF DECALCIFICATION
4 TYPES
-Acids
-Chelating agents
-Ion exchange resins
-Electrophoresis
ACIDS- most commonly used
• Nitric acid
ex. Aqueous Nitric Acid, Formol Nitric Acid
-Develop a yellow color due to formation of nitrous acid AID:
-5-10% most commonly used decalcifying agent
Examples: Perenyi's fluid
• 5% formic acid
-Best general decalcifying agent
- Recommended for small pieces of bones and teeth handle
• 1% HCI/ Von Ebner's fluid
-For urgent biopsies
-Recommended for teeth and small pieces of bones
-Inferior decalcifying agent
ACIDS
• 5% Tricholoroacetic acid
-For small spicule of delicate tissues
-Does not require washout
-also a fixative
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• Chromic acid/ flemming's fluid
-For minute bones
-Decalcifying and fixing agent genic
• Perenyi's fluid
-Decalcifying agent & tissue softener
• CHELATING AGENT
-Combine with Ca, Fe, Mg sults
-Recommended for detail microscopic studies
-Principle: binds with calcium
1. Cal-ex
2. EDTA
TEST FOR COMPLETE DECALCIFICATION
-to know if there is no calcium
• Physical/ mechanical
-Touch
-Pliability
-Resistance to fingernail
-Needing
• Radiological method or X-Ray method-most sensitive
-very expensive. Films used= fixatron, x-omat, Kodak film
• Chemical method (Calcium Oxalate Test).
-simple, reliable, recommended for routine purposes
-Clear fluid- completely decalcified, without calcium
-Cloudy-repeat decalcification, with calcium
• TISSUE SOFTENER for unduly hard tissues that may damage the microtome knives
1. Perenyi's fluid
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2. 4% aq. Phenol
3. Molliflex
4.2% HCI
5. 15% HCl in 70% alcohol
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