South Valley University
Name; Chama Kabaso
Student No. 201912010459
Module; CELLULAR PATHOLOGY
Assignment; 7
Question; Outline the advantages and
disavantages of any 4 decalcification methods
used in histopathology.
Lecturer; Mr Kabasiya Musipili
Due Date; 19th September 2020
Decalcification is the technique of removing calcium from bone or other calcified tissue. In
order to obtain satisfactory paraffin sections of bone, inorganic calcium must be removed from
the organic collagen matrix, calcified cartilage, and surrounding tissues. Calcified tissue must be
decalcified before processing or the tissue can be sectioned. Decalcification of bone is often an
essential and important step during tissue processing. This technique helps to remove calcium
salts from the tissue without affecting the morphology of the tissue and staining. Removal of
calcium salt can be done with the help of acid, chelating agents, ion-exchange resin and
electrolysis method. Any successful decalcification process is depends on the assessment of
consistency of the tissue, volume of the tissue, proper washing and fixation of tissue. In addition,
right selection of decalcifying agent and exact determination of the end point of decalcification
process are also important.
ACID DECALCIFICATION
Acid decalcifiers can be divided into two groups: strong (inorganic) eg e.g. nitric, hydrochloric
and weak (organic) acid. Eg formic, acetic, picric
ADVANTAGES
Acids quickly and easily dissolve calcium salts
DISADVANTAGES
Dedalcifier with higher concentration of acids acts rapidly and affects tissue staining the
most
Tissue left in acid too long will lose nuclear staining
When heat is used with strong acids during decalcification, swelling of tissue and
possible digestion of bone collagen will occur
When not rinsed in water prior to processing, acids will continue to decalcify tissue; will
also prevent possible chemical reactions with subsequent reagents and contamination of
processor reagents
CHELATING METHOD
Chelating agents are organic compounds that possess the property of binding certain metals
The chelating agent generally used for decalcification is ethylenediaminetetraacetic acid
(EDTA).
ADVANTAGES
The process does not damage tissues or their stainability
When time permits, EDTA is an excellent bone decalcifier for enzyme staining, and
electron microscopy
DISADVANTAGES
It is confined to small specimens and teeth
though pH 8 and above gives optimal binding, the higher pH may damage alkali-sensitive
protein linkages
It is a slow process
ION-ECHANGE METHOD
This method involves the use of formic acid over a layer of ammoniated salt of sulfonated resin.
Ammonium ions from the resin are exchange for calcium ions this keeps a solution free of
calcium and speed up the reaction
DISADVANTAGES
Extent of decalcification cannot be measured by chemicals method
ELECTROLYTIC METHOD
this method uses a mixture formic and hydrochloric acid placed in an apparatus. The bone is
attached to an anode and the current is passed through the solution. The calcium ion with their
associated positive charge are rapidly attracted from the anode and the current is passed through
the solution
ADVANTAGE
Most bone specimen can be calcified rapidly with this method usually within 2-6hours
DISADVANTAGES
Because of heat generated by is method there is strong potential for the total loss of
cellular detail and stain ability; hence rarely used
REFERENCES
1.Bancroft’s Theory and practice of Histological techniques 7 th Edition
2 Beltrami, C.A., Fabris, G., Marzola, A., et al., 1975. Staining of gastrin cells with lead
hematoxylin. Histochemical Journal 7, 95.
3.Carazzi, D., 1911. Eine neue Hämatoxylinlösung. Zeitschrift für wissenschaftliche
Mikroskopie und für mikrosko-pische Technik 28, 273.
4.Carson, F.L., 1997. Histotechnology: a selfinstructional text. American Society for Clinical
Pathology, Chicago, 6, 93
5.Cole, E.C., 1943. Studies in hematoxylin stains. Stain Technology 18, 125
6.Delafield, J., cited by Prudden, J.M., 1885. Zeitschrift für wissenschaftliche. Mikroskopie und
für mikroskopische Technik 2, 228.
7.Ehrlich, P., 1886. Fragekasten. Zeitschrift für wissenschaft-liche. Mikroskopie und für
mikroskopische Technik 3, 150.
8.Feldman, A., Dapson, R., 1985. Newsletter, Winter.ANATECH.
9.Feldman, A., Dapson, R., 1987. Newsletter, Winter.ANATECH.
