HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE

LESSON 3: DECALCIFICATION
MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD

OUTLINE B. UNIQUE CHARACTERISTICS

I. Decalcification
A. Characteristics of a Good Decalcifying ● Stable
Agent ● Easily available
B. Unique Characteristics ● Inexpensive
C. Factors Affecting Rate of Decalcification
● Easy to prepare
II. Four Types of Decalcifying Agents
A. Acid
B. Chelating Agents C. FACTORS AFFECTING
C. Ion Exchange Resin RATE OF DECALCIFICATION
D. Electrophoresis
Concentration
I. DECALCIFICATION ● Less concentrated = slow acting
Tissue-to-volume Ratio
Purpose: ● 1:20
● Removal of calcium and lime salts ○ 1 parts tissue and 20 parts decalcifying
● Done after fixation and before dehydration and agent
impregnation Temperature
● Calcium might interfere with accurate evaluation ● Must be well regulated
and examination Mechanical Agitation
○ Calcium should be removed in the tissue ● It is the key to a rapid acting or rapid decalcifying of
samples because it can potentially the sample
interfere with the accurate evaluation and Size and Consistency of Tissue Sample
examination of the metabolic bone ● Large, thick, rigid = longer decalcification process
disease, differentiate mineralized bone
from osteoid, and morphometric III. FOUR TYPES OF DECALCIFYING AGENTS
measurement of the tissue sample
Significance: A. ACID
● Facilitate normal cutting of tissue in sectioning
● Prevent obscuring microanatomical detail of tissue

Organs that require decalcification:

● Bones
○ Calcium is present in the bones
● Tuberculous Lungs
○ Calcified lungs due to TB bacteria
● Arteriosclerotic Vessels
○ Accumulated lipids, calcium, cellular debris
in the lumen of the blood vessels
Nitric Acid
● Teeth
● 5-10%
● Routine or most common decalcifying agent
In order to facilitate good cutting and evaluation of your
● Fastest decalcifying agent in the market now
tissue samples, they should undergo removal of calcium
● Imparts yellow coloration to the tissue sample
and lime salts first.
through nitrous acid formation
○ Remedy:
A. CHARACTERISTICS OF A GOOD DECALCIFYING
■ Add urea or Sodium
AGENT
Thiosulfate/sulfate
● Do not cause cell destruction ■ 70% ROH (DHD)
● Rapid, cheap, inexpensive ● ROH is an alkyl alcohol
○ Should also render best and accurate ● Variation/s:
result ○ 10% Aqueous Nitric Acid Solution
● Safe ○ Formol-Nitric Acid
● Readily available ○ Perenyi’s Fluid
■ Acts as tissue softener
■ Nitric acid + chromic acid + ROH

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 ○ Phloroglucinol-Nitric Acid Common brand:
■ Fastest agent (simple or ● Cal-Ex and Versene
compound) ○ Contains Na2EDTA
● Simple solution = 1
ingredient/component only C. ION EXCHANGE RESIN
● Compound solution = 2 or
more ingredients/
components
Hydrochloric Acid
● Provides good nuclear staining at 1%
● Slower and causes more distortion compared to
nitric acid
● Variation/s:
○ Von Ebner’s Fluid Principle:
■ For teeth and small bones ● Increase solubility
■ HCL + 36% NaCl + distilled water ○ Uses formic acid with TSE : Formic acid of
Formic Acid 1:20-30
● Good for routine decalcification of post-mortem ● Remove calcium ions from formic acid containing
research tissues, small pieces of bone and teeth, decalcifying solutions
ISH staining ● Not recommended for hydrochloric acid and nitric
● If the formic acid contains a large amount of nitric acid fluid containing mineral acids
acid, it produces better nuclear staining and less Decalcification Extent:
tissue distortion ● Can be measured using physical method by simply
● Variation/s: bending or poking the tissue sample and/or X-ray
○ Formic Acid method
○ Formic Acid-Sodium Citrate Solution
Trichloroacetic Acid D. ELECTROPHORESIS
● Best for small bone spicules
● Good nuclear staining
● Slow, weak
Chromic Acid (Flemming’s Fluid)
● Best for minute bone spicules
Sulfurous Acid
● Best for minute pieces of bone
● Weak
Citric Acid Citrate Buffered Solution
● pH: 4.5
Principle:
● Attracting calcium ions going to the cathode part of
B. CHELATING AGENTS
the agarose gel
Advantage:
● Time is shortened due to heat and electrolytic
reaction produced in the process
○ Shorter time is needed to remove calcium
ions or calcium salts
○ It utilizes 88% formic acid
Principle:
Procedure:
● Use of other salts to form complexes with Calcium
● Used for small bone fragments
salts for ease of removal
Decalcification Extent:
Procedure:
● Best Measured Using:
● Utilized in Immunohistochemistry and enzyme
○ Physical or Mechanical Test
staining with the help of electron microscope
○ X-ray or Radiologic Method
Duration:
○ Chemical Method
● 1-3 weeks for small tissue samples
● 6-8 weeks for longer and dense bones
pH:
● 7-7.4
○ Should be maintained at this pH in order to
work properly

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