Chapter 8 Flashcards
the removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen, and surrounding tissues of the bone.
bone decalcification
the removal of calcium ions from
a bone or calcified tissue through a histological process that makes them flexible and easier to cut
decalcification
Decalcification enables the histotechnologist to cut soft sections of the bone using the _______, so that they can be processed like any other soft tissue of the body
microtome
Decalcification is a lengthy procedure, as bone pieces have to be left in the decalcifying agent for
______ or _______, depending on the size of the tissue
several days or even weeks
thickness of specimen
2-3mm
a satisfactory fixative for bone
but where the preservation of bone marrow is important
buffered formalin
3 main types of decalcifying agents
strong mineral acids
weaker organic acids
chelating agents
the most widely used agents for routine decalcification of large amounts of bony tissues because they are stable, readily
available, and relatively inexpensive as compared to other decalcifying agents
acid decalcifying agents
the most rapid in action but if used longer than necessary will rapidly cause a loss of nuclear staining and can macerate tissues
strong acids (hydrochloric or nitric acid)
concentrations of strong acids (hydrochloric or nitric acid)
up to 10%
The most common and the fastest decalcifying agent used so far, utilized both as a simple solution
or combined with other reagents.
nitric acid
This may be used as simple aqueous solutions with recommended concentrations of 5-10%.
nitric acid
It is a very rapid decalcifying agent, producing minimal distortion and is, therefore, recommended for routine purposes
nitric acid
The disadvantages are inhibiting nuclear stains and destroying tissues, especially in concentrated solutions
nitric acid
Nitric acid should be prevented by not combining it with ______ or ______
formaldehyde or alcohol
FORMULA:
○ Concentrated nitric acid 10 mL
○ Distilled water added up to 100 mL
aqueous nitric acid solution (10%)
decalcification time of aqueous nitric acid solution (10%)
12-24 hours
ADVANTAGES:
- It is rapid in action
- It produces minimum distortion of tissues
- It produces good nuclear staining (although less than in slower acting agents)
- The acid may be easily removed by 70% alcohol
- It is recommended for urgent biopsy, and for needle and small biopsy specimens to permit rapid diagnosis within 24 hours or less
- It can be used for large or heavily mineralized cortical bone specimen if decalcification progress is carefully monitored by a decalcification endpoint test
aqueous nitric acid solution (10%)
DISADVANTAGES:
-Prolonged decalcification may lead to tissue distortion
-It can seriously damage tissue stainability
- It imparts a yellow color with nitrous acid, thereby impairing the staining reaction of the tissue
- Old nitric acid solution is particularly damaging and should be replaced with fresh stock solution
- Strong acids tend to be more damaging to tissue antigens for immunohistochemical staining, and enzymes may be totally lost
aqueous nitric acid solution (10%)
FORMULA:
○ Concentrated nitric acid 10 mL
○ Strong formaldehyde, 40% 5 mL
○ Distilled water 85 mL
formol-nitric acid
decalcification time for formol-nitric acid
1-3 days
Advantages:
- It is rapid-acting; hence, is recommended for urgent biopsies
- Nuclear staining is relatively good
- It produces less tissue destruction than 10% aqueous nitric acid
formol-nitric acid
DISADVANTAGE:
The yellow color imparted by nitrous acid formation will impair staining reaction of the cell.
formol-nitric acid
FORMULA:
○ Nitric acid 10% 40 mL
○ Chromic acid 0.5% 30 mL
○ Absolute ethyl alcohol 30 mL
perenyi’s fluid
Mix shortly before use. Chromic acid must be collected for proper disposal.
perenyi’s fluid
decalcification time for perenyi’s fluid
2-7 days
ADVANTAGES:
- It is recommended for routine purposes
- It decalcifies and softens tissues at the same time
- Nuclear and cytoplasmic staining is good
- Maceration is avoided due to the presence of chromic acid and alcohol
perenyi’s fluid
DISADVANTAGE:
It is a slow decalcifying agent for dense bones; hence, is not recommended for urgent diagnosis
perenyi’s fluid
FORMULA:
○ Concentrated nitric acid 10 mL
○ Phloroglucin 1g
○ Nitric acid 10% 100 mL
phloroglucin-nitric acid
(To be added after disappearance of dense white fumes formed by combining the first two ingredients.)
phloroglucin-nitric acid
decalcification time for phloroglucin-nitric acid
12-24 hours
ADVANTAGE:
It is the most rapid decalcifying agent so far, recommended for urgent cases
phloroglucin-nitric acid
DISADVANTAGES:
- Nuclear staining is poor
- Prolonged decalcification produces extreme tissue distortion
- Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours
phloroglucin-nitric acid
inferior compared to nitric acid in its role as a decalcifying agent because of its slower action and greater distortion of tissue
produced on the decalcified section.
hydrochloric acid (HCl)
hydrochloric acid (HCl) produces good nuclear staining and if used in _________, may be recommended for surface decalcification of the tissue blocks
1% solution with 70% alcohol
FORMULA:
○ Saturated aqueous solution of NaCl 50 mL
○ 36% concentrated hydrochloric acid 8 mL
○ Distilled water 50 mL
von ebner’s fluid
ADVANTAGES:
- It permits relatively good cytologic staining
- It is a moderately rapid decalcifying agent
- It does not require washing out before dehydration
- It is recommended for teeth and small pieces of bone
von ebner’s fluid
DISADVANTAGE:
The extent of decalcification cannot be measured by a chemical test
von ebner’s fluid
WEAK ACIDS such as ______ are popular and are widely used for decalcification
formic acid
Organic acids such as ______ and ______ are better suited to bone marrow, since they are not as harsh. However, they act more slowly on dense cortical bone
acetic and formic acid
Other acids such as ________ have also been used
trichloroacetic acid (TCA)
_______ and _______ are not used alone as decalcifying agents, but are found as components of Carnoy’s and Bouin’s fixatives.
Picric acid and acetic acid
These fixatives may act as incidental, albeit, weak decalcifiers, and can be used in urgent cases when there is only minimal calcification
carnoy’s and bouin’s fixatives
a moderate-acting decalcifying agent which produces better nuclear staining with less
tissue distortion, and is safer to handle than nitric acid or hydrochloric acid
formic acid
It is recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations
formic acid
Formic acid can be used as a ________ or combined with formalin or with a buffer
simple 10% aqueous solution
It is slower than the strong acid agents, but it is much gentler in action and less likely to interfere with nuclear staining
formic acid
______ in a 10% concentration is the best all-around decalcifier
formic acid
________ is the only weak acid used extensively as a primary decalcifying agent. Addition of citrate probably accelerates decalcification by chelating the calcium as it is liberated from the bone
formic acid
FORMULA:
○ Formic acid (Sp. grav. 1.20) 10 mL
○ Normal saline 10% 90 mL
formic acid
decalcification time for formic acid
2-7 days
It may be used both as a fixative and decalcifying agent
formic acid
It requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue
formic acid
FORMULA:
○ Aqueous sodium citrate 20% 50 mL
○ Formic acid 45% 50 mL
formic acid-sodium citrate solution
decalcification time for formic acid-sodium citrate solution
3-14 days
It is recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes
formic acid-sodium citrate solution
DISADVANTAGES:
- It is relatively slow; hence, is not recommended for routine purposes and for dense tissues
- It requires neutralization with 5% sodium sulfate
formic acid-sodium citrate solution
FORMULA:
○ Trichloroacetic acid 5 g
○ Formal saline 10% 95 mL
trichloroacetic acid
decalcification time for trichloroacetic acid
4-8 days
It does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration
trichloroacetic acid
DISADVANTAGES:
- It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone
- It is very slow-acting; hence, is not recommended for urgent examinations
trichloroacetic acid
a very weak decalcifying solution suitable only for minute pieces of bone
sulfurous acid
FORMULA:
○ Chromic acid % 15 mL
○ Osmium tetroxide 4 mL
○ 2% Glacial acetic acid 1 mL
chromic acid
environmental toxin and is highly corrosive to skin and mucous membrane
chromic acid
substances which combine with calcium ions and other salts (e.g. iron and magnesium deposits) to form weakly dissociated complexes and facilitate removal of calcium salt
chelating agents
The most common chelating agent in the market is __________, with the commercial name of Versene, recommended only for detailed microscopic studies
EDTA salt
