Decalcification removal of _________ ions from a bone or calcified tissue through a histological process that makes them flexible and easier to cut
calcium
main object of decalcification in a surgical pathology laboratory
bones
A satisfactory fixative for bone
buffered formalin
- zinc formalin mixtures, B5, formol-acetic alcohol (Davidson’s fixative), or Bouin’s solution for bone marrow preservation
Done after fixation and before impregnation, to ensure and facilitate the normal cutting of sections and to prevent obscuring the microanatomic detail of such sections by bone dust and other cellular debris
Decalcification
True or False
- cartilage does not require any softening, except if some calcified areas are present because they are composed of insoluble keratin filaments
True
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
True or False
- nuclear and cytoplasmic detail are compromised if specimens are exposed for too long to acidic decalcifying agents, which can extract RNA and remove the purine and pyrimidine bases from DNA
True
Three main types of decalcifying agents
- Strong mineral acids
- Weak acids
- Chelating agents
Most common chelating agent
ethylene diamine tetra acetic acid (EDTA) salt
What metallic ions do EDTA binds?
calcium and magnesium
Used as an anticoagulant and water softener
EDTA
Decalcifying agent preferred for the preservation of nuclear DNA or if histochemical methods for nucleic acids or enzyme activities are intended
EDTA
True or False
- EDTA is faster than decalcification by acids and it is suitable for urgent specimens
False
- EDTA is longer than decalcification by acids (very slow but very gentle), not suitable for urgent specimens
Decalcifying agent that is more appropriate for research applications where very high-quality morphology is required or particular molecular elements must be preserved for techniques such as IHC, FISH or PCR
EDTA
Tissues decalcified in EDTA solutions should not be placed directly into _____________, because this will cause residual EDTA to precipitate in the alcohol and within the tissue
70% alcohol
Recommended ratio of fluid to tissue volume in decalcification
20 to 1
Recommended temperature during decalcification?
room temperature range of 18°C to 30°C
During decalcification, at what temperature where impaired nuclear staining of Van Gieson’s stain for collagen fiber happens?
37°C
During decalcification, at what temperature where tissue will undergo complete digestion within 24-48 hours?
55°C
This determination technique where the point at which all the calcium has been removed performs manipulation, bending, probing or trimming of the specimen to “feel” for remaining calcified areas
Physical tests
- - generally considered to be unreliable
- mechanical damage can occur during bending or probing and small deposits of calcium can easily be missed
- weighing the specimen after rinsing and blotting has also been described and an effective method for large specimens
True or False
- Physical test causes false positive microfractures leading to misdiagnosis
True
- pricking, slicing, bending or squeezing tissue can disrupt soft tumor from the bone or cause false positive microfractures of fine trabeculae, leading to a potential misdiagnosis
Where do you perform chemical test using litmus paper?
at the discarded decalcifying agent
Cloudiness in the discarded decalcifying agent indicates?
presence of cloudiness indicates that there is still calcium found in the solution; solution must remain clear for it to be completed
Chemical test is unsuitable for solutions containing over __% acid
over 10% acid
