Decalcification, Microtomy and Embedding

Question 1

11. Mention two types of decalcification techniques

Answer 1

Acid decalcification: hydroxyapatite is the hard compound found in bones composed of calcium, phosphate and hydroxide. This is soluble in acidic environment and can be removed by hydrochloric acid, nitric acid or formic acid. The first two are strong acids and can lead to over decalcification and maceration. Therefore, formic acid is commonly used as its more gentle and preserves tissue architecture. It is used in ion-exchange resin which is formic acid over a layer of ammoniated salt of sulphonated resin, the resin absorbs the calcium and prevents it from being re-absorbed by the tissue.
Advantages of this method include well preserved cellular detail, faster decalcification, elimination of daily solution change and resin can be reused. Electrolyte method also uses formic acid but mixed with HCl in a glass jar, however it needs to be slightly warmed up and this increase in temperature may cause tissue destruction and is not commonly used.
Chelating agents: examples include EDTA which chelates calcium ions present in solution, this method is useful when bones need to be stained with different stains as maintains the best architecture, however, it is slower acting and requires several weeks at room temperature to achieve decalcification.

Question 2

How do you check for end-point decalcification

Answer 2

• Mechanical: Probing the specimen with a needle or pin and scraping the section surfaces. This is commonly used but it is inaccurate and can create histological artefacts and is also subjective.
• Chemical: this depends on precipitation of calcium oxalate, 5 mls of the used decalcifying fluid is made neutral to litmus paper with concentrated ammonium hydroxide. Another 5 mls of saturated ammonium oxalate solution is added to the decalcifying fluid. The resulting solution is mixed well and allowed to stand for 30 minutes.
Results: Turbidity – production of calcium oxalate; calcium still present.
No turbidity – decalcification complete.
The decalcifying fluid must be changed after each chemical check.
• Radiography: most accurate method of determining the completeness of decalcification. Visual evidence that demineralisation is complete. Cannot be used with metallic fixed tissue because the metal will render the specimen radiopaque (Refers to structures that are dense and resist the pas-sage of x-rays).

Question 3

13. What factors influence rate of decalcification?

Answer 3

• Temperature: the higher the temperature the faster the rate of decalcification but may also result in damage to tissues and increase in decaying rate.
• Method used
• Concentration: more concentrated solutions decalcify more rapidly but more harmful to the tissue.
• Volume of solution: volume of fluid to the volume of the tissue -20 to 1.
• Agitation: increase the exchange of fluids within as well as around tissues.
• Size: ideally sections should be 3mm thick for decalcification
• Solution: Aqueous solutions are used Alcoholic solution – aid in preventing undue swelling of the tissue inefficient decalcifying agent.

Question 4

14. How to orient the following specimen:
    a) Tubular structures
    b) Skin biopsies
    c) Intestine, gallbladder and other tissues
    d) multiple tissue pieces

Answer 4

a) Tubular structures, e.g. arteries, veins, fallopian tube and vas deferens: cross sections of the wall and lumen should be visible.
b) Skin biopsies: embed shave biopsies on edge and punch biopsies on their side so that all the epi-dermis, dermis and hypodermis are visible
c) Intestine, gallbladder and other epithelial tissue: orient so the epithelial surface is cut last, mini-mizing compression and distortion of this layer.
d) Multiple tissue pieces: place side by side with the epithelial surfaces facing in the same direction.
e) Tissues with a wall such as cysts are embedded on edge.

Question 5

15. What is the routine maintenance on microtomes?

Answer 5

• Cleaning the microtome to remove any debris.
• Lubricating on a routine schedule as recommended by the manufacturer.
• The maintenance done should be documented accordingly.

Question 6

1.	Troubleshooting of microtomy:
•	Soft sections 
•	Incomplete sections 
•	Curved ribbons 
•	Chattering 
•	Excessive compression 
•	Sections will not form ribbons 
•	Lengthwise scratches or splits

Answer 6

• Soft sections – insufficient dehydration or clearing
• Incomplete sections – incomplete impregnation of the tissue with paraffin wax, incorrectly em-bedded tissue, sections cut superficially, aggressive trimming.
• Curved ribbons – the block edges are not on equal level, the blade is blunt, excessive paraffin wax and tissue varies in consistency due to improper orientation of the block.
• Chattering - when the section has thick and thin zones parallel to blade edge, blade or block are poorly secured in placed, these should be tightened to avoid any needless movements, very steep clearance angle or calcified areas in tissue can also cause chattering – reduce angle and for decal-cifying the block should be rehydrate by placing in 5% HCL solution, over-dehydration, and the block would need to be placed in xylene, alcohol and water and start the process again to re-wax the specimen.
• Excessive compression – dull blade, paraffin wax too soft for the tissue, high clearance angle
• Sections will not form ribbons - paraffin block may be too hard for sectioning and so the tissue should be heated and re-embedded in lower melting point paraffin, debris on the knife edge or the clearance angle may be too high or too little and it should be adjusted to the optimal angle.
• Lengthwise scratches or splits – defect in the knife edge or a hard particle in the block.