Decalcifying Solution, Formic Acid 5%, Aqueous

SOLUTION:

Additionally Needed:

For storage requirements and expiration date refer to individual bottle labels.

APPLICATION:  

Newcomer Supply Decalcifying Solution, Formic Acid, 5% Aqueous, provides a moderate rate of decalcification while maintaining excellent cellular morphology.  This solution is a general purpose decalcifier and is suitable for all bone specimen types from sternal or iliac crest bone marrow biopsies (light bone) to femoral head and long bone sections (compact bone).

METHOD: 

Fixation:  Formalin 10% Phosphate Buffered (Part 1090)

1. See Procedure Note #1.

Technique:  Paraffin sections cut at 5 microns on adhesive slides
Solutions: All solutions are manufactured by Newcomer Supply, Inc.

PROCEDURE: 

1. Fix bone specimen in fixative of choice, for a length of time sufficient for specimen size and type.

1. See Procedure Notes #2 and #3.

2. Wash fixed specimen in running tap water for 10 minutes.
3. Submerge fixed bone segment(s) in container of Decalcifying Solution, Formic Acid 5%, Aqueous that adequately covers the specimen. A 20:1 ratio is recommended.

1. See Procedure Notes #4 and #5.

4. Check the specimen regularly for adequate solution coverage during the decalcification process for optimal decalcifying reaction.  Decalcification time will vary and is dependent on size and weight of bone.
   1. Check light bone samples every 30 to 60 minutes; check compact bone samples every 1 to 2 hours.
   2. Bone marrow or light bone biopsies, on the average, will decalcify in 4 to 6 hours.
   3. A 3 mm thick section of femoral head, on the average, will decalcify in 8 to 24 hours.
5. Check completion of decalcification with Decalcification End Point Set (Part 1051) regularly to deter over-decalcification and loss of cellular morphology.

1. See Procedure Note #6.

6. Wash the specimen in running tap water when decalcification is judged to be complete.  Suggested time for small samples is 30-60 minutes; larger bones 1-4 hours or according to laboratory protocol established times.

1. Additional trimming of decalcified bone can occur at this stage to a size and thickness suitable for tissue processing.

7. Proceed with laboratory tissue processing procedure for bone specimens.
8. Trim block(s) and section the processed, paraffin embedded bone; if block trimming or sectioning is impaired due to bone hardness, surface decalcification is recommended.
9. Perform surface decalcification by soaking the paraffin block with exposed tissue surface side down in Decalcifying Solution, Formic Acid 5%, Aqueous for 15-60 minutes. Rinse block thoroughly with distilled water to remove corrosive acids and re-section.

1. See Procedure Note #7.

PROCEDURE NOTES:

1. Other fixatives that provide satisfactory results for bone specimens are:  AZF Fixative (Part 1009), B-5 Fixative Modified, Zinc Chloride (Part 1015), Bouin Fluid (Part 1020), Zamboni Fixative (Part 1459) and Zinc Formalin Fixative (Part 1482).
2. If possible, reduce the overall size of a larger bone specimen by bisecting or cutting the bone into smaller pieces and remove any excess attached soft tissue or skin for faster fixation.  A maximum bone thickness of 3-5 mm is recommended.
3. To ensure optimal staining results, adequate fixation of bone is essential before exposing specimen to decalcification solution.
4. Decalcification solution should be in contact with all specimen surfaces.  If multiple pieces are in one container, ensure that pieces are separated and/or suspended and not in direct contact or stacked on top of each other.  Change the solution at least daily and never add to or mix fresh solution with old.
5. Decalcification can be enhanced with the use of low speed agitation with either a stir bar/stir plate or rotator/shaker.
6. Decalcification end-point testing can also be accomplished through specimen radiography. Physical testing (probing or bending) of the bone is not recommended.
7. Surface decalcification removes only a thin layer of residual calcium from the tissue block surface.  This will allow only a few calcium-free sections to be obtained. Repeating the surface decalcification process for additional sections may be required.

REFERENCES:

1. Bancroft, John D., and Marilyn Gamble. Theory and Practice of Histological Techniques. 6th ed. Oxford: Churchill Livingstone Elsevier, 2008. 338-343.
2. Luna, Lee G. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York: Blakiston Division, McGraw-Hill, 1968. 6-11.
3. Urban, Ken. “Routine Decalcification of Bone.” Laboratory Medicine 12.4 (1981): 207-212.
4. Villanueva, Anthony. “Experimental Studies in Demineralization and Its Effects on Cytology and Staining of Bone Marrow Cells.” The Journal of Histotechnology 9.3 (1986): 155-161.
5. Modifications developed by Newcomer Supply Laboratory.