TECHNICAL - PROCEDURES & INSTRUCTIONS

Instructions for Using RDO:

Topics:

Multipurpose Decalcifier Usage
Vitro Diagnostic Use
Technical Endpoint Indicator
Decalcification Time Chart

RDO MULTIPURPOSE DECALCIFICATION TECHNIQUE

RDO is a remarkably effective multipurpose decalcifier that can be tailored to suit your specific lab routine. As with most acids, nuclei acids in the cell can become subject to ribonuclease digestion, resulting in a loss of basophilic properties. Careful monitoring should be used with RDO to avoid over decalcification. Most decalcification occurs in approximately 4-6 hours or less, depending on the thickness and density of specimens. Overnight decalcification should be avoided. Full Strength RDO is used for hard compact bone, ie. femur heads. RDO may be diluted with tap water to permit multipurpose use. Dilution with tap water is recommended for mildly calcified specimens such as bone marrow biopsies (core). In either case, the standard procedure should be to check the specimen every ½ -1 hour for mildly calcified specimens and every 1-2 hours for compact bone to determine the endpoint of decalcification.

Techniques for Optimal Decalcification with RDO

Frequent mild agitation or swirling of the specimen in solution will enhance even penetration and decrease the exposure time of the tissue to the acid solution. This will also minimize over decalcification of the outer tissue or bone before sufficient core decalcifying is achieved.

Dilute stock solution of RDO with tap water. Dilution factors will be dependent upon thickness and density of a specimen. Dilution will allow you to slow down the decalcification process to suit you specific lab routine.

To avoid over decalcification, check specimens at regular intervals for an endpoint. Every ½ - 1 hour for mildly calcified specimens and every 1-2 hours for compact bone.

Reducing temperature of the decalcifying solution to approximately 20 ° C will promote histochemical staining in procedures such as H & E, Masson’s, Van Gieson’s, and Azure-Eosin.

To remove sediment, RDO may be filtered, if desired, without altering its effectiveness.

To achieve optimal performance, do not reuse RDO. Since the nature of a decalcifying agent is to release calcium ions from the bone into the acid solution, as the solution becomes saturated with calcium ions, the decalcification process will slow down.

Addition of an alcoholic solution can aid in preventing undue swelling and hydrolysis of the tissue. It will, however, slow down the decalcifying agent. An 8:2 ratio of stock RDO (8) to 80% alcohol (2) can serve as a standard range for this method.

Rinse specimens thoroughly in running tap water after decalcification.

References:

A BRIEF ATLAS OF HISTOLOGY: Thomas & Robert Leeson, W.B. Saunder Co. Philadelphia/London/Toronto, 1979.

ATLAS OF NORMAL HISTOLOGY: Mariano S.H. DiFiore, Lea & Febrger, Philadelphia.
LABORATORY MANAGEMENT CONSULTANTS: S. Brown, St. Louis. 1995

MANUAL FOR HISTOLOGIC TECHNICIANS 3rd. EDITION: Ann Preece, Little, Brown & Co. Boston, 1959, 1969, 1972

THEORY AND PRACTICE OF HISTOTECHNOLOGY 2nd EDITION: Dezna Sheehan, Barabara Hrapchak, Battle Press / Columbus Richland.

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RDO IN VITRO DIAGNOSTIC USE

RDO is a very rapid bone decalcifier. The acid component of RDO reacts with calcium in mineralized tissues to form soluble calcium salts. The little extra care required in its use will result in routine sections with superb histological detail and staining characteristics in a fraction on the time required with other decalcifiers.

Tissues should be thoroughly fixed before decalcification. Most standard fixatives can be used prior to RDO use. To insure adequate fixation and decalcification, specimens should be trimmed to less than 1 cm thickness. FORMALIN FIXATION AND RDO DECALCIFICATION SHOULD NOT BE COMBINED. Hydrochloric acid (active ingredient of RDO) and formaldehyde vapors have been reported to form a potent carcinogen, bischloromethyl ether. Prior fixation with formalin is permissible. Brief washing in water before RDO decalcification is advised.
Do not use metallic equipment/cassettes for decalcification. RDO corrodes most metals after long periods of exposure. Decalcification tissues may be placed in metallic equipment after washing.

DO NOT OVER DECALCIFY. RDO action is very rapid. DO NOT leave bone specimen in RDO for several days as required with most commercial decalcifiers. MOST SPECIMENS CAN BE DECALCIFIED IN FOUR HOURS OR LESS. Use adequate volume of RDO to tissue; a 20:1 volume ration of RDO tissue is recommended. The key determinations for time required for decalcification are size and density of the specimen. Most mature bones of 1 cm size are decalcified in 4-6 hours; smaller cancellous bone in 2-4 hours. Bone biopsies are decalcified in 30-60 minutes. Teeth and entire femur heads may require overnight treatment. If RDO action is too rapid, dilute with distilled or deionized water. Good results have been obtained on bone marrow biopsies with a 3:1 dilution (RDO to water). Determine the point of decalcification using standard methods (ie. X-ray, flexibility, chemical analysis).

Proceed with routine processing and embedding. Washing tissue prior to processing is optional.

Overexposure to RDO can result in poor hematoxylin staining. If this occurs, satisfactory results can be obtained by treating deparafinizing slides to hematoxylin with aqueous saturated lithium carbonate (1-2 minutes) or 10% aqueous sodium bicarbonate (6-8 hours). Poor histological detail/artifacts (swelling, fragmentation) can also occur from excess decalcification. Hemosiderin is not removed by RDO.

STORAGE AND DISPOSAL

Store at room temperature. Keep container closed at all times. Store only in a glass or plastic container. Do not use metal containers as RDO will irreversibly corrode aluminum, nickel, and some stainless steel equipment.

After long periods of storage some change of color or an increase of suspended precipitate may occur. These are normal occurrences and do not affect the decalcifying potential of RDO. The precipitate may be allowed to settle or removed by filtration; however, neither action is necessary.

RDO is biodegradable as received and may be disposed of down regular city sewer systems with water flush. Dispose of according to federal, state, and local regulations. Care should be taken to insure the finish of chrome plated plumbing fixtures.

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DECALCIFICATION ENDPOINT INDICATOR TECHNICAL MEMO

COMPONENTS:
Solution A, Ammonium Hydroxide
Solution B, Ammonium Oxalate

APPLICATIONS:
This procedure will help avoid one of the more common causes of loss of specimen morphology: over decalcification. It helps you limit the process to just what is needed to maintain specimen quality at a higher level.
The following simple test is designed to tell you two things:
When your decalcification solution is exhausted and needs to be changed.
When your specimen is not decalcified enough.

METHOD:
Pipette 5 ml solution A into suitably sized flask or tube.
Add 5 ml of solution B
Add 5 ml of solution from the bottom of decalcification vessel. (Avoid picking up particulates in the pipette.)
Let stand 15 minutes.
If the test aliquot is clear, decalcification is completed and solution is still usable.
If cloudy, the solution is exhausted, indicated by the precipitated calcium oxalate, and decalcification is not complete. Change the fluid and proceed, testing frequently.

REFERENCES:

Luna, L.G.; Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts, 1st Edition, Johnson Printers, Maryland, 1992.

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DECAL TIME CHART

All tissues should be checked at least halfway through the time given.
12... = Check regularly for desired endpoint
Decalcification times prepared by Mary Parker, Durham, NC.