TECHNICAL - AVAILABLE REPRINTS

Emory University Hospital Histology Manual:

Decalcification Using RDO
Processing Bone Marrow Biopsies for Hematopathology

Microtime Georgia State Newsletter:

Method to Facilitate Sectioning Incompletely Decalcified Bone

Parafin and Plastic Techniques:

Preparation of Bone Marrow Specimens

H.I.S.T.O. Iowa State Newsletter:

Bone Marrow Biopsies

Animal Tissue Techniques:

Decalcification

DECALCIFICATION USING RDO

Adapted from the Standard Operating Procedures manual of the Histology Laboratory Department of Anatomic Pathology at EMORY UNIVERSITY HOSPITAL, Atlanta, Ga.

Principle:

Before any bone or other calcified tissue can be processed and sectioned conventionally, the calcium must be removed. This process is called "decalcification". Failure to decalcify results in torn, ragged sections and damage to the cutting edge of the microtome knife. The principle underlying the action of acid decalcifying agents involves the solubility of metallic salts. Calcium in bones is mainly carbonate and phosphate salts, which are only slightly soluble in water. An acid acts to release calcium from its combination with these anions and effects an ion exchange to give a soluble calcium salt. The calcium ions are effectively removed from the bone and remain in the decalcification solution.

Reagent:

RDO Rapid Decalcifier (Apex Engineering Products Corporation)

Precautions:

Wear gloves and protective clothing during decalcification procedures
Perform procedure in a well ventilated area

Take caution not to spill on stainless or Formica counters

DO NOT MIX RDO WITH SOLUTIONS CONTAINING FORMALDEHYDE. POTENTIAL FOR THE RELEASE OF TOXIC GAS EXISTS.

Procedure:

Specimens should be well fixed prior to RDO decalcification.

Specimens should be trimmed to less than 1/8 inch thickness for efficient, expedient, and complete decalcification.

Wash specimens thoroughly in tap water to remove as much fixative as possible.
Place specimens in RDO and check periodically for complete decalcification by bending to check flexibility.

After decalcification, rinse specimen to remove excess RDO.

Process specimens with routine workload.

Disposal:

RDO as a whole is considered a non-hazardous and biodegradable material and may be disposed of down regular city sewers with a water flush according to federal, state, and local regulations.

References:

Sheehan, D., Hrapchak, B., "Theory and Practice of Histotechnology, 2nd Ed. ", Mosby, St. Louis, pp. 89-96, 1980
Apex Engineering Products Supplemental Instruction Sheet

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PROCESSING BONE MARROW BIOPSIES FOR HEMATOPATHOLOGY

Adapted from the Standard Operating Procedures of the Histology Laboratory Department of Anatomic Pathology at EMORY UNIVERSITY HOSPITAL, Atlanta, GA

PRINCIPLE:
When architectural features of both bone and marrow are important in diagnosis, aspirated bone marrow is usually obtained with sternal puncture needle. Smears are made and the remainder is processed as a biopsy specimen.

PRECAUTIONS:
Wear gloves and protective clothing during decalcification procedures.

Perform procedures under a fume hood.

DO NOT MIX FORMHALDEHYE WITH RDO. POISONOUS GAS IS LIBERATED.

PROCEDURE:
Bone marrow biopsies are collected by the Hematology Laboratory Staff and placed immediately into B5 fixative (to which 1.0ml of formaldehyde is added just prior to use). The time of collection is recorded on the specimen container and at the top of the Pathology requisition. The specimen is assigned an accession number, which is recorded on the requisition and the specimen container.

The following steps are performed by the histotech:

The histotech assigned to bone marrows will check the gross room throughout the day for arrival of any bone marrow specimens.
Fixation time in B5 is 1-2 hours.
Fill out a gross sheet for bone marrow listing:
Surgical number
The patient’s name
Record "bone marrow biopsy" in the TYPE OF TISSUE column
Record "BM" number in CASSETTE IDENTIFICATION column
Record the number of pieces of bone marrow in CASSETTE IDENTIFICATION column
Record the location code in the LOCATION column
After fixation, wash in tap water for 2-3 minutes (decant used B5 into labeled water container).
Decalcify specimen in RDO solution by pouring filtered RDO (approx. 20cc) into specimen container.
Decal time is 20 to 40 minutes.
Note: Some biopsies require less time than others, depending on the amount of bone present; check biopsies after 20 minutes.

Biopsy is checked for decalcification by bending to check flexibility.
Place specimen into blue cassette labeled with both surgical and bone marrow numbers (decant used RDO into proper drain with water flush).
Wash in running tap water for 1 hour.
Place in 10% buffered fomalin until ready to load on processor.
The evening histotech will load specimens onto processor.
Note: Some biopsies may arrive too late to complete this procedure in entirety. Store in 10% buffered fomalin overnight to be completed the following day.

Bone marrow biopsy sections must be air-dried completely before heat-affixation at 60 degrees C oven for 40 minutes.

REFERENCES:
Beard, C., et al, "ACHIEVING TECHNICAL EXCELLENCE IN LYMPH NODE SPECIMENS: AN UPDATE", LABORATORY MEDICINE, VOL 16, No. 8, Aug. 1985, 468-475

Beard, C., Bowling, M., "TECHNICAL FACTORS IN EVALUATION OF LYMPH NODE BIOPSIES", Laboratory of Pathology, National Cancer Institute, National Institute of Health, Bethesda, Md.

Santoianni, R., Hammami, A., "NUCLEAR BUBBLING: AN OVERLOOKED ARTIFACT", JOURNAL OF HISTOTECHNOLOGY, Vol. 13, No. 2, June 1990, 135-136

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METHOD TO FACILITATE SECTIONING INCOMPLETELY DECALCIFIED BONE

Bone biopsies are sometimes difficult to obtain and uncomfortable for the patient. Therefore, incomplete decalcification before processing can create problems if the bone sections cannot be achieved without loss of tissue, and require re-decalcification, which takes precious time.

Ideally, the specimen should not be processed until decalcification is complete but as we know, many times the histologist is rushed by the pathologist or clinician to produce section in an inadequate period of time. Sections of bone that have been hurriedly processed and fail to section because of incomplete decalcification may be placed in a decal solution for a short period of time (this will vary with the density and size of the bone), washed in ammonia water, rechilled, and sectioned in the same day without having to remove from the block and reprocess. Small pieces of bone such as bone marrow biopsies take only a short soak of about 30 minutes and will section easily. Larger pieces also will section but take a longer soak. The depth of effectiveness is minimal and if levels are needed, the block may have to be reintroduced to the decal solution more than once.

RDO is one of the decal solutions, which produce the fastest results. There are other rapid decalcifiers that are on the market and may work as well. This is a solution to an uncontrollable problem, but the best method is to do it right the first time. Necessity is the mother of invention but you cannot beat "the right way, baby." Most assuredly the patient will this, "uh huh!"

Editor's Note: Given a Pathology Residency Program at Emory University Hospital, we have specimens submitted for processing which, at times, should have been submitted for decalcification. After we've "crunched" through and faced the block, and have determined that reprocessing and redecalcification may not be necessary, we will soak the block on plain ice if the specimen is not too hard or in a standard formic acid/formaldehyde decal solution or RDO if more hard treatment is necessary. We have had success in acquiring good quality sections and stains even without an ammonia rinse.

Not only is this a helpful technical tip from Shirley, but it shows how far a little "TLC" during sectioning can go... to separate the histotech from the histohack.

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PARAFFIN AND PLASTIC TECHNIQUES FOR PREPARATION OF BONE MARROW SPECIMENS

Aspirated Marrow Units

1. On the floor, about 5ml of marrow aspirate is placed into a lavender top B-D vacutainer containing 0.07ml 15% EDTA and mix the contents quickly and thoroughly.
   
2. After arriving in the laboratory, the contents of the B-D tube are then filtered through either templefiber or Histowrap recovering only units.
   
3. The units are wrapped in the templefiber or Histowrap and placed in a bottle containing working B-5 fixative. The units are fixed for 1 hour.
   
4. The wrapped units are placed in a plastic processing cassette (do not use metal) and stored in 70% ethyl alcohol until processing.
   

Needle Biopsy of the Marrow

1. On the floor, the biopsy material in placed in a bottle containing working B-5 fixative.
   
2. After arriving in the laboratory, the biopsy is fixed for a minimum of 30 minutes; 1 hour preferable.
   
3. After fixation, rinse the biopsy in tap water.
   
4. The specimen is placed in RDO decalcifying solution for 15-30 minutes.
   
5. When decalcification is complete, the specimen is wrapped in lens paper, placed in a plastic processing cassette (no metal) and washed in running tap water for 30 minutes.
   
6. Process as usual.
   

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BONE MARROW BIOPSIES
By: Becky Scholes, HTL, MT (ASCP)

H.I.S.T.O.
The Official Newsletter of the Iowa Society for Histotechnology
Tec Tips

I acquired this decalcification procedure after the pathologists complained of poor detail with nitric acid and various commercial products. Dr. Mahoney must like it - he kissed me on the cheek! Thanks to Jan Graham in Ft. Dodge for the procedure.

Aspirate:

1. Allow aspirate to clot in petri dish.
2. Transfer clot to specimen container filled with 10% buffered formalin.
3. Process as usual.
   

Bone Core Fixation:

1. Immediately fix acetic formalin for a minimum of one hour.
2. Fix overnight if brought in later than 2 hours end of the workday.
3. Decalcify first thing in the morning.
   

Bone Core Decalcification:

1. Place fixed core and label in a plastic cassette (RDO discolors metal). Decalcify in dilute RDO for 1 hour. Do NOT leave overnight.
2. Test for decalcification by gently checking for pliability.
3. Rinse in running tap water for approximately 2 minutes.
   

Processing:

1. If the biopsy came in afternoon, process with rest of specimens.
2. If the biopsy is decalcified in the morning, hand process in 120cc plastic specimen cups, 15 minutes in each container, starting with 70% ethanol or speed process through processor, 15 minutes each station with heat and vacuum, starting with the first dehydrant station.
   

Microtoming:

1. Cut both aspirate and bone cores at 2 microns.
2. Place 3 levels on one slide. Cut an extra slide of the last level and set aside on the back of the water bath in case a special stain is requested.
   

Staining:

1. Hematoxylin approximately 5 minutes. Bone core 1 minute (Check under microscope before counterstaining).
2. Eosin for 10 dips, dehydrate, clear, and coverslip.
   

Discussion:
Over decalcification will result in poor or indifferent histological detail and staining characteristics. Less than one hour is usually not sufficient.

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ANIMAL TISSUE TECHNIQUES
Fourth Edition Gretchen L. Humason
Decalcification

Calcium deposits may be so heavily concentrated in the tissue that they may interfere with sectioning and result in torn sections and nicks on the knife-edge. If deposits are sparse, overnight soaking of blocked tissue in water will soften the deposits sufficiently for sections. Heavy deposits may be removed by any of several methods, but do not leave tissue in any of the fluids longer than necessary.

If any doubt arises about the completion of decalcification, check for calcium by the following method:

To 5ml of the solution containing the tissue, add 1ml of 5% sodium or ammonium oxalate. Allow standing for 5 minutes. If precipitate forms, decalcification is not complete. A clear solution indicates it is complete. Sticking needles in the tissue to check hardness is a sloppy technique that can damage cells.

An excellent decalcifying fluid, RDO, can be purchased by the gallon. After using RDO for several years, I recommend it as superior to other solutions. Its rapidity of action is remarkable and quality of staining and histological detail following its use is excellent. Old bones cut down to 1 cm in thickness, if possible, require a 6-hour treatment; small and young pieces only 1-2 hours. Teeth will require overnight and up to 18-24 hours. Do not over decalcify; this detracts from the staining quality. Decalcifying may be followed by brief washing in water, but this is not necessary. Fixation and decalcification may be combined in a mixture of 1 part undiluted formalin with 9 parts RDO.

Manufacture's Note

The combination of RDO and formalin is discouraged, but should always be done under a fume hood to ensure the removal of potentially harmful vapors. Always follow the suggested directions for use. Please contact Karen Kohout at APEX ENGINEERING PRODUCTS with any questions you may have about the use of RDO.

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