Decalcification in Surgical Pathology

Introduction

Decalcification is a routine procedure with the purpose of making a calcified tissue compatible with the embedding media for cutting a micro slide and the subsequent staining. The most ubiquitous media is paraffin wax. Decalcification adjusts the hard substance of bones to the softness of paraffin. Other media, as resins, do not require decalcification at all.

Bones are the main object of decalcification in a surgical pathology laboratory, but other specimens, such as calcified tumors, calcified heart valves, and atheroma plagues require this procedure. The variety of calcified specimens in surgical pathology requires nonstandard approaches to the process.

For more than one hundred years of using decalcification, many manuals and numerous articles have been devoted to decalcification methodology.1, 4,7 The most comprehensive material on decalcifying techniques, which includes comparison old methods with current ones, is in The Journal of Histotechnology.3This review evaluates the quality of histological presentation (cellular damage and interference with staining) after the use of different decalcifying agents. Although this article was written more than ten years ago, there is very little change since then in methodology, limited to some additional decalcification solutions and minimal advances in microwave-assisted decalcification.6

The literature about decalcification is enormous. The focus of most reviews and studies, however, has been on the histotechnological aspect living outside the specifics of the procedure in the surgical pathology laboratory, as well as the other components of specimen processing that precede decalcification. There are particularities that are determined by a variety of the pathology material. This review tries to fill this gap by examining how the principles of decalcification can be applied to the realities of surgical pathology practice. The article incorporates also my personal experience.

WHO is responsible for decalcification in the surgical pathology laboratory?

Traditionally, histotechs are in charge of decalcification, for understandable reasons. They are most interested in smooth microtomy; they are also on the receiving end of pathologists’ complains about defects in staining; they can make a proper choice of decalcification mode for immunochemistry (IHC) antigenicity and enzyme histochemistry preservation. This is correct in research, but the situation of specimen workflow in a modern surgical pathology laboratory, especially in an academic facility, is different.

The variety of clinical situations in a surgical pathology laboratory generates diverse pathological material. Different specimens arrive, sometimes simultaneously, requiring triage of processing. The pressure of turnaround time (TAT) should also be taken into account. In my experience, the grossing person should be in charge of decalcification with complete responsibility for the outcome, at least in large surgical pathology laboratories with a representative volume of calcified specimens. Bones and other calcified specimens are different, but histotechs do not have complete information about the specimen. The grossing person has it from the requisition form and hands on knowledge of the specimen’s specifics. For example, needle bone biopsies, including bone marrow cores, require differentiated handling in fixation and decalcification. Femoral heads with clinical diagnoses of degenerative arthritis and avascular necrosis need different mode of decalcification. The grossing person has more working space and time flexibility to monitor the decalcification process. Of course, the grossing person should be knowledgeable in histotechnology. Co-operation between the grossing person, histotechnologists, and pathologists is essential.

WHAT does precede decalcification?

Fixation must precede decalcification, without exception. A poorly fixed bone cannot be protected from the deleterious effects of decalcification reagents. Fixation is intended to maintain more or less the cellular integrity of the tissue. Calcium and other minerals are not stones in the field that just have to be collected and removed. Minerals are incorporated in the bone tissue. By removing them we inevitably destroy to some extent the tissues structure (osteoid matrix template) that should be protected by fixation. In a calcified heart valve or a calcified lymph node calcium is a “guest.” The tissue/organ structure is maintained during decalcification, but cellular structure is vulnerable.

There are two formulas in grossing processing of calcified specimens: fixation– section- decalcification and section-fixation-decalcification (cut, fix, decalcify). The second formula is the most widely accepted, although numerous deviations occur from this protocol. Unless some safety considerations, for example of AIDS or TB, dictate preliminary fixation, bone cutting in the fresh state assures better bone/soft tissue relationships, provides even fixation, and decreases turnaround time.6 There are, of course, exceptions, for example in the case of a metallic prosthesis that is cemented in the extremity. There is no way to cut the bone properly without decalcification after preliminary fixation. Grossing a long bone specimen with osteosarcoma after chemotherapy can be mentioned as another exception.

The uniformity of the section’s thickness is always desirable, but it becomes a serious requirement with implementation of microwave- assisted processing. It is unreasonable to make a section too thin (less than 3 mm). Uniform 3-4-5 mm sections are optimal for decalcification with or without microwave- accelerated processing. There are many technical details pertaining to how to get a uniform representative bone section.5

For large bone specimens, the old “golden”(though unwarranted) standard ratio of fixative/tissue of 20: 1 is not achievable and is in any case unnecessary if a modern tissue processor is used, as is the case in the vast majority of facilities. Rivers of formalin stream through laboratories, requiring time consuming and expensive safety measures. However, a femoral head can be in formalin for a month, remaining completely unfixed (red) in the center, independently of fixative/bone ratio. This is also a justification of the cut, fix, decalcify formula for grossing bones in surgical pathology.

HOW is decalcification carried out?

Methods of decalcification

Decalcification is a chemical process. The goal is to extract calcium and other minerals. By essence, the procedure is demineralization of the specimen. Calcium in bones, as well as in pathologic calcification, is in carbonate and phosphate salts together with smaller amounts of other mineral salts, which are almost insoluble. The principle of decalcification is simple: strong mineral acids, such as 10% hydrogen chloride (HCl), or weak organic acids, such as 5-10% formic acid (HCOOH), form soluble calcium salts in an ion exchange that moves calcium in the decalcification solution. The same final effect makes 14% ethylene diaminatetraacetic acid-EDTA (C10H12N2O8· 2Na)-that sequesters metallic ions, including calcium, in aqueous solutions as a chelating factor.

Acid solutions and chelating agents are the most popular, although there are other ways of decalcification, including electrolytic ionization and the use of ion-exchange resins. Despite the physical methods having definite advantages, especially in the speed of processing, the general surgical pathology practice does not use them, for many reasons. They require closer monitoring, cleaning and maintenance of the equipment involved. Surgical pathology practice requires simple, easily manageable, and, yes, cheap methods of decalcification.

Most surgical pathology laboratories use commercial decalcification solutions. The variety of brands that is offered by manufacturers brings only an illusion of choice, because they are variations of the same basic components with more or less disclosed recipes. For example, BBC Biochemical keeps the composition of the decalcifier RapidCal· Immuno™ undisclosed.

Although routine staining can be completely satisfactory after 5% nitric acid, this reagent is not appropriate owing to the fast speed at which it works making it difficult to monitor. EDTA, with its gradual “biting” calcium ions is too slow. For example, the human temporal bone decalcification takes 4 to 7 month (!) that in microwave can be reduced to 3-6 weeks, but this practice is not, of course, the surgical pathology realm.

There are three main groups of commercially available decalcification solution:

Strong acid or weak      acids

  1. Strong  or weak acids with EDTA
  2. Fixative +      decalcifier  in one solution

Table 1 presents the principle of choice a decalcification solution in bone gross processing. It summarizes the general approach to chemical decalcification using examples of commercially available solutions, without endorsing any of them, because this requires comparative studies under similar conditions. The mentioned solutions or brands do not reflect the variety of offers by manufacturers and, of course, recipes in manuals.

Table 1. Bone decalcification solutions in surgical pathology

“+” optimal, “+/-“satisfactory, “-“ negative result

 

Type of bone specimen Strong acid(hydrochloric acid like RDO Strong acid +EDTA like Surgipath Decalcifier II® Weak acid(Formic acid)Like Immunocal™ Weak acid +EDTA like Formical-2000™
Compact (cortical)bone

+

+/-

Spongy (cancellous)bone

+/-

+

+/-

+/-

Bone needle biopsy

+/-

+

+

If it comes to a choice in cases of pathological calcium deposits in a heart valve or in a lymph node, semiconductor- grade hydrochloric acid with EDTA, for example in Decal® or Surgipath Decalcifier II®, provides mild decalcification. This decalcifier is 3-4 times slower than the RDO type solution.

By the way, at NSH Mark Ostermeier told at the awards banquet that RDO stood for the names of his parents, Donald and Rosemary Ostermeier who were the inventors of the product that was originally designed to clean pipes.

A combination of a fixative (for instance, formaldehyde) with a decalcifier (for instance, formic acid and EDTA), such as Formical-4™, is intentionally omitted in the table. In combined formalin/ acid decalcifier solutions, both penetrate fast. Formalin’s penetration is 12 times faster than binding, which in turn is about four times faster than cross-linking.2 This means that acid is starting its work before formalin fixation is complete. Decalcification with fixation is an option, although it should be used with some reservations. It will not be simultaneous because the penetration coefficient of acids is always higher than that of fixatives. Needle biopsies are vary greatly by consistency of the bone and subjects are often fragile, such that any rush with decalcification before complete fixation is unreasonable. Bone marrow biopsies usually arrive in the laboratory at different points in the fast fixative (Zn formalin) process, which necessitates time monitoring of fixation. It is unwise to impose on them an additional fixation uncertainty by using a combination of fixative and decalcifier.

EDTA’s ubiquity, from industry to medicine (chelating therapy in mercury and lead poison, Versenate for atherosclerosis, etc.) makes it welcomed with some justification as a “magic” decalcifier. Below are discussed some EDTA issues in order to underscore details important for decalcification in a surgical pathology laboratory.

EDTA does not work in formic acid with pH 3 as a decalcifier. The best chelating ability is at a pH higher than 8 when the tissue maceration starts. The optimal pH is 7-7.6, so it is the necessity to maintain this narrow window. In decalcification, acids provide ions, but EDTA is responsible for their distribution to the “customer”, that is the saturated calcium elimination solution. As in a good production company, all departments should work in concert, without bottle- neck areas. In combined solutions, for example formic acid + EDTA, such an area is EDTA’s behavior. EDTA works too slowly under pH 5, owing to insolubility, but after pH 8 the solution becomes tissue unfriendly, due to alkaline sensitive protein bonds. Loaded with undistributed calcium, the solution can precipitate at the bone surface, which requires more intensive agitation and more intensive post- decalcification rinsing. In short, a buffered acid decalcifier with EDTA comes with a tag of more careful monitoring and, yes, with an additional price. This dive in EDTA’s physical chemistry is detailed only to show that this in principle excellent decalcifier requires careful handling. This also cast doubts about the rationality of mixing acids with EDTA.

As opposed to research with enzyme histochemistry, immunohistochemistry, in-situ-hybridization, electron microscopy, and other methods, the practical surgical pathology use of these methods is relatively limited. In general, three groups of decalcification solution mentioned above completely satisfy the surgical pathology practice. The overall immunophenotypic and morphologic results are acceptable for the vast majority of diagnoses. The emphasis on IHC in surgical pathology determines the choice of decalcification method. The faster calcium is removed by strong acids, the less damage is to tissue antigenicity, with the exception of immunoglobulin. The tissue structure is maintained better with a carefully monitored, slower decalcification. Histotechnologists know from experience that, if routine staining is bad, then IHC will also be impaired.3 However, only EDTA decalcification completely satisfies the requirements for an in- situ hybridization (ISG) procedure. The same can be said about enzyme histochemistry. The Ethylenediaminetetraaxetic Acid (14%) reagent, which is offered by Decal®, should be reserved for these occasions. EDTA works slowly, but it is the best for these studies.

By the way, with EDTA we do not have problems with pale nuclei H&E staining because “over-decalcification” is practically impossible, normal hematoxylin nuclei and  no eosin over- staining.

In fact, even two decalcifiers can satisfy the basic requirements of the general surgical pathology practice: strong acid, such as hydrochloride and weak acid, such as formic acid. The artisan art is in managing them properly. As in internal medicine, the physician develops personal experience in the administration of limited brands of medications, with some individualization of their use.

Microwave decalcification

The ubiquitous microwave oven is a multipurpose instrument in the histology laboratory. Microwave acceleration of the decalcification process is desirable. The manufacturers offer some options, from stand- alone portable device (TissueWave™ 2 Thermo Scientic®) to a special program in general microwave processor, such as Milestone’s PATHOS.

With speed, the problem comes of time and temperature monitoring; however, this is solvable by using different programs. Employment of this methodology requires triage of calcified materials and specific grossing technique for uniformity of section. Facilities with high volume of calcified material can benefit from the use of microwave assisted decalcification. It seems reasonable to equip a specialized Bone Grossing Table, like the one offered by TBJ. Inc. with a portable microwave decalcification instrument for effective monitoring, as well as for chemical safety considerations. It seems that this is the future of decalcification procedure before it will be replaced by advances in histotechnology techniques.

“Light decalcification”

Pathologists sometimes use the confusing firm, “light decalcification”, to warn the grossing person or histotechnologists, that the specimen should not be overexposed to decalcification solution, in order to prevent cellular or IHC staining damage. In practice, light decalcification means the necessity of more careful monitoring or employment of a weak organic mineral acid, such as formic acid, which does not prevent overexposure in any case. “Over- decalcification” is the common term for overexposure, although this is not accurate, because you cannot take out more calcium than exists in the tissue. Pathologist should be relieved from everyday involvement in such technical procedure as decalcification. The grossing person or histotechnologist ought to have enough information and be sufficiently qualified to make decisions in this regard. However, surgical pathologists should be equipped to oversee the procedure responsibly. This is one of the goals of the current article.

Agitation

Periodic agitation is useful, but rarely employed nowadays. An appropriate container should make possible to take off salts from the surface. Sometimes, especially in a biopsy or examination of a compact bone tumor, it is a good option to use in addition a soft brush. It helps to get rid also of bone dust, if it has not been done immediately after the cut. A Compact Interdental Brush, as used in dentistry, is a good tool for this purpose. The technology of vacuum variable pressure microwave assisted decalcification can be helpful in facilitating agitation.

Endpoint of decalcification

Monitoring the endpoint is crucial in the decalcification procedure. Poorly infiltrated by paraffin wax tissues are brittle; they are difficult to section of a regular microtome as well as to stain properly. The goal is to stop the process to avoid overexposure.  We do not fight calcium to the last ion but try only to make the calcified tissue adjusted to the microtome blade, type of paraffin, and, yes, to the skills of the histotechnologists.  Overnight decalcification is undesirable but was and still is commonly used. Sometimes it is a necessity, for example in complex odontoma that requires 2-3 days of decalcification.

In a case of acid decalcification (formic acid, HCl, or nitric acid) “over-decalcification” can cause damage nucleic acids , when the nuclei will appear pale and in some cases do not stain at all.

Methods of endpoint determination

Every laboratory has its own method for the decalcification endpoint determination. Some are technically advanced (X-ray), others are sophisticated (chemical, weighing) or there are simple mechanical tests of plausibility and “permeability” by probing.

Radiography, as an ideal, can be used in specialized facilities, especially when big fragments of bone are involved, for example in osteocarcomas after chemotherapy. A portable device Faxitron can provide reliable determination of the endpoint of decalcification. However, this method is too inconvenient and expensive for a regular surgical pathology laboratory.

In the chemical method, a mix of acid decalcification solution (RDO, for instance) with 5% ammonium oxalate (the usual test solution) and 5% ammonium hydroxide is used. A milky precipitate signals that decalcification is incomplete.9 This test is cumbersome and useless in the every- day practice of surgical pathology when many samples are placed in the decalcification solution simultaneously. It is definitely more objective than the bubble method which includes the observation of carbon- dioxide bubbles at the surface of the bone during acid decalcification.10

Weight loss/ gain is a reliable method of determination of the endpoint of decalcification, especially for non-acid decalcifier, such as EDTA.8 However, single EDTA use is a rarity in surgical pathology. This method requires careful blotting and weighing in milligrams of the bone section. It is difficult to catch the point when the section starts to gain weight again after stopping losing it, which is evidence that water goes back after the calcium leaves. However, at least it is closer to the surgical pathology practice of the simultaneous processing of samples in batches.

While considering through the different methods of determination of the decalcification endpoint, with some reluctance, but adherence to the realities of practice, the simple method of pliability should be mentioned as the most acceptable. As any subjective determination, filing of “pliability” is very vague. If the bone bends without resistance, the endpoint is reached. With some experience gathered by obligatory feedback from histotechs and pathologists, the grossing person or histotechnologist can develop the skill that satisfies the practice requirement.

Some histotechnologists press the bone with a fingernail. A retractable indentation occurs without resistance if the area is decalcified. Poking with a needle, which some pathologists like to do is unreliable and very much “barbaric.” Of course, sharp objects should not be used.

The good method, in my opinion, is to use a plastic cone like instrument with a pin at the end that makes retractable indentations on the surface without damaging the specimen, although this requires some training and experience (Figure 1.).

 

This device is especially useful in heterogeneous bone specimens and in pathological calcifications when pliability cannot be used. There may be different designs of the device, but the principle of retractable indentation as a test of the endpoint of decalcification can be used in practice.

In for some extraordinary diagnostic situations, a more precise method of determination of the decalcification endpoint should be in the possession of every surgical pathology laboratory. In my opinion, weight loss/ gain is the most common for different decalcifiers, including EDTA if it were used for some purposes, for example in enzyme studies. The person responsible for decalcification should be trained in this method, which is not technically simple.

Microwave assisted decalcification with different programs can effectively contribute to endpoint decalcification monitoring. It is time to bring “educated electronic” in charge of this crucial for the outcome the histological slide process.

Rinsing

This procedure was once highly regarded. Rinsing was carried out for hours, even overnight, and could cause tissue swelling. The current practice tries the opposite approach by minimizing rinsing, sometimes even skipping this time consuming process. The effects of the swelling are sometimes overestimated, but there is no reason to contaminate the tissue processor with acids or continue the protein hydrolysis by residual acid. A reasonable 10—30 minutes rinsing is appropriate, especially if EDTA is used to get rid of a precipitate that occurs on the surface. 11Sometimes careful rinsing is very important, as in the case of calcifying odontogenic tumors (Pindborg) when acid prevents amyloid detection. If the decalcified section cannot be placed in the tissue processor after rinsing for any reason, it should be placed back in formalin or preferably, in 70% ethanol.

Decalcification on block

Sometimes, more often than ought to be, the bone goes in the tissue processor under- decalcified or even undecalcified. Of course, reprocessing is an option, but histotechnologists occasionally try to decalcify on the block.

The most popular procedure is to put the block face down in RDO (usually on the gauze) for 10- 30 minutes to soak. Acid removes a few micrometers of calcium from the tissue surface, permitting only a few sections. Another method uses the Softening Solution (MasterTech, Easy Cut, American Master Tech Scientific, Inc.), which combines hydrochloric acid with Polysorbate 20, making the surface of under decalcified bone softer and cuttable. Or, while the block is still in the chuck, after a rough trim, the face of the block is wiped with MasterTech. After exposure, the histotechnologist’s skill is essential. Rinsing in water or ammonia is appropriate.

It is obvious that decalcification on a block is a choice between two “bads:” the block remains undecalcified and every next trim requires repetition of the procedure, with inevitable loss of material, except targeted decalcification:

in cases of patchy calcifications in tiny core bone biopsy, when decalcifying the entire core carries a danger of destroying other components of the specimen;

in core breast biopsies with micro calcifications (the main reason for the biopsy), when a grossly recognizable area of macro calcification sometimes is present; this fragment might pop out during cutting if it were not mildly decalcified;

lungs with areas of calcification should be decalcified on the block; the same is true for ovaries, as well as for brain tissue when, for example in meningioma, psammomas have a diagnostic value.

A cotton tip moistened in a decalcifier can be applied to the area of interest. A better option, however, is an angle- cut sponge moistened in decalcifying softening solution and applied directly to the calcified area. A cassette with a block is placed on the side facing the moistened sponge on a handle for a certain time. The end-point of decalcification is a problem in this situation. Some experimentation with calcified heart valves can give an approximate time of exposure of the moistened sponge. These occasions are rare and the procedure is time- consuming but rewarding.

Some recommendations from surgical pathology practice

Bone marrow biopsies arrive as a rule in Zn formalin, the commonly used fixative that replaced B-5. Fixation and decalcification require monitoring for a certain time. Most of the cores are similar (8 gauge), although some of them, especially in children, are with minimal or without any calcifications. Monitoring of the decalcification endpoint is essential, because the exposure to the decalcifier lasts three hours. The general rule is: less decalcification is better, because bone structure is not a consideration. A careful rinsing is desirable. 10% formic acid without any additions is the best, especially for IHC, although the iron store is diminished. It works slower than a strong acid, but affords an advantage for monitoring the decalcification endpoint of samples arriving at different times. Actually, Immunocal is a buffered 10 % formic acid. Some laboratories just prepare in house unbuffered 10% formic acid with positive results. I have read about positive results with Chelator•Cal™  from BBC Biochemical http://www.bbcus.com/products.html?pc=43&pid=209, but I haven’t had my experience with this reagent.   

Curetted bone material is usually heterogeneous and therefore requires separation any non-calcified fragments, because every one of them is valuable. In osteonecrosis, the area of interest might be at one edge of the core biopsy. After fixation of 2-3 hours in formalin, the calcified material should be wrapped and placed in a cassette for 1-2 hours in a solution such as Immunocal™ (formic acid), with monitoring every 30 minutes. Ideally, a core of bone biopsy should be monitored until it floats but usually the specimen is wrapped and it is on the bottom of the container.

In osteosarcoma, a large bone specimen after chemotherapy has some decalcification particularities. After the longitudinal slab is completely fixed (1-2 days), uncalcified areas of the slab should be separate from those that will need decalcification maintaining their place on the map for calculations of the percentage of tumor necrosis. Sections for decalcification should include the periphery of the tumor at the junction with all layers of the bone. In grossing osteosarcoma in long bones after chemotherapy, I would recommend that the following sequence of processing be followed:

a/ ink the areas of interest; b/ cut the bone specimen in the fresh  in half (or more if the bone is thicker than 3 cm) longitudinally while maintaining all bone/ soft tissue relationships for evaluation of the general picture; c/ take the necessary photographs; d/ put the halves (or more slabs) in fixative for 1-2 days; e/ rinse; f/ put the slabs  in decalcification solution (this is the point of deviation from a standard procedure cut, fix, decalcify); g/ final grossing cuts; h/ final decal in the processing cassette. The goal is to attain an accurate calculation of the percentage of necrosis of the tumor after chemotherapy, because the diagnosis is known already. This can be achieved only if the sections for calculation are more or less even in thickness. There is inevitably loss of bone marrow and fragile necrotized tissue during sawing even with a fine saw.

A complex odontoma, as well as a compound odontoma, requires thin cutting of the specimen, long decalcification, careful rinsing and cleaning of the surface. A tissue softener (MasterTech type) is useful before making the final cut on the microtome.

Nasal sinuses debris that is collected by vacuum aspiration in a Soft Tissue Trap container often contains small, thin bone fragment. The specimen is placed in a Surgipath Decalcifier II® type solution for 1-2 hours after fixation.

It is a waste of time to put toenails in decalcification solution, because they are composed of insoluble keratin filaments. After fixation, depending on the amount of adjusted soft tissue, the nail should be placed in a 5%  sodium hydroxide (NaOH) solution or a 10% ammonia solution for alkaline hydrolysis, with vigorous rinsing off of the soapy water when the nail becomes pliable.  By the way, cartilage does not require any softening, except if some calcified areas are present.

Gout specimens can be problematic. If any crystalline material is seen on gross examination, it’s better to pick it out with a needle, suspend it in a drop of water or alcohol, and look at it with a polarizing system. If the characteristic negatively birefringent crystal needles are seen with this approach, the diagnosis in general is clear. Gouty tissue – (that is tissue containing monosodium urate crystals) – should be fixed in absolute alcohol, and processed into xylene and paraffin directly. The section with gout suspicion should begin to undergo automated processing from 70% alcohol. Urate crystals, particularly in large deposits (tophi), often survive formalin fixation and routine processing anyway. If a gout specimen is received in formalin (uric acid is even more soluble in formalin than in water), it can be recovered by drying the fixative on a slide in an oven and mounting it unstained for Diff-Quick stain. Before starting decalcification in soft-tissue chondroma with calcification, in post-traumatic ligamentous calcification or in chondrocalcinosis (pseudo gout), it is a good idea to make a smear slide for red filter under polarized light to exclude gout (uric acid crystals are needle shaped and bright yellow).

If for some reason a decalcified histology block needs to be additionally decalcified, “go back to water (xylene or whatever “clearant” you use → graded alcohols → water) and place in decal. The results will be slightly of less quality than if the tissue was correctly decalcified in the first place, but you will be able to section it (René J. Buesa).”
Last words

The surgical pathology laboratory works as an “assembly line.”  There is a necessity to triage calcified specimens by preliminary fixation, size, and type of calcifications; otherwise any meaningful monitoring of decalcification process becomes impossible. Every laboratory should develop a protocol for different kinds of specimens. Compromises inevitably occur due to turnaround time considerations, but these should still adhere to the principles of histotechnology.

Contemporary technology has significantly changed our attitude toward decalcification in surgical pathology practice. Big microtome “mamma” knifes disappeared at the end of eighties, as well as microtome knife sharpening and honing. Easily replaced microtome blades with a better quality of the cutting edge, different embedding materials, and some histotechnologists tricks have made under decalcified blocks sometimes more a nuisance than a problem. Nevertheless, appropriate decalcification is a necessity, because, if the histotechnologist even solves the cutting problem (a pain in the neck at the level of art), it comes at the price of the quality of the slide, especially in term of staining. Sometimes a diagnostically valuable feature disappears unnoticed by the pathologist.

New polymer resins as embedding materials, new procedures (for example cryotomy of undecalcified bones for enzyme histochemistry) are coming into the histotechnology that might make decalcification obsolete. However, decalcification procedure will remain a part of surgical pathology practice for the foreseeable future.

Decalcification is accompanied by urban legends. For example, I ask the participants of my workshop on bone grossing: “Does Coca-Cola have decalcification ability?” Usually, I have many positive responses. Just in case, I experimented with Coca-Cola decalcification with a negative result. Will decalcification outlast soda drinks?

References

1. Brain Edward B. Preparation of decalcified Sections. C.C. Thomas, Springfield, IL 1966.

7. Buesa RJ. Histology without formalin? Annals of Diagnostic Pathology.  2008; 12 (6): 387-96.

4. Callis G., Starchi D. Decalcification of Bone: Literature Review and Practical Study of Various Decalcifying Agents, Methods, and Their Effects on Bone Histology. The Journal of Histotechnology. 1998; 21:49-58.

2. Dickson Glenn R. Methods of calcified tissue preparation, Elsevier, 1984

6. Dimenstein IB. Bone grossing techniques: helpful hints and procedures. Annals of Diagnostic Pathology. 2008; 12: 191-198.

5. Gruntz SL, English V. Expedient Bone Throughput Using Microwave Decalcification. HistoLogic. 2011; XLIV (1):1.

3. Hikins David W.L. Calcified tissue CRC Press, 1989

10. Hornbeck C. Emmanual J., Bloebaum RD. A comparative study of three paraffin media for preparing large decalcified bone sections. The  Journal of Histotechnology. 1986; 9: 227-229.

8. Rosen AD. End-point determination in EDTA decalcification using ammonium oxalate. Stain Technol. 1981; 56: 48-49.

9. Urban K. Routine decalcification of bone.  Lab Med. 1981; 12: 207-212.

11. Villanueva A. R. Post-Decalcification Treatment of Bone Specimen.  HistoLogic. 1982; Volume XII Number 3 p.181.

The article “Decalcification in Surgical Pathology” is published in AAPA’s  The Cutting Edge Journal. AAPA 2012. Vol. 2. 3: 21-23

 

 

 

2 Responses to Decalcification in Surgical Pathology

  1. Anu says:

    Wow Professor……. i wish all our teachers could write this well!
    I’ll remember more about decalcification because i read it here. otherwise, I was dreading reading up on it. Thank you.

  2. EMMANUEL.I. says:

    it was a wonderful write up. you are awesome professor it helped me alot

Leave a Reply

Your email address will not be published. Required fields are marked *

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <s> <strike> <strong>