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The Impact of Decalcification on Staining


Different types of cancers frequently metastase to bone tissue. Treatment planning decisions are often based upon histology and special staining of these distant sites of disease. These decisions may rely on the outcome of immunohistochemistry, in situ techniques or molecular testing. Decalcification of the bone tissue is required to get quality histological slides. There are many decalcification reagents on the market. This presentation discusses the pros and cons of decalcification methods and how the laboratory should handle these specimens to best help the patient.

Learning Objectives:

  1. Review the common decalcification methods.
  2. Discuss the downstream effects of decalcification methods on staining.
  3. Describe the process of validation and reporting.

Loralee McMahon is the Immunohistochemistry Supervisor in Surgical Pathology at the University of Rochester Medical Center, Rochester, NY. She has worked as a histotechnologist for about 18 years. Her previous job was the Histology Supervisor at a Dermatopathology Lab in the DC area. Previous positions in histology included doing general histology and advanced staining for Alzheimer’s disease and Orthopaedics research.

She holds a Bachelor’s Degree in Biology and a Master’s degree in Medical Management. Loralee is ASCP certified as a Histotechnologist and has a New York State license as a Medical Technologist. During the summer she teaches a Histology Techniques course for a Medical Technician program.

She has been a contributory author on articles that have been published most recently in the following journals: The Journal of Histotechnology, Applied Immunohistochemistry and Molecular Morphology, International Journal of Clinical and Experimental Pathology, American Journal of Clinical Pathology, and Human Pathology

Webinar Transcription

The Impact of Decalcification on Staining

Today’s topic, we're going to be talking about staining and decalcification. Specifically, the effect that decal has on the tissues that you get in the laboratory on a daily basis, and any of the downstream testing that may need to be done on these samples.

The type of testing that can be done on formalin formalin-fixed paraffin-embedded samples seems to increase every day, so it's very important as a histo tech that we look at the impact that this decal might have on a tissue. And it's not as simple as it used to be 10 to 15 years ago. And it can be a hotly discussed topic within the lab.

So our learning objectives for today is just very simply a brief overview of some common decal methods. Discussion of some of the downstream effects of decal on tissue and staining. And we’ll touch a little bit on validation issues that you may have come a crossed.

What is Bone?

First of all, what is bone? Besides the sample you get in the lab that no one likes to cut, it can be difficult and present a challenge.

Bone is a specialized type of connective tissue that is made up of minerals, collagen, and cells in the marrow. And it's frequently the site of metastasis and cancers. You will definitely encounter bone in the histo lab quite frequently, and it's very important to be able to process these samples like you would routine tumor samples so that the pathologist can get an adequate section to make a diagnosis.

And what is decalcification? Quite simply it's the process of removing calcium ions from the tissue. This process is what makes the bone soft enough so that you can cut it and obtain a thin section for pathological diagnosis. And these tissues cannot be sectioned routinely like you would a liver or any other organ in the body without some type of decal. You can cut on decalcified bone, but it requires special processing and a special type of knife. And that progressing is usually done in plastics or resins, and it takes a quite a length of time. It's not routinely done in the histology lab, and probably not the best for turnaround time.

Purpose of Decalcification

Like I mentioned, the main purpose of decal is to allow a histo tech to get a thin, nice section for diagnosis. This picture shows you an example of well-preserved and well decalcified bone marrow. The staining itself looks good. It's bright, and clear, and crisp and you can see nuclear detail and the hematoxylin looks a nice purple color. There's no rips or tears in the section. Most pathologists would be happy with this kind of section.

Bone can arrive to the histology laboratory in several ways. You can get a small bone marrow biopsy, or a small biopsy of a bone lesion that they took from a patient. And sometimes you can get larger long bones or femoral heads from a diseased piece of bone that is removed from a patient surgically. Both of these specimens need to be processed for diagnosis, and they do require decalcification. That process is necessary in order to obtain a section so that the pathologist can give a diagnosis to the patient.

This is just an example of a specimen received in the lab that contains some cells and marrow, and trabecular bone. And in order to get a section like this you need to properly decal the section first.

Why is Decalcification Important?

And why is decal important, why do we care so much about it? Because cancer metastasizes to the bone in 68% of breast, prostate, and lung cancers. So that means that 68% of people with those types of cancers have a good chance of getting a bone mets down the road, and those biopsies will most likely end up in the histology lab. It is up to the pathologist to determine if this cancer is from a previous cancer or if it's a new site of primary cancer. And it's up to the histo tech to produce a good section in the lab that allows the pathologist to give this diagnosis.

It's also very important for the diagnosis and staging of lymphomas and leukemias. You’ll get a lot of bone marrow biopsies into the lab that are for this purpose. Without a good section and other downstream ancillary testing that pathologists may need they can't render a good, confident diagnosis. And like I mentioned before, all these patients with the mets from a previous cancer are going to need to have treatment planning that’s based upon the type of cancer that they have, and based upon the staining that you might do in the laboratory.

For example, metastatic breast cancer. These are photos of a patient that had several bone lesions that were found on CAT scan, or CT scan, and they subsequently biopsied. The patient had a history of breast cancer years before and had undergone mastectomy, but showed up at screening CAT scan with these bone lesions. And they biopsied them and sent them to the lab. And the pathologist decided, through the staining and the processing that the histo techs did, that these lesions from the original breast cancer. Now the patient is eligible for a specific treatment plan. And this was based upon the IHC stains that were performed in the lab, and ERO in this case confirmed that the bone lesion was a breast primary that was estrogen receptor positive. So the choice of decal really makes a difference in this case.

Prostate cancer also accounts for about 90% metastasis to the bone in men. It's the leading cause of death for these patients. So as you can see, it is very important not only to get a nice H&E stain, but it's also important to get a good section for immunohistochemical staining, or IHC staining. This is a prostate-specific antigen IHC, or a PSA stain. It was done on the bone lesion to confirm that the cancer in the bone was actually from the prostate.


So now that you see how important the decal process is, we can talk a little bit about the methods that are commonly used in the histology lab. There's the chemical method of decal. This happens when calcium salts are dissolved. And then ionized in a strong acid, thus it removes the calcium from the tissue, making it softer so that it can be cut.

Nitric acid and hydrochloric acids work quickly to decal, so they work very rapidly at getting the calcium ions out of the tissue. They can damage the morphology of delicate tissue that’s left too long, and they can also make ancillary testing and staining unsuccessful, especially if this subsequent testing is on DNA or RNA, as it can damage the DNA or RNA on the tissue.

Strong acids, such as nitric acid and hydrochloric acid must be monitored pretty closely when you’re doing a decal process so that you don’t run the risk of over-decalcifying the tissue. Stomach acid is also pretty common as an acid decal and it's a little more gentle than some of the nitric and hydrochloric acids that are out there. It also means that it will be a little bit slower, so the results may take a little bit longer to get to where you need to be for progressing and sectioning.

There's also a chelating method of decal. This is where calcium ions will bind to the agent and they're removed from the tissue. And I think the most commonly used one in my experience has been EDTA. It's an organic compound. It's very slow at working, but it's very gentle. And it tends to have a lot less damage to the DNA and the RNA in the tissue. So NC2 harbortization, cytogenetics, molecular testing, enzyme histochemstry, any DNA/RNA analysis that you need to perform is usually successful on these tissues. And I have found that most histology labs are using these two types of decal, either a chelating or an acid decal, or some combination. It depends on the specimens that are being received in the lab and what the end results is.

Choosing a Method

So when deciding which method to choose from, the lab needs to decide what's most important; is it speed, is it quality, is it a balance. It's a balance between the two, so ideally you need to find that balance. You need to find a balance between speed and quality. Speed is very important for turnaround times and for sick patients that need diagnoses quickly. It's also important to keep the pathologists out of the lab, because if they're waiting for their bone to come out two and days they're going to start to get impatient.

Quality also needs to be there as well so that the pathologist has a decent to be able to render the diagnosis. Speed is great, but if you end up with poor quality you’re not helping the patient. So finding this balance can be a challenge in the lab, because the fast turnaround times are so important nowadays. All the labs are monitored for their turnaround times, and it is very important that these patients’ diagnoses as quickly as possible. Our lab struggles with this. It's not impossible to find a balance between speed and quality, but I think that everyone in the group, the lab, the pathologist, the clinician need to understand what exactly goes into the process of decalcifying the tissue, cutting the tissue, and then doing any type of ancillary staining. It's not as simple as just dumping the specimen into a decal solution and processing it the next day.

It's also important to note that decal methods will vary from lab to lab. There's no one size fits all, and I'm not recommending one over the other. The decal method chosen for the lab should fit the workflow, it should fit the specimens that come into the lab, and it should fit the patients that come into the lab.

You should also make an attempt to standardize it within the lab, because if you’re going to be validating any of decalcified tissues with any of your staining, the closest you can get to standardizing it in the lab is going to make your validation easier and more reproducible. But with any process in the histology laboratory, that’s kind of difficult.

There's a lot of decal solutions on the market and they all claim to be the best. There's a lot of brand names out there, but I looked up a lot of the brand names and kind of came up with this group of decal solutions. So there's EDTA solutions out there, there's high percentages of formic acid with a little bit of fixation in there. And there's also hydrochloric acid. And then there's acids that I have ion exchange resins in them, and formic acid. And there's also a lot of labs out there that maybe using a home brew, maybe something that they discovered from an article in a journal where it worked well, and they tried it the lab and seems to work well for them. I find that it may be necessary for the lab to have maybe one or two different types of decal solutions in the lab to make everyone happy.

What Should the Lab Do?

So with all these decal solutions out there, and all these methods, what do you? Which one do you choose? And it can be very difficult to come up with the right answer. What I would do in this case is ask a pathologist. They're the experts in their field. They know what downstream testing might be needed for different diseases. They talk to the clinicians about the patients so they know what might be needed in the future for these patients. The pathologists also are pretty good at keeping up on all this new testing that’s coming out. It might benefit you also to check out some of this new testing. Get online, look up the instructions for use. See if there's any special requirements for the tissues, such fixation length or type of decal. We know from some of the predictive markers, ER/PR and HER2, that there are some guidelines that are very specific to those.

You should also look at the workflow in the lab. Is it more beneficial that the histo tech monitors decal? Maybe the pathologist’s assistants that are taking the specimens can more easily monitor the decal process. Maybe the pathologist does it if you’re in a smaller lab and the pathologist is able to do this for you. What works best and gives you the best results is what you need to go with. Maybe you need different types of decal based upon the specimen, or based upon the patient’s history. This can create some issues, but it can give everybody what they need. And this is what we do in our lab, we rely a lot on the patient history and a lot on the pathologist’s assistant to decide which decal solution the specimen is going to go into.

So there's some questions you need to ask before placing the tissue into fixative or into decal, and the number one question I think is what's the patient’s history. Does this patient have a history of cancer? The patient’s history is what's going to drive the downstream testing beyond the H&E, beyond that routine stain that you get in the lab.

Things to Think About

So, some things to think about. Treat each sample individually. We're in the age of personalized medicine, so how about treating each sample as personalized. What works for one sample may not work for another. The size of the sample matters. You know that the size matters when you try to fix a specimen. A big sample is not going to fix as well as a small sample. Bigger tissues may take a longer time to decal. The type specimen matters. Femoral heads and bone marrows may need different protocols. You have a hard, long bone, and then you have a bone marrow which is a little bit softer. And bone marrows are often used for lots of downstream testing. So it matters what type of specimen it is.

And the desired speed, this is always an argument in our lab as to why the bone marrows aren’t out, or why the decals take so long, it's how fast you need the results. This is the most difficult part of the process. You want fast results, but you also want good results. And going back to treating each sample individually, look at the patient’s history. Does the patient have a history of cancer?

Which Type to Use

So if you were to do a literature search on types of decal and the effects that decal has on specimens, you would probably get a lot of differing opinions. At least when I was preparing this presentation, and when our lab was going through our decal struggles, we did a lot of literature searches, and we found a lot of different opinions. And I’ve been in seminars where people would say that they know that their stain works well with acid decal no matter what. And then someone across the room would say I know that my stain works well and I use EDTA. So I think in the hands of different laboratories you need to test it within your lab. Some of the literature that I found, the most recent one I found was in February of last year, that said that there was a marked decrease in Ki67 and p53 IHC staining after two hours in an acid decal, and there was some effect on the ER/PR and HER2 staining. And there's been a lot of publications out there about adverse events that acid decalcification has on RNA and DNA studies, and many manufacturers actually recommend that you don’t use their testing on acid decalcified tissue. Obviously, if you’ve validated it in your lab and you can prove that it works then you’re okay.

And I’ve seen a lot of literature that compares several different decalcification methods with IHC and NC2 harbortization, and DNA and RNA testing, and it seems to be that ETDA, as we discussed, is the best method for these. It preserves the DNA better than some of the acid methods. The issue that come into play with EDTA, as we discussed before, that it's a little bit slow. But if your end result is FISH or DNA analysis it may be your best option.

If you were to receive a bone specimen and the patient’s history is a suspected mets, what would you do? How would you treat this specimen? I would ask questions, find out what the pathologist or the clinician may want to test later on. This can be the most challenging aspect of it. The pathologist and the clinician want fast, accurate results, but they may not understand just what it takes to get there, and you don’t want to rush it.

Ewing’s sarcoma is an example of one of these dual tested samples, where you need CD99, or you need IHC stain in order to confirm the Ewing sarcoma diagnosis. You also need a molecular test for rearrangement, so you have to be able to do both, so the decal method is very important. An acid may be okay for the IHC, but not for the molecular tests, so you need to consider both testing options in this case.

This is a different kind of an example of a decal struggle. These are tissue blocks, and the center block has a little bit tiny bit of decal right in the middle of the block. And this can make it difficult to section; although, not impossible, but it can make it difficult to section. And what if it's in the middle of the tumor, which often happens with breast cancers. You will get some calcifications in the middle of your breast cancer sample and you won't be able to section the sample very well. You also need to consider that this patient is probably going to need ER/PR/HER2, and maybe some HER2/FISH, are there any requirements for that, the type of decal? This particular specimen has already been processed, it's already been embedded, so your options are limited here. One of the things that we can do is---one of the things that this presents us with is under decalcification, or no decalcification in a sense. You can't get a section. The section will not adhere to the slide, you’re going to lose tissue morphology because of the torn-up section. And if you do any type of IHC most likely the heat retrieval step will just blow the tissue off the slide. So what you can do is try to cut it. You’ll end up usually with a shredded mess that is hard, if not impossible to pick up to get a good section. But something to cut it will probably nick your blade and drive most histo techs crazy.

Since this tissue has already been processed your options are to run it back, run it back through your xylenes and alcohols, and back into decal. And you can decal the specimen from that point, which can take some time, or you can do what some labs do, and I know our lab does it, which is surface decal. You take the block, you place it in a decal solution for a short period of time to soften the surface so that you can take a section. Does this effect the staining, maybe. I think if you’re careful and only place the block in decal for a short period of time you’re probably okay, but who knows. Testing it or validating it may be worth it if you’re suspicious of the results, if you think that the decal has affected the staining. But I would be smart about it as well. Don’t dump the block in decal and go home for the evening, and come back in the next day. You’re trying to limit the exposure to decal, especially if you suspect it has an effect on the downstream staining.

Over decalcification can present a problem to the histo tech as well. It will be easy to cut, it will be nice and soft but too long in decal and you’re going to start to lose nuclear detail. You will get swelling of the tissue, especially if the fixation was not complete. If the fixation is not complete and you throw it in decalcification you may end up with some necrosis in your tissue. Too long you can lose the glycogen in the tissue, and you could end up with no staining at all, and even make your H&E look bad. Mostly H&Es are unaffected by the length that they are in decal, as long it's not too short or too long, and as long as the fixation was complete. Like I said, if the fixation was not complete and you plop the tissue into decal you could end up with some necrosis. Some labs have counter acted this by using a decal that contains some fixative in it. This will allow for fixation and decalcification to occur simultaneously, and it can help speed up the process.

So this picture is over decalcified tissue. You can see that it's kind of a muddy looking H&E. You don’t see any real good blues, purples or pinks like you would on a normal H&E stain. There's no nuclear detail and generally the stain is just not good. And at this point I think the tissue is probably lost and you’re going to end up with the pathologist that’s not very happy.

Comparison of Methods

So a quick rundown of a comparison of the methods. You have EDTA or acid-based decal, or a combination of both. EDTA is slow and gentle. And acids, when they're used properly, are quick, and they have good morphology but may affect other testing. So the question to ask is what testing is needed after decal.

Immunohistochemistry, or IHC stains, these stains are the basis for treatment decisions for many types of cancers. If you think about ER/PR, HER2, P16, PD-L1, all of these new markers that come out are often the source of treatment planning for patients. Some published data suggests that decal doesn’t affect IHC, that it can actually enhance the staining, kind of begin the retrieval steps. Others state that it can hinder the staining and that it most likely antibody dependent. One antibody may be affected by it, while others are not. That’s they recommend you testing it in your own hands. I recommend reading the package inserts out of these some of the FDA-cleared tests. More often than not, these FDA-cleared tests do not include a cohort of decalcified samples. So when they got their FDA clearance they did not use decalcified tissue. They usually recommend that you do not do it on decal tissue, or that you perform a validation to prove that it does work with your lab’s decal method.

This is an example of metastatic lung cancer to the bone. Napsin-A is a popular stain, IHC stain, to help support the diagnosis that the bone lesion is a long primary. This patient might also be eligible for PD-L1 staining. That is very big right now, in my lab at least, and I’ve seen it actually advertised on television, so it's important to consider your decal method. If your decal is affecting the staining you could end up giving a false negative or a false positive to a pathologist.

Predictive markers, like ER/PR, HER2, HER2/FISH, and PD-L1, like I just mentioned, are FDA-cleared kits and they were not performed on decalcified samples. Manufacturers recommend that you do not perform these tests on decalcified tissue. We struggled with this, because what if that’s your only source of diagnostic material, what do you do? You can't refuse the specimen. I don’t think that’s an option. What can you do instead to get around this? Our lab has taken to the habit of taking the bone marrow and separating, if there's any tissue or blood clot, from part of the bone marrow, very gently teasing that off of the bone core, and placing that piece in regular routine fixative, and then taking the bone core and putting it through the decal process. So that way you have one sample that gets processed without decal. And more often times than not, that tissue or blood clot that came from that biopsy specimen will continue tumor cells, as you can see from the photo, that the testing can be performed on, and that you won't have to worry about the adverse effects of decal. This could also be the basis for your validation, if you were performing a validation. To see if decal affects your staining, you could take a thank you sample and divide it in half, put one in decal and one in routine progressing, do the same staining on both samples and compare your results and see if you got the results. We did this for quite a few biopsies in our lab for HER2, and we found that HER2/FISH worked about 50% of the time. And we went back and looked at how the samples were decalcified. And it seemed to be related to the type of decal and the length of time that it was in the decal. So this was another reason to push the histology lab to maybe change their method of decal and push a different way to process these samples.

Enzyme histochemistry is done in our lab routinely on bone marrow biopsies for our hematopathology group. Acid decal inhibits esterases and TrAP staining, but EDTA does not. It seems to work quite well. Our lab uses EDTA on most of the bone marrows now so that we can get an esterase stain for our - - pathologist. The only bad part about this is that it takes a little bit longer, so when a pathologist is standing there asking you for their bone marrow because they need to render a diagnosis quite quickly you have to come back to them and say it's in EDTA, it's not quite ready yet. It's going to take a little while. They're usually okay with that if you can give them a decent looking esterase stain.

But the patient sample comes to the lab and it needs NC2 harbortization, or cytogenic or molecular studies, most of the testing requires that these tissues are not decalcified in any strong acids. Strong acids tend to adversely affect the tissue, it degrades the nucleic acids in the tissue. Most package inserts that you will find are going to recommend against putting tissue in decal. This, again, can create an issue if it's the only source of diagnostic material. Sometimes it may be worth going back to a previous sample if you have the luxury of that. If the patient came to the lab prior to getting the bone biopsy, go back and do samples on the original biopsy, or the original diagnostic material that was not decalcified. Have a conversation with the clinician, see if you can obtain another sample that does not have—or that has not been decalcified that may not have bone in it.


So validation, validation is a huge problem. You have to do it. It's a The College of American Pathologists (CAP) requirement, and it's good patient care. You want to prove that what you’re doing in the lab works, it works with your tissue, it works with your decal method, and it works on the tissues that you stain. This can be difficult if you get samples from outside institutions and you’re not sure what kind of decal they used. How do you do a validation on decalcified tissues? I suggest as simply as possible, like I mentioned before, using two different decal methods, or a using a decal method and a non-decal method on the same tumor.

Is there any reporting issues? The CAP and other inspection agencies may have requirements for you to report that tissue was decalcified. And if you perform testing on it and the testing is not approved for decalcified tissue, they want you to report that in the report. I would talk to your inspectors when they come through. Use your keyway or compliance group, and review all applicable guidelines for reporting. Look at all the sources of inspectors, anybody that comes into our lab that is inspecting you and ask them, ask them what to do, how to report it, and what's required, and make sure that you fulfil those requirements.

The CAP recommends that you test a sufficient number of tissues to ensure that our IHC assays is reproducible and consistent. Just like they want you to validate all your immunos that are not decalcified, they also want you to validate your immunos that are decalcified. The recommendation states that the medical director can determine the size and the extent of the validation. And it should be done predictive markers and non-predictive markers, so basically everything that you have in the laboratory.

How do you do it? It's the same way you validate any other procedure. You’re going to compare it to the currently validate procedure. So if you have a marker that’s validated on non-decalcified tissue, stain some tissues that are similar that have been decalcified and see if your results match. You could take the same sample, like I said before, divide it in half, process one with decal and process the other without, stain for the same marker, and then compare the results. You could get another lab to help you. If you have a partner lab out there that is willing to help you, you guys could change tissues, you could do decal, you could stain some of their decalcified specimens, you could have your tissues sent there and they could stain them, compare your rates and see if you can get a 90% concordance rate. And I recommend if you come across an antibody that does not work, if you can't get a 90% concordance rate, that you don’t offer it on decalcified samples. I think it's better to not do the testing than to give out incorrect results.

The CAP checklist also has a requirement that a disclaimer be present on reports for HER2 testing. And it mentions HER2 specifically, but I think it's probably a good practice to report it on any tissue that has been decalcified, and then done stains on. It says that you need to put this in there as a disclaimer saying that you did this test on a decalcified tissue, and it has not been validated. You should also check with any other inspection agencies that come into the lab that may be applicable to you. I'm in New York so we have the New York State Department of Health comes in to inspect us, and if you’re a Joint Commission, they may have some other recommendations or requirements that you need to follow.


So, just in summary, bone is the site of mets for many cancers, so you are frequently going to see these biopsies in your lab. And decalcification is necessary for quality sections and proper diagnosis. The type of decal that you use should work for the lab, work for the workflow of the lab, and should also benefit the patient. You have to be able to give a good result, a confident result that you know what your tissues and your staining is working properly. And as always, the tests should be validated prior to use for patient diagnosis. It's a requirement from the inspection agencies, and it's also good patient care.

And that’s all I have for today. If there's any questions?


I am wondering if there's any impact on IHC staining if non-decalcified tissue blocks are soaked in 10% formic as a surface decal after rough trimming, but before sectioning.


So I talked a little bit about that, where you often get a breast sample that has been processed. It has a little bit of calcium in it that you didn’t see, the pathologist assistant did not see when they were grossing it, but you’re cutting into it and you find oh no, there's this piece of calcium in here, and it makes it very difficult section. So you take your sample and you put it in formic acid. I tend to think that a short time in formic acid to get a section is not going to affect the IHC staining. I can't say for certain that it's not going to affect the IHC staining, but I think if you can avoid it you probably should, but I know that our lab does it. And I have not seen, or it's not been brought to my attention that they think that this is affecting any of the staining, especially since it's usually a short period of time, 5, 10 minutes and you’re cutting and able to cut a few sections.

How long do you suggest leaving the block in decal for a surface decal, and how long do you rinse it in water?


I would leave it probably 5, 8 minutes and then try. And then if you need to go a little bit longer you can go a little bit longer. And definitely give it a good rinse, a good 5 minutes in running water and see if that works for you.

In our research lab we are performing ISH for viral RNA in whole guinea pig ear and whole primate ear bone sections, and I'll soon be doing the same for primate nasal bone on turbinates also. We're using EDTA, and then she mentions a product. It does indeed take longer to decalcify, but we notice the salts or sediment is rubbing the epithelium off some of the sections. Is there a recommendation to reduce this from happening? And she asks for products, of course, that you recommend. But maybe we can do that off-line, but is there any recommendation that you have to reduce this from happening?


I would say, when I was working in orthopedics research we would do a lot of mouse, and rat, and rabbit tissues in like whole bones and stuff, so we would decal for a long time in EDTA. And we would change it frequently. So I think that if you change the EDTA out frequently you’re going to reduce the amount of sediment that’s in that solution so that you won't experience that.

A comment from you, if you would. Immunocal works well for IHC, but we don’t see staining with ISH for RNA in the same specimens. Any comments?


I have to look and see, but I think that’s an acid decal, so you’re probably going to run into some issues with that type of decal, because it's an acid-based decal, when you start doing ISH in any type of DNA or RNA analysis. That’s been my experience as well.

Can you recommend a book that will cover all the aspects of IHC techniques?


General IHC techniques? I would say probably a good place to start would be the histology book by—the self-instructional text by Freeda Carson, that has some IHC in there. And also without being vendor specific, some of the vendors out there that are big players in the IHC world will give you some educational materials that talk a lot about IHC. There's some pretty good ones out there.

For a typical 2 cm X 2 cm bone, how long would it take to decalcify in EDTA?


I’d have to check with the histo lab because they do a lot of this for me. I know that the acid—we used to do the acid decal on these bone marrows and it took a few hours. I believe that there are now currently with the EDTA they're leaving them overnight. But those biopsies are not put into EDTA until the evening hours. So it's not like a 24-hour period, but it may be closer to 8 to 12 hours. I hope that helps. I can probably get more detail if you need it.

And what about times on surface floating for blocks in decal solution?


I would say 5, 8 minutes and give it a try. And then soak it a little bit more if it doesn’t work. But I would try not to go much longer than that.

What about rapid decal solutions that stop decal automatically, is it okay to leave the specimen in the decal solution past the point where decal has stopped? Are there adverse effects of this?


Well I would say that I don’t know. I don’t know much about decal—I mean I imagine that the decal solution would eventually become saturated with the calcium ion so that no more decalcification would take place. But I don’t know how you would know what that point is. By the size of the specific, I don’t know if the particular manufacturer gives you recommendations. If the decal has stopped I don’t think there would be any adverse effects to the tissue if you left it in there a little longer, but again, I don’t know how you would tell if there's like an indicator that would let you know that the decal process has gone to completion.

How long, with the acid type decalcifiers, how long would a piece of tissue need to be in solution before adverse effects start to show on the H&E and IHC?


With acid decalcification it would take a while, I think it would take a long time for that to happen. Normally acid decal you kind of put it in, and then you check every, you know, you check after two hours. And then you’re checking, obviously, this depends on which acid decal you’re using. If it's a strong hydrochloric acid, or if it's formic, they may be a little different. But if you have a strong acid you want to check it after two hours, and then check it again after two hours. I would imagine that if you left a sample in a strong acid overnight you might experience some adverse effects to IHC. And definitely going to see something if you’re doing NC2 harbortization. You may not get any results at all.

Should a validation be done for every antibody that the lab uses? There can be some difficultly acquiring specific cases of tissue, et cetera, for every antibody.


Well, we struggle with this as well, because we have a large menu, so it would probably take me a long time to get through all the required antibodies and the required decals. So my advice to you is to look at the types of biopsies that you get. Do you get a lot of lung biopsies, do you get that maybe have cartilage, or not cartilage, but bone in them. Or do you get a lot of bone mets that come from prostate cases, or breast cases? So your menu of antibodies can be greatly reduced if you’re only staining those tissues with the most commonly used antibodies. So like I said in the presentation, bone and prostate cancers, and lung cancers often metastasize to the bone. It's a big part of the metastasis to the bone. So you would want to make sure that your antibodies for P63, or Napsin, or PSA, or ER/PR, HER2, the ones that you use on those breast, prostate, and lung cancers work in the bone. So you don’t necessarily have to do your entire menu, but maybe a subset of it to be specific as to what you get. If you are in a derm path lab you may never see a decal specimen, or very few decal specimens, so it all depends on what your case mix is.

How long should fixation occur prior to decalcification, tissue specific or general time?


Well, if you have a breast core you need to follow the guidelines, the American Society of Clinical Oncology (ASCO) CAP guidelines of 8 hours minimum, 6 to 8 hours minimum. I would say that I would follow your routine guidelines for your tissues in the lab for fixation. And then after that time has been achieved, then put them in decal. Our lab does add a little bit of fixation to their EDTA decal. So we decal for a certain period of time, and then we plop that bone marrow into our EDTA fixative that has fixative in it. We have a home brew. And it kind of helps complete the fixation, and then start the decal process.

When using EDTA for larger bone specimens over extended time, even when changing the solution frequently, do you keep it on a rocker or stir plate? Also, do you use it at room temperature or heated?


We used to keep it on a stir plate. So we would have a little piece of plastic, something that fit into the container that would elevate the specimen off the surface of the container, and then we’d put a stir bar in that. And we would stir that EDTA while it was decaling and change it out daily at room temperature. We did at that point in my career had a microwave processer, so we could microwave decal. Again, this was a research lab, so we kind of played with a lot of different things. We did take those bones that we thought maybe were close to being decalcified and we’d stick them in the microwave and kind of elevate the temperature a little bit to the help the decal process kind of go to completion. But I don’t think you would have to do the entire decal process at an elevated temperature.

Do you recommend fixing bone after decaling it? We fix our bone in 10% MBF, then decalcify in formic acid.


I don’t think you would need to if you’ve fixed your bone for a length of time and then you’ve done your decal, I think putting it back into fixative is not necessary.

Can you discuss common tests or methods for determination of decalcification endpoints?


This is also a constant topic our laboratory. You know, I think that the best and most reliable way is to get in there and feel the specimen, kind of bend it, make sure that it's pliable. There are, you can take a pointer and gently poke it to make sure you can get into the sample a little bit; although, you want to be very gentle by doing this. You don’t want to risk breaking the specimen or destroying any part of the specimen. You could also x-ray the sample if you have access to that. We have in our laboratory, as you saw those blocks that I had x-rayed, we have a small x-ray machine that’s for the tissue samples that come into the lab, so we can actually x-ray the sample to see if all the bone has gone, or all the calcium has gone from the bone. And they don’t by any stretch x-ray every biopsy that comes through. They basically just go in, feel it, make sure that it's pliable and maybe poke it with a pointer.

Our lab uses three different decals. Do we have validate all of them?


The hard answer is yes. I would say you would have to, but look at why you use the three different decals. There has to be a reason, so if you use three different decals there has to be an endpoint that you’re looking for, and why would you use three different decals? Are you doing NC2 harbortization on these tissues? And I would say, again, like I said before for a previous question, don’t stain your whole menu, just stain those tests that you frequently see are done on bone. And if you’re only doing a handful of antibodies on your acid decalcified samples then I think you could greatly reduce the size and the scope of the validation and still cover your bases.

We get tissue from labs all over the place. How do you handle this?


Well, we get tissues from labs from all over the place, and we also send some tissues out for some testing that we don’t do here. So I’ve got tissues from some labs where I wasn’t sure of the decal. So I would call and I would ask. And if they wanted HER2/FISH on these samples I’d say what kind of decal did you use. And if they told me they used an acid decal, usually what I do is I say okay, I'll give it a try but I'm not going to promise that it's going to work. I think that’s it's dependent on how long they leave it in decal. So, and we struggle with this, because we get tissues from all over the place. On our test menu that we hand out to clients, we say please if you’re going to be doing any DNA, RNA, NC2 harbortization, to not decalcify the tissue, or at least discuss it with the pathologist prior to this. And I’ve sent samples out where they’ve refused the tissue because they said it was decalcified, and that wasn’t part of their testing validation. So I think it's a struggle. I think that the more people know about decal, especially the clinicians and the pathologists, that it will help understanding it. But you can't ignore the fact that a lot of cancers metastasize to the bone. And if these patient’s biopsies, and that’s all they have is this biopsy from the bone, it's very important that we get the testing done right. So it's very important to know what kind of decal it was placed in. And like I said, give it a try, if it doesn’t work it doesn’t work, but at least you gave it a try.

If I use acid for decalcification, how much will affect the immunoreactivity?


I don’t know. I think it's antibody dependent. I’ve done some literature researches and I’ve done a lot of stains on decalcified bone. And I’ve seen that the IHC usually works if the decal is within reason. I’ve done immunos on EDTA decal mouse tissue, mouse bone that’s been in EDTA for two weeks and it's worked. But I’ve also done some antibodies that did not work so well on those, so I don’t know if it's the antibody or the decal. So I would say that those are the types of antibodies you need to test if you’re unsure that it works. If you’re lucky to be able to get a piece of tonsil and you can cut it in half and throw half in decal for three days and see if it still stains for Ki67 after three days. I think that would be some way to kind of monitor it and maybe guide you, and assisting as to how long—at what point is the immunoreactivity going to be gone? Take a piece of tonsil, throw it in decal, take it out, take a piece out, process it, keep going. Go for a week, go for a month and see what you can get. There's a lot of different opinions out there if you do a literature search and look at the journal articles.

The last question, when trimming down large bones we have used everything from hammer and chisel, electric Stryker saws, table saws, and band saws, and even hand-held saws. Do you know of an anchoring device which helps to hold the bone in place for hand-held Stryker saws in preparation for decal?


Right, I don’t know what these are called, but I know I’ve seen them in garages, and maybe some mechanics out there know. It's like a vice that you attach to a table. Or if you had a work bench you would attach this to a table. And they actually put the bone in there and to hold it, to hold it in place, because we struggle with this also. We have hand-held saws, we have disgustingly an old meat saw that you would get from like a deli. We have one that you get from your local home improvement stores. And we've had some people get injured on these because they don’t use the proper protective equipment, so it is a struggle. But I think the thing that has worked the best is using that vice, that they can actually put the bone in there and hold it, and then be able trim like femoral head and be able to get a good nice section. You can also, we've done things such as decaled very large pieces in a large amount of decal. Unfortunately, those were done in an acid decal, so they were just looking for primary diagnosis and we didn’t do any subsequent protein or DNA analysis on the patient. But we have done that and stained very large pieces and put them in huge vats of acid decal, and it's works quite well. But I think if you look into that vice type thing, I think you’ll be surprised. It's pretty good.


And in fact, thank you. What a great community this is. Comment, we have two comments. I think it's called an angle clamp. Thank you to Jennifer for that. And Lilla says vice grips with gauze on each side works great.

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