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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.
- Review the common decalcification methods.
- Discuss the downstream effects of decalcification methods on staining.
- 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
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