Dermatopathology for the Histology Lab

CLIFFORD M. CHAPMAN, BS, MS, HTL(ASCP), QIHC(ASCP): 

What I'd like to first do is to review the learning objectives for today, and we want to, at the end of this session we want all the participants to understand how specimens for dermatopathology are surgically grossed, processed and embedded to retain proper orientation in the final microscope slide; we also want you to be able to understand the histology of skin specimens; and to understand how to recognize and successfully handle all types of dertmatopathology specimens received in your laboratory.

As a starting point, we must understand that skin specimens for dermatopathology are among the most difficult to handle in the path lab because pathologists must be able to see the dermal-epidermal junction in each tissue section.  There are some exceptions to that, however, 98% of the time the pathologist needs to see that dermal-epidermal junction, or DE junction as it's referred to, so that every skin specimen that you receive must be accurately oriented during both grossing and embedding.  It's also important to note that when you receive a full thickness skin specimen, it contains three tissue types; it contains epidermis, dermis, and adipose tissue.  Epidermis is usually very hard, it has keratin on the surface, it's been made even harder during the tissue processing process; dermis is a bit softer, being comprised of connective tissue; and adipose tissue is softer still with adipose or fat cells comprising that layer.  So really what you have are three tissue types in one block, which makes it difficult to cut if not properly embedded, and also difficult to cut if you cut at too fast a microtome speed.  So we'll address these issues later on in the webinar.  Also as you may notice in your laboratory as we do in ours, specimens gets smaller all the time, you will receive many, many tiny specimens not just derm path specimens, small core biopsies, FNAs, things like that we'll discuss those as well.  So these issues are really just for starters and we'll dig into everything else.

Now in dermatopathology it’s important that we assist the pathologist because the pathologist has hundreds of possible diagnoses to differentiate from.  When one receives a specimen, it comes with a requisition and the differential diagnosis is indicated on it, however, it could be one of hundreds, so we must provide the pathologist with optimal microscope slides, and that's really our goal today to understand how to do that.

Malignant Melanoma

Let's look at the facts regarding malignant melanoma as that's one of the most critical specimens, if not the most critical specimens that you'll receive in your dermatopathology laboratory or in the derm section of your hospital laboratory. 

Firstly, in 2003, malignant melanoma affected 50,000 Americans.  That number was up to over 68,000 in 2009, and over 70,000 in 2013.  And these caused the majority of skin cancer related deaths.

Secondarily, we should note that the five year survival rates are 96% for localized malignant melanoma, but only 14% for metastatic melanoma. 

Then this incidence of malignant melanoma in the US is increasing at approximately 6% per year.

From a medical legal point of view, malignant melanoma is one of the most common legally contested diagnoses yielding malpractice.

And when I first started, failure to diagnose the malignant melanoma is actually the most common malpractice claim against the pathologist.

And finally, you need to understand that you are a member of the healthcare team that helps to cure malignant melanoma.  The cure for malignant melanoma is to complete removal of the cancer cells.  And we’ll see as we go through the webinar that it's very important not only to make the diagnosis, but to determine if all the cancer cells have been removed. 

And then I provide you with the references for those particular statistics.

Skin Histology

Now in order to make optimal dermatopathology slides, we must understand skin histology.  On this slide here we have a schematic representation of the three layers of skin that we talked about, and as we spoke about the dermal-epidermal junction is of the utmost importance in making an accurate diagnosis by the pathologist.  This is schematically the epidermis so the outer layer, is hair coming out, is the epidermis sitting on a basement membrane.  And then below the epidermis is the dermis.  And then below still is the adipose layer.

If we look at a schematic of a higher power, we can see that basal cells which are indicated here by the red circular cells, sitting on the basement membrane, those will grow and divide and differentiate and push up toward the surface of the skin.  And they'll differentiate firstly into squamous cells, which will flatten out to form granular cells which make protein that is on the surface of the skin and helps to seal the skin, protect it against moisture loss, protect it against bacteria and viruses.

Now the basal layers grow and differentiate so that if a basal cell grows and differentiate or grows out of control, does not differentiate, stays as a basal cell, it results in a basal cell carcinoma.  Similarly, these blue cells which depict squamous cells, if those grow and divide out of control, it will be a squamous cell carcinoma.  Now both squamous cell and basal cell generally speaking will stay in the location that they arise in.  Very rarely do they metastasize in the patient and continue to grow and cause troubles.  However, this cell depicted right here, the brown melanocyte right in the middle, the melanocytes are a wandering cell, their job is to move through the epidermis between epidermal cells and infuse them with melanin to protect against UVlight, so by their nature they're a wandering cell.  So if they begin to grow and divide out of control, they may first stay localize within the epidermis but if they're not removed, and they continue to grow, they can grow down into the dermis and then eventually into the adipose layer.  And at each one of these junctions, if they encounter blood vessels or lymphatic vessels, they may penetrate the vessels, get into the bloodstream of a lymphatic stream and be transported to other parts of the body where they metastasize in other organ systems such as the brain, the lung, liver, and that's ultimately deadly for the patient.  So one of our major jobs as histologists is to help not only diagnose if there's a melanoma, but to ensure that it's completely and properly removed.

Continuing with the schematics, if we look at a hair follicle, each hair follicle has structures associated with it.  Here is the hair shaft itself, accompanied by an arrector pili muscle which will pull that hair erect when necessary.  We have a nerve associated with the hair follicle, sebaceous glands, eccrine glands, and then you can see that at the bulb of the hair follicle, there may be melanin containing cells in the base of that as well and that can also be a source for a melanoma. 

H&E Stain

Now if we switch over and begin to look at H&E stain, haemotoxylin and eosin stain slide just to give you an idea of the relative thicknesses of the layers, here's the epidermis, which is quite thin, 7 to 12 cell layers thick.  You can see the dermis is somewhat thicker, comprised the area from here to here, and then the bottom layer, the adipose layer, and you can also see the base of many hair follicles throughout.

Continuing with H&E stains slides, we have a high power of a normal piece of skin.  We have the epidermis, again 7 to 12 cell layers thick with these projections which are called rete ridges, which help anchor the epidermis into the dermis.  Upon higher magnification, you can see a high mag of the epidermis, and insider or comprised within the epidermis these pigmented cells, brown pigmented cells, and those are melanocytes.  These ones happen to be normal melanocytes.

Again, back to low power, you can see there is one hair follicle here, with an associated sebaceous glands down at the base.  And at a higher power, we can see epidermis with associated hair follicles, one there, one there, and sebaceous glands which secrete sebum, which travels up the hair shaft, and provides the oily substance on your skin to help keep your skin soft and supple and seal out bacteria and viruses.  And then deeper in the dermis are eccrine glands which make sweat through the tubules that make their way up to the surface of the skin that provide you with the cooling mechanism.  So you can see full thickness here as well, epidermis is the dermal layer, and these open spaces down at the base are adipose cells which are empty due to the fact that the adipose and fat is removed and solubilized during tissue processing through the alcohols.

Skin Specimen Preparation

We’ll begin now, we have an understanding of basic skin histology, let's look at how we receive and prepare skin specimens.  The first step after receiving is to perform surgical grossing, get the specimens ready for tissue processing.  And of course we want to make sure that this is done very safely using personal protective equipment and proper ventilation.  Grossing personnel are also relied upon to be very accurate and to confirm accuracy of information on the requisition, and also primarily to be aware of specimens that will need inking of surgical margins.  In our laboratory, we ink all ellipses, assuming that they're done for an incision.  It can’t hurt to ink a specimen.  We use a 0.5% acetic acid solution to fix the inks to the specimen before we begin to surgically cut them.  And then we provider general grossing instructions to all grossing personnel as well as specific instructions for derm path specimens.

Now when working in dermatopathology as a surgical grossing tech you have to learn a new language, and this language has to be clear and concise.  A few of the terms that you may see are a flat pigmented lesion is referred as a macule.  If the lesion is raised, whether it’s pigmented or not, it could be referred to as a papule or a large, a plaque.  Sometimes specimens are received, many times ellipses, and they may have an ulceration or erosion in them, referred to as an ulcer.  Again sometimes within an elliptical specimen you may find a nodule, or a cyst like structure being contained within.  And finally polyps refer to fibro-epithelial polyps.  Many times they're removed for cosmetic reasons, they're almost always benign, usually ink is applied at the very base of the polyps so that you can see where it was attached to the patient.  Clear and concise surgical grossing terminology is indeed a must for accurate gross descriptions.

Continuing with specimen types that may be received in the laboratory, there are a few basic types, between four and five.  Punch specimens are exactly that, they’re cylindrical pieces of tissue.  Maybe done as a biopsy of a larger lesion, or simply an excision of a small lesion.  So that's why it's important to in the gross description when you describe a punch specimen to describe it exactly as that, received a punch specimen or a cylindrical punch specimen and do not refer to it as a punch biopsy because the punch could be being done as a biopsy, or being done as an excision and that is really up to the clinician to determine that designation.

Two millimeter punches, we receive them, we keep them as one piece.  I will show you the procedure and how we check them under the microscope after embedding to make sure that the orientation is correct.  Punches that are 3 to 5 mm in diameter fall into this classification where they would simply be bisected into two pieces.  And punch specimens that are 6 mm or larger are inked, they’re treated as excisions and cut into three pieces usually referred to as ends and body.  Those are usually done as an excision so we don't take any chances. 

And here's a good tip for you.  You may receive in your laboratory two punch specimens in one bottle.  We put them into separate cassettes, we'll called them specimen 1, specimen 2, or A1, A2, because it's possible that the clinician may have taken two separate biopsies or two separate punch specimens from two distinct areas on the patient.  And if you don't differentiate the two specimens in some way, that can be a problem if one of those turns out to be a melanoma and the clinician's office can't remember which one is which.  So we like to put them in separate cassettes.  If you don’t want to do that, you should at least ink them two separate colors so you can differentiate between the two of them.  That may be a medical/legal issue that can come back in the future.

We talked about 2 mm punches, they're very troublesome, they're very small, simply measuring 2 mm in diameter many times they're 2 mm deep as well so they're difficult to embed properly.  So the procedure in the laboratory is to first of all mark and note that the cassette contains a 2 mm punch, and then embedding personnel are to embed it as they see correct.  And then also put a sticker on the cassette so that when it goes to microtomy the histo tech who is doing the microtomy can go ahead and make surface cuts.  As soon as they see a specimen, they pick it up on a glass slide, they can bring it to the microscope in the laboratory and you can see that the condenser location on the microscope is underneath the stage, and here is the condenser right here.  You basically want to rack the condenser down, pull it down. 

Then what you can do it you can take that microscope slide that has a section on it, unstained, right off the water bath and it will look here in this fashion.  So the light diffracts through and it allows you to see the proper orientation or improper orientation of the 2 mm punch.  You can see the epidermis, you can see the dermis, and you can see the overall orientation is correct for the punch.  So this is what the specimen looked like upon first cut right off the water bath, and this is what it looked like once it was stained with the H&E.  It's exactly correct.  The reason to do this is that if the punch is missed embedded, if It's embedded with the epidermis down let's say, that first cut you want to save that and determine if that's incorrect, then you still have plenty of tissue to go back, melt it down and re-embed it.  If you simply just forge ahead and cut the specimen then you'll cut through the epidermis or tangentially and then the pathologist may not be able to make a diagnosis based on the incorrect orientation of the punch in the paraffin block.

Continuing with surgical grossing, so we have punch specimens, we have elliptical specimens; they are usually done as an excision of a tumor.  As I mentioned at our laboratory we always ink ellipses, so here we see some yellow epidermis with the brown macular on the surface, and we've inked the dermis and surgical margins blue, and then we'll go ahead and bread loaf the ellipse.  If a benign condition is indicated, for example if they suspect a cyst, it's acceptable to ink, bread loaf and then just submit the central piece for review by the pathologist but you need to make sure that you save the remainder of the tissue just in case it winds up being some kind of tumor.

Continuing with specimen types, so we've had a punch, we've had an eclipse, a shave specimen, and again I refer to it as a shave specimen, not a shave biopsy, they can be done as either, a biopsy or an excision.  So you have to check the requisition.  If a benign condition is indicated, you can simply bisect or trisect or quadrisect the shave.  If a cancer is indicated, you want to ink that first, and then pretty much treat it as an ellipse with ends and bodies. 

And additional specimen type is an aggregate, and an aggregate simply results from the scraping or curating of a lesion, you wind up with multiple fragments.  They're very difficult to orient, it's virtually impossible to orient all the pieces, so what we try to do or we instruct the histologist to pick out one or two of the biggest pieces and try to orient them correctly, put them on edge so that they get a correct dermal-epidermal junction orientation.

And finally we'll get specimens that we simply refer to as an irregular fragment.  We may not be able to tell which end is up, it might be a good idea to bring that and show it to a pathologist.  It's always a good idea to make a diagram so that you can go back and determine the correct orientation.

And then finally a fibroepithelial polyp or polyp refer to, also known as skin tags.  They're usually benign, but again we like to apply a drop of ink at the excision point just so the pathologist knows kind of which end is up and which end is relative to the patient.

The final most complicated specimen is an oriented ellipse, this is an ellipse that comes with some kind of orientation on it, and this schematic I've given you, this is a suture.  And the specimens are oriented by the clinician, they can use sutures, they might use notches, they might use ink to indicate a 12 o'clock position, or a superior position.  You always want to make sure that you check the requisition for such notations or diagrams.  Sometimes if it's a notch or a slit, it may not be evident by just looking at the specimen.  You want to make sure that you read the requisition.  And also once you've determine where the orientation is, whether it's a suture or an ink mark, you want to leave it in place while you prepare the specimen for the cassette.  And you also want to note in the gross description which cassette contains the orientation marker. 

You can see here that we will after inking we will actually do a double ink, red and blue ink to the surgical margins.  And then we'll go ahead and bread loaf the ellipse and make sure that we put each end, here's B1, here's B2, make sure you put those in separate cassettes because it is now an orient ellipse, and you can see that we've got four pieces of body left, we'll put two in each cassette.  We try to limit the number of pieces of tissue in a cassette to one or two, in some cases we may allow three but the more pieces of tissue you put in, the more chance you have of kind of miss embedding and not getting everything embedded on the same level.

Now providing you with kind of an actual preparation, this is a model that we used for demonstration purposes.  We have an ellipse, which is being measured, the length, the width, the depth, and then the description of I think you can see right here, there's a black macule present on the surface of the epidermis.  We'll measure the size of that as well.  Note whether it's centrally located or off center or adjacent to a margin.  And then simply go through and bread loaf the ellipse usually placed on the epidermis and then cut through with a surgical blade to provide uniformed pieces, ideally 2 mm thick. 

Here is an actual specimen, that one, the bottom part had blue ink applied to it.  The top has had red ink applied to it.  This diagram shows how that is applied blue and then red.  And then you can see on the final microscope slide the pathologist will be able to see the dermal-epidermal junction, they'll be able to see this blue area which are the tumor cells, and then they'll be able to see blue margin, and red margin, so that for each slide assessment, it's not only is the tumor present, but does it touch any of the surgical margin.  And if it does, you have to presume, the presumption is that there are tumor cells left in the patient which you now need to go back and remove further.  So the orient of the ellipse allows you to go back to the patient and just re-excise the area that has a positive margin.  So if it were positive on the blue margin, you could leave the red margin alone.

Okay, we have some various I wouldn’t say unusual but maybe rare specimens, maybe something a little bit different that you would receive, you'll receive them routinely in the laboratory, but you may not receive them frequently specimens for assessment of alopecia, which is hair loss, the pathological loss of hair.  You can see here in this patients, this is lots of hair loss going on.  And the rule here is to use the Headington procedure which is to basically in the surgical grossing procedure to cut parallel to the dermal-epidermal junction.  This is one of the few exceptions to the rule of preserving the dermal-epidermal junction.  And you can see why in this H&E, you can see that you've got one, two, three hair follicles, and these follicles are the ones that are located closer to the epidermis so they're somewhat smaller.  They have sebaceous glands associated with them.  When you go deeper into the dermis, here are the same hair follicles, one, two, and three, now associated with eccrine glands which are deeper in the dermis.  This is important because the pathologist as part of the evaluation, they count the number of hair follicles in the specimen and do the calculation to figure out number of hair follicles per square millimeter.  And this helps determine based on the patient demographic whether they've got pathological hair loss going on or it may be hereditary.

This H&E shows a section from a positive case of alopecia.  This is a hair follicle which has degenerated.  You can see it's not big and fat and plump like this one.  It's depleted.  Here's another one that's being depleted and you can also notice this fine infiltrate throughout the interstitium which indicates that there's an inflammatory process going on and there's some kind of infiltration of inflammatory cells. 

As we've mentioned, this procedure is referred to as the Headington procedure, and from the surgical grossing point of view, these specimens are virtually always punch specimens.  They're usually 3 or 4 mm punches, so the Headington procedure wants to identify roughly 1 mm or about in the middle of this punch specimen to cut it parallel to the epidermis.  Once that cut's been made to apply ink to the cut services, here's one piece, here's the other piece, put them in separate cassettes, and then in that fashion then the pathologist will be able to see cross sections of hair follicles, they'll be able to count hair follicles and make the diagnosis. 

Again, it's important to check the requisition to see if the Headington procedure applies, you've got to look for terms on the requisition such as alopecia, hair loss, hair thinning, traction, and baldness.  Those are all kind of key words that would indicate Headington.  Make sure to identify the cassettes so that the embedding team in the morning knows that it's a Headington case and to embed it properly, and that will indeed ensure proper embedding technique.

Specimen for immunofluorescence

Additional specimens that you may receive in your laboratory may be designated for immunofluorescence.  Now whether you do the procedure in your laboratory or you forward them to another part of your hospital or institution, to make sure that these specimens arrive in the proper fixative, which is very different than formalin fixative.  Specimens indicated for direct immunofluorescence of skin biopsies need to go into a medium called Michel's medium.  Sometimes it's called immunofluorescence transport medium, sometimes it's called Zeus medium, but this is basically a special precipitating buffer.  It's not formaldehyde.  In fact, if the tissue does go into 10% formalin, it will render it not usable for the DIF or the Direct Immunofluorescence procedure.  So you've got to really make sure that when the specimens arrive in your lab, they look different. We purposely make them look different.  You can see what this, where the blue arrow is is a formalin bottle with a yellow label on it.  It looks very much different than the red arrow specimen, which is a smaller bottle, it's got a blue top on it.  So that visually you want these to look very different so that when people receive them in the laboratory, and should they wind up going into the grossing area by mistake, you want your grossing team to be able to say, well, wait a second, this doesn’t belong here, we need to send this to the immuno lab.  So it's very, very important.  It's an additional challenge when you work at a reference laboratory because all your clients may not be using your bottles, you may get a different bottle so that's why it's important to put double eyeballs on this, both in terms of accessioning personnel who receive the specimen, and grossing personnel who will be opening up the bottle to ensure that it does not get forwarded into formaldehyde because the final result which you can see here is a distinct immunofluorescence pattern. 

This top micrograph where I've got the blue arrow on, this is staining of the dermal-epidermal junction with fluoresceinated antibodies.  And this bottom micrograph is intracellular staining of the epidermis.  The different types and patterns of staining are very important in what the pathologist uses to dictate and determine what the diagnosis will be.

Alright, I don't know about you and your team, but over the years I have noticed a steady increase in the number of very tiny and very small specimens received in the laboratory whether they be skin biopsies, whether they be needle biopsies, curating spine needle aspirates, so each laboratory must develop ways to handle very tiny specimens.  And there are several solutions to this issue, all of them have advantages and disadvantages.  I will tell you that sponges, the blue sponges, they're easy to use, however, we do not prefer them because they tend to retain processing fluids and also with very tiny biopsies they can slip into and kind of work their way into the sponge which makes them very difficult to remove the following morning or the subsequent embedding process to grab them with your forceps and pull them out.  You can also damage the pieces by doing that.

Another solution is to use mesh cassettes or biopsy cassettes, and these are a good solution, they've got a fine mesh on them.  However, you should be advised that some of them, because their holes are so small and the mesh is small, it's possible they become air locked especially if you stack the cassettes too tightly in the processing rack.  If they become air locked, it inhibits or sometimes prevents certain processing stations from moving into the cassette, infiltrating and penetrating the tissue and then moving back out again, so you can wind up with processing issues.

You can also use papers, histo wraps where you can spot tiny specimens out on a piece of paper, wrap it up, put the paper in the cassette.  But again in the morning you've got to unwrap it and grab it with forceps and fiddle around with it some more, you could break it so that's the disadvantage of that.

In our laboratory, we use HistoGel which is depicted here.  HistoGel is a compound that is liquid when heated to 60 degrees.  It can be dispensed on top of a tiny specimen.  And you can kind of add two or three or four drops and it builds up a gelatin button on top of the specimen and then it cools to room temperature and it becomes almost like Jell-o, so you can imagine a Jell-o mold that's got gelatin on the outside and the specimen on the inside.  The advantage of that is that the specimen itself will never be lost.  It's trapped inside the gel.  You don’t have to touch it again with forceps.  In the morning you can simply lift the gelatin button out, put it in place.  The disadvantage is that it's labor intensive, you've got to make sure you heat up the HistoGel before you use it.  And when you spot it out, you will not be able to orient the tissue inside the gelatin, however, the next morning during embedding, or after the embedding process, you’re able to identify the tissue.  And if you want to go and cut some of the gelatin away, some of the HistoGel away and give it a flat surface to ensure proper orientation, you can do that. 

As an example, I'll provide you with one particular case of a minute specimen. The specimen was described as a less than 0.1 fragment of tissue and it certainly was very tiny, so it was embedded in HistoGel, prepared, and was really so small the next morning we couldn't really find it, see it visually.  So we basically went ahead and cut 30 serial section slides, stained them all, and on slides number 5, 6, and 7 we were able to locate the piece of tissue which we additionally, one of those slides we decolorized and restained with a PAS stain.  You can see the positive fungal hyphae within the specimen.  So we were able to make a diagnosis with a very tiny piece of tissue.  Again, it's labor intensive but the bottom line is the specimen that doesn’t go missing and you don't have to handle it anymore with forceps.

Bone specimens

Why are we talking about bone specimens in a derm lecture?  Well, if you receive dermatopathology specimens you may also receive oral specimens which might be specimens of tongue or gum or mucosa but you may also receive parts of jaw, you may receive parts of teeth that may have tumors in them that you'll be expected to process and get something on a microscope slide.  So when dealing with bone, you want to make sure your first decision is what to use for a decalcifying agent.  Because we're a reference laboratory, we are not under the time constraints that some of you may be under in a hospital where you’re getting bone marrows.  So we're able to use 10% formic acid, in 10% formalin, which is not buffered, and this solution not only decalcifies the bone but it also continues to fix it so it results in this excellent bone histology that you see, and results in relatively easy cutting.  If you are in a hospital, you may have to use a rapid decalcifying agent, which can decalcify in a matter of hours instead of the formic acid from formalin which is actually going to be some days or even a week by the time it's all done.  But either way, whatever you pick for a decalcifying agent, two principles remain the same.  One is that the decalcifying solution which here is depicted in the schematic by the blue solution, that does get exhausted as it's used, so when you're doing your decalcification, unless it's a small, let's say unless it's a small bone marrow, tiny piece of bone marrow in let's say 100 mL container, other than that, any specimen bottle that it's received in you simply can’t just pour out the formalin, pour it on decal and put it on a shelf and leave it there for three days.  It's got to be changed every day because as the specimen decalcifies, the decalcifying solution is pulling out calcium and it's becoming exhausted.  So our procedure in the laboratory is to check all bone specimens every 24 hours, and replace anything that's not decalcified, replace the decalcification solution.

In addition, we agitate the solution.  You want to make sure that the decal solution is moving all the time.  In this schematic I've got a stir bar down at the bottom of the beaker.  I've got the cassette suspended so that the solution can pass through the cassette and have easy access to the specimen.  If you have multiple specimens, what we use as an orbital shaker, they each had individual specimen bottle, we put the bottle on a shaker and put that on a slow speed to continue agitation. 

And then the final step of handling in processing bones is to make sure of the decalcification endpoint, and there are a couple of different ways to do that.  One is a chemical method, you can use to determine that there's no more calcium present in the decalcifying solution.  You can use your manual determination where you just pick up the specimen and squeeze it, try to bend it.  That works, it's not as good as the chemical method, but it does work.  And then once the specimens decalcified, you need to rinse the decalcifying solution out of it, put it back into formalin and let it fix a little bit more, and then process on a large size process, don't use a rapid. 

And then after cutting the specimen the next morning during microtomy, we use gelatin coated slides to pick up the specimens.  It helps the bone specimens to adhere to the glass slide and get a good staining.

 And you can see in this micrograph, high power micrograph, the histology is quite excellent.  This is trabecula bone, which has this pink osteoid on the surface and you can see the location of osteoblasts which are making bone, there's one, there's two.  And then osteoclasts which are multi-nucleated cells which eat up the calcified bone to release calcium into the blood stream to maintain your calcium level.  And of course, fine histology, excellent histology out here in the bone marrow.  So the results are quite good and you too can get good results for your bone specimens.

You may receive specimens, patients that are suspected of having gout.  They may go to the dermatologist and complain of painful lesions within their skin because gout is caused by the precipitation of uric acid in the bloodstream.  So it may precipitate in joint areas in which case you may get a fine needle aspirate.  But it may also build up in just lesions inside the skin.  So if you are assessing specimens for the presence of urate crystals, for patients suspected of having gout, the clinician has to be educated to UVlight, you'll be able to see these urate crystals.

Tissue processing is straightforward.  You want to make sure that you don't overprocess. 

And then embedding you want to make sure that the epidermis is away from the embedder, and they're using a 40, 30 to 45 degree angle. 

In addition you want to place the number of panel of the cassette to the left so that when you load it into the microtome, you load it with the number of panel to the right, which ensures that you're cutting from dermis to epidermis. 

Embedding we talked about, make sure you indicate what a 2 mm punch may be in or something small. 

And then you want to standardize your work envelope to make sure that your blocks are moving  left to right, and that the only labeled slides that you have at the time of cutting are the ones for the one block that you’re working on.  Done blocks wind up over here.  If blocks need to be removed, they can go to the penalty box and be held out but you need to discard the slide to make sure it's not on the bench while you’re working on another case.

Microtomy for derm path, make sure that you get full sections.  This is an incomplete section.  Here's a complete section showing inked margin and epidermis. 

And then chatter, you want to make sure to minimize that.  It can be affected by fixative.  It can be affected by overprocessing but most importantly it can be affected by speed of cutting during microtomy.  Faster is not better.  You want to use a slow speed for skin specimens so that you don’t tear the epidermis away from the dermis. 

Everyone has their own H&E stain protocols.  You can use Harris or Gill's hematoxylin, Eosin-Phloxine, different bluing reagents.  If you're deparaffinizing using xylene substitutes, make sure that you're 100% alcohol stations are indeed 100% because some substitutes are very water intolerant. 

So you should be able to get the perfect H&E which is excellent nuclear detail, three shades of eosin, three shades of pink.

And you can get great results to show squamous cell carcinomas.

Basal cell carcinomas is a low power is normal epidermis in the lesion itself. 

And of course the one that's most troublesome that we need to look about are malignant melanoma.  On the lower power you can see some cells happening down here, down deep in the dermis, and on the high power you can see that they are melanocytic and they are malignant melanomas.

Nail specimens are taken for one of two reasons for fungal, the question of fungal infection, or question melanoma.  Here's a positive PAS .

Because the PAS stain will make sure and show not only with or without diastase, but it will show you fungal particles on the surface of skin, and within nail specimens.

So why is this all important?  Because your histology laboratory is a member of the healthcare team that helps to cure skin cancer.  If you have any questions in the future, you can contact me at this e-mail address, which is available, you can go back online and find it, or you can make a little note of it right now.  And if you want to incorporate any of the procedures that I've talked about, you may want to look at the most recent book that I've been able to publish with Dr. Dimenstein, Dermatopathology Laboratory Techniques.  Easily available from Amazon, just a couple of clicks, that's how everybody gets everything these days. 

So in closing I would like to thank you all for listening today and participating in the webinar.  I wish you success in implementing any of these techniques into your laboratory, and I'll hand over the webinar to Rick so that we can answer some questions. 

Questions and Answers

MODERATOR:  First is the stacking of the melanocytes normal?

DR. CHAPMAN:  Stacking or staining?

MODERATOR:  It says stacking, but we can look at it either way.

DR. CHAPMAN:  Okay.  So I think it's probably in reference to the micrograph that I showed that had a bunch of melanocytes kind of stacking up and grouping together.  Yes, that can be normal.  People that have freckles or pigmented... let see, macules on their skin, that's perfectly normal, it's perfectly benign.  The melanocytes can kind of group up and as long as they stay put, much as a congenital nevus, if they stay in place, everything is fine.  So the thing to be concerned about from a patient's point of view and from your own point of view is if you've got lesions, you've got a freckle that's now changed in color or it's bleeding or it looks like it's branching out, it has a rough edge, you need to see your dermatologist at that point.

MODERATOR:  Thank you very much.  And we do have a number of questions here but if you have some questions, send then into the Q&A box or the chat box.  We'll find them and ask them. 

Next question for you Cliff.  For histologists, skin histology is important.  Distinguishing layers of the skin in terms of structure and function helps with embedding.  What are the key pathological examples affecting skin and epidermal derivatives that the histologist should recognize?

DR. CHAPMAN:  I think that the histologist should be able to recognize when you receive an ellipse and they're big pieces of tissue, and you'll be able to see in the block visually that there's a lesion on top, there's like a bump or a lesion.  And what you should be able to recognize that when you put that block in your microtome and you cut it, when you're asking your slides and you’re laying it out on the water bath, you want to make sure that you've got a full face.  You want to make sure you've got the full epidermis and the full lesion because if you submit a slide to a pathologist and it's not full faced they're obliged to get levels.  And that just makes the case longer to sign out.  So we actually let our histologist look at those, look at the blocks, look at the ribbons on the water bath and they can decide to take a level if they want, and make another slide to ensure that they get a full face.  So that's what the histologist should be able to understand.

MODERATOR:  Okay, good.  Thank you.  Do you think it's best to put the B1 and B2 end pieces into another cassette?  Sometimes we get the entire specimen in one cassette.

DR. CHAPMAN:  Yes, standard practice from my point of view is when an ellipse is done, if it is an unoriented ellipse, both the end pieces can go into one cassette but they should go into a separate cassette as "the ends."  And then remainder of that ellipse should go into cassettes designated as the body.  If it is an oriented ellipse, then each end piece should get its own cassette to ensure proper orientation and should be diagram, written out and diagrammed by the grossing personnel. 

MODERATOR:  Okay, thank you.  Have you ever cut the pinch biopsies into three sections when doing cut parallels?  We at times would also cut the second piece in half vertically.

DR. CHAPMAN:  Okay, this refers to the need, the ultimate way, and this refers to the Headington procedure where you're cutting parallel to the epidermis.  The best scenario is when the clinician submits two punches, one you can do Headington, and one you can do vertically.  But as the participant points out in the question, you can the punch is big enough, you could trisect it, embed two of the pieces via the Headington procedure, and then the third piece vertically.  We don’t do that in this laboratory but it certainly can be done.

MODERATOR:  Wonderful, thank you.  We use a dye methylene blue on the processor.  We found it helps with locating tiny specimens when embedding.  It does not show on the H&E slide or interfere with marking dyes.  Do you use a dye on the processor?

DR. CHAPMAN:  The answer is yes, we don’t use methylene blue, we use eosin.  A little bit of eosin in the last alcohol before xylene and that imparts a slight pink tinge to these skin specimens, so you’re able to see a little more clearly upon embedding.  I think the current methodology is to add 5 mL, just 5 mL to a gallon of the last 100% alcohol.

MODERATOR:  Okay, good.  Thank you.  We have time for one, maybe two more questions.  For a specimen that have been placed in HistoGel to the IHC test showing non-specific uptake or false positive staining, with that said, should the lab tech inform the pathologist that the specimen was processed with HistoGel?

DR. CHAPMAN:  Yeah, the answer to the question is no.  The HistoGel has no effect on any immunohistochemistry staining and also it really has no effect on an H&E stain as well.  You may see remnants of the gel but they’re basically cleared and may be slightly pink but they have no effect on H&E staining, no effect on special stains, and no effect on immunohistochemistry staining.

MODERATOR:  Excellent.  Thank you.  Let me just everyone's attention quickly to the chat box.  Susanna has out the information that Cliff was talking about earlier on his last slide.  She's put that into the chat box so that you can copy and paste that, Cliff's e-mail address, thank you very much for that, Cliff certainly, and also the name of what I’m sure is an excellent book, knowing Cliff, into the chat box.

The last question that we have time for today Cliff is, what option do we have if a microwave can’t be used for a bone specimen?

DR. CHAPMAN:  The option is, and if you go in the Journal of Histotechnology archives, someone has talked about using heat and microwaves to kind of speed up decalcification of bone.  I'll be honest with you, I’m not a fan of heating any specimens more than they need to be heated.  All tissue is protein and the more you heat protein, the hotter it gets, physically hotter it gets.  So I mean other than extending the calcification times, that's what I would do.  I would just extend the calcification times or you could go to a more aggressive decalcifier if you're in more of a hurry.