Fixation on Histology

Nail Protocol: From H&E to IHCs Getting Nails to Stick

  

General Anatomic PathologyAnytime I hear a histotech talk about cutting nails it’s usually universally negative. It can be a battle to cut a nail block and create suitable sections. There is so much to consider to increase your success rate, like how to face in, if or how to soften the nail and what additives to use in the waterbath. When a section finally does get mounted onto a slide, all too often it will wash away. This vicious cycle can be downright soul killing. Once finally triumphant in this process, celebration ensues…until the pathologist sends back a request for immunohistochemistry (IHC) on the same nail (what do they think we are, some expert level wizards?). Fear not! I have some solutions. although I can hear the doubt in your mind already.

I work in a lab that specializes in skin, hair and nails where we cut several nail specimens per day in all shapes and sizes. Below I have outlined our protocol for getting nails to stick (nearly) every time after many trials and tribulations.

Pre-Processing:
In pre-processing, nails are sent to us in formalin or as clipping in a baggie which we then process in formalin. Lab assistants identify nails asking for fungal interpretation and pre-order a periodic acid-schiff (PAS) and H&E stains on the case. If the requisition mentions anything about “pigment”, “dark color” or flat out says possible melanoma, a panel of H&E levels x3, PAS, Fontana Masson, Melan-A and (preferentially expressed antigen in melanoma (PRAME) are automatically ordered. Typically, we will get small nail clippings submitted but we also get entire nails sent in, when this happens, our grossers section these nails at 3-4 mm. No preemptive softening of nails is used unless it cannot be avoided. We do this because no matter what, the techs will use a softener and since IHCs may be involved, we want to use as little as possible. We submit these nail specimens on a biopsy run in our microwave processors and they process as well as one could hope a keratin specimen would. That is to say, the nail pieces are still hard bits of keratin that are now slightly pink from the eosin we put in our processors. Other than that, the nails remain mostly unchanged.

Embedding:
I’m surprised at how many labs ignore this next bit but believe me, your life will be easier when cutting nails if the specimen is embedded at a 45˚ angle to the blade. Bones are not the only hard tissue we histotechs have to cut! Embedding nails at a 45˚ angle help prevent the pieces from popping out of the block, helps preserve the microtome blade and makes for smooth and complete sections to mount onto slides.

Microtomy:
Facing-
In this lab, we face nails at 5um. I know, tedious, but if embedded correctly it doesn’t take as long as it sounds. The benefit to facing in at a low micron is smaller nail fragments are not faced through or ripped out of the block from aggressive facing and larger nails pieces can get a full face without chunking out.  A full face is so important when looking for possible melanoma in a specimen.

Softening-
Here is where I’m going to lose a few people. We soften our nails using… [Pause for dramatic effect] Decal B … [Cue histotech outrage] I can hear you all roaring in unison “There’s no calcium in nails! Decal won’t work!!”. Whelp guess what?! Decal DOES work, and when used appropriately it works in a way that doesn’t sacrifice antigenicity for IHC slides unlike many traditional nail softeners. We’ve verified antigenicity preservation using skin specimens stained with IHCs without use of decal, post -treated with decal and then stained again. Here’s how we use decal for our nail specimens. After getting a full face on the block, each microtomy station has a small plastic specimen cup filled with decal B. The faced block is floated in decal (specimen side down) for 20-30 minutes. We’ve left specimens in Decal B overnight for particularly unruly specimens but 30 minutes max for specimens that require IHC. Afterwards, the block is removed from decal, rinsed in water and placed onto wet ice to soak for another 15-20 minutes. Do not forget to rinse the block! Decal can be corrosive to the metal faces on the microtome!

Microtomy-
Decal B doesn’t penetrate too far into the nails when soaked for a short period of time. There is usually only 1 good ribbon with 15-20 sections you can get when using this method. This is why we make sure the block is cut on the same microtome it was faced into on. We cut our ribbons at 3-4 microns. For IHCs, 3 microns if at all possible. The thinner the nail piece, the less the tissue edges will resist fluid deposition onto the slide. With our IHC platform fluid is directly dispensed onto the slide surface and cutting at thinner section helped circumvent nails washing off. All sections should be mounted onto a fresh charged slide. Old slides will cause tissue loss! If they have been laying around for a while, toss them (or at least only use them for H&E tissue slides). Our slides retain some water behind the sections so we draw the water off with a kimwipe. Finally, we place all of the slides in the oven at 65˚C for 45 minutes to an hour. If water is left behind the section while the slides are in the oven it will prevent adhesion to the slide. After baking, slides are sent for H&E, deparaffinization for special stains or for IHC staining.

If we’ve gone through all of this and the nail still falls off, we will repeat everything from facing, softening and microtomy with one small change. A TINY pinch of gelatin is added to the waterbath and mixed before floating the ribbon. We try to avoid this as gelatin causes some slight background staining. Our pathologists prefer a cleaner slide to read.  While we histotechs joke that we are not wizards (contrary to the belief of many pathologists) we are incredibly capable scientists, who when challenged, typically rise to the occasion! This method definitely isn’t perfect but it helps to alleviate a bit of the guesswork involved in cutting tricky nails!

Here are a few examples using this method:

1.)Nail Protocol Example

H&E                                                                           Melan-A

2.)Nail Protocol ExampleH&E                                                                           PAS


This is a good nail protocol but it isn’t perfect! Here is an example of a slide with some tissue displacement of a large nail specimen that was deemed interpretable by our pathologists:

3.)Nail Protocol ExampleH&E                                                                   Fontana Masson





Written by Ariel Liberda, BS




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